Bovine babesiosis in India: Estimation of prevalence by systematic review and meta analysis.

Bovine babesiosis is a serious threat to the livestock sector especially in tropical countries like India. Understanding the epidemiology of the disease in the country is essentially important in strategizing the available methods to effectively control the disease. Keeping this as the background, the present study was undertaken to estimate the pooled prevalence of bovine babesiosis in India. The relevant literature pertaining to bovine babesiosis was identified and a total of 49 studies published between 1983 and 2018 were included in the final systematic review and meta-analysis. Meta-analysis was conducted using meta-package of R software and prevalence estimates were calculated. Bovine babesiosis was reported from 21 states of India with pooled prevalence estimate of 6% (95% CI = 4%-9%) using random effect model. Zone wise analysis revealed highest pooled prevalence in the west zone and north zone (8%) followed by east zone (7%), central zone (6%), south zone (4%) and northeast zone (4%). The results of meta-analysis indicated high variability between studies. In addition, the pooled seroprevalence was high (29%) compared to prevalence of active infection (5%) of bovine babesiosis in India. Further, the pooled prevalence estimate of B. bigemina infection in India was more (7%) compared to B. bovis infection (1%). The estimation of prevalence of active infection and seroprevalence separately will helps to understand the actual disease prevalence in the country. The study indicated the wide prevalence of bovine babesiosis in India which urges for immediate mitigation strategies.

Animal disease surveillance: Its importance & present status in India.

Animal disease surveillance encompasses systematic collection of long-term data on disease events, risk factors and other relevant parameters followed by analyzing the same with reference to temporal and spatial characteristics to arrive at a conclusion so that necessary preventive measures can be taken. In India, the animal disease surveillance is done through National Animal Disease Reporting System, which is a web-based information technology system for disease reporting from States and Union Territories with the aim to record, monitor livestock disease situation and to initiate the preventive and curative action in a swift manner during disease emergencies. National Animal Disease Referral Expert System is a dynamic geographic information system and remote sensing-enabled expert system that captures an incidence of 13 economically important livestock diseases from all over the country and also provides livestock disease forecasting. The laboratories under State and Central governments, several research institutes under the Indian Council of Agricultural Research and veterinary colleges are involved in livestock disease diagnosis including zoonotic diseases. An integrated surveillance system is necessary for early detection of emerging/zoonotic diseases in humans. This review provides information on disease reporting and surveillance systems in animal health sector and the need for One Health approach to improve and strengthen the zoonotic disease surveillance system in India.

An inhibition enzyme immuno assay exploring recombinant invariant surface glycoprotein and monoclonal antibodies for surveillance of surra in animals.

The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.

Cytokine gene expression and pathology in mice experimentally infected with different isolates of Trypanosoma evansi.

Aim of the present study was to assess the cytokine gene expression in liver, kidney and spleen and histopathological changes in mice infected with buffalo and dog isolates of Trypanosoma evansi. Forty-four Swiss albino mice was divided into eleven groups of four mice each and injected subcutaneously with 1 × 105 trypanosomes of buffalo and dog isolate to twenty mice each, four mice served as control. Mice were examined for clinical signs, blood smear for trypanosome counts. Blood for PCR, liver, kidney, spleen, heart, lung, testis and abdominal muscle for histopathology and liver, kidney, spleen for cytokine gene expression studies, were collected. Mice showed dullness, lethargy, hunched back, sluggish movements on D4 and D5 in buffalo and dog isolate, respectively. Parasite count in blood varied between the two isolates of T. evansi. By PCR, trypanosome DNA was detected on D1 and D2 for buffalo and dog isolate, respectively. Splenomegaly was observed in mice infected with buffalo isolate but not with dog isolate. Histopathological changes were observed in liver, kidney, spleen and heart of mice but no changes in testis and abdominal muscles. Blood vessels of liver, heart, lung showed presence of trypanosomes in mice infected with buffalo isolate but not for dog isolate. Cytokine gene expression of IL-2, IL-4, IL-6, IL-12, TNF-α and IFN-γ increased in liver, kidney and spleen in both these isolates. However, the buffalo isolate exhibited pronounced increase in cytokine gene expression when compare to dog isolate of T. evansi. Anti-inflammatory cytokine gene IL-10 showed 50-60 and 10-20 folds increment in buffalo and dog isolates, respectively. This is the first report of IL-4, IL-6, IL-10 and IL-12 cytokine changes in mice infected with T. evansi. A variation in pathogenicity between buffalo and dog isolates was recorded indicating buffalo isolate of T. evansi remained more pathogenic in mice.

Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis.

Trypanosomosis or surra is caused by the haemoflagellate parasite, Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis.

Prevalence of Neospora caninum antibodies in dairy cattle and water buffaloes and associated abortions in the plateau of Southern Peninsular India.

A seroprevalence study of bovine neosporosis was conducted among 1,927 dairy cattle and 341 water buffaloes from Karnataka and Andhra Pradesh states in plateau of southern peninsular India by employing competitive enzyme-linked immunosorbent assay. Overall, 12.61 and 9.97 % sera samples were found positive for the presence of Neospora caninum antibody, respectively, among cattle and water buffaloes. Out of 1,927 sera samples from cattle, 912 and 1,015 samples were collected from unorganized and organized herds, respectively. The cattle screened were of upgraded Holstein-Friesian and water buffaloes were of graded Surti breed. Significantly (p < 0.05) higher prevalence was found in the cattle in unorganized herds (16.66 %) in comparison to organized herds (8.96 %). The highest seroprevalence was recorded in the age group of 4 years and above in both type of cattle herds and water buffaloes. There was a significant variation of seroprevalence (p < 0.05) observed between different age groups of cattle. The rate of seroprevalence increased with the increment in the age of the animals suggesting a possibility of horizontal mode of transmission of the infection from the environment. The percentage of abortion history was more in seropositive group (51.65 %) in comparison to the seronegative group (5.84 %) and the seropositive cattle were 8.84 times more likely to experience abortion than the seronegative cattle. The occurrence of abortion among different age groups varied significantly (p < 0.05). The findings revealed the presence of neosporosis in the southern peninsular India among cattle and water buffaloes and a strong association between the seroprevalence and abortion.

Prevalence of Toxoplasma gondii, Leptospira spp., and Coxiella burnetii-associated antibodies in dairy cattle with reproductive disorders.

Background and Aim: In cattle dairy farms, abortions and other reproductive problems due to major infectious diseases are overlooked, and identifying their causative agents is very challenging without a confirmatory diagnosis. Further, a prevalence study in animals will provide important hints of pathogen reservoirs and provide necessary direction to disease burden with appropriate control and biosecurity measures at the farm level. This study aimed to estimate the prevalence of Toxoplasma gondii antibodies in dairy cattle associated with reproductive problems along with coexisting antibodies against abortifacient zoonotic (Coxiella burnetii and Leptospira spp.) pathogens. Materials and Methods: Cattle sera (n = 246) from dairy farms (n = 35) situated in different locations in India were screened for anti-T. gondii and C. burnetii antibodies with enzyme-linked immunoassay and Leptospira spp. antibodies with microscopic agglutination test. Results: The overall prevalence of 11.4% (95% confidence intervals [CIs]: 7.99%-15.96%) antibodies in cattle associated with reproductive problems (p < 0.021) with farm-level seropositivity of 43% was observed. Further, on analysis of screened sera, 49.8% (95% CI: 42.6%-55%) and 77.6% (95% CI: 72%-82.4%) of samples were found to be positive for C. burnetii and Leptospira spp. antibodies, respectively. Moreover, the seropositivity of 91.9% (226/246) for at least one of the screened zoonotic pathogens was observed, indicating antibodies against either of these organisms in association with reproductive disorders (p < 0.005). The percentage of cattle found to have T. gondii antibodies was only 1.8%, whereas 11.5% and 41.6% of cattle were found to have C. burnetii and Leptospira spp. antibodies, respectively. Nevertheless, the predominantly mixed infections observed were of Leptospira and C. burnetii (34.5%), followed by all three infections (4.9%); toxoplasmosis and leptospirosis (3.5%); and toxoplasmosis and Q fever (2.2%). Conclusion: The serological detection of antibodies against these pathogens in cattle may have significant implications for the livestock industry and public health, suggesting the need for continuous surveillance and monitoring of these infections to prevent their spread.

Mining the pervasiveness of surra in different animal species of Northeastern states of India: Assam, Mizoram and Tripura.

