FIELD OF THE INVENTION
This invention relates to the field of prevention of domestic ruminant against Mycobacterium subspecies paratuberculosis (hereinafter referred to as Mycobacterium paratuberculosis or M. paratuberculosis). The invention pertains to a novel vaccine with a new strain of M. paratuberculosis, method of culture, the final vaccine formulation along with adjuvants compositions containing those M. paratuberculosis as antigen and process for the preparation of the novel vaccine formulation, and methods for prevention and control of Johne’s Disease in Asian continent preferably in India among its domestic ruminants.
BACKGROUND OF THE INVENTION:
Johne’s Disease is a chronic enteric inflammatory granulomatous disease which was first observed by Johne & Frothingham in 1895. It is caused by Mycobacterium paratuberculosis, which infects in cattle, sheep and goats. It has also been reported to infect in horses, pigs, deer and alpaca (Naser et al. 2000, Ghadiali et al. 2004, Singh et al. 2005a). As such, the disease is prevalent in all domestic ruminants across the globe. The significant impact of this disease on global economy has been well documented (Johnson- Ifearulundu and Kaneene, 1997, McKenna et al. 2006). Annual losses to the US cattle alone, where nearly 40% of the herds are said to be affected (Jones, 1989; Stabel, 1998), have been estimated to be staggering $ 0.2 to 1,5 billion (Ott et al. 1999; Chi et al. 2002), with an estimated cost per cow of $ 800-8000 per year. Johne’s Disease has also been said to be the most prevalent and costly diseases of dairy and beef cattle in US (Collins, 1997). In Australia, the death rate of sheep is twice as normal with a 6.4% decrease in gross margin (Anonymous, 2005b) and the annual loss per head is thought to be $ 39,000 for diary and $ 37,000 for beef animals. In Spain, the cost of treatment of animals afflicted with JD has been estimated to be $120 per sheep and $60 per diary head. The prevalence of Johne's Disease (hereinafter JD) has also been found to increase in countries of Australia, Newzealand, Netherlands, Belgium, Austria, England, Germany, United States, Iceland, Italy, Slovenia, Canada, Czech Republic, Spain, Switzerland, Denmark, Uruguay including India. It has been a priority to control JD in India. JD is highly endemic in the Northern Parts of India. Samples from cattle, buffaloes, sheep and goats from around the country have been shown to be positive in a variety of assays (Pandey 1940, Rao 1950, Anonymous 1950-60, Rajagopalan 1950, Chowdhury 1980,
Kulshreshtha et al. 1980, Paliwal et al. 1984, Reddy et al. 1984, Dabardgoo and Muley 1987, Kalla 1988, Ramkumar 1988, Tripathy et al. 1999, Goswamy et al. 2000, Kumar 2002, Sivakumar et al. 2003, Kumar 2004, Singh et al. 2005b, Singh et al. 2005c, Vohra et al.2006, Singh et al. 2007) and also referred to as ‘silent killers’ for livestock industry. The disease spreads in utero, through colostrums or milk and via faecal route. Infection takes place within first year, but symptoms are mostly at an adult stage. Quality (fat, and protein content) and quantity of milk are affected; besides, it leads to further immune suppression of the animals and to infertility. Immune suppression makes the animals further susceptible to other diseases leading to general ill health (Clarke 1997, Tripathi et al. 2002). Poor condition and productivity of the afflicted animals lead to premature culling in prime productive age and poor body condition at slaughter. The bacteria can get transmitted even through pasteurized milk. It is also capable of resisting normal UV exposure and during chlorine concentration method of water purification. It can survive 11 months in bovine faeces, as well as in river water. Thus, Mycobacetirum paratuherculosis is a serious threat to human life as well, control of which is immediate need of the hour. In humans, M.paratuberculosis bacilli have been found in tissues examined from Crohn's disease patients indicating possible zoonotic transmission from infected dairy products to humans.
Vaccines have been developed to fight Johne’s Disease (hereinafter JD). Simple inactivated or killed vaccines have been developed, and live vaccines are also in vogue. Subunit vaccines those differentiate vaccinated and infected animals have also been generated. Such vaccines in live attenuated, inactivated or killed form have been developed from various strains isolated based on epidemiology in other countries in the Western world. These vaccines are not able to counter indigenous strains of Mycobacetirum paratuherculosis prevalent in India and countries like SAARC countries including other Asian countries as well, where no vaccination regime against JD exists till date. Although, vaccination has reduced incidence of JD to an extent in many countries, they provide incomplete protection and do not prevent future infection.
Since, no indigenous vaccine against Johne’s Disease has been systematically developed in India till date, the inventors in this application have developed an indigenous vaccine against Johne’s Disease which is capable of preventing against of most prevalent strain of Mycobacetirum avium paratuherculosis in the Indian subcontinent and the rest of Asian countries among others. This particular strain is a novel strain which is having an unique genotype that does not exist in any other country, being referred to as the ‘S5 Indian Bison Type’ strain. The inventors have also confirmed that this particular strain afforded better and more rapid protection to local ruminants in India, compared to the very few other commercial vaccines those widely used in other countries at present. Inventors have also produced a better and simplified cost effective diagnosis technique to identify the particular strain through ELISA and PCR techniques.
OBJECTS OF THE INVENTION
One object of the invention is to provide an immunogenic vaccine composition for prevention against Johne’s Disease in ruminants caused due to Mycobacetirum avium paratuberculosis.
One other object of the invention is to provide a vaccine composition immunogenic against Johne’s Disease and identify, isolate processes to isolate and sequencing the said genotypic strain responsible for new serotypic specificity causing Johne’s Desease.
Another object of the invention is to provide a simple and easy method of large scale cultivation of organism Indian bison type S5’ strain of M.paratuberculosis, capable for production of a vaccine formulation against Johne’s disease with novel adjuvants along with the antigen in presence of other pharmaceutically acceptable carriers.
One other object of the invention provides a method of preventing and control of Johne's disease in domestic ruminants. The method includes administering to a mammal a vaccine composition against M. paratuberculosis. The vaccine composition includes an antigen derived from the ‘Indian bison type S5’ strain of M.paratuberculosis.
Yet another aspect of the invention is to provide a novel process of preparation of the vaccine formulation with novel adjuvants against Johne’s Disease.
The invention also provides new diagnostic techniques to identify infection of M.paratuberculosis and to identify the particular strain responsible for infection.
SUMMARY OF THE INVENTION
According to one embodiment of the invention the invention claims, a novel vaccine composition for prophylaxis against Johnes Disease caused due to infection of
Mycobacterium avium paratuherculosis S5 strain in domestic ruminants including but not limited to cattle, sheep, goat, and other domestic ruminants.
Another embodiment of the invention provides novel vaccine composition comprising Mycobacterium avium paratuherculosis S5 strain (MAP-S5) as the antigen formulated with montanide oil or aluminium hydroxide gel as the adjuvant in Phosphate buffered diluent and thiomersal as preservative.
One another embodiment of the invention provides a method of adaptation of MAP-S5 in liquid culture through fermentation in presence of novel media components including specialized broth , OADC supplement and a mixture of five antibiotics along with presence of 2.5% glycerol, or 0.4% Tween 80 upto 50 liter fermentation flasks at a time.
Yet another embodiment of the invention describes method of inactivation of the novel vaccine composition against Johnes disease comprising MAP-S5 strain.
