Introduction
Salmonellosis is the leading cause of gastroenteritis [1] and septicemia [2] in humans worldwide, and the second most frequently reported zoonotic disease in many countries [3]. Worldwide, it is the most common pathogen causing foodborne illnesses [4,5], that are directly linked to the consumption of poultry or poultry products [6]. The widespread commercial trade in animal- derived food products, the lack of surveillance of the Salmonella serotype distribution among farming communities, especially in Zimbabwe, and the level of antimicrobial resistance (AMR) in many countries are of global importance [2,4] and warrants further study. It is estimated that approximately 93 million enteric infections and 155,000 deaths are attributed to Salmonella each year [5,6]. Although the presence of Salmonella in commercial poultry units, its meat, and poultry products has been studied [4,7] its prevalence in live commercial poultry units in Zimbabwe has not been elucidated. The microbial quality of animal-based products in many farms is questionable. In the developing world many times it is difficult to link disease outbreaks and their source. In addition, the various poultry and other animal products [9,10] supposedly harboring microorganisms like Salmonella are enormous. Furthermore, few countries have a viable surveillance system that estimates the burden of salmonellosis in human populations [2,11]. In emerging countries such as Zimbabwe, poultry meat is the most important and least expensive source of animal protein [12]. Most of the predominant species are non-typhoidal [1] and they are responsible for both animal and human salmonellosis [13]. Nevertheless, outbreaks of food-borne diarrhoea are common which are associated with transference and consumption of raw meat or animal products. Several cases of human Salmonellosis are attributed to the consumption of poultry meat because poultry is an important reservoir of this microorganism [14]. The colonization of birds by these microorganisms begins mostly at the farm level [14]. Therefore, there is a need to carry out research to vindicate the production of these animal-based products.
Contamination of broiler meat usually occurs during the various stages of chicken slaughter and processing [15] and, all edible tissues are potentially at risk of contamination from sources both within and outside the animal. The most commonly isolated Salmonella serotypes worldwide were Salmonella enteritidis (65% of isolates), Salmonella typhimurium (12%), and Salmonella newport (4%) [13]. Birds become infected by horizontal transmission through infected litter, feces, feed, water, dust, fluff insects, equipment, fomites, diseased chicks, and salmonella-contaminated rodents [10]. Other domestic animals, wild birds, and personnel can transmit Salmonella species. to broiler chickens throughout the rearing period. Vertical transmission from parent flocks results mostly from ovarian transmission or through the eggshell after laying [16,17]. Regardless of the entry point salmonellosis is an economically significant disease, especially in third world countries. Unfortunately, healthy chickens can be infected with Salmonella species without showing any clinical symptoms, therefore many farmers do not know that their chickens are infected [18]. It is essential to take into account the problem of contamination of livestock both for its impact on public health and for its significant economic repercussions [19]. The AMR in Salmonella species has become a major problem in public health worldwide [13]. It is known that in developing countries the rise of AMR is increasingly under surveillance, but in many African countries, this problem remains very complex. This is because of the paucity of information available on bacterial resistance. It has been demonstrated that the widespread and indiscriminate use of antibiotics in veterinary medicine, is one of the major contributors to the development and spread of AMR [20], resulting in prolonged illness, disability, and death. There is thus a need to investigate the prevalence of Salmonella species in intact animals to prevent spoilage within the food chain. Therefore, this study aimed to determine the prevalence of Salmonella species in broilers at Africa University and to show the level of AMR of Salmonella species among the common antibiotics.
Materials and Methods
Study site
The experiment was carried out at Africa University Farm located at longitude E 0320 36 .036, latitude S180 53.786, and altitude 1,104 m above sea level. The area receives an average annual rainfall above 800 mm with an annual average temperature of 19°C. Summer and winter temperatures vary widely but fall within a range of 16°C–39°C, and from 6°C to 20°C respectively. The area is a savanna dominated by Brachystegia tree and Hyperhenia grass species. In this region, specialized farming is common involving both crop and animal production.
