Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

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.

Occurrence, molecular characterization and antimicrobial-resistance pattern of Staphylococcus species isolates from buck semen.

Staphylococcus aureus is one of the most prevalent pathogens, and a causative agent of a variety of infections in humans and animals. Most studies concentrated on characterization of staphylococcus isolates and its antimicrobial resistance from various illness of veterinary importance, but there is no specific study that is available on isolates from reproductive tract of small ruminants and especially its semen. Hence, in the current study, a total of 48 semen samples were collected from healthy bucks of different breeds to investigate the occurrence of S. aureus. Antimicrobial resistance and virulence of the Staphylococcus isolates were determined to assess the adverse effects of them on buck fertility. The bacterial isolates were tentatively confirmed as Staphylococcus spp. based on the Gram's staining, growth on Mannitol salt agar and catalase test. Overall, 75% (n = 36) of the samples were positive for Staphylococcus spp. from the total 48 buck semen ejaculates from different breeds and among them 23 (63.89%) were coagulase-negative (CoNS) and 13 (36.11%) were coagulase-positive Staphylococcus (CoPS) strains. The species identified by molecular characterization are S. aureus, S. chromogenes, S. haemolyticus, S. sciuri, S. simulans, and S. epidermidis from buck semen. Further, these isolates exhibited varying degrees of multidrug resistance genotypically as well as phenotypically. The presence of antibiotic resistance and virulence genes may pose a potential threat to reproductive health of animals, the animal handlers and livestock keepers, while simultaneously highlighting the need for vigilant monitoring of these isolates at the time of semen cryopreservation.

Molecular detection of important abortion-causing microorganisms in preputial swab of breeding bucks using PCR-based assays.

Infectious diseases and aetiological agents related to female reproductive systems were extensively covered compared to its male counterpart. There needs a proper study to bridge this gap, where microflora and infectious agents of both male and female reproductive are mutually intelligible. With this study, we aimed to evaluate the microbial contamination of the preputial cavity and also screened for abortion-causing agents which are zoonotic as well. In goats, such types of abortions are caused by Brucella melitensis, Chlamydophila, Campylobacter and Coxiella etc. One of the major sources of contamination of semen is the preputial cavity, which is exposed to the external environment leading to spread of infection into the female via semen straws or by natural service. In the current study, good quality bucks (n = 32, Barbari = 12, Jamunapari = 10, Jakhrana = 10) which were routinely used for semen collection were screened for their preputial swabs, for the presence of the above pathogens. For detection of Brucella melitensis, OMP31 based TaqMan® probe real-time PCR assay was used, and for Chlamydia, 16srRNA gene based SYBR® green real-time PCR assay was employed for detection of Chlamydophila abortus. While for Campylobacter spp. and Coxiella burnetii, 16srRNA gene based conventional PCR and Trans-PCR were used, respectively. In the current study, of the screened preputial swabs, none of them showed positive for Brucella and Coxiella, but of the screened 32 samples 17 showed positive for Chlamydia (53.13%) and two (6.25%) showed positive for Campylobacter spp. The current study emphasizes on the farms and laboratories which were regularly involved in screening of brucellosis also often overlook the other potential non-brucella pathogens, causing abortions eventually incurring severe economic losses to the goat keepers.

Evaluation of newly developed 'six recombinant secretary proteins based 'cocktail ELISA' and 'whole cell lysate' based 'indigenous ELISA' and tissue microscopy' with 'Gold standard' histo-pathology for the diagnosis of Johne's disease in slaughtered goats and buffaloes.

Three screening tests {(newly developed, six recombinant secretory proteins based 'cocktail ELISA', in-house robust 'indigenous ELISA' based on semi-purified protoplasmic antigens and tissue microscopy were evaluated with 'Gold standard', histo-pathology for the diagnosis of Johne's disease in goats and buffaloes. Serum and tissues {mesenteric lymph nodes and intestines) were driven from farmer's goats (n = 77) and buffaloes (n = 40) slaughtered for harvesting meat and farm goats (n = 77), died and necropsied. Twenty seven (35%) goats and 23 (57.5%) buffaloes were positive in all the four tests. Of 134 tissues screened by histo-pathology, 79.8% MLN and 76.8%, intestines, were positive for MAP infection. In tissue microscopy, 55.2 and 52.3%, goats and buffaloes were positive, respectively. Of 117 sera screened by i_ELISA, 58.4 and 70.0%, goats and buffaloes were positive, respectively. Whereas, c_ELISA detected 55.8 and 62.5%, goats and buffaloes, positives, respectively. Twelve tissues (70.5%) of goats necropsied were positive, both in tissue microscopy and histo-pathology. Most significant gross findings were serous atrophy of the fat and mild to moderate, diffuse thickening of terminal ileum, especially at ileo-caecal junction with or without transverse / longitudinal corrugations. In histo-pathology grade III and IV lesions were significantly low as compared to grade I and II. Of the four tests used for screening 268 samples, histo-pathology was most sensitive (78.3%), followed by i_ELISA (62.3%), c_ELISA (58.9%) and tissue microscopy (58.9%). Between two ELISA tests, c_ELISA using six recombinants secretory proteins, had higher specificity as compared to i_ELISA.