One another embodiment of the invention include efficiacy studies of the vaccine composition in domestic ruminants thereby also enabling a method of diagnosis of infection of MAP-S5 in domestic ruminants.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure no. 1: Physical appearance of MAP ‘S5Tndian Bison Type strain after 80 days post inoculation on Herrold’s Egg Yolk Medium without malachite green for the preparation of working seed bank.
Figure No. 2: Sterility testing of Liquid Media on Nutrient Agar (A) and Blood Agar (B). Figure No.3: Comparative growth curve of MAP S5 growing in different batches of liquid media.
Figure No.4: Growth Curve of MAP-S5 strain in liquid media: scale-up upto 50 liters.
Figure No.5: Status of Body weight in sheep and goat vaccinated with BioJD-Oil.
Figure No.6: Status of Body Weight in Sheep and Goat vaccinated with BioJD-Gel.
Figure No. 7: Status of Body Weight in Cattle with BioJD-Oil.
Figure No. 8: Sero monitoring in Sheep and Goat vaccinated with BioJD-Oil.
Figure No. 9: Sero-monitoring in Sheep and Goat vaccinated with Bio-JD-Gel.
5
Figure No. 10 : Sero-monitoring in Cattle with BioJD-Oil.
Figure No. 11: Sero-monitoring in Cattle with BioJD-Gel.
Figure No. 12: Status of Cell Mediated Immune Response.
DETAILED DESCRIPTION OF THE INVENTION
Example 1: Isolation and characterization of S5 strain of Mycobacterium avium paratuherculosis.
Several strains of Mycobacterium paratuherculosis have been isolated from cattle, sheep, goats and blue bulls by the inventors for over two decades. Mycobacterium avium paratuherculosis (hereinafter MAP) is a facultative, anaerobic bacterium, which once inside the host resides and replicates mostly in macrophages. The “Mycobacterium avium paratuherculosis S5 strain” was isolated by CIRG, Makdhoom is referred as the ‘Indian bison’ type. This particular isolate is different from the previously described type isolated from US bisons (Whittington et al. 2001) and is a unique genotype not found elsewhere in the World. Restriction Fragmant Length Polymorphism (RFLP) analysis have been used to differentiate sheep and cattle types and isolates from other ruminants to give similar profiles to cattle isolates. Charecterizaton of this particular genotype have been studied through microscopy, culture, ELISA and PCR and PCR-RE for genotype mapping of MAP. This particular strain, S5 isolated from goats have been confirmed to be prevalent in endemic in North Indian wild and domestic ruminant population. Also published data (Singh et al.2007) using faecal and milk samples showed high prevalence of this genotype among diary herds. These assays were also found to react in experiments where samples from Crohn’s disease patients in India were used (Singh et al. 2005a). Phylogenetic analysis of the Indian bison type S5 strain have been performed which have identified that this particular sequence is unique and different from the ‘American bison type strain’.
Biovet has lawfully obtained the S5 strain under a grant of exclusive licence from CIRG, Makhdoom upon a mutual agreement entered on 23rd Day of August 2011. The strain has also been deposited by CIRG to National Labaratory at IMTECH, Chandigarh a constituent unit of CSIR, and VTCC which is a constituent unit of ICAR.
The product claimed under this invention is a vaccine effective to prevent and control both domestic and wild ruminants against Johne’s Disease. An effective formulation of the vaccine is prepared using heat inactivated the Indian bison type strain of
6
Mycobacterium avium paratuberculosis as the antigen along with Montanide oil as adjuvant, in presence of phosphate buffer at pH 6.5 to 7.5. Thiomersal is used as a preservative.
Example 2: Preparation of Seed
The invention also provides a novel process of manufacture a water-in-oil adjuvanted vaccine against Johne’s disease using bacteria grown in liquid medium in fermentor. Herrold’s Egg Yolk solid seed medium (HEYM) containing Mycobactin J was inoculated with MAP-S5 strain and incubated at 36° ±1°C for seed preparation and seed thus prepared were stored over vapor phase of liquid nitrogen (LN2). Purity was confirmed by Ziehl-Neelsen’s method by examining microscopically for acid-fast organisms that have the morphological characteristics of M.paratuberculosis.
Seed bank has been prepared after harvesting and pooling from 50 square bottles (250 mL) and stored over a vapour phase of liquid nitrogen in LN2 storage container. Sterile glycerol have been used as Cryo-preservative. Quality Control check for Seed bank is provided below:
• Sterility - Sterility had complied as per sterility protocol.
• Purity - Complied by Ziehl-Neelsen’s method and examined microscopically for acid- fast organisms that have the morphological characteristics of M. avium subsp. paratuberculosis. No adventitious organism was observed.
• Molecular characterization: -Whole genome sequencing is done by Genotypic Technology (P) Ltd, Bangalore. The pure MAP S5 DNA isolation report and genomic library have also been prepared.
Example 3: Method of Adaptation of Mycobacterium avium paratuberculosis
Adaptation in liquid culture by fermentation technique: Two sets of liquid media with a volume of 500mL each one with 2.5% v/v Glycerol and another with 0.4% tween80v/v in 2 liter bottles (Schott Duran) were prepared to study the dispersion of cells during suspension culture. Liquid medium was formulated with 4.7 gm per 100 ml broth* (details provided below) with OADC [Oleic Acid, Albumin (Bovine), Dextrose, Catalase] supplement with Mycobactin J (2 mg per liter), and a mixture of 5 antibiotics namely
7
Polymixin B, Amphotericin B, Nalidixic Acid, Trimethiopin, Azolectin in an amount of
0. 5 ml per liter.
*Broth composition per liter:
Ammonium Sulphate 0.5 gm,
L-glutamic acid 0.55 gm,
Sodium Citrate 0.1 gm,
Pyridoxine 0.006 gm,
Biotin 0.005 gm,
Disodium Phosphate 2.5 gm,
Monopotassium phosphate I gm,
Ferric Ammonium Citrate 0.04 gm,
Magnesium Sulphate 0.05 gm,
Calcium Chloride 0.005 gm,
Zinc Sulphate 0.001 gm,
Copper Sulphate 0.001 gm.
OADC Supplement to prepare 1 liter medium:
Bovine albumin fraction V 5.00 gm
Dextrose 2 gm
Catalase 0.004 gm
Oleic Acid 0.05 gm
Sodium Chloride 0.850 gm
Distilled Water 100 ml
The prepared media were inoculated with 2-5 ml of MAP-S5 strain (harvested from solid culture 250ml square bottle pooled into 20 ml normal saline) respectively. The cultures were incubated at 36°±1°C under constant stirring at low rotation per minute (RPM) 50- 100 RPM. The initial OD value was taken at wavelength 600nm in UV spectrophotometer. Growth curve was also monitored at every 15 days interval. The prepared media (sterile) were inoculated with MAP-S5 strain and initial OD value has been read at wavelength 600 nm in UV spectrophotometer (EL1CO). Growth curve was being monitored at every 15 days interval till 75th day. No further trial with medium with Tween 80 has been undertaken as the dispersion of cells in both cultures was found uniform but growth medium containing Glycerol showed better and promising results in comparison with growth in Tween 80 medium.
Culture Media Volume (liter) Seed
used Day OD Observation
Liquid culture media with 2.5% Glycerol. 0.5 From
solid
culture 0th day 0.092 Fast growth Cell dispersion was found uniform.
15th day 0.481
30th day 0.560
45th day 60th day 75th day 0.671
0.693
0.752
Liquid culture media with 0.4% Tween 80. 0.5 From
solid
culture 0th day 0.304 Growth Observed but comparatively slow growth with respect to growth seen with 2.5% glycerol.
Cell dispersion was found uniform.
15th day 0.485
30th day 0.545
45th day 0.595
60th day 0.629
75th day 0.630
Liquid culture media with 2.5% Glycerol and mixture of 5 antibiotics. 1.5 From
solid
culture 0th day 0.025 Cell dispersion was found uniform
15th day 0.558
30th day 0.609
45th day 0.648
60th day 0.667
Liquid culture media with 2.5% Glycerol and mixture of 5 antibiotics. 2.0 From
solid
culture 0th day 0.036 Cell dispersion was found uniform
15th day 0.586
30th day 0.614
45th days 0.688
60th day 0.698
Liquid culture media with 2.5% Glycerol and mixture of 5 antibiotics. 50 From
solid
culture 0th day 0.079 Fast growth
Cell dispersion was found uniform even in the 50 liter batch, when compared to 0.5-2 liter bottle culture(s).
15th day 0.541
30th day 1.059
45th day 60th day 0.969
0.980