Bird management
A batch system is employed at the study site and an all –in –all out system was in place at the time of conducting this study. Birds were housed in deep litter fowl runs with a capacity of 1,000–5,000 broilers. Housing and management of fowl runs were such that each run would be allowed to run for 42 days and rested for 14 days. Brooding which lasted for 28 days was done in guards within each fowl run. Lighting and temperature were managed according to Irvine’s [21] guidelines. Wood shavings were the preferred bedding material and were only removed after 42 days, unless for spoiled areas within the guard and run. These would be hand removed and replaced with new and fresh bedding material. Feeding and watering were done manually according to Profeeds [22] guidelines, and both were provided ad libitum. A heating source attached to a rotating fan was used for temperature control and humidity was maintained below 60%. Five successive batches separated by a week were used in the current study. The study adhered to the ARRIVE guidelines for handling live animals, in compliance with measures to ensure the humane treatment of live animals during sample collection were strictly followed. The research adhered to institutional, national, and international guidelines for the ethical use of animals in research. A qualified veterinarian collected the samples from animals.
Collection and preparation of samples
Fifteen rectal swab samples were collected every 14 days according to Totton et al. [23] from each batch using sterile swab sticks (Oxoid, Cheshire, England) on days 2, 16, 30, and 44, placed in labeled buffered peptone water broth universal bottles [7] and transferred to the laboratory for bacterial culture and isolation. The samples were transported to the laboratory immediately after collection before overnight incubation at 37°C.
Laboratory isolation and identification of Salmonella
Isolation and identification of Salmonella was done and the flow chart is in Figure 1. The incubation process took 6 days.
On the first day, swabs were placed in peptone water at 37°C overnight followed by the addition of 0.1 ml of the peptone water into 10 ml of Rappaport vissiliadis (RV) media and incubated overnight at 37°C on the second day. The RV was used because it is selective for Salmonella species. A loopful of inoculated RV media was streaked on xylose lysine deoxycholate (XLD) (Merck) on the third day. The XLD is an indicator media for Salmonella [14] and single colonies of the suspected Salmonella were picked from the XLD agar on the fourth day, at which time they were inoculated into the biochemical media and enriched with 10 ml of buffered peptone water. Spherical transparent red halo colonies with typical black centers on XLD were selected as presumptive Salmonella colonies [7]. The inoculated peptone water and biochemical test agars were then incubated at 37°C overnight. On day five, 1 ml of the peptone water was extracted and diluted in 9 ml of saline water for each sample to give three serial dilutions, using a micropipette 0.1 ml of the final dilution was introduced on plate count agar discs, and was then spread evenly over the plate using a glass rod. The presumptive Salmonella isolates were subjected to biochemical tests using commercially available media (Oxoid, UK). Briefly, a loopful of colonies was stabbed into Citrate agar, Lysine iron agar (LIA), Urease agar, and Motility agar, and incubated at 37°C overnight [24]. On day six, the plate count agar was retrieved from the incubator to enumerate the resultant individual colonies.
Data collection
Each colony-forming unit was counted from the plate count agar after 24 hours of incubation according to the method of Isnawaida et al. [25] using a colony counter (Scan 300, Saint Nom, France) and recorded as colony-forming units per gram (cfu/gm). The antibiotic susceptibility of the isolates was evaluated using the disc diffusion technique. To meet the 0.5 McFarland turbidity requirements, isolated colonies were stirred for a few minutes in sterile saline water-filled tubes until a smooth suspension was observed. An aliquot from the smoothed suspension was swabbed uniformly across the Muller Hinton agar plates 'surface, which were allowed to dry at room temperature, after which eight antibiotic discs were tested for susceptibility according to Procura et al. [26]. Eight antibiotic discs with ten grams each of Ampicillin, Cloxacillin, Gentamycin, Kanamycin, Neomycin, Penicillin, Sulphamethoxazole, and 30 g of Erythromycin, were introduced into each plate positioned at least 15 mm apart to prevent overlapping inhibitory zones. The plates were incubated overnight at 37°C. The plates were removed from the incubator and the inhibition zones (zones of clearance) were measured around the antibiotic disc. The diameter of inhibition zones was interpreted as being resistant or sensitive in accordance with the Clinical and Laboratory Standards Institute [27].
