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ABSTRACT Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes located within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the cytotoxicity of C. difficile may be modulated by genomic variants outside the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between cytotoxicity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model cytotoxicity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci and changes in cytotoxicity. Combined, these data support a model of C. difficile disease wherein cytotoxicity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections, including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Finally, we highlight the intersection of loci identified by the GWAS for different phenotypes and clinical severity. This strategy to identify overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes. IMPORTANCE Clostridioides difficile has two major disease-mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study, we demonstrate via multiple approaches that genomic variants outside the PaLoc are associated with changes in cytotoxicity. These genomic variants may provide new avenues of exploration in the hunt for novel disease-modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical for clinical infection, such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and, as such, vary in their appropriateness for certain bacterial genome-wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important for both bacterial function and human clinical disease.

ABSTRACT Between October 2016 and June 2017, a C57BL/6J mouse colony that was undergoing a pre- and perinatal methyl donor supplementation diet intervention to study the impact of parental nutrition on offspring susceptibility to disease was found to suffer from an epizootic of unexpected deaths. Necropsy revealed the presence of severe colitis, and further investigation linked these outbreak deaths to a Clostridium difficile strain of ribotype 027 that we term 16N203. C. difficile infection (CDI) is associated with antibiotic use in humans. Current murine models of CDI rely on antibiotic pretreatment to establish clinical phenotypes. In this report, the C. difficile outbreak occurs in F1 mice linked to alterations in the parental diet. The diagnosis of CDI in the affected mice was confirmed by cecal/colonic histopathology, the presence of C. difficile bacteria in fecal/colonic culture, and detection of C. difficile toxins. F1 mice from parents fed the methyl supplementation diet also had significantly reduced survival (P < 0.0001) compared with F1 mice from parents fed the control diet. When we tested the 16N203 outbreak strain in an established mouse model of antibiotic-induced CDI, we confirmed that this strain is pathogenic. Our serendipitous observations from this spontaneous outbreak of C. difficile in association with a pre- and perinatal methyl donor diet suggest the important role that diet may play in host defense and CDI risk factors. IMPORTANCE Clostridium difficile infection (CDI) has become the leading cause of infectious diarrhea in hospitals worldwide, owing its preeminence to the emergence of hyperendemic strains, such as ribotype 027 (RT027). A major CDI risk factor is antibiotic exposure, which alters gut microbiota, resulting in the loss of colonization resistance. Current murine models of CDI also depend on pretreatment of animals with antibiotics to establish disease. The outbreak that we report here is unique in that the CDI occurred in mice with no antibiotic exposure and is associated with a pre- and perinatal methyl supplementation donor diet intervention study. Our investigation subsequently reveals that the outbreak strain that we term 16N203 is an RT027 strain, and this isolated strain is also pathogenic in an established murine model of CDI (with antibiotics). Our report of this spontaneous outbreak offers additional insight into the importance of environmental factors, such as diet, and CDI susceptibility.

When looking to improve animal welfare, it is essential to understand the viewpoints of stakeholders in the industry. Previous research has engaged stakeholders such as farmers, veterinarians, and the public to better understand future dairy industry directions. However, the use of perspective-taking as a tool to overcome farmer’s focus on current barriers to the industry has not been explored. Therefore, the aims of this study were to have farmers take the perspective of the cow, to elicit: (1) farm elements that are essential to create a cow-centric dairy system, and (2) how this system can be achieved in the next 50 years. To do this, we employed participatory methods (photo elicitation, timelining) to generate discussion with 12 New Zealand dairy farmers. Pasture access is frequently cited as an integral component for promoting good welfare in dairy cows, thus, this cohort of farmers was considered a good case study as they all had experience managing cows in grazing systems. Participants were asked to create a cow-centric farm design, and a pathway to implementation. Following thematic analysis of the multiple data sources, the results are presented under the following three themes: (1) cow-centric farm designs (environmental considerations, cow handling and care), (2) timeline to achieve and sequence of implementation (“low hanging fruit” and long-term investments), and (3) assumptions and resources needed (including financial considerations, technology, farmer buy-in, regulations and enforcement and other stakeholder involvement). By better understanding how current industry practice aligns, and also differs, from what the cow would want, we can work towards future management systems that incorporate the requirements of all stakeholders, including the cow.

