Pathogen surveillance and cross-sectional studies are critical epidemiological tools for investigating infectious agents and are essential for informing public health interventions (1–3). In the United States (U.S.), for instance, the National Animal Health Monitoring System (NAHMS) routinely conducts nationwide assessments to evaluate the health and management practices of various domestic animal populations (4). In fulfilling this role, NAHMS frequently employs surveillance methodologies, as demonstrated by its most recent 2021–2022 nationwide study of the U.S. swine industry (5). Within the cattle sector, NAHMS has successfully conducted four such national studies to date (6). Similarly, in China, surveillance efforts have led to the identification of 46 hydrogen sulfide (H₂S)-negative Salmonella serovars from a range of human, animal, and environmental sources (7).

H₂S-negative Salmonella serovars are considered “atypical” because “typical” Salmonella isolates produce H₂S (8). In recent years, the global incidence of H₂S-negative Salmonella isolates has increased (7), prompting increased calls for enhanced surveillance to support their detection (9, 10). Unfortunately, these atypical variants often go undetected in traditional Salmonella isolation procedures due to their deviation from the conventional phenotypic characteristics of the bacteria (11, 12). Specifically, H₂S production on various culture media, manifested as “black colonies,” has long served as a key phenotypic marker for identifying Salmonella and distinguishing it from other members of the Enterobacteriaceae family (7, 8, 13, 14). However, the absence of this trait in H₂S-negative strains increases the likelihood of false negatives during routine culture-based identification, unless detection methods that do not rely on H₂S production, such as those reviewed by Yang et al. (15), are employed.

Furthermore, these atypical strains have been associated with clinical diseases and outbreaks (16, 17). In Salmonella, the phs, cys, and asr operons are key genomic regions involved in H₂S production (17). The H₂S-negative phenotype is primarily attributed to missense mutations in the phs operon, which plays a central role in the reduction of thiosulfate to H₂S (12). Additional mutations, including those in the cysJ gene, have also been documented (17). In some isolates, mutations have been identified in other genes such as tetR (a putative transcriptional regulator of the TetR family), moaC (involved in molybdenum cofactor biosynthesis), and sph (streptomycin phosphotransferase) (12). Multiple studies across the globe have reported the isolation of these atypical Salmonella serovars from live animals and animal-derived products (8, 9, 14, 18, 19). However, aside from the identification of an H₂S-negative Salmonella Cerro isolate from cattle and cattle farm environments in the U.S. (20), no other such isolates have been reported from bovine sources or their environments. This is particularly notable given that cattle are well-established reservoirs of Salmonella (21).

In the U. S., cattle production accounts for the largest share of total cash receipts from agricultural commodities, making it the most significant sector in U.S. agriculture (22). The two primary production systems in the U.S. beef industry are cow–calf operations and cattle-feeding operations (23). Compared to cow–calf operations, cattle-feeding operations are higher-risk ventures that economically favor large-scale establishments, whereas cow–calf systems are more adaptable for small-scale, part-time farmers (24). These cow–calf operations are widely distributed across the country (25), with the southeastern U.S. serving as home to approximately one-third of all cow–calf producers (26). This makes the region an ideal geographic area for conducting research on cow–calf populations.

Cow–calf operations focus on producing annual calf crops, which are typically weaned or sold at ~6 months of age, either as stockers (weaned calves up to 1 year of age) or as yearlings destined for cattle-feeding operations (27). These systems serve as an important source of income for many part-time farmers, who often rely on local extension services to manage their animals.

Despite the critical role cow–calf operations play in U.S. agriculture, research specifically focused on rural cow–calf systems and their contribution to the prevalence of key pathogens, such as Salmonella, remains limited. This gap is partly due to their underrepresentation in national surveillance studies, which often overlook rural operations because of geographic and demographic constraints.

To address this gap, we conducted a study during a scheduled animal extension activity in rural Alabama, as part of the Cooperative Extension Program (CEP) (28), that involved deworming and vaccinating local cow–calf herds. The objective of this study was to investigate the presence of H₂S-negative Salmonella in cattle feces, as well as serovar diversity, antimicrobial resistance (AMR) profiles, and overall epidemiology of Salmonella among cow–calf farms in the southeastern U.S.

