Abstract: Background and Objectives: The objective of the study was to determine the occurrence of Salmonella and their antimicrobial susceptibility pattern in poultry feeds, faeces, drinking water and litters sampled from Teaching and Research Poultry Farm and Wilson Poultry Farm located in Maiduguri the capital city of Borno State, Nigeria. Materials and Methods: The study design was a cross sectional study. A total of 120 samples were collected with 60 samples each from the two locations. Fifteen samples each from poultry faeces; drinking water, feeds and litters were collected using convenience sampling technique. About 10 g each of the samples from faeces, feeds and litters were weighed. Similarly, 10 mL of water sample were equally measured. Faeces, feeds and litters were collected in plastic bags and drinking water in aseptic sample bottles and then labeled properly. Isolation and identification of Salmonella were performed according to standard bacteriological protocol. Susceptibility testing of Salmonella isolates to 10 different antimicrobial agents was carried out using disc diffusion method on Muller-Hinton agar. Data generated were analyzed using descriptive statistics with Microsoft Excel version 2010. Results: The overall prevalence of Salmonella in poultry samples from Teaching and Research Poultry Farm was found to be 31.67%. The highest prevalence of 53.33% was obtained from faeces followed by litters (33.33%) and 20.00% was recorded for samples obtained from feeds and drinking water, respectively. The overall prevalence of Salmonella in poultry samples from Wilson Poultry Farm was found to be 46.67%. Higher prevalence of 80.00% was obtained for samples from litters, followed by faeces (66.67%), while 26.67 and 13.3% were associated with drinking water and feeds, respectively. The resistance and sensitivity pattern were displayed by 24 Salmonella isolates to 10 anti-microbial agents. Septrin (SXT), Amplicin (PN) and Ceporex (CEP) had the highest resistance of 5 (10.64%), 4 (8.51%) and 4 (8.51%) to Salmonella isolates, while Ciproflox (CPX) showed no resistance (0.00%). The Salmonella isolates recorded higher sensitivity to Ciproflox (CPX), 12 (25.53%), Tarivid (OFX), 10 (21.28%), Augmentin (AU) and Reflacine (PEF), 9 (19.15%). Conclusion: From the results of this study, it has been demonstrated that the Salmonella present in poultry feeds, faeces, drinking water and litters may pose a risk to humans who are poultry consumers.
INTRODUCTION
The members of the genus Salmonella are Gram-negative, motile, facultative anaerobic bacilli belonging to the family Enterobacteriacae1. Salmonella comprised of two central species; Salmonella enterica and Salmonella bongori. Salmonella is a leading cause of food-borne mortality and morbidity in United States and other parts of the world2.
The occurrence of Salmonella species has become one of the major health challenges not only in the study area but in several parts of Nigeria3. Salmonellosis ranges in severity from self-limiting gastroenteritis to septicaemia4. The severity of the salmonellosis depends heavily on host susceptibility and the virulence of the serovars1. It is characterized clinically by one or more of the three major syndromes, septicaemic, acute and chronic enteritis which infect both humans and animals with millions of illness reported worldwide5,6. Salmonellosis is one of the leading causes of bacterial gastroenteritis in humans and is responsible for over 1.4 million illnesses annually in the USA7. Although, Salmonella species also cause clinical diseases in a variety of animal species, many domestic and wild animals become colonized and shed these bacteria in their faeces with apparent sign of illness. If ingested either through direct exposure to faeces or through faecal contamination of food or water, dominant Salmonella serovars can subsequently cause disease in humans and other animals8.
The infected birds may serve as transport vehicle for transmission of Salmonella species to humans through the consumption of poultry meat and eggs. Similarly, multi-drug resistance of certain pathogenic strains of Salmonella to antimicrobial agents may serve as a public health risk to humans. Antibiotic resistance among S. species is a well confirmed phenomenon. Animals are the primary source of zoonotic Salmonella9. The effective understanding of poultry Salmonella and information on their identifiable characteristics will enable significant improvement in their control strategies. Therefore the study was conducted to determine the occurrence of Salmonella in poultry associated with their accessories and to demonstrate the antimicrobial susceptibilities of the Salmonella isolates to ten different antimicrobial agents.
