COMPLETE AFTER PROVISIONAL
LEFT ON 2 4 JUL 2001
FORM 2

THE PATENTS ACT, 1970
THE COMPLETE SPECIFICATION
(See section 10)
1. THE PROCESS FOR MANUFACTURING PHARMACEUTICAL COMPOSITION FOR PROVIDING PASSIVE IMMUNITY TO CANINE
2. Cadila Pharmaceuticals Limited, IRM House, Off C.G. Road, Navrangpura, Ahmedabad- 380009, Gujarat, India, an Indian company.
3. The.following specification particularly describes and ascertains the nature of this invention and the manner in which it has to be performed.

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FIELD OF INVENTION
The objective of the present invention is to provide a process of manufacturing a pharmaceutical composition for providing passive immunity to canine.
The further objective of the present invention is to provide passive immunity against canine distemper, canine parvovirus, infectious canine hepatitis, canine laryngotraceitis, canine parainfluenza type II, and canine leptospirosis.
The further objective of the present invention is to provide a pharmaceutical composition with lower incidence of serum sickness, allergic reaction etc.
The further objective of the present invention is to provide a pharmaceutical composition with higher amount of immunoglobulins and antibodies.
The further objective of the present invention is to provide a process with improved yield.
The further objective of the present invention is to provide a pharmaceutical composition in a solid dosage from so that it is easy to transport.
The further objective of the present invention is to reduce the amount of pharmaceutical composition to be administered as a single dosage form.
BACKGROUND OF THE INVENTION
Canine hepatitis, Leptospirosis, parvovirus infections, Distemper are major cause of death as an epidemic. Active immunity to these diseases is provided by vaccination in advance. However, it is not always practicable. This is more so when an outbreak of epidemic infections takes place. During the outbreak to prevent dogs from acquiring infections, the only measure left is to passively immunize them.

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Mainly canines ( dogs ) suffer from following diseases:
1. Canine Distemper
2. Canine Parvo
3. Infectious Canine Hepatitis ( CAV -1)
4. Canine Laryngotracheitis ( CAV - II )
5. Canine Parainfluenza Type II
6. Canine Leptospirosis
Diseases like canine parvovirus, canine distemper, infectious canine hepatitis, canine laryngotracheitis, and canine parainfluenza type II are viral in nature whereas canine leptospirosis is bacterial in nature. Important features of these include:
• They all are highly infectious and having high mortality rate. Today, due to owners' awareness and vaccination, though the incidence is around 30% of pet population, the death rate among the infected animals is around 80%.
• They occur often as mixed infection making diagnosis difficult and by the time the major one is controlled, the other one comes up and damages further.
• The major occurance is of Parvo infection, followed by Distemper and Hepatitis. The other three often complicate the clinical picture.
• The diagnostic facilities are almost non-existing (leaving vet to his judgement).
• High Fever is a common feature.
There is no specific treatment available for these viruses. All the treatment given is symptomatic in nature. The antibiotics (intravenous chloramphemicol, enrofloxacin, gentamicin, etc.) are used for treating and preventing the secondary bacterial complications. The saline/dextrose infusions for combating the dehydration due to diarrhoea and vomition (for at least 5 days). Antipyretics, analgesics, antispasmodics, antiemetics, liver extract + vitamin B complex + vit C injections, styptics for controlling intestinal haemorrhage, etc. Dehydration and weakness following the disease are major worry for the owner, during which secondary complications can occur.

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All above diseases are severe and fatal in nature. For prophylaxis there are no drug available in market. But there is some ( Modified / Inactivated ) vaccine available in market. The closest product is Homoserum which is manufactured by Pasteur Merieux, but not marketed or available in India. However,-Homoserum is a serum volume requirements for injection much higher and hence uncomfortable for the doctor and the animal. Also, the number of bleedings possible from the dog (source of the bulk active) is fewer, being a serum, hence cost of manufacturing is higher in case of Homoserum.
US patent 5807,551 describes a method to provide artificial passive immunity to birds by administration of superconcentrated antibodies to hatching eggs or to post-hatch birds. The patent describes the method to concentrate antibodies present in the serum.
All knowm methods of providing passive immunity uses specific antibodies or immunoglobulins derived from serum. The present invention describes a method of producing and using plasma rich in antibodies and immunoglobulins for use as providing passive immunity.
.REFERENCES:
1. US patent no. 5807551
Method to provide artificial passive immunity in birds.
Reynolds Donald L.
Iowa State University Research Foundation, Inc.

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SUMMARY OF THE INVENTION
The present invention provides a process for manufacturing pharmaceutical composition for providing passive immunity to canine.
According to present invention, the said composition is prepared by raising antibodies to these infectious agents in dogs, and isolating these antibodies containing homoplasma with very high antibody levels. The isolated antibody containing plasma is lyophilized under critical conditions and stored at 4-8 degree C before use. It is reconstituted by addition of diluent before use and given parenterally.
The composition so prepared is found to be stable and effective in providing passive immunity.
DESCRIPTION OF THE INVENTION
According to the present invention is described a process of manufacturing pharmaceutical composition to provide passive immunity to canine.
The mild strains of the viruses mentioned and bacterium Leptospira are injected into healthy dogs. The dogs are then reared for about 3-4 months, giving them enough time to allow them to develop resistance against those specific viruses. Then they are challenged by the virulent strains of the viruses and bacteria. Those dogs that have enough antibody titre to fight back the viruses will survive. The blood is taken out from such dogs every 3-4 months (the dog is not killed, but bled). Through centrifugation, plasma is separated which contains antibodies of all these viruses and bacteria. The antibody titre so obtained in the plasma will be good enough to fight back the disease, once the clinical signs have set in. The plasma is then lyophilized. The product is made available in vials in powder form, along with 10 ml water/diluent.

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EXAMPLE 1:
1. Select the dog of about 1 and half to two years of age preferably males , weighing between 30 - 50 kgs.
2. Screen the dog for Rabies antibodies, if present discard the dog.
3. Screen the dog for worms and if persist deworming is done.
4. Screen the dog for antibodies against following : Select the dogs which shows high titer.

> Canine distemper virus
> Canine parvo virus
> Infectious Canine Hepatitis Virus ( CAV -1 )
> Canine Parainfluenza Type- II
> Infectious Canine Laryngotracheitis Virus ( CAV - II )
> Leptospira icterohemorrhagica

5. Immunize the dog on 7th or 8th day with immunizing virus - bacterial antigen mixture i.e. 1.5 ml deep intramuscularly.
6. The second , third and fourth vaccination is done at the interval of 8 to 10 days.
7. After 8 to 10 days of last vaccination test bleeding is done to check the antibody titer of individual virus and bacteria.
8. After the last vaccination , dogs are challenged with virulent mixture of challenge viruses® 106TCID 50/ 0.25 ml per virus and for bacteria @ 106 cells/0.25 ml.
9. After 8 days of challenge check the serum of each dog for presence of any virus , no virus must be detected in the serum. Those dogs which withstand the virulent challenge are used for continuous plasma production.

10. Test bleeding is done to check the antibody titer of individual virus and bacteria.
11. The dog showing high titer are now bled by " Plasmapheresis" . wherein the erythrocytes and cellular components from blood are separated from the plasma which is retained outside and the cellular components are retransfused back into the animal body.
12. Now pool the plasma from all the dogs and again check the antibody titer of individual virus and bacteria.

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13. Methods of Detection of Antibody:
a) Canine distemper virus = 1. Haemagglutination Test using Human
"0"+ve RBCs.
2. Serum Neutralization Test
3. Complement Fixation Test

b) Canine parvo virus = Haemagglutination Test using Pig RBCs.
c) Infectious Canine Hepatitis Virus = Haemagglutination Test using Human
"0" +ve RBCs.
d) Canine Parainfluenza Type- II = Haemagglutination Test using Guinea Pig RBCs
e) Canine Laryngotracheitis Virus = Passive Haemagglutination Test
f) Leptospira icterohemorrhagica = Agglutination lysis test.
14. If the titer of plasma bulk is having protective titer then, it is checked for sterility,
pH , Clarity , Total Protein.
15. Then aseptically formulation is done by adding Gentamicin @ 0.2 mg / ml as a
preservative.
16. Then aseptically fill the 10 ml Glass vial @ 3.5 ml / Vial and is lyophilized. 17. Final product Quality control tests are done i.e.
a) Antibody titer
b) Sterility
c) Viable Virus Infection
d) Total Protein
e) Clarity
f) Pyrogen
g) Abnormal Toxicity h) Moisture Content
17. Store at 2° - 4° C temperature.
The above process can be represented in flow chart below:

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The canine homoplasma is the blood constituent of the dog blood i.e. blood without the RBCs , WBCs and Platelets, but has fibrin which is inactivated and antibodies against above diseases.
The homoplasma so prepared was subjected for stability and other studies.
Homoplasma is a translucent straw coloured fluid, soluble in distilled water/normal saline 5% dextrose saline, totally sterile, can be given intravenously, in a saline drip, sub-cutaneously or deep intramuscularly. It is moderately hygroscopic, lyophilized power which has to be reconstituted in distilled water before administration. It is stable at room temperature between 26°C to 30°C for about 5 days and between 2°C to 8°C it is stable for 2 years beyond which only 2 log titre reduction was observed in preceding 6 months.
In a test sample size of 40 vials, twenty were kept at room temperature and 20 at 2°C to 8°C for 2 years. These were tested for their total serum titres (log titres) every six months, and were found to be quite stable till two years. The titres reduced by 2 logs after one and half years i.e. during the third trial, those vials kept between 26°C and 30°C were tested every day @ two vials per day and the 5th day onwards recorded a steady decrease in titres @ one log to 2 logs per day, after reconstitution.
This canine homoplasma comprises of 3.5 ml canine homologus immune plasma with 0.2 ml Gentamicin (80mgs/2ml) as final concentration lyophilized in sterile conditions, in a 10ml vial, to be reconstituted with 10 ml sterile distilled water. The pH of this homoplasma is between 6.9 to 7.1, tested on reconstitution every six months, during the above trial.

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This canine homoplasma is made up of a mixture of passive antibodies, which have no target-organs and have short half lives comprising of 48 to 72 hours in cases where active infection persists, but maximum life of such passive antibodies is 12 to 15 days when given in healthy animals as a prophylactic measure. The antibodies being protein particles, immunoglobulin in composition, they undergo disintegration process and are voided out of the animal's body as non-infective protein complexes.
Due to this short half-life, the homoplasma is non-teratogenic, non-toxic in reaction, it contains no pyrogens or known microorganisms. It is not known to react with any other drug. It has been proven non-toxic, when given intraperitoneally in Guinea pigs and swiss albino mice.
Six guinea pigs, 12 mice and six rabbits were inoculated 0.5ml each of the Canine homoplasma intraperitoneally and observed for adverse reactions if any for a total period of three months. Three guinea-pigs, three rabbits and six mice were sacrificed after 8 days and organs histopathologically screened for inflammation, in duration/any abnormality, etc. No such defects were noted. Similarly after 3 months when rest of the animals were sacrificed and similarly observed no. defects were noted. In rare cases of reaction, antihistamines such as dexamethazone is advised, especially when given in a drip-form or when given intravenously the same is followed as in case of other similar immuno-therapies. The homoplasma on reconstitution is quite a translucent, straw coloured fluid in appearance, and on reconstitution trials with different fluids such as sterile distilled water, 0.89% normal saline solution and 5% dextrose saline solution all at room temperature it demonstrated total dissolution of all lyophilized particles, repeatedly.

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Therefore, it was concluded that the canine homoplasma is non-toxic and non-pyrogenic in reaction. It demonstrates good solubility and being a- homologous blood-plasma product it is totally excreted by the body as immune, complexes or disintegrated by the body in the normal course of time and cannot be teratogenic in origin.
The sterility of the product was evaluated after reconstitution, wherein about 20-30 vials were tested at random from a batch of 300 vials for their sterility by inoculating 0.1 ml of the reconstituted canine homoplasma in sterile pertone water for general aerobic organisms, 0.1 ml on Sabarouds agar plate for detection of fungi, 0.1 ml in thioglycollate agar for spore-forming bacteria and 0.1 ml in MDCK and vero cells for detection of viable viruses if any. All were incubated at 37°C except the Sabarouds agar plates, which were kept at room temperature, for about 8 days. There was no growth observed what so ever, on any of the media indicated above, proving the sterility of the canine homoplasma and deeming it fit for intravenous injection.
This canine homoplasma was also given to 4 pups intraperitoneal^ @ 5 ml neat serum to 3 pups subcutaneously in the flank region @ 5 ml neat serum and 4 4 pups . intravenously @ 5 ml diluted with 10 ml of sterile distilled water. None of the pups received the 'serotherapy-mandatory' dexamethazone/ betamethazone 0.5 ml. None of the above pups showed any immediately or delayed reaction, even after 8 days post-trial, proving it to be a very safe injectable homologous preparation..

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Once the standardization of the Immunizing (attenuated) virus mixture was perfected and the standardization of the challenge (Virulent) virus was completed, the clinical trials for testing the efficiency of the final product was undertaken, first on an experimental basis and later on, on canine patients admitted in various stages of sickness.
EXPERIMENTAL TRIALS:
The Experimental trials were formulated with prophylaxis and curative treatments in mind, groups were made for each type of trial i.e. pups, below one year's age, pregnant bitches, (the stage of pregnancy not being fixed) and dogs older than one year but not above five years. These animals were randomly selected with no differentiation in sex, size, breed etc. Most of the dogs were non-discript/mongrel type, the rest being crossbreeds both of whom have little chance of acquiring antibodies against any of the selected trial diseases, since none were vaccinated. Each group is shown below for prophylactic experimentation and therapeutic trials, the adults were both male and female selected at random for the trials, except in the pregnant groups.
These dogs were brought, dewormed and serologically tested for rabies antibodies, as previously stated in the chapter on "Selection and of care of dogs". Therefore in total there were 120 dogs, in both the trials, put together. The fluctuations in the serum titres of all the dogs are mentioned at the end of the chapter on Experimental trials, wherein the table depicts the entire summary of the exDerimentation.

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Prophylactic-therapy trials with Canine Homoplasma:
All the dogs in this group were administered the canine homoplasma after 10 days observation period was over; at the rate of 2 ml per kg body weight subcutaneously except the three control pups in Group 1. All pups weighed between 1.5-3 kgs.

Group 1 (Control) 3 pups as control were treated with normal canine plasma 2ml/ kg body weight, e.g. canine serum without those antibodies present in the canine homoplasma -t hese were challenged with virulent virus mixture after 8 days post-inoculation (pi).
Group 2 6 pups were given canine homoplasma 2 ml per kg body weight once and were challenged with virulent virus mixture after about 30 hours, pi.
Group 3 8 pups were given canine homoplasma 2 ml/ kg body weight once and were challenged after 4 day pi.
Group 4 7 pups were given the Canine homoplasma as in group 3 and challenged after 10th day pi.
All the above four groups were challenged on different days with 2.5ml plus 0.5 ml of virulent virus mixture deep intramuscularly and observed daily for symptoms of the diseases, which commenced after about 18-24 hours in case of Group 1 which had not received the Canine homoplasma at all. This group showed sings of tremors of the extremities anorexia, high fever (104°C to 105°C), bloody foetid diarrhoea, in all the 3 pups, shaking of the head, wailing and in general a very soggy and shaggy appearance. All the pups died between about 3-5 days despite supportive therapy.
The pups in Group 4, who were challenged after 10th day pi. started showing some fo the above symptoms on 7th day pi but much milder in nature. These pups showed high fever (103° - 104°) mild, tremors, slight diarrhoea but no blood. Only five pups out of seven recovered after about 3-4 days of supportive treatment. Total recovery time to attain normalcy for the five surviving pups was 10-13 days. (One very weak pup weighing less than 1.5 kgs died after 3 days). One pup suffered paralysis of hind legs and lingered on for an about 20 days after which it died.

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The supportive treatment consisted of normal saline 15 ml/kg body weight plus 2 ml polybion in the saline and 5 ml neohepatex or any liver extract which is injectable through the intravenous route. This entire therapy is given twice or thrice a day, as the need arises, but not more than thrice in case of pups.
Six pups in Group 3 after the 6 days p.i. showed only dullness and depression and mild fever (103°C) for about 2-3 days, after which they recovered and started feeding on their own. Total time taken to attain normalcy was about 8 days for all 8 pups. No supportive treatment was given.
In case of Group 2 all the six pups looked very wobbly and sick after 7 days pi. but they did not have fever or diarrhoea and did not stop feeding and looked more alert than those in Group 3. These pups recovered within three days without treatment and were normal in appearance and behaviour.
Apart from the protection afforded to these pups by the Canine homoplasma, these pups must be having their own maternal antibodies also, though smaller in quantity since these pups were strays from unvaccinated stray dams. As observed from the group 1, only maternal antibodies are not sufficient to overcome a virulent challenge, maternal antibodies have to be supplemented with concentrated antibodies from the Canine homoplasma. It was therefore concluded that the Canine Homoplasma worked best for pups within 8 days of giving the challenge i.e. the antibodies half-life in the homoplasma was about 8-10 days after which the antibodies started disintegrating very rapidly. The pups are the most susceptible groups of canines, when challenged within about 30 hours of administering the homoplasma (group 2), due to the maximum titres of the passive antibodies the pups did not exhibit much of symptoms and recovered very rapidly.

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Trial with Pregnant bitches: 12 numbers
Pregnant bitches, six in the initial stages of pregnancy - about 3-4 weeks pregnancy and six in late stages about 6-8 weeks (total 62-65 days is the gestation period in bitches) pregnancy were selected at random. Five bitches from the initial stages and five of the late stages of pregnancy i.e. total 10 bitches were given Canine Homoplasma @ 2 ml/kg body weight, except one bitch in the initial and other in late stages of pregnancy, which were segregated as control bitches. These two bitches were given normal canine plasma negative for the antibodies present in Canine homoplasma. All the twelve bitches were challenged with the virulent virus 2.5 ml plus 0.5 ml, deep intramuscularly and observed for the symptoms of the diseases and distress signs of abortion if any. The ten trial bitches were challenged, 48 hours after homoplasma was given.
Control bitches:
The two bitches one in early and other in late pregnancy which were not given any Canine Homoplasma, showed bloody foetid, diarrhoea, haematoemesis, high fever (104°C - 106°C), tonic convulsion of the extremities/head and succumbed before abortion within 35 - 40 hours of challenge being administered, i.e. they died due to the acute forms of the main diseases i.e. the Canine Distemper and the Canine Parvovirus. Supportive treatment was started immediately after onset of first symptom i.e. about 18-20 hours after challenge. This early onset was thought to be due to the extreme stress, which depletes the immune system that the pregnancy creates, which was not found in the pups.

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Early Pregnancy Group
All five bitches in this group showed anorexia as the first symptom after about 48 hours pi, had fever (104°C to 105°C), one bitch had very high fever (106°) and tonic convulsions on the head and extremities. This one bitch also started bleeding profusely per oss and aborted, due to which a litter of six pups was lost, despite starting the supportive therapy six hours after exhibition of first symptom. The bitch took more than 3 days to recover after the constant supportive therapy administration. The other six bitches recovered after supportive therapy administration within 15 to 20 hours, did not bleed nor did they abort, but showed dullness, anorexia, mild temperature of 103°C, roughness of body coat and in general debility.
Late Pregnancy Group
The five bitches in this group started showing the first symptoms after about 48 hours pi i.e. the same time as above group. Three bitches in this group were about 58-60 days pregnant and became anorexic, there was mild reddish discharge per oss, they exhibited breathlessness and one bitch after delivery six, normal pups was unable to walk properly (semi-paralytic) and had mild fever (103°), was dull for about 36-38 hours, but recovered after supportive therapy. The fever inability to walk, breathlessness in this bitch was attributed to eclamsia, which is due to calcium depletion during pregnancy, rather than any effect of challenge virus. The other two bitches also delivered, but remained dull and weak, could not clean their own pups, and had to be helped. They remained dull for the next two days but recovered after supportive therapy. The two bitches which were 50 - 55 days pregnant, remained dull, anorexic, rough coated and mild discharge tinged with blood continued till the bitches delivered. Both the litters were healthy and the pups survived. All the five bitches recovered after supportive therapy and delivered normally, without any loss of pups.

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Therefore it can be concluded that this type of prophylactic passive antibody therapy not only does not interfere with the pregnancy or parturition, but also helps the animals to withstand the onslaught of the virulent (challenge) viruses as well. It can also be concluded that when given at the beginning of the second stage of pregnancy, it is highly beneficial not only for the pregnant bitch but also for the litter, which has excellent chances of survival due to high maternal antibody titres and continuous flow of antibodies for the first eight days through milk.
Trials with adult dogs (males): 12 numbers
Similar to above were two control dogs and ten experimental dogs, all between two and five years old, seronegative for all the viruses mentioned in the challenge viruses.
Group I : Five dogs challenged 30 hours after receiving the Canine
Homoplasma.
Group II : Five dogs were challenged after seven days of receiving the
Canine Homoplasma.
Group III (Control): Instead of Canine homoplasma normal canine plasma was
administered to these 2 dogs which were challenged 30 hrs after grouping them randomly along with Group I.
To all dogs showing symptoms from the three groups, symptomatic supportive therapy was administered but the control group dogs succumbed 3 day pi. These dogs showed typical symptoms of CPV and CD viruses (as described in B) more prominently than others. The dogs in Group I exhibited mild aneroxia, dullness, fever and uneasiness but recovered within 48 hrs. pi, whereas those in Group II were all anorexic, had mild fever (103°C) and were dull in general. One dog after 3 days showed high fever (105.5°) and displayed convulsions and died suddenly. Such type of deaths are observed in cases of acute CPV infections wherein heart block occurs, due to infection of CPV in cardiac muscles. One dog showed slight blood in urine,

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anorexia, 106°C fever, and roughness of coat for about five-six days but recovery was slow. All 4 dogs normalized after 8-10 days pi.
From the above it can be concluded that the trend of recovery follows the half-life of antibodies, i.e. those dogs challenged within 30 hours of receiving the Canine homoplasma stood a better chance of survival than those who were infected at later stages, when the half life of most of the antibodies were at an ebb.
Advantages offered by the present invention over homoserum are:
1. Homoplasma yields 4 times more volume than homoserum.
2. Homoplasma has higher concentration of antibodies.
3. Homoserum has many specific and non specific inhibitors and factors which causes the Allergic Reaction, while Homoplasma causes least reaction and which are tolerable.
4. Homoplasma has more IgG than Homoserum. This nonspecific IgG is the most helpful during recovery of the patient from sickness, as no IgG is lost -whatever the body produces is ultimately used in the body - building mechanism.
Therapeutic/ Curative Trials with Canine Homoplasma
• Trials with 40 Pups:
The 40 pups in the age group between 5 months to 8 months, weighing between 1.5 kg to 3 kg were divided as follows at random :
Group 1: 4 pups as plasma controls were administered normal canine plasma @ 10 ml/kg body weight. This normal plasma did not contain the antibodies found in the canine - Homoplasma preparation. These were challenged with 2.5 ml plus 0.5 ml of the virulent virus mixture after 48 hrs.

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Group 2: 6 pups were given Canine Homoplasma 10 ml/kg body weight only once before challenge with virulent virus after 4 days.
Group 3:12 pups were given virulent virus mixture 2.5 ml plus 0.5 ml deep intramuscularly on day one and after 48 hours were given the 10ml/kg body weight of the canine Homoplasma. Four of the pups in this group did not show full symptoms whereas 8 pups showed severe diarrhoea with blood after about 36 hours.
Group 4:18 pups were given 2.5 ml plus 0.5 ml mixture of the virulent virus mixture on day one and were treated after 72 hours of showing a variety of severe symptoms, with supportive treatment along with 10ml/kg body weight of canine Homoplasma. All the pups were bled for titres before start of experiment and after the treatment at least once.
In case of group 1 all the four pups which were challenged succumbed about 48 hours after the challenge dose, the predominant symptoms were those of canine parvovirus, canine distemper and canine hepatitis, since these are fatal diseases of pups. They manifest in adult dogs as severe symptomatic diseases resulting into fatality due to secondary complications mainly.
In case of group 2, three pups did not show any symptoms and were quite normal in their appearance and behaviour. One pup passed about 13 times liquid foetid blood tinged diarrhoea and appeared as if it would succumb, but once administered with 30 ml of glucose saline and 10ml/g body weight of Canine homoplasma, the pup recovered within 30 hours and started feeding after about 48 hours. The other two pups looked dull, depressed did not feed and had 104°C temperature. Both were constipated and remained in the above condition till about 48 hours after which they recovered but slowly, without any further supportive medication except the Canine homoplasma.

