Introduction

The Salmonella spp. is identified in several species which causes one of the most important zoonotic diseases worldwide (Koochakzadeh et al., 2015, Juffo et al., 2017). Salmonella spp. is one of the important causes of diarrhea in foals which are more prevalent enteric pathogens in foals between 1 and 3 month of age (Olivo et al., 2016). Salmonella can cause acute and chronic diarrhea disease, neonatal bacteremia, or subclinical colonization in apparently healthy horses (manship et al., 2019). Salmonella spp. is a bacillus of 0.7-1.5×2-5 µm diameters, facultative anaerobic and non-sporulating (Bustos et al., 2016). In addition to the sole host-adapted serovar, Salmonella enterica serovar abortus-equi, some ubiquitous serovars of the bacterium,including Salmonella enterica serovar Typhimurium (ST) and Salmonella enterica serovar enteritidis (S. enteritidis), are frequently isolated from horses (Ahmed et al., 2012; Zahraei Salehi et al., 2012; Juffo et al., 2017).

Salmonella enterica subspecies enteric serovar abortus-equi (S. abortus-equi) is frequently reported as a cause of abortion in mares and neonatal septicemia and polyarthritis in Asian and African countries (Bustos et al., 2020, Glandolfo et al., 2018). It is well known as the etiological agent of equine abortion (Wang et al., 2019). Although S. enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with the clinical disease (Leon et al., 2016). Various factors may affect microbial balance often leading to disturbances that may result in debilitating conditions such as colic and laminitis. Among those factors are high carbohydrate nutrition, medical substances, animal-related factors, pathological conditions and stress (Uzal and Diab 2015; Garber et al., 2020).

The detrimental impact of the carrier state, either persistently or transiently, is due to veiled shedding of the bacteria and therefore the likelihood of prolonged propagation of the infection to the susceptible hosts. Further, zoonotic potential of the bacterium is assumed as a serious challenge in public health sector (Martelli et al., 2018).

Although selective media are incorporated in phenotypic protocols of Salmonella isolation, the problems related to the sensitivity and specificity of the methods may limit their usage in the epidemiological investigations. Moreover, these procedures are time-consuming and laborious (Singer et al., 2006). Recently, development of genotypic techniques for direct detection of Salmonella in feces and food samples is regularly reported (Zahraei Salehi et al., 2005; Singer et al., 2006; Moganedi et al., 2007). Molecular assays provide more efficiency in less time in comparison with the phenotypic methods of Salmonella isolation (Paião et al., 2013).

Nowadays, emergence and dissemination of antimicrobial resistance is a matter of worrisome worldwide not only for public but also for veterinary medicine. Current studies have documented multi-drug resistance (MDR) among Salmonella serovars isolated from human and animals, which may impede the effectiveness of antibiotic therapy (Ahmed et al., 2012; Zahraei Salehi et al., 2012; Leona et al., 2018). S. enterica is an important cause of health care-associated infection in veterinary hospitals with outbreaks of multi-drugs resistant (Burgess et al., 2018).

The most controversial antimicrobial agents prescribed in the cases of equine Salmonellosis are ceftiofur and gentamycin. Given that they do not necessarily kill all the Salmonella organisms in the gut, latent carrier state may be the outcome of antibiotics utilization following recovery. Moreover, the imbalance and dis-ruption in normal intestinal microbial flora following the consumption of antibiotics may also prompt the colonization of Salmonella in the gut. This is because of the combat between pathogens and normal flora for the nutrients and attachment sites (Leona et al., 2018). Popularity of horses and horse-racing as a recreation and favorable sport and paucity of information regarding Salmonella colonization in horses in the West of Iran encouraged us to aim this study towards a timely manner survey on the prevalence of latent equine salmonellosis, with the focus on the serotypes and antimicrobial resistance patterns of the isolates in Kurdistan province of Iran.

Materials and Methods

A total of 130 horses, categorizing in four age groups, were randomly enrolled in the research. Table 1 shows the season and gender variables in relation to the age groups. An approximate of 50 gr feces was collected from each animal in the sterile glass bottles in rectal palpation and delivered to the laboratory within maximum of five h under cold conditions.

The specimens were screened for Salmonella based on the phenotypic procedure introduced by López-Martín et al., (2016). The cell culturing, immunoassay and polymerase chain reactions are the current methods to detect these pathogenic agents (Zhang 2019). Initially, the homogenization of an approximate of 5 gr individual fecal sample was carried out in 90 mL Buffered Peptone Water (BPW, Merck, Germany) and followed by the incubation at 37°C for 18-24 h. Further, 25 μL of the pre-enrichment media was inoculated into 10 mL Rappaport Vassiliadis Enrichment (RV, Merck, Germany) broth and incubated for 15-18 h at 41.5-42°C. A loop-full of the previous enrichment medium was streaked onto Hektoen Enteric (HE, Merck, Germany) agar, then, incubated for 24 h at 37°C. Finally, a presumptive colony to Salmonella (green colony with dark center) from each plate was purified on MacConkey (MAC, Merck, Germany) agar. The biochemical identification was based on the Gram staining, TSI, IMViC, and Urea reactions. The isolates were transferred to the Faculty of Veterinary Medicine, Tehran University (Tehran, Iran) for serotyping using commercial antisera.

In parallel, molecular detection of Salmonella was fulfilled from each pooled fecal sample. The overnight suspensions of the individual fecal samples incubated in Tetrathionate (TT, Merck. Germany) broth were used to prepare a 10^-1 aliquot. The aliquot was transferred to a microtube containing 500 μL Brain and Heart Infusion (BHI, Merck, Germany) broth. Following the incubation at 37°C for 3 h, the tubes were centrifuged at 12000 g for 5 min and the supernatants were discarded. Adding 200 μL deionized distilled water, the pellets were vortexed, boiled for 15 min, and centrifuged same as the previous step. The supernatant was collected as DNA repertoire (Alinovi et al., 2003).

The PCR (BIORad T100, USA) for detection of InvA gene was carried out in 25 μL volume containing 12.5 μL 2X ready-to use PCR master mix (CinnaGen, Iran), 9.1 μL of deionized distilled water, 2 μL (50 ng) of template DNA, and 0.7 μL of each primer. The sequence of the primer pair was ST139:5ʹ-GTGAAATTATCGCCACGTTCGGGCAA-3ʹ and ST141:5ʹ-TCATCGCACCGTCAAA GGGAACC-3ʹ. The thermal condition of the reaction was the same as introduced by Rahn et al., (1992). The used positive and negative controls were Salmonella Typhimurium ATCC1730 and DNA-free master mix, respectively. The PCR products were electrophoresed on 1.2% agarose gel (CinnaGen, Iran). Further, PCR-positive samples were phenotypically re-analyzed for the Salmonella confirmation once more.

Likewise, agar disk diffusion method was applied to determine the antimicrobial resistance profile of the isolates, in accordance with the CLSI guidelines (CLSI 2013). The used antibiotic disks included ampicillin (A), chloramphenicol (C), enrofloxacin (E), florfenicol (F), gentamicin (G), kanamycin (K), nalidixic acid (Na), nitrofurantoin (Ni), streptomycin (S), sulphamethoxazole (Su), and tetracycline (T).

The statistical association between the proportional morbidity of Salmonella and the variables was analyzed using SPSS software (version 21.0). Based on Kolmogorov-Smirnov normality test, parametric t-test, and non-parametric Man-Whitney U test were used to investigate the significant differences between groups for the measured analyses. A P-value less than 0.05 was considered as statistical significance.

Results

Of the 130 fecal samples, two Salmonella isolates were recovered in routine bacteriological method, representing to an overall incidence of 1.53%. In comparison, Salmonella was detected in 10 fecal samples (7.69%), producing a 284 bp amplicon in PCR reaction (Figure 1). As the positive samples in phenotypic method were also detected in molecular approach, it can be implied that the sensitivity of culture method was 20% of the PCR method. The two Salmonella isolates were recognized as ST based on the Kauffman-White Scheme (1,4,5,12:i:1,2). Besides, despite the re-cultivation of PCR-positive samples, no Salmonella spp. was isolated. Generally, the highest prevalence of Salmonella fecal shedding was among ≥1-5≤ years-old animals, male sex, and in summer. Despite this, no statistical association was observed between the frequencies of fecal carriers and variables including age, gender, and season (P≥0.05). Table 2 represents the proportional morbidity of Salmonella in regards to the analyzed variables. Antimicrobial susceptibility testing revealed resistance to ampicillin (it is from the same family of penicillin that is used to treat or prevent many different types of bacteria such as Salmonella), tetracycline, streptomycin, and sulphamethoxazole, and chloramphenicol in the both isolates, whilst resistance to gentamicin in only one isolate. No resistance was observed against nalidixic acid, nitrofurantoin, enrofloxacin, florfenicol, and kanamycin.

Table 1. Demographic information of the studied population

Table 2. Prevalence of Salmonella fecal shedding in related to age, sex and season in the present study.

Figure 1. Agarose gel electrophoresis of PCR products with invA gene primers (284 bp). M: 100 bp DNA Ladder (CinnaGen, Iran), PC: positive control (Salmonella Typhimurium ATCC1730). NC: negative control. Lanes 1-9: field samples.

Discussion