Trypanosoma evansi is a flagellated, extracellular haemoprotozoan parasite infecting a wide range of mammalian hosts including dromedaries, cattle, equines and dogs cause disease surra. Carrier animals with sub-clinical infection cause significant monetary losses to livestock holders and therefore detection of infection status using molecular diagnostic techniques becomes important in order to control the disease. In the current study cattle, buffalo, goat, pig and dog samples from three northeastern states of India-Assam, Mizoram and Tripura were screened to determine the prevalence of surra. A total of 1702 samples including 795 from Assam, 678 from Mizoram and 229 from Tripura were screened by CATT/T. evansi test out of which 16.8%, 27.1% and 22.3% samples in respective states were found to have antibodies against T. evansi. DNA detection of T. evansi by PCR amplification targeting VSG gene revealed the molecular prevalence of surra in Assam, Mizoram and Tripura as 8.5%, 7.5% and 4.4% respectively. The analysis of amplified partial VSG sequences showed 99% similarity within an animal species whereas 86-94% similarity was observed among different species of animals revealing the homogeneity. The study established the prevalence of surra in different species of animals in the three northeastern states of India-Assam, Mizoram and Tripura and this study is the first report of T. evansi infection in pig and goat from India. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12639-021-01392-z.

Seroprevalence of Trypanosoma evansi in cattle and analysis of associated climatic risk factors in Mizoram, India.

Surra, a haemoprotozoan parasitic disease even in subclinical form poses a challenge in terms of diagnosis and management to animal health practitioners and policy makers as well; eventually imparting financial loss to the livestock holders. A systematic study was designed to assess the seroprevalence of surra in cattle and associated climatic risk factors, by collecting 480 serum samples across the eight districts of Mizoram during 2017-2019. The apparent and true seroprevalence detected by card agglutination test was 37.08% (CI at 95%: 32.88-41.49) and 36.59% (CI at 95%: 32.4-40.99) whereas by recombinant Variable Surface Glycoprotein based indirect ELISA was 41.88% (CI at 95%: 37.5-46.3) and 40.35% (CI at 95%: 36.02-44.76) respectively. Climate parameters which influence vector population were extracted from their respective database and were correlated with seroprevalence data. Linear discriminant analysis revealed that air temperature, relative humidity and diurnal temperature range, leaf area index and soil moisture as significant risk factors discriminating seropositive and seronegative data sets classified by indirect ELISA. This study is the first report on seroprevalence of surra in cattle of Mizoram and the situation demands deployment of intervention strategies in order to assess the endemicity of the disease and thereby preventing the economic losses.

Development of an antigen ELISA using monoclonal antibodies against recombinant VSG for the detection of active infections of Trypanosoma evansi in animals.

Trypanosoma evansi, a haemo-flagellated protozoan parasite causes chronic wasting disease in a wide range of animals. For its diagnosis, blood smear examination is useful in clinical cases for direct identification of the parasite but in latent infection the carrier animals are difficult to screen out by conventional blood smear test. Harboring low level of parasites and showing no symptom, the carrier animals for surra can act as a source of infection. The level of parasitaemia fluctuates, especially during latent infection; moreover the antibodies which are not found early in the infection may persist even after recovery or chemotherapy. In the present study a double antibody sandwich ELISA exploring, monoclonal antibodies and hyperimmune serum, raised against recombinant variable surface glycoprotein has been developed to detect circulating trypanosome antigens. The developed antigen detection ELISA (Ag-ELISA) was evaluated using 652 blood samples collected from cattle, buffalo, equine and camel. The statistical analysis of the data showed diagnostic sensitivity and specificity at 97.4% and 96.4% respectively, with a positive-negative cut-off OD value >0.28. Furthermore, the detection limit of the assay was found to 7.15 trypanosomes per mL. The present finding revealed that the developed assay can be exploited as a potential diagnostic test in the detection of circulating trypanosome antigens and also can be used as a population screening test for multiple animal species for detection of active infection for further treatment and control of the disease.

Seroepidemiology of peste des petits ruminants in sheep and goats of southern peninsular India.

This paper presents the results of a seroepidemiological study carried out between July 2006 and March 2007 to detect the presence of antibodies to peste des petits ruminants (PPR) virus in randomly collected serum samples from sheep and goats in southern peninsular India. The authors used a competitive enzyme-linked immunosorbent assay with a monoclonal antibody developed against a neutralising epitope of the haemagglutinin (HA) protein of the virus. A total of 1,492 sheep sera and 2,068 goat sera collected from the six southern Indian states were screened. It was determined that 41.35% of the sheep sera and 34.91% of the goat sera were positive for the presence of antibody. The study indicated an extensive endemicity of the disease in these states, which is attributed to the agro-climatic conditions and the migration of livestock.

Antigen detection ELISA: A sensitive and reliable tool for the detection of active infection of surra.

