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Peste Des Petits Ruminants Virus Infection of Goats in Bangladesh: Pathological Investigation, Molecular Detection and Isolation of the Virus

M. A. Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

I. Shadmin
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

M. Noor
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

R. Parvin
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

M. R. Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Peste des petits ruminants (PPR), an economically important morbillivirus infection of sheep and goats, is widely distributed in sub-Saharan Africa, Middle East and western and southern Asia including Bangladesh. A small flock of Black Bengal goats contracted PPR following introduction of new animals. A pathological investigation was conducted on the outbreak, the viral RNA corresponding to F gene was detected by RT-PCR and the virus was isolated in Vero cells. Out of 37 goats 19 (51 %) developed clinical disease, of which 5 (13.5 %) died. Goats under one year of age had highest morbidity and mortality with typical signs and lesions of PPR. Viral RNA could be detected in mesenteric and bronchial lymph node tissues. Typical cytopathic effects (CPE) in Vero cells following inoculation of lymph node tissue homogenate were visible at the third passage. However, the replication of virus in cell culture was detected by RT-PCR at the first and second passage in the absence of visible CPE. RT-PCR appears to be a very useful and sensitive tool not only for the detection of PPR virus in clinical samples but also for monitoring the growth of virus in cell culture following inoculation.

Keywords: PPR, Infection, Goats, Bangladesh, Pathological investigation, Molecular detection, Isolation of the virus

Location: Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Start Date: 01-07-2007

End Date: 31-07-2007

Program Area: Pest Management

Commodity: Goat

Objectives:

1. The present paper reports an investigation of a PPR outbreak in a goat flock in Bangladesh, including pathological observations, molecular detection and isolation of the virus.

Methodology:

The study was conducted on a natural outbreak of the disease in July 2007 in a flock of 37 Black Bengal goats raised by the Department of Animal Breeding and Genetics of Bangladesh Agricultural University, Mymensingh for experimental purposes. Detailed history and clinical features of the outbreak recorded. Pathological investigation - Routine necropsy was performed on dead goats. Gross pathological changes were recorded. The samples of trachea, lung, spleen, lymph node, rumen, intestine, kidney, heart and liver containing lesions were collected for histopathological study and fixed in 10% neutral buffered formalin. Pieces of mesenteric and bronchial lymph nodes were collected aseptically and stored at -70ºC for molecular detection and isolation of the virus. Formalin-fixed tissues were processed for paraffin embedding, sectioned and stained with haematoxylin and eosin for histopathological study following standard procedures. Molecular detection of the virus - A reverse transcription polymerase chain reaction (RT-PCR) was adopted for the detection of PPR virus. The reaction was carried out with a PPRV-specific primer set (PPRVF1b: 5´-AGT ACA AAA GAT TGC TGA TCA CAG T-3′ and PPRVF2d: 5´-GGG TCT CGA AGG CTA GGC CCG AAT A-3′) originally designed by Forsyth and Barrett (1995) to amplify a 448-bp cDNA product from the F gene spanning from the nucleotide position 760 to 1207. A lyophilized live PPR vaccine, produced by the Livestock Research Institute (LRI), Mohakhali, Dhaka, Bangladesh, was used as the positive control. RNA was extracted from the reconstituted vaccine or tissue homogenate from the field samples using RNeasy Mini Kit (Qiagen, Germany) according to manufacturer’s protocol. The RT-PCR was performed with Qiagen One- Step RT-PCR kit (Qiagen, Germany). The thermocycling profile was as follows: reverse transcription at 50ºC for 30 min, initial denaturation and activation of polymerase at 94ºC for 15 min, followed by 35 cycles of denaturation, annealing and extension at 94ºC for 1 min, 50ºC for 1 min and 72ºC for 2 min, respectively, and final elongation at 72ºC for 7 min. The RT-PCR products were analysed by electrophoresis on 1.5% agarose gel stained with ethidium bromide. Isolation of the virus - PPR virus was isolated in Vero cells from mesenteric and bronchial lymph node tissues of affected animals. The lymph nodes were macerated with sterile mortar and pestle to prepare a 20% (w/v) tissue homogenate in Dulbecco’s phosphate buffered saline (PBS)-A. The suspension was clarified by centrifugation (800g) for 10 min, the supernatant was collected in fresh Falcon tube and treated with gentamycin 500 μg/ml for 30 min at room temperature. The treated suspension was stored in aliquots at -70°C until used. Prior to use, the suspension was passed through a syringe filter of 0.2 μm pore size and the filtrate was used as the inoculum. Confluent monolayers of Vero cells were prepared in 25 sq cm flasks following standard subculture procedure using Dulbecco’s modified Eagle’s medium (MEM) enriched with 5% fetal calf serum. For isolation of the virus 200 μl of each sample was inoculated in duplicate flasks. The cells were examined daily for cytopathic effects (CPE). Each sample was subjected to three blind passages irrespective of the appearance of CPE. After each passage the cell culture supernatant was tested by RT-PCR for the detection of PPR virus as described above.

Source of Information: The Bangladesh Veterinarian (2011) 28(1) : 1 – 7

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Classically, the growth of PPR virus in cell culture is confirmed by virus neutralization test with known antiserum. In the present study, RT-PCR was employed to ascertain the growth of PPR virus in cell culture. This technique could detect the virus in cell culture supernatant before the appearance of visible CPE. Although the positive RT-PCR result after the first passage could be due to the residual inoculum, an increased concentration of the RT-PCR products in the second passage, yet in the absence of visible CPE, would confirm active replication of the virus in cell culture. The concentration of RT-PCR products further increased after the third passage when virus-induced CPE was also obvious. Hence, RT-PCR can be used not only for the detection of PPR virus in clinical samples but also for monitoring the growth of virus in cell culture following inoculation.

Knowledge Management: Journal