An Abattoir-Based Study on the Prevalence of Salmonella Fecal Carriage and ESBL Related Antimicrobial Resistance from Culled Adult Dairy Cows in Wuhan, China

The objective of this study was to estimate the fecal carriage of Salmonella spp. among culled adult dairy cows presented to an abattoir in Wuhan, China and to evaluate their antimicrobial resistance profiles. Rectal swabs from 138 culled cows were cultured. Laboratory analysis involved the identification of Salmonella, the susceptibility assessment and the presence of Extended Spectrum β-lactamases and mcr genes in the isolates. An overall prevalence of Salmonella of 29.0% was recorded with 63.4% (26/41) and 2.4% (1/41) of the isolates identified as S. Typhimurium and S. Dublin, respectively. The occurrence of Salmonella was higher (odd ratios: 3.3) in culled cows originating from the northeast zone of China than cows originating from the central and north zones. Twenty multi-drug resistant strains (resistant to three or more antimicrobial agents) were detected (48.8%) and overall, a high resistance to ampicillin (36/41) and tetracycline (15/41) was observed. Extended Spectrum β-lactamases phenotypes were found in 7/41 isolates, of which all contained the blaCTX-M resistance gene, and no mcr genes were found by polymerase chain reaction. The high prevalence of Salmonella fecal carriage and antimicrobial resistance may contribute to an increased risk of Salmonella transmission to food.


Introduction
Salmonellosis is a bacterial zoonotic disease that affects a wide range of domestic animals, including chickens, pigs, and cattle, as well as humans [1][2][3]. The disease causes fever, diarrhea, dehydration, and even death among vulnerable people such as children, the elderly,

Antimicrobial Resistance Test
A detailed description of the percentage of the 41 Salmonella isolates' resistance to nine agents is shown in Table 2. Detailed information on the antimicrobial resistance and susceptibility profiles of the 41 isolates can be found in the Supplementary Material.

ESBL Characterization
ESBL-producing Salmonella was found in seven (17.1%; 95% CI: 7.2, 32.1) of the isolates and nine (22.0%; 95% CI: 10.6, 37.6) isolates possessed the bla CTX-M gene. No bla TEM , bla SHV , bla KPC , mcr-1, or mcr-2 were detected in any of the 41 Salmonella isolates. Eight of the nine bla CTX-M positive isolates were cultured from cows sourced from the north and northeast zones of China.

Discussion
Salmonella was cultured from approximately 30% of the sampled culled adult dairy cows at the abattoir in Wuhan, China. The prevalence recorded from this study was slightly higher than that reported in other studies from the USA (25%; 3%) and Italy (0%) in culled dairy cows [22][23][24]. In China, a prevalence of approximately 20% was reported in cattle sampled at multiple abattoirs and it was postulated that the feces and the hides posed a high risk for contamination with Salmonella [13]. Yang [25] found 13 Salmonella isolates from 78 pieces of beef in marketplaces in Shaanxi and Xu [26] reported the prevalence of Salmonella in beef increased recently (9.30-15.79%) in southern China. The high prevalence of Salmonella in feces in this research may be related to the place of origin.
With data collected by the National Foodborne Disease Outbreaks Surveillance System, Salmonella was reported to be responsible for 13.2% of all human foodborne cases in China in 2013 [12]. The per capita consumption of beef (predominantly sourced from beef cattle, culled dairy cows, and sometimes buffalo) is predicted to increase in China [9], with intensification of domestic production systems and international importation required to meet this demand [27]. Culled dairy cows comprise a large part of beef production in many countries. Currently, for example, in Canada and the USA, approximately 30% of beef production is sourced from culled dairy cows [28,29]. The high prevalence of Salmonella detected in the current study is a potential risk to humans, as well as increasing the risk of cross-contamination of carcasses within the abattoir environment [13]. There is a need to determine the occurrence of Salmonella within China's dairy farms and to investigate potential risk factors so that appropriate mitigation strategies can be developed and implemented [13,30,31].
From this study, about 50% of the isolated Salmonella demonstrated multi-drug resistance. The recorded multi-drug resistance in this study was higher than an earlier study conducted with cattle in China (0% and 35%, respectively) [6,13] but was lower than that found in dry milk-related infant food (75%) [32]. The antimicrobials with the highest resistance were ampicillin and tetracycline. These antibiotics belonged to the two most commonly used antibiotic groups in 2018 in the Chinese livestock industries, including chicken and pigs [33]. This finding is similar to that reported in culled dairy cows in California [22] and is likely linked to the global long-term use of these antibiotics for promoting growth and controlling mastitis in dairy cows [34].
The findings of this study showed high antimicrobial resistance towards ampicillin (AMP) and tetracyclines (TET). From this study, polymyxin B (PB) and imipenem (IPM) were observed to have the least resistance. These findings are in agreement with which the findings of others [25,35,36]. PB had been used as a therapeutic drug and feed additive since the 1980s in China. Its usage is expected to reach 16 500 tonnes by the year 2021 [37]. IPM, one of the carbapenems, is widely used against multi-resistant Gram-negative pathogens in human medicine, especially strains producing ESBL [38]. The resistance profiles towards PB and IPM are of public health importance as the encoding genes responsible for resistance are located on the mobile genetic elements [37]. Our study showed a low occurrence of the resistance genes (ESBL and mcr) in Salmonella. The absence of the mcr gene in the tested Salmonella isolates is likely a result of the ban on the use of polymyxin for promoting the growth of livestock in China in 2016 [39]. The monitoring of resistance genes in bacterial isolates is important to provide evidence on the reduction in the use of antimicrobials [16]. There is a need to determine and assess antimicrobial usage patterns in dairy farms in China, as has been undertaken in other countries [22,40].
In this study, we only collected fecal swabs from culled dairy cows at the abattoir but did not collect samples from other sites, such as the hides, which have been shown to be heavily contaminated with Salmonella in another study in China [13]. We were unable to characterize the distribution of Salmonella isolates based on the origin source of the cows due to the small sample size. Both the lack of detailed data on farms and of information on transport duration and condition of transportation were further limitations to this study, as others have highlighted the importance of these factors on shedding/carriage of Salmonella [41,42]. Our study highlights the potential hazard to food safety arising from Salmonella carriage by culled dairy cows processed at an abattoir. There is a need to determine the occurrence and carriage of Salmonella among dairy cows in China across the different regions to identify potential risk factors for infection so that appropriate on-farm control measures can be developed.