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In case of group 3 wherein the pups were infected and the later on treated after 48 hours with canine Homoplasma irrespective of the degree of symptoms, the experiments stimulates the situation wherein all the animals who come in contact with infected environment and animals wherein the animals have to undergo passive immunization irrespective of their immune status to prevent any major calamity in terms of mortality. Most of the infected cases come late to the clinics i.e. after acute onset of the symptoms as in this group also, wherein five of the eight pups started with symptoms after 20 hours of the inoculation with the virulent virus. All the pups with severe symptoms such as 105°C - 106°C fever, vomination, bloody diarrhoea, icterus, subnormal body temperature, convulsions, incordination of movements etc were all administered 10 ml/kg body weight canine Homoplasma intravenously after the usual treatment with 0.5 ml of decadron to prevent anaphylaxis/ allergy if any. All the pups weighed less than five kg and therefore they were administered between 40 to 50 ml of the canine Homoplasma.
There was complete recovery in. all the eight pups within 48 hours of administration and the other four who received the same showed no symptoms at all. The four pups from this group were dull, depressed, had bloody diarrhoea and succumbed suddenly, such cases are seen most commonly in CPV infections.
In case of group 4, out of the 18 pups which were treated after about 3 days the onsetof the acute disease, only about eleven survived, from these eleven, seven pups were about 8-10 months old, whereas the other four were more than 3 months and less than 7 months old i.e. they were big enough to overcome immunologically the onslaught of the virulent virus till about 72hrs from the 7 which died, these pups were about three and half months to five months old, but smaller in size, weighing between two to four Kgs died within 24 to 60 hrs before receiving the treatment. The 11 which survived showed typical symptoms of mixed infection i.e. high fever (104° -106°C) dysentery, vomition, laced with blood, convulsions, etc leading to prostration.

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After intensive treatment with supportive therapy along with 10 ml/kg body weight of Canine homoplasma, did these pups show signs of slow recovery. Only the 7 older pups recovered and started feeding after about 48 hrs, the other pups which weighed less took about 3 days to recover and additional dose of 10 ml/kg body weight of Canine homoplasma was required.
• Trials with 20 pregnant bitches:
20 pregnant bitches were acquired in the early and late stages of pregnancy and divided into three trial groups - i}The early pregnancy group wherein there were eight bitches, out of which three were primi-para and fine with at least 3 gestations over ii} The second group comprised of the control group wherein one early and one late pregnant bitch would be inoculated with the virulent viruses only and the, iii} third group of 10 late pregnant bitches. All the bitches in the 3 groups were inoculated with the virulent virus mixture of 2.5 ml plus 0.5 ml, deep intramuscularly and observed for onset of the symptoms, which commenced after about 22 to 30 hrs later. All the bitches were given supportive therapy treatment and 0.5ml of decadron before serotherapy as a routine practice.
o The eight bitches in this group showed either mild vomition or mild diarrhoea, one of the primipara bitch was dull, anorexic and other four bitches had 103° to 104°C temperature, but no other symptoms exhibited. All the bitches were administered Canine homoplasma @ 10 ml/kg b.wt. alongwith 15 ml/Kg b. wt. Glucose saline intravenously within 12 hrs of showing various symptoms. Three of the pregnant bitches showed dramatic recovery and only one primi-para bitch aborted resulting into a loss of four pups which were dead, the bitch continued to vomit several times even after abortion. In case of this bitch the Canine homoplasma was repeated after about 24 hours only once. Seven bitches recovered totally after 48 hrs of supportive treatment with only one dose of Canine homoplasma, whereas one bitch took more than five days to come to normalcy.

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o Both the control bitches were not given the Canine homoplasma, only supportive treatment intravenously alongwith normal canine plasma, and both the bitches aborted after about 26-30 hrs post-inoculation with virulent virus, both succumbed to severe viral onslaught, showing mixed infections, the dominant symptoms being bloody, foetid diarrhoea, lacrimation, constant vomition and abortion. The late pregnant bitch died after 36 hours and the early pregnant bitch died after 40 hrs. In this group a total of nine pups were lost during abortion.
o In this group of 10 bitches who also started displaying symptoms 22 to 30 hrs post inoculation, one group of four bitches was treated at about 22 hrs i.e. immediately after the onset of symptoms with 10ml/kg b.wt. of Canine homoplasma and 15 ml per kg b.wt. of glucose saline. Out of these bitches two could not withstand the onslaught of the virulent virus mixture and aborted, these were given intensive supportive treatment with added liver tonic 5 ml in the above glucose saline and both the bitches had to be given 10 ml/kg body wt additional dose of Canine homoplasma after 18 hrs of the first dose and the supportive therapy was administered every six hours sin a drip form. A total of eleven pups was lost during abortion.
Three bitches although they showed milder symptoms of only vomition, dullness and lacrimation, continued to show these symptoms for about 3 days after the initial C. Homoserum dose. These were given only one dose, since their condition did not worsen any further and they did not abort. The other five bitches reacted by displaying, diarrhoea with blood, vomition laced with spots of blood, dullness, anorexia, severe lacrimation, incordination of movements, two bitches even showed yellow tinges to the mucosa. For these bitches treatment was delayed to 24 to 30 hrs after onset of symptoms and the Canine homoplasma administered with full precautions by dissolving 10 ml/kg b.wt in about 50 ml glucose saline and giving it as slow intravenous. The same was repeated on 3 bitches after about 30 hrs. Two of the above five recovered within 6 hrs of administration of Canine homoplasma,

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although they were very weak, but did not abort, one of the -remaining two recovered slowly, thus had to be administered the second dose of Canine homoplasma after 24 hrs of the first dose.
From the above it can be safely concluded that 'the Canine homoplasma can safely be given during early or late pregnancy, which is due to the fact that since it is formulated from the same homologous species i.e. the canine, the rate of reaction or allergy/anaphylaxis is minimal. Unless and until the dogs are in the last stages, with the virus/viruses having proliferated throughout the body tissues, the antibody or immuno-therapy has a dramatic effect as can be demonstrated from the experiments above.
As stated in the previous trials, these dogs were inoculated with the virulent virus mixture and it took about 24 - 36 hrs for the onset of the symptoms to initiate, since a stray adult dog has a fully developed immune system, ready to react strongly. These dogs were divided into three groups, wherein only i} 2 dogs were held in the control group which was not to be given supportive only, ii} The group was treated after about 6 - 10 hrs of the onset of symptoms and the last iii} group was treated after 24-30 hrs after the onset of symptoms.
i} The two control dogs initially showed symptoms of dullness, depression, anorexia followed by rise in body temperature to about 104.6°C to 105.8° for the first two days and third day onwards suffered from severe gastro-enteritis with copious bloody diarrhoea and vomition with blood clots, one dog died within six hours of the onslaught and the other succumbed after bout 36 hrs of the initial symptoms and in addition to above, also showed deep yellow mucous membranes, lacrimation and death due to severe dehydration.

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ii} In this group of 8 dogs, five were treated after 6 hrs of the onset of the symptoms, with 10ml/kg b.wt of Canine homoplasma and glucose saline @ 15 ml/kg b.wt and observed for signs of recovery. One male dog who showed an initial rise in temperature to about 104°C had copious stools which were black bloody with clots and foetid odour after which the dog could not support itself and collapsed. Thus dog was being given glucose saline and Canine homoplasma when he died, having subnormal temperature. This dog displayed typical signs and clinical symptoms of Canine parvoviral gastroenteritis. It is not common for adult dogs to succumb to the parvoviral disease and therefore it seems that either the dog was immuno compromised, or had a heavy worm load or a concurrent infection was present, whereby the dog could not sustain the onslaught of the fresh viral infection.
Out of the remaining four dogs, three showed typical symptoms of infectious Canine Hepatitis which is quite common in adult dogs, with a rapid development of icterus, coughing, lacrimation from the eyes, whitish yellow stools, pale mucos-membranes and anorexia. These dogs received the Canine homoplasma in the prescribed proportions with a glucose saline drip and after another 10 - 12 hrs recovered enough to drink water on their own. After another six hours they had bread and milk and were on their way to recovery. The fifth dog-a bitch apart from showing the mild gastro enteritis also vomited several times, had sub-normal temperature of about 100°C and was quite dull but recovered within 10-12 hrs of the prescribed treatment with Canine homoplasma and started feeding within another 3 hrs.
Three dogs were treated after 10 hrs of the onset of the symptoms which had become very acute with diarrhoea, vomition, pale yellow mucosa and two dogs showed total recumbency. All the three dogs were treated with the prescribed Canine homoplasma with glucose saline, but considering the acuteness of the disease they were administered by the show intra-venous route. After about 2 hrs later the vomition and diarrhoea stopped, but weakness, icterus and recumbency persisted, therefore glucose saline @ 15 ml/kg, b.wt alongwith neophepatex 5 ml

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total, was administered. Weakness, anorexia and subnormal temperature of 100°C persisted at times even tonic muscular spasms were observed, therefore another dose of Canine homoplasma was repeated after 24 hrs along with 50 ml glucose salien in drip form, this helped to relieve the symptoms drastically and after about 24-30 hrs the three dogs were drinking water and moving around, overcoming their recumbancy. From the 8 dogs in this group one died and the rest recovered from very critical conditions of the virulent viral attack. All the 7 dogs were feeding and behaving normally on the third day of the experiment.
iii) In group there were 10 dogs treated after 24-30 hrs of onset of the symptoms - all the dogs after 24 hrs of purging, vomition, high interns, coughing etc were in a bad shape, most of them were dull, depressed, dehydrated, 8 dogs had tonic convulsions, two dogs were only vomiting blood and were gaping for breadth, due to lowered haemoglobin levels, and fluid loss. In these two dogs, it was not possible to locate the veins and therefore the Canine homoplasma was given subcutaneously in the flank regions -i.e. in one flank the Canine homoplasma and in another side about 200 ml of dextrose salien with neohepatex 5 ml. After about two hours one dog recovered sufficiently to lick some water while in a sitting posture, but was unable to support himself on all four legs. The other dog during the same time was recumbent but alive. The former dog was given 200 ml glucose saline plus 5 ml neohepater after about 3 hrs in the flank region once again, but died after about 8 hrs of start of the treatment due to cardiac and respiratory arrest. The later dog which was recumbent died in a lingering manner without any further symptoms after about 6 hrs of the onset of symptoms. The eight dogs with convulsions were treated in a similar manner and were given 200 ml glucose/ dextrose saline and 5 ml neohepatex every three hrs and Canine homoplasma after the initial dose was given after 10 hrs interval. Three dogs showed rapid improvement and could drink water, stand up and take a few steps before falling. In these three dogs, the saline treatment was stopped after 6 hrs (two treatments) and only one more dose of Canine homoplasma was administered after 10 hrs alongwith the other dogs. Out of the remaining five

25
dogs two dogs lingered on with dullness, anorexia, subnormal temperatures and severe debility. After two days these dogs developed icterus and correa, despite a third dose of C. Homoserum given to all the eight dogs after 30 hrs. Both these dogs died of severe dehydration and respiratory failure on the fourth day, despite concentrated efforts with supportive therapy. These deaths were due to extensive tissue damage and collapse of the immune defence mechanisms, wherein intensive efforts to save their lives failed, due to lack of administration of treatment in the proper time (early treatment) i.e. treatment was given too late. Only three dogs survived, but were slow to recover from their acute onset, due to delay in the treatment.
All the three, after about 8 days showed correa, incordination of movement and erratic intake of feed. These three dogs were given a fourth dose of Canine homoplasma on the eighth day when the supportive treatment was stopped and the dogs after a further three days observation period were declared fit. This Canine homoplasma was given @ 2 ml/kg b.wt as a precautionary measure, to neutralise residual circulating antigens if any. There were reports by several clinicians who were given trial samples, that the residual symptoms of weakness, incordination of movements, correa, erratic feeding etc reduced considerably when at the end of the treatment period one dose of Canine homoplasma @ 2ml/kg b.wt. was administered.
Considering the above experimentation, it can be concluded that all the viruses and the bacteria administered as an virulent virus mixture before various treatment intervals follow a pattern of universal truth - i.e. prevention is better than cure, and that early treatment simulates a near preventive measure. The fact that the viruses and the bacteria involved are all celulolytic in nature, cause extensive tissue damage in very short periods of time especially, during an acute onset, means that treatment if any should be initiated as early as possible to avoid extensive and irrevocable tissue damage which leads to a total breakdown of the body-mechanisms and culminates in death.

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From the table, a conclusive pattern of survival can be noted, wherein the antibodies concentrated in the Canine Homoplasma have helped the affected dogs to recover rapidly.
The blood is taken for antibody serum titre after the observation periods, since there is no titre detectable in the unvaccinated dogs. The titres can be detected only when challenged, till then the antibodies are dormant and not activated by the memory forming B-lymphocytes. This conclusion was drawn after checking the titres of several pups and adult dogs. The pups had generally negligible titres of maternal antibodies, which totally disappeared on challenge.
The most common types of antibodies encountered were against the Canine distemper, canine parvovirus, infectious canine hepatitis, and least against Canine leptospira infections. The other two disease's antibodies, although fatal for dogs, were not commonly encountered. In case of the experimental trials, the average serum titres represent only those antibodies which are in circulation after the neutralization of the challenge virus. In case of pups these titres were low due to the late response of the immature immune system, whereas in case of the adult dogs and pregnant animals, the titres were much higher due to the response of a fully developed immune system. It is also felt that there must have been an add on titre due to the amnestic B-lymphocytic stimulation in dogs who had suffered from some of the diseases earlier in life and survive their onslaught.

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CLINICAL TRIALS OF CANINE HOMOPLASMA
Clinical trials were carried out on dogs/ pups/pregnant bitches which were brought by owners for either admission to the attached animal hospital or to the out patient departments or to the private clinics of the practising' veterinary doctors. Most of the animals brought by the owners were already suffering from acute stages of the disease/s or were in the incubation stages, whereby routine treatment generally aggravated their conditions. Any dog, whether adult or pup cannot survive when brought in the last stages of any nearly fatal sickness. These clinical trials were carried out over a period of three years from 1986 to 1989 and in the results represented here the dogs have been broadly classified in three susceptible groups i.e. Pups from 2 week to 12 months, B. Adult dogs from 1 year to 6 years and C. Bitches in various stages of pregnancy i.e. first (early pregnancy) transistor, and last trimester (late pregnancy), and recently parchurited (delivered) bitches. A. total of A 571 pups, B. 337 adult dogs and C. 297 bitches were administered the serotherapy with canine homoplasma, either in smaller or larger doses, i.e. in total the clinical trials of 1200 dogs are recorded in this study. For the sake of convenience, only the outstanding or complicated case histories are mentioned and discussed, otherwise a generalized clinical picture of these cases is presented and evaluated. Since most of the dogs were brought by the owners in a very serious condition, it was difficult to persuade the owners to allow the animal to be bled even for 5 ml blood, for fear that the animal may collapse and die. Therefore, serum titres were not taken in all the cases as done in the experimental trials. Those serum titres that were evaluated gave a fairly accurate diagnosis of the situation the animal was in. Most of the dogs, especially the pups were infected before primary vaccination was administered and therefore their titres represented the actual virulent strength of the viral infection. The serum titres in case of clinical trials were those of the disease which demonstrated first onset symptoms and the maximum symptoms i.e. Canine Parvo virus, Canine Distemper and varying numbers of animals showed icterus, typical in Infectious Canine Hepatitis/ Leptospirosis.

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Clinical Trials in 571 pups :
In case of pups the most fatal disease is the Canine distemper (CD) followed by the Canine parvovirus, infectious Canine Hepatitis. Canine Para Influenza, Canine Laryngotracheitis and lastly the Canine Leptospirosis. The common symptom in all these diseases is rising body temperature to about 103° - 105° C. The most outstanding symptom of CD in pups is cannot support itself, discharges from eyes and nose, twitching of muscles, at times convulsions, wailing, shaking of head and wobby gait.
In case of Parvo virus the symptoms are bloody diarrhoea, only blood clots fall instead of faecal matter, and pup also vomits blood, there may be blood in urine and generalized acute dehydration, followed by coma and death. In the cardiac form, there was sudden death, here the pups do not show any of the symptoms described above, only fall to the ground dead. This was seen also as a mixed form, i.e. the pup showed initially dysentery, was treated for about 2-3 days and suddenly died, at times towards the end of the treatment, while recovering. In these cases death was diagnosed as due to heart block, on post-mortum.
In case of mixed infections in pups mortality rate was higher and can be attributed to the fact that pups not only have weakly developed immune system, but due to their tender age, smaller size and body weight, they weaken easily due to dehydration. In pups all the vital organs such as the liver, spleen, kidneys, heart and even brain are still immaturely developed and cannot sustain the onslaught of the disease.

29
They even do not respond to the treatment and have to be given ready to immunize supplements such as Canine homoplasma, which gave heartening results in this category. About 10 ml (1 vial) homoplasma along with the full supportive therapy has to be administered every 18-24 hours. The pups were given supportive therapy comprising of 20-10.0 ml dextrose (5%) saline @ 20 ml/kg body weight, 0.5 ml to 2 ml of Neohepatex a crude liver extract and 0.5 to 1 ml of polybion - a vitamin B-complex supplement with Co-enzyme A.
Pups weighing within 1 kg, were those who suffered acutely and more than 20% dehydration by body weight was observed resulting into death. These mortality cases comprised of about 126 pups i.e. 22% most of these were from those litters, the dams of which were not vaccinated during epidemics in certain localities of the Mumbai city. In these cases the dams were either not vaccinated and were unable to transmit any immunity to the pups, whereas in some cases it was seen that the dame was already incubating the disease and the new born pups contracted the same, while suckling the dam within few days of birth. The other type of succeptible group of pups was the one which was about 1/4 to 2 months old unvaccinated, recently sold to new owners and due to infection in the environment, the pups caught the virulent viruses, the onset of such disease was not only very sudden but the disease spread • in the body rapidly with fatal consequences, at times even before treatment could be initiated.
The lowest mortality was encountered amongst pups in the groups which was slightly older i.e. between 2 to 6 months here the mortality was only 28 pups i.e. 5%. These pups weighed between 1 kg to 6 kg body weight and could be categorized as recently vaccinated pups with infection, and unvaccinated pups with infection. The 28 pups which died in this older age group, was mainly because some had been brought to the hospital and private clinics in very serious conditions and no amount of treatment could save these.

Amongst the first groups pups, the vaccination may have been given during the disease incubation stages and the disease flared up, or the vaccine may have been partially neutralized as in cases of residual maternal antibody activity and during an acute onslaught of the virus, resulted in inadequate protection. Another reason is that vaccination produces a stress and if given durjng epidemics in the locality, the antibody is not produced in sufficient quantities at short-notice and disease manifestation sets in to overtake the antibody production. Out of such 221 cases treated there was an overall response of 90% i.e. 199 pups recovered after the Canine homoplasma was administered to them with supportive therapy every 18 hours. Out of these 221 pups only 12 pups were required to be given three doses of Canine homoplasma on alternate days, the rest of the pups showed a dramatic break in the acute disease symptoms and recovered to about 80% after the second day of treatment. Some of these pups had suffered from mixed Canine distemper and parvovirus infection and these few pups showed residual symptoms of shaking their heads periodically, incoordination in gait, etc; but had resumed feeding normally as well as regained other normal body processes. In all the cases treated with Canine homoplasma, fever 104° C to 106°C was the first symptom to come down to 101° to 102° and in case of CPV, the dysentry was the first symptom to stop.
Mortality Percentage in Pups

Pups Mortality in Pups Overall Mortlaity
Pups < 1 kg Pups > 1 kg
517 126 28 154
22% 5% 27%
Overall recovery after treatment with Canine homoplasma was 73%.
In case of infectious canine hepatitis and canine para-influenza, the symptoms of jaundice, and acute respiratory distress and congestion of the chest were first to be relieved, within a span of 24 to 30 hours. Those cases, which showed dysentery, were invariably more serious than other cases, because apart from the disease aspect, severe dehydration and loss of blood were the other debilitating factors.

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Amongst the unvaccinated infected group the response was less dramatic, since there were no antibodies in their system and as soon as passive antibodies were introduced, the body defences reacted and partial virus neutralization occurred. These pups had to be given at least two more doses of the Canine homoplasma on alternate days to overcome and neutralize the circulating viruses. Similarly out of 571 pups infected in total, 417 survived and recovered resulting into 73% survival rate in treated animals and 27% mortality rate in weak and advanced cases which could not be saved. Since these were the owner-based pups and the owners refused to allow withdrawal of blood for titration, serum titres are not presented.
Clinical Trials in 337 adult dogs
In case of adult dogs the diseases showed no clear cut symptoms, dogs were presented starting with high fever (104° to 106°C) anorexic, dull, depressed at times very weak and since no differential diagnosis could be made, the treatment in such cases was terramycin - 2 ml (500 gms) and 5% dextrose saline @ 25 ml/kg body weight. If the animal after 24 - 48 hrs started showing any other symptoms, treatment was administered accordingly or if no response to above treatment was felt, then in addition Canine homoplasma @ 10 ml/kg body weight, was administered, because all the six diseases mentioned are initiated by high fever and above symptoms. Generally after the first dose the fever and other symptoms drastically reduced indicating that the animal was incubating any of the above diseases. The advantage of Canine homoplasma is that it also works in cases of non-specific diarrhoea and fever, resulting into total and rapid recovery.

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This may be due to the fact that during the generation of specific Antibodies a lot of non-specific IgG is also released into circulation as a humeral response of the immune system, apart from the cell mediated specific response. Since no ammonium sulphate precipitation is followed, during manufacture of Canine homoplasma, this IgG remains intact and helps in more rapid recovery. This IgG is not measurable since during the immunological tests known/ specific antigens react with the specific antibodies, but in the blood stream of the animals body, this IgG makes its presence felt and attaches itself to other antigens/ antigen-cell receptors etc.
Young adults about 13-15 months old were the worst affected and brought into the hospital / clinics quite debilitated, unconscious in conditions, especially true of dogs belonging to the smaller breeds category such as the pomoranians, pekenese, terriers, spitz and miniature breeds. The body weight of these dogs was hardly between 2 kgs and 5 kgs, therefore it was a hindrance for the much needed supportive therapy, since the Canine homoplasma had to be given half intravenously and half subcutaneously, since the dosage was 10 ml/kg body weight for the curative measures. In this group only two dogs out of 337 treated died due to adverse serum reaction, i.e. due to allergic causes, after administration of Canine homoplasma.
These two dogs were treated previously for about five days in a private clinic and when they did not respond to treatment, they were brought as emergency cases to the animal hospital having erythematous eruptions and on giving strong remedial antihistamine, went into a coma and died due to respiratory collapse. These are know as idiotypic serum reaction, wherein due to previous treatment with serum the dog is sensitised and idiotypic antibodies are formed, resulting into a type II hypersensitivity reaction, wherein severe complement erythrocytelysis occurs and the dog goes in a comatose condition due to histamine release etc.

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There were 33 cases which were presented with a mixed type of infection, i.e. severe jaundice with the belly of the dog totally yellow, body skin yellowish gray, discharge from the eyes, mild greyish diarrhoea, twitching of the ears, face and limbs periodically, 104°C constant fever, not responding to ordinary antibiotics and supportive therapy. Nine of these dogs which were totally dehydrated, slipped in a come and eventually succumbed even after treatment with Canine homoplasma and improved supportive therapy.
Table : Mortality Percentage in Adult Dogs.

Total Mortality in dogs Overall
Total Idiotypic dogs Mixed infection Dysentry dogs Mortality
337 2 9 6 17
6.7% 30.3% 20.2% 5.1%
Overall mortality in adult dogs was 5.1% and overall recovery after treatment with Canine homoplasma was 94.9%.
There were about 54 dogs which were presented with subnormal (99° - 100°) temperature, acute dysentery, vomiting laced with blood clots, difficulty in breathing, twitching of limbs etc. In 33 cases there was remarkable stoppage of bloody diarrhoea, cessation of vomition, twitching of limbs and normalization of body temperature on administration of first Canine homoplasma dose.
Out of 54 presentations only 6 dogs died. Five of the recovering animals had to be given another dose of Canine homoplasma for speeding up the process of recovery i.e. to neutralize those antigen particles which are released in circulation after about 24 - 30 hours during the phase of recovery from acute onset of disease (i.e. out of 337 adult dogs only 17 died and 320 recovered).

