Comparison of culture-based methods for detecting <i>Campylobacter jejuni</i> and <i>Campylobacter hyointestinalis</i> in dairy cattle feces

<i>Campylobacter</i> is the most commonly reported cause of bacterial gastroenteritis in humans. Although cattle are recognized as a potential reservoir for several <i>Campylobacter</i> spp., most detection standards primarily target thermotolerant species, notably <i>Campylobacter jejuni</i> and <i>Campylobacter coli</i>, possibly underestimating the prevalence of others. This study evaluated the performance of different culture-based methods for detecting <i>Campylobacter</i> spp. in fecal samples collected rectally from dairy cows in a single commercial research herd across four time points. Six combinations of analyses were tested, involving either direct culture or enrichment broths (Preston and Bolton) paired with selective agar media (modified charcoal cefoperazone deoxycholate agar or Preston). Incubation was performed at 37°C under microaerobic conditions to support growth of non-thermotolerant species. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Only <i>C. jejuni</i> and <i>Campylobacter hyointestinalis</i> were detected. While <i>C. jejuni</i> prevalence declined over time, <i>C. hyointestinalis</i> remained consistently high throughout the study period (June-August 2024). The performance of tested detection methods varied significantly between species. Enrichment with Bolton broth increased the odds of detecting <i>C. hyointestinalis</i> by over 3,000-fold compared to the direct culture (odds ratio [OR] = 3,075; 95% confidence interval [CI]: 272-34,651). For <i>C. jejuni</i>, enrichment with Preston broth increased the odds of detection by more than eightfold (OR = 8.52, 95% CI: 3.16-22.9). Detection was primarily influenced by broth; selective agar had no independent effect. These findings emphasize the importance of method selection in the detection of <i>C. hyointestinalis</i> and <i>C. jejuni</i>, suggesting that <i>C. hyointestinalis</i> may be more prevalent in cattle than previously assumed.IMPORTANCE<i>Campylobacter</i> bacteria commonly cause gastrointestinal illness in humans and are frequently found in animals such as cattle. Detecting these bacteria in animal samples is important for understanding their occurrence and potential relevance to food safety. Many commonly used laboratory methods focus on <i>Campylobacter</i> spp. that grow under specific conditions, which may limit the detection of other species. This study compared several culture-based methods for the isolation of <i>Campylobacter</i> spp. from fecal samples collected from dairy cattle. Species identification was subsequently performed using MALDI-TOF MS. The findings show that detection varied, depending on the culture method and the <i>Campylobacter</i> spp., highlighting the potential impact of method choice on surveillance outcomes.