Experimental design
A cross-sectional study design was formulated to determine Salmonella in chickens. Raw samples were collected at different time intervals in a completely randomized design with two factors (batch and time). Five batches were used (1–5) and samples were collected over time; 2, 14, 30, and 44 days. Fifteen samples were taken from each batch at every stage making a total of 60 samples per batch and 300 samples for all batches.
Statistical analysis
Statistical comparisons of the type of salmonella and AMR rates among the different Salmonella species were analyzed using ANOVA GLM of SPSS version 16 in the following model:
Y ijk=µ+ Bi + Tj + BTij + ejk
Figure 1.
Laboratory workflow for isolation and identification of Salmonella species from Cobb 500 broilers at Africa University. PCA=plate count agar; RV=Rappaport vissiliadis; XLD=xylose lysine deoxycholate.
where y=response variable (cfu, Salmonella), µ=mean common to all variables, Bi=batch effect (i=1,2,3,4,5), Tj=effect of time of collection (j=2 d, 14 d, 30 d, 44 d), BTij=interaction between batch and time, Ej=error term. Means were separated using the Tukey HSD test.
Results
Cultured samples were subject to a plate count and the results are shown in Table 1. The number of coliform counts decreased with time for all batches, however, significant differences were observed among batches (p < 0.05). Batches 1 and 2 consistently showed higher coliform counts over time (p < 0.05) while batches 3–5 did not show any pattern, overall, the CFU counts declined with time for all batches. Coliform counts decreased by approximately 50% from day 16 for all batches (Fig. 2). The results of the biochemical test conducted for specific Salmonella species are presented in Table 2. Salmonella enterica, S. typhi, and S. typhimurium were the identified serotypes in the sample populations. The citrate test is based on the ability of bacteria to utilize citrate as a sole source of carbon and bromothyl blue is used as a pH indicator after citrate utilization. Salmonella enterica showed a positive reaction, whereas serotypes S. typhi and S. typhimurium showed negative reactions. LIA is recommended for use in differentiating certain members of the Enterobacteriaceae, which produce hydrogen sulfide and lead to the decarboxylation or deamination of lysine, S. enterica and S. typhi were positive to LIA while S. typhimurium was not. Some bacteria produce the enzyme urease which hydrolyses urea with the formation of ammonia (alkaline) in the media that results in a color change of the indicator, phenol red. Salmonella species did not hydrolyze urea and therefore showed no color change. All bacteria were motile and gave a diffuse spreading growth that was recognizable even by the naked eye. A greater proportion of the birds carried S. enterica (74%), followed by S. typhi (17%), and lastly S. typhimurium (9%). Since the samples were sub-cultured in RV broth as well as XLD agar which select against most coliforms the percentage amount of salmonella in the samples relative to other bacteria was not estimated. A sensitivity analysis of a homogenous salmonella sample to selected antibiotics was conducted and the results are shown in Figure 3. Of the eight antimicrobials used, the bacteria only showed sensitivity to kanamycin, this implies that only kanamycin is currently able to eradicate the bacteria. Antibiotics of the penicillin family (penicillin and ampicillin) proved to be ineffective against the bacteria. Erythromycin showed intermediate sensitivity, meaning the anticipated effect of the antimicrobial was not fully achieved.
Table 1.
Least square means of Salmonella (cfu/gm) in broiler chickens at Africa University Farm.
| Batches | Time (days) | SE | p values | |||||
| 2 | 16 | 30 | 44 | B | T | BT | ||
| 1 | 80.33a | 55.00a | 19.66a | 12.66a | 2.33 | 0.001 | 0.001 | 0.13 |
| 2 | 74.00b | 50.67b | 16.33b | 8.33b | 2.33 | 0.001 | 0.001 | 0.21 |
| 3 | 79.67a | 42.33d | 21.33a | 12.67a | 2.33 | 0.001 | 0.001 | 0.31 |
| 4 | 60.67c | 50.00b | 16.67b | 8.33b | 2.33 | 0.001 | 0.001 | 0.22 |
| 5 | 77.33a | 48.00c | 18.33ab | 6.67b | 2.33 | 0.001 | 0.001 | 0.34 |
abcdColumn means are significantly different at p < 0.05.