Table -1: Growth Curve in Glycerol and Tween 80 liquid culture of S5 strain.
Example 4: Method of Inactivation
Three trial batches (Batch I, II, and III of 4000, 3300 and 2500 doses respectively) of vaccine have been prepared using Montanide oil as adjuvant and Thiomersal as preservative. Fully grown MAP-S5 culture was harvested, pooled and heat inactivated at 72°C for two hours.
Parameter Date of inactivation Observation
Constant temperature v/s different time interval:
Temperature: 72°C for 2 hours Time intervals: 00:00 hr, 00:30 hr, 01:00 hr, 01:30 hr and 02:00hr. Batch-1:09.04.2012 Batch-11: 09.04.2012 Batch-Ill: 10.04.2012 Culture samples inoculated on HEYM solid medium.
Growth observed in 00:00hr.in batch I, II, and III.
00:30hr., 01:30hr.and 02:00hr samples: No growth observed till date in all 3 batches.
Constant time v/s temperature differential:
Time constant: 02.00hrs. temperature differential: 65°C, 72°C, 75°C. Batch-1: 24.04.2012 Batch-2: 25.04.2012 Batch-3: 26.04.2012 Culture samples inoculated on HEYM solid medium.
No growth observed till date
Table -2: Inactivation parameters

Group Observation
Day OD at 560 nm observed
Test Sample: 5 ml of inactivated JD antigen inoculated in 500 ml culture media (Middlebrook 7H9 broth). 0th 0.086
yth 0.086
15th 0.085
21st 0.081
30th 0.084
Control Sample: 0.5 ml of live JD antigen MAP-S5 in 500 ml culture. 0th 0.018
yth 0.067
15th 0.220
21st 0.309
30th 0.424
Table -3: Comparison between inactivated samples and live samples

Example 5: Method of Preparation of Vaccine Composition
Fully grown MAP-S5 culture was harvested and inactivated at 72°C for two hours. After completion of inactivation, culture was washed for 3 times with sterile normal saline and pelleted. 5.0 mg antigen per dose on dry weight basis as one dose was taken and formulated with oil adjuvant i.e. Montanide oil (Seppic, France) at a range of about 40%-50% v/v, 10%w/v thiomersal solution at 1 ml/litre to the final volume and Phosphate buffer diluent (PBD) to an extent to make up final volume.
Pooled antigen were mixed thoroughly in PBD and required quantity of thiomersal was added to the “antigen-PBD mixture”, and mixed thoroughly. This bulk antigen mixture was added to the required quantity of montanide oil with continuous stirring and emulsified with homogenizer to make water in oil emulsion. Completion of emulsification is the given final vaccine composition. After emulsification, the bulk vaccine was filled into 100 ml sterile polypropelene (PP) vials and Stoppard with rubber stopper (nitrile) followed by sealing with aluminium seals. The formulated bulk vaccine was stored at 2°-8°C. The prepared vaccine was tested and verified to comply with all standard specifications like pH, color, emulsion, sterility, safety, efficacy and stability (Table 4.3). Efficacy was tested by checking the degree of shedding of M. paratuherculosis in fecal samples of vaccinated animals by Ziehl-Neelsen’s staining method. Antibody titer in blood samples were monitored by ELISA method.
Ingredients Concentration
Montanide Oil (adjuvant) 40%-50% v/v
MAP-S5 Antigen 2.5 mg.dw*/dose
10% Thiomersal w/v lml/L of PBD
PBD (Phosphate Buffer Diluent) q.s. to final formulated volume

