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Caprine brucellosis due to Brucella melitensis is an important zoonotic disease. The present study was carried out to address the lack of a comprehensive study on the status of caprine brucellosis in Puducherry, India using serological and molecular tests in goats and to assess the seroprevalence in human risk groups of the aforementioned region to ascertain the public health significance of the disease. Seroprevalence in 120 goats was found to be zero, 3.33% and 18.33% by Rose Bengal agglutination Test (RBT), Standard Tube Agglutination Test (STAT) and Immunoglobulin G Indirect Enzyme Linked Immune Sorbant Assay (IgG iELISA) respectively. Of the 120 goat genital swabs screened, while conventional polymerase chain reaction (PCR) detected genus specific 16S rRNA and Brucella melitensis specific omp2 genes in 17.50% and 5.00% of samples respectively, the OMP31TaqMan® real time PCR with a positive detection of 40.0 % was both the most sensitive and specific for detection of Brucella melitensis. The study provides insight into the optimization of diagnostic tests following cluster wise sampling for brucellosis in goats. The strain of Brucella melitensis in Puducherry was found to be Biovar 3 based upon suggestive results of Restriction Fragment Length Polymorphism (RFLP) of omp2 gene product. Seroprevalence by IgG iELISA was 33.33 % in 30 samples from human subjects. Serological evidence of caprine brucellosis in goats and human subjects and molecular detection of Brucella melitensis in Puducherry, India warrants regular screening, surveillance and reporting of disease in goats and human risk groups.IMPORTANCEBrucellosis is an important zoonotic pathogen causing abortions in domestic animals as well as posing risk to livestock keepers and handlersControl of this disease requires proper surveillance programme, and in this study the peninsular coastal region of India was sampled by a cluster method and evaluated for brucellosis in goats as well as people at riskThis study compared the diagnostic sensitivity of various serological and molecular tests and found that the OMP31 gene TaqMan probe based Real Time assay to be highly sensitive in diagnosing brucellosisSimilarly, in human subjects exposed to risk factors like animal handlers, veterinarians, livestock keepers the sero-positivity was 33.33% by ELISA

Caprine intestinal diseases associated with clostridia are generally caused by Cpa and Etx encoded alpha (α) and epsilon (ε) toxinotypes of Clostridium perfringens type D respectively. A recent study on goat enterocolitis, demonstrated that the incidence of Clostridium perfringens type-D toxinotype and beta 2 toxins is high, suggesting its role in enterocolitis and many other diseases of goats affecting intestinal tract. Considering this scenario, the present prevalence study was planned to screen the goat intestinal tissues for the presence of the epsilon toxin and beta 2 toxin. Tissue sections from enterotoxaemia suspected cases in 189 goats were collected and epsilon-toxin was demonstrated by immuno-histochemically and toxinotyping multiplex polymerase reaction in 19 animals and beta 2 toxin in 19 animals by multiplex polymerase reaction. Immuno-reactivity to epsilon toxin was detected maximum in distal ileum of goat intestine and this toxin was produced by Clostridium perfringens type D. It suggests that immunohistochemistry is a confirmatory tool for detection of bacterial toxin especially epsilon toxin where isolation and characterisation of bacteria is not possible. Here, we have reported a strong association between ε-toxin (epsilon) and beta-2 toxin in causing disorders of intestine in goats. In addition, we have explored the possible role of cpb2 positive isolates of C. perfringens and their pathogenic effects in causing enterotoxaemia. These determinants help in the understanding of the pathogenesis of enterotoxaemia in goats which needs to be further investigated.