Trypanosomosis, an endemic disease in Asia, America (central and south) and Africa causes havoc economical loss in livestock industry. The carrier animals which are symptomless and harbours low level of parasites can act as a source of infection. The level of parasitaemia fluctuates, especially during the latent infection; moreover the antibodies which are not found early in the infection may persit even after recovery or chemotherapy. The parasitological and/or serological tests always can not detect current infection or carrier animals. Hence, in the present study double antibody sandwitch antigen detection ELISA (Ag-ELISA) is developed to detect circulating trypanosomes. The new assay has been evaluated using 554 field samples comprising bovine and camel. The diagnostic sensitivity and specificity of the new assay was found to be 97.4% and 99.0% respectively, with a Cohen's kappa value of 0.96. The developed assay could detect 11.5 Trypanosoma evansi per mL from the experimentally infected blood, buffy coat and purified T. evansi samples. The findings revealed that the developed assay can be exploited as a potential diagnostic tool in the detection of active trypanosomal infection.

Development and evaluation of recombinant antigen and monoclonal antibody based competition ELISA for the sero- surveillance of surra in animals.

Trypanosoma evansi, a haemoflagellated protozoan parasite, is responsible for chronic as well as the acute debilitating disease called surra in a wide range of herbivores and carnivores including domestic and wild animals. Since the parasite is having wide host range, there is a need for diagnostic test which can detect the T. evansi specific antibody in different species of animals for generating sero-surveillance data. In the present study we developed and evaluated competitive enzyme immunoassay using monoclonal antibodies (MAbs) raised against recombinant variable surface glycoprotein (rVSG) of T. evansi. The immunoreactivity of the developed MAbs (IgG3-subtype) was evaluated by immunoblot as well as ELISA and subsequently used in the development and standardization of competitive ELISA (C-ELISA). Further, the serological data generated from the C-ELISA using reference samples constituting true positive or surely infected (35), true negative (45), sero-positive (225) and sero-negative (215) samples and was analyzed statistically. The true positivity/negativity was determined by thin blood smear examination and diagnostic PCR assay, While, seropositivity/seronegativity of the reference samples was determined through standard reference tests. The data showed the diagnostic sensitivity of 92.6% and specificity of 96.4% with Cohen's kappa value of 0.88. In order to determine the utility of C-ELISA in detecting T. evansi antibodies in different species of animals, the assay was further evaluated with 1361 field sera sample comprising bovine, horse, donkey and camel. Since the C-ELISA described herein has showed high sensitivity and specificity, this single test can be explored in the sero-surveillance of T. evansi in a wide range of animals.

Flagellar antigen based CI-ELISA for sero-surveillance of surra.

Trypanosoma evansi causes a disease known as 'surra' in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive-negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen's Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra.

Immediate and delayed effects of successful percutaneous transvenous mitral commissurotomy on global right ventricular function in patients with isolated mitral stenosis.

Global right ventricular function of the pressure-overloaded right ventricle in patients with mitral stenosis and pulmonary hypertension after successful percutaneous transvenous mitral commissurotomy (PTMC) has not been well-defined. With the use of a recently developed Doppler method for estimating right ventricular function in human beings, we studied 25 consecutive patients with isolated rheumatic mitral stenosis before, immediately after (mean, 40+/-12 h) and at a mean follow-up of 11.5 months after PTMC. Immediately after percutaneous mitral commissurotomy, there was a significant increase in mitral valve area (P = 0.000017) along with a decrease in mean pulmonary pressure (P = 0.001). The index was not affected immediately after successful PTMC (0.70+/-0.25 vs., 0.58+/-0.18; P = 0.06); however, at follow-up of about one year, the index showed a significant decrease (0.697+/-0.28 vs. 0.380+/-0.13; P = 0.0008, n = 24). The change in the index was characterised by a significant prolongation of the right ventricular ejection time, with a decrease in the isovolumic intervals. The Doppler index of combined right ventricular function was significantly correlated to the mean pulmonary artery pressure (r = 0.695, P<0.001) and systolic pulmonary artery pressure (r = 0.60, P = 0.007) before PTMC and also immediately after the procedure; however, at follow-up, the index had no correlation with the Doppler estimated pulmonary artery systolic pressure (r = 0.07). Despite a larger mitral valve area following PTMC, right ventricular isovolumic indices remain abnormal on mid-term follow-up, although global function tends to normalise in two-thirds of the patients.

Congenitally unguarded tricuspid valve orifice with a giant right atrium and a massive clot in an asymptomatic adult.