Table 4.1: Formulation of JD vaccine in montanide oil as adjuvant
Ingredients Concentration
2% Aluminium Hydroxide gel 1.00-1.25 mg AI+++/dose
MAP-S5 Antigen 2.5 mg.dw*/dose
10% Thiomersal w/v lml/L of PBD
PBD (Phosphate Buffer Diluent) q.s. to final formulated volume

Properties Specifications Results
Batch I Batch II Batch III
pH 7.2- 7.6 7.42 7.51 7.48
Color Off white, opaque oil emulsion. Complies. Complies Complies
Emulsion No phase separation at 2-8 °C. Complies. Complies Complies
Sterility Should complies as per I.P. 2010 Complies. Complies Complies
Safety Should comply in target animals. Complies
Efficacy Variation in degree of shedding of MAP in fecal samples of infected animals (Acid-fast staining). Antibody response by ELISA Method.
Non-shedding of MAP in fecal samples of Non-infected animals.
Persistence of protective antibody in non-infected animals. Result shown
Stability Real time at 2-8°C Result shown
Table 4.3 : Standard specifications of JD vaccine Example 6: Animal Trial(s)

To estimate the efficacy of the vaccine, each batch of vaccine, total nine batches which include vaccine formulations with montanide oil (Bio-JD oil™) as well as vaccine formulations with gel (Bio-JD gel™) were tested in sheep, goat, cattle (including kids and calves) kept at Biovet premises. Fecal and serum samples were collected for monitoring the vaccine response and even the body weight of the animals were recorded. The details pertaining to the different batches of vaccines and the experimental trials conducted are mentioned below:
i. Batch I (Bio-JD oil):
Date of vaccination 03/04/2012
Targeted animal species Cattle (N=8) Goat (N=l) and Sheep (N=l)
No. of animals 10
Age 4-11 months
Location Experimental shed, Biovet Pvt. Ltd., Bangalore

Group Dose of vaccine Species Animal
ID Age
(month) Sex Body weight (Kg) (pre and post vaccination)
O'"
Day 30lh
Day 60,h
Day 90th
Day 120"’
day ISO'"
day 180,h
day 210,h
day 270"1
day 360lh
day
Group
I 1 ML Cattle BPLI08 4 M 36 37 59 60 3 65.5 94.4 94.5 83.5 OW ow
Cattle BPL099 7 M 30 34 55.5 53.3 63 70.5 70.8 67.5 OW ow
Cattle BPLI09 7 M 30 32 died - - - - - - -
Group
II 2 ML Cattle BPL079 II M 69 72 OW OW OW ow OW OW OW ow
Cattle BPLI02 5 M 54 58 79.5 79.8 88 5 94.5 95 OW OW ow
Cattle BPL083 8 M 47 50 71.5 76.3 84 91 5 91.5 84.5 ow ow
Group
III 1 ML Sheep STS
003 4 F 21 25 24.4 24.3 25.3 28.5 28.3 29.4 30.9 33
Goat* STG
Oil 6 F 12.69 25.9 23.8 23.6 25 25 27.9 25.9 27.5 29
Control Unvaccinated Cattle BPL
097 9 M 62 68 88 89 98 98 OW NA NA NA
Cattle BPL
103 9 M 60 65 69 70.3 78 78 OW NA NA NA

Table 5.1.2: Monitoring of vaccine response by body weight of animal [OW = over weight, NA: Not Available]
Grou
P Dose
or
vaccin
e Animal
species Animal ID Acid Fast Staining (pre and post vaccination)
0th
Day 30th
day 60th
day 90th
day 120th
day 150th
day 180t
h
day 270t
h
day 360t
h
day
Group
I 1 Ml. Cattle BPL108 +++
+ +++ +++ ++ + + Neg Neg Neg
Cattle BPL099 +++
+ +++ +++ ++ + + Neg Neg Neg
Cattle BPLI09 +++
+ +++ Died Died Died Died Died Died Died
Group
II 2 ML Cattle BPL079 +++
+ +++ +++ ++ ++ ++ + + +
Cattle BPL 102 +++
+ +++ +++ ++ + + ++ + +
Cattle BPL083 +++
+ +++ +++ ++ ++ ++ + + +
Group
III 1 ML Sheep STS 003 Neg Neg Neg + Neg Neg Neg Neg Neg
Goat* STG Oil Neg Neg Neg + Neg Neg Neg Neg Neg
Contr
ol Cattle BPL 097 +++
+ ++++ +++ ++++ ++++ +++ + NA NA
Cattle BPL 103 +++
+ ++++ +++ ++++ ++++ +++ ++ NA NA

[Neg: Negative, NA: Not Available]
Group Animal
ID Average O.D. (pre and post vaccination)
0 DPV 30
DPV 60
DPV 90
DPV 120
DPV 150
DPV 180
DPV 270
DPV 360
DPV
Vaccinated BPL079 0.915 0.826 2.18 1.6 2.85 2.01 1.95 1.401 1.01
BPL083 0.91 0.91 2.271 2.241 3.1 2.34 2.12 1.901 1.23
BPL099 0.511 0.73 2.118 2.781 3.2 2.593 2.039 1.91 1.41
BPL 102 0.37 0.492 2.07 2.85 2.6 2.45 1.727 1.52 1.42
BPL 108 0.52 0.76 2.385 2.92 2.89 2.34 2.246 1.51 1.21
BPL 109 0.42 0.487 Died - - - - - -
Control BPL 097 0.9 0.92 1.02 1.08 1.08 1.023 1.078 NA NA
BPL 103 0.93 0.95 1.05 1.09 1.07 1.03 1.18 NA NA
Table 5.1.4: Monitoring of seroconversion indirect ELISA [ NA: not available; DPV: days post vaccination],

ii. Batch II: Bio-JD oil
Date of vaccination 10/04/2012
Targeted animal species Sheep (N=8) and Goat (N=2)
No. of animals 10
Age 2-5 months
Location Experimental shed, Biovet Pvt. Ltd., Bangalore
Table 5.2: Details of JD vaccine Batch 11 for efficacy studies

