FIELD OF THE INVENTION:
The present invention relates to a novel COFEB (Complement Fixation Test for Equine Babesiosis) buffer and kit useful in detection of Babesia equi antibodies.
BACKGROUND:
COFEB - kit is intended for detection of the antibodies of Babesia equi (B. equi), a ptotozoan parasite which is transmitted by ticks. It infects red blood cells of the equines (including horses, donkeys and mules) and induces high fever, anaemia, jaundice, haemoglobinurea and the infection may terminate into death. However, the animals that survive the attack, on recovery may become permanent source of infection to other animals. Although morbidity and mortality rates, have not been adequately worked out in the country, Malhotra et al. (1978) conducted serological survey of healthy equines in some parts of Haryana, U.P. and Rajasthan and reported the prevalence rate of B. equi as high as 50.1 per cent. There are sporadic reports of equine babesiosis in the country; however, outbreaks of the disease causing 17.5% mortality and few cases of abortions have also been recorded (Gautam and Dwivedi, 1976).
The clinical cases of B. equi infection can be diagnosed by demonstrating the parasites inside the erythrocytes in the peripheral blood smears. But this method fails to detect latent infection/carrier animals. However, serological tests can help in detecting such stages of the infection. These serological tests include complement fixation test (CFT) [Frerichs et al., 1969a], indirect fluorescent antibody test (IFAT) [Madden and Holbrook, 1968], monoclonal antibody based competitive inhibition enzyme linked immunosorbent assay (C-ELISA) [Knowles et al., 1991] and capillary agglutination test (CAT) [Malhotra et al., 1978]. Office International des Epizootics (OIE) is the international body competent to recommend test procedures on the subject, recommended CFT as a primary test for detection of equine piroplasmosis (OIE, 1996). At present, CFT is the only officially approved test in many countries like U.S.A., Japan, Australia etc. and is being used for testing the horses prior to the importation into these countries. As per the guidelines of world trade organisation
(WTO), every coutnry has to follow the OIE recommendations to satisfy the quarantine certifications for the international movement of equines for sports (Asian games, Olympic games. Commonwealth games etc.), race, show, trade and other purposes. Here it is significant that to promote trade and exportation of Indian horses abroad, OIE recommended tests are very much essential.
Besides India has a sizeable population of equines, i.e. 2.00 million comprising of 0.80 million horses, 1.00 million donkeys and 0.20 million mules. For transportation of essential commodities and military cargo in the Himalayan region, the role of mules and ponies are extremely essential. The horses are widely used for patrolling in different States and central police forces. Besides, in rural and semi urban areas of northern India donkeys, ponies and mules are extensively used for draught purposes. All these animals are susceptible to B.equi infection. Hence there is a need to develop an equally accurate and less costly diagnostic kit to detect 6. equi infection and which could be conducted without involving foreign exchange.
Diagnosis of B. equi infection in the country is generally made by blood smear examination of the suspected animals. This method can detect clinical cases only and can not detect carrier animals. CFT is the recommended test by OIE for detection of carrier animals. But CFT is very tidious test involves import of costly materials and also requires expertise. Hence, it can not be widely used in India for routine diagnostic test at different parts of country where such tests have to be carried out.
At present, there is no diagnostic test or kit, which is easy to use requiring low skill to perform and economical for detection of 8. equi infection in equines. Keeping in view of the above facts, a simple cost effective, easy to use, COFEB-kit is an
Indigenous improvement of OIE recommended CFT with novelty that the kit is totally indigenous, cost effective, easy to access and requires low technical skill for performance.
Detailed description of the invention :
Accordingly, the invention provides a new COFEB buffer useful as a diluent in complement fixation test comprising :

