Rajamani Barathidasan

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed ‘pigeon PMV-1’ (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

The first case of angioinvasive pulmonary aspergillosis in a Himalayan Griffon Vulture ( Gyps himalayensis )

We present the first case of angioinvasive pulmonary aspergillosis caused by Aspergillus fumigatus in a Himalayan Griffon vulture (Gyps himalayensis). We describe in detail the clinical, gross, histopathological and mycological findings. Clinical signs of weakness, emaciation, dyspnea, incoordination, and inability to fly were observed in this bird before succumbing to the illness, despite supportive care. Grossly, several yellowish circumscribed, raised, miliary nodules were observed on the surface of lungs, air sac membranes, trachea, pericardium, aorta, pulmonary artery and kidneys. Histologically, granulomatous pneumonia and airsacculitis, and angioinvasion of fungal hyphae were noticed. Aspergillus fumigatus organism was isolated from the lungs and air sacs of the bird. This seems to be the first report of angioinvasive pulmonary aspergillosis in birds of prey, and a rare finding in avian species.

Upregulated Myc Expression in N-Methyl Nitrosourea (MNU)-induced Rat Mammary Tumours

BACKGROUND The most common incident cancer and cause of cancer-related deaths in women is breast cancer. The Myc gene is upregulated in many cancer types including breast cancer, and it is considered as a potential anti-cancer drug target. The present study was conducted to evaluate the Myc (gene and protein) expression pattern in an experimental mammary tumour model in rats. MATERIALS AND METHODS Thirty six Sprague Dawley rats were divided into: Experimental group (26 animals), which received the chemical carcinogen N-methyl nitrosourea (MNU) and a control group (10 animals), which received vehicle only. c-Myc oncoprotein and its mRNA expression pattern were evaluated using immunohistochemistry (IHC) and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively, in normal rat mammary tissue and mammary tumours. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control for semi-quantitative RT-PCR. RESULTS Histopathological examination of mammary tissues and tumours from MNU treated animals revealed the presence of premalignant lesions, benign tumours, in situ carcinomas and invasive carcinomas. Immunohistochemical evaluation of tumour tissues showed upregulation and heterogeneous cellular localization of c-Myc oncoprotein. The expression levels of c-Myc oncoprotein were significantly elevated (75- 91%) in all the tumours. Semi-quantitative RT-PCR revealed increased expression of c-Myc mRNA in mammary tumours compared to normal mammary tissues. CONCLUSIONS Further large-scale investigation study is needed to adopt this experimental rat mammary tumour model as an in vivo model to study anti-cancer strategies directed against Myc or its downstream partners at the transcriptional or post-transcriptional level.

Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

ABSTRACT Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

Detection and partial genetic characterisation of a novel variant of Avian nephritis virus in Indian poultry flocks showing diverse clinical signs.

Avian nephritis virus (ANV) infects poultry flocks worldwide, but no confirmed cases have been reported from India so far. In the current study, disease investigation was carried out in 21 broiler flocks at different parts of India with clinical signs of nephritis, uneven and stunted growth, diarrhoea, reduced body weight, and mortality up to 9.72%. Out of the 21 flocks screened, two were found positive for ANV in RT-PCR assay. BLAST analysis revealed that the ANV of Indian origin was closely related to ANV-1 strains reported from Japan, Hungary and China. However, comparison of a small portion (~12% of nucleotides, i.e. ~60 nts, common site for ANV-1 and ANV-3, position 2200-2260 of ORF 1a gene) of the Indian ANV sequence with ANV-3 sequences revealed 89-93% identities with different ANV-3 isolates. Phylogenetically, ANV-1 forms three clades, and the Indian ANV clustered under clade II. This study confirms the existence of ANV in Indian poultry flocks and is the first report on the molecular detection and genetic characterisation of ANV from India.

Molecular detection and characterization of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinical samples of commercial poultry flocks in India

Although the existence of infectious laryngotracheitis virus (ILTV) in India was first reported in 1964, no reports are available regarding its molecular detection and characterization. The present study was aimed to detect and characterize ILTV from recent respiratory disease complex (RDC) outbreaks of commercial poultry flocks in different parts of the country by using envelope glycoprotein G gene (US4 gene) based PCR and sequencing. A total of thirty two flocks with a history of RDC were investigated. Overall, all the strains/breeds of birds and all ages of birds are equally susceptible and depending on the severity, the clinical signs and gross lesions were varied. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. The phylogenetic analyses of eight representative sequences in the present study deciphered that Indian ILT viruses are closely related to chicken embryo origin vaccine strains of Italy, USA, China and Brazil.

