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Natural Peste Des Petits Ruminants Virus Infection in Black Bengal Goats: Virological, Pathological and Immuno Histochemical Investigation

Emdadul Haque Chowdhury
Professor
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Ataur Rahman Bhuiyan
Department of Livestock Services, Dhaka, Bangladesh.

Mohammad Mushfiqur Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Mohammad Sahinur Alam Siddique
Department of Livestock Services, Dhaka, Bangladesh.

Mohammad Rafiqul Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Abstract/ Executive Summary:

Peste des Petits Ruminants (PPR), also known as Goat Plague, occurs in goats, sheep and related species. It is caused by a morbillivirus in the family Paramyxoviridae. In Bangladesh PPR is endemic and it causes serious economic losses. Pathology of PPR has been reported in different goat and sheep breeds from natural and experimental infections. Field results are better indicators of pathogenicity of the circulating virus. The severity of the disease varies with species, breed and immune status of the host. Pathological investigations of natural outbreaks of PPR in Black Bengal goats are very limited. The current investigation was aimed at describing pathology and antigen localization in natural PPR infections in Black Bengal goats. A total of 28 outbreaks were investigated clinically and virologically. Average flock morbidity and mortality were 75% and 59%, respectively, with case fatality rate of 74%. Necropsy was conducted on 21 goats from 15 outbreaks. The major gross lesions were congestion of gastrointestinal tract, pneumonia, engorged spleen, and oedematous lymphnodes. Histopathological examination revealed severe enteritis with denudation of intestinal epithelium, severe broncho-interstitial pneumonia with macrophages within lung alveoli and extensive haemorrhages with depletion of lymphoid cells and infiltration of macrophages in the sinuses of spleen. In lymph nodes, the cortical nodules were replaced by wide sinusoids with severe depletion of lymphocytes, infiltration of mononuclear cells and some giant cells in sub-capsular areas and medullary sinuses. PPR virus antigen was found in pneumocytes and alveolar macrophages in lungs. Viral RNA could be detected by RT-PCR in 69 out of 84 nasal swab, 59 out of 84 blood and 21 out of 21 lymph node samples. Sequence analyses revealed closeness of Bangladeshi strains with other recent Asian isolates. Natural outbreaks of PPR in Black Bengal goats in Bangladesh resulted in 75% and 59% flock morbidity and mortality, respectively, with a case fatality rate of 74%. The striking histo-morphologic diagnosis of PPR was acute pneumonia and severe gastro-enteritis.

Keywords: PPR, Natural outbreak, Pathology, Antigen detection, Nucleic acid detection

Location:

Start Date: 00-12-2008

End Date: 00-12-2010

Program Area: Pest Management

Commodity: Goat

Objectives:

To investigate on the pathology and antigen localization in natural PPR infections in Black Bengal goats.

Methodology:

A total of 28 PPR outbreaks in goat from different parts of the country during December, 2008 – December, 2010 were included in this study. The size of the affected flocks varied from 6 to 805 with a total of 1264 goats. All the farms were in contact with veterinarians, who observed daily for morbidity, mortality and clinical signs and recorded body temperature of the affected goats. The veterinarians were in contact with the investigators and filled in the prescribed clinical examination form. From 15 outbreaks 21 dead goats were subjected to necropsy at the necropsy room of veterinary clinics or at the Department of Pathology, Bangladesh Agricultural University, Mymensingh. From the remaining 13 outbreaks, only nasal and blood smeared filter papers were received from the veterinarians with completed clinical examination form. The blood samples were collected from jugular vein by aseptic means. A few drops of blood were poured on the filter paper, from the base towards the tip, until it was completely soaked. Nasal swabs were collected with sterilized swab sticks, which were smeared on the filter paper. Smeared filter papers were air-dried avoiding direct sunlight and preserved in labelled sterilized Eppendorf tube. The papers and tissues were stored at −70°C at the laboratory until analysis. Necropsy Necropsies were performed on 21 goats of 15 outbreaks that were found dead at farms. None of them received any supportive treatment prior to death. These goats died 24 hours to 7 days after onset of clinical signs. Tissue samples from various organs were collected at necropsy and fixed in 10% neutral buffered formalin. Tissues from bronchial lymph node of dead animals were also collected aseptically for detection of viral RNA by RT-PCR. Histopathology Histopathology was performed on one goat from each of the 15 outbreaks, where necropsy was conducted. Formalin-fixed samples were processed and stained as described. Briefly, the fixed tissues were trimmed and further fixed for 24 hrs. Tissues were kept in running tap water overnight to wash out formalin. The tissues were dehydrated in ascending grades of alcohol (50%, 70%, 80%, 95%) and three changes of absolute alcohol for one hour in each. Sections were cleared in chloroform by two changes, 1½ hours for each. Immunohistochemistry The tissues were sectioned with a microtome at 5 μm thickness. Sections were allowed to spread on warm water bath (37°C) and taken on poly-L-lysine-coated slides (Thermoshandon). Afterwards the procedure was as follows: (i) The sections were deparaffinized in fresh xylene (four changes, 5 minutes in each) and rehydrated through descending grades of alcohol (three changes in absolute alcohol, three minutes in each; 95% alcohol for 3 min; 80% alcohol for 3 min; 70% alcohol for 3 min and 50% for 3 min) followed by washing in running tap water for 20 minutes and in distilled water for 5 min. (ii) Sections were placed in a Coplin Jar containing 0.1% trypsin (Sigma-Aldrich) in Tris buffer, pH 7.8 (10 mM) and incubated for 10 minutes at 37°C in water bath, followed by washing in chilled PBS (0.01 M) in another Coplin jar (2 changes, 5 min in each). (iii) The trypsinized sections were fixed in air-free disposable cover plates and stacked in a immunostaining rack (Thermo Shandon). (iv) Endogenous tissue peroxidase was inactivated by applying 0.3% H₂O₂ to the sections at room temperature for 10 minutes followed by washing in PBS. (v) The sections were incubated with 100 μl blocking solution (non-immune goat serum) for 10 min, and washed again in PBS for 10 minute. (vi) The sections were incubated with primary antibody 1: 50 dilution of mAb (clone 38–4) against N protein overnight at 4°C in moist chamber (mAb was a kind gift from PPR Reference Laboratory, CIRAD France). (vii) After incubation with primary antibody, the sections were washed in PBS (three washes, each for 3 minutes). (viii) Remaining reaction was developed according to instruction of Histostain® plus Kits (LAB-SA Detection System, Invitrogen). (ix) The reaction was visualized by adding AEC (3-Amino-ethylcarbazole), 3 drops per section with incubation for 10 minutes.

Source of Information: BMC Veterinary Research 2014, 10:263,http://www.biomedcentral.com/1746-6148/10/263

Article Link (Online Journal): http://download.springer.com/static/pdf/532/art%253A10.1186%252Fs12917-014-0263-y.pdf?originUrl=http%3A%2F%2Fbmcvetres.biomedcentral.com%2Farticle%2F10.1186%2Fs12917-014-0263-y&token2=exp=1458375021~acl=%2Fstatic%2Fpdf%2F532%2Fart%25253A10.1186%25252Fs129

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The striking histo-morphological diagnosis was acute pneumonia and severe gastro-enteritis. Since Bangladesh is a PPR endemic country, stamping out is not practiced. Therefore, early supportive treatment based on the pathological findings, could reduce the mortality of PPR virus infected goats which will be economic for the poor farmers. A detailed experimental pathological study of Black Bengal goats infected with different isolates is required.

Knowledge Management: Journal