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Clinical Trials in 297 Pregnant bitches
Pregnancy as stated earlier causes great stress in the body and on the immune system of the bitch. During this period all the body systems contributes towards the development of the foetus, its immune system development etc particularly towards the late pregnancy period, the immune system of the bitch during this phase is weakened and totally unprepared to fight the disease/ antigen onslaught if any. Antibiotic therapy is generally avoided in pregnancy due to its aberrant action on the foetus. Whatever is injected parentrally into the body is secreted through the milk in pregnancy. Therefore to save the litter if the bitch is passively immunised about a week before delivery then the litter of the bitch not only has a good antibody titre but
retains the antibodies much longer, as the bitch's milk contains higher concentrations of secretary/ neutralizing antibodies. Only in rare cases would these passive homologous antibodies have idiotypic action, causing abortion. This is a risk to be taken when the environment is heavily contaminated or the bitch is in contact with other dogs who are actively suffering from any of the high-risk viral diseases.
There were about 131 bitches in early pregnancy, which were presented in the hospital and private clinics. From these 131, 82 were in contact actively with infected animals in their neighbour hoods and had started showing mild diseases symptoms, which could have been due to any of the above viruses. The most common symptoms included high fever (104° -106°) anorexia, reddish discharge per oss, five bitches had yellowish tinge on their hairless parts of the body. One older bitch had loose stools without blood.
The five bitches which had yellowish tinge to the skin, started vomition a day after admitting tot he hospital and were given Canine homoplasma about four days after onset of symptoms.

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Table : Mortality Percentage in Pregnant Bitches:

Total No. of Bitches Mortality in Early Pregnancy Pregnant Bitches Late Pregnancy Overall Mortality
297 4(1.34%) 1(0.33%) 5(1.68%)
Nos. of litters lost 2 Litters of 11 pups 7 litters of 32 pups 9 litters of 43 pups
Overall recovery after treatment with Canine Homoplasma was 98.32%.
' • ■ y
Two of the bitches which were vomiting at least 12-14 times a day aborted and had
subnormal temperatures, turned toxic and succumbed after 4-5 days of abortion. A
total of 11 pups were lost from the two. The other three stopped vomition and
regained normal body temperature after 24 hrs treatments with Canine homoplasma.
Therefore although there was only two fatalities amongst this group of five infected bitches, two precious litters totalling 11 pups was lost. The yellowish body tinge faded away after about a week and the 3 bitches were feeding normally about the same time as above. The older bitch with loose foetid smelling stools was lost. The yellowish body tinge faded away after about a week and the 3 bitches were feeding normally about the same time as above. The older bitch with loose foetid smelling stools was given the C homoplasma a day after the symptoms and therefore " recovered without any. further complications. The other 49 bitches in early pregnancy, showed varied symptoms, but 12 bitches showed mixed distemper and parvovirus symptoms and at times loose sanguinous stools. All the twelve were administered canine Homoserum about 24-48 hrs after onset of symptoms and except for one very young white bitch, a primipara about one year two months old, who collapsed and died. On post mortem heart muscle and intestinal contents showed presence of Canine Parvovirus (CPV) and the liver had haemorrhaegic patches. The other was about two and half years old with a congenitally enlarged heart and could not withstand the stress of pregnancy and distemper, which was later on isolated from the Urinary bladder.

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The 166 bitches in late pregnancy were admitted or brought to the hospital/ clinic in various stages of emergency, wherein they were administered first - aid and Canine homoplasma. 16 bitches were brought to 3 private clinics with severe eclamsia, wherein calcium and glucose saline was administered with decadron and Canine homoplasma as a precaution, since some of these bitches showed abdominal contractions. All were administered Canine homoplasma within first 24 hrs of the concentrations, but still six bitches aborted and six litters, (collectively 42 pups) were lost. No mortality was noticed in these bitches which eventually recovered, but pups could not be saved.
From 80 bitches which were on the verge of delivery and got infected, all were administered Canine homoplasma within 24-48 hrs after delivery. Only one bitch died of toxic complications with the litter unborn. There were about 42 bitches which gave birth to about 338 pups form 42 litters, out of which only 32 pup succumbed to infection, that too because they were too small to inject the Canine homoplasma. They weighed between 40-65 grm only. The larger pups weighing between 80-100 gms were given 0.5 ml subcutaneously Canine homoplasma and the (mother) bitch of the litter was given at the rate of 10 ml/kg b.wt. the full dose, so that should could secrete the antibodies in the milk and save herself and her litter. From the other 38 who showed symptoms of mild fever (103-105°C) off feed, vomition and weakness of the hind limbs, unable to support themselves, etc were put on high supportive therapy mixed with Canine homoplasma, these bitches required another dose about one week later, bud did not abort. They delivered 8-10 days after the start of therapy and were given the second dose just after delivery, so that there was sufficient protection for themselves as well as their litters. The overall recovery rate was 98.32%.

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It can therefore be concluded that in case of pregnancy, if the Canine homoplasma is given in time, it was capable of saving not only the life of the bitch but also was able to sustain the litter. Therefore it is suggested that the Canine homoplasma may be , given to the pregnant bitch two weeks before and one week after delivery to ensure a healthy dam and litter.

Group & No. No. Range of Serum titres %
No. of dogs Survived Dead ' - Recovery
Dead Survived
Prophylact 38 10 CPV :0-:128 128-1024 79.95
trials (48) CD :0-:128 ICH :0-:80 CPV2 :0-:80 CPI :0-:128 LH :0-:128 64-1024 40 - 640 20 - 320 20-512 16-512
a. Pups (24) 19 5 CPV:0-:128 CD :0-:128 ICH :0-:80 CPV2:0-:80 CPI :0-:128 LH :0-:32 1:128-10241:64-5121:40-3201:40-3201:20-2561:16-128 79.16
Group 1 (3) - 3 CPV :0- - -
Control • 1:128 CD :0-:64 ICH :0-:40 CPV2:0-:20 CPI :0-:0 LH :0-:0
Group 2 (6) 6 CPV-CD-ICH-CAV2-CPI-LH- :256-:512:128-:512:40-:320:40-:80:128-:256:64-:128 100
Group 3(8) 8. CPV-CD-ICH-CAV2-CPI-LH- :256-:1024:64-:512:80-:320:40-:320:128-:256:64-:128 100

B. Pregnant 10 2 CPV :0-: 128 :256-:1024 83.33
Bitches (12) CD :16-:128 ICH : 10-:40 CAV2 :20-:40CPI :0-:128 LH :0-:128 :128-: 1024 : 40-:640 :20-:80 : 64-:256 :64-:512 '
Control (2) 2 CPV: 0-:128 CD: 16-:64 ICH : 10^:40 ' CAV2 :20-10" CPI :0-:128 LH :0-:128 V
Early 5 - CPV- :256-:1024 100
Pregnant CD- :256-:1024
(5) ICH-CAV2-CPI-LH- :80-:640 :40-:80 :64-:128 :64-:256
Late 5 - CPV- :256-:1024 100
Pregnant CD- :128-:512
(5) ICH-CAV2-CPI-LH- :40-:640 :20-:80 :64-:256 :128-:512
C. Adult 9 3 CPV:0-:128 :256-:1024 75
Dogs (12) CD: 8-: 128 ICH : 0-:80 CAV2 :0-:80 CPI : 0-:64 LH :0-:128 :128-:512 :160-:640 : 20-:80 :64-:512 : 80-:256
Group 1 (5) 5 CPV-CD-ICH-CAV2-CPI-LH- :256-:1024:128-:512:160-:640:80-:160:256-:512:128-:256 100

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Group 2 (5) 4 . 1 CPV:0-:128 CD: 32-: 128 ICH : 0-:40 CAV2 :0-:80 CPI: 0-:64 LH :0-:128 :256-:1024 :128-:512 :160-:640 : 20-:80 : 64-: 128 : 80-: 128 80
Group 3 (2) - 2 CPV :0-:8 - -
Controlt - CD :8-:128 ICH : 10^:80, , CAV2 :0-:40 CPI : 4-:64 *--LH : 0-:8 »

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Therapeutic trials in dogs (40)

Group & No. No. Range of Serum titres 0//o
No. of dogs Survived Dead Recovery
A. Pups 25 15 CPV:0-:128 :4-:512 62.5
(40) CD 3p-: 128 ICH :t)-:320 CAV2 :0-:40 CPI : 0-:64" ■ LH :0-:16 :0-:1024:0-:1280:0-:80:0-:512:0-:64
Group 1 (4) - 4 CPV :0-:64 - -
Control CD : 0-:32 ICH :10-:40 CAV2 :10-:40 CPI : 0-:16 LH :0-:16
Group 2 (6) 6 CPV-CD-ICH-CAV2-CPI-LH- :4-:512:32-:1024: 10-: 1280:0-:80:0-:512:8-:64 100
Group 3 8 4 CPV:0-:128 :10-:512 66.6
(12) CD : 0-:64 ICH : 0-:320 CAV2 :0-:10 CPI : 0-:64 LH : 0-:0 :0-:256:0-:640:0-:40:0-:128:0-:8
Group 4 11 7 CPV:0-:128 :8-:512 61.1
(18) CD :0-:128 ICH :10-:40 CAV2 :0-:10 CPI : 0-:8 LH : 0-:0 :0-:1024:0-:320:20-:20:32-:256:0-:0

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B. Pregnant 18 2 CPV :0-:64 :0-:1024 90
Bitches (20) CD : 0-:32 ICH : 0-:40 CAV2 :0,:0 CPI : 0-:4 .. LH : 0-:8 :10-:1024:0-:320:0-:40:0-:256:0-:128
Early 8 - CPV- :10-:512 100
Pregnant CD- :40-:-1024
(8) ICH-CAV2-CPI-LH- :0-:160 :0-:40 :0-:128 :0-:64
Late 10 - CPV- :0-:1024 100
pregnant CD- :10-:256
(10) ICH-CAV2-CPI-LH- :0-:320 :0-:40 :0-:256 :80-:128
Control (2) 2 CPV :0-:64 CD : 0-:32 ICH : 0-:40 CAV2 :0-:0 CPI : 0-:4 LH : 0-:8
C. Adults 13 7 CPV:0-:128 :4-:1024 65
(20) CD: 0-:128 ICH :20-:80 CAV2 :0-:40 CPI :0-:128 LH : 0-:256 :0-:512:10-:320:0-:80:0-:256:0-:128
Group 1 (2) - 2 CPV :0-:32 - -
Control CD :0-:128 ICH :20-:80 CAV2 :0-:40 CPI : 0-:64 LH : 0-:32

42

Group 2 (8) 7 1 CPV :16-:128CD : 0-:128ICH :20-:40 :32-:512 :64-:512 :40-:320 :40-:80 87.5
CAV2 :0-:40 CPI : 8-:64 LH : 0-:256 :64-:256 :8-:128 i.
Group 3 6 4 CPV :8:128 '* •4-: 1024.. 60,
(10) CD: 4-: 128 ICH :40-:80 CAV2 :0-:40 CPI : 16- :(P:51-2. , :10-:320 :0-:10 :0-:64
:128 :0-:128
._. LH : 0-:32
Total no. of dogs in experimental trials : 88 Nos.
From the above table it is seen that only the surviving animals have attained the requisite titres. In this experimental group the pups, had no titres initially and developed titres on giving the C homoplasma as in case of the prophylactic group or developed the titres after virulent virus infection as in case of therapeutic trials.
Only in case of a small percentage (2-3%) adults had a negligible titre for CD/CPV, . but otherwise were clean i.e. no initial titres. In case of the pregnant bitches, only 2 bitches had initial titres for CD (1:10 and 1:40) and one had titres for C. Leptospira, i.e. 1:80, but did not demonstrate live bacteria, through urine, this was considered to be due to pregnancy stress.

43
Table depicting the Clinical Trials : Conclusive evidence of efficacy of Canine homoplasma:

Groups & No. No. % Range of Serum titers
No. of dogs survive Dead Recove
d ry
Survived Dead
A. 571 417 154 73% CPV: 16-1024 16-:256
Pups CD: 257-1024. 16-:128
ICH : 40-640. , CAV1: 64-256 CAV2: 40-160 LH : 32-256 20-:40 8-:128 10-:40 4-:64
B. 337 320 17 94.9% CPV: 64-1024 32-: 128
adults CD: 128-1024 ICH: 40-1280 CAV1: 64-256 CAV2: 80-320 LH: 64-512 16-:128 20-:40 32-: 128 20-:40 64-: 128
C. 297 292 5 98.32% CPV: 128-1024 16-: 128
Pregnant CD: 128-1024 32-:256
bitches ICH :80-1280 CAV1 :64-256 CAV2 :40-160 LH : 64-256 10-:40 . 16-:64 10-:20 32-: 128
No. of - 9 i.e. 43 -
litters lost pups
Total No. of dogs : 1205 Nos.
CPV - The HI titre of 1:256 is considered as protective, the range of titres i.e. lowest
and highest is mentioned.
CD - the titre of 1:128 is considered as protective, the range of the titres is depicted
in the table.
ICH - The CFT titres of 1:80 is considered protective the range is as above
CPI - The HI titres of 1:128 is protective, the range is as above.
CAV2 - the CFT titre of 1:80 is protective, the range is as above.
LH - The agglutination titre of 1:128 is considered as protective, the range is as
above.

44
• The titres of the litters could not be measured since, these were born dead or died immediately after birth.
• Amongst the pups, out of 41 surviving pups only 54 could be bled and 82 pups out of 154 dead could be bled for titres, the owners refused permission for bleeding.
• Out of 320 adults only 32 could be bled for titres and .out of the 17 dead about 10 dogs could be bled, all were owned dogs.
• Out of 292 surviving bitches, since there was negligible difference in the titres of the early and late pregnant bitches, these were not differentiated but given as single titres.
From the above tables it is seen that some of the dead dogs have high titre to some diseases which is became they have no circulating antigens for these diseases, due to which there is no neutralization or complex formation by the antibody. The dog can show low titres, which means that there is active neutralization of the antigen occurring after giving the Canine homoplasma. Lower antigen titres antibody of one disease and high antibody titres of other would mean that the former antigen is out of the tissuecells and is undergoing neutralization by the antibody, whereas only high antibody titres would mean that either the antigen has still not come out of the tissue, as in case of incubation, or the animal does not have that particular antigen/disease. The dog which become weak physically cannot cope with the infection i.e. invasion of the disease agent immunologically, and generally succumbs during an acute onslaught, although his antibody titres are quite high.

45
In case of pups, the serotherapy with Canine homoplasma acts as a boon, since along with their own depleting antibodies, these antibodies give them the neutralizing power and therefore high recovery rate. In case of the pregnant bitches the antibodies being homologous in nature are readily accepted, resulting into natural delivery and no abortion. In adult dogs also, some residual antibodies, which are not sufficient to overcome acute infections and an amnesic response takes about 2 to 4 days to bring about antibody production, therefore the Canine homoplasma turns out to be a boonf or such animals.
The recovery rate is a definite conclusion of the efficacy of the Canine homoplasma's role in neutralizing active infection.

46
We claim:
1. The process for manufacturing pharmaceutical composition for providing passive
immunity to canine comprising of following steps:
a) Selecting an animal,
b) giving an injection of antigen of organism to animal,
c) giving booster dose of antigen,
d) checking antibody titre,
e) removing plasma from the animals,
f) adding antibiotic to the plasma,
g) filling plasma into glass vial, h) lyophilization.

2. The animal as claimed in claim 1 can be canine.
3. The canine as claimed in claims 1 and 2 can be a dog.
4. The organism as claimed in claim 1 is canine distemper, canine parvovirus, infectious canine hepatitis, canine laryngotraceitis, canine parainfluenza type II, and canine Leptospirosis.
5. The antibiotic as claimed in claim 1 is selected from broad spectrum antibiotic.
6. The antibiotic as claimed in claim 1 and 5 is gentamicin.
7. The method as claimed in claim 1 and herein described in example 1.
Dated this 23rd of July, 2001

Dr. Bakulkesh Mafatlal Khamar
COMPLETE AFTER PROVISIONAL
LEFT ON 2 4 JUL 2001
FORM 2

THE PATENTS ACT, 1970
THE COMPLETE SPECIFICATION
(See section 10)
1. THE PROCESS FOR MANUFACTURING PHARMACEUTICAL COMPOSITION FOR PROVIDING PASSIVE IMMUNITY TO CANINE
2. Cadila Pharmaceuticals Limited, IRM House, Off C.G. Road, Navrangpura, Ahmedabad- 380009, Gujarat, India, an Indian company.
3. The.following specification particularly describes and ascertains the nature of this invention and the manner in which it has to be performed.

2
FIELD OF INVENTION
The objective of the present invention is to provide a process of manufacturing a pharmaceutical composition for providing passive immunity to canine.
The further objective of the present invention is to provide passive immunity against canine distemper, canine parvovirus, infectious canine hepatitis, canine laryngotraceitis, canine parainfluenza type II, and canine leptospirosis.
The further objective of the present invention is to provide a pharmaceutical composition with lower incidence of serum sickness, allergic reaction etc.
The further objective of the present invention is to provide a pharmaceutical composition with higher amount of immunoglobulins and antibodies.
The further objective of the present invention is to provide a process with improved yield.
The further objective of the present invention is to provide a pharmaceutical composition in a solid dosage from so that it is easy to transport.
The further objective of the present invention is to reduce the amount of pharmaceutical composition to be administered as a single dosage form.
BACKGROUND OF THE INVENTION
Canine hepatitis, Leptospirosis, parvovirus infections, Distemper are major cause of death as an epidemic. Active immunity to these diseases is provided by vaccination in advance. However, it is not always practicable. This is more so when an outbreak of epidemic infections takes place. During the outbreak to prevent dogs from acquiring infections, the only measure left is to passively immunize them.

3
Mainly canines ( dogs ) suffer from following diseases:
1. Canine Distemper
2. Canine Parvo
3. Infectious Canine Hepatitis ( CAV -1)
4. Canine Laryngotracheitis ( CAV - II )
5. Canine Parainfluenza Type II
6. Canine Leptospirosis
Diseases like canine parvovirus, canine distemper, infectious canine hepatitis, canine laryngotracheitis, and canine parainfluenza type II are viral in nature whereas canine leptospirosis is bacterial in nature. Important features of these include:
• They all are highly infectious and having high mortality rate. Today, due to owners' awareness and vaccination, though the incidence is around 30% of pet population, the death rate among the infected animals is around 80%.
• They occur often as mixed infection making diagnosis difficult and by the time the major one is controlled, the other one comes up and damages further.
• The major occurance is of Parvo infection, followed by Distemper and Hepatitis. The other three often complicate the clinical picture.
• The diagnostic facilities are almost non-existing (leaving vet to his judgement).
• High Fever is a common feature.
There is no specific treatment available for these viruses. All the treatment given is symptomatic in nature. The antibiotics (intravenous chloramphemicol, enrofloxacin, gentamicin, etc.) are used for treating and preventing the secondary bacterial complications. The saline/dextrose infusions for combating the dehydration due to diarrhoea and vomition (for at least 5 days). Antipyretics, analgesics, antispasmodics, antiemetics, liver extract + vitamin B complex + vit C injections, styptics for controlling intestinal haemorrhage, etc. Dehydration and weakness following the disease are major worry for the owner, during which secondary complications can occur.

4
All above diseases are severe and fatal in nature. For prophylaxis there are no drug available in market. But there is some ( Modified / Inactivated ) vaccine available in market. The closest product is Homoserum which is manufactured by Pasteur Merieux, but not marketed or available in India. However,-Homoserum is a serum volume requirements for injection much higher and hence uncomfortable for the doctor and the animal. Also, the number of bleedings possible from the dog (source of the bulk active) is fewer, being a serum, hence cost of manufacturing is higher in case of Homoserum.
US patent 5807,551 describes a method to provide artificial passive immunity to birds by administration of superconcentrated antibodies to hatching eggs or to post-hatch birds. The patent describes the method to concentrate antibodies present in the serum.
All knowm methods of providing passive immunity uses specific antibodies or immunoglobulins derived from serum. The present invention describes a method of producing and using plasma rich in antibodies and immunoglobulins for use as providing passive immunity.
.REFERENCES:
1. US patent no. 5807551
Method to provide artificial passive immunity in birds.
Reynolds Donald L.
Iowa State University Research Foundation, Inc.

5
SUMMARY OF THE INVENTION
The present invention provides a process for manufacturing pharmaceutical composition for providing passive immunity to canine.
According to present invention, the said composition is prepared by raising antibodies to these infectious agents in dogs, and isolating these antibodies containing homoplasma with very high antibody levels. The isolated antibody containing plasma is lyophilized under critical conditions and stored at 4-8 degree C before use. It is reconstituted by addition of diluent before use and given parenterally.
The composition so prepared is found to be stable and effective in providing passive immunity.
DESCRIPTION OF THE INVENTION
According to the present invention is described a process of manufacturing pharmaceutical composition to provide passive immunity to canine.
The mild strains of the viruses mentioned and bacterium Leptospira are injected into healthy dogs. The dogs are then reared for about 3-4 months, giving them enough time to allow them to develop resistance against those specific viruses. Then they are challenged by the virulent strains of the viruses and bacteria. Those dogs that have enough antibody titre to fight back the viruses will survive. The blood is taken out from such dogs every 3-4 months (the dog is not killed, but bled). Through centrifugation, plasma is separated which contains antibodies of all these viruses and bacteria. The antibody titre so obtained in the plasma will be good enough to fight back the disease, once the clinical signs have set in. The plasma is then lyophilized. The product is made available in vials in powder form, along with 10 ml water/diluent.

6
EXAMPLE 1:
1. Select the dog of about 1 and half to two years of age preferably males , weighing between 30 - 50 kgs.
2. Screen the dog for Rabies antibodies, if present discard the dog.
3. Screen the dog for worms and if persist deworming is done.
4. Screen the dog for antibodies against following : Select the dogs which shows high titer.

> Canine distemper virus
> Canine parvo virus
> Infectious Canine Hepatitis Virus ( CAV -1 )
> Canine Parainfluenza Type- II
> Infectious Canine Laryngotracheitis Virus ( CAV - II )
> Leptospira icterohemorrhagica

5. Immunize the dog on 7th or 8th day with immunizing virus - bacterial antigen mixture i.e. 1.5 ml deep intramuscularly.
6. The second , third and fourth vaccination is done at the interval of 8 to 10 days.
7. After 8 to 10 days of last vaccination test bleeding is done to check the antibody titer of individual virus and bacteria.
8. After the last vaccination , dogs are challenged with virulent mixture of challenge viruses® 106TCID 50/ 0.25 ml per virus and for bacteria @ 106 cells/0.25 ml.
9. After 8 days of challenge check the serum of each dog for presence of any virus , no virus must be detected in the serum. Those dogs which withstand the virulent challenge are used for continuous plasma production.

10. Test bleeding is done to check the antibody titer of individual virus and bacteria.
11. The dog showing high titer are now bled by " Plasmapheresis" . wherein the erythrocytes and cellular components from blood are separated from the plasma which is retained outside and the cellular components are retransfused back into the animal body.
12. Now pool the plasma from all the dogs and again check the antibody titer of individual virus and bacteria.