(Table Removed)
The invention also provides a kit useful for diagnosis of Babesia equi antibodies in equine sera, the said kit comprising: a) a buffer as described above; b) standard positive antigen prepared from erythrocytes of donkeys infected with an isolate of Babesia equi; c) standard negative antigen prepared from healthy eiythroc3rtes of a healthy donkey; d) standard positive sera prepared from a donkey experimentally infected with Babesia equi ; e) standard negative sera prepared from a healthy donkey; f) complement prepared from sera of guinea pigs; and g) haemolysin raised in rabbits against sheep erythrocytes.
Further, the invention provides a method of preparing a kit as described above the said method comprising: a) providing a buffer as described above: b) providing a standard positive antigen prepared from erythrocytes of donkeys infected with an isolate of Babesia equi; c) providing a standard negative antigen prepared from healthy erythrocytes of a healthy donkey; d) providing a standard positive sera prepared from a donkey experimentally infected with Babesia equi; e) providing a standard negative sera prepared from a healthy donkey; f) providing a complement prepared from sera of guinea pigs; and g) providing a haemolysin raised in rabbits against sheep erythrocytes.
Methodologies of the preparation of the reagents of the COFEB - kit:
i) Buffer
Conventional veronal Buffer (pH = 7.2)
Veronal buffer is used as a diluent for various biologicals/immunolbiologicals, in complement fixation test . The ingredients of veronal buffer contains sodium barbitone (C8H11N5O3Na) and diethyl barbituric acid (C8H12N2O3) apart from Sodium chloride (NaCl), Magnesium chloride hexa hydrate (MgCl2.6H2O), Calcium chloride anhydrous (CaCl2) and distilled water.
The disadvantages of veronal buffer are that sodium barbitone and diethyl barbituric acid are categorized under narcotics and hence difficult to access. They are not available indigenously and have to be always imported at high cost.
New Alternate Buffer [molarity = 0.148 M, pH=6.9 to 7.4]:
We found that the veronal buffer could successfully be replaced with an alternate buffer which is totally indigenous does not involve narcotics and at least five times cheaper than veronal buffer. After trying various permutations and combinations we finalized the following composition as an improved equivalence to veronal buffer:
a) Sodium chloride (NaCl) 8 gm
b) Potassium chloride (KCl) 0.2 gm
c) Di-sodium hydrogen phosphate (Na2 HPO4 ) 1.15 gm
d) Potassium di-hydrogen phosphate (KH2PO4) 0.2 gm
e) Calcium chloride dihydrate (CaCl2.2H2O) 0.037 gm
f) Magnesium chloride hexa hydrate (MgCl2.6H2O) 0.168 gm
g) Distilled water 1000 ml
This buffer is hereafter referred to as "COFEB buffer" or "C.B".

Data in Table -1 provide the comparative economic advantage of our alternate buffer over the veronal buffer in CFT.
Table-1 The comparative cost of veronal buffer and COFEB-buffer(for one litre)

(Table Removed)
il) Antigens:
a) Standard Positive antigen:
The standard positive antigen was prepared from an Indian isolate of B. equi following Frerichs et al. (1969b). It was raised in three splenectomised donkeys (non-descript breed, one year old) at different occasions. Each donkey received a
tranfusion of 200 ml blood from a donor carrier animal. Experimentally infected animals were bled from the jugular vein at the peak of parasitaemia (more than 70%) and 1-2 litres of blood was taken in a flask containing heparin as an anticoagulant @ 20 I.U. per ml of blood. The erythrocytes were separated out by centrifugation from the blood at 800 g at 4°0 for 20 minutes. The pellet thus formed was washed three times in COFEB buffer as described above. The erythrocytes were then lysed by suspending in chilled distilled water in a ratio of 1:9 for over night at 4°C. Thereafter, the suspension was centrifuged at 40,000g at 4°C for 30 minutes. The brown coloured pellet appearing at the buttom was collected.The presence of the parasites in the pellet was cofirmed by examining Giemsa's stained smear prepared from the pellet. Upon confirmation of the organisms, the pellet was washed two times with COFEB buffer by centrifugation at 40,000 g at 4°C for 30 minutes each. The pellet was finally suspended in COFEB buffer @ lgm/2ml and homogenized in Dounce glass homogeniser for 10 minutes. Polyvinyl pyrrolidone was added as stablizer @ l%w/v and was mixed thoroughly. Then it was titrated and freeze dried under vacuum in hermetically sealed neutral glass ampoules. The ampoules were kept at 4°C till used. b) Standard negative antigen:
Standard negative antigen has been included for better interpretation of the results. Standard negative antigen was prepared from the erythrocytes collected from the blood of a healthy donkey free from B.equi infection, following the same procedure as used for preparation of standard positive antigen.
iii) Standard Sera
Standard positive and negative sera to B.equi are not commercially available in India.
Positive sera was prepared from the donkeys experimentally infected with B. equi. For non-descript breed donkeys aged around one year were utilized. These donkerys were screened serologically and microscopically for B. equi infected donkey blood (parasitaemia 60% - 70%). These donkerys were serologically monitored routinely for B.equi by CFT. The sera was collected when the titer was 1:26 or more and pooled. This was used as standard positive sera.
The standard negative sera was prepared from the four new born foals prior to colostrums feeding, whose mothers were negative for B.equi antibodies when tested by CFT. These foals were screened serologically and microscopically for B.equi and found negative.
Both standard positive and negative sera were inactivated at 62°C for 45 minutes to destroy the complement present in sera. The sera were freeze dried in hermetically sealed glass ampoules by conventional method.
iv) Complement:
Sera of guinea pig was used as complement. Ready to use complement-freeze dried, fresh or stored in deep freeze at -20°C or below is not available commercially within the country. So, complement also is to be either imported or stored in deep freeze. Thus, deep freeze is required for the storage of complement and power failure can cause decrease in the titer of the complement. We prepared complement which could be easily stored at 40°C in common refrigerator with consequent advantage of low storage cost.
Procedure for preparation of ready to use complement::
The blood was taken from twelve healthy guinea-pigs (weighing not less than 1/2 kg. Body weight each). The sera was harvested, centrifuged at 4°C and was pooled. It was freeze dried in hermetically sealed neutral glass ampoules under vacuum as described earlier and used as complement. The entire operation was carried out in a cold chain (4°C). The complement was titrated. The ampoules were kepto at 4°C till used. The shelf life of such presented complement is more than one year. Other advantage of our complement is that in freeze -dried form, it gives reliably constant titer, which may not be possible by storing complement at - 20°C. This stability in titer value of lyophilized complement has opened the avenues for making the availability of the complement at commercial level, which at present is missing. It does not require additional storage facilities. As it is in freeze-dried form, it is easy to handle also.
v) Haemqiysin:
Availability of haemolysin in India is like that of complement and needs to be imported. Haemolysin was raised in rabbits against sheep erythrocytes. The blood was collected from four healthy young sheep. The RBCs were separated out and washed in C.B. Washed sheep erythrocytes were inoculated into three healthy rabbits (each weighing not less than 1.5 kg.) as per the following immunisation schedule: Table 2. Immunisation schedule for raising haemolysin in rabbits against
sheep red blood cells.