The current study was designed to analyse the effects of experimental induction of enterotoxaemia through intra-duodenal inoculation of C. perfringens type D culture isolated from spontaneous outbreaks in goats. Twenty goats (6–9 month age) were divided into four groups and C. perfringens type D culture was inoculated intra-duodenally as per following: Group-I (whole cultures-WC), group-II (culture supernatant-CS), group-III (washed cells-WS), and group-IV (uninfected control-C). The treated animals were sacrificed after 72 h post infection (hpi), and necropsy showed gross changes including haemorrhages and congestion in the ileal and colon mucosa, pulmonary congestion and edema in lung. Kidney, brain and spleen exhibited severe to moderate congestion. Microscopic changes like haemorrhages, degenerative and necrotic changes in the mucosal epithelium of intestine and haemorrhages in kidney parenchyma were observed in the H&E stained sections. Lung alveolar sacs were filled with proteinaceous fluid. Immunohistochemistry revealed positive immunolabelling for etx (epsilon toxin) in the mucosa of intestine in WC and CS group. Control animals did not exhibit any significant gross or microscopic changes. PCR amplification of DNA extracted from intestinal tissues of WC and CS groups showed positive for etx gene demonstrating the production of epsilon toxin. Transcriptional responses in experimental groups were assessed by quantitative reverse transcription real time PCR (qRT-PCR). Genes including IL-1β and IL2 showed up-regulation in all the experimental groups (WC, CS&WS). Specifically the toxin-based experimental groups (WC&CS) showed up-regulation of the gene responsible for chemotaxis viz. IL-8, while the washed cells group (WS) showed higher transcriptional response to Cathepsin-L (Cat-L) gene denoting the acute inflammatory response due to neutrophil elastase activity. These results take a cue on the evolving nature of the enterotoxaemia in goats due to various strains circulating in the field. The host response and its modulation due to the novel enterotoxaemia strains throws light on the current challenges in efficient control of the disease in goats., Clostridium perfringens; Enterotoxaemia; Goat; Interleukin-8; Transcriptional response.

The role of various pathogenic agents affecting gastro-intestinal tract of neonatal goat kids was studied. Intestinal tissue samples of 142 necropsied kids died due to enteritis, pneumo-enteritis, tapeworm’s infestations and septicemia were collected for detection of toxinotypes of Clostridium perfringens, enteropathogenic E. coli (EPEC), Group-A rotavirus (GARV) and bovine coronavirus (BCV). C. perfringens toxinotyping was done by multiplex PCR using primers for cpa, cpb, cpb2, etx and iap gene. Further, identification of EPEC was done using SYBR green based real-time PCR assay targeting bfpA gene. Detection of GARV and BCV was done by one-step RT-PCR (osRTPCR). The incidence of C. perfringens was 16.20% (47.83% toxinotype A and 52.17% toxinotype D), with b2- toxin present in 30.43% of the samples. Incidence of EPEC in 0-1 and 1–3 month diarrhoeic kids was 36.62% and 25.35%, respectively. For viral aetiology, 11.97% were positive for GARV and 9.86% positive for BCV. Mixed infection of C. perfringens and EPEC was seen in 12.68% cases, while it was 2.11% for C. perfringens + GARV and 1.40% for C. perfringens + BCV. Similarly, the mixed occurrence of EPEC+GARV was 10.56% and that of EPEC+BCV was 7.04%. The combined infection of EPEC+GARV+BCV was 0.70%. The results suggested that C. perfringens type A and type D are the common toxinotypes affecting neonatal goat kids, with β2 toxin being an additional potential virulence factor. SYBR green based real-time PCR assay can be used as quick lab-based technique for efficient screening of EPEC from enteritis affected kids.Group-A rotavirus and bovine coronavirus appeared to be instrumental in causing mixed infection enteritis in kids.

Coenurus cerebralis is the larval stage of Taenia multiceps, which infects the muscles and brain of goats and, to a lesser extent, sheep. The resulting cerebral and non-cerebral infections caused by the larval form (metacestode) of this cestode are commonly known as coenurosis. A weak emaciated carcass of five months old female goat, on necropsy, revealed numerous parasitic cysts (n = 56, grossly visible) in the visceral cavity including heart, diaphragm, thoracic cavity, abdominal cavity and pelvic inlet. A large number of variable sized parasitic cysts were also observed embedded in the pericardium and myocardium causing functional damage to the heart. The parasite caused extensive tissue damage at gross and microscopic levels in the heart including traumatic destruction of the myocardium with degenerative and necrotic changes and infiltration of mononuclear cells. On parasitological examination, the cysts were identified as Coenurus cerebralis, as the scolices had characteristic four suckers and a rostellum with a double crown of hooks. Further confirmation was done using polymerase chain reaction targeting specific ND1 and CO1 genes. Phylogenetic analysis of CO1 and ND1 genes showed a major branch comprising two clades of T. multiceps grouped as separate entities with the first clade showing T. multiceps/Coenurus cerebralis native CIRG strain (cerebral) being placed in proximity to T. multiceps/Coenurus cerebralis CIRG strain (non-cerebral/visceral) compared to the Chinese strains of T. multiceps. The phylogenetic analysis of ND1 and CO1 genes of C. cerebralis of cerebral and non-cerebral isolates revealed close proximity but expressed in two different disease forms (i.e., visceral coenurosis and neural coenurosis) which indicated that they were very close divergent from a common ancestor. On the basis of the observations it was concluded that goat died due to cardiac dysfunction resulting from severe systemic infection of metacestode of T. multiceps was closely related to isolate that caused neural coenurosis in another goat. Based on the sequencing analysis and phylogenetic information, the possible differences in the clinical manifestation (neural or visceral) could be attributed to the pathogenesis.