Species Animal
ID Age
(month) Sex Body weight (Kg) (Pre and post vaccination )
0"1
Day 7th Day JO'"
Day 60'"
Day 90'"
Day 120'"
Day 150'"
Day iso"1
Day 240“’
Day 270"1
day 360lh
day
Goat STG
0012 3 F 10.73 11.65 10.44 9.4 9.91 13.5 11.3 14 12.2 13 15.75
Goat STG
0013 2.5 F 6.81 6.8 7.09 Died Died Died Died Died Died Died Died
Sheep STS 004 5 F 11.19 12.83 12.5 14.22 16.34 20.5 20 20.5 23.8 20.59 20.7
Sheep STS 005 4 M 8.91 9.97 10.26 12.6 14.53 21.5 21.35 21.8 23 25.56 28.7
Sheep STS 006 4 F 10.25 11 11.28 12.75 13.9 18 5 17.02 18.59 19 20 21.7
Sheep STS007 3 F 8.92 9.31 9 83 12 13.9 198 19 20 22 23.8 26.81
Sheep STS 008 2 F 7.23 7.59 7.82 9 9.13 11.7 11.8 11.9 13 13 14
Sheep STS009* 3 F 7.61 8.12 8.7 10.84 12.4 16.3 14.85 15 16.3 18.25 21.52
Sheep STS 010 3 F 4.39 8.63 8.94 10.59 12.34 13.4 13.2 13.8 14 15.8 17
Sheep STS0I1 2 F 5.75 4.51 4.9 6.4 7.17 10.14 10.7 10.8 12.8 14.95 16

e 5.2.1: Monitoring of vaccine response by body weight
Animal
ID Age
(month) Acid Fast Staining (pre and post vaccination in days)
species Sex Qth
Day yih
Day so"1
Day 60th
Day 90th
Day 120th
Day 150th Day 180th
Day 270th
Day 360,h
day
Goat STG
0012 3 F + + + Neg Neg Neg Neg Neg Neg Neg
Goat STG
0013 2.5 F + + + Neg Neg Neg Neg Neg Neg Neg
Sheep STS 004 5 F ++++ +++ ++ + ++ ++ + + + + +
Sheep STS 005 4 M ++ ++ + + ++ ++ ++ + + + +
Sheep STS 006 4 F +++ +++ + ++ ++ ++ Neg Neg Neg Neg
Sheep STS007 3 F +++ +++ ++ + Neg Neg Neg Neg Neg Neg Neg
Sheep STS 008 2 F +++ +++ + + + + + + Neg Neg
Sheep STS009* 3 F ++++ +++ + + + + + + + +
Sheep STS 010 3 F ++++ +++ ++ ++ + + Neg Neg Neg Neg
Sheep STS0I1 2 F ++ ++ ++ ++ + + Neg Neg Neg Neg
Table 5.2.2: Monitoring of vaccine response by fecal shedding of MAP

Group Animal
species Animal
ID Age
(month) Sex Average O.D. (Days post vaccination)
0
DPV 10
DPV 30
DPV 90
DPV 120
DPV 150
DPV 180
DPV 270
DPV 360
DPV
Vaccinated Goat STG
0012 3 F 0.25 0.32 0.46 0.91 1.2 1.25 1.21 1.04 0.89
STG
0013 2.5 F 0.24 0.31 0.46 0.95 1.21 1.21 1.1 1.03 0.90
Sheep STS
004 5 F 0.14 0.27 0.31 0.76 0.35 0.4 0.41 0.43 0.39
STS
005 4 M 0.262 0.20 0.939 1.23 0.82 0.89 0.82 0.70 0.69
STS
006 4 F 0.29 0.158 0.673 0.945 1.04 1.2 1.13 1.05 0.93
STS007 3 F 0.24 0.138 0.44 1.22 0.732 0.83 0.85 0.84 0.79
STS
008 2 F 0.95 0.30 0.703 1.18 0.91 0.96 0.96 0.97 0.91
STS
010 3 F 0.51 0.21 0.496 1.166 1.351 1.35 1.35 1.42 0.72
STS0I1 2 F 0.28 0.11 0.262 2.39 1.38 1.39 1.39 1.32 0.77
Control Sheep STS009 3 F 0.51 0.417 0.381 0.44 0.379 0.48 0.58 0.73 0.62
Table 5.2.3: Monitoring of seroconversion by Indirect ELISA.

iii. Batch III: Bio-JD Gel
Manufacturing date of vaccine 18/05/12
Date of vaccination 29/05/12
Targeted animal species Sheep (N=l) and Goat (N=3)
No. of animals 4
Age 2-6 months
Location Experimental shed, Biovet Pvt. Ltd., Bangalore
Table 5.3: Details of Bio-JD vaccine Batch III for efficiacy studies

Group Animal
species Animal
ID Body weight (Kg) (pre and post vaccination)
0
DPV 30
DPV 60
DPV 90
DPV 120
DPV 150
DPV 210
DPV 270
DPV 360
DPV
Vaccinated Sheep STS 012 7.78 9.13 9.91 8.68 11.4 12.27 15.21 18 22
Goat STG 014 14.42 16.9 17.47 19.42 24 24 22.25 29 35
Goat STG 016 8.6 10.6 11.05 12.91 18.8 19.5 22.3 27.5 30
Control Goat STG 015 7.74 8.74 8.95 10.25 13.24 13.6 13.65 21.7 22.3
Table 5.3.1: Monitoring by Body Weight

Microscopic Examination (pre and post vaccination in days)
Group species ID 0DPV 30
DPV 60
DPV 90 DPV 120
DPV ISO
DPV 180
DPV 270
DPV 360
DPV
Sheep STS
012 ++++ +++ +++ ++ Neg Neg Neg Neg Neg
Vaccinated Goat STG
014 ++++ +++ +++ + + + + + +
Goat STG
016 ++++ +++ +++ ++ + + Neg Neg Neg
Control Goat STG
015 ++ ++ ++ +++ +++ +++ +++ +++ +++
Table 5.3.2: Monitoring by faecal smear microscopy