(Table Removed)
* i/v = intra-venous, s/c = sub-cutaneous
On 15th day of inoculation, the rabbits were test bled and the-sera v;as harvested. The sera having a titer more than 1:8000 were pooled and freeze dried in hermetically sealed neutral glass ampoules and stored at 4°C. Validation of COFEB-kit
Four tests were performed to validate the COFEB-kit and to establish its superiority and advantage over OIE recommended conventional CFT. These tests and their results are as follows:
1. The 6. equi antigen prepared by us for COFEB-kit was tested with the
imported B. equi and B. caballl sera. The test gave specific reaction against B. equ'i
antigen only in the complement fixation test. There was no cross reaction with S.
cajba///antigen. Similarly, the standard positive sera prepared by us for COFEB-kit
also gave monospecific reaction in OFT.with the imported B. egui antigen only.
2. Four healthy donkeys identified as 6. equi negative by blood smear
examination were found positive for 6. equi infection when tested at different
occasions by COFEB-kit. When these animals were stressed by splenectomy,
succumbed to infection and developed 70 to 80% parasitaemia, thus indicating the
ability of the kit for detection of latent infection/carrier animals.
3. Similarly, four healthy donkeys which were negative by blood smear examination as well as by COFEB kit, when splenectomised, did not succumb to infection and remained negative for 6. equi infection for 2 weeks. However, v/hen these animals received intra-venous infection of 150-200 ml. blood collected from the COFEB-Kit positive but apparently healthy animals, shov/ed high rise of parasitaemia; thus, confirming the validation of the developed kit.
4. A total of 688 equine serum samples collected from different parts of the country during different seasons when tested simultaneously by COFEB-kit and conventional OFT gave identical results as presented in the table 3. These tests were performed under different climatic conditions to establish the reliability and suitability of the COFEB-kit under varied climatic conditions. The significant advantage of COFEB-kit is that it could be safely stored at 4°C using ordinary refrigerator which is most useful in all developing countries.
Under less stringent storage conditions, for less costing and better adaptability to the conditions of the developing countries, the COFEB-kit has the capability to emerge as an international standard to test equines for B.equi infections. The improved test kit developed herein has immediate advantage to India for periodic and regular monitoring of equine population for B.equi infection and control the disease.
Table 3: Comparative result of conventional complement fixation test (CFT) & the invented COFEB-kit(complement fixation test for equine babesiosis-kit).
(Table Removed)
References:
Frerichs, W.M.; Holbrook, A.A. and Johnson,A.J.(1969a) Equine piroplasmosis : complement fixation titres of horses infected with Babesia caballi. American Journal of Veterinary Research 30 (5) : 697-702.
Frerichs, W.M.; Holbrook.A.A. and Johnson,A.J(1969b) Equine piroplasmosis: Production of antigens for tlie connplement fixation test. Annerican Journal of Veterinary Research 30 (8): 1337-1341.
Gautam, O.P. and Dwivedi, S.K.(1976) Equine babesiosis : A severe outbreak in a stud farm at Hisar. Indian Veterinary Journal 53 : 546-551.
Knowles, D.P.: Perryman,L.E. and Kappmeyer, L.S.(1991) Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody based competitive inhibition en2yme linked immunosorbent assay. Journal of Clinical Microbiology 29 : 2056-2058.
Madden, P.A. and Holbrook,A.A.(1968) Equine piroplasmosis : Indirect fluorescent antibody test for Babesia caballi. American Journal of Veterinary Research 29:117-123.
Malhotra, D.V.; Banerjee, D.P. and Gautam, O.P.(1978) Prevalence of latent cases of Babesia equi infection in some parts of north west India as measured by the capillary agglutination test. Equine Veterinary Journal 10(l):24-26.
OIE (1996) Equine Piroplasmosis. In : Manual of standards for diagnostic tests and vaccines List A and B diseases of mammals, birds and bees. Office international des epizooties, Paris, France pp. 420- 425.