MATERIALS AND METHODS
Study area: The study area is Maiduguri, Borno state which is located in the north-eastern, arid zone of Nigeria. It has an area of about 69,436 km2 and lies within latitude 11°50' 46" N and longitude10 13°08' 29" E. It is found within the Savannah with low records of rainfall. The area is in the tropical continental north with dry period of 4-8 months, October-May, followed by a short rainy season from late June to early October. The Borno state has boundaries with Chad to the northeast, Cameroon to the east and Adamawa state to the southwest. Agriculture and livestock farming is the mainstay of the State economy.
Study design: The study design was a cross sectional study.
Duration of the study: The duration of the study was for a period of 9 months beginning from October-June, 2018.
Sampling and sample collection: A total of 120 samples were collected from Teaching and Research Poultry Farm, University of Maiduguri and Wilson Poultry Farm Fori Ward, Maiduguri. Sixty samples were collected from each of the two locations. Fifteen samples each from poultry faeces, drinking water, feed and litter were collected using convenience and not convenient technique. About 10 g each, of the sample from faeces, feeds and litters were weighed. Similarly, 10 mL of water sample were equally measured. Faeces, feeds and litters were collected in plastic bags, while drinking water was collected in aseptic sample bottles and were labeled accordingly, kept in a cool box containing ice blocks and then transported to the Veterinary Microbiology Laboratory, Department of Veterinary Microbiology, University of Maiduguri, where they were processed immediately.
Isolation and identification of Salmonella: About 90 mL of normal physiological saline was added to the 10 g of each sample and was mixed thoroughly about sieved. One milliliter of each sieved (or filtered) sample solution was inoculated into Selenite F broth and incubated at 37°C for 18-24 h. A loopful of the broth culture was sub-cultured onto Deoxycholate citrate agar (DCA), incubated at 37°C for 24-48 h. This was subsequently sub cultured onto nutrient agar slants, incubated for 18-24 h and then stored at 4°C until required. The Gram’s-staining techniques was performed on pure black colonies of Salmonella11. The biochemical characterization was carried out using Triple Sugar Iron (TSI), Simmon Citrate, Urease and Indole tests to check for phenotypic changes produced by Salmonella within the media12.
Antimicrobial susceptibility testing of Salmonella isolates: Susceptibility testing of Salmonella isolates to 10 different antimicrobial agents was carried out through disc diffusion method, to determine their antimicrobial susceptibility pattern. The antibiotics include: Ciprofloxacin, Streptomycin, Pefloxacin, Amoxicillin, Ofloxacin, Apramycin, Ceftiofur and Cefotaxime. The isolate of Salmonella were subcultured into Muller-Hinton broth (Oxoid-CMO405) and then incubated at 37°C for 24 h. Serial dilution was carried out in the ratio of 2:4 of the broth and distilled water to reduce the degree of turbidity of the broth culture. About 1 mL was dispensed onto Muller-Hinton agar and the excess was discarded. Antibiotic discs were applied aseptically to the surface of the plate. This was incubated at 37°C for 24 h under aerobic condition to observe for zones of inhibition, which was measured with the aid of a calibrated ruler11,13.
Statistical analysis: Data generated were analyzed into tables, charts and figures using simple percentages and descriptive statistics.
RESULTS
Distribution of Salmonella in samples collected from the teaching and research poultry farm, University of Maiduguri: The number of positive samples was found to be 19. Highest prevalence rate of 53.33% was obtained for samples from faeces, followed by litters (33.33), whereas, low prevalence rate of 20.00% was recorded for samples obtained from feeds and drinking water, respectively. The overall prevalence of Salmonella in poultry samples from University of Maiduguri teaching and research poultry farm was found to be 31.67% (Table 1).