Figure 2.
Coliform counts (cfu/gm) in broiler chickens at Africa University.
Table 2.
Biochemical test results for Salmonella species at Africa University Farm.
| Biochemical tests | Salmonella enterica | Salmonella typhi | S. typhimurium |
|---|---|---|---|
| Citrate | + | − | − |
| Lysine iron agar | + | + | − |
| Urea agar | − | − | − |
| Motility agar | + | + | + |
Figure 3.
Sensitivity, analysis for Salmonella species to Ampicillin (AMP), Cloxacillin (CLO), Erythromycin (ERY) Gentamycin (GEH), Kanamycin (KAN), Neomycin (NEO), Penicillin (PEN), and Sulphamethoxazole (SMX) at Africa University, Zimbabwe.
Discussion
The total bacterial counts were high in young birds and decreased as the birds were growing, similar results were obtained by Ansari-Lari et al. [16] and Bokaie et al. [28] and the variation may be attributed to many factors including climatic, management and biosecurity measures. In addition, newly hatched birds are prone to bacterial infections. However, with time, the natural flora serves to reduce the accumulation of constituent species through ecosystem interaction in the gut, therefore eliminates unfavorable bacteria [2,29,30]. Eventually, bacterial species levels start to decline until they reach an optimal sustainable level. We could not find a plausible reason for the high cfu counts in batches 1 and 2, because birds were raised under the same conditions by the same personnel, possibly they carried over infection from the hatchery. The use of antibiotics may contribute to a reduction in microbial populations over time; however, Salmonella species have shown antibiotic resistance [8] in the past decade [31], and their use is deemed unfavorable [19].
The prevalence of Salmonella in the current study is not surprising, Ansari-Lari et al. [24] reported prevalence rates of approximately 64% in Iran. The order of prevalence of Salmonella species in the current study is consistent with Ansari-Lari et al. [16] where S. enterica and S. typhimurium are the frequently identified serovars. However, Le Bouquin et al. [32] showed that S. hadar, S. anatum, and S. mbandaka were more prevalent in broiler flocks in France, nonetheless, the prevalence rate was similar for S. enterica and S. typhimurium. The prevalence of Salmonella could also be attributed to flock size and the use of therapeutic or sub-therapeutic doses of antibiotics [24] which is a common practice in Zimbabwe. It is possible that the frequent visits by poultry workers in and out of the fowl runs could introduce the bacteria. This is in agreement with Le Bouquin et al. [32] who showed that the risk of flock contamination decreased in proportion to the number of visits by the poultry farmer. The increased need in the amount of food, water, labor, and materials is a source of infection [33] in large flocks. It is plausible that Salmonella prevalence decreased with time for all batches, thus risks of Salmonellosis were low, and hence chickens were safe for human consumption.
Salmonella enterica is responsible for pullorum disease in chickens [34]. Unfortunately, in growing and mature fowl, infected chickens may not exhibit any signs and exhibit no physical symptoms. Because of this, the disease survives in perpetuity in a carrier state resulting in persistent infection and transmission to eggs or progeny [34]. However, in the current study, the evaluated animals were commercial broilers and, its possible transmission to eggs or progeny was not possible. The effects of S. enterica on the hen’s reproductive tract is worrisome from an animal health and breeding perspective, as it is known to contaminate the albumen, vitelline membrane, and possibly the yolk, yet egg-sanitizing practices are only superficial, therefore, decreasing pathogen contamination becomes ineffective. Salmonella species can be typhoidal and human-restricted (S. typhi and S. paratyphi A) or non-typhoidal having a broad host range and predominantly causing gastroenteritis (S. typhimurium and S. enteritidis). In this aspect, Salmonella species and their management in broiler production should be based on their potential to cause typhoid (in humans) or their impacts on broiler growth (non-typhoidal). In other instances, S. typhimurium has been observed to be typhoidal in which case its management involves both animal and public health concerns. However, Jung et al. [12] reported that the species is non-typhoidal but is the major cause of human foodborne infections posing a global threat to public health. Typhoid fever is a life-threatening infection caused by S. typhi. Results from the current study confirm the possibility of this bacterium in broiler meat posing a health threat to consumers. Salmonellosis is positively associated with the warmer seasons, particularly summer [35] and this is attributed to higher temperatures that increase the transmission of Salmonella as well as increased bacterial proliferation.
