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Experimental Pathogenesis of Pullorum Disease in Chicks by Local Isolate of Salmonella Pullorum in Bangladesh

Dr. Emdadul Haque Chowdhury
Professor
Dept. of Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

This study was undertaken to observe the experimental pathogenesis of locally isolated Salmonella enterica subspecies enterica serovar Pullorum in chicks. Fifty chicks were experimentally infected by the oral route with 2 x 10⁷ (CFU) units of Salmonella Pullorum organisms reconstituted in 0.5 ml of sterile phosphate buffer saline (PBS), PH 7.2 and 50 chicks were given only 0.5 ml of sterile PBS as control. Observations were made on clinical signs, gross pathology, and reisolation of S. Pullorum from different organs and blood, histopathological lesions, detection of antibody levels and detection of S. Pullorum by PCR at different time intervals of experimental period. Five birds were randomly selected and sacrificed on 6 hrs before inoculation and 6 hrs, 12 hrs, 24 hrs, 2 days, 3 days, 1^stweek, 2^nd, 3^rd and 4^thweeks of post infection (PI). The clinical signs of infected chicks were depression, loss of appetite, huddled together, loss of feed and water intake, reduced mean body weights, ruffled feathers, diarrhoea, laboured breathing and pasty vent. The highest gross lesion was (84%) unabsorbed and coagulated yolk and the lowest lesion was (32%) pericarditis and necrotic foci/ nodules in heart. Microscopically, the liver showed congestion, focal necrosis with multifocal infiltration of histiocytes in liver parenchyma. Salmonella organisms were reisolated from different organs and blood at 12 hrs PI. The antibody titre increased gradually and the highest titer was 7275.717 ± 5087.24 at 4 wks PI. In rapid plate agglutination test, the positive result was found from one wk of PI with the sera of infected birds. At 12 hrs PI Salmonella was detected by PCR from 20% liver and 20% lung samples of infected birds and no Salmonella was isolated from control group. The orally inoculated Salmonella Pullorum organisms produced lesions in digestive tract, invaded digestive tracts and entered to blood and seeded to different organs in different time intervals and ultimately produced clinical signs, gross and microscopic tissue lesions with immunological response.

Keywords: Pathogenesis, Pullorum disease, ELISA, PCR, Chicks

Location: Dept. of Pathology, Bangladesh Agricultural University, Mymensingh

Start Date: 00-00-2009

End Date: 00-00-2011

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

The present study was undertaken to study the experimental pathogenesis and pathology in chicks (immature birds) with locally isolated Salmonella Pullorum.

Methodology:

Chicks: 100 day-old (Salmonella Pullorum seronegative) chicks of Isa Brown breed were purchased from Nourish Hatchery Ltd. for this experimental inoculation. The birds were divided into 2 groups. One group was used as experimental infection and other was kept as control. Experimental infection: Chicks were reared in experimental shed with optimum brooding temperature and they were given commercial feed and water ad libitum throughout the study period. Fifty chicks were experimentally infected by the oral route with 2 x 10⁷ (CFU) unit of Salmonella enterica sub. enterica serovar Pullorum organisms in 0.5 ml of sterile phosphate buffer saline (PBS), pH 7.2 (Roy et al., 2001). The control birds received only 0.5 ml PBS without bacteria. Clinical signs: Clinical signs of chicks after experimental infection were observed and recorded up to the end of the experiment. Sample collection: Five birds were randomly selected and sacrificed on 6 hrs before inoculation and 6 hrs, 12 hrs, 24 hrs, 2 days, 3 days, 1^stweek, 2^nd, 3^rd and 4^thweeks of post infection. A total of 50 birds were used for the control group, and were necropsized on scheduled dates as in the infected groups. Bacterial sample: The bacteriological samples (crop, liver, lung, heart, cecum, blood, bile and yolk or spleen) were collected at different time interval described in the experimental design with pre-enriched in buffered peptone water in sterile poly bags. ELISA and rapid plate agglutination (RPA) sample: The serum samples were collected during necropsy at different time interval described in the experimental design for ELISA and Rapid Plate Agglutination (RPA) test (Islam et al., 2006; Haider et al., 2007a) Histopathological sample: Liver, lungs, heart, spleen, intestine, etc. were collected in 10% buffered- formalin at different time interval described in the experimental design for histopathological examination. PCR sample: Liver and lungs were collected in 50% buffered- glycerol in sterile poly bags at different time interval described in the experimental design and preserved in - 80⁰C for PCR (Haider et al., 2007b). Gross pathology: The postmortem examination of all the birds performed as per schedule. At necropsy, gross tissue changes were observed and recorded carefully following method described by Wray and Davis, 2001. Reisolation of S. Pullorum from different organs and blood: S. Pullorum was reisolated from different organs (one gm) and blood (one ml) samples by standard methods (Cheesbrough, 2000). Detection of antibody levels by ELISA: An enzyme–linked immunosorbent assay (ELISA) with antigen coated ELISA kits (FLOCKSCREEN^TMGuild Hay Ltd. UK) was used (Solano et al., 2000). The obtained optical density (OD) with sera was measured at 550 nm with an ELISA reader (Toso Ltd. MRP-A4i, Tokyo, Japan). Detection of antibody by rapid plate agglutination (RPA): Rapid plate agglutination (RPA) test was done with Salmonella Pullorum antigen (Yamane et al., 2000). Two drop sera samples of different time interval were mixed with an equal volume of antigen on a plate, and typical agglutination occurred within 2 min. was taken as a positive result. Histopathological study: The representative formalin–fixed tissue samples were trimmed, processed, sectioned and stained performed by standard procedure (Luna, 1968). Detection of S. Pullorum by PCR: The preserved liver and lung tissue samples were used for the detection of S. Pullorum by PCR according to standard procedure (Haider et. al., 2007b). Statistical analysis: Significance of different groups was determined by using MSTAT computer software program. The differences in the increase or decrease of the ELISA antibody titre of chicks of different groups at different week were analyzed by analysis of variance in a Completely Randomized Design (CRD) using an MState computer package program. Significant differences between means were identified by Least Significant Differences (LSD).

Source of Information: J. Bangladesh Agril. Univ. 10(1): 87–94, 2012, ISSN 1810-3030

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In this study, 8% mortality was found in chicks from 3 to 7 days of PI. Clinical signs were observed from 24 hrs PI and in all birds. The gross and histopathological lesions were observed from 72 hrs and 2wk PI, respectively to 3 wks PI and the highest lesions were found from 1wk to 2 wks PI and 2-3 wks PI, respectively. Gross and histopathological lesions were variable in different birds at different time intervals. The highest number of S. Pullorum was 16.14x10⁸ CFU/gm of liver tissues reisolated at 72 hrs PI. Blood was positive for S. Pullorum at 12 hrs PI. ELISA titre gradually increased from 1 wk PI and at the same time infection decreased. S. Pullorum was detected by PCR from 12 hrs PI in liver and lung. At 2 wks PI, the chicks recovered from clinical signs gradually from experimental infection with S. Pullorum. In short, the pathogenesis of experimental pullorum disease in chicks was, after inoculation of Salmonella Pullorum by oral administration, the organism localized in digestive tract, from where entered to blood and localized in different tissues at different time intervals producing lesions and immunological response. However, for useful application of the present research findings further studies should be focused on the serotyping and molecular characterization and identification of immunogenic variation and development of vaccine with isolated Salmonella.

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