ABSTRACT: There is growing evidence that animal personality is linked to a range of productivity traits in farm animals, including dairy cattle. To date, the methodology for assessing personality traits of dairy cattle is time consuming and often requires a test arena, which limits the opportunity for commercial farms to use personality traits of dairy cattle for individualized management. This study investigated whether personality traits of pastured dairy cattle, scored in short behavioral tests, are associated with daily behavioral patterns and milk production. Cows (n = 87) were exposed twice to each of 5 behavioral tests, where their responses to novel or putatively stressful situations were scored on an ordinal scale for investigative and reactive behavior toward a novel object and a novel human after exiting the milking parlor, response to restraint in a crush, avoidance distance from an approaching human in the paddock, and response to milking (step-kick behavior). Most behavior test scores were consistent over the 2 test repeats (using repeatability estimates, Mann-Whitney U test of difference between repeats, and Spearman rank correlation). Behavior test scores were subjected to a principal components analysis that revealed intertest relationships in 3 factors of correlated sets of test scores, interpreted as personality traits (fearful of humans, calm-investigative, and reactive to milking). Regression analyses determined how these traits were associated with daily grazing, ruminating, and lying behaviors, and milk production (after controlling for cow age, breed, lactation status, group, and climate variables). Cows that were more fearful of humans (high avoidance distance, reactive toward the novel human) had reduced lying time compared with cows that scored low on this trait. Cows that were more calm (during restraint) and investigative (toward the novel object) had greater grazing time, which likely contributed to their greater milk production compared with cows that scored low on this trait. Cows that were more reactive to milking produced less milk than cows that scored low on this trait. These results indicate that individual differences in daily behavior patterns and milk production of dairy cattle are associated with personality traits of cows, measured using several short behavioral tests. These methods may be useful for characterizing grazing dairy cattle on commercial farms, which could aid in understanding individual behavior patterns and provide opportunities for individualized management.

ABSTRACT: Separation of the cow and calf shortly after birth is a common practice on commercial dairy farms around the world, but there are emerging concerns about this practice among citizens and other stakeholders. Continuous improvement of on-farm management practices in collaboration with dairy sector stakeholders increases the likelihood that farming systems evolve in a way that is consistent with societal expectations. Few commercial dairy farms provide extended cow-calf contact, and there is little understanding of how dairy farmers view this practice. This study examined the views of New Zealand dairy farmers toward providing cow-calf contact, particularly the barriers to adopting such a system in a seasonal-calving pasture-based dairy system. Standard farm practice in New Zealand is to remove the calf from the cow around 24 h (but could be up to 48 h) after birth. These conventional farmers (n = 63) were randomly selected from the database of all dairy farmers in New Zealand and telephone-interviewed using a semistructured interview format. Their responses to questions about providing cow-calf contact (defined as contact beyond the standard practice of 48 h) were analyzed using thematic analysis. Three major themes of concern were identified by these farmers about providing cow-calf contact as follows: (1) poor animal welfare, especially the risk of mastitis in the dam, inadequate colostrum for the calf, increased stress from delayed separation, and lack of shelter for calves while outdoors with the cow; (2) increased labor and stress on staff; and (3) system-level changes required, including infrastructure and herd management. Many of these concerns stemmed from challenges related to the nature of large-scale seasonal-calving pasture-based dairy systems, where a large number of calves are born in a short period of time and may be exposed to inclement weather in late winter in some areas. Several small-scale farmers (n = 4) providing cow-calf contact for longer than standard practice of 48 h were also interviewed; all permitted contact for at least 4 wk. These farmers also felt that animal welfare and health were important, and that this was promoted in their cow-calf contact systems. Concerns about colostrum and mastitis, for example, were not raised by these farmers, but they did agree that additional infrastructure and shelter were important considerations for cow-calf contact systems. Some conventional farmers expressed cognitive dissonance in that they theoretically preferred cow-calf contact but could not see it being realistic or practical to implement. Farmers currently providing longer cow-calf contact may be a useful resource for better understanding of how practical and economical cow-calf contact systems could be adopted on commercial pastoral dairy farms.

In pasture-based systems, cows may spend several hours away from the paddock and may also walk several kilometres to meet daily milking requirements; this could lead cows to experience time constraints for grazing, ruminating and lying time in the paddock. This study investigated how walking distance and time spent away from the paddock affected daily behavioural patterns (i.e., grazing, ruminating and lying time) and milk yield. Dairy cows were managed in three rotationally grazed groups (n = 29 cows each) on the same farm and milked twice daily. A triaxial ear tag accelerometer on each cow recorded daily duration of grazing and ruminating, and a leg-based accelerometer recorded the daily lying time, for 13 days. GPS collars on four cows per group recorded the daily walking distance and total time away from the paddock for the group. A mixed repeated measures model tested how time off-paddock and walking distance affected the daily behavioural patterns; age, breed, milk yield and maximum ambient temperature were used as covariates with group as the observational unit. A second similar model tested how these factors affected milk yield. Walking distance and time spent away from the paddock were not correlated. When daily walking distance increased (to a maximum of 4 km/d), cows spent more time grazing and less time ruminating, but lying time was not affected. This result may, in part, be related to the greater energy expenditure demands for walking longer distances and milk production. When time away from the paddock increased (to a maximum of 4 h/d), cows spent less time lying, but grazing and ruminating times were not affected. Milk yