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13. Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India
Raghavendra Sumanth Pudupakam, Shobana Raghunath, Meghanath Pudupakam and Sreenivasulu Daggupati
Veterinary World, 10(3): 348-352
Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates.
Materials and Methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established.
Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains.
Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.
Aim: The present investigation was conducted to study the association between udder morphology and in vitro activity of milk leukocytes in high yielding crossbred cows.
Materials and Methods: A total of 48 healthy high yielding crossbred cows were selected for the study. The udder configuration and teat/udder morphology were recorded before milking. Milk samples (100 ml/cow) were collected aseptically. Milk somatic cell counts (SCC) and milk differential leukocyte counts were performed microscopically. Milk leukocytes (viz., neutrophils, lymphocytes, and macrophages) were isolated from milk samples by density gradient centrifugation. Phagocytic index (PI) of milk neutrophils and macrophages were evaluated by colorimetric nitro blue tetrazolium assay. Lymphocytes proliferation response was estimated by MTT assay and expressed as stimulation index.
Results: There was a significant (p<0.01) positive correlation between milk SCC with mid teat diameter, teat base diameter and significant (p<0.05) negative correlation between milk SCC and the height of the teat from the ground. Milk SCC was found to be significantly (p<0.01) lower in bowl-shaped udder and higher (p<0.01) in pendulous type. Milk macrophage percentage was positively (p<0.01) correlated with udder circumference. PI of milk neutrophil was negatively (p<0.01) correlation between teat base diameter, and PI of milk macrophages was found to be positively (p<0.01) correlated with teat apex diameter. Both PI of milk neutrophils and macrophages was found to be significantly (p<0.01) lower in the animals having flat and round teat and pendulous type of udder. In vitro PI of milk neutrophils was found to be significantly (p<0.01) lower in flat teat. In vitro PI of milk macrophages was found to be significantly (p<0.01) lower in the round and flat teats compared to pointed and cylindrical teats.
Conclusion: Udder risk factors such as teat shape and size, teat to floor distance, udder shape, and size may decrease the in vitro activity of milk leukocytes hence facilitates the incidence intramammary infections.
Aim: The study was undertaken to detect the clinical signs, postmortem lesions of embryonated duck plague (DP) infected eggs, and histopathological changes of chorioallantoic membrane (CAM) in non-descriptive ducks of West Bengal with special reference to standardize nested polymerase chain reaction (PCR).
Materials and Methods: After postmortem of suspected carcasses, samples were collected for virus isolation and identification through specific pathogen free (Khaki Campbell) embryonated duck eggs. PCR was also done as confirmatory test after doing postmortem of duck embryos. DP specific nested PCR was standardized for better confirmation of the disease. Sensitivity of nested primers was also tested for DP virus.
Results: Gross, postmortem and histopathological changes were prominent in dead embryos. First set of primer was able to detect 602 bp fragments of DNA polymerase gene of duck enteritis virus from infected CAM. Subsequently, a DP specific nested PCR which was very much sensitive for very small amount of viral genome was successfully standardized. After NCBI blast nucleotide sequence of nested PCR product (Accession No. HG425076) showed homology with the sequences data available in GenBank.
Conclusion: The study concludes that PCR assay is very much helpful to diagnose DP disease and developed nested PCR is a double confirmatory diagnostic tool for DP.
10. Hemato-biochemical alterations and urinalysis in dogs suffering from benign prostatic hyperplasia
M. R. Das, R. C. Patra, R. K. Das, P. K. Rath and B. P. Mishra
Veterinary World, 10(3): 331-335
Aim: The study was designed to evaluate the hemato-biochemical alterations, urinalysis along with histomorphological and histological changes of prostate glands in dogs affected with benign prostatic hyperplasia (BPH) in and around Bhubaneswar, Odisha, India.
Materials and Methods: In toto, 445 dogs presented to the Teaching Veterinary Clinical Complex of the College of Veterinary Sciences and Animal Husbandry, one Government Veterinary Hospital and two pet clinics in and around Bhubaneswar screened for the presence of BPH. Most of the 57 dogs were 6 years and above as reported by the owners. Only 57 dogs found positive for BPH basing on the presence of typical clinical signs subjected for a detailed hemato-biochemical study. Most of the 57 dogs were 6 years and above as reported by the owners. Routine and microscopic urinalyses were done as per the routine procedure. Histomorphological evaluations of prostate glands were done through manual rectal palpation. Histological examinations of prostate tissue sections of two dead dogs were conducted with routine hematoxylin and eosin stain.
Results: The study revealed about 12.8% (57/445) of dogs was suffering from BPH. Typical clinical signs - such as passing small thin tape-shaped feces, holding tail away from backward, tenesmus, and straining during urination and defecation - were seen in most of the cases. Urine samples of affected dogs were positive for glucose, occult blood, and protein. A significant decrease in lymphocytes and increase in eosinophil counts in dogs with BPH was recorded. Serum biochemical analysis showed a nonsignificant increase in creatinine and blood urea nitrogen with a significant decrease in total protein, albumin, globulin, A:G ratio. Histology of prostate glands collected during postmortem was characterized by fibrosis of prostate gland, and hyperplasia of the acinar epithelium.