This cross-sectional study targeted rural, small-scale cow–calf operations, a population often underrepresented in national-level surveillance due to geographic and demographic constraints. The most recent cow–calf study, organized by the National Animal Health Monitoring System (NAHMS) in the U.S., took place in 2017 (51). That survey reported a Salmonella prevalence of 1.0% at the herd level and 4.4% at the animal level among cow–calf populations (51). Alabama, one of the major cow–calf states included in the NAHMS survey, recorded 20,004 beef operations as of December 2017 (51).

Although our study does not match the scale of national surveillance efforts, the Salmonella prevalence we observed, 27.8% at the herd level and 3.5% at the animal level, is notably high, given our sample size of 311 animals. This discrepancy may arise from methodological differences, some of which are discussed below.

National studies typically rely on stratified sampling strategies designed to generate broad, generalizable inferences across states (52). In contrast, our investigation focused on multiple herds within a defined subregion of a single state, specifically targeting rural, small-scale cow–calf operations. Additionally, inherent sampling biases arising from farm selection criteria and reliance on owner consent were not controlled in this study. National-level surveillance efforts often offer incentives and resources that help reduce the impact of such limitations.

Moreover, findings from localized studies may reflect regional and time-dependent factors, as noted in earlier research (53). For example, cattle in the southern U.S. have been shown to shed Salmonella in feces at higher rates than those in the northern regions (54). Seasonal variation may also influence shedding; previous studies have reported increased Salmonella prevalence in dairy herds during the summer months (55). Since our study was conducted as a one-time, cross-sectional survey in the spring, future studies conducted in other seasons could provide valuable insights into seasonal trends in Salmonella shedding among these populations.

Additional confounding factors likely contributed to the observed prevalence. Notably, poor biosecurity practices were evident across several farms. For instance, none of the operations required visitors to wear boot covers, a basic yet critical preventive measure. Based on these observations, we recommend increased outreach through veterinary extension services and biosecurity training programs targeted at rural cattle producers. Such efforts could substantially reduce Salmonella transmission risk.

Finally, public education regarding the importance of research participation is essential in these communities. Engaging rural farmers in ongoing disease surveillance through community-centered outreach can enhance participation and enable more comprehensive studies that better capture the burden of disease among underrepresented cattle populations.

The higher frequency of Salmonella shedding observed among adult cattle (3.8%) compared to calves (2.6%) in our study was not statistically significant. Although sampling bias related to animal age may have influenced this outcome, a similar study investigating clinical salmonellosis in dairy cattle reported a markedly higher prevalence rate of 49.3% in adults and 23.8% in calves (56). In the same study, the prevalence of fecal Salmonella shedding in apparently healthy cattle was 10.1% for adults and 5.4% for calves (56). This age-related dynamic in Salmonella shedding may be partly attributed to the passive immunity that calves acquire through colostrum, which offers temporary protection against infection (57, 58). In our study, calves were not classified as either pre-weaned or weaned, limiting our ability to assess this effect more precisely. Future research that specifically distinguishes between pre-weaned and weaned calves would provide a more comprehensive understanding of Salmonella shedding dynamics in this age group.

Nevertheless, the high frequency of Salmonella shedding among adult cattle may be associated with waning immunity, underlying health conditions, or physiological stressors such as pregnancy. Additionally, adult cattle outnumbered calves in all the herds visited, resulting in a smaller calf sample size. This sampling imbalance may also have contributed to the apparent disparity in Salmonella shedding between age groups.

Our findings contrast with those reported by Cummings et al. (59), who observed a higher prevalence of Salmonella in calves compared to adult cattle. A potential explanation for this discrepancy is that all of the animals in their study were clinically ill and admitted to a veterinary hospital, a setting that may have influenced infection rates. The role of age as a risk factor for Salmonella infection in cattle remains inconclusive. In a previous study, a recent investigation also found no significant association between age and Salmonella shedding (60).

