Survey of Salmonella serovars in broilers and laying breeding reproducers in Eastern Algeria

Background: Avian salmonellosis affects the poultry industry in underdeveloped and in developed countries. The aim of this study was to identify the most common Salmonella serovars in broilers and laying breeding reproducers in Eastern Algeria according to the ISO 6579 method. Methodology: A total of 294 samples were obtained from two flocks of 10,000 broilers and laying breeding reproducers. Samples included livers and spleens, drag swabs of bottom boxes of young chickens, cloacal swabs, and faecal samples of chickens. Additional samples were also taken from water, feed and dusty surfaces. Results and conclusions: Only the cloacal swabs, poultry faeces and samples from dusty surfaces were positive for Salmonella Typhimurium and Salmonella Livingstone with a detection rate of 12% and 1.6% respectively. The results showed evidence of legislative failure regarding biosafety within the poultry industry in the area of Batna, Eastern Algeria.


Introduction
Salmonellosis is the main collective food-borne disease of human beings, caused by many serotypes of Salmonella enterica.Poultry products and derivatives have been implicated in Salmonella zoonoses in both developed and underdeveloped countries.Pullorum disease with Salmonella Pullorum represented a serious poultry health problem until huge resources were used to limit its expansion, but these efforts allowed the emergence of other Salmonella [1].The development of food-borne disease in humans caused by S. Typhimurium, and S. Enteritidis following the consumption of contaminated eggs and egg products is mainly due to lapses in hygiene practices and the propagation of these bacteria in the poultry chain, especially in layers.This work was undertaken to investigate Salmonella serovars in the parental laying poults and broiler chicken poultry industry, to identify the prevalent Salmonella species, and to determine the bacteria's sensitivity to antimicrobial agents.

Materials
Two poultry houses each containing 10,000 parental broiler chickens and laying poults (Hubbard F15 and Highline breeders) were studied.They received locally manufactured feed, and the feed and drinking water were automatically distributed.The animals were apparently healthy and no symptoms were observed.

Methods
Sampling was conducted according to the SNA Hatcheries guide for detection of Salmonella in breeding reproducers [2].Sixteen livers and spleens were aseptically removed from day-old chickens and 120 cloacal swabs and 120 faecal samples were collected from the laying poults and 18 week-old broilers.Also collected were 4 x 200 ml of water aseptically from the watering system [3], 4 x 500 g of food obtained randomly from a bag on a truck during unloading [4], 24 swipes of building surfaces obtained using sterile tissue "chiffonnettes" at the level of rearing surfaces [5] and 4 bottom boxes cleaned with sterile tissue pre-moistened [6].All samples were immediately inoculated in 225 Buffered Peptone Water (BPW) bottles.Care was taken to prevent contamination by using aseptical methods.The samples were labelled and transported to the Laboratory of Microbiology where the analysis was performed during the four hours post sampling.

Microbiological analysis
Solid samples were transferred aseptically to sterile Stomacher bags and homogenized in 100 ml of (BPW) using a Seward 400 circulator Stomacher unit Brinkmann USA prior to static incubation at 37°C for 18-24 hours.The samples were next inoculated in Rappaport-Vassiliadis medium (RV) and Selenite Cysteine Broth (SCB) (Flucka) at the ratio of 1/10 and incubated at 43°C and 37°C respectively for 24 hours.Brillant Green Agar and Hektoen agar (Biomerieux) were streaked with culture broth.Suspected Salmonella colonies on agar plates were inoculated on triple sugar iron agar (TSI) and incubated for 24 hours at 37°C.Suspect Salmonella cultures from TSI were tested biochemically using API20E strips (Biomerieux-France).Identification of Salmonella-positive cultures was confirmed by the slide agglutination test using poly O and poly H diagnostic antisera (Institut Pasteur) [7].

Results
No Salmonella pathogens were found in water but contamination from coliforms exceeded acceptable values, in all cases being above 25 X 10 3 CFU of Total Coliforms (Table 1); the food was also free from pathogens, especially Salmonella.Salmonella was recovered from 2.39 % of broiler breeders.The seven Salmonella from the faecal samples, the nest box, roosts, and litter were serotyped as S.Typhimurium.The only Salmonella isolate from cloacal swabs was S. Livingstone (Table 2).Susceptibility tests showed that all the S. Typhimurium isolates were resistant to ampicillin, ticarcillin, chloramphenicol and trimethoprim/sulfametoxazole but sensitive to other antibiotics

Discussion
The absence of Salmonella from food suggests that the food processing is well handled [9].
The high coliform count indicates that bacterial loads at other sources were not in compliance with Algerian legislation.The water sampled did not contain pathogenic micro-organisms (Salmonella and staphylococci); however, Algerian legislation on the drinking water also requires that the number of microorganisms in the drinking water of birds should be less than 100 CFU/ml for total bacteria and 50 CFU/ml for coliforms.The results (Table 1) showed that the coliform counts in the farms were above the acceptable level.One of the main consequences of fecal contamination of drinking water is the increased risk of enteric infection by pathogens such as Salmonella [3].There is a clear link between the high degree of contamination by Escherichia coli and the presence of Salmonella, but in this case Salmonella were not isolated at significant levels from broilers and laying reproducers as has been reported previously by several authors [10].
The cloacal swabs collected from the first building of six-week-old broiler reproducers did not carry Salmonella, and the laying reproducers produced only one isolate of S. Livingstone.Low recovery of Salmonella in these cases may be related to the early age of the birds.
Four Salmonella isolates were recovered from the poultry faeces sampled in the first building of broilers but no Salmonella were recovered from level surfaces.As a consequence, we followed the SNA hatcheries guidelines and doubled the number of samples to seek Salmonella in the second building [2].After this second isolation, we detected more isolates of S. Typhimurium on the litter faeces.This finding is similar to that of of Aeron et al. [11].
The litter faecal material of the building, sampled using drag swabs, shows a high level of contamination by Salmonella [5,10].This can lead to contamination of the poultry carcasses from the feathers and feet of birds and therefore litter is an excellent indicator of poultry contamination by Salmonella [5].
The antibiotic sensitivity patterns we found were similar to results obtained by Leon-Velarde et al. [15], essentially consisting of resistance to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline, the profile of S. Typhimurium DT104.However, due to the absence of facilities in our laboratory for phage typing or PCR, we are unable to investigate these results further.

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Our legislation defines general measures for prevention of contamination in poultry breeding and recommends systematic livestock incineration following positive bacteriology with a rate greater than 2% (for Salmonella other than S. Enteritidis and S. Typhimurium, which are considered as major pathogens) followed by serologic confirmation.The buildings we investigated presented a higher prevalence of S. Typhimurium (12%) and were not in compliance with the law, illustrating the need for more strategies to prevent poultry infection.Some strategies that have been proposed in the literature include competitive exclusion of the bacteria by a probiotic Lactobacillus acidophilus [13] and vaccination programs [14].

Conclusion
This study revealed that the prevalence rate of Salmonella in broilers and laying breeding reproducers in Batna were 12% and 1.6% for S. Typhimurium and S. Livingstone respectively.The poultry faeces and litter were the only samples contaminated with these pathogens.The existence of S. Typhimurium in poultry and in the environment of broiler reproducers is a potential danger to public health.Measures must be observed to reduce the risk  of contamination of poultry and to prevent infection by consumption of avian products.

Table 1 .
Microbiological analysis of the water.

Table 2 .
Distribution of Salmonella in broiler and layer breeding reproducers and living surfaces.

Table 3 .
Prevalence of Salmonella serovars in broiler and layer breeding reproducers.