7
13. Methods of Detection of Antibody:
a) Canine distemper virus = 1. Haemagglutination Test using Human
"0"+ve RBCs.
2. Serum Neutralization Test
3. Complement Fixation Test

b) Canine parvo virus = Haemagglutination Test using Pig RBCs.
c) Infectious Canine Hepatitis Virus = Haemagglutination Test using Human
"0" +ve RBCs.
d) Canine Parainfluenza Type- II = Haemagglutination Test using Guinea Pig RBCs
e) Canine Laryngotracheitis Virus = Passive Haemagglutination Test
f) Leptospira icterohemorrhagica = Agglutination lysis test.
14. If the titer of plasma bulk is having protective titer then, it is checked for sterility,
pH , Clarity , Total Protein.
15. Then aseptically formulation is done by adding Gentamicin @ 0.2 mg / ml as a
preservative.
16. Then aseptically fill the 10 ml Glass vial @ 3.5 ml / Vial and is lyophilized. 17. Final product Quality control tests are done i.e.
a) Antibody titer
b) Sterility
c) Viable Virus Infection
d) Total Protein
e) Clarity
f) Pyrogen
g) Abnormal Toxicity h) Moisture Content
17. Store at 2° - 4° C temperature.
The above process can be represented in flow chart below:

8
The canine homoplasma is the blood constituent of the dog blood i.e. blood without the RBCs , WBCs and Platelets, but has fibrin which is inactivated and antibodies against above diseases.
The homoplasma so prepared was subjected for stability and other studies.
Homoplasma is a translucent straw coloured fluid, soluble in distilled water/normal saline 5% dextrose saline, totally sterile, can be given intravenously, in a saline drip, sub-cutaneously or deep intramuscularly. It is moderately hygroscopic, lyophilized power which has to be reconstituted in distilled water before administration. It is stable at room temperature between 26°C to 30°C for about 5 days and between 2°C to 8°C it is stable for 2 years beyond which only 2 log titre reduction was observed in preceding 6 months.
In a test sample size of 40 vials, twenty were kept at room temperature and 20 at 2°C to 8°C for 2 years. These were tested for their total serum titres (log titres) every six months, and were found to be quite stable till two years. The titres reduced by 2 logs after one and half years i.e. during the third trial, those vials kept between 26°C and 30°C were tested every day @ two vials per day and the 5th day onwards recorded a steady decrease in titres @ one log to 2 logs per day, after reconstitution.
This canine homoplasma comprises of 3.5 ml canine homologus immune plasma with 0.2 ml Gentamicin (80mgs/2ml) as final concentration lyophilized in sterile conditions, in a 10ml vial, to be reconstituted with 10 ml sterile distilled water. The pH of this homoplasma is between 6.9 to 7.1, tested on reconstitution every six months, during the above trial.

9
This canine homoplasma is made up of a mixture of passive antibodies, which have no target-organs and have short half lives comprising of 48 to 72 hours in cases where active infection persists, but maximum life of such passive antibodies is 12 to 15 days when given in healthy animals as a prophylactic measure. The antibodies being protein particles, immunoglobulin in composition, they undergo disintegration process and are voided out of the animal's body as non-infective protein complexes.
Due to this short half-life, the homoplasma is non-teratogenic, non-toxic in reaction, it contains no pyrogens or known microorganisms. It is not known to react with any other drug. It has been proven non-toxic, when given intraperitoneally in Guinea pigs and swiss albino mice.
Six guinea pigs, 12 mice and six rabbits were inoculated 0.5ml each of the Canine homoplasma intraperitoneally and observed for adverse reactions if any for a total period of three months. Three guinea-pigs, three rabbits and six mice were sacrificed after 8 days and organs histopathologically screened for inflammation, in duration/any abnormality, etc. No such defects were noted. Similarly after 3 months when rest of the animals were sacrificed and similarly observed no. defects were noted. In rare cases of reaction, antihistamines such as dexamethazone is advised, especially when given in a drip-form or when given intravenously the same is followed as in case of other similar immuno-therapies. The homoplasma on reconstitution is quite a translucent, straw coloured fluid in appearance, and on reconstitution trials with different fluids such as sterile distilled water, 0.89% normal saline solution and 5% dextrose saline solution all at room temperature it demonstrated total dissolution of all lyophilized particles, repeatedly.

10
Therefore, it was concluded that the canine homoplasma is non-toxic and non-pyrogenic in reaction. It demonstrates good solubility and being a- homologous blood-plasma product it is totally excreted by the body as immune, complexes or disintegrated by the body in the normal course of time and cannot be teratogenic in origin.
The sterility of the product was evaluated after reconstitution, wherein about 20-30 vials were tested at random from a batch of 300 vials for their sterility by inoculating 0.1 ml of the reconstituted canine homoplasma in sterile pertone water for general aerobic organisms, 0.1 ml on Sabarouds agar plate for detection of fungi, 0.1 ml in thioglycollate agar for spore-forming bacteria and 0.1 ml in MDCK and vero cells for detection of viable viruses if any. All were incubated at 37°C except the Sabarouds agar plates, which were kept at room temperature, for about 8 days. There was no growth observed what so ever, on any of the media indicated above, proving the sterility of the canine homoplasma and deeming it fit for intravenous injection.
This canine homoplasma was also given to 4 pups intraperitoneal^ @ 5 ml neat serum to 3 pups subcutaneously in the flank region @ 5 ml neat serum and 4 4 pups . intravenously @ 5 ml diluted with 10 ml of sterile distilled water. None of the pups received the 'serotherapy-mandatory' dexamethazone/ betamethazone 0.5 ml. None of the above pups showed any immediately or delayed reaction, even after 8 days post-trial, proving it to be a very safe injectable homologous preparation..

11
Once the standardization of the Immunizing (attenuated) virus mixture was perfected and the standardization of the challenge (Virulent) virus was completed, the clinical trials for testing the efficiency of the final product was undertaken, first on an experimental basis and later on, on canine patients admitted in various stages of sickness.
EXPERIMENTAL TRIALS:
The Experimental trials were formulated with prophylaxis and curative treatments in mind, groups were made for each type of trial i.e. pups, below one year's age, pregnant bitches, (the stage of pregnancy not being fixed) and dogs older than one year but not above five years. These animals were randomly selected with no differentiation in sex, size, breed etc. Most of the dogs were non-discript/mongrel type, the rest being crossbreeds both of whom have little chance of acquiring antibodies against any of the selected trial diseases, since none were vaccinated. Each group is shown below for prophylactic experimentation and therapeutic trials, the adults were both male and female selected at random for the trials, except in the pregnant groups.
These dogs were brought, dewormed and serologically tested for rabies antibodies, as previously stated in the chapter on "Selection and of care of dogs". Therefore in total there were 120 dogs, in both the trials, put together. The fluctuations in the serum titres of all the dogs are mentioned at the end of the chapter on Experimental trials, wherein the table depicts the entire summary of the exDerimentation.

12
Prophylactic-therapy trials with Canine Homoplasma:
All the dogs in this group were administered the canine homoplasma after 10 days observation period was over; at the rate of 2 ml per kg body weight subcutaneously except the three control pups in Group 1. All pups weighed between 1.5-3 kgs.

Group 1 (Control) 3 pups as control were treated with normal canine plasma 2ml/ kg body weight, e.g. canine serum without those antibodies present in the canine homoplasma -t hese were challenged with virulent virus mixture after 8 days post-inoculation (pi).
Group 2 6 pups were given canine homoplasma 2 ml per kg body weight once and were challenged with virulent virus mixture after about 30 hours, pi.
Group 3 8 pups were given canine homoplasma 2 ml/ kg body weight once and were challenged after 4 day pi.
Group 4 7 pups were given the Canine homoplasma as in group 3 and challenged after 10th day pi.
All the above four groups were challenged on different days with 2.5ml plus 0.5 ml of virulent virus mixture deep intramuscularly and observed daily for symptoms of the diseases, which commenced after about 18-24 hours in case of Group 1 which had not received the Canine homoplasma at all. This group showed sings of tremors of the extremities anorexia, high fever (104°C to 105°C), bloody foetid diarrhoea, in all the 3 pups, shaking of the head, wailing and in general a very soggy and shaggy appearance. All the pups died between about 3-5 days despite supportive therapy.
The pups in Group 4, who were challenged after 10th day pi. started showing some fo the above symptoms on 7th day pi but much milder in nature. These pups showed high fever (103° - 104°) mild, tremors, slight diarrhoea but no blood. Only five pups out of seven recovered after about 3-4 days of supportive treatment. Total recovery time to attain normalcy for the five surviving pups was 10-13 days. (One very weak pup weighing less than 1.5 kgs died after 3 days). One pup suffered paralysis of hind legs and lingered on for an about 20 days after which it died.

13
The supportive treatment consisted of normal saline 15 ml/kg body weight plus 2 ml polybion in the saline and 5 ml neohepatex or any liver extract which is injectable through the intravenous route. This entire therapy is given twice or thrice a day, as the need arises, but not more than thrice in case of pups.
Six pups in Group 3 after the 6 days p.i. showed only dullness and depression and mild fever (103°C) for about 2-3 days, after which they recovered and started feeding on their own. Total time taken to attain normalcy was about 8 days for all 8 pups. No supportive treatment was given.
In case of Group 2 all the six pups looked very wobbly and sick after 7 days pi. but they did not have fever or diarrhoea and did not stop feeding and looked more alert than those in Group 3. These pups recovered within three days without treatment and were normal in appearance and behaviour.
Apart from the protection afforded to these pups by the Canine homoplasma, these pups must be having their own maternal antibodies also, though smaller in quantity since these pups were strays from unvaccinated stray dams. As observed from the group 1, only maternal antibodies are not sufficient to overcome a virulent challenge, maternal antibodies have to be supplemented with concentrated antibodies from the Canine homoplasma. It was therefore concluded that the Canine Homoplasma worked best for pups within 8 days of giving the challenge i.e. the antibodies half-life in the homoplasma was about 8-10 days after which the antibodies started disintegrating very rapidly. The pups are the most susceptible groups of canines, when challenged within about 30 hours of administering the homoplasma (group 2), due to the maximum titres of the passive antibodies the pups did not exhibit much of symptoms and recovered very rapidly.

14
Trial with Pregnant bitches: 12 numbers
Pregnant bitches, six in the initial stages of pregnancy - about 3-4 weeks pregnancy and six in late stages about 6-8 weeks (total 62-65 days is the gestation period in bitches) pregnancy were selected at random. Five bitches from the initial stages and five of the late stages of pregnancy i.e. total 10 bitches were given Canine Homoplasma @ 2 ml/kg body weight, except one bitch in the initial and other in late stages of pregnancy, which were segregated as control bitches. These two bitches were given normal canine plasma negative for the antibodies present in Canine homoplasma. All the twelve bitches were challenged with the virulent virus 2.5 ml plus 0.5 ml, deep intramuscularly and observed for the symptoms of the diseases and distress signs of abortion if any. The ten trial bitches were challenged, 48 hours after homoplasma was given.
Control bitches:
The two bitches one in early and other in late pregnancy which were not given any Canine Homoplasma, showed bloody foetid, diarrhoea, haematoemesis, high fever (104°C - 106°C), tonic convulsion of the extremities/head and succumbed before abortion within 35 - 40 hours of challenge being administered, i.e. they died due to the acute forms of the main diseases i.e. the Canine Distemper and the Canine Parvovirus. Supportive treatment was started immediately after onset of first symptom i.e. about 18-20 hours after challenge. This early onset was thought to be due to the extreme stress, which depletes the immune system that the pregnancy creates, which was not found in the pups.

15
Early Pregnancy Group
All five bitches in this group showed anorexia as the first symptom after about 48 hours pi, had fever (104°C to 105°C), one bitch had very high fever (106°) and tonic convulsions on the head and extremities. This one bitch also started bleeding profusely per oss and aborted, due to which a litter of six pups was lost, despite starting the supportive therapy six hours after exhibition of first symptom. The bitch took more than 3 days to recover after the constant supportive therapy administration. The other six bitches recovered after supportive therapy administration within 15 to 20 hours, did not bleed nor did they abort, but showed dullness, anorexia, mild temperature of 103°C, roughness of body coat and in general debility.
Late Pregnancy Group
The five bitches in this group started showing the first symptoms after about 48 hours pi i.e. the same time as above group. Three bitches in this group were about 58-60 days pregnant and became anorexic, there was mild reddish discharge per oss, they exhibited breathlessness and one bitch after delivery six, normal pups was unable to walk properly (semi-paralytic) and had mild fever (103°), was dull for about 36-38 hours, but recovered after supportive therapy. The fever inability to walk, breathlessness in this bitch was attributed to eclamsia, which is due to calcium depletion during pregnancy, rather than any effect of challenge virus. The other two bitches also delivered, but remained dull and weak, could not clean their own pups, and had to be helped. They remained dull for the next two days but recovered after supportive therapy. The two bitches which were 50 - 55 days pregnant, remained dull, anorexic, rough coated and mild discharge tinged with blood continued till the bitches delivered. Both the litters were healthy and the pups survived. All the five bitches recovered after supportive therapy and delivered normally, without any loss of pups.

16
Therefore it can be concluded that this type of prophylactic passive antibody therapy not only does not interfere with the pregnancy or parturition, but also helps the animals to withstand the onslaught of the virulent (challenge) viruses as well. It can also be concluded that when given at the beginning of the second stage of pregnancy, it is highly beneficial not only for the pregnant bitch but also for the litter, which has excellent chances of survival due to high maternal antibody titres and continuous flow of antibodies for the first eight days through milk.
Trials with adult dogs (males): 12 numbers
Similar to above were two control dogs and ten experimental dogs, all between two and five years old, seronegative for all the viruses mentioned in the challenge viruses.
Group I : Five dogs challenged 30 hours after receiving the Canine
Homoplasma.
Group II : Five dogs were challenged after seven days of receiving the
Canine Homoplasma.
Group III (Control): Instead of Canine homoplasma normal canine plasma was
administered to these 2 dogs which were challenged 30 hrs after grouping them randomly along with Group I.
To all dogs showing symptoms from the three groups, symptomatic supportive therapy was administered but the control group dogs succumbed 3 day pi. These dogs showed typical symptoms of CPV and CD viruses (as described in B) more prominently than others. The dogs in Group I exhibited mild aneroxia, dullness, fever and uneasiness but recovered within 48 hrs. pi, whereas those in Group II were all anorexic, had mild fever (103°C) and were dull in general. One dog after 3 days showed high fever (105.5°) and displayed convulsions and died suddenly. Such type of deaths are observed in cases of acute CPV infections wherein heart block occurs, due to infection of CPV in cardiac muscles. One dog showed slight blood in urine,

17
anorexia, 106°C fever, and roughness of coat for about five-six days but recovery was slow. All 4 dogs normalized after 8-10 days pi.
From the above it can be concluded that the trend of recovery follows the half-life of antibodies, i.e. those dogs challenged within 30 hours of receiving the Canine homoplasma stood a better chance of survival than those who were infected at later stages, when the half life of most of the antibodies were at an ebb.
Advantages offered by the present invention over homoserum are:
1. Homoplasma yields 4 times more volume than homoserum.
2. Homoplasma has higher concentration of antibodies.
3. Homoserum has many specific and non specific inhibitors and factors which causes the Allergic Reaction, while Homoplasma causes least reaction and which are tolerable.
4. Homoplasma has more IgG than Homoserum. This nonspecific IgG is the most helpful during recovery of the patient from sickness, as no IgG is lost -whatever the body produces is ultimately used in the body - building mechanism.
Therapeutic/ Curative Trials with Canine Homoplasma
• Trials with 40 Pups:
The 40 pups in the age group between 5 months to 8 months, weighing between 1.5 kg to 3 kg were divided as follows at random :
Group 1: 4 pups as plasma controls were administered normal canine plasma @ 10 ml/kg body weight. This normal plasma did not contain the antibodies found in the canine - Homoplasma preparation. These were challenged with 2.5 ml plus 0.5 ml of the virulent virus mixture after 48 hrs.

18 ^
Group 2: 6 pups were given Canine Homoplasma 10 ml/kg body weight only once before challenge with virulent virus after 4 days.
Group 3:12 pups were given virulent virus mixture 2.5 ml plus 0.5 ml deep intramuscularly on day one and after 48 hours were given the 10ml/kg body weight of the canine Homoplasma. Four of the pups in this group did not show full symptoms whereas 8 pups showed severe diarrhoea with blood after about 36 hours.
Group 4:18 pups were given 2.5 ml plus 0.5 ml mixture of the virulent virus mixture on day one and were treated after 72 hours of showing a variety of severe symptoms, with supportive treatment along with 10ml/kg body weight of canine Homoplasma. All the pups were bled for titres before start of experiment and after the treatment at least once.
In case of group 1 all the four pups which were challenged succumbed about 48 hours after the challenge dose, the predominant symptoms were those of canine parvovirus, canine distemper and canine hepatitis, since these are fatal diseases of pups. They manifest in adult dogs as severe symptomatic diseases resulting into fatality due to secondary complications mainly.
In case of group 2, three pups did not show any symptoms and were quite normal in their appearance and behaviour. One pup passed about 13 times liquid foetid blood tinged diarrhoea and appeared as if it would succumb, but once administered with 30 ml of glucose saline and 10ml/g body weight of Canine homoplasma, the pup recovered within 30 hours and started feeding after about 48 hours. The other two pups looked dull, depressed did not feed and had 104°C temperature. Both were constipated and remained in the above condition till about 48 hours after which they recovered but slowly, without any further supportive medication except the Canine homoplasma.

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In case of group 3 wherein the pups were infected and the later on treated after 48 hours with canine Homoplasma irrespective of the degree of symptoms, the experiments stimulates the situation wherein all the animals who come in contact with infected environment and animals wherein the animals have to undergo passive immunization irrespective of their immune status to prevent any major calamity in terms of mortality. Most of the infected cases come late to the clinics i.e. after acute onset of the symptoms as in this group also, wherein five of the eight pups started with symptoms after 20 hours of the inoculation with the virulent virus. All the pups with severe symptoms such as 105°C - 106°C fever, vomination, bloody diarrhoea, icterus, subnormal body temperature, convulsions, incordination of movements etc were all administered 10 ml/kg body weight canine Homoplasma intravenously after the usual treatment with 0.5 ml of decadron to prevent anaphylaxis/ allergy if any. All the pups weighed less than five kg and therefore they were administered between 40 to 50 ml of the canine Homoplasma.
There was complete recovery in. all the eight pups within 48 hours of administration and the other four who received the same showed no symptoms at all. The four pups from this group were dull, depressed, had bloody diarrhoea and succumbed suddenly, such cases are seen most commonly in CPV infections.
In case of group 4, out of the 18 pups which were treated after about 3 days the onsetof the acute disease, only about eleven survived, from these eleven, seven pups were about 8-10 months old, whereas the other four were more than 3 months and less than 7 months old i.e. they were big enough to overcome immunologically the onslaught of the virulent virus till about 72hrs from the 7 which died, these pups were about three and half months to five months old, but smaller in size, weighing between two to four Kgs died within 24 to 60 hrs before receiving the treatment. The 11 which survived showed typical symptoms of mixed infection i.e. high fever (104° -106°C) dysentery, vomition, laced with blood, convulsions, etc leading to prostration.

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After intensive treatment with supportive therapy along with 10 ml/kg body weight of Canine homoplasma, did these pups show signs of slow recovery. Only the 7 older pups recovered and started feeding after about 48 hrs, the other pups which weighed less took about 3 days to recover and additional dose of 10 ml/kg body weight of Canine homoplasma was required.
• Trials with 20 pregnant bitches:
20 pregnant bitches were acquired in the early and late stages of pregnancy and divided into three trial groups - i}The early pregnancy group wherein there were eight bitches, out of which three were primi-para and fine with at least 3 gestations over ii} The second group comprised of the control group wherein one early and one late pregnant bitch would be inoculated with the virulent viruses only and the, iii} third group of 10 late pregnant bitches. All the bitches in the 3 groups were inoculated with the virulent virus mixture of 2.5 ml plus 0.5 ml, deep intramuscularly and observed for onset of the symptoms, which commenced after about 22 to 30 hrs later. All the bitches were given supportive therapy treatment and 0.5ml of decadron before serotherapy as a routine practice.
o The eight bitches in this group showed either mild vomition or mild diarrhoea, one of the primipara bitch was dull, anorexic and other four bitches had 103° to 104°C temperature, but no other symptoms exhibited. All the bitches were administered Canine homoplasma @ 10 ml/kg b.wt. alongwith 15 ml/Kg b. wt. Glucose saline intravenously within 12 hrs of showing various symptoms. Three of the pregnant bitches showed dramatic recovery and only one primi-para bitch aborted resulting into a loss of four pups which were dead, the bitch continued to vomit several times even after abortion. In case of this bitch the Canine homoplasma was repeated after about 24 hours only once. Seven bitches recovered totally after 48 hrs of supportive treatment with only one dose of Canine homoplasma, whereas one bitch took more than five days to come to normalcy.

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o Both the control bitches were not given the Canine homoplasma, only supportive treatment intravenously alongwith normal canine plasma, and both the bitches aborted after about 26-30 hrs post-inoculation with virulent virus, both succumbed to severe viral onslaught, showing mixed infections, the dominant symptoms being bloody, foetid diarrhoea, lacrimation, constant vomition and abortion. The late pregnant bitch died after 36 hours and the early pregnant bitch died after 40 hrs. In this group a total of nine pups were lost during abortion.
o In this group of 10 bitches who also started displaying symptoms 22 to 30 hrs post inoculation, one group of four bitches was treated at about 22 hrs i.e. immediately after the onset of symptoms with 10ml/kg b.wt. of Canine homoplasma and 15 ml per kg b.wt. of glucose saline. Out of these bitches two could not withstand the onslaught of the virulent virus mixture and aborted, these were given intensive supportive treatment with added liver tonic 5 ml in the above glucose saline and both the bitches had to be given 10 ml/kg body wt additional dose of Canine homoplasma after 18 hrs of the first dose and the supportive therapy was administered every six hours sin a drip form. A total of eleven pups was lost during abortion.
Three bitches although they showed milder symptoms of only vomition, dullness and lacrimation, continued to show these symptoms for about 3 days after the initial C. Homoserum dose. These were given only one dose, since their condition did not worsen any further and they did not abort. The other five bitches reacted by displaying, diarrhoea with blood, vomition laced with spots of blood, dullness, anorexia, severe lacrimation, incordination of movements, two bitches even showed yellow tinges to the mucosa. For these bitches treatment was delayed to 24 to 30 hrs after onset of symptoms and the Canine homoplasma administered with full precautions by dissolving 10 ml/kg b.wt in about 50 ml glucose saline and giving it as slow intravenous. The same was repeated on 3 bitches after about 30 hrs. Two of the above five recovered within 6 hrs of administration of Canine homoplasma,

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although they were very weak, but did not abort, one of the -remaining two recovered slowly, thus had to be administered the second dose of Canine homoplasma after 24 hrs of the first dose.
From the above it can be safely concluded that 'the Canine homoplasma can safely be given during early or late pregnancy, which is due to the fact that since it is formulated from the same homologous species i.e. the canine, the rate of reaction or allergy/anaphylaxis is minimal. Unless and until the dogs are in the last stages, with the virus/viruses having proliferated throughout the body tissues, the antibody or immuno-therapy has a dramatic effect as can be demonstrated from the experiments above.
As stated in the previous trials, these dogs were inoculated with the virulent virus mixture and it took about 24 - 36 hrs for the onset of the symptoms to initiate, since a stray adult dog has a fully developed immune system, ready to react strongly. These dogs were divided into three groups, wherein only i} 2 dogs were held in the control group which was not to be given supportive only, ii} The group was treated after about 6 - 10 hrs of the onset of symptoms and the last iii} group was treated after 24-30 hrs after the onset of symptoms.
i} The two control dogs initially showed symptoms of dullness, depression, anorexia followed by rise in body temperature to about 104.6°C to 105.8° for the first two days and third day onwards suffered from severe gastro-enteritis with copious bloody diarrhoea and vomition with blood clots, one dog died within six hours of the onslaught and the other succumbed after bout 36 hrs of the initial symptoms and in addition to above, also showed deep yellow mucous membranes, lacrimation and death due to severe dehydration.