Group Animal
species Animal
ID Age
(month) Sex Average O.D. (Days post vaccination)
0
DPV 10
DPV 30
DPV 90
DPV 120
DPV 150
DPV 180
DPV 270
DPV 360
DPV
Vaccinated Goat STG
0012 3 F 0.25 0.32 0.46 0.91 1.2 1.25 1.21 1.04 0.89
STG
0013 2.5 F 0.24 0.31 0.46 0.95 1.21 1.21 1.1 1.03 0.90
Sheep STS
004 5 F 0.14 0.27 0.31 0.76 0.35 0.4 0.41 0.43 0.39
STS
005 4 M 0.262 0.20 0.939 1.23 0.82 0.89 0.82 0.70 0.69
STS
006 4 F 0.29 0.158 0.673 0.945 1.04 1.2 1.13 1.05 0.93
STS007 3 F 0.24 0.138 0.44 1.22 0.732 0.83 0.85 0.84 0.79
STS
008 2 F 0.95 0.30 0.703 1.18 0.91 0.96 0.96 0.97 0.91
STS
010 3 F 0.51 0.21 0.496 1.166 1.351 1.35 1.35 1.42 0.72
STS0I1 2 F 0.28 0.11 0.262 2.39 1.38 1.39 1.39 1.32 0.77
Control Sheep STS009 3 F 0.51 0.417 0.381 0.44 0.379 0.48 0.58 0.73 0.62

Table 5.3.3: Monitoring of Seroconversion by indirect ELISA, iv. Batch IV: Bio-JD gel
Manufacturing date of vaccine 18/05/12
Date of vaccination 29/05/12
Targeted animal species Sheep (N=l) and Goat (N=3)
No. of animals 4
Age 2-6 months
Location Experimental shed, Biovet Pvt. Ltd., Bangalore

Table 5.4: Details of Bio-JD vaccine Batch IV for efficacy studies.
Group Animal
species Animal
ID Body weight (Kg) (pre and post vaccination)
0
DPV 30
DPV 60
DPV 90
DPV 120
DPV 150
DPV 210
DPV 270
DPV 360
DPV
Vaccinated Sheep STS 012 7.78 9.13 9.91 8.68 11.4 12.27 15.21 18 22
Goat STG 014 14.42 16.9 17.47 19.42 24 24 22.25 29 35
Goat STG 016 8.6 10.6 11.05 12.91 18.8 19.5 22.3 27.5 30
Control Goat STG 015 7.74 8.74 8.95 10.25 13.24 13.6 13.65 21.7 22.3

Table5.4.1: Monitoring of Body weight.
Group Animal
species Animal
ID Microscopic Examination (pre and post vaccination in days)
0 DPV 30
DPV 60
DPV 90 DPV 120
DPV 150
DPV 180
DPV 270
DPV 360
DPV
Vaccinated Sheep STS
012 ++++ +++ +++ ++ Neg Neg Neg Neg Neg
Goat STG
014 ++++ +++ +++ + + + + + +
Goat STG
016 ++++ +++ +++ ++ + + Neg Neg Neg
Control Goat STG
015 ++ ++ + + +++ +++ +++ +++ +++ +++

Table 5.4.2: Monitoring of MAP shedding by fecal smear microscopy
Group Animal ID Antibody titer (pre and post vaccination in days)
0 DPV 30
DPV 60 DPV 90 DPV 120 DPV ISO DPV 180 DPV 270 DPV 360 DPV
vaccinated STS 012 0.27 0.45 0.95 1.21 1.3 1.31 1.29 0.91 0.90

STG 014 0.25 0.48 0.89 1.24 1.34 1.29 1.32 0.90 0.97
STG 016
i 0.28 0.60 1.02 1.3 1.52 1.35 1.32 1.2 0.81
Control | STG 015
1 0.27 0.31 0.34
. 0.35 0.45 0.52 0.42 0.43 0.48
Table 5.4.3: Monitoring of seroconversion

v. Batch V: Bio-JD-Gel
Date of vaccination 20/11/12
Targeted animal species Calves (N=5), Goat (N=l) and sheep (N=l)
No. of animals 7 (all vaccinated)
Location Experimental shed, Biovet Pvt. Ltd., Bangalore
Table 5.5: Details of Bio-JD vaccine Batch V for efficacy studies.

SI.
No. Animal
ID Antibody titer in OD values (pre and post vaccination in days)
0 DPV 30 DPV 60 DPV 90 DPV 120 DPV
1 BPL 18? 0.43 0.64 0.95 1.29 1.36
2 BPL 183 0.33 0.75 0.95 1.30 1.37
3 HBPL 181 0.54 0.69 1.10 1.35 1.36
4 BPL 176 0.25 0.44 0.85 1.43 1.43
5 BPL 179 0^52 0.43 0.83 1.33 1.37
6 STS 013 0.33 0.48 0.90 1.38 1.39
7 STG 012 0.31 0.46 0.93 1.25 1.35
Table 5.5.1: Monitoring of Seroconversion

vi. Batch VI: Bio-JD-Gel:
Manufacturing date of vaccine 06/11/2012
Date of vaccination 15/12/12
Targeted animal species Cattle
No. of animals 7 (vaccinated=4, control=3)
Location Experimental shed, Biovet Pvt. Ltd., Bangalore

Group Animal ID Antibody titer (days post vaccination)
0 DPV 30 DPV 60 DPV 90 DPV 120 DPV
Vaccinated BPL 177 0.25 0.49 0.73 1.25 1.33
BPL 178 0.33 0.49 0.72 1.25 1.26
BPL 182 0.37 0.43 0.83 1.25 1.26
BPL 167 0.33 0.49 0.79 1.13 1.26
Control BPL 186 0.25 0.35 0.38 0.44 0.46
BPL 187 0.35 0.33 0.45 0.48 0.47
BPL 168 0.29 0.30 0.35 0.39 0.43
Table 5.6.1: Monitoring of Seroconversion

vii) Batch VII (BioJD-oil) and Batch VIII (BioJD-Gel)
Manufacturing date of vaccine TB-BioJd-Oil 12004 (Dec 12) and TB-BioJd-Gel 12001 (Nov 12)
Date of vaccination 20/12/2012
Targeted animal species Sheep (N=2) and Goat (N=13)
No. of animals 15
Age 1 -3 years
Location Experimental shed, Biovet Pvt. Ltd., Bangalore