Interestingly, with regard to antimicrobial resistance, two of the four tetracycline-resistant isolates in our study were recovered from calves. Previous studies have shown that calves are more likely to harbor resistant Escherichia coli strains than adults (61, 62). This pattern has been consistently reported over decades across diverse geographic regions and farming practices (61). One explanation offered is the unique composition of the intestinal microbiome in young calves, which may favor the persistence of multidrug-resistant strains (61). While the presence of tetracycline-resistant Salmonella in calves, as observed in our study supports this hypothesis, further research is needed to substantiate this relationship, especially regarding Salmonella isolates from cattle.

Similar to age, the sex of cattle was not significantly associated with Salmonella shedding. However, just as there were more adults than calves in our sampled herds, there were also more females than males, which likely introduced sampling bias. These limitations undoubtedly influenced our findings. Therefore, the lack of significant associations between age or sex and Salmonella shedding in cow–calf operations in this study should be interpreted cautiously, taking these methodological constraints into account.

The association between diarrhea and fecal shedding of Salmonella was also investigated in this study. It is important to emphasize that the diarrheic state of animals cannot be conclusively attributed to either nutritional or infectious causes, as both factors can influence fecal consistency in cattle. However, all animals sampled appeared clinically healthy, and most farms maintained their herds on grass pastures, supplementing with Bermuda grass hay and soy hull pellets twice daily, along with ad libitum access to water. Sampling occurred at various times of the day, including before, during, and after feeding.

Despite these variables, fecal shedding of Salmonella among diarrheic animals was the only statistically significant parameter in our study. This finding aligns with earlier studies that have linked bovine enteritis to Salmonella shedding, a common clinical manifestation of salmonellosis in adult cattle (63). Similar associations have been documented in other research (64, 65). Notably, some non-diarrheic animals in our study were also found to be shedding Salmonella, supporting the notion that asymptomatic carriers can serve as a persistent source of infection (54). These subclinical shedders pose a risk of undetected transmission, emphasizing the importance of maintaining strict hygiene protocols for farmworkers handling both symptomatic and asymptomatic animals.

Our study also demonstrated that individual animals can shed multiple Salmonella serovars simultaneously, a phenomenon supported by previous research (66). This underscores the importance of selecting more than one presumptive colony during Salmonella isolation, when resources allow. However, this recommendation must be balanced with practical limitations, as dominant serovars may be repeatedly isolated due to the often indistinguishable colony morphology on selective agar media. The number of colonies selected for further analysis should therefore be guided by the researcher's discretion, resource availability, and specific study objectives.

A noteworthy observation from our study was the prevalence of Salmonella Thompson, Salmonella Hadar, and Salmonella Braenderup—serovars not commonly associated with cattle globally (67). Nonetheless, Salmonella Thompson and Salmonella Braenderup have been isolated from bovine lymph nodes (68), which are significant reservoirs of Salmonella and may contribute to human infection (69), particularly when these infected lymph nodes are inadvertently incorporated into ground beef (68). Recent studies have shown that a considerable proportion of human salmonellosis cases linked to beef are associated with ground beef products (70).

In addition to direct transmission through meat, these serovars have also been implicated in environmental contamination, leading to outbreaks. For example, a recent multistate outbreak of Salmonella Braenderup in the U.S. affected 551 individuals across 34 states (71–73). Traceback investigations and comparative genomic analyses linked the outbreak strain to a water source used on a vegetable farm (71). These findings highlight the potential for Salmonella-infected cattle feces to contaminate nearby water bodies through runoff, posing broader public health risks. Research suggests that reintroduction events by host animals may contribute to the persistent contamination of farm water systems (74).

As expected, the presence of the tet(A) gene in Salmonella Hadar isolates conferred phenotypic resistance to tetracycline. This finding is consistent with previous studies that identified tet(A) as the most prevalent tetracycline resistance gene in Salmonella (75). Interestingly, although aminoglycoside resistance genes such as aph(3″)-Ib, aph(6)-Id, aac(6′)-Iy, and aac(6′)-Iaa were detected in several isolates, none exhibited phenotypic resistance to aminoglycosides. This discrepancy aligns with earlier research in which resistance genes were identified in 37 Salmonella isolates without corresponding phenotypic resistance (76). These findings suggest that resistance genes may be functionally inactive or “silenced” due to regulatory mutations, disrupted expression pathways, or the presence of cryptic or non-functional elements (77, 78). In particular, the aac (6′)-Iaa gene has been previously reported as a cryptic gene (79).