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ii} In this group of 8 dogs, five were treated after 6 hrs of the onset of the symptoms, with 10ml/kg b.wt of Canine homoplasma and glucose saline @ 15 ml/kg b.wt and observed for signs of recovery. One male dog who showed an initial rise in temperature to about 104°C had copious stools which were black bloody with clots and foetid odour after which the dog could not support itself and collapsed. Thus dog was being given glucose saline and Canine homoplasma when he died, having subnormal temperature. This dog displayed typical signs and clinical symptoms of Canine parvoviral gastroenteritis. It is not common for adult dogs to succumb to the parvoviral disease and therefore it seems that either the dog was immuno compromised, or had a heavy worm load or a concurrent infection was present, whereby the dog could not sustain the onslaught of the fresh viral infection.
Out of the remaining four dogs, three showed typical symptoms of infectious Canine Hepatitis which is quite common in adult dogs, with a rapid development of icterus, coughing, lacrimation from the eyes, whitish yellow stools, pale mucos-membranes and anorexia. These dogs received the Canine homoplasma in the prescribed proportions with a glucose saline drip and after another 10 - 12 hrs recovered enough to drink water on their own. After another six hours they had bread and milk and were on their way to recovery. The fifth dog-a bitch apart from showing the mild gastro enteritis also vomited several times, had sub-normal temperature of about 100°C and was quite dull but recovered within 10-12 hrs of the prescribed treatment with Canine homoplasma and started feeding within another 3 hrs.
Three dogs were treated after 10 hrs of the onset of the symptoms which had become very acute with diarrhoea, vomition, pale yellow mucosa and two dogs showed total recumbency. All the three dogs were treated with the prescribed Canine homoplasma with glucose saline, but considering the acuteness of the disease they were administered by the show intra-venous route. After about 2 hrs later the vomition and diarrhoea stopped, but weakness, icterus and recumbency persisted, therefore glucose saline @ 15 ml/kg, b.wt alongwith neophepatex 5 ml

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total, was administered. Weakness, anorexia and subnormal temperature of 100°C persisted at times even tonic muscular spasms were observed, therefore another dose of Canine homoplasma was repeated after 24 hrs along with 50 ml glucose salien in drip form, this helped to relieve the symptoms drastically and after about 24-30 hrs the three dogs were drinking water and moving around, overcoming their recumbancy. From the 8 dogs in this group one died and the rest recovered from very critical conditions of the virulent viral attack. All the 7 dogs were feeding and behaving normally on the third day of the experiment.
iii) In group there were 10 dogs treated after 24-30 hrs of onset of the symptoms - all the dogs after 24 hrs of purging, vomition, high interns, coughing etc were in a bad shape, most of them were dull, depressed, dehydrated, 8 dogs had tonic convulsions, two dogs were only vomiting blood and were gaping for breadth, due to lowered haemoglobin levels, and fluid loss. In these two dogs, it was not possible to locate the veins and therefore the Canine homoplasma was given subcutaneously in the flank regions -i.e. in one flank the Canine homoplasma and in another side about 200 ml of dextrose salien with neohepatex 5 ml. After about two hours one dog recovered sufficiently to lick some water while in a sitting posture, but was unable to support himself on all four legs. The other dog during the same time was recumbent but alive. The former dog was given 200 ml glucose saline plus 5 ml neohepater after about 3 hrs in the flank region once again, but died after about 8 hrs of start of the treatment due to cardiac and respiratory arrest. The later dog which was recumbent died in a lingering manner without any further symptoms after about 6 hrs of the onset of symptoms. The eight dogs with convulsions were treated in a similar manner and were given 200 ml glucose/ dextrose saline and 5 ml neohepatex every three hrs and Canine homoplasma after the initial dose was given after 10 hrs interval. Three dogs showed rapid improvement and could drink water, stand up and take a few steps before falling. In these three dogs, the saline treatment was stopped after 6 hrs (two treatments) and only one more dose of Canine homoplasma was administered after 10 hrs alongwith the other dogs. Out of the remaining five

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dogs two dogs lingered on with dullness, anorexia, subnormal temperatures and severe debility. After two days these dogs developed icterus and correa, despite a third dose of C. Homoserum given to all the eight dogs after 30 hrs. Both these dogs died of severe dehydration and respiratory failure on the fourth day, despite concentrated efforts with supportive therapy. These deaths were due to extensive tissue damage and collapse of the immune defence mechanisms, wherein intensive efforts to save their lives failed, due to lack of administration of treatment in the proper time (early treatment) i.e. treatment was given too late. Only three dogs survived, but were slow to recover from their acute onset, due to delay in the treatment.
All the three, after about 8 days showed correa, incordination of movement and erratic intake of feed. These three dogs were given a fourth dose of Canine homoplasma on the eighth day when the supportive treatment was stopped and the dogs after a further three days observation period were declared fit. This Canine homoplasma was given @ 2 ml/kg b.wt as a precautionary measure, to neutralise residual circulating antigens if any. There were reports by several clinicians who were given trial samples, that the residual symptoms of weakness, incordination of movements, correa, erratic feeding etc reduced considerably when at the end of the treatment period one dose of Canine homoplasma @ 2ml/kg b.wt. was administered.
Considering the above experimentation, it can be concluded that all the viruses and the bacteria administered as an virulent virus mixture before various treatment intervals follow a pattern of universal truth - i.e. prevention is better than cure, and that early treatment simulates a near preventive measure. The fact that the viruses and the bacteria involved are all celulolytic in nature, cause extensive tissue damage in very short periods of time especially, during an acute onset, means that treatment if any should be initiated as early as possible to avoid extensive and irrevocable tissue damage which leads to a total breakdown of the body-mechanisms and culminates in death.

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From the table, a conclusive pattern of survival can be noted, wherein the antibodies concentrated in the Canine Homoplasma have helped the affected dogs to recover rapidly.
The blood is taken for antibody serum titre after the observation periods, since there is no titre detectable in the unvaccinated dogs. The titres can be detected only when challenged, till then the antibodies are dormant and not activated by the memory forming B-lymphocytes. This conclusion was drawn after checking the titres of several pups and adult dogs. The pups had generally negligible titres of maternal antibodies, which totally disappeared on challenge.
The most common types of antibodies encountered were against the Canine distemper, canine parvovirus, infectious canine hepatitis, and least against Canine leptospira infections. The other two disease's antibodies, although fatal for dogs, were not commonly encountered. In case of the experimental trials, the average serum titres represent only those antibodies which are in circulation after the neutralization of the challenge virus. In case of pups these titres were low due to the late response of the immature immune system, whereas in case of the adult dogs and pregnant animals, the titres were much higher due to the response of a fully developed immune system. It is also felt that there must have been an add on titre due to the amnestic B-lymphocytic stimulation in dogs who had suffered from some of the diseases earlier in life and survive their onslaught.

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CLINICAL TRIALS OF CANINE HOMOPLASMA
Clinical trials were carried out on dogs/ pups/pregnant bitches which were brought by owners for either admission to the attached animal hospital or to the out patient departments or to the private clinics of the practising' veterinary doctors. Most of the animals brought by the owners were already suffering from acute stages of the disease/s or were in the incubation stages, whereby routine treatment generally aggravated their conditions. Any dog, whether adult or pup cannot survive when brought in the last stages of any nearly fatal sickness. These clinical trials were carried out over a period of three years from 1986 to 1989 and in the results represented here the dogs have been broadly classified in three susceptible groups i.e. Pups from 2 week to 12 months, B. Adult dogs from 1 year to 6 years and C. Bitches in various stages of pregnancy i.e. first (early pregnancy) transistor, and last trimester (late pregnancy), and recently parchurited (delivered) bitches. A. total of A 571 pups, B. 337 adult dogs and C. 297 bitches were administered the serotherapy with canine homoplasma, either in smaller or larger doses, i.e. in total the clinical trials of 1200 dogs are recorded in this study. For the sake of convenience, only the outstanding or complicated case histories are mentioned and discussed, otherwise a generalized clinical picture of these cases is presented and evaluated. Since most of the dogs were brought by the owners in a very serious condition, it was difficult to persuade the owners to allow the animal to be bled even for 5 ml blood, for fear that the animal may collapse and die. Therefore, serum titres were not taken in all the cases as done in the experimental trials. Those serum titres that were evaluated gave a fairly accurate diagnosis of the situation the animal was in. Most of the dogs, especially the pups were infected before primary vaccination was administered and therefore their titres represented the actual virulent strength of the viral infection. The serum titres in case of clinical trials were those of the disease which demonstrated first onset symptoms and the maximum symptoms i.e. Canine Parvo virus, Canine Distemper and varying numbers of animals showed icterus, typical in Infectious Canine Hepatitis/ Leptospirosis.

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Clinical Trials in 571 pups :
In case of pups the most fatal disease is the Canine distemper (CD) followed by the Canine parvovirus, infectious Canine Hepatitis. Canine Para Influenza, Canine Laryngotracheitis and lastly the Canine Leptospirosis. The common symptom in all these diseases is rising body temperature to about 103° - 105° C. The most outstanding symptom of CD in pups is cannot support itself, discharges from eyes and nose, twitching of muscles, at times convulsions, wailing, shaking of head and wobby gait.
In case of Parvo virus the symptoms are bloody diarrhoea, only blood clots fall instead of faecal matter, and pup also vomits blood, there may be blood in urine and generalized acute dehydration, followed by coma and death. In the cardiac form, there was sudden death, here the pups do not show any of the symptoms described above, only fall to the ground dead. This was seen also as a mixed form, i.e. the pup showed initially dysentery, was treated for about 2-3 days and suddenly died, at times towards the end of the treatment, while recovering. In these cases death was diagnosed as due to heart block, on post-mortum.
In case of mixed infections in pups mortality rate was higher and can be attributed to the fact that pups not only have weakly developed immune system, but due to their tender age, smaller size and body weight, they weaken easily due to dehydration. In pups all the vital organs such as the liver, spleen, kidneys, heart and even brain are still immaturely developed and cannot sustain the onslaught of the disease.

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They even do not respond to the treatment and have to be given ready to immunize supplements such as Canine homoplasma, which gave heartening results in this category. About 10 ml (1 vial) homoplasma along with the full supportive therapy has to be administered every 18-24 hours. The pups were given supportive therapy comprising of 20-10.0 ml dextrose (5%) saline @ 20 ml/kg body weight, 0.5 ml to 2 ml of Neohepatex a crude liver extract and 0.5 to 1 ml of polybion - a vitamin B-complex supplement with Co-enzyme A.
Pups weighing within 1 kg, were those who suffered acutely and more than 20% dehydration by body weight was observed resulting into death. These mortality cases comprised of about 126 pups i.e. 22% most of these were from those litters, the dams of which were not vaccinated during epidemics in certain localities of the Mumbai city. In these cases the dams were either not vaccinated and were unable to transmit any immunity to the pups, whereas in some cases it was seen that the dame was already incubating the disease and the new born pups contracted the same, while suckling the dam within few days of birth. The other type of succeptible group of pups was the one which was about 1/4 to 2 months old unvaccinated, recently sold to new owners and due to infection in the environment, the pups caught the virulent viruses, the onset of such disease was not only very sudden but the disease spread • in the body rapidly with fatal consequences, at times even before treatment could be initiated.
The lowest mortality was encountered amongst pups in the groups which was slightly older i.e. between 2 to 6 months here the mortality was only 28 pups i.e. 5%. These pups weighed between 1 kg to 6 kg body weight and could be categorized as recently vaccinated pups with infection, and unvaccinated pups with infection. The 28 pups which died in this older age group, was mainly because some had been brought to the hospital and private clinics in very serious conditions and no amount of treatment could save these.

Amongst the first groups pups, the vaccination may have been given during the disease incubation stages and the disease flared up, or the vaccine may have been partially neutralized as in cases of residual maternal antibody activity and during an acute onslaught of the virus, resulted in inadequate protection. Another reason is that vaccination produces a stress and if given durjng epidemics in the locality, the antibody is not produced in sufficient quantities at short-notice and disease manifestation sets in to overtake the antibody production. Out of such 221 cases treated there was an overall response of 90% i.e. 199 pups recovered after the Canine homoplasma was administered to them with supportive therapy every 18 hours. Out of these 221 pups only 12 pups were required to be given three doses of Canine homoplasma on alternate days, the rest of the pups showed a dramatic break in the acute disease symptoms and recovered to about 80% after the second day of treatment. Some of these pups had suffered from mixed Canine distemper and parvovirus infection and these few pups showed residual symptoms of shaking their heads periodically, incoordination in gait, etc; but had resumed feeding normally as well as regained other normal body processes. In all the cases treated with Canine homoplasma, fever 104° C to 106°C was the first symptom to come down to 101° to 102° and in case of CPV, the dysentry was the first symptom to stop.
Mortality Percentage in Pups

Pups Mortality in Pups Overall Mortlaity
Pups < 1 kg Pups > 1 kg
517 126 28 154
22% 5% 27%
Overall recovery after treatment with Canine homoplasma was 73%.
In case of infectious canine hepatitis and canine para-influenza, the symptoms of jaundice, and acute respiratory distress and congestion of the chest were first to be relieved, within a span of 24 to 30 hours. Those cases, which showed dysentery, were invariably more serious than other cases, because apart from the disease aspect, severe dehydration and loss of blood were the other debilitating factors.

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Amongst the unvaccinated infected group the response was less dramatic, since there were no antibodies in their system and as soon as passive antibodies were introduced, the body defences reacted and partial virus neutralization occurred. These pups had to be given at least two more doses of the Canine homoplasma on alternate days to overcome and neutralize the circulating viruses. Similarly out of 571 pups infected in total, 417 survived and recovered resulting into 73% survival rate in treated animals and 27% mortality rate in weak and advanced cases which could not be saved. Since these were the owner-based pups and the owners refused to allow withdrawal of blood for titration, serum titres are not presented.
Clinical Trials in 337 adult dogs
In case of adult dogs the diseases showed no clear cut symptoms, dogs were presented starting with high fever (104° to 106°C) anorexic, dull, depressed at times very weak and since no differential diagnosis could be made, the treatment in such cases was terramycin - 2 ml (500 gms) and 5% dextrose saline @ 25 ml/kg body weight. If the animal after 24 - 48 hrs started showing any other symptoms, treatment was administered accordingly or if no response to above treatment was felt, then in addition Canine homoplasma @ 10 ml/kg body weight, was administered, because all the six diseases mentioned are initiated by high fever and above symptoms. Generally after the first dose the fever and other symptoms drastically reduced indicating that the animal was incubating any of the above diseases. The advantage of Canine homoplasma is that it also works in cases of non-specific diarrhoea and fever, resulting into total and rapid recovery.

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This may be due to the fact that during the generation of specific Antibodies a lot of non-specific IgG is also released into circulation as a humeral response of the immune system, apart from the cell mediated specific response. Since no ammonium sulphate precipitation is followed, during manufacture of Canine homoplasma, this IgG remains intact and helps in more rapid recovery. This IgG is not measurable since during the immunological tests known/ specific antigens react with the specific antibodies, but in the blood stream of the animals body, this IgG makes its presence felt and attaches itself to other antigens/ antigen-cell receptors etc.
Young adults about 13-15 months old were the worst affected and brought into the hospital / clinics quite debilitated, unconscious in conditions, especially true of dogs belonging to the smaller breeds category such as the pomoranians, pekenese, terriers, spitz and miniature breeds. The body weight of these dogs was hardly between 2 kgs and 5 kgs, therefore it was a hindrance for the much needed supportive therapy, since the Canine homoplasma had to be given half intravenously and half subcutaneously, since the dosage was 10 ml/kg body weight for the curative measures. In this group only two dogs out of 337 treated died due to adverse serum reaction, i.e. due to allergic causes, after administration of Canine homoplasma.
These two dogs were treated previously for about five days in a private clinic and when they did not respond to treatment, they were brought as emergency cases to the animal hospital having erythematous eruptions and on giving strong remedial antihistamine, went into a coma and died due to respiratory collapse. These are know as idiotypic serum reaction, wherein due to previous treatment with serum the dog is sensitised and idiotypic antibodies are formed, resulting into a type II hypersensitivity reaction, wherein severe complement erythrocytelysis occurs and the dog goes in a comatose condition due to histamine release etc.

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There were 33 cases which were presented with a mixed type of infection, i.e. severe jaundice with the belly of the dog totally yellow, body skin yellowish gray, discharge from the eyes, mild greyish diarrhoea, twitching of the ears, face and limbs periodically, 104°C constant fever, not responding to ordinary antibiotics and supportive therapy. Nine of these dogs which were totally dehydrated, slipped in a come and eventually succumbed even after treatment with Canine homoplasma and improved supportive therapy.
Table : Mortality Percentage in Adult Dogs.

Total Mortality in dogs Overall
Total Idiotypic dogs Mixed infection Dysentry dogs Mortality
337 2 9 6 17
6.7% 30.3% 20.2% 5.1%
Overall mortality in adult dogs was 5.1% and overall recovery after treatment with Canine homoplasma was 94.9%.
There were about 54 dogs which were presented with subnormal (99° - 100°) temperature, acute dysentery, vomiting laced with blood clots, difficulty in breathing, twitching of limbs etc. In 33 cases there was remarkable stoppage of bloody diarrhoea, cessation of vomition, twitching of limbs and normalization of body temperature on administration of first Canine homoplasma dose.
Out of 54 presentations only 6 dogs died. Five of the recovering animals had to be given another dose of Canine homoplasma for speeding up the process of recovery i.e. to neutralize those antigen particles which are released in circulation after about 24 - 30 hours during the phase of recovery from acute onset of disease (i.e. out of 337 adult dogs only 17 died and 320 recovered).

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Clinical Trials in 297 Pregnant bitches
Pregnancy as stated earlier causes great stress in the body and on the immune system of the bitch. During this period all the body systems contributes towards the development of the foetus, its immune system development etc particularly towards the late pregnancy period, the immune system of the bitch during this phase is weakened and totally unprepared to fight the disease/ antigen onslaught if any. Antibiotic therapy is generally avoided in pregnancy due to its aberrant action on the foetus. Whatever is injected parentrally into the body is secreted through the milk in pregnancy. Therefore to save the litter if the bitch is passively immunised about a week before delivery then the litter of the bitch not only has a good antibody titre but
retains the antibodies much longer, as the bitch's milk contains higher concentrations of secretary/ neutralizing antibodies. Only in rare cases would these passive homologous antibodies have idiotypic action, causing abortion. This is a risk to be taken when the environment is heavily contaminated or the bitch is in contact with other dogs who are actively suffering from any of the high-risk viral diseases.
There were about 131 bitches in early pregnancy, which were presented in the hospital and private clinics. From these 131, 82 were in contact actively with infected animals in their neighbour hoods and had started showing mild diseases symptoms, which could have been due to any of the above viruses. The most common symptoms included high fever (104° -106°) anorexia, reddish discharge per oss, five bitches had yellowish tinge on their hairless parts of the body. One older bitch had loose stools without blood.
The five bitches which had yellowish tinge to the skin, started vomition a day after admitting tot he hospital and were given Canine homoplasma about four days after onset of symptoms.

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Table : Mortality Percentage in Pregnant Bitches:

Total No. of Bitches Mortality in Early Pregnancy Pregnant Bitches Late Pregnancy Overall Mortality
297 4(1.34%) 1(0.33%) 5(1.68%)
Nos. of litters lost 2 Litters of 11 pups 7 litters of 32 pups 9 litters of 43 pups
Overall recovery after treatment with Canine Homoplasma was 98.32%.
' • ■ y
Two of the bitches which were vomiting at least 12-14 times a day aborted and had
subnormal temperatures, turned toxic and succumbed after 4-5 days of abortion. A
total of 11 pups were lost from the two. The other three stopped vomition and
regained normal body temperature after 24 hrs treatments with Canine homoplasma.
Therefore although there was only two fatalities amongst this group of five infected bitches, two precious litters totalling 11 pups was lost. The yellowish body tinge faded away after about a week and the 3 bitches were feeding normally about the same time as above. The older bitch with loose foetid smelling stools was lost. The yellowish body tinge faded away after about a week and the 3 bitches were feeding normally about the same time as above. The older bitch with loose foetid smelling stools was given the C homoplasma a day after the symptoms and therefore " recovered without any. further complications. The other 49 bitches in early pregnancy, showed varied symptoms, but 12 bitches showed mixed distemper and parvovirus symptoms and at times loose sanguinous stools. All the twelve were administered canine Homoserum about 24-48 hrs after onset of symptoms and except for one very young white bitch, a primipara about one year two months old, who collapsed and died. On post mortem heart muscle and intestinal contents showed presence of Canine Parvovirus (CPV) and the liver had haemorrhaegic patches. The other was about two and half years old with a congenitally enlarged heart and could not withstand the stress of pregnancy and distemper, which was later on isolated from the Urinary bladder.

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The 166 bitches in late pregnancy were admitted or brought to the hospital/ clinic in various stages of emergency, wherein they were administered first - aid and Canine homoplasma. 16 bitches were brought to 3 private clinics with severe eclamsia, wherein calcium and glucose saline was administered with decadron and Canine homoplasma as a precaution, since some of these bitches showed abdominal contractions. All were administered Canine homoplasma within first 24 hrs of the concentrations, but still six bitches aborted and six litters, (collectively 42 pups) were lost. No mortality was noticed in these bitches which eventually recovered, but pups could not be saved.
From 80 bitches which were on the verge of delivery and got infected, all were administered Canine homoplasma within 24-48 hrs after delivery. Only one bitch died of toxic complications with the litter unborn. There were about 42 bitches which gave birth to about 338 pups form 42 litters, out of which only 32 pup succumbed to infection, that too because they were too small to inject the Canine homoplasma. They weighed between 40-65 grm only. The larger pups weighing between 80-100 gms were given 0.5 ml subcutaneously Canine homoplasma and the (mother) bitch of the litter was given at the rate of 10 ml/kg b.wt. the full dose, so that should could secrete the antibodies in the milk and save herself and her litter. From the other 38 who showed symptoms of mild fever (103-105°C) off feed, vomition and weakness of the hind limbs, unable to support themselves, etc were put on high supportive therapy mixed with Canine homoplasma, these bitches required another dose about one week later, bud did not abort. They delivered 8-10 days after the start of therapy and were given the second dose just after delivery, so that there was sufficient protection for themselves as well as their litters. The overall recovery rate was 98.32%.

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It can therefore be concluded that in case of pregnancy, if the Canine homoplasma is given in time, it was capable of saving not only the life of the bitch but also was able to sustain the litter. Therefore it is suggested that the Canine homoplasma may be , given to the pregnant bitch two weeks before and one week after delivery to ensure a healthy dam and litter.

Group & No. No. Range of Serum titres %
No. of dogs Survived Dead ' - Recovery
Dead Survived
Prophylact 38 10 CPV :0-:128 128-1024 79.95
trials (48) CD :0-:128 ICH :0-:80 CPV2 :0-:80 CPI :0-:128 LH :0-:128 64-1024 40 - 640 20 - 320 20-512 16-512
a. Pups (24) 19 5 CPV:0-:128 CD :0-:128 ICH :0-:80 CPV2:0-:80 CPI :0-:128 LH :0-:32 1:128-10241:64-5121:40-3201:40-3201:20-2561:16-128 79.16
Group 1 (3) - 3 CPV :0- - -
Control • 1:128 CD :0-:64 ICH :0-:40 CPV2:0-:20 CPI :0-:0 LH :0-:0
Group 2 (6) 6 CPV-CD-ICH-CAV2-CPI-LH- :256-:512:128-:512:40-:320:40-:80:128-:256:64-:128 100
Group 3(8) 8. CPV-CD-ICH-CAV2-CPI-LH- :256-:1024:64-:512:80-:320:40-:320:128-:256:64-:128 100

B. Pregnant 10 2 CPV :0-: 128 :256-:1024 83.33
Bitches (12) CD :16-:128 ICH : 10-:40 CAV2 :20-:40CPI :0-:128 LH :0-:128 :128-: 1024 : 40-:640 :20-:80 : 64-:256 :64-:512 '
Control (2) 2 CPV: 0-:128 CD: 16-:64 ICH : 10^:40 ' CAV2 :20-10" CPI :0-:128 LH :0-:128 V
Early 5 - CPV- :256-:1024 100
Pregnant CD- :256-:1024
(5) ICH-CAV2-CPI-LH- :80-:640 :40-:80 :64-:128 :64-:256
Late 5 - CPV- :256-:1024 100
Pregnant CD- :128-:512
(5) ICH-CAV2-CPI-LH- :40-:640 :20-:80 :64-:256 :128-:512
C. Adult 9 3 CPV:0-:128 :256-:1024 75
Dogs (12) CD: 8-: 128 ICH : 0-:80 CAV2 :0-:80 CPI : 0-:64 LH :0-:128 :128-:512 :160-:640 : 20-:80 :64-:512 : 80-:256
Group 1 (5) 5 CPV-CD-ICH-CAV2-CPI-LH- :256-:1024:128-:512:160-:640:80-:160:256-:512:128-:256 100

39

Group 2 (5) 4 . 1 CPV:0-:128 CD: 32-: 128 ICH : 0-:40 CAV2 :0-:80 CPI: 0-:64 LH :0-:128 :256-:1024 :128-:512 :160-:640 : 20-:80 : 64-: 128 : 80-: 128 80
Group 3 (2) - 2 CPV :0-:8 - -
Controlt - CD :8-:128 ICH : 10^:80, , CAV2 :0-:40 CPI : 4-:64 *--LH : 0-:8 »

40
Therapeutic trials in dogs (40)

Group & No. No. Range of Serum titres 0//o
No. of dogs Survived Dead Recovery
A. Pups 25 15 CPV:0-:128 :4-:512 62.5
(40) CD 3p-: 128 ICH :t)-:320 CAV2 :0-:40 CPI : 0-:64" ■ LH :0-:16 :0-:1024:0-:1280:0-:80:0-:512:0-:64
Group 1 (4) - 4 CPV :0-:64 - -
Control CD : 0-:32 ICH :10-:40 CAV2 :10-:40 CPI : 0-:16 LH :0-:16
Group 2 (6) 6 CPV-CD-ICH-CAV2-CPI-LH- :4-:512:32-:1024: 10-: 1280:0-:80:0-:512:8-:64 100
Group 3 8 4 CPV:0-:128 :10-:512 66.6
(12) CD : 0-:64 ICH : 0-:320 CAV2 :0-:10 CPI : 0-:64 LH : 0-:0 :0-:256:0-:640:0-:40:0-:128:0-:8
Group 4 11 7 CPV:0-:128 :8-:512 61.1
(18) CD :0-:128 ICH :10-:40 CAV2 :0-:10 CPI : 0-:8 LH : 0-:0 :0-:1024:0-:320:20-:20:32-:256:0-:0

41

B. Pregnant 18 2 CPV :0-:64 :0-:1024 90
Bitches (20) CD : 0-:32 ICH : 0-:40 CAV2 :0,:0 CPI : 0-:4 .. LH : 0-:8 :10-:1024:0-:320:0-:40:0-:256:0-:128
Early 8 - CPV- :10-:512 100
Pregnant CD- :40-:-1024
(8) ICH-CAV2-CPI-LH- :0-:160 :0-:40 :0-:128 :0-:64
Late 10 - CPV- :0-:1024 100
pregnant CD- :10-:256
(10) ICH-CAV2-CPI-LH- :0-:320 :0-:40 :0-:256 :80-:128
Control (2) 2 CPV :0-:64 CD : 0-:32 ICH : 0-:40 CAV2 :0-:0 CPI : 0-:4 LH : 0-:8
C. Adults 13 7 CPV:0-:128 :4-:1024 65
(20) CD: 0-:128 ICH :20-:80 CAV2 :0-:40 CPI :0-:128 LH : 0-:256 :0-:512:10-:320:0-:80:0-:256:0-:128
Group 1 (2) - 2 CPV :0-:32 - -
Control CD :0-:128 ICH :20-:80 CAV2 :0-:40 CPI : 0-:64 LH : 0-:32

42

Group 2 (8) 7 1 CPV :16-:128CD : 0-:128ICH :20-:40 :32-:512 :64-:512 :40-:320 :40-:80 87.5
CAV2 :0-:40 CPI : 8-:64 LH : 0-:256 :64-:256 :8-:128 i.
Group 3 6 4 CPV :8:128 '* •4-: 1024.. 60,
(10) CD: 4-: 128 ICH :40-:80 CAV2 :0-:40 CPI : 16- :(P:51-2. , :10-:320 :0-:10 :0-:64
:128 :0-:128
._. LH : 0-:32
Total no. of dogs in experimental trials : 88 Nos.
From the above table it is seen that only the surviving animals have attained the requisite titres. In this experimental group the pups, had no titres initially and developed titres on giving the C homoplasma as in case of the prophylactic group or developed the titres after virulent virus infection as in case of therapeutic trials.
Only in case of a small percentage (2-3%) adults had a negligible titre for CD/CPV, . but otherwise were clean i.e. no initial titres. In case of the pregnant bitches, only 2 bitches had initial titres for CD (1:10 and 1:40) and one had titres for C. Leptospira, i.e. 1:80, but did not demonstrate live bacteria, through urine, this was considered to be due to pregnancy stress.

43
Table depicting the Clinical Trials : Conclusive evidence of efficacy of Canine homoplasma:

Groups & No. No. % Range of Serum titers
No. of dogs survive Dead Recove
d ry
Survived Dead
A. 571 417 154 73% CPV: 16-1024 16-:256
Pups CD: 257-1024. 16-:128
ICH : 40-640. , CAV1: 64-256 CAV2: 40-160 LH : 32-256 20-:40 8-:128 10-:40 4-:64
B. 337 320 17 94.9% CPV: 64-1024 32-: 128
adults CD: 128-1024 ICH: 40-1280 CAV1: 64-256 CAV2: 80-320 LH: 64-512 16-:128 20-:40 32-: 128 20-:40 64-: 128
C. 297 292 5 98.32% CPV: 128-1024 16-: 128
Pregnant CD: 128-1024 32-:256
bitches ICH :80-1280 CAV1 :64-256 CAV2 :40-160 LH : 64-256 10-:40 . 16-:64 10-:20 32-: 128
No. of - 9 i.e. 43 -
litters lost pups
Total No. of dogs : 1205 Nos.
CPV - The HI titre of 1:256 is considered as protective, the range of titres i.e. lowest
and highest is mentioned.
CD - the titre of 1:128 is considered as protective, the range of the titres is depicted
in the table.
ICH - The CFT titres of 1:80 is considered protective the range is as above
CPI - The HI titres of 1:128 is protective, the range is as above.
CAV2 - the CFT titre of 1:80 is protective, the range is as above.
LH - The agglutination titre of 1:128 is considered as protective, the range is as
above.

44
• The titres of the litters could not be measured since, these were born dead or died immediately after birth.
• Amongst the pups, out of 41 surviving pups only 54 could be bled and 82 pups out of 154 dead could be bled for titres, the owners refused permission for bleeding.
• Out of 320 adults only 32 could be bled for titres and .out of the 17 dead about 10 dogs could be bled, all were owned dogs.
• Out of 292 surviving bitches, since there was negligible difference in the titres of the early and late pregnant bitches, these were not differentiated but given as single titres.
From the above tables it is seen that some of the dead dogs have high titre to some diseases which is became they have no circulating antigens for these diseases, due to which there is no neutralization or complex formation by the antibody. The dog can show low titres, which means that there is active neutralization of the antigen occurring after giving the Canine homoplasma. Lower antigen titres antibody of one disease and high antibody titres of other would mean that the former antigen is out of the tissuecells and is undergoing neutralization by the antibody, whereas only high antibody titres would mean that either the antigen has still not come out of the tissue, as in case of incubation, or the animal does not have that particular antigen/disease. The dog which become weak physically cannot cope with the infection i.e. invasion of the disease agent immunologically, and generally succumbs during an acute onslaught, although his antibody titres are quite high.

45
In case of pups, the serotherapy with Canine homoplasma acts as a boon, since along with their own depleting antibodies, these antibodies give them the neutralizing power and therefore high recovery rate. In case of the pregnant bitches the antibodies being homologous in nature are readily accepted, resulting into natural delivery and no abortion. In adult dogs also, some residual antibodies, which are not sufficient to overcome acute infections and an amnesic response takes about 2 to 4 days to bring about antibody production, therefore the Canine homoplasma turns out to be a boonf or such animals.
The recovery rate is a definite conclusion of the efficacy of the Canine homoplasma's role in neutralizing active infection.

46
We claim:
1. The process for manufacturing pharmaceutical composition for providing passive
immunity to canine comprising of following steps:
a) Selecting an animal,
b) giving an injection of antigen of organism to animal,
c) giving booster dose of antigen,
d) checking antibody titre,
e) removing plasma from the animals,
f) adding antibiotic to the plasma,
g) filling plasma into glass vial, h) lyophilization.

2. The animal as claimed in claim 1 can be canine.
3. The canine as claimed in claims 1 and 2 can be a dog.
4. The organism as claimed in claim 1 is canine distemper, canine parvovirus, infectious canine hepatitis, canine laryngotraceitis, canine parainfluenza type II, and canine Leptospirosis.
5. The antibiotic as claimed in claim 1 is selected from broad spectrum antibiotic.
6. The antibiotic as claimed in claim 1 and 5 is gentamicin.
7. The method as claimed in claim 1 and herein described in example 1.
Dated this 23rd of July, 2001

Dr. Bakulkesh Mafatlal Khamar
COMPLETE AFTER PROVISIONAL
LEFT ON 2 4 JUL 2001
FORM 2

THE PATENTS ACT, 1970
THE COMPLETE SPECIFICATION
(See section 10)
1. THE PROCESS FOR MANUFACTURING PHARMACEUTICAL COMPOSITION FOR PROVIDING PASSIVE IMMUNITY TO CANINE
2. Cadila Pharmaceuticals Limited, IRM House, Off C.G. Road, Navrangpura, Ahmedabad- 380009, Gujarat, India, an Indian company.
3. The.following specification particularly describes and ascertains the nature of this invention and the manner in which it has to be performed.

2
FIELD OF INVENTION
The objective of the present invention is to provide a process of manufacturing a pharmaceutical composition for providing passive immunity to canine.
The further objective of the present invention is to provide passive immunity against canine distemper, canine parvovirus, infectious canine hepatitis, canine laryngotraceitis, canine parainfluenza type II, and canine leptospirosis.
The further objective of the present invention is to provide a pharmaceutical composition with lower incidence of serum sickness, allergic reaction etc.
The further objective of the present invention is to provide a pharmaceutical composition with higher amount of immunoglobulins and antibodies.
The further objective of the present invention is to provide a process with improved yield.
The further objective of the present invention is to provide a pharmaceutical composition in a solid dosage from so that it is easy to transport.
The further objective of the present invention is to reduce the amount of pharmaceutical composition to be administered as a single dosage form.
BACKGROUND OF THE INVENTION
Canine hepatitis, Leptospirosis, parvovirus infections, Distemper are major cause of death as an epidemic. Active immunity to these diseases is provided by vaccination in advance. However, it is not always practicable. This is more so when an outbreak of epidemic infections takes place. During the outbreak to prevent dogs from acquiring infections, the only measure left is to passively immunize them.

3
Mainly canines ( dogs ) suffer from following diseases:
1. Canine Distemper
2. Canine Parvo
3. Infectious Canine Hepatitis ( CAV -1)
4. Canine Laryngotracheitis ( CAV - II )
5. Canine Parainfluenza Type II
6. Canine Leptospirosis
Diseases like canine parvovirus, canine distemper, infectious canine hepatitis, canine laryngotracheitis, and canine parainfluenza type II are viral in nature whereas canine leptospirosis is bacterial in nature. Important features of these include:
• They all are highly infectious and having high mortality rate. Today, due to owners' awareness and vaccination, though the incidence is around 30% of pet population, the death rate among the infected animals is around 80%.
• They occur often as mixed infection making diagnosis difficult and by the time the major one is controlled, the other one comes up and damages further.
• The major occurance is of Parvo infection, followed by Distemper and Hepatitis. The other three often complicate the clinical picture.
• The diagnostic facilities are almost non-existing (leaving vet to his judgement).
• High Fever is a common feature.
There is no specific treatment available for these viruses. All the treatment given is symptomatic in nature. The antibiotics (intravenous chloramphemicol, enrofloxacin, gentamicin, etc.) are used for treating and preventing the secondary bacterial complications. The saline/dextrose infusions for combating the dehydration due to diarrhoea and vomition (for at least 5 days). Antipyretics, analgesics, antispasmodics, antiemetics, liver extract + vitamin B complex + vit C injections, styptics for controlling intestinal haemorrhage, etc. Dehydration and weakness following the disease are major worry for the owner, during which secondary complications can occur.

4
All above diseases are severe and fatal in nature. For prophylaxis there are no drug available in market. But there is some ( Modified / Inactivated ) vaccine available in market. The closest product is Homoserum which is manufactured by Pasteur Merieux, but not marketed or available in India. However,-Homoserum is a serum volume requirements for injection much higher and hence uncomfortable for the doctor and the animal. Also, the number of bleedings possible from the dog (source of the bulk active) is fewer, being a serum, hence cost of manufacturing is higher in case of Homoserum.
US patent 5807,551 describes a method to provide artificial passive immunity to birds by administration of superconcentrated antibodies to hatching eggs or to post-hatch birds. The patent describes the method to concentrate antibodies present in the serum.
All knowm methods of providing passive immunity uses specific antibodies or immunoglobulins derived from serum. The present invention describes a method of producing and using plasma rich in antibodies and immunoglobulins for use as providing passive immunity.
.REFERENCES:
1. US patent no. 5807551
Method to provide artificial passive immunity in birds.
Reynolds Donald L.
Iowa State University Research Foundation, Inc.

5
SUMMARY OF THE INVENTION
The present invention provides a process for manufacturing pharmaceutical composition for providing passive immunity to canine.
According to present invention, the said composition is prepared by raising antibodies to these infectious agents in dogs, and isolating these antibodies containing homoplasma with very high antibody levels. The isolated antibody containing plasma is lyophilized under critical conditions and stored at 4-8 degree C before use. It is reconstituted by addition of diluent before use and given parenterally.
The composition so prepared is found to be stable and effective in providing passive immunity.
DESCRIPTION OF THE INVENTION
According to the present invention is described a process of manufacturing pharmaceutical composition to provide passive immunity to canine.
The mild strains of the viruses mentioned and bacterium Leptospira are injected into healthy dogs. The dogs are then reared for about 3-4 months, giving them enough time to allow them to develop resistance against those specific viruses. Then they are challenged by the virulent strains of the viruses and bacteria. Those dogs that have enough antibody titre to fight back the viruses will survive. The blood is taken out from such dogs every 3-4 months (the dog is not killed, but bled). Through centrifugation, plasma is separated which contains antibodies of all these viruses and bacteria. The antibody titre so obtained in the plasma will be good enough to fight back the disease, once the clinical signs have set in. The plasma is then lyophilized. The product is made available in vials in powder form, along with 10 ml water/diluent.

6
EXAMPLE 1:
1. Select the dog of about 1 and half to two years of age preferably males , weighing between 30 - 50 kgs.
2. Screen the dog for Rabies antibodies, if present discard the dog.
3. Screen the dog for worms and if persist deworming is done.
4. Screen the dog for antibodies against following : Select the dogs which shows high titer.

> Canine distemper virus
> Canine parvo virus
> Infectious Canine Hepatitis Virus ( CAV -1 )
> Canine Parainfluenza Type- II
> Infectious Canine Laryngotracheitis Virus ( CAV - II )
> Leptospira icterohemorrhagica

5. Immunize the dog on 7th or 8th day with immunizing virus - bacterial antigen mixture i.e. 1.5 ml deep intramuscularly.
6. The second , third and fourth vaccination is done at the interval of 8 to 10 days.
7. After 8 to 10 days of last vaccination test bleeding is done to check the antibody titer of individual virus and bacteria.
8. After the last vaccination , dogs are challenged with virulent mixture of challenge viruses® 106TCID 50/ 0.25 ml per virus and for bacteria @ 106 cells/0.25 ml.
9. After 8 days of challenge check the serum of each dog for presence of any virus , no virus must be detected in the serum. Those dogs which withstand the virulent challenge are used for continuous plasma production.

10. Test bleeding is done to check the antibody titer of individual virus and bacteria.
11. The dog showing high titer are now bled by " Plasmapheresis" . wherein the erythrocytes and cellular components from blood are separated from the plasma which is retained outside and the cellular components are retransfused back into the animal body.
12. Now pool the plasma from all the dogs and again check the antibody titer of individual virus and bacteria.

7
13. Methods of Detection of Antibody:
a) Canine distemper virus = 1. Haemagglutination Test using Human
"0"+ve RBCs.
2. Serum Neutralization Test
3. Complement Fixation Test

b) Canine parvo virus = Haemagglutination Test using Pig RBCs.
c) Infectious Canine Hepatitis Virus = Haemagglutination Test using Human
"0" +ve RBCs.
d) Canine Parainfluenza Type- II = Haemagglutination Test using Guinea Pig RBCs
e) Canine Laryngotracheitis Virus = Passive Haemagglutination Test
f) Leptospira icterohemorrhagica = Agglutination lysis test.
14. If the titer of plasma bulk is having protective titer then, it is checked for sterility,
pH , Clarity , Total Protein.
15. Then aseptically formulation is done by adding Gentamicin @ 0.2 mg / ml as a
preservative.
16. Then aseptically fill the 10 ml Glass vial @ 3.5 ml / Vial and is lyophilized. 17. Final product Quality control tests are done i.e.
a) Antibody titer
b) Sterility
c) Viable Virus Infection
d) Total Protein
e) Clarity
f) Pyrogen
g) Abnormal Toxicity h) Moisture Content
17. Store at 2° - 4° C temperature.
The above process can be represented in flow chart below:

8
The canine homoplasma is the blood constituent of the dog blood i.e. blood without the RBCs , WBCs and Platelets, but has fibrin which is inactivated and antibodies against above diseases.
The homoplasma so prepared was subjected for stability and other studies.
Homoplasma is a translucent straw coloured fluid, soluble in distilled water/normal saline 5% dextrose saline, totally sterile, can be given intravenously, in a saline drip, sub-cutaneously or deep intramuscularly. It is moderately hygroscopic, lyophilized power which has to be reconstituted in distilled water before administration. It is stable at room temperature between 26°C to 30°C for about 5 days and between 2°C to 8°C it is stable for 2 years beyond which only 2 log titre reduction was observed in preceding 6 months.
In a test sample size of 40 vials, twenty were kept at room temperature and 20 at 2°C to 8°C for 2 years. These were tested for their total serum titres (log titres) every six months, and were found to be quite stable till two years. The titres reduced by 2 logs after one and half years i.e. during the third trial, those vials kept between 26°C and 30°C were tested every day @ two vials per day and the 5th day onwards recorded a steady decrease in titres @ one log to 2 logs per day, after reconstitution.
This canine homoplasma comprises of 3.5 ml canine homologus immune plasma with 0.2 ml Gentamicin (80mgs/2ml) as final concentration lyophilized in sterile conditions, in a 10ml vial, to be reconstituted with 10 ml sterile distilled water. The pH of this homoplasma is between 6.9 to 7.1, tested on reconstitution every six months, during the above trial.

9
This canine homoplasma is made up of a mixture of passive antibodies, which have no target-organs and have short half lives comprising of 48 to 72 hours in cases where active infection persists, but maximum life of such passive antibodies is 12 to 15 days when given in healthy animals as a prophylactic measure. The antibodies being protein particles, immunoglobulin in composition, they undergo disintegration process and are voided out of the animal's body as non-infective protein complexes.
Due to this short half-life, the homoplasma is non-teratogenic, non-toxic in reaction, it contains no pyrogens or known microorganisms. It is not known to react with any other drug. It has been proven non-toxic, when given intraperitoneally in Guinea pigs and swiss albino mice.
Six guinea pigs, 12 mice and six rabbits were inoculated 0.5ml each of the Canine homoplasma intraperitoneally and observed for adverse reactions if any for a total period of three months. Three guinea-pigs, three rabbits and six mice were sacrificed after 8 days and organs histopathologically screened for inflammation, in duration/any abnormality, etc. No such defects were noted. Similarly after 3 months when rest of the animals were sacrificed and similarly observed no. defects were noted. In rare cases of reaction, antihistamines such as dexamethazone is advised, especially when given in a drip-form or when given intravenously the same is followed as in case of other similar immuno-therapies. The homoplasma on reconstitution is quite a translucent, straw coloured fluid in appearance, and on reconstitution trials with different fluids such as sterile distilled water, 0.89% normal saline solution and 5% dextrose saline solution all at room temperature it demonstrated total dissolution of all lyophilized particles, repeatedly.

10
Therefore, it was concluded that the canine homoplasma is non-toxic and non-pyrogenic in reaction. It demonstrates good solubility and being a- homologous blood-plasma product it is totally excreted by the body as immune, complexes or disintegrated by the body in the normal course of time and cannot be teratogenic in origin.
The sterility of the product was evaluated after reconstitution, wherein about 20-30 vials were tested at random from a batch of 300 vials for their sterility by inoculating 0.1 ml of the reconstituted canine homoplasma in sterile pertone water for general aerobic organisms, 0.1 ml on Sabarouds agar plate for detection of fungi, 0.1 ml in thioglycollate agar for spore-forming bacteria and 0.1 ml in MDCK and vero cells for detection of viable viruses if any. All were incubated at 37°C except the Sabarouds agar plates, which were kept at room temperature, for about 8 days. There was no growth observed what so ever, on any of the media indicated above, proving the sterility of the canine homoplasma and deeming it fit for intravenous injection.
This canine homoplasma was also given to 4 pups intraperitoneal^ @ 5 ml neat serum to 3 pups subcutaneously in the flank region @ 5 ml neat serum and 4 4 pups . intravenously @ 5 ml diluted with 10 ml of sterile distilled water. None of the pups received the 'serotherapy-mandatory' dexamethazone/ betamethazone 0.5 ml. None of the above pups showed any immediately or delayed reaction, even after 8 days post-trial, proving it to be a very safe injectable homologous preparation..

11
Once the standardization of the Immunizing (attenuated) virus mixture was perfected and the standardization of the challenge (Virulent) virus was completed, the clinical trials for testing the efficiency of the final product was undertaken, first on an experimental basis and later on, on canine patients admitted in various stages of sickness.
EXPERIMENTAL TRIALS:
The Experimental trials were formulated with prophylaxis and curative treatments in mind, groups were made for each type of trial i.e. pups, below one year's age, pregnant bitches, (the stage of pregnancy not being fixed) and dogs older than one year but not above five years. These animals were randomly selected with no differentiation in sex, size, breed etc. Most of the dogs were non-discript/mongrel type, the rest being crossbreeds both of whom have little chance of acquiring antibodies against any of the selected trial diseases, since none were vaccinated. Each group is shown below for prophylactic experimentation and therapeutic trials, the adults were both male and female selected at random for the trials, except in the pregnant groups.
These dogs were brought, dewormed and serologically tested for rabies antibodies, as previously stated in the chapter on "Selection and of care of dogs". Therefore in total there were 120 dogs, in both the trials, put together. The fluctuations in the serum titres of all the dogs are mentioned at the end of the chapter on Experimental trials, wherein the table depicts the entire summary of the exDerimentation.

12
Prophylactic-therapy trials with Canine Homoplasma:
All the dogs in this group were administered the canine homoplasma after 10 days observation period was over; at the rate of 2 ml per kg body weight subcutaneously except the three control pups in Group 1. All pups weighed between 1.5-3 kgs.

Group 1 (Control) 3 pups as control were treated with normal canine plasma 2ml/ kg body weight, e.g. canine serum without those antibodies present in the canine homoplasma -t hese were challenged with virulent virus mixture after 8 days post-inoculation (pi).
Group 2 6 pups were given canine homoplasma 2 ml per kg body weight once and were challenged with virulent virus mixture after about 30 hours, pi.
Group 3 8 pups were given canine homoplasma 2 ml/ kg body weight once and were challenged after 4 day pi.
Group 4 7 pups were given the Canine homoplasma as in group 3 and challenged after 10th day pi.
All the above four groups were challenged on different days with 2.5ml plus 0.5 ml of virulent virus mixture deep intramuscularly and observed daily for symptoms of the diseases, which commenced after about 18-24 hours in case of Group 1 which had not received the Canine homoplasma at all. This group showed sings of tremors of the extremities anorexia, high fever (104°C to 105°C), bloody foetid diarrhoea, in all the 3 pups, shaking of the head, wailing and in general a very soggy and shaggy appearance. All the pups died between about 3-5 days despite supportive therapy.
The pups in Group 4, who were challenged after 10th day pi. started showing some fo the above symptoms on 7th day pi but much milder in nature. These pups showed high fever (103° - 104°) mild, tremors, slight diarrhoea but no blood. Only five pups out of seven recovered after about 3-4 days of supportive treatment. Total recovery time to attain normalcy for the five surviving pups was 10-13 days. (One very weak pup weighing less than 1.5 kgs died after 3 days). One pup suffered paralysis of hind legs and lingered on for an about 20 days after which it died.

13
The supportive treatment consisted of normal saline 15 ml/kg body weight plus 2 ml polybion in the saline and 5 ml neohepatex or any liver extract which is injectable through the intravenous route. This entire therapy is given twice or thrice a day, as the need arises, but not more than thrice in case of pups.
Six pups in Group 3 after the 6 days p.i. showed only dullness and depression and mild fever (103°C) for about 2-3 days, after which they recovered and started feeding on their own. Total time taken to attain normalcy was about 8 days for all 8 pups. No supportive treatment was given.
In case of Group 2 all the six pups looked very wobbly and sick after 7 days pi. but they did not have fever or diarrhoea and did not stop feeding and looked more alert than those in Group 3. These pups recovered within three days without treatment and were normal in appearance and behaviour.
Apart from the protection afforded to these pups by the Canine homoplasma, these pups must be having their own maternal antibodies also, though smaller in quantity since these pups were strays from unvaccinated stray dams. As observed from the group 1, only maternal antibodies are not sufficient to overcome a virulent challenge, maternal antibodies have to be supplemented with concentrated antibodies from the Canine homoplasma. It was therefore concluded that the Canine Homoplasma worked best for pups within 8 days of giving the challenge i.e. the antibodies half-life in the homoplasma was about 8-10 days after which the antibodies started disintegrating very rapidly. The pups are the most susceptible groups of canines, when challenged within about 30 hours of administering the homoplasma (group 2), due to the maximum titres of the passive antibodies the pups did not exhibit much of symptoms and recovered very rapidly.

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Trial with Pregnant bitches: 12 numbers
Pregnant bitches, six in the initial stages of pregnancy - about 3-4 weeks pregnancy and six in late stages about 6-8 weeks (total 62-65 days is the gestation period in bitches) pregnancy were selected at random. Five bitches from the initial stages and five of the late stages of pregnancy i.e. total 10 bitches were given Canine Homoplasma @ 2 ml/kg body weight, except one bitch in the initial and other in late stages of pregnancy, which were segregated as control bitches. These two bitches were given normal canine plasma negative for the antibodies present in Canine homoplasma. All the twelve bitches were challenged with the virulent virus 2.5 ml plus 0.5 ml, deep intramuscularly and observed for the symptoms of the diseases and distress signs of abortion if any. The ten trial bitches were challenged, 48 hours after homoplasma was given.
Control bitches:
The two bitches one in early and other in late pregnancy which were not given any Canine Homoplasma, showed bloody foetid, diarrhoea, haematoemesis, high fever (104°C - 106°C), tonic convulsion of the extremities/head and succumbed before abortion within 35 - 40 hours of challenge being administered, i.e. they died due to the acute forms of the main diseases i.e. the Canine Distemper and the Canine Parvovirus. Supportive treatment was started immediately after onset of first symptom i.e. about 18-20 hours after challenge. This early onset was thought to be due to the extreme stress, which depletes the immune system that the pregnancy creates, which was not found in the pups.

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Early Pregnancy Group
All five bitches in this group showed anorexia as the first symptom after about 48 hours pi, had fever (104°C to 105°C), one bitch had very high fever (106°) and tonic convulsions on the head and extremities. This one bitch also started bleeding profusely per oss and aborted, due to which a litter of six pups was lost, despite starting the supportive therapy six hours after exhibition of first symptom. The bitch took more than 3 days to recover after the constant supportive therapy administration. The other six bitches recovered after supportive therapy administration within 15 to 20 hours, did not bleed nor did they abort, but showed dullness, anorexia, mild temperature of 103°C, roughness of body coat and in general debility.
Late Pregnancy Group
The five bitches in this group started showing the first symptoms after about 48 hours pi i.e. the same time as above group. Three bitches in this group were about 58-60 days pregnant and became anorexic, there was mild reddish discharge per oss, they exhibited breathlessness and one bitch after delivery six, normal pups was unable to walk properly (semi-paralytic) and had mild fever (103°), was dull for about 36-38 hours, but recovered after supportive therapy. The fever inability to walk, breathlessness in this bitch was attributed to eclamsia, which is due to calcium depletion during pregnancy, rather than any effect of challenge virus. The other two bitches also delivered, but remained dull and weak, could not clean their own pups, and had to be helped. They remained dull for the next two days but recovered after supportive therapy. The two bitches which were 50 - 55 days pregnant, remained dull, anorexic, rough coated and mild discharge tinged with blood continued till the bitches delivered. Both the litters were healthy and the pups survived. All the five bitches recovered after supportive therapy and delivered normally, without any loss of pups.

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Therefore it can be concluded that this type of prophylactic passive antibody therapy not only does not interfere with the pregnancy or parturition, but also helps the animals to withstand the onslaught of the virulent (challenge) viruses as well. It can also be concluded that when given at the beginning of the second stage of pregnancy, it is highly beneficial not only for the pregnant bitch but also for the litter, which has excellent chances of survival due to high maternal antibody titres and continuous flow of antibodies for the first eight days through milk.
Trials with adult dogs (males): 12 numbers
Similar to above were two control dogs and ten experimental dogs, all between two and five years old, seronegative for all the viruses mentioned in the challenge viruses.
Group I : Five dogs challenged 30 hours after receiving the Canine
Homoplasma.
Group II : Five dogs were challenged after seven days of receiving the
Canine Homoplasma.
Group III (Control): Instead of Canine homoplasma normal canine plasma was
administered to these 2 dogs which were challenged 30 hrs after grouping them randomly along with Group I.
To all dogs showing symptoms from the three groups, symptomatic supportive therapy was administered but the control group dogs succumbed 3 day pi. These dogs showed typical symptoms of CPV and CD viruses (as described in B) more prominently than others. The dogs in Group I exhibited mild aneroxia, dullness, fever and uneasiness but recovered within 48 hrs. pi, whereas those in Group II were all anorexic, had mild fever (103°C) and were dull in general. One dog after 3 days showed high fever (105.5°) and displayed convulsions and died suddenly. Such type of deaths are observed in cases of acute CPV infections wherein heart block occurs, due to infection of CPV in cardiac muscles. One dog showed slight blood in urine,

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anorexia, 106°C fever, and roughness of coat for about five-six days but recovery was slow. All 4 dogs normalized after 8-10 days pi.
From the above it can be concluded that the trend of recovery follows the half-life of antibodies, i.e. those dogs challenged within 30 hours of receiving the Canine homoplasma stood a better chance of survival than those who were infected at later stages, when the half life of most of the antibodies were at an ebb.
Advantages offered by the present invention over homoserum are:
1. Homoplasma yields 4 times more volume than homoserum.
2. Homoplasma has higher concentration of antibodies.
3. Homoserum has many specific and non specific inhibitors and factors which causes the Allergic Reaction, while Homoplasma causes least reaction and which are tolerable.
4. Homoplasma has more IgG than Homoserum. This nonspecific IgG is the most helpful during recovery of the patient from sickness, as no IgG is lost -whatever the body produces is ultimately used in the body - building mechanism.
Therapeutic/ Curative Trials with Canine Homoplasma
• Trials with 40 Pups:
The 40 pups in the age group between 5 months to 8 months, weighing between 1.5 kg to 3 kg were divided as follows at random :
Group 1: 4 pups as plasma controls were administered normal canine plasma @ 10 ml/kg body weight. This normal plasma did not contain the antibodies found in the canine - Homoplasma preparation. These were challenged with 2.5 ml plus 0.5 ml of the virulent virus mixture after 48 hrs.

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Group 2: 6 pups were given Canine Homoplasma 10 ml/kg body weight only once before challenge with virulent virus after 4 days.
Group 3:12 pups were given virulent virus mixture 2.5 ml plus 0.5 ml deep intramuscularly on day one and after 48 hours were given the 10ml/kg body weight of the canine Homoplasma. Four of the pups in this group did not show full symptoms whereas 8 pups showed severe diarrhoea with blood after about 36 hours.
Group 4:18 pups were given 2.5 ml plus 0.5 ml mixture of the virulent virus mixture on day one and were treated after 72 hours of showing a variety of severe symptoms, with supportive treatment along with 10ml/kg body weight of canine Homoplasma. All the pups were bled for titres before start of experiment and after the treatment at least once.
In case of group 1 all the four pups which were challenged succumbed about 48 hours after the challenge dose, the predominant symptoms were those of canine parvovirus, canine distemper and canine hepatitis, since these are fatal diseases of pups. They manifest in adult dogs as severe symptomatic diseases resulting into fatality due to secondary complications mainly.
In case of group 2, three pups did not show any symptoms and were quite normal in their appearance and behaviour. One pup passed about 13 times liquid foetid blood tinged diarrhoea and appeared as if it would succumb, but once administered with 30 ml of glucose saline and 10ml/g body weight of Canine homoplasma, the pup recovered within 30 hours and started feeding after about 48 hours. The other two pups looked dull, depressed did not feed and had 104°C temperature. Both were constipated and remained in the above condition till about 48 hours after which they recovered but slowly, without any further supportive medication except the Canine homoplasma.

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In case of group 3 wherein the pups were infected and the later on treated after 48 hours with canine Homoplasma irrespective of the degree of symptoms, the experiments stimulates the situation wherein all the animals who come in contact with infected environment and animals wherein the animals have to undergo passive immunization irrespective of their immune status to prevent any major calamity in terms of mortality. Most of the infected cases come late to the clinics i.e. after acute onset of the symptoms as in this group also, wherein five of the eight pups started with symptoms after 20 hours of the inoculation with the virulent virus. All the pups with severe symptoms such as 105°C - 106°C fever, vomination, bloody diarrhoea, icterus, subnormal body temperature, convulsions, incordination of movements etc were all administered 10 ml/kg body weight canine Homoplasma intravenously after the usual treatment with 0.5 ml of decadron to prevent anaphylaxis/ allergy if any. All the pups weighed less than five kg and therefore they were administered between 40 to 50 ml of the canine Homoplasma.
There was complete recovery in. all the eight pups within 48 hours of administration and the other four who received the same showed no symptoms at all. The four pups from this group were dull, depressed, had bloody diarrhoea and succumbed suddenly, such cases are seen most commonly in CPV infections.
In case of group 4, out of the 18 pups which were treated after about 3 days the onsetof the acute disease, only about eleven survived, from these eleven, seven pups were about 8-10 months old, whereas the other four were more than 3 months and less than 7 months old i.e. they were big enough to overcome immunologically the onslaught of the virulent virus till about 72hrs from the 7 which died, these pups were about three and half months to five months old, but smaller in size, weighing between two to four Kgs died within 24 to 60 hrs before receiving the treatment. The 11 which survived showed typical symptoms of mixed infection i.e. high fever (104° -106°C) dysentery, vomition, laced with blood, convulsions, etc leading to prostration.

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After intensive treatment with supportive therapy along with 10 ml/kg body weight of Canine homoplasma, did these pups show signs of slow recovery. Only the 7 older pups recovered and started feeding after about 48 hrs, the other pups which weighed less took about 3 days to recover and additional dose of 10 ml/kg body weight of Canine homoplasma was required.
• Trials with 20 pregnant bitches:
20 pregnant bitches were acquired in the early and late stages of pregnancy and divided into three trial groups - i}The early pregnancy group wherein there were eight bitches, out of which three were primi-para and fine with at least 3 gestations over ii} The second group comprised of the control group wherein one early and one late pregnant bitch would be inoculated with the virulent viruses only and the, iii} third group of 10 late pregnant bitches. All the bitches in the 3 groups were inoculated with the virulent virus mixture of 2.5 ml plus 0.5 ml, deep intramuscularly and observed for onset of the symptoms, which commenced after about 22 to 30 hrs later. All the bitches were given supportive therapy treatment and 0.5ml of decadron before serotherapy as a routine practice.
o The eight bitches in this group showed either mild vomition or mild diarrhoea, one of the primipara bitch was dull, anorexic and other four bitches had 103° to 104°C temperature, but no other symptoms exhibited. All the bitches were administered Canine homoplasma @ 10 ml/kg b.wt. alongwith 15 ml/Kg b. wt. Glucose saline intravenously within 12 hrs of showing various symptoms. Three of the pregnant bitches showed dramatic recovery and only one primi-para bitch aborted resulting into a loss of four pups which were dead, the bitch continued to vomit several times even after abortion. In case of this bitch the Canine homoplasma was repeated after about 24 hours only once. Seven bitches recovered totally after 48 hrs of supportive treatment with only one dose of Canine homoplasma, whereas one bitch took more than five days to come to normalcy.

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o Both the control bitches were not given the Canine homoplasma, only supportive treatment intravenously alongwith normal canine plasma, and both the bitches aborted after about 26-30 hrs post-inoculation with virulent virus, both succumbed to severe viral onslaught, showing mixed infections, the dominant symptoms being bloody, foetid diarrhoea, lacrimation, constant vomition and abortion. The late pregnant bitch died after 36 hours and the early pregnant bitch died after 40 hrs. In this group a total of nine pups were lost during abortion.
o In this group of 10 bitches who also started displaying symptoms 22 to 30 hrs post inoculation, one group of four bitches was treated at about 22 hrs i.e. immediately after the onset of symptoms with 10ml/kg b.wt. of Canine homoplasma and 15 ml per kg b.wt. of glucose saline. Out of these bitches two could not withstand the onslaught of the virulent virus mixture and aborted, these were given intensive supportive treatment with added liver tonic 5 ml in the above glucose saline and both the bitches had to be given 10 ml/kg body wt additional dose of Canine homoplasma after 18 hrs of the first dose and the supportive therapy was administered every six hours sin a drip form. A total of eleven pups was lost during abortion.
Three bitches although they showed milder symptoms of only vomition, dullness and lacrimation, continued to show these symptoms for about 3 days after the initial C. Homoserum dose. These were given only one dose, since their condition did not worsen any further and they did not abort. The other five bitches reacted by displaying, diarrhoea with blood, vomition laced with spots of blood, dullness, anorexia, severe lacrimation, incordination of movements, two bitches even showed yellow tinges to the mucosa. For these bitches treatment was delayed to 24 to 30 hrs after onset of symptoms and the Canine homoplasma administered with full precautions by dissolving 10 ml/kg b.wt in about 50 ml glucose saline and giving it as slow intravenous. The same was repeated on 3 bitches after about 30 hrs. Two of the above five recovered within 6 hrs of administration of Canine homoplasma,

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although they were very weak, but did not abort, one of the -remaining two recovered slowly, thus had to be administered the second dose of Canine homoplasma after 24 hrs of the first dose.
From the above it can be safely concluded that 'the Canine homoplasma can safely be given during early or late pregnancy, which is due to the fact that since it is formulated from the same homologous species i.e. the canine, the rate of reaction or allergy/anaphylaxis is minimal. Unless and until the dogs are in the last stages, with the virus/viruses having proliferated throughout the body tissues, the antibody or immuno-therapy has a dramatic effect as can be demonstrated from the experiments above.
As stated in the previous trials, these dogs were inoculated with the virulent virus mixture and it took about 24 - 36 hrs for the onset of the symptoms to initiate, since a stray adult dog has a fully developed immune system, ready to react strongly. These dogs were divided into three groups, wherein only i} 2 dogs were held in the control group which was not to be given supportive only, ii} The group was treated after about 6 - 10 hrs of the onset of symptoms and the last iii} group was treated after 24-30 hrs after the onset of symptoms.
i} The two control dogs initially showed symptoms of dullness, depression, anorexia followed by rise in body temperature to about 104.6°C to 105.8° for the first two days and third day onwards suffered from severe gastro-enteritis with copious bloody diarrhoea and vomition with blood clots, one dog died within six hours of the onslaught and the other succumbed after bout 36 hrs of the initial symptoms and in addition to above, also showed deep yellow mucous membranes, lacrimation and death due to severe dehydration.

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ii} In this group of 8 dogs, five were treated after 6 hrs of the onset of the symptoms, with 10ml/kg b.wt of Canine homoplasma and glucose saline @ 15 ml/kg b.wt and observed for signs of recovery. One male dog who showed an initial rise in temperature to about 104°C had copious stools which were black bloody with clots and foetid odour after which the dog could not support itself and collapsed. Thus dog was being given glucose saline and Canine homoplasma when he died, having subnormal temperature. This dog displayed typical signs and clinical symptoms of Canine parvoviral gastroenteritis. It is not common for adult dogs to succumb to the parvoviral disease and therefore it seems that either the dog was immuno compromised, or had a heavy worm load or a concurrent infection was present, whereby the dog could not sustain the onslaught of the fresh viral infection.
Out of the remaining four dogs, three showed typical symptoms of infectious Canine Hepatitis which is quite common in adult dogs, with a rapid development of icterus, coughing, lacrimation from the eyes, whitish yellow stools, pale mucos-membranes and anorexia. These dogs received the Canine homoplasma in the prescribed proportions with a glucose saline drip and after another 10 - 12 hrs recovered enough to drink water on their own. After another six hours they had bread and milk and were on their way to recovery. The fifth dog-a bitch apart from showing the mild gastro enteritis also vomited several times, had sub-normal temperature of about 100°C and was quite dull but recovered within 10-12 hrs of the prescribed treatment with Canine homoplasma and started feeding within another 3 hrs.
Three dogs were treated after 10 hrs of the onset of the symptoms which had become very acute with diarrhoea, vomition, pale yellow mucosa and two dogs showed total recumbency. All the three dogs were treated with the prescribed Canine homoplasma with glucose saline, but considering the acuteness of the disease they were administered by the show intra-venous route. After about 2 hrs later the vomition and diarrhoea stopped, but weakness, icterus and recumbency persisted, therefore glucose saline @ 15 ml/kg, b.wt alongwith neophepatex 5 ml

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total, was administered. Weakness, anorexia and subnormal temperature of 100°C persisted at times even tonic muscular spasms were observed, therefore another dose of Canine homoplasma was repeated after 24 hrs along with 50 ml glucose salien in drip form, this helped to relieve the symptoms drastically and after about 24-30 hrs the three dogs were drinking water and moving around, overcoming their recumbancy. From the 8 dogs in this group one died and the rest recovered from very critical conditions of the virulent viral attack. All the 7 dogs were feeding and behaving normally on the third day of the experiment.
iii) In group there were 10 dogs treated after 24-30 hrs of onset of the symptoms - all the dogs after 24 hrs of purging, vomition, high interns, coughing etc were in a bad shape, most of them were dull, depressed, dehydrated, 8 dogs had tonic convulsions, two dogs were only vomiting blood and were gaping for breadth, due to lowered haemoglobin levels, and fluid loss. In these two dogs, it was not possible to locate the veins and therefore the Canine homoplasma was given subcutaneously in the flank regions -i.e. in one flank the Canine homoplasma and in another side about 200 ml of dextrose salien with neohepatex 5 ml. After about two hours one dog recovered sufficiently to lick some water while in a sitting posture, but was unable to support himself on all four legs. The other dog during the same time was recumbent but alive. The former dog was given 200 ml glucose saline plus 5 ml neohepater after about 3 hrs in the flank region once again, but died after about 8 hrs of start of the treatment due to cardiac and respiratory arrest. The later dog which was recumbent died in a lingering manner without any further symptoms after about 6 hrs of the onset of symptoms. The eight dogs with convulsions were treated in a similar manner and were given 200 ml glucose/ dextrose saline and 5 ml neohepatex every three hrs and Canine homoplasma after the initial dose was given after 10 hrs interval. Three dogs showed rapid improvement and could drink water, stand up and take a few steps before falling. In these three dogs, the saline treatment was stopped after 6 hrs (two treatments) and only one more dose of Canine homoplasma was administered after 10 hrs alongwith the other dogs. Out of the remaining five

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dogs two dogs lingered on with dullness, anorexia, subnormal temperatures and severe debility. After two days these dogs developed icterus and correa, despite a third dose of C. Homoserum given to all the eight dogs after 30 hrs. Both these dogs died of severe dehydration and respiratory failure on the fourth day, despite concentrated efforts with supportive therapy. These deaths were due to extensive tissue damage and collapse of the immune defence mechanisms, wherein intensive efforts to save their lives failed, due to lack of administration of treatment in the proper time (early treatment) i.e. treatment was given too late. Only three dogs survived, but were slow to recover from their acute onset, due to delay in the treatment.
All the three, after about 8 days showed correa, incordination of movement and erratic intake of feed. These three dogs were given a fourth dose of Canine homoplasma on the eighth day when the supportive treatment was stopped and the dogs after a further three days observation period were declared fit. This Canine homoplasma was given @ 2 ml/kg b.wt as a precautionary measure, to neutralise residual circulating antigens if any. There were reports by several clinicians who were given trial samples, that the residual symptoms of weakness, incordination of movements, correa, erratic feeding etc reduced considerably when at the end of the treatment period one dose of Canine homoplasma @ 2ml/kg b.wt. was administered.
Considering the above experimentation, it can be concluded that all the viruses and the bacteria administered as an virulent virus mixture before various treatment intervals follow a pattern of universal truth - i.e. prevention is better than cure, and that early treatment simulates a near preventive measure. The fact that the viruses and the bacteria involved are all celulolytic in nature, cause extensive tissue damage in very short periods of time especially, during an acute onset, means that treatment if any should be initiated as early as possible to avoid extensive and irrevocable tissue damage which leads to a total breakdown of the body-mechanisms and culminates in death.

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From the table, a conclusive pattern of survival can be noted, wherein the antibodies concentrated in the Canine Homoplasma have helped the affected dogs to recover rapidly.
The blood is taken for antibody serum titre after the observation periods, since there is no titre detectable in the unvaccinated dogs. The titres can be detected only when challenged, till then the antibodies are dormant and not activated by the memory forming B-lymphocytes. This conclusion was drawn after checking the titres of several pups and adult dogs. The pups had generally negligible titres of maternal antibodies, which totally disappeared on challenge.
The most common types of antibodies encountered were against the Canine distemper, canine parvovirus, infectious canine hepatitis, and least against Canine leptospira infections. The other two disease's antibodies, although fatal for dogs, were not commonly encountered. In case of the experimental trials, the average serum titres represent only those antibodies which are in circulation after the neutralization of the challenge virus. In case of pups these titres were low due to the late response of the immature immune system, whereas in case of the adult dogs and pregnant animals, the titres were much higher due to the response of a fully developed immune system. It is also felt that there must have been an add on titre due to the amnestic B-lymphocytic stimulation in dogs who had suffered from some of the diseases earlier in life and survive their onslaught.

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CLINICAL TRIALS OF CANINE HOMOPLASMA
Clinical trials were carried out on dogs/ pups/pregnant bitches which were brought by owners for either admission to the attached animal hospital or to the out patient departments or to the private clinics of the practising' veterinary doctors. Most of the animals brought by the owners were already suffering from acute stages of the disease/s or were in the incubation stages, whereby routine treatment generally aggravated their conditions. Any dog, whether adult or pup cannot survive when brought in the last stages of any nearly fatal sickness. These clinical trials were carried out over a period of three years from 1986 to 1989 and in the results represented here the dogs have been broadly classified in three susceptible groups i.e. Pups from 2 week to 12 months, B. Adult dogs from 1 year to 6 years and C. Bitches in various stages of pregnancy i.e. first (early pregnancy) transistor, and last trimester (late pregnancy), and recently parchurited (delivered) bitches. A. total of A 571 pups, B. 337 adult dogs and C. 297 bitches were administered the serotherapy with canine homoplasma, either in smaller or larger doses, i.e. in total the clinical trials of 1200 dogs are recorded in this study. For the sake of convenience, only the outstanding or complicated case histories are mentioned and discussed, otherwise a generalized clinical picture of these cases is presented and evaluated. Since most of the dogs were brought by the owners in a very serious condition, it was difficult to persuade the owners to allow the animal to be bled even for 5 ml blood, for fear that the animal may collapse and die. Therefore, serum titres were not taken in all the cases as done in the experimental trials. Those serum titres that were evaluated gave a fairly accurate diagnosis of the situation the animal was in. Most of the dogs, especially the pups were infected before primary vaccination was administered and therefore their titres represented the actual virulent strength of the viral infection. The serum titres in case of clinical trials were those of the disease which demonstrated first onset symptoms and the maximum symptoms i.e. Canine Parvo virus, Canine Distemper and varying numbers of animals showed icterus, typical in Infectious Canine Hepatitis/ Leptospirosis.

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Clinical Trials in 571 pups :
In case of pups the most fatal disease is the Canine distemper (CD) followed by the Canine parvovirus, infectious Canine Hepatitis. Canine Para Influenza, Canine Laryngotracheitis and lastly the Canine Leptospirosis. The common symptom in all these diseases is rising body temperature to about 103° - 105° C. The most outstanding symptom of CD in pups is cannot support itself, discharges from eyes and nose, twitching of muscles, at times convulsions, wailing, shaking of head and wobby gait.
In case of Parvo virus the symptoms are bloody diarrhoea, only blood clots fall instead of faecal matter, and pup also vomits blood, there may be blood in urine and generalized acute dehydration, followed by coma and death. In the cardiac form, there was sudden death, here the pups do not show any of the symptoms described above, only fall to the ground dead. This was seen also as a mixed form, i.e. the pup showed initially dysentery, was treated for about 2-3 days and suddenly died, at times towards the end of the treatment, while recovering. In these cases death was diagnosed as due to heart block, on post-mortum.
In case of mixed infections in pups mortality rate was higher and can be attributed to the fact that pups not only have weakly developed immune system, but due to their tender age, smaller size and body weight, they weaken easily due to dehydration. In pups all the vital organs such as the liver, spleen, kidneys, heart and even brain are still immaturely developed and cannot sustain the onslaught of the disease.

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They even do not respond to the treatment and have to be given ready to immunize supplements such as Canine homoplasma, which gave heartening results in this category. About 10 ml (1 vial) homoplasma along with the full supportive therapy has to be administered every 18-24 hours. The pups were given supportive therapy comprising of 20-10.0 ml dextrose (5%) saline @ 20 ml/kg body weight, 0.5 ml to 2 ml of Neohepatex a crude liver extract and 0.5 to 1 ml of polybion - a vitamin B-complex supplement with Co-enzyme A.
Pups weighing within 1 kg, were those who suffered acutely and more than 20% dehydration by body weight was observed resulting into death. These mortality cases comprised of about 126 pups i.e. 22% most of these were from those litters, the dams of which were not vaccinated during epidemics in certain localities of the Mumbai city. In these cases the dams were either not vaccinated and were unable to transmit any immunity to the pups, whereas in some cases it was seen that the dame was already incubating the disease and the new born pups contracted the same, while suckling the dam within few days of birth. The other type of succeptible group of pups was the one which was about 1/4 to 2 months old unvaccinated, recently sold to new owners and due to infection in the environment, the pups caught the virulent viruses, the onset of such disease was not only very sudden but the disease spread • in the body rapidly with fatal consequences, at times even before treatment could be initiated.
The lowest mortality was encountered amongst pups in the groups which was slightly older i.e. between 2 to 6 months here the mortality was only 28 pups i.e. 5%. These pups weighed between 1 kg to 6 kg body weight and could be categorized as recently vaccinated pups with infection, and unvaccinated pups with infection. The 28 pups which died in this older age group, was mainly because some had been brought to the hospital and private clinics in very serious conditions and no amount of treatment could save these.

Amongst the first groups pups, the vaccination may have been given during the disease incubation stages and the disease flared up, or the vaccine may have been partially neutralized as in cases of residual maternal antibody activity and during an acute onslaught of the virus, resulted in inadequate protection. Another reason is that vaccination produces a stress and if given durjng epidemics in the locality, the antibody is not produced in sufficient quantities at short-notice and disease manifestation sets in to overtake the antibody production. Out of such 221 cases treated there was an overall response of 90% i.e. 199 pups recovered after the Canine homoplasma was administered to them with supportive therapy every 18 hours. Out of these 221 pups only 12 pups were required to be given three doses of Canine homoplasma on alternate days, the rest of the pups showed a dramatic break in the acute disease symptoms and recovered to about 80% after the second day of treatment. Some of these pups had suffered from mixed Canine distemper and parvovirus infection and these few pups showed residual symptoms of shaking their heads periodically, incoordination in gait, etc; but had resumed feeding normally as well as regained other normal body processes. In all the cases treated with Canine homoplasma, fever 104° C to 106°C was the first symptom to come down to 101° to 102° and in case of CPV, the dysentry was the first symptom to stop.
Mortality Percentage in Pups

Pups Mortality in Pups Overall Mortlaity
Pups < 1 kg Pups > 1 kg
517 126 28 154
22% 5% 27%
Overall recovery after treatment with Canine homoplasma was 73%.
In case of infectious canine hepatitis and canine para-influenza, the symptoms of jaundice, and acute respiratory distress and congestion of the chest were first to be relieved, within a span of 24 to 30 hours. Those cases, which showed dysentery, were invariably more serious than other cases, because apart from the disease aspect, severe dehydration and loss of blood were the other debilitating factors.

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Amongst the unvaccinated infected group the response was less dramatic, since there were no antibodies in their system and as soon as passive antibodies were introduced, the body defences reacted and partial virus neutralization occurred. These pups had to be given at least two more doses of the Canine homoplasma on alternate days to overcome and neutralize the circulating viruses. Similarly out of 571 pups infected in total, 417 survived and recovered resulting into 73% survival rate in treated animals and 27% mortality rate in weak and advanced cases which could not be saved. Since these were the owner-based pups and the owners refused to allow withdrawal of blood for titration, serum titres are not presented.
Clinical Trials in 337 adult dogs
In case of adult dogs the diseases showed no clear cut symptoms, dogs were presented starting with high fever (104° to 106°C) anorexic, dull, depressed at times very weak and since no differential diagnosis could be made, the treatment in such cases was terramycin - 2 ml (500 gms) and 5% dextrose saline @ 25 ml/kg body weight. If the animal after 24 - 48 hrs started showing any other symptoms, treatment was administered accordingly or if no response to above treatment was felt, then in addition Canine homoplasma @ 10 ml/kg body weight, was administered, because all the six diseases mentioned are initiated by high fever and above symptoms. Generally after the first dose the fever and other symptoms drastically reduced indicating that the animal was incubating any of the above diseases. The advantage of Canine homoplasma is that it also works in cases of non-specific diarrhoea and fever, resulting into total and rapid recovery.

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This may be due to the fact that during the generation of specific Antibodies a lot of non-specific IgG is also released into circulation as a humeral response of the immune system, apart from the cell mediated specific response. Since no ammonium sulphate precipitation is followed, during manufacture of Canine homoplasma, this IgG remains intact and helps in more rapid recovery. This IgG is not measurable since during the immunological tests known/ specific antigens react with the specific antibodies, but in the blood stream of the animals body, this IgG makes its presence felt and attaches itself to other antigens/ antigen-cell receptors etc.
Young adults about 13-15 months old were the worst affected and brought into the hospital / clinics quite debilitated, unconscious in conditions, especially true of dogs belonging to the smaller breeds category such as the pomoranians, pekenese, terriers, spitz and miniature breeds. The body weight of these dogs was hardly between 2 kgs and 5 kgs, therefore it was a hindrance for the much needed supportive therapy, since the Canine homoplasma had to be given half intravenously and half subcutaneously, since the dosage was 10 ml/kg body weight for the curative measures. In this group only two dogs out of 337 treated died due to adverse serum reaction, i.e. due to allergic causes, after administration of Canine homoplasma.
These two dogs were treated previously for about five days in a private clinic and when they did not respond to treatment, they were brought as emergency cases to the animal hospital having erythematous eruptions and on giving strong remedial antihistamine, went into a coma and died due to respiratory collapse. These are know as idiotypic serum reaction, wherein due to previous treatment with serum the dog is sensitised and idiotypic antibodies are formed, resulting into a type II hypersensitivity reaction, wherein severe complement erythrocytelysis occurs and the dog goes in a comatose condition due to histamine release etc.

33
There were 33 cases which were presented with a mixed type of infection, i.e. severe jaundice with the belly of the dog totally yellow, body skin yellowish gray, discharge from the eyes, mild greyish diarrhoea, twitching of the ears, face and limbs periodically, 104°C constant fever, not responding to ordinary antibiotics and supportive therapy. Nine of these dogs which were totally dehydrated, slipped in a come and eventually succumbed even after treatment with Canine homoplasma and improved supportive therapy.
Table : Mortality Percentage in Adult Dogs.

Total Mortality in dogs Overall
Total Idiotypic dogs Mixed infection Dysentry dogs Mortality
337 2 9 6 17
6.7% 30.3% 20.2% 5.1%
Overall mortality in adult dogs was 5.1% and overall recovery after treatment with Canine homoplasma was 94.9%.
There were about 54 dogs which were presented with subnormal (99° - 100°) temperature, acute dysentery, vomiting laced with blood clots, difficulty in breathing, twitching of limbs etc. In 33 cases there was remarkable stoppage of bloody diarrhoea, cessation of vomition, twitching of limbs and normalization of body temperature on administration of first Canine homoplasma dose.
Out of 54 presentations only 6 dogs died. Five of the recovering animals had to be given another dose of Canine homoplasma for speeding up the process of recovery i.e. to neutralize those antigen particles which are released in circulation after about 24 - 30 hours during the phase of recovery from acute onset of disease (i.e. out of 337 adult dogs only 17 died and 320 recovered).

34
Clinical Trials in 297 Pregnant bitches
Pregnancy as stated earlier causes great stress in the body and on the immune system of the bitch. During this period all the body systems contributes towards the development of the foetus, its immune system development etc particularly towards the late pregnancy period, the immune system of the bitch during this phase is weakened and totally unprepared to fight the disease/ antigen onslaught if any. Antibiotic therapy is generally avoided in pregnancy due to its aberrant action on the foetus. Whatever is injected parentrally into the body is secreted through the milk in pregnancy. Therefore to save the litter if the bitch is passively immunised about a week before delivery then the litter of the bitch not only has a good antibody titre but
retains the antibodies much longer, as the bitch's milk contains higher concentrations of secretary/ neutralizing antibodies. Only in rare cases would these passive homologous antibodies have idiotypic action, causing abortion. This is a risk to be taken when the environment is heavily contaminated or the bitch is in contact with other dogs who are actively suffering from any of the high-risk viral diseases.
There were about 131 bitches in early pregnancy, which were presented in the hospital and private clinics. From these 131, 82 were in contact actively with infected animals in their neighbour hoods and had started showing mild diseases symptoms, which could have been due to any of the above viruses. The most common symptoms included high fever (104° -106°) anorexia, reddish discharge per oss, five bitches had yellowish tinge on their hairless parts of the body. One older bitch had loose stools without blood.
The five bitches which had yellowish tinge to the skin, started vomition a day after admitting tot he hospital and were given Canine homoplasma about four days after onset of symptoms.

35
Table : Mortality Percentage in Pregnant Bitches:

Total No. of Bitches Mortality in Early Pregnancy Pregnant Bitches Late Pregnancy Overall Mortality
297 4(1.34%) 1(0.33%) 5(1.68%)
Nos. of litters lost 2 Litters of 11 pups 7 litters of 32 pups 9 litters of 43 pups
Overall recovery after treatment with Canine Homoplasma was 98.32%.
' • ■ y
Two of the bitches which were vomiting at least 12-14 times a day aborted and had
subnormal temperatures, turned toxic and succumbed after 4-5 days of abortion. A
total of 11 pups were lost from the two. The other three stopped vomition and
regained normal body temperature after 24 hrs treatments with Canine homoplasma.
Therefore although there was only two fatalities amongst this group of five infected bitches, two precious litters totalling 11 pups was lost. The yellowish body tinge faded away after about a week and the 3 bitches were feeding normally about the same time as above. The older bitch with loose foetid smelling stools was lost. The yellowish body tinge faded away after about a week and the 3 bitches were feeding normally about the same time as above. The older bitch with loose foetid smelling stools was given the C homoplasma a day after the symptoms and therefore " recovered without any. further complications. The other 49 bitches in early pregnancy, showed varied symptoms, but 12 bitches showed mixed distemper and parvovirus symptoms and at times loose sanguinous stools. All the twelve were administered canine Homoserum about 24-48 hrs after onset of symptoms and except for one very young white bitch, a primipara about one year two months old, who collapsed and died. On post mortem heart muscle and intestinal contents showed presence of Canine Parvovirus (CPV) and the liver had haemorrhaegic patches. The other was about two and half years old with a congenitally enlarged heart and could not withstand the stress of pregnancy and distemper, which was later on isolated from the Urinary bladder.

36
The 166 bitches in late pregnancy were admitted or brought to the hospital/ clinic in various stages of emergency, wherein they were administered first - aid and Canine homoplasma. 16 bitches were brought to 3 private clinics with severe eclamsia, wherein calcium and glucose saline was administered with decadron and Canine homoplasma as a precaution, since some of these bitches showed abdominal contractions. All were administered Canine homoplasma within first 24 hrs of the concentrations, but still six bitches aborted and six litters, (collectively 42 pups) were lost. No mortality was noticed in these bitches which eventually recovered, but pups could not be saved.
From 80 bitches which were on the verge of delivery and got infected, all were administered Canine homoplasma within 24-48 hrs after delivery. Only one bitch died of toxic complications with the litter unborn. There were about 42 bitches which gave birth to about 338 pups form 42 litters, out of which only 32 pup succumbed to infection, that too because they were too small to inject the Canine homoplasma. They weighed between 40-65 grm only. The larger pups weighing between 80-100 gms were given 0.5 ml subcutaneously Canine homoplasma and the (mother) bitch of the litter was given at the rate of 10 ml/kg b.wt. the full dose, so that should could secrete the antibodies in the milk and save herself and her litter. From the other 38 who showed symptoms of mild fever (103-105°C) off feed, vomition and weakness of the hind limbs, unable to support themselves, etc were put on high supportive therapy mixed with Canine homoplasma, these bitches required another dose about one week later, bud did not abort. They delivered 8-10 days after the start of therapy and were given the second dose just after delivery, so that there was sufficient protection for themselves as well as their litters. The overall recovery rate was 98.32%.

37
It can therefore be concluded that in case of pregnancy, if the Canine homoplasma is given in time, it was capable of saving not only the life of the bitch but also was able to sustain the litter. Therefore it is suggested that the Canine homoplasma may be , given to the pregnant bitch two weeks before and one week after delivery to ensure a healthy dam and litter.

Group & No. No. Range of Serum titres %
No. of dogs Survived Dead ' - Recovery
Dead Survived
Prophylact 38 10 CPV :0-:128 128-1024 79.95
trials (48) CD :0-:128 ICH :0-:80 CPV2 :0-:80 CPI :0-:128 LH :0-:128 64-1024 40 - 640 20 - 320 20-512 16-512
a. Pups (24) 19 5 CPV:0-:128 CD :0-:128 ICH :0-:80 CPV2:0-:80 CPI :0-:128 LH :0-:32 1:128-10241:64-5121:40-3201:40-3201:20-2561:16-128 79.16
Group 1 (3) - 3 CPV :0- - -
Control • 1:128 CD :0-:64 ICH :0-:40 CPV2:0-:20 CPI :0-:0 LH :0-:0
Group 2 (6) 6 CPV-CD-ICH-CAV2-CPI-LH- :256-:512:128-:512:40-:320:40-:80:128-:256:64-:128 100
Group 3(8) 8. CPV-CD-ICH-CAV2-CPI-LH- :256-:1024:64-:512:80-:320:40-:320:128-:256:64-:128 100

B. Pregnant 10 2 CPV :0-: 128 :256-:1024 83.33
Bitches (12) CD :16-:128 ICH : 10-:40 CAV2 :20-:40CPI :0-:128 LH :0-:128 :128-: 1024 : 40-:640 :20-:80 : 64-:256 :64-:512 '
Control (2) 2 CPV: 0-:128 CD: 16-:64 ICH : 10^:40 ' CAV2 :20-10" CPI :0-:128 LH :0-:128 V
Early 5 - CPV- :256-:1024 100
Pregnant CD- :256-:1024
(5) ICH-CAV2-CPI-LH- :80-:640 :40-:80 :64-:128 :64-:256
Late 5 - CPV- :256-:1024 100
Pregnant CD- :128-:512
(5) ICH-CAV2-CPI-LH- :40-:640 :20-:80 :64-:256 :128-:512
C. Adult 9 3 CPV:0-:128 :256-:1024 75
Dogs (12) CD: 8-: 128 ICH : 0-:80 CAV2 :0-:80 CPI : 0-:64 LH :0-:128 :128-:512 :160-:640 : 20-:80 :64-:512 : 80-:256
Group 1 (5) 5 CPV-CD-ICH-CAV2-CPI-LH- :256-:1024:128-:512:160-:640:80-:160:256-:512:128-:256 100

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Group 2 (5) 4 . 1 CPV:0-:128 CD: 32-: 128 ICH : 0-:40 CAV2 :0-:80 CPI: 0-:64 LH :0-:128 :256-:1024 :128-:512 :160-:640 : 20-:80 : 64-: 128 : 80-: 128 80
Group 3 (2) - 2 CPV :0-:8 - -
Controlt - CD :8-:128 ICH : 10^:80, , CAV2 :0-:40 CPI : 4-:64 *--LH : 0-:8 »

40
Therapeutic trials in dogs (40)

Group & No. No. Range of Serum titres 0//o
No. of dogs Survived Dead Recovery
A. Pups 25 15 CPV:0-:128 :4-:512 62.5
(40) CD 3p-: 128 ICH :t)-:320 CAV2 :0-:40 CPI : 0-:64" ■ LH :0-:16 :0-:1024:0-:1280:0-:80:0-:512:0-:64
Group 1 (4) - 4 CPV :0-:64 - -
Control CD : 0-:32 ICH :10-:40 CAV2 :10-:40 CPI : 0-:16 LH :0-:16
Group 2 (6) 6 CPV-CD-ICH-CAV2-CPI-LH- :4-:512:32-:1024: 10-: 1280:0-:80:0-:512:8-:64 100
Group 3 8 4 CPV:0-:128 :10-:512 66.6
(12) CD : 0-:64 ICH : 0-:320 CAV2 :0-:10 CPI : 0-:64 LH : 0-:0 :0-:256:0-:640:0-:40:0-:128:0-:8
Group 4 11 7 CPV:0-:128 :8-:512 61.1
(18) CD :0-:128 ICH :10-:40 CAV2 :0-:10 CPI : 0-:8 LH : 0-:0 :0-:1024:0-:320:20-:20:32-:256:0-:0

41

B. Pregnant 18 2 CPV :0-:64 :0-:1024 90
Bitches (20) CD : 0-:32 ICH : 0-:40 CAV2 :0,:0 CPI : 0-:4 .. LH : 0-:8 :10-:1024:0-:320:0-:40:0-:256:0-:128
Early 8 - CPV- :10-:512 100
Pregnant CD- :40-:-1024
(8) ICH-CAV2-CPI-LH- :0-:160 :0-:40 :0-:128 :0-:64
Late 10 - CPV- :0-:1024 100
pregnant CD- :10-:256
(10) ICH-CAV2-CPI-LH- :0-:320 :0-:40 :0-:256 :80-:128
Control (2) 2 CPV :0-:64 CD : 0-:32 ICH : 0-:40 CAV2 :0-:0 CPI : 0-:4 LH : 0-:8
C. Adults 13 7 CPV:0-:128 :4-:1024 65
(20) CD: 0-:128 ICH :20-:80 CAV2 :0-:40 CPI :0-:128 LH : 0-:256 :0-:512:10-:320:0-:80:0-:256:0-:128
Group 1 (2) - 2 CPV :0-:32 - -
Control CD :0-:128 ICH :20-:80 CAV2 :0-:40 CPI : 0-:64 LH : 0-:32

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Group 2 (8) 7 1 CPV :16-:128CD : 0-:128ICH :20-:40 :32-:512 :64-:512 :40-:320 :40-:80 87.5
CAV2 :0-:40 CPI : 8-:64 LH : 0-:256 :64-:256 :8-:128 i.
Group 3 6 4 CPV :8:128 '* •4-: 1024.. 60,
(10) CD: 4-: 128 ICH :40-:80 CAV2 :0-:40 CPI : 16- :(P:51-2. , :10-:320 :0-:10 :0-:64
:128 :0-:128
._. LH : 0-:32
Total no. of dogs in experimental trials : 88 Nos.
From the above table it is seen that only the surviving animals have attained the requisite titres. In this experimental group the pups, had no titres initially and developed titres on giving the C homoplasma as in case of the prophylactic group or developed the titres after virulent virus infection as in case of therapeutic trials.
Only in case of a small percentage (2-3%) adults had a negligible titre for CD/CPV, . but otherwise were clean i.e. no initial titres. In case of the pregnant bitches, only 2 bitches had initial titres for CD (1:10 and 1:40) and one had titres for C. Leptospira, i.e. 1:80, but did not demonstrate live bacteria, through urine, this was considered to be due to pregnancy stress.

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Table depicting the Clinical Trials : Conclusive evidence of efficacy of Canine homoplasma:

Groups & No. No. % Range of Serum titers
No. of dogs survive Dead Recove
d ry
Survived Dead
A. 571 417 154 73% CPV: 16-1024 16-:256
Pups CD: 257-1024. 16-:128
ICH : 40-640. , CAV1: 64-256 CAV2: 40-160 LH : 32-256 20-:40 8-:128 10-:40 4-:64
B. 337 320 17 94.9% CPV: 64-1024 32-: 128
adults CD: 128-1024 ICH: 40-1280 CAV1: 64-256 CAV2: 80-320 LH: 64-512 16-:128 20-:40 32-: 128 20-:40 64-: 128
C. 297 292 5 98.32% CPV: 128-1024 16-: 128
Pregnant CD: 128-1024 32-:256
bitches ICH :80-1280 CAV1 :64-256 CAV2 :40-160 LH : 64-256 10-:40 . 16-:64 10-:20 32-: 128
No. of - 9 i.e. 43 -
litters lost pups
Total No. of dogs : 1205 Nos.
CPV - The HI titre of 1:256 is considered as protective, the range of titres i.e. lowest
and highest is mentioned.
CD - the titre of 1:128 is considered as protective, the range of the titres is depicted
in the table.
ICH - The CFT titres of 1:80 is considered protective the range is as above
CPI - The HI titres of 1:128 is protective, the range is as above.
CAV2 - the CFT titre of 1:80 is protective, the range is as above.
LH - The agglutination titre of 1:128 is considered as protective, the range is as
above.

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• The titres of the litters could not be measured since, these were born dead or died immediately after birth.
• Amongst the pups, out of 41 surviving pups only 54 could be bled and 82 pups out of 154 dead could be bled for titres, the owners refused permission for bleeding.
• Out of 320 adults only 32 could be bled for titres and .out of the 17 dead about 10 dogs could be bled, all were owned dogs.
• Out of 292 surviving bitches, since there was negligible difference in the titres of the early and late pregnant bitches, these were not differentiated but given as single titres.
From the above tables it is seen that some of the dead dogs have high titre to some diseases which is became they have no circulating antigens for these diseases, due to which there is no neutralization or complex formation by the antibody. The dog can show low titres, which means that there is active neutralization of the antigen occurring after giving the Canine homoplasma. Lower antigen titres antibody of one disease and high antibody titres of other would mean that the former antigen is out of the tissuecells and is undergoing neutralization by the antibody, whereas only high antibody titres would mean that either the antigen has still not come out of the tissue, as in case of incubation, or the animal does not have that particular antigen/disease. The dog which become weak physically cannot cope with the infection i.e. invasion of the disease agent immunologically, and generally succumbs during an acute onslaught, although his antibody titres are quite high.

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In case of pups, the serotherapy with Canine homoplasma acts as a boon, since along with their own depleting antibodies, these antibodies give them the neutralizing power and therefore high recovery rate. In case of the pregnant bitches the antibodies being homologous in nature are readily accepted, resulting into natural delivery and no abortion. In adult dogs also, some residual antibodies, which are not sufficient to overcome acute infections and an amnesic response takes about 2 to 4 days to bring about antibody production, therefore the Canine homoplasma turns out to be a boonf or such animals.
The recovery rate is a definite conclusion of the efficacy of the Canine homoplasma's role in neutralizing active infection.

46
We claim:
1. The process for manufacturing pharmaceutical composition for providing passive
immunity to canine comprising of following steps:
a) Selecting an animal,
b) giving an injection of antigen of organism to animal,
c) giving booster dose of antigen,
d) checking antibody titre,
e) removing plasma from the animals,
f) adding antibiotic to the plasma,
g) filling plasma into glass vial, h) lyophilization.

2. The animal as claimed in claim 1 can be canine.
3. The canine as claimed in claims 1 and 2 can be a dog.
4. The organism as claimed in claim 1 is canine distemper, canine parvovirus, infectious canine hepatitis, canine laryngotraceitis, canine parainfluenza type II, and canine Leptospirosis.
5. The antibiotic as claimed in claim 1 is selected from broad spectrum antibiotic.
6. The antibiotic as claimed in claim 1 and 5 is gentamicin.
7. The method as claimed in claim 1 and herein described in example 1.
Dated this 23rd of July, 2001

Dr. Bakulkesh Mafatlal Khamar