Occurrence of enteric viruses causing clinical diarrhea in small ruminants in northern Indian plains: a reverse transcription PCR based molecular study.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Enterocolitis in goats associated with enterotoxaemia in the perspective of two toxins: Epsilon toxin and beta-2 toxin - An immunohistochemical and molecular study.

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.

Comparative molecular characterization and phylogenetic analysis of cerebral and non-cerebral coenurosis in Indian goats.

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.

Detection of the presence of Coxiella burnetii in a case of goat abortion: a first report from India.

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.

Comparative diagnostic evaluation of OMP31 gene based TaqMan® real-time PCR assay with visual LAMP assay and indirect ELISA for caprine brucellosis.

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.