COMPARATIVE DIAGNOSTIC STUDY OF AVIAN SALMONELLOSIS IN SALAHALDEEN PROVINCE

288 COMPARATIVE DIAGNOSTIC STUDY OF AVIAN SALMONELLOSIS IN SALAHALDEEN PROVINCE B. S. Noomi Assist Prof. Coll. of Vet. Med. Univ. of Tikrit vetbashar1981@gmail.com ABSTRACT The aims of this study were to detection of dominant Salmonella species that caused poultry infection Salahaddin province , and evaluation local prepared and manufactured serological kits that used in diagnosis of poultry salmonellosis, for this purpose 100 diarrheatic hen checked by culture methods, PCR, ELISA, Whole blood agglutination test and Slide agglutination test. The results showed that Salmonella isolation from hen in rate 34% , intestine is most suitable site for Salmonella isolation and Salmonella. typhimurium is most dominant spp. The sensitivity of ELISA test was 76.4%, while for other used tests were 100%. The specificity of ELISA test, Whole blood agglutination test, slide agglutination test for S. typhimurium and slide agglutination test for S enteritidis were: 80.3%, 86.3%,77.9% and 66.6% respectively.


‫اقية‬ ‫العر‬ ‫اعية‬ ‫الزر‬ ‫العموم‬ ‫مجمة‬
. Salmonellosis is an infectious disease of humans and animals, it occur due to infection by Salmonella and main species of them: Salmonella enterica, and Salmonella.Bongori (18).Avian can infected by Salmonella vertically and horizontally via contamination food, water and hatching eggs, as well as infection may be occur via birds, rodent, insects, and even infected farm workers (26).Many factors influence with occurrence of avian salmonellosis, include host age, genetic, stress factors from environmental, treatment with antimicrobial and anti-inflammatory drugs and other infections (8).Main clinical signs of Salmonella in poultry were depression, somnolence, weakness, loss of appetite, drooping wings, breathing or gasping, diarrhea and dehydration.In some cases lameness, swelling of joint and blindness may be occur (5).Salmonellosis diagnosed by: -Direct staining of samples by gram stain, this methods is not specific because of Salmonella morphology share with other Gram negative bacteria (12).
-Culture methods: it is highly specialized methods but because of interrupted bacterial shedding, it gave false negative result (24).
-Whole blood agglutination test: this test can performed directly in field (18).
-ELISA: This test used for detection of IgG against Salmonella, it is more sensitive than other serological tests (17).

MATERIALS AND METHODS
This study performed in salahaldeen governorate in period from January to march-2018, in multi flocks.
-Sample: 100 diarrheatic hens from flocks contain 50000 hens, from each hen intestine, liver, spleen, and gallbladder were taken for bacterial isolation and blood for serology tests -Culture methods : all samples cultivation in peptone water (HIMEDIA-INDIA) (pre enrichment medium) incubated in 37 o C for 24h, after that sub culturing on Selenite F broths (HIMEDIA-INDIA) as liquid selective enrichment medium and incubation on 43 o C for 24h.then sub culturing on selective sold media (Xylose lysine deoxycholate agar, Brilliant green agar and MacConkey agar) and incubation on 37 o C for 24h (18).A group of biochemical tests applied according to (20).These tests were used for recognition of isolate in genus and species .Antibiotic sensitivity test: applied according to (2).
-Serotyping of Salmonella isolate: performed by using Salmonella antisera (pro-lab Diagnostics-USA) which consist from two groups: Salmonella Polyvalent Somatic O Antisera and Salmonella Polyvalent flagellar H Antisera.These tests were used for confirmation species of Salmonella.
-PCR test: DNA template Prepared by reactivation of bacteria by culturing in brainhart infusion broth, then DNA extraction by boiling lysis method and according to (23).Compound of reaction: as in Table 1.Thermo cycler programs as ias in Table 2 -Slide agglutination test (for detection of S. typhimurium) preformed in the current study by preparation of somatic and flagellar antigen for S.typhimurium.Somatic antigen prepared by using heating 100C and according to (13).While flagellar antigen prepared by using formalin (BDH -England) and according to (4).
-Slide agglutination test (for detection of S. enteritidis) preformed in the current study by preparation of whole bacteria antigen by sonication of S. enteritidis bacteria for 50 minutes at intervals in a water-cooled sonicator(40 MHZ/second) and according to (15).

RESULTS AND DISCUSSION
According to colony morphology, biochemical tests and result of PCR, Salmonella was isolated from hen in rate 34% (34:100) figure (1) (the case considered as positive case if Salmonella isolated from intestine or liver or spleen or gallbladder).In the current study Salmonella isolation was in high ratio in compare with other studies(1;15; 19; 21).That's may be due to types of samples, in our study the samples taken from clinically infected hens while other studies applied as survey.In the current study Salmonella isolation rate from intestine, gallbladder, spleen and liver were 91.1%, 55.8%, 32.% and 11.7% respectively.Table 3 In the current study positive isolation case gave negative results in ELISA test (effect in sensitivity).That's due to early stage of infection or low efficiency of immune system (14).As well as ELISA kit used in current study detected IgG which appeared after 10 days from infection (20).When compare between whole blood agglutination test (for detection of S. gallinarum and S. Pullorum) and bacterial culture showed that the sensitivity, specificity, positive predictive values and negative predictive values of whole blood agglutination test were 100%, 86.3%, 75% and 76% respectively.As describe in Table7.In compare between slide agglutination test (for detection S. typhimurium) and bacterial culture showed that the sensitivity, specificity, positive predictive values and negative predictive values of slide agglutination test were 100%, 77.9%, 73.6%,100% respectively.As describe in Table 8.Appearance of positive result in serology tests that showed negative results in culture is due to interval bacterial shedding, or low number of bacteria, or treated with antibiotic(2).There are many factors lead to false positive results in serology test (low specificity) which due to cross reaction with other similar bacteria, vaccination, carrier birds and endemic area (2).
In current study showed difference in sensitivity and specificity of serological tests.That's may be due to types of antigen used in serological test (whole cell or parts of bacteria).The low specificity of slide agglutination tests is due to cross reaction between salmonella with other bacteria particularly Enterobacteriaceae (2).

Figure 1 :
Figure 1: Electrophoresis on 2 % a garose gel and ethidium bromide staining, showing the results of PCR procedures.M: DNA marker, CP control positive, CN: control negative, wells 1-8 positive samples of Salmonella which showed band in size 284 bp.

INTRODUCTION
Salmonella is gram negative bacteria, non capsulated, non sporulated , motile by Peritrichous flagella except S. gallinarum and S. pullorum (19).The genus Salmonella belonged to Enterobacteriaceae, which have more than 2500 serotypes based on 16SrRNA sequence analysis

Table 1 . Compounds used in preparation of Reaction Mixture Compounds used in preparation of Reaction Mixture Volume (microliters) Reference Taq PCR Master Mix KIT: Which contain Taq DNA Polymerase (2.5 Unit), PCR Buffer with 3mM MgCL2, 200μMdNTP 25 (Qiagen, Germany).
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Table 3 . Isolation of salmonella according to organ
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