Distribution of Salmonella in samples collected from Wilson Poultry Farm, Fori Ward Maiduguri: Twenty eight positive samples were obtained. Higher prevalence rate of 80.00% was obtained for samples from litters, followed by faeces (66.67%) whereas, low prevalence rate of 26.67% and 13.3% was obtained for samples from drinking water and feeds, respectively. The overall prevalence of Salmonella in poultry samples from Wilson Poultry Farm was found to be 46.67% (Table 2).
Biochemical characterization of Salmonella isolates: About 44 samples were found to be positive for citrate utilization test, 47 samples each were found to be positive with urease and triple sugar iron tests (TSI) (Table 3).
Citrate utilization and urease test for Salmonella isolates: Of the total number of positive samples (47) obtained, 44 Salmonella isolates were found to utilize citrate with characteristic change in colour from green to blue.
All the 47 positive isolates of Salmonella were subjected to urease test were found to be urease positive with characteristic change in colour from amber to pinkish.
Triple sugar iron test for isolates of Salmonella: All the 47 samples showed the production of hydrogen sulfide (H2S), while 23 of the total positive samples of Salmonella isolates exhibited gas production. All the positive samples also showed red slope, whereas, 30 of the total positive samples showed a yellow butt. All samples exhibiting these characteristics were found to be Salmonella.
Antimicrobial susceptibility test for Salmonella isolates: The resistance and sensitivity pattern were displayed by 24 Salmonella isolates to 10 antimicrobial agents. Septrin (SXT), Amplicin (PN) and Ceporex (CEP) had the highest resistance of 5 (10.64%), 4 (8.51%), 4 (8.51%) to Salmonella isolates, followed by Nalidixic Acid (NA)-3 (6.38%), Gentamicin (CN)-3 (6.38%), Streptomycin (S)-2 (4.26%), Reflacine (PEF)-2 (4.26%), Tarivid (OFX)-2 (4.26%) and Augmentin (AU)-1 (2.13%), respectively, 0 (0.00%) was found in Ciproflox (CPX).
| Table 1: | Distribution of Salmonella isolates in poultry samples from University of Maiduguri Teaching and Research Poultry Farm, Nigeria |
| Fc: Faeces, Fd: Feeds, Lt: Litters, Dw: Drinking water | |
| Table 2: | Distribution of Salmonella isolates in poultry samples from Wilson Poultry Farm Fori Ward Maiduguri, Nigeria |
| Fc: Faeces, Fd: Feeds, Lt: Litters, Dw: Drinking water | |
| Table 3: | Biochemical characterization of Salmonella isolates from poultry samples in Maiduguri, Nigeria |
| DCA: Deoxycholate citrate agar, TSI: Triple sugar iron agar | |
| Table 4: | Antimicrobial susceptibility test for Salmonella isolates from poultry samples in Maiduguri, Nigeria |
| Fig. 1: | Distribution of Salmonella isolated from two different poultry farms based on sample distribution |
The highest intermediate value were 3 (6.38%) for Nalidixic Acid (NA), Ceporex (CEP) and Streptomycin (S), followed by 2 (4.26%) for Septrin (SXT) and Augmentin (AU), followed by 1 (2.13%) for Amplicin (PN) and Reflacine (PEF), 0 (0.00%) were found in Ciproflox (CPX) and Tarivid (OFX). The Salmonella isolates were sensitive to Ciproflox (CPX), 12 (25.53%), Tarivid (OFX), 10 (21.28%), Augmentin (AU) and Reflacine (PEF), 9 (19.15%), Gentamicin (CN), 8 (17.02%), Amplicin (PN) and Streptomycin (S), 7 (14.89%), Nalidixic Acid (NA), 6 (12.77), Ceporex (CEP) and Septrin (SXT), 5 (10.64), respectively (Table 4).
Salmonella isolated from two different poultry farms based on sample distribution: Of the samples collected, the highest prevalence of Salmonella recorded was found with the samples obtained from faeces (38%) followed by litters (36%), whereas, drinking water and feed had the lowest prevalence rate of 15% and 11%, respectively (Fig. 1).