Abortion is a major cause of economic loss to the goat industry. Coxiella burnetii the causative agent of Q fever is an important zoonotic agent known to be prevalent worldwide. In the present investigation, we detected the presence of Coxiella burnetii by the modified Ziehl Neelsen method of staining and its DNA by trans-PCR assay in the placenta obtained from the aborted goat. We also ruled out other common causes of abortion in this case. Based on a literature survey, this is the first report on the direct detection of Coxiella burnetii from an aborted goat to be reported from India.

In the present study, out of 1156 neonatal goat kids, 238 showing clinical diarrhea were used for detection of toxinotypes of Clostridium perfringens, Enteropathogenic E. coli (EPEC), Group A rotavirus (GARV) and Bovine coronavirus (BCV). Isolation and toxinotyping of isolates were done by multiplex Polymerase chain reaction (PCR) using primers for cpa, cpb, cpb2, etx and iap genes. For EPEC, isolation and identification were done using bfpA gene and SYBR green based real time PCR (qPCR). GARV and BCV were detected, by one-step RT-PCR (osRT-PCR). The incidence of C. perfringens was 15.13% with 75% isolates toxinotype A, 25% type D and 61.11% of isolates carrying the β2-toxin gene. The incidence of EPEC was 68.07% based on qPCR, whereas 21.85% were positive for GARV and 15.97% for BCV by osRT-PCR. There was mixed infection of C. perfringens and EPEC in 11.76% and 3.78% for C. perfringens and GARV and 2.1% of C. perfringens and BCV. EPEC and GARV was 19.74% and EPEC plus BCV positivity was 11.34%. GARV and BCV was 5.88%, and 4.20% had mixed infection of EPEC, GARV and BCV. Of the total diarrheic kids sampled, 0.84% had mixed infection of C. perfringens, GARV, BCV and EPEC. On the basis of the above findings, it may be concluded that isolation, multiplex PCR and real time PCR facilitated the characterization of circulating C. perfringens toxinotypes and EPEC in goats reared under semi-arid conditions. The importance of enteritis caused by GARV and BCV and their role in mixed infection in goats requires extensive screening and pathogenicity studies to associate the symptoms with disease., U populaciji od 1156 neonatalnih jarića 238 je pokazivalo kliničke znakove proljeva. Od njih su uzeti uzorci izmeta za dokazivanje toksinskih tipova bakterije Clostridium perfringens, enteropatogenih sojeva bakterije E. coli (engl. enteropathogenic E. coli, EPEC), rotavirusa skupine A (engl. group A rotavirus, GARV) i goveđeg koronavirusa (engl. bovine coronavirus, BCV). Izdvajanje i toksinska tipizacija izolata provedeni su višestrukom lančanom reakcijom polimerazom (PCR) upotrebom početnica za gene cpa, cpb, cpb2, etx i iap. Izdvajanje i identifikacija EPEC-a provedeni su pretragom na gen bfpA i PCR-om u stvarnom vremenu, uz upotrebu SYBR zelenila (qPCR). Za dokaz rotavirusa skupine A i goveđeg koronavirusa upotrijebljena je RT-PCR (osRT-PCR). Incidencija bakterije C. perfringens iznosila je 15,13 %. Od toga je 75 % izolata pripadalo toksinskom tipu A, 25 % tipu D, dok je 61,11 % izolata imalo gen za toksin β2. Incidencija EPEC-a iznosila je 68,07 %, a 21,85 % pretraženih uzoraka bilo je pozitivno na GARV te 15,97 % na BCV. Mješovita infekcija bakterijom C. perfringens i EPEC-om utvrđena je u 11,76 % uzoraka, C. perfringens i GARV u 3,78 % te C. perfringens i BCV u 2,1 % pretraženih uzoraka. Mješovita infekcija EPEC-om i GARV-om utvrđena je u 19,74 %, a EPEC-om i BCV-om u 11,34 %. Mješovita infekcija rotavirusom i koronavirusom bila je ustanovljena u 5,88 %, a mješovita infekcija EPEC-om, GARV-om i BCV-om u 4,20 % uzoraka. Od ukupnog broja pretražene jaradi s proljevom u njih 0,84 % dokazana je mješovita infekcija bakterijom C. perfringens, rotavirusom skupine A, goveđim koronavirusom i EPEC-om. Na osnovi prikazanih rezultata može se zaključiti da izdvajanje, višestruki PCR te PCR u stvarnom vremenu omogućuju karakterizaciju i praćenje kolanja toksinskih tipova bakterija C. perfringens i EPEC-a u koza uzgajanih u sušnim uvjetima. Važnost enteritisa uzrokovanog rotavirusom skupine A i goveđim koronavirusom te njihova uloga kod mješovitih infekcija u koza zahtijevaju pojačan nadzor i istraživanje patogenosti radi povezivanja kliničkih znakova s ustanovljenom bolešću.

