Title of Invention	A KIT FOR DIAGNOSIS OF BRUCELLOSIS
Abstract	The present invention relates to a kit for diagnosis of brucellosis in small ruminants, preferably sheep and goat. The kit described in the present invention is an ELISA kit, preferably indirect ELISA kit. The present invention also relates to a process to obtain such a kit. Fig: 1-4.

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FIELD OF THE INVENTION The present invention relates to a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. The kit described in the present invention is an ELISA kit, preferably indirect ELISA kit for sero-diagnosis of brucellosis in sheep and goats. The present invention also relates to a process to obtain such a kit. BACKGROUND OF THE INVENTION AND PRIOR ART Goat and sheep production is becoming increasingly important in developing nations of the world like India. About five million households in the country are engaged in the rearing of small ruminants (sheep, goats) and other allied activities. According to FAO 2000, India has a population of 57.6 million sheep and 122.53 million goats, which constitutes about 5.31% and 17% of world sheep and goat population respectively. Their contribution to the economy through production of fibre, meat, skins and milk is 5.4% of GNP of agriculture sector. The strategies for development of small ruminant sector need to be identified and prioritized based on the epidemiological studies, genetic biodiversity, etc.(17th livestock census, 2003). Diseases are major cause of economic losses and inefficient production in goats. Brucellosis is an important reproductive disease in sheep and goats characterized by abortion, stillbirths and reproductive failure. Free grazing and movement with frequent mixing of flocks of sheep and goats also contribute to the high prevalence and wide distribution of brucellosis in these animals in India. The changing and fast growing dairy and meat industry in India has resulted in intensified trade and animal movements and provide a new and increased risk in spreading the infection. The disease is endemic at lower level in sheep and goats but lesser in prevalence than in cattle. The abortion and still birth in sheep and goats was found to be 11.3% and 1.5% respectively and estimated losses would be Rs. 10,000 million. Moreover, the disease is underreported or overlooked and misdiagnosed because of the difficulty in diagnosis and the absence and lack of experience with laboratory testing. Brucellosis is almost invariably transmitted to man from infected domestic animals. Bracelet abortus, B. melitensis and B. suits are pathogenic for man. Though B. smelliness is more infectious to man than B. abortus and in general is the dominant causative agent of brucellosis. The disease caused by infection with B. abortus is indistinguishable from that by B. melitensis and may be equally severe (Dokuzoguz et al 2005). Although brucellosis in livestock and transmission of infection to the human population has been significantly decreased following the instigation of effective vaccination-based control and prevention programmes in parts of the world, it remains an uncontrolled problem in regions of high endemic such as the Mediterranean, Middle East, Africa, Latin America and parts of Asia (Corbel, 1997; Refai 2002). Of main concern in India is B. melitensis and B. abortus. B. melitensis transmitted by goats and sheep and related animals is most virulent for man. Since, brucellosis transmitted from small ruminants poses a higher risk to humans than transmitted from cattle; efforts are needed to diagnose the disease in goats and sheep using sensitive screening tests. Increasing demand for dairy products and protein, changing agricultural methods, and increased trade and movement of animals has caused concerns that the prevalence may increase. Therefore, there is an urgent need for the strict implementation of a control policy not only for cattle but also for small ruminants. Given the potentially huge economic and medical impact, a control policy could be cost-effective (Roth et al. 2003.) India has already a policy for the control of brucellosis in dairy cattle (Renukaradhya et al. 2002) and such policy in small ruminants helps a great extent to minimize the disease in man and reduce the economic losses. Diagnostic test options for brucellosis in sheep and goat At present evidence of Brucella infection is provided by: i) Isolation of cultures using selective media. ii) Serological tests. iii) Nucleic acid detection methods. i) Isolation of cultures using selective media: Though