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.

Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

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.

Bacteriophages: A possible solution to combat enteropathogenic Escherichia coli infections in neonatal goats.

Due to awareness and benefits of goat rearing in developing economies, goats' significance is increasing. Unfortunately, these ruminants are threatened via multiple bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). In goat kids and lambs, EPEC causes gastrointestinal disease leading to substantial economic losses for farmers and may also pose a threat to public health via the spread of zoonotic diseases. Management of infection is primarily based on antibiotics, but the need for new therapeutic measures as an alternative to antibiotics is becoming vital because of the advent of antimicrobial resistance (AMR). The prevalence of EPEC was established using bfpA gene, uspA gene and Stx1 gene, followed by phylogenetic analysis using Stx1 gene. The lytic activity of the isolated putative coliphages was tested on multi-drug resistant strains of EPEC. It was observed that a PCR based approach is more effective and rapid as compared to phenotypic tests of Escherichia coli virulence. It was also established that the isolated bacteriophages exhibited potent antibacterial efficacy in vitro, with some of the isolates (16%) detected as T4 and T4-like phages based on gp23 gene. Hence, bacteriophages as therapeutic agents may be explored as an alternative to antibiotics in managing public, livestock and environmental health in this era of AMR.

Chemotactic factor inducing Interleukin-8 (IL8) gene is transcriptionally elevated in experimental enterotoxaemia in goats caused by Clostridium perfringens type D.

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-1ß 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.

Molecular etiopathology of naturally occurring reproductive diseases in female goats.

AIM: The aim of the present study was to investigate the molecular etiopathology of occurrence of reproductive diseases in female goats. Reproductive diseases in goats account for major economic losses to goat farmers in terms of valuable loss of offspring and animal productivity. MATERIALS AND METHODS: A total of 660 female genitalia were examined for pathological conditions (macroscopic and microscopic lesions). The etiopathological study was carried out for the presence of pathogenic organisms such as Brucella, Chlamydia, and Campylobacter in the uterus and ovary. Based on the microscopic lesions, suspected samples were subjected to diagnostic polymerase chain reaction (PCR) for various etiological agents employing 16srRNA genus specific primers for Campylobacter and Chlamydophila and OMP31 gene-based PCR for Brucella melitensis and nested PCR using ITS-1 gene primers for Toxoplasma gondii. For Brucella suspected samples, immunohistochemistry (IHC) was also performed. RESULTS: In studied female genitalia, 108 (16.30%) showed gross abnormalities with overall 23.32% occurrence of pathological conditions (macroscopic and microscopic lesions). Pathological involvement of the uterus was the highest 68 (62.96%), followed by the ovaries 27 (25%) and other organs. Major uterine condition observed was endometritis (5.60%). In uterine infections, 35 (5.30%) samples were found positive for Campylobacter spp., 12 (1.81%) samples for B. melitensis, and 3 (0.45%) samples were positive for Chlamydophila spp. Among the samples positive for B. melitensis by PCR, 3 were found positive by IHC also. Corynebacterium ovis was detected by PCR using specific primers in a case of hydrosalpinx. It was concluded that many pathological lesions in female genitalia of functional significance play a major role in infertility in goats. CONCLUSION: The present study concluded that many pathological lesions in female genitalia of functional significance play a major role in infertility in goats.

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.

Ross River Virus (RRV) infection in horses and humans: a review.

A fascinating and important arbovirus is Ross River Virus (RRV) which is endemic and epizootic in nature in certain parts of the world. RRV is a member of the genus Alphavirus within the Semliki Forest complex of the family Togaviridae, which also includes the Getah virus. The virus is responsible for causing disease both in humans as well as horses. Mosquito species (Aedes camptorhynchus and Aedes vigilax; Culex annulirostris) are the most important vector for this virus. In places of low temperature as well as low rainfall or where there is lack of habitat of mosquito there is also limitation in the transmission of the virus. Such probability is higher especially in temperate regions bordering endemic regions having sub-tropical climate. There is involvement of articular as well as non-articular cells in the replication of RRV. Levels of pro-inflammatory factors viz., tumor necrosis factor-alpha (TNF-α); interferon-gamma (IFN-γ); and macrophage chemo-attractant protein-1 (MAC-1) during disease pathogenesis have been found to be reduced. Reverse transcription-polymerase chain reaction (RT-PCR) is the most advanced molecular diagnostic tool along with epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detecting RRV infection. Treatment for RRV infection is only supportive. Vaccination is not a fruitful approach. Precise data collection will help the researchers to understand the RRV disease dynamics and thereby designing effective prevention and control strategy. Advances in diagnosis, vaccine development and emerging/novel therapeutic regimens need to be explored to their full potential to tackle RRV infection and the disease it causes.