Table 5.7: Details of Bio-JD vaccine Batch VII and Batch VIII for efficacy studies
S.
No. Initial
Group New
Group Animal
species Animal ID Age Sex 0 DPV 60
DPV 90
DPV
1 Vaccinated Oil
vaccine Goat JD-CIRG 03 5 Months F 14.3 17.6 18
2 Vaccinated Goat JD-CIRG 05 5 Months F 17.75 18 18.9
3 Control Goat JD-CIRG 06 5 Months M 24.5 31 31.5
4 Vaccinated Goat JD-CIRG 07 7 Months F 20 22.8 23.2
5 Vaccinated Sheep STS-002 14 Months F 24.5 24.5 24.9
6 Vaccinated Gel
vaccine Goat STG-002 11 Months F 16 31.3 31.5
7 Control Goat STG-004 15 Months F 26 27.95 28.1
8 Vaccinated Goat STG-005 17 Months F 25 26 26.3
9 Vaccinated Goat STG-008 17 Months F 19.7 23.2 23.5
10 Control Goat STG-009 18 Months F 18.1 19.8 20.1
11 Vaccinated Goat STG-011 18 Months F 22.8 25.9 26.1
12 Control Control Goat JD-CIRG 02 6 Months F 27.7 20 21

13 Control Goat JD-CIRG 04 7 Months F 18 20 20.5
14 Control Goat STG-003 15 Months F 30.4 33.5 34
15 Control Sheep STS-001 17 Months M 39 - -
Table 5.7.1: Monitoring by Body Weight

Initial Group New
Group Animal ID 0 DPV 30 DPV 60 DPV 90 DPV 180 DPV
Vaccinated Oil
vaccine JD-CIRG 03 ++ ++ ++ + +
Vaccinated JD-CIRG 05 ++ ++ + Neg Neg
Control JD-CIRG 06 + + Neg Neg Neg
Vaccinated JD-CIRG 07 ++ ++ + + +
Vaccinated STS-002 Neg Neg Neg Neg Neg
Vaccinated Gel
vaccine STG-002 Neg Neg Neg Neg Neg
Control STG-004 ++ ++ + + +
Vaccinated STG-005 ++ ++ + Neg Neg
Vaccinated STG-008 + + + + +
Control STG-009 Neg Neg Neg Neg Neg
Vaccinated STG-011 Neg Neg Neg Neg Neg
Control Control JD-CIRG 02 + + + + +
Control JD-CIRG 04 ++ ++ ++ ++ ++
Control STG-003 +• + + ++ +
Control STS-001 + + NA NA NA
Table 5.7.2: Monitoring by MAP shedding by faecal smear microscopy

Initial Group New Animal ID Antibody titer (days post vaccination)
Group 0 DPV 30 DPV 60 DPV 90 DPV 120 DPV 150
DPV 180 DPV
Vaccinated JD-CIRG 03 0.48 0.56 0.95 1.25 1.5 1.54 1.5
Vaccinated JD-CIRG 05 0.56 0.78 1.02 1 23 1.55 1.5 1.51
Control Oil
vaccine JD-CIRG 06 0.64 0.89 1.25 1.45 1.59 1.6 1.59
Vaccinated JD-CIRG 07 0.49 0.6 0.99 1.23 1.52 1.54 1.56
Vaccinated STS-002 0.46 0.55 0.98 1.33 1.55 1.56 1.52
Vaccinated STG-002 0.46 0.59 0.95 1.35 1.54 1.55 1.54
Control STG-004 0.64 0.79 1 23 1.44 1.55 1.55 1.56
Vaccinated Gel STG-005 0.34 0.55 0.75 1.24 1.45 1,54 1.54
Vaccinated vaccine STG-008 0.64 0.98 1.25 1.23 1,54 1.62 1.56
Control STG-009 0.55 0.72 1.12 1.52 1.57 1.61 1.59
Vaccinated STG-011 0.54 0.75 1.32 1.42 1.52 1.63 1.62
Control JD-CIRG 02 0.51 0.52 0.53 0.55 0.54 0.56 0.58
Control Control JD-CIRG 04 0.45 0.5 0.51 0.52 0.54 0.56 0.52
Control STG-003 0.33 0.35 0.42 0.45 0.42 0.46 0.49

Table 5.7.3: Monitoring of Seroconversion
viii) Batch IX: BioJD-Gel
Date of vaccination 24/01/13
Targeted animal species Calves
No. of animals 10
Age 3-15 months
Location Experimental shed, Biovet Pvt. Ltd., Bangalore
Table 5.8: Details of Bio-JD vaccine Batch IX for efficacy studies

Group Animal ID Antibody titer (days post vaccination)
0 DPV 30 DPV 60 DPV 90 DPV
Vaccinated BPL 158 0.25 0.43 0.56 1.29
BPL 188 0.28 0.46 0.62 1.35
BPL 157 0.33 0.44 0.68 1.55
BPL 160 0.52 0.87 1.05 1.43
BPL 162 0.57 0.86 1.10 1.46
BPL 163 0.35 0.61 0.75 1.25
BPL 167 0.35 0.54 0.69 1.26
BPL 168 0.35 0.55 0.81 1.25
Control BPL 166 0.36 0.47 0.36 0.38
BPL 180 0.53 0.58 0.56 0.60
Table 5.8.1: Monitoring of Seroconversion