DISCUSSION
Salmonella is an enteric pathogen that is shed predominantly in faeces resulting in faecal contamination of feed and water14,15. Salmonellosis is a major public health concern and continues to have a serious economic importance in the poultry industry in all countries16. With the great expansion of the poultry industry, the wide spread occurrence of the avian salmonellosis has ranked it as one of the most important egg-borne bacterial diseases of poultry.
The present study was conducted to determine the prevalence and antimicrobial susceptibility of Salmonella associated with poultry accessories in Maiduguri, Borno state. Out of the total of 120 samples examined for Salmonella, 47 tested positive for Salmonella. The overall prevalence of Salmonella in this study was found to be 78.33%, which was higher than 43.6% reported in 5 different regions of Nigeria17. Furthermore, the prevalence of 30.00% was recorded for faecal samples, followed by 28.33% for litters, while drinking water and feeds recorded 11.67 and 8.33%, respectively. The prevalence reported in this study is higher than those documented for laying hens in EU countries, with overall prevalence of zoonotic Salmonella serovars of 2.5%. The high prevalence observed in this study may be attributed to lack of implementation of control programmes on poultry farms and differences in terms of Salmonella status among countries but could be influenced by housing system, local environmental conditions, sample types, collection seasons, laying period, isolation methodologies and culture media.
The highest Salmonella prevalence per type of poultry sample found in faeces is 30.00%, which was closely followed by litters, 28.33%, then drinking water and feeds had the prevalence of 11.67 and 8.33%, respectively. The 30.00% prevalence for faecal samples found in this study is higher than 11% reported in chicken faeces from Ibadan18. The 28.33% prevalence of Salmonella associated with litters is higher than 15.63% earlier reported19. On the contrary, the prevalence reported for feed and drinking water in this present study were lower when compared with previous studies19. Moreover, the rate of Salmonella isolation from feed and water samples could be an indication of poor sanitation, handling and contamination along the poultry production chain as well as cross contamination, which calls for serious concerns. Although water was the least contaminated of the four samples, it is of particular concern since birds are exposed to microbial load whenever they drink water.
In most cases, unhygienic packaging could serve as a source of contamination in feed. Contamination may also occur during processing, transport and distribution of compounded feed mixture. The prevalence of Salmonella in feed appeared to be quite low. Salmonella contamination is not uniformly distributed in the feed and to detect the level of contamination accurately, several hundred samples are required20.
Further investigations could be useful to clarify the sources of infections and factors leading to the widespread isolation of Salmonella in Nigerian poultry farms. Antimicrobial resistance was determined for ten commonly used agents on fifteen Salmonella isolates. In this study, 29.16% resistance to Augmentin has been reported, which is lower than 100% resistance of Salmonella from poultry sources21.
Of the total samples subjected to antimicrobial susceptibility test, resistance to Nalidixic acid was 41.66%, Gentamicin 33.33% and Augmentin was 29.16%. Sensitivity to Ciproflox was found to be 100%, Tarivid, 41.66% Reflacine 29.16% and Streptomycin and Ampicillin were 25.00%. The development of quinolone-resistant Salmonella strains, especially those of poultry meat origin which is gradually leading to an epidermic22-24. Nalidixic acid is able to develop resistance quite rapidly. Several studies have also shown that resistance to Nalidixic acid has been on the increase.
CONCLUSION
The overall prevalence of Salmonella was 78.33% obtained from faeces, feeds, litters and drinking water. This study has shown high sensitivity to Ciproflox (100%) and Tarivid (41.66%), whereas high resistance was recorded for Nalidixic acid (41.66%), Gentamicin (33.33%) and Augmentin (29.16%). The relatively high prevalence could be linked to poor infrastructure and low biosecurity measures on poultry farms in the study area and can form the basis for instituting effective intervention strategies.
ACKNOWLEDGMENTS
The authors wish to acknowledge the contributions of the technologists in Microbiology Laboratory, Department of Veterinary Microbiology, University of Maiduguri.