Brucellosis is one of the leading causes of abortion in domestic animals that imposes costs on both economy and society. The disease is highly zoonotic and poses risk to animal handlers due to its zoonotic nature. It causes stillbirth, loss of kids and abortion in last term of pregnancy. Reproductive damage includes infertility in does and orchitis and epididymitis in breeding bucks, which result in high financial losses to farmers and the agriculture industry as a whole. It requires highly sensitive and specific assays to diagnose the disease at field level. In the current study, a visual loop-mediated isothermal amplification (LAMP) assay and the TaqMan® real-time PCR were developed with high sensitivity and specificity. For the TaqMan® probe, real-time PCR primers were developed using Omp31 gene as target and primers were designed using discontiguous conserved sequences of Omp31 gene. The Omp31 probes were designed by attaching 6-FAM reporter dye at the 5′ end and BHQ-1 quencher at the 3′ end. Published primers were used for visual LAMP assay targeting the Omp25 gene. Sensitivity of the standardized visual LAMP assay and TaqMan® real-time PCR assay was determined by serial dilution of positive Brucella melitensis DNA (102 to 10−4 ng) obtained from standard culture. The TaqMan® probe real-time assay can detect as low as 100 fg of B. melitensis DNA, whereas culture from vaginal swab washings has a limit of detection (LOD) of only 1 cfu/ml. Similarly, the visual LAMP assay can detect as low as 10 fg of B. melitensis DNA as compared to an LOD of 30 cfu/ml from culture of vaginal swab washings. Both assays were compared with serological tests (serum tube agglutination test (STAT) and indirect enzyme-linked immunosorbent assay (iELISA)) for diagnostic sensitivity and specificity. Diagnostic sensitivities and specificities for TaqMan® real-time PCR vs. LAMP assays were 98 and 100% vs. 100 and 97.8%, respectively. Results of visual LAMP assay indicated that LAMP is a fast, specific, sensitive, inexpensive and suitable method for diagnosis of B. melitensis infection under field conditions. On the other hand, Omp31 TaqMan® probe real-time assay can be used in conjunction with the other field-based diagnostic tests due to its high specificity.

Caseous lymphadenitis (CLA) is a chronic and sub-clinical disease of sheep and goats with high prevalence in domestic animals. It is caused by Corynebacterium pseudotuberculosis biovar ovis. An outbreak of CLA in superficial and visceral forms was observed in Barbari and Jakhrana goat herds maintained at ICAR-CIRG, Makhdoom (UP) in July, 2016. A total of 147 goats was affected by the infection of external lymph nodes and two cases in Barbari goats showed internal abscess on necropsy. Biopsy pus samples were collected and identified the organism after Gram's staining. All biopsy samples were inoculated to blood agar and subjected to biochemical (catalase, oxidase and nitrate) tests. Isolation of DNAfrom culture and 16S rRNAgene-based PCR for C. ovis was carried out for confirmatory diagnosis. Gross and histopathological studies were conducted in cases of internal abscess. Antibiotic sensitivity tests were conducted for all the isolates obtained during the study. External lymph nodes were predominantly affected involving pre-scapular and post-scapular (94), pre-femoral (44) and mandibular lymph nodes (9). After incising abscesses, white thick pus oozed out. In Grams ’stained smears, the organism was pleomorphic rods arranged in Chinese letters. On blood agar, round, raised, greyish colonies were observed and biochemical examination showed catalase positive and oxidase negative. The 16S rRNA genebased PCR confirmed the presence of C. ovis. In two cases, visceral abscess was observed with oozing pus from affected lungs. Histopathology of affected lung tissue showed infiltration of inflammatory cells including neutrophils, lymphocytes and macrophages within infected tissue and concentric fibrous layers separated by inspissated caseous exudate. Based on morphology, biochemical tests and PCR, causal organism was confirmed as C. ovis and based on 16 antibiotics tested for sensitivity, ciprofloxacin was the only antibiotic found to be effective against isolates.