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Pathology of Fowl Paratyphoid and Molecular Detection of Its Pathogen in Layer Chickens

J. Alam
Department of Animal Science, Faculty of Agriculture, Exim Bank Agricultural University Bangladesh, Chapainawabganj-6300, Bangladesh.

T. Chakma
Department of Pathobiology, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

M. S. Islam
Department of Microbiology and Public Health, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

M. T. Islam
Department of Pathobiology, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

M. A. H. N. A. Khan
Department of Pathology, Faculty of Veterinary Medicine, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

M. T. Islam
Institute of Biotechnology and Genetic Engineering Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

M. G. Haider
Department of Pathobiology, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

Abstract/ Executive Summary:

The study was aimed to ascertain the pathology of fowl paratyphoid and molecular detection of its causal agent (Salmonella spp) in chickens. Pathological and swab samples were collected from layers in Gazipur district, Bangladesh. For observing the gross and microscopic lesions of different organs necropsy and histopathology were done, and to isolate and identify the Salmonella spp, different bacteriological tests and Polymerase Chain Reaction (PCR) was performed. Swabs from 150 chickens showed 66% of salmonellosis. Gram’s staining of isolated bacteria showed pink-colored rod-shaped bacilli. In biochemical tests, Salmonella fermented dextrose, maltose, xylose, arabinose, dulcitol, mannitol except for lactose and sucrose. Investigation of gross lesions at necropsy revealed hemorrhage and congestion in the intestine, liver, spleen, and ovaries. Necrotic foci were found in the liver and spleen, and button-like ulceration in cecal tonsils as well. Microscopic lesions included hemorrhage and focal necrosis in the liver and spleen. Congestion and infiltrations of inflammatory cells were observed in the small intestine. The ovary was hemorrhagic and there was the infiltration of heterophils. Biochemically positive and isolated Salmonella organisms were confirmed by PCR method using invA and IE1 primers. The final results showed that a total of 91.7% Salmonella suspected cultures were confirmed as Salmonella Enteritidis.

Keywords: Samonella, Histopathology, Gram stain, Hemorrhage, Inflammatory.

Location: Gazipur district, Bangladesh

Start Date: 00-09-2016

End Date: 00-06-2017

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To observe the prevalence of PT Salmonella in chickens at Gazipur and to study the gross and histopathological changes in tissues in chickens as well.

Methodology:

A total of 150 (one hundred fifty) poultry samples including 30 sick birds on the basis of clinical signs of PT, 70 apparently healthy birds, and 50 dead birds from 10 commercial layer farms were randomly collected from different areas of Gazipur district, Bangladesh during the period of September’16 to June’17. At the Post mortem examination, a systemic dissection was made along with that the gross changes in different tissues were recorded. After necropsy representative samples like lungs, liver, spleen, heart, kidney, caecal tonsils, intestine and ovary were collected in 10% buffer formalin for histopathology. Liver, spleen, lungs, and intestinal swab samples were collected from 150 chickens during necropsy. The total swab samples were 600. Aseptic cotton swabs were used and all the swabs were collected in test tubes containing 10 ml tetrathionate broth (TTB) according to methods as described previously. after collection, at first, all the bacteriological samples were incubated for 24 hours in TTB. The samples were primarily cultured in Nutrient agar and then sub-cultured in the Salmonella-Shigella (SS) agar, XLD agar, Triple sugar iron (TSI) agar, Brilliant green agar (BGA) and Eosine methylene blue (EMB) agar to get desired single colony. The presumptive colonies of suspected bacteria in various media were characterized microscopically using Gram’s stain. For the separation of motile and non-motile bacteria, a motility test was performed using the hanging drop slide method. Nine important sugars such as glucose, sucrose, lactose, mannitol, dulcitol, arabinose, inositol, xylose, and maltose were used for the sugar fermentation test. The biochemical identification of PT Salmonella was performed using inositol and TSI agar slant. Tissue samples collected from the intestine, liver, lungs, spleen, heart, and ovary were fixed in 10% neutral buffered formalin and further processed for histopathological staining with hematoxylin and eosin stains. Photomicrography of stained tissues was taken using a photomicrographic camera. From the isolated pure culture, the genomic DNA of PT Salmonella was extracted using DNA extracting kits. Extracted DNA was amplified using primers invA and IE1 targeting for the gene of Salmonella Spp. using commercial PCR kits in gene amplification PCR system 9600 Thermocycler. The thermal profile of PCR for InvA gene was 94°C for 5 minutes (initial denaturation), 94°C, for 20 seconds (denaturation), 50°C for 30 seconds (annealing), 72°C for 30 seconds (elongation), and 72°C for 7 minutes (final extension), and the holding temperature was 4°C. Separation of amplified products was done by electrophoreses on 1.5% agarose gel containing 5µg per ml ethidium bromide with a 100 bp ladder as molecular weight marker.

Source of Information: Ann. Bangladesh Agric. (2020) 24 (2) : 47-57

Article Link (Online Journal): DOI: https://doi.org/10.3329/aba.v24i2.55783

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Fowl paratyphoid is generally a subclinical infection of all domestic poultry and game birds. Salmonella Enteritidis is one of the many causal agents of this disease which has zoonotic importance. The histopathology of fowl paratyphoid, isolation, and molecular detection of its causal agent as Salmonella spp. were conducted in this study. The prevalence of salmonellosis was found at 66%. The gross and histopathological lesions were shown the severity of paratyphoid infections in chickens. The severity may be enhanced by the combination of other diseases like colibacillosis, mycoplasmosis, and mycotoxicosis in densely poultry populated areas. Besides, this PT Salmonella can cause huge public health hazards in the experimental areas.

Knowledge Management: Journal