isolation is a gold standard test for diagnosis of brucellosis, it requires selective media, is time consuming and very hazardous requiring BSL- II cabin to work. ii) Serological tests: A) Rose Bengal Plate Test (RBPT): In routine tests, anti-brucella antibodies are detected in serum or milk. The most widely used serum-testing procedures for diagnosis of Brucella infection in sheep and goats are the buffered Brucella antigen tests (RBPT). Though RBPT has high sensitivity it has very low specificity. But it is still recommended for screening flocks and individual animals. B) Serum Agglutination Test (SAT): In order to measure the antibody and to know the class of antibody (IgG/ IgM), RBPT positive sera are submitted to SAT. It is often the last of all the available tests to detect diagnostic antibody concentrations in the accusative stage of the disease and after abortion. In the chronic stages, agglutinins have often disappeared while other tests remain positive. There are many reports of infected animals that give less than diagnostically significant results in the SAT. As SAT may yield both false negative and false positive results (Corbel et al. 1997), it effectively detects brucellosis only on a herd basis. However, serum agglutination test (SAT) is not considered reliable for use in small ruminants (OIE-2006). Q Enzyme Linked Immunosorbent Assays (ELISA): Some success has been achieved in the past few years with ELISA that use various smooth lipopolysaccharide (LPS) antigens. These tests promise increased sensitivity and specificity, but a great deal of work is still required on standardization of reagents (Gupta and Vihan 2001). Mikolon et al. (1998) recommended both RBPT and milk ELISA as good screening tests for detection of brucellosis in goat sera and milk samples respectively. MRT is not used as it is highly unreliable in diagnosing small ruminant brucellosis. Similarly, use of Elisa and RBPT for screening brucella infection in vaccinated and infected flocks has also been reported (Marin et al., 1999). iii) Nucleic acid detection methods: PCR based diagnoses are widely used to detect brucella genus or multiplex PCR to detect brucella species in samples. Though PCR is sensitive but not suitable for large scale screening. In India, vaccination is not yet practiced for sheep and goats. So interference of vaccine titre is ruled out. PDADMAS has developed an AB-ELISA kit of international standard for diagnosis of brucellosis in cattle. A total of 1,31,574 sera samples were screened from different parts of the country and seroprevalence was found to be 20%. Based on the seroscreening using the kit, a control programmer for brucellosis was formulated (Renukaradhya et al. 2002). The kits have generated revenue to the tune of 5.5 lakes. Such kind of epidemiological screening is not done in the case of small ruminants. Only limited reports by few workers are available in particular geographical locations. (Singh et al.2000; Murrain et al., 2000). To formulate a control strategy for our country, a large scale screening of 1.8 million sera samples is required for a target population of 57.6 million sheep and 122.53 million goats in the country. To screen such large number of sera samples, ELISA test is very useful than any other methods like isolation, serological or Nucleic acid detection methods (PCR). Though tests like PCR give promising results, they are expensive and not recommended for large scale use. As ELISA kits are robust and are highly sensitive, they are best suited for such large scale screening. To evolve epidemiology of brucellosis in sheep and goats in the country, such tests are urgently required. With this existing situation, a need was felt for the development of indirect ELISA kit for diagnosis of brucellosis in sheep and goats. The present disclosure overcomes the limitations of prior art and discloses a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. OBJECTIVES OF THE INVENTION The main objective of the present invention is to obtain a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. Another main objective of the present invention is to obtain a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat, wherein said kit is an indirect ELISA kit. Yet another main objective of the present invention is to develop a process to obtain a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. STATEMENT OF THE INVENTION Accordingly, the present invention relates to a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Fig. 1: Flowchart for extraction of Smooth Lipopolysaccharide antigen. Fig. 2: Antigen Titration Graph; Antigen dilution Vs OD values. Fig. 3: Flow chart for Elisa protocol. Fig. 4: Graph depicting positive cut-off for alias. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. In another embodiment of the present invention, the kit is an indirect ELIS A kit wherein said kit comprises: i) antigen coated plates; ii) blocking-cum-dilution buffer; iii) control sera; iv) washing buffer; v) conjugate; vi) chromomeric substrate; and vii) stopping agent. In yet another embodiment of the present invention, said antigen coated plates are coated with purified smooth lipopolysaccharide, obtained from Brucella species, preferably from antigenic strain of Brucella abortus. In yet another embodiment of the present invention, said blocking-cum-dilution buffer comprises a buffered saline, a surfactant and a blocking agent which reduces background cooler. In still another embodiment of the present invention, said buffered saline is selected from a group comprising trips buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline; said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 4-(l,l,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate. preferably polyethylene glycol sorbitan monolaurate and said blocking agent is selected from a group comprising bovine serum albumin-fraction V, bovine gelatin, skim milk powder and fontal calf serum, preferably bovine gelatin. In still another embodiment of the present invention, said buffer comprises buffered saline of pH in the range of 7-7.5, about 0.01 - 0.1 % surfactant, and about 1 - 2.5% a blocking agent, preferably buffered saline of pH 7.2, 0.05% surfactant and 2% blocking agent. In still another embodiment of the present invention, said control sera comprises of positive control serum, moderate positive control serum and negative control serum. In still another embodiment of the present invention, said buffered saline is selected from a group comprising tris buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline, said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 2-(l,l,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate, preferably polyethylene glycol sorbitan monolaurate. In still another embodiment of the present invention, said washing buffer comprises buffered saline of pH in the range of 7-7.5 and about 0.01 - 0.1 % surfactant, preferably buffered saline of pH of 7.2 and 0.05% surfactant. In still another embodiment of the present invention, said conjugate is selected from a group comprising rabbit anti-goat Gigs conjugated with alkaline phosphatase, rabbit anti-sheep IgG conjugated with alkaline phosphatase, rabbit anti-sheep IgG conjugated with horse radish peroxidase (HRP) and rabbit anti-goat IgG conjugated with HRP, preferably rabbit anti-goat HRP conjugate. In still another embodiment of the present invention, said chromogenic substrate is selected from a group comprising 2,2'-azino-bis3-ethylbenzthiazoline-6-sulphonic acid (ABTS), o-phenylene diamine di-hydrochloride (OPD), 5-amino salicylic acid (5-AS) and 3,3',5,5'-tetramefhylbenzidine (TMB), preferably OPD. In still another embodiment of the present invention, said stopping agent is selected from a group comprising 0.1M citric acid, 1M sulphuric acid and 2M sulphuric acid, preferably 1M sculptures acid. In still another embodiment of the present invention, said kit has diagnostic sensitivity of about 92 - 100%, preferably about 98% and diagnostic specificity of about 92 -100%, preferably about 95%. The present invention also relates to a blocking-cum-dilution buffer comprising a buffered saline, a surfactant and a blocking agent for detecting antigen-antibody interaction(s). In another embodiment of the present invention, said buffered saline is selected from a group comprising tris buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline; said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 4-(l,l,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate. preferably polyethylene glycol sorbitan monolaurate and said blocking agent is selected from a group comprising bovine serum albumin-fraction V, bovine gelatin, skim milk powder and foetal calf serum, preferably bovine gelatin. In yet another embodiment of the present invention, said buffer components include buffered saline of pH ranging from about 7 to 7.5, preferably about 7.2; surfactant at a concentration ranging from about 0.01 to 0.1 %, preferably about 0.05 %; and blocking agent at a concentration ranging from about 1 to 2.5 %, preferably about 2.0 %; components are at a ratio ranging from about 1: 0.0001-0.001: 0.01-0.025, preferably