The prevalence of AMR in the current study is not surprising although alarming. As reported by Abayneh et al. [36], resistance is a result of indiscriminate use of antimicrobial agents by unskilled practitioners in both the veterinary and public health sectors, this is becoming common in SSA and the world over [36]. Ansari-Lari et al. [16] have shown a positive association between antibiotic use and Salmonella prevalence, possibly because the phage types of Salmonella species have shown drug resistance. Resistance to penicillin and ampicillin in livestock is a major concern for veterinary medicine and public health [37,38]. Without proper interventions, the spread of resistant bacteria could compromise animal health, food safety, and human medicine. A one health approach [39,40] involving antimicrobial stewardship, biosecurity measures, surveillance, and public education is essential to mitigate the impact of AMR and ensure sustainable antibiotic use. Although we did not test the resistance of each Salmonella species against the antimicrobials, it has been shown that DT104 is currently the dominant penta-resistant clone of S. typhimurium, many other phage types (DT29, DT204, DT193, and DT204C) of this serovar have also be seen with multi-drug resistance [12,41]. In addition, more than 90% of food contamination is caused by S. typhimurium [12], therefore this species is of particular concern to human health. The intermediate sensitivity of ERY in the current study means that either the bacteria are developing resistance or the antimicrobial is gaining control of the bacteria. In this case, further studies are necessary to confirm the direction and magnitude of this sensitivity, since our study was only limited to one farm. Antibiotic resistance of Senterica and S. typhimurium in raw chicken meat [8] is an ongoing study with unfavorable results [42]. To reduce the development of AMR in poultry Jung et al. [12] proposed a 4–12-week course of antibiotics which should include either a third-generation cephalosporin or fluoroquinolone, in addition to the use of alternative antimicrobial treatments [43] suggested for future research.
Conclusion
Three salmonella species; Salmonella enterica, S. typhi as well and S. typhumirum were identified in broiler chickens at Africa University. Salmonella enterica is a concern for animal scientists while S. typhi and S. typhimurium are a public health concern. The levels of infestation could not be attributed to age or time of sampling although there is evidence that the number of colony-forming units of the Salmonella species are highest in chickens below the age of 7 days, gradually declining in the second week and stabilizing from day 16 until slaughter. The bacteria showed resistance to antibiotics of the penicillin family (namely penicillin G and ampicillin). There is an urgent need to control Salmonella in live broiler production and further reduce its prevalence in fresh or processed broiler meat. The control strategies evolve around eliminating the sources of salmonella as well as proper antibiotic use. It is also evident that the older the birds are, the less chance of Salmonella prevalence, and further research should look at the economics of a prolonged production cycle maybe back to eight-week cycles. A multifaceted approach combining biosecurity, vaccination, probiotics, antibiotic stewardship, and surveillance is essential to controlling Salmonella in broiler production and minimizing AMR risks. Implementing these measures ensures safer poultry production, reduces public health risks, and preserves antibiotic efficacy for future generations.
Acknowledgments
This study was carried out in collaboration with the Mutare Provincial Veterinary Authorities. The project was supported financially by the Africa University Farm.
Conflict of interest
The authors declare no conflicts.
Ethical approval
The study was carried out according to ARRIVE guidelines.
Author contributions
SBF: Writing - Original draft preparation, Data curation, WS: Conceptualization, Methodology, Formal analysis, Visualization, Investigation, Supervision, Writing- Reviewing and Editing, WDD: Writing- Original draft preparation, Writing- Reviewing and Editing.