CLAIMS:
1. A method for preparing complement fixation test based (COFEB) kit useful for diagnosis of Babesia equi antibodies in equine sera, said method comprise of the steps .
a) Providing COFEB buffer consisting of
-Sodium chloride (NaCI) 7.8 to 8.2 gm
-Potassium chloride (KCI) 0.15 to 0.25 gm
-Di-sodium hydrogen phosphate(Na2HP04) 1.0 to 1.20 gm
-Potassium di-hydrogen phosphate (KH2PO4) 0.15 to 2.5 gm
-Calcium chloride dihydrate(CaCl22H20) 0.035 to 0.040 gm
-Magnesium chloride hexa hydrate (MgCl2SH2O) 0.150 to 0.175 gm
-Distilled water 1000 ml
b) Providing a standard freeze dried positive antigen, consisting of 1 to 8 units of Babesia equi antigen.
c) Providing standard freeze dried negative antigen consisting of 25 to 100 micro litres of erythrocytes of healthy donkey free from Babesia equi infection.

d) Providing of 1 to 10 units of freeze dried anti bodies to Babesia equi prepared from the donkeys experimentally infected with Babesia equi organisms.
e) Providing 50 to 100 micro liters of standard freeze dried negative sera prepared from a donkey free from Babesia equi infection.
f) Providing of 1 to 8 units of freeze dried guinea pig complement.
g) Providing freeze dried rabbit sera containing 1 to 10 units of haemolysin against sheep R.B. Cs and
h) Collecting the materials of steps (a) to (g) to obtain the kit.
2. A method as claimed in claim 1 wherein COFEB buffer consists preferably of::
-Sodium chloride (NaCI) 8 gm
-Potassium chloride (KCI) 0.2 gm
-Di-sodium hydrogen phosphate(Na2HPO4) 1.15 gm
-Potassium di-hydrogen phosphate (KH2PO4) 0.2 gm
-Calcium chloride dihydrate(CaCl22H2O) 0.037 gm
-Magnesium chloride hexa hydrate (MgCl26H2O) 0.168 gm
-Distilled water 1000 ml
3 A method as claimed in claim 1 wherein standard positive freeze dried antigen, used is preferably 2- 4 complement fixing units of antigen prepared from the red blood cells of donkey infected experimentally with Babesia equi.
4. A method as claimed in claim 1 wherein standard freeze dried negative antigen preferably consists of erythrocytes prepared preferably from 1.5 ml peripheral blood of donkey free from Babesia equi infection.
5. A method as claimed in claim 1 wherein preferably 2- 4 complement fixing units of freeze dried anti bodies to Babesia equi prepared from the donkeys experimentally infected with Babesia equi or ganisms

6 A method as claimed in claim 1 wherein standard freeze dried negative sera preferably consists of 50 microlitres volume .prepared from a donkey free from Babesia equi infection .
7 A method as claimed in claim 1 wherein freeze dried guinea pig serum preferably consists of 2 to4 units of the complement.
8 A method as claimed in claim 1 wherein freeze dried rabbit sera preferably consists of 8 units of haemolysin against sheep R.B. Cs.
9 A method for preparing a complement fixation test based (COFEB) kit substantially as herein described and illustrated.