A novel recombinant Meq protein based dot-ELISA for rapid and confirmatory diagnosis of Marek’s disease induced lymphoma in poultry

Mareks disease (MD), is an economically important virus disease of poultry throughout the world. In this study, we for the first time reports development of a novel dot enzyme-linked immunosorbent assay (dot-ELISA) for the confirmatory diagnosis of lymphoma caused by Mareks Disease Virus (MDV). Suspected lymphoma tissue extracts from the diseased birds were used for the Meq oncoprotein antigen detection, which is expressed specifically in MDV lymphomas. Recombinant Meq oncoprotein was expressed using Expresso™ Rhamnose Sumo Cloning and Expression system and the hyperimmune serum was raised against it, which was used later while developing dot-ELISA. The dot-ELISA exhibited higher specificity (92%) in diagnosing MD lymphomas as compared to conventional PCR (40%), where later assay is unable to differentiate disease development (lymphoma) and/or infection. The developed dot-ELISA proved to be a specific, rapid and inexpensive technique detecting MDV lymphomas in poultry. Of the note, this new assay could be opted as a valuable diagnostic tool in the resource poor countries andcould further be used to differentiate from other tumor causing viruses in poultry.

Cytological and immunocytological detection and differentiation of Marek’s disease and lymphoid leucosis in poultry

AbstractMarek’s disease (MD) and lymphoid leucosis (LL) are the major diseases causing lymphoid tumors in chickens accounting for high economical losses. Gross examination could not yield definite diagnosis owing to their similar presentation of lesions. Thus present work was aimed for diagnosis and differentiation of MD and LL by utilizing simple cytology and novel immunocytology techniques. Cytological examination was carried out on slides with tumor touch imprints stained by simple Giemsa staining. The diagnosis was mainly achieved based on morphology of cell population. In the present study, out of a total of 595 cases examined, 502 cases had pleomorphic lymphocytic cell population suggestive of MD and 53 cases had uniform lymphocytic/lymphoblast cell population suggestive of LL, while the rest 40 cases remained inconclusive. A definitive diagnosis was achieved after performing immunocytology using specific antibodies that revealed 518 cases had reactivity for Meq oncoprotein specific for MD and 77 cases showed immunoreactivity for IgM in transformed B-cells confirming LL. The technique of immunocytology which has been useful for detecting human viral pathogens and MD in poultry has been applied for the first time as a novel, simple, rapid and inexpensive technique that could be used as an alternate test to effectively detect and differentiate MD and LL in poultry.

Intussusception of jejunum in pullet naturally infected with coccidiosis: A case report

This paper describes a rare case of jejunal intussusception in a pullet that suffered from severe diarrhea and succumbed to death. Based on necropsy and microscopic examination, the recorded case of intussusception was found to be associated with severe coccidial enteritis. It is suggested on the basis of present findings and earlier published reports that, intussusception in birds could be attributed to conditions that causes reduced feed intake, hyperirritability, and increased intestinal motility.

Rare case of chronic pulmonary aspergillosis in emu chicks (Dromaius novaehollandiae)

Aspergillus spp. are opportunistic pathogens which cause pulmonary aspergillosis in human, animals and birds that are immunocompromised. Seven emu chicks of different ages ranging from 2 weeks to 7 weeks were presented for postmortem examination and diagnosis. Necropsy revealed granulomatous pneumonia in all the chicks. Multiple granulomas of varying sizes (1–4 mm) were also seen in the serosa of proventriculus, gizzard and kidneys. Direct microscopic examination of 10% KOH treated, lactophenol cotton blue stained smears from crushed nodules showed segments of branching fungal hyphae and conidia characteristic of aspergillus. Tease mounts wet smear preparation from the culture revealed conidiophores and conidia characteristic for Aspergillus fumigatus. Histopathologically, multifocal granulomas were seen in the lungs with central area of necrosis, numerous giant cells, mononuclear infiltration and sparse hyphae in the periphery. The frequent reports on aspergillosis in different avian species require a special attention to undertake necessary prophylactic and therapeutic measures to minimize economic losses.