about 1 : 0.0005: 0.02. In still another embodiment of the present invention, said buffer reduces background colour and detects antigen-antibody interaction(s) in disease(s) selected from a group comprising brucellosis, infectious bovine rhinotracheitis and leptospirosis. The present invention also relates to a process to obtain a blocking-cum-dilution buffer, wherein said process comprises mixing a buffered saline, a surfactant and a blocking agent at a ratio ranging from about 1 : 0.0001-0.001 : 0.01-0.025, preferably about 1 : 0.0005 : 0.02. In still another embodiment of the present invention, said buffered saline is selected from a group comprising tris buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline; said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 4-(l,l,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate, preferably polyethylene glycol sorbitan monolaurate and said blocking agent is selected from a group comprising bovine serum albumin, bovine gelatin, skim milk powder and fontal calf serum, preferably bovine gelatin. In still another embodiment of the present invention, said buffer components include buffered saline of pH ranging from about 7 to 7.5, preferably about 7.2; surfactant at a concentration ranging from about 0.01 to 0.1 %, preferably about 0.05 %; and blocking agent at a concentration ranging from about 1 to 2.5 %, preferably about 2.0 %; components are at a ratio ranging from about 1: 0.0001-0.001: 0.01-0.025, preferably about 1 : 0.0005: 0.02. The present invention also relates to a kit for diagnosis of diseases comprising a blocking-cum-dilution buffer. In still another embodiment of the present invention, said kit is an indirect ELISA kit and said diseases are selected from a group comprising brucellosis, infectious bovine rhinotracheitis (IBR) and leptospirosis, preferably brucellosis in ruminants selected from a group comprising cattle, sheep and goat. In still another embodiment of the present invention, said buffered saline is selected from a group comprising tris buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline, said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 2-(l,l,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate, preferably polyethylene glycol sorbitan monolaurate and said blocking agent is selected from a group comprising bovine serum albumin, bovine gelatin, skim milk powder and fontal calf serum, preferably bovine gelatin. In still another embodiment of the present invention, said blocking-cum-dilution buffer components include buffered saline of pH ranging from about 7 to 7.5, preferably about 7.2; surfactant at a concentration ranging from about 0.01 to 0.1 %, preferably about 0.05 %; and blocking agent at a concentration ranging from about 1 to 2.5 %, preferably about 2.0 %; components are at a ratio ranging from about 1: 0.0001-0.001: 0.01-0.025, preferably about 1 : 0.0005: 0.02. In still another embodiment of the present invention, said kit comprises: i) antigen coated plate(s); ii) control sera; iii) conjugate; iv) washing buffer; v) chromogenic substrate; and vi) stopping agent. In still another embodiment of the present invention, said antigen coated plate(s) are coated with purified smooth lipopolysaccharide obtained from Brucella species, preferably from antigenic strain of Brucella abortus; said control sera is selected from a group comprising positive control serum, moderate positive control sera and negative control sera; said conjugate is selected from a group comprising rabbit anti-goat IgG conjugated with alkaline phosphatase, rabbit anti-sheep IgG conjugated with alkaline phosphatase, rabbit anti-sheep IgG conjugated with horse radish peroxidase (HRP) and rabbit anti-goat IgG conjugated with HRP, preferably rabbit anti-goat HRP conjugate; said washing buffer comprises buffered saline of pH of about 7-7.5 and about 0.01 - 0.1 % surfactant, preferably buffered saline of pH of about 7.2 and about 0.05% surfactant; said chromogenic substrate is selected from a group comprising 2,2'-azino-bis3-ethylbenzthiazoline-6-sulphonic acid (ABTS), o-phenylene diamine di-hydrochloride (OPD), 5-amino salicylic acid (5-AS) and 3,3',5,5'-tetramethylbenzidine (TMB), preferably OPD; said stopping agent is selected from a group comprising about 0.1M citric acid, about 1M sulphuric acid and about 2M sulphuric acid, preferably about 1M sulphuric acid; said kit has diagnostic sensitivity of about 92 -100%, preferably about 98% and diagnostic specificity of about 92 -100%, preferably about 95%. The present invention relates to a kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. The kit described in the present invention is an ELISA kit, preferably indirect ELISA kit. The present invention also relates to a process to obtain such a kit. The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the invention. EXAMPLE 1: Standard strain Standard antigenic strain of B. abortus S99 which was maintained at PD ADMAS for production of Brucella diagnostic kits was revived and confirmed by biochemical tests and PCR, cloning and sequencing (Shone et al. 2007). EXAMPLE 2: Large scale bacterial culturing The pure colonies were picked up from Riposte Agar plates and suspended in 10 ml of sterile PBS. After overtaxing, the bacterial suspension was overlaid on tryptose agar in Roux flasks. Thirty flasks were simultaneously inoculated from the same master plate to provide the identical bacterial population originating from a single colony. After one hour adsorption, Roux flasks were inverted and incubated for 72 hours at 37°C. The purity of the culture in every flask was confirmed by Gram's staining. To each flask, 30 ml of 2% phenol saline was added, gently agitated and incubated at same conditions mentioned above. The suspensions were pooled, centrifuged at 14,000 rpm at 4° C for 20 minutes. The centrifugation was repeated and pellets were carefully collected, weighed and used for antigen extraction. To obtain 5 gm wet weight of bacteria, thirty Roux flasks were harvested. EXAMPLE 3: Smooth Lipopolysaccharide Antigen extraction 5g wet weight of brucella cells was suspended in 17 ml of distilled water and heated to 66°C and 19 ml of 90% (v/v) phenol at 66°C was added. The mixture was stirred continuously at 66°C for 15 minutes, cooled and centrifuged at 10,000 rpm for 15 minutes at 4°C. The brownish phenol in the bottom layer was aspirated with a long microtip and large cell debris was removed by filtration (using a Whatman No.l filter). The LPS was precipitated by the addition of 50 ml cold methanol containing 0.5 ml methanol saturated with sodium acetate. After 2 hours' incubation at 4°C, the precipitate was removed by centrifugation at 10,000rpm for 10 minutes. The precipitate was stirred with 8 ml of distilled water for 18 hours and centrifuged at 10,000rpm for 10 minutes. The collected supernatant solution was kept at 4°C. This step was repeated for best recovery of antigen. Next, 0.8 g of trifluroacetic acid was added to the 16 ml of crude LPS, stirred for 10 minutes and the precipitate was removed by centrifugation. The translucent supernatant solution was concentrated and dialyzed against distilled water (two changes of at least 4000 ml each) and then freeze dried. From 5g wet weight of bacterial cells, 10 ml of smooth Lipopolysaccharide was extracted. (Fig.l) EXAMPLE 4: Raising of hyper immune sera Anti-Brucella sLPS hyper immune sera was raised in two brucellosis-negative bannur breed female adult sheep. Equal quantities of Freund's complete adjuvant and sLPS (500 ul) was mixed and injected subcutaneously. After 10 days, equal quantities of sLPS and Freund's incomplete adjuvant was injected s/c. The injections were repeated at weekly intervals for another 3 weeks. One week after the last booster, the animals showed a titre of 1:2560. A large quantity of blood was collected, sera was separated and lyophilized. This undiluted, hyper immune sera was considered as strong positive control. Pre-immune sera was used as the negative control. The moderate positive control was prepared by diluting strong positive sera with pre-immune sera at 1:500 dilutions. EXAMPLE 5: Titration of antigen The optimum concentration of antigen was standardized by checker board titration. Serial dilutions of the antigen were titrated against 1:100 and 1:200 dilution of hyperimmune serum. The OD values were plotted on a graph. The point where there was sharp fall on the line graph (1:500) was taken as the optimum dilution of antigen (Fig. 2). EXAMPLE 6: Standard ELISA protocol The polysorb microtiter plates were coated with 1:500 dilution of sLPS antigen at lOOul per well in carbonate-bicarbonate buffer (pH 9.6) and incubated 37°C for 1 hour. Antigen coated plates were washed three times with washing buffer (PBS containing 0.05 % Tween20) pH 7.2. Test and control sera diluted in PBST (1:100) containing 2% gelatin was added to respective wells (lOOul) of the plates in duplicates (test sera) and quadruplicate (controls) and incubated at 37°C for lhr. The plates were then washed as mentioned earlier. The anti-goat HRP conjugate (Pierce, Germany), diluted (1:8000) in PBST buffer was added to all the