Conclusion: High rate of the prevalence of BPH in dogs poses an alarming condition which if diagnosed at an early stage can certainly prolong the longevity of the dogs.
microbial ecology and hematological parameters of broiler fed cassava waste
pulp fermented with Acremonium charticola
Turrini Yudiarti, Isroli Isroli, Endang Widiastuti and Fatan Dwi Putra
Aim: The aim of this study was to evaluate the effect of feeding Acremonium charticola-fermented cassava pulp (AC-FCP) on the intestinal microbial ecology and hematological indices of broiler chickens.
Materials and Methods: A total of 240 male Lohman day-old-chicks were randomly allotted to one of the four experimental diets including control diet, control diet + antimicrobials (neomycin; 300 mg/kg diet), diet containing AC-FCP (16 g/100 g diet) or diet containing AC-FCP + antimicrobials. At day 28, the birds from each pen were blood sampled, sacrificed and immediately the internal organs were removed and weighed. Digesta were obtained from the ileum and cecum.
Results: Birds fed AC-FCP had lower (p<0.05) coliform bacteria count in the ileal digesta than birds fed control diet or control diet + antimicrobials. Butiric acid was higher (p<0.05) in the cecal content of birds fed AC-FCP than in other birds. Propionic acid was also higher in AC-FCP fed birds than in other birds although statistically not significant. The percentages of lymphocytes and heterophils were higher (p<0.05) and tended (p=0.07) to be lower, respectively, in broilers fed control diet than in other birds. The birds provided control diet had lower (p<0.05) heterophils to lymphocytes ratio compared to those receiving AC-FCP or AC-FCP + antimicrobials. Serum total protein and globulin were higher (p<0.01) in birds fed control diet or control diet + antimicrobials compared to AC-FCP or AC-FCP + antimicrobials fed birds. Serum albumin was lower (p<0.01) in AC-FCP birds than that in other birds. There was a tendency (p=0.09) that birds fed AC-FCP diet had lower total serum cholesterol than other birds.
Conclusion: Feeding AC-FCP has potential to improve the intestinal health and protect the birds from acute infections.
8. Molecular characterization of velogenic viscerotropic Ranikhet (Newcastle) disease virus from different outbreaks in desi chickens
V. S. Dhaygude, G. K. Sawale, M. M. Chawak, N. R. Bulbule, S. D. Moregaonkar and D. S. Gavhane
Veterinary World, 10(3): 319-323
ABSTRACT Aim: Diagnosis of velogenic viscerotropic Ranikhet disease from six different flocks of desi chicken in and around Mumbai by gross and histopathological examination, isolation of virus and molecular methods.
Materials and Methods: A total of 25 carcasses (varying between 2 and 6 carcasses from each flock) of six different flocks of adult desi chicken were subjected to necropsy examination for diagnosis of the disease during the span of a year (2014-2015). After thorough gross examination, the tissue samples were collected and processed for virus isolation and histopathological examination. The 20% tissue homogenate was inoculated into 9-day-old specific pathogen free (SPF) embryonated eggs. Mean death time (MDT) of embryos after inoculation and intracerebral pathogenicity index (ICPI) were used to judge velogenic nature of the virus. Newcastle disease virus (NDV) was isolated from six cases and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting the partial fusion protein gene of the viral genome.
Results: A total of 25 carcasses (varying between 2 and 6 carcasses from each flock) of six different flocks of desi chicken were presented for postmortem examination to Department of Veterinary Pathology, Bombay Veterinary College, Parel, Mumbai during 2014-2015. The gross and histopathological examination revealed lesions suggestive of viscerotropic velogenic form of the Newcastle disease (ND). The 20% tissue homogenate was inoculated into 9-day-old embryonated eggs from SPF chicken. NDV was isolated from six cases and confirmed by RT-PCR targeting the partial fusion protein gene. MDT of all the isolates was <60 h which indicated velogenic nature of the virus. ICPI of the isolates ranged between the 1.63 and 1.78. In four out of six outbreaks concurrent moderate to heavy infection of Ascardii galli in one flock and Railetina spp. in three flocks was also noted. In this study, viscerotropic velogenic form of ND was confirmed in all six outbreaks by gross and microscopic examination, virus isolation and RT-PCR.
Conclusion: In this study, viscerotropic velogenic form of ND was confirmed in all six outbreaks by gross and microscopic examination, virus isolation and RT-PCR. Nowadays, vaccine strains Lasota, B1 and F strains are used widely in India to control the infection of NDV. However, virulent NDV strains are still isolated frequently in the birds under backyard and also in commercial venture which demonstrates that NDV remains an on-going threat to commercial as well as backyard poultry flocks.
Keywords: mean death time, Newcastle disease virus, reverse transcriptase polymerase chain reaction, specific pathogen free, velogenic.