Congenitally unguarded tricuspid valve orifice, a variant of tricuspid valve dysplasia, is a rare malformation with protean manifestations. This report describes an asymptomatic adult who, on echocardiographic examination ordered in view of an abnormal 12-lead surface electrocardiogram and plain chest X-ray, was found to have an unguarded tricuspid valve orifice with a giant right atrium (12 x 10 cm), intense spontaneous echo contrast and a large right atrial clot.

Noncompaction of left ventricular myocardium in the presence of calcific aortic stenosis in an adult.

We describe an adult patient with a hitherto unreported association of severe aortic stenosis with extensive noncompaction of the left ventricular myocardium without any hypertrophy; however, there was severe left ventricular systolic dysfunction in the presence of a normal-sized left ventricular cavity on two-dimensional echocardiography. This condition was differentiated from persistence of embryonic intramyocardial sinusoids by selective coronary angiography.

Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance.

Trypanosoma evansi, a haemoflagellate, causes "surra" an important chronic wasting disease of a wide range of wild and domestic herbivorous and carnivorous animals including cattle, buffaloes, camels, horses, etc. The untreated recovered animal can act as a carrier without exhibiting the disease symptoms and can be a source of infection to healthy animals. The diagnosis and subsequent treatment of the carrier animals is helpful to curb the disease. As the parasitaemia in carrier animals is very scanty, the conventional blood smear examination, which is widely practiced in the field, cannot detect such condition. For this purpose improved diagnostics are very much useful for mass sero-screening test such as ELISA. In the present study, the VSG of T. evansi was expressed in prokaryotic system (E. coli) and thereafter its immunoreactivity has been evaluated in immuno blot and enzyme immuno assay. The expressed protein showed 95.6% sensitivity, 98.0% specificity and 0.93 Cohen's kappa value, when compared with standard antigens. The developed antigen has also been validated with field serum samples from bovine, camel and horse collected from different states of India. The data showed that the developed recombinant antigen can be a diagnostic tool to detect carrier animals as well as control of the disease.

PCR based diagnosis of trypanosomiasis exploring invariant surface glycoprotein (ISG) 75 gene.

The invariant surface glycoprotein (ISG-75) gene of Trypanosoma evansi buffalo isolate from Karnataka state in India was sequenced and analyzed to elucidate its relationship with other isolates/species. The sequenced ISG-75 gene was also explored to device a polymerase chain reaction (PCR) strategy for the diagnosis of trypanosomiasis in carrier animals. The six cloned ISG gene sequences revealed the open reading frame (ORF) of 1572 and 1527 nucleotide (nt) encoding a polypeptide of 523 and 508 amino acids (aa) respectively and belongs ISG-75 gene family. Sequence analysis revealed 91-100% and 65-99% similarity at nt and aa levels, respectively with other isolates/species and belongs to the RoTat 1.2 strain. The diagnostic PCR based on ISG-75 sequence amplifies a 407 bp product specifically from the different T. evansi isolates and could detect 0.04 pg and 1.2 ng of DNA from purified trypanosomes and T. evansi infected rat blood samples respectively. Subsequently the PCR detected 0.02 and 0.27 trypanosomes ml(-1) respectively, from purified trypanosomes and T. evansi (buffalo isolate) infected rat blood. By the developed PCR assay trypanosomal nucleic acid was detected in experimental rats and buffalo on 24 h post infection (p.i.) and 3rd day post infection (d.p.i.), respectively. The developed ISG-75 gene based PCR assay could be useful in detection of carrier status of trypanosomiasis in animals.

Expressed truncated N-terminal variable surface glycoprotein (VSG) of Trypanosoma evansi in E. coli exhibits immuno-reactivity.

The variant surface glycoprotein (VSG) of trypanosome is an important part of its body surface coat, which is expressed in early, middle and late stages of infection contributing a major diagnostic value. In the present study, the 5' end of the partial VSG gene sequences (681 bp) encoding N-terminal protein of RoTat 1.2 VSG (227 amino acid) was amplified, cloned into pET32a vector, and expressed in prokaryotic system. The fused His-tagged expressed VSG protein (43 kDa) of the Trypanosoma evansi was characterized in SDS-PAGE and immunoblotting using hyperimmune/immune sera raised against buffalo, dog, lion and leopard isolates of T. evansi. The expressed protein remained immunoreactive with all the sera combinations. The animals immunized with whole cell lysate or recombinant protein showed similar antibody reactions in ELISA and CATT (Card Agglutination Test for Trypanosomiasis). This study suggests the expressed recombinant truncated VSG is having its importance for its possible use in sero-diagnosis of surra.