wells (lOOul volumes) and incubated for lhr at 37°C on orbital shaker. After washing, freshly prepared OPD solution containing 5mg OPD tab in 12.5ml of distilled water and 50 ul of 3% H2O2 was added and kept for color development till 10 min. Finally; the enzyme-substrate reaction was stopped by adding IM H2SO4 in 50ul volumes. The color development was read at 492nm using an ELISA microplate reader (Fig. 3). The results were interpreted by calculating percent positively (PP) as follows: PP= Mean OD of the test sera sample X 100 Median OD of the strong positive sera Sera samples showing PP values of 54 and above were considered brucella positive. EXAMPLE 7: Diagnostic cut-off A total of 3000 sera samples from sheep and goats were subjected a U.S.D.A approved kit (VMRD Inc.U.S.A). Of these, 200 positive and 200 negative sera were tested by laboratory standardized indirect ELISA test and values were plotted on line graph. The point of intersection of the two lines was taken as the cut-off percentage positively in the indirect ELISA test (Fig. 4). EXAMPLE 8: Comparatives results of 1000 sera tested A total of 374 and 626 sera samples from goats and sheep respectively were subjected to RBPT and I ELISA tests. In goats, 18 (4.84%) and 28 (7.48%) were found to be positive. Similarly in sheep, 37 (5.91%) and 54 (8.626%) were positive. On overall comparison it was found that sensitivity of indirect ELISA was found to be higher (8.20%) than RBPT (5.50%). Advantages of ELISA: 1. The use of highly purified smooth lipopolysaccharide antigen in indirect ELISA (instead of crude antigen) is responsible for the increased sensitivity of the test. 2. Also, it is possible to screen large no. of samples (40 samples/plate) simultaneously and can be calculated using computer linked ELISA reader. Goat and sheep population of the country is 122.53 and 57.6 millions respectively. Brucellosis is an important reproductive disease in sheep and goats characterized by abortion, stillbirths and reproductive failure and were found to be 11.3% and 1.5% respectively. The disease is under reported or overlooked and misdiagnosed because of lack awareness and non availability of suitable laboratory tests. Also brucellosis transmitted from small ruminants poses a higher risk to humans than transmitted from cattle. Therefore efforts are needed to diagnose the disease in goats and sheep using sensitive screening tests. India has already a policy for the control of brucellosis in dairy cattle and such policy in small ruminants helps a great extent to minimize the disease in man and reduce the economic losses. Diagnostic test options for brucellosis in sheep and goats include isolation of causative organism, serological tests like RBPT, SAT and ELISA and by molecular techniques. There are few reports of sero-prevalence studies of brucellosis in small ruminants. In the present study indirect ELISA has been standardized using smooth lipopolysaccahride antigen from a standard strain B. abortus S99 Antigen dilution of 1:500 was taken as the optimum by checker board titration. AnXi-Brucella sLPS hyperimmune sera was raised in two brucellosis-negative adult sheep. Pre-immune sera was used as the negative control. The moderate positive control was prepared by diluting strong positive sera with pre-immune sera at 1:500 dilutions. ELISA protocol has been standardized using laboratory standardized (modified) antigen extraction protocol, and blocking buffer. Sera samples showing Percentage Positive values of 54 and above were considered brucella positive. A total of 374 and 626 sera samples from goats and sheep respectively were subjected to RBPT and I ELISA tests. On overall comparison it was found that sensitivity of indirect ELISA was found to be higher (8.20%) than RBPT (5.50%). Cost of testing / sample is Rs.20.25 and 10.25 inclusive and excluding of equipment and establishment charges respectively. References Corbel M J. (1997). Brucellosis: an overview. Emerg Infect Dis. 3: 213-21. Dokuzoguz B, Ergonul O, Baykam N, Esener H, Kilic S, Celikbas A (2005). Characteristics of B. melitensis versus B. abortus bacteraemias. J Infect. 50: 41-5. Gupta V K and Vihan V S (2001). A document on Caprine brucellosis. CIRG Technical bulletin No. 17. Central Institute for Research on Goats. Makhdoom, P.O. Farah, Mathura-281122. Livestock census (2003) Government of India, Ministry of Agriculture, Department of Animal Husbandry Dairying and fisheries, http://dahd.nic.in/census.htm. Manual of diagnostic tests and vaccines for terrestrial animals. Chapter 2.3.1. bovine brucellosis Marin C M, Moreno E, Moriyon I, Diaz R and Blasco J M (1999). Clinical and Diag. Lab. Immunol., 6: 269-272. Mikolon A B, Gardner I.A, Hietala S K, Anda J H D, Pestana E C, Hennager S G and Edmondson A J (1998). J. Clin. Microbiol., 36: 1716-1722. Mrunalini N, Rama Sastry P, Pandarinadh G N and Ramakriishna Rao (2000). Ind Vet. J., 11: 932-935. Shome Rajeswari, Devine M, Shome B R, Desai G S, Patil S, Bhure S K and K. Prabhudas (2007). Cloning and sequencing of partial regions in chromosome II of B. abortus and chromosome I of B. melitensis (Accepted for publication in Indian Veterinary Journal) Refai M. (2002). Incidence and control of brucellosis in the Near East region. Vet Microbiol. 90: 81-110. Renukaradhya G J, Igloo S and Rajasekhar M. (2002). Epidemiology, zoonotic aspects, vaccination and control/eradication of brucellosis in India. Vet Microbiol. 90: 183-95. Roth F, Siesta J, Orchid D, Chimed-Ochir G, Hutton G and Cursive O (2003). Human health benefits from livestock vaccination for brucellosis: case study. Bull World Health Organ. 81: 867-76. Singh S V, Agarwal G S, Basra H V, Gupta V K and Singh N (2000). Ind. J. NiM. Sci., 70: 154-156. We claim: 1) A kit for diagnosis of brucellosis in small ruminants preferably sheep and goat. 2) The kit is an indirect ELISA kit wherein said kit comprises: i) antigen coated plates; ii) blocking-cum-dilution buffer; iii) control sera; iv) washing buffer; v) conjugate; vi) chromogenic substrate; and vii) stopping agent. 3) The kit as claimed in claim 2, wherein said antigen coated plates are coated with purified smooth lipopolysaccharide, obtained from Brucella species, preferably from antigenic strain of Brucella abortus. 4) The kit as claimed in claim 2, wherein said blocking-cum-dilution buffer comprising a buffered saline, a surfactant and a blocking agent which reduces background colour. 5) The blocking-cum-dilution buffer as claimed in claim 4, wherein said buffered saline is selected from a group comprising tris buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline; said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 4-(1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate. preferably polyethylene glycol sorbitan monolaurate and said blocking agent is selected from a group comprising bovine serum albumin-fraction V, bovine gelatin, skim milk powder and foetal calf serum, preferably bovine gelatin. 6) The blocking-cum-dilution buffer as claimed in claim 4, wherein said buffer comprises buffered saline of pH in the range of 7-7.5, about 0.01 - 0.1 % surfactant, and about 1 - 2.5% a blocking agent, preferably buffered saline of pH 7.2, 0.05% surfactant and 2% blocking agent. 7) The kit as claimed in claim 2, wherein said control sera comprising of positive control serum, moderate positive control serum and negative control serum. 8) The washing buffer as claimed in claim 2, wherein said buffered saline is selected from a group comprising tris buffered saline, tris-HCl and phosphate buffered saline, preferably phosphate buffered saline, said surfactant is selected from a group comprising polyethylene glycol sorbitan monolaurate, 2-(l,l,3,3-tetramethylbutyl) phenyl-polyethylene glycol and polyethylene sorbitan mono palmitate, preferably polyethylene glycol sorbitan monolaurate. 9) The washing buffer as claimed in claim 2, wherein said washing buffer comprises buffered saline of pH in the range of 7-7.5 and about 0.01 - 0.1 % surfactant, preferably buffered saline of pH of 7.2 and 0.05% surfactant. 10) The kit as claimed in claim 2, wherein said conjugate is selected from a group comprising rabbit anti-goat IgG conjugated with alkaline phosphatase, rabbit anti-sheep IgG conjugated with alkaline phosphatase, rabbit anti-sheep IgG conjugated with horse radish peroxidase (HRP) and rabbit anti-goat IgG conjugated with HRP, preferably rabbit anti-goat HRP conjugate. 11) The kit as claimed in claim 2, wherein said chromogenic substrate is selected from a group comprising 2,2'-azino-bis3-ethylbenzthiazoline-6-sulphonic acid (ABTS), o-phenylene diamine di-hydrochloride (OPD), 5-amino salicylic acid (5-AS) and 3,3',5,5'-tetramethylbenzidine (TMB), preferably OPD. 12) The kit as claimed in claim 2, wherein said stopping agent is selected from a group comprising 0.1M citric acid, 1M sulphuric acid and 2M sulphuric acid, preferably 1M sulphuric acid. 13) The kit as claimed in claim 2, wherein said kit has diagnostic sensitivity of about 92 - 100%, preferably about 98% and diagnostic specificity of about 92 -100%, preferably about 95%.

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