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Development of Slide Micro-agglutination System for the Rapid Diagnosis of Salmonella Infection in the Chicken

R. Parvin
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

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

M. Asaduzzaman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

M. G. Haider
Livestock Research Institute (LRI), Mohakhali, Dhaka, Bangladesh.

N. Jannat
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

The cultural method to identify avian Salmonella infections is laborious and expensive, thus a rapid, sensitive and cost-effective method for the diagnosis of salmonellosis is anticipated. S. pullorum was isolated and a stained Salmonella somatic antigen was prepared. The protein concentration of stained antigen was measured and adjusted to 3000 ng/µl. Preservative was used to increase its shelf life. Slide agglutination tests were carried out with un-diluted and diluted anti-sera having known ELISA titre and end point agglutination titre was determined. The standard curves were generated using 2 and 10 fold dilutions of sera to determine the titre of the unknown field sera. It was observed that the newly developed stained somatic antigen produced distinct tiny clumps with positive anti-sera of Salmonella spp. The present slide agglutination system was found to be easy, sensitive, reliable, cost and time effective and needs very small amount of antigen, sera and as well as accessories. This method may be used to screen the Salmonella infections in the poultry farms and to calculate the titre of the anti-salmonella antibody in the infected and vaccinated chickens under farm conditions. A test kit containing stained antigen along with positive and negative control sera may be prepared for commercial uses. Keywords: Avian salmonellosis, rapid diagnosis, colour antigen, slide agglutination test.

Keywords: Chicken, Salmonella Infection, Micro-agglutination

Location: Department of Pathology, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To prepare stained colored Salmonella antigen with a local isolate and development of slide micro agglutination system for the rapid diagnosis of Salmonella infection in chickens in the field condition.

Methodology:

The locally isolated S. pullorum was used for production of the Salmonella colored antigen. Test tubes containing samples on nutrient broth were incubated for 24 hrs at 37°C. From the nutrient broth, subcultures were also made on Brilliant Green agar, Salmonella Shigella agar, MacConkey agar, EMB agar, TSI agar, LB agar and nutrient agar, and incubated at 37°C for over night. The identification of the organisms was performed by the tests as described by different literature [15, 16, 17, and 18]. On the basis of colony and staining characters the organisms were isolated and identified. The organism was further confirmed by PCR as described in [19]. A conical flask was taken, containing 50 ml of LB broth. Single colony of Salmonella pullorum was inoculated into the culture. Then the flasks were placed in incubation for 48 hrs. 50 ml broth culture was divided into two conical flasks containing each 25ml. Then tetrazolium salt was added aseptically in the amount of 0.5% in each broth culture and incubated one flask for 2 hrs and another flask for 24 hrs. After incubation, 0.5% phenol was added and incubated for 1 hr and 2 hrs, respectively. The stained broth suspension was filtered through sterile gauge and poured in to eppendorf tube. Afterwards, these were centrifuged at 16000 rpm for 15 minutes, supernatant was decanted and cells were suspended in 0.5% phenolized saline, 0.5% fomalinized saline and 0.09% sodium azide. The suspension was mixed by vigorous vortex with a few sterile small pieces of glass and transferred it another eppendorf tube. Then the solution was stored at 4°C as neotetrazolium stained antigen for future use. The total protein concentration of the stained antigen was measured by the Folin Phenol method of Lowry [20]. For this, 20 μl of stained antigen and 20 μl chicken sera were placed on a sterile glass slide by a micropipette and mixed thoroughly by stirring with tips. The results were read within 1 minute. In positive case, granules (agglutinates) were formed rapidly due to combination of homologous antigen and antibody which was seen during rocking. In the absence of antibody, no such granules (agglutinates) were formed. Positive and negative sera were run parallel in each time. The known positive sera were obtained from the Department of Pathology, Bangladesh Agricultural University, Mymensingh that was raised. The sera were then diluted and the results of diluted sera were observed. The known ELISA titers of positive sera ranging from 676 to 1, 2000 were selected for the 10 fold or 2 fold dilutions. The agglutination titer of each diluted sera was compared with the known ELISA titer and plotted in standard curves. Co-relation curve was prepared using 10 fold and 2 fold diluted sera to determine the antibody titer. A total of 180 sera from 7 flocks and 4 birds (3 dead and 1 live) from one flock were donated by a commercial hatchery of Bangladesh. Necropsy and histopathology was done on the selected bird sample and the findings were recorded. The slide micro-agglutination test was performed with the newly developed salmonella colored antigen. ELISA titers of field sera were determined by the help of standard curve. For easy dispensing of salmonella colored antigen to the field users a package was prepared. The prepared package contains 1ml stained antigen in a glass vial, positive serum, negative serum and information sheet. Materials not supplied but are necessary are glass slide for slide agglutination test, dropper, and stirrer.

Source of Information: IRJALS,Research Paper, ISSN: 1839-8499, May – 2012 ,Volume – 1, Issue – 2 , Article #01

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The tetrazolium stained Salmonella antigen from a local isolate was successfully developed which will help to detect salmonella infection in the chicken flocks in field condition within a very short time. A kit was organized named “BAU-Path S Antigen Kit” contain 1ml of developed stained antigen that was sufficient for 50 test, positive serum, negative serum and information sheet. However; cautions have to be taken with the reading of the results. With increase time, the stained antigen may react with the non-specific antibody present in the serum. Therefore, results within one minute were suggested as confirmed case of salmonellosis. The antigen should be shaking before use. Always keep away from the light.

Knowledge Management: Journal