A contrasting phenomenon was observed with ciprofloxacin and levofloxacin. Despite the absence of known quinolone resistance genes, such as qnr, qep, or aac(6′)-Ib-cr (80), and no impactful point mutations detected, all isolates exhibited intermediate resistance to these fluoroquinolones. Resistance to quinolones is multifaceted and is commonly associated with mutations in genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) (80). In our study, however, the intermediate resistance observed may instead be attributed to the activity of multiple efflux pumps, particularly those belonging to the SOS-box family, including mdtABC-tolC, emrAB-tolC, and acrAB-tolC (81). The emrA and emrB genes specifically encode efflux systems capable of expelling fluoroquinolones, thereby contributing to reduced susceptibility (81). Although some resistance patterns were shared among serovars isolated from different farms, further studies are needed to understand the dynamics of antimicrobial resistance and selection in small-scale farming systems.

To the best of our knowledge, this study is the first to report the isolation of an H₂S-negative Salmonella Typhimurium strain from cattle feces. Globally, H₂S-negative Salmonella Typhimurium isolates have been reported from various animal and human sources. For instance, these isolates have been recovered from retail chicken and pork products in Japan and China (8, 18), while a national surveillance effort in China identified H₂S-negative Salmonella Typhimurium in humans and an unspecified livestock source (7). More recently, the monophasic variant of this serovar exhibiting the H₂S-negative phenotype was isolated from raw chicken samples in Portugal (9), and in Sweden, the monophasic variant was implicated in a human salmonellosis outbreak (16). However, none of these reports documented the isolation of this atypical serovar from cattle feces. In the U.S., H₂S-negative Salmonella has previously been detected in cattle, but the isolate belonged to the Salmonella Cerro serovar (20). The rarity of these atypical isolates likely reflects their ability to evade detection by conventional culture methods, although emerging evidence suggests that they are more widespread than previously assumed.

In terms of genetic mechanisms, the synonymous mutation observed in the cysI gene of our isolate is unlikely to have functional consequences, as such mutations are generally considered neutral (82). However, the missense mutation in the phsC gene may have functional implications, as such changes can affect protein stability and protein-protein interactions at the cellular level (83). Of the operons involved in H₂S production in Salmonella, the phs operon, which includes the phsA, phsB, and phsC genes, is considered the most critical (14). Mutations in this operon, particularly in the phsA, have been reported to result in premature stop codons (9, 14), which truncates the thiosulfate reductase enzyme required for H₂S production (12). Similar missense mutations in the phs and cys operons have also been described in previous studies (7, 14, 17). Nonetheless, it remains uncertain whether the phsC mutation identified in our isolate alone is sufficient to account for the H₂S-negative phenotype observed. Further functional studies are needed to clarify the impact of these mutations on Salmonella metabolism and detectability.

We acknowledge that biases associated with our sampling strategy, particularly in relation to age group and sex, represent limitations of this study. These biases were largely due to the voluntary participation of farmers, which constrained our ability to randomize the study population. This challenge has also been noted in similar surveillance efforts (56). Despite these constraints, our study provides important insights into a largely underrepresented population of rural cattle farms and reports, for the first time, the isolation of an H₂S-negative Salmonella Typhimurium serovar from cattle feces. Given the observed prevalence of Salmonella shedding, we recommend that extension services actively promote improved hygiene practices and biosecurity measures within cow–calf operations.

Furthermore, expanded studies involving larger sample sizes and broader geographic coverage are needed to provide a comprehensive overview of Salmonella prevalence and diversity in U.S. cattle and to better assess their public health implications. In conclusion, through an integrative approach combining classical microbiology, antimicrobial resistance profiling, and whole-genome sequencing, this study underscores the significant role that rural cow–calf herds may play as reservoirs for Salmonella.