Observations:
a) Monitoring of vaccine response by body weight:
The body weight of the animals was recorded before vaccination (Oth day) and later on 30th, 60th, 90th, 120th, 150th, 180th, 210th, 270th and 360th days post vaccination. All the animals showed an increase in body weight post vaccination (Figure Nos 5, 6 and 7).
b) Monitoring of vaccine response by microscopic examination of fecal smear:
Faecal samples of the animals were collected periodically and examined microscopically after acid fast staining. MAP shedding was recorded before vaccination (Oth day) and later on 30th, 60th, 90th, 120th, 150th, 180th, 270th and 360th days post vaccination. All the animals showed decrease in the MAP shedding and most of the animals become negative by end of one year.
c) Monitoring of seroconversion by indirect ELISA:
Serum samples of the animals were checked by performing Indirect ELISA using purified protoplasmic antigen (PPA) of MAP. Antibody titer against MAP was recorded before vaccination (Oth day) and later on 30th, 60th, 90th, 120th, 150th, 180th, 270th and 360th days post vaccination. All the animals showed increase in the antibody titer post vaccination (Figure Nos. 8, 9, 10, and 11).
d) Evaluation of cellular immune response: Cell Mediated Immune (CMI)
response: Interferon gamma (IFNy) was evaluated for 10 animals (calves) after vaccination with JD gel vaccine BioJD-Gel (including 2 control animals) using Bovigam assay Kit. It was observed that, the CMI response is found in both the vaccinated and control group. However the response in vaccinated animals was more intense post vaccination. The details are mentioned below (Figure No. 12):
Group Animal ID O. D. Values (Days Post vaccination)
0DPV 60 DPV
Vaccinated
(Calves) BPL 158 0.322 1.133
BPL 188 0.378 1.217

BPL 157 0.460 1.234
BPL 160 0.381 1.492
BPL 162 0.524 1.508
BPL 163 0.376 1.271
BPL 167 0.441 1.233
BPL 168 0.299 0.311
Control
(Calves) BPL 166 0.480 0.461
BPL 180 0.333 0.388
Table 6: Cell Mediated Immune Response of Bio-JD Gel.

References:
1. Shanker H, Singh SV, Singh PK, Singh AV, Sohal JS, Greenstein RJ (2009). Presence, characterization, and genotype profiles of Mycobacterium avium subspecies paratuberculosis from unpasteurized individual and pooled milk, commercial pasteurized milk, and milk products in India by culture, PCR, and PCR-REA methods. International Journal of Infectectious Diseases, 14: 121-126.
2. Singh N, Vihan VS, Singh SV, Gupta VK (1998). Prevalence of Johne’s disease in organized goat herds. Indian Journal of Animal Sciences, 68: 41-42.
3. Singh SV, Singh PK, Singh AV, Sohal JS, Gupta VK, Vihan VS (2007a). Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘bison type’ with commercial vaccine for the control of Capri-paratuberculosis in India. Vaccine, 25: 7102-7110.
4. Singh SV, Singh AV, Singh PK, Gupta VK, Kumar S, Vohra J (2007b). Seroprevalence of
paratuberculosis in young kids using ‘bison type’, Mycobacterium avium subsp.
paratuberculosis antigen in plate ELISA. Small Ruminant Research, 70: 89-92.
5. Singh AV, Singh SV, Makharia GK, Singh PK, Sohal JS (2008). Presence and
characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn’s disease and in the healthy human population in India.
International Journal of Infectious Diseases, 12; 190-197.
6. Singh PK, Singh SV, Singh AV, Sohal JS (2009). Variability in susceptibility of different Indian goat breeds with respect to natural and experimental infection of Mycobacterium avium subspecies paratuberculosis. Indian Journal of Small Ruminant Research, 15: 35-43.
7. Singh SV, Singh PK, Singh AV, Sohal JS, Sharma MC (2010). Therapeutic effects of a
new ‘Indigenous Vaccine’ developed using novel native ‘Indian Bison type’ genotype of Mycobacterium avium subspecies paratuberculosis for the control of clinical Johne’s disease in naturally infected goatherds in India. Veterinary Medicine International,
doi: 10.4061/2010/351846.
7. Sohal J S, Sheoran N, Narayanasamy K, Brahmachari V, Singh S V, Subodh S (2009). Genomic analysis of local isolate of Mycobacterium avium subspecies paratuberculosis. Veterinary Microbiology, 134: 375-382.
8. Singh, S. V., Singh, P. K., Singh, M. K., Singh, A. V. , Sohal, J. S., 2011. Therapeutic potential of Johne's disease vaccine: A follow up post vaccination study in a goatherd of endangered Jamunapari breed, naturally infected with Mycobacterium avium subspecies paratuberculosis. International Journal of Livestock Production, 2 (13): 192-204. DOI: 10.5897 / IJLP10. 023

We Claim:
1. A vaccine formulation for prophylaxis against Johnes Disease in domestic ruminants comprising :
a. inactivated Mycobacterium avium paratuherculosis strain S5 (MAP-S5) as antigen in an amount of 2.5 mg dry weight per dose;
b. adjuvants selected from Montanide oil in an amount of 40% to 50% v/v and 2% Aluminium Hydroxide gel in an amount of 1.00-1.25 mg Al+++per dose;
c. 10% thiomersal as preservative; and
d. Phosphate buffered diluent in an amount of 1 ml per liter (quantity sufficient to final volume).
2. The vaccine formulation of claim 1, wherein the MAP-S5 antigen is adapted in liquid culture with novel media scale-up upto 50 liters of fermentation culture, in presence of 2.5% of glycerol, and a mixture of 5 antibiotics.
3. The vaccine formulation of claim 1, wherein the MAP-S5 antigen is adapted in liquid culture with novel media scale-up upto 50 liters of fermentation culture, in presence of 0.4% Tween 80, and a mixture of 5 antibiotics.
4. The vaccine formulation of claims 2 and 3, wherein the antibiotics are Polymixin B, Amphotericin B, Nalidixic Acid, Trimethiopin, Azolectin in an amount of 0.5 ml per liter.
5. The vaccine formulation of claim 1 wherein the vaccine formulation is heat inactivated in at a temperature range of 65 °C to 72°C for 2 hours.
6. The vaccine formulation of claim 1 wherein the pH of the vaccine formulation ranges from 6.8 to 7.8, preferably from 7.2 to 7.6.
7. The vaccine formulation of claim 1, wherein the domestic ruminants includes but not limited to sheep, cattle and goat, any other domestic ruminants infected by MAP-S5 strain.
8. The vaccine formulation of claim 1, wherein the vaccine is a stable off-white, opaque oil emulsion at 2°C -8°C.
9. A method of diagnosis for detecting infection of domestic ruminants by MAP-S5 strain through monitoring of microscopic examination of faecal smear, or monitoring of seroconversion by indirect ELISA method.
10. The method of claim 9, wherein the the domestic ruminants includes but not limited to sheep, cattle and goat, any other domestic ruminants susceptible of infection by MAP-S5 strain.