Study Design
This was an abattoir surveillance study conducted from June to July 2019 in Wuhan investigating the prevalence of Salmonella in fecal samples collected from adult culled dairy cows from Chinese dairy farms. The selected abattoir processed most of the culled dairy cows for local consumption in Wuhan. The source population included all culled dairy cows at the abattoir during the study time and these originated from the north (Inner Mongolia and Hebei Provinces), northeast (Heilongjiang, Jilin, and Liaoning Provinces), northwest (the Ningxia Hui Autonomous Region), central (Henan and Hubei Provinces), and east (Shandong Province) zones of China (Figure 1).

Study Populations and Selection of Study Subjects
A total of 138 culled adult dairy cows were sampled in this study. All sampled cows were Holstein Friesians, the predominant dairy breed within the dairy industry in China. A total of 3-5 animals from the 6-10 animals processed each day were selected based on their originating zones. Data on the provinces of origin were recorded prior to sampling. An animal's age was assessed by examination of their incisors, and then, categorized into four groups (2.5, 3.5, 4.5, and ≥5 years old).

Collection of Fecal Swabs
Feces were collected post stunning by inserting two cotton swabs into the rectum and rotating against the rectal wall. These were then immediately placed into a sterile tube containing 10 mL of buffered peptone water (BPW) (Hopebio Co., Ltd., Qingdao, China), stored on ice, and transported to the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University, Wuhan, where they were processed on the day of collection.

Isolation and Identification
The culture and isolation of Salmonella on the fecal swabs were conducted according to the international standard methods: EN-ISO 6579:2002/A1: 2007: Amendment 1: Annex D. Briefly, pre-enrichment with BPW was done at 37°C for 18-20 h and thereafter, 100 µl from the pre-enrichment homogenate was inserted on a modified semi-solid Rappaport-Vassiliadis (MSRV) medium (Hopebio Co., Ltd., Qingdao, China), while 1 mL of culture was transferred into 9 mL of Mueller-Kauffmann Tetrathionate novobiocin (MKTTn) broth (Hopebio Co., Ltd., Qingdao, China). Both preparations were then incubated at 41.5 and 37°C, respectively, for selective enrichment. After 48 h, suspected growth on the MSRV media and aliquots from all MKTTn Broth were streaked onto Xylose Lysine Deoxycholate (XLD) agar (Hopebio Co., Ltd., Qingdao, China), which was incubated aerobically at 37 • C for 24 h. At least three typical-looking colonies of Salmonella on each XLD agar plate were selected and were confirmed by performing a polymerase chain reaction (PCR) with the primers for the Salmonella enterotoxin (Stn) gene [43]. After this, Salmonella isolates with different colony morphologies but from the same sample were taken and purified for further molecular identification.

Characterization Using Rapid Molecular Detection Methods
The confirmed Salmonella were characterized using a PCR according to serotype-specific genes to detect S. Typhimurium, S. Enteritidis, S. Agona, S. Dublin, and S. Infantis [44][45][46] (Table 3). Table 3. Primers of the Salmonella serotype genes used in this study.

Antibiotic Susceptibility Test
Antibiotic susceptibility of the Salmonella isolates was determined by the broth microdilution method according to the guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI) [47,48]. A total of 9 antimicrobials representing 9 classes that are commonly used in animals and humans were used. This included AMP, TET, GEN, IPM, and SXT with recommended cut-off values by CLSI [48] and florfenicol (FFC), enrofloxacin (ENR), and ceftiofur (CEF), which had recommended cut-off values by CLSI M31-A3 [47]. The interpreted result of PB was unavailable in the recent CLSI criteria; therefore, a PB ≥ 4 µg/mL, recognized by CLSI in 1981 [49], was considered resistant. AMP, TET, GEN, ENR, FFC, and CEF were selected as these are frequently used in veterinary clinical food animal practice. Susceptibility to PB was also tested, as it can still be used for therapeutic purposes in China, although it has been officially banned for use as a growth-promoter [21]. Escherichia coli ATCC 25,922 was used as the quality control strain. Strains resistant to three or more antimicrobial agents were considered as being MDR.

Statistical Analysis
Data sorting was done in Microsoft Excel (Microsoft Office 2017) and thereafter analyzed using the statistical software SPSS version 22.0 (SPSS, Inc., Chicago, IL, USA). The prevalence of Salmonella at the animal level was estimated. The associations between age groups, locations, and patterns of resistance were determined by calculating odd ratios and their 95% CI. A Chi-square test was used to compare the prevalence between the age groups and locations and the results were interpreted at p value ≤ 0.05.

Conclusions
From this abattoir surveillance, it is concluded that the prevalence of Salmonella spp. among adult culled dairy cows was 29%. A high proportion (approximate 50%) of the isolates showed multiple-drug resistance. There is a need to determine the occurrence of Salmonella in dairy farms and the potential risk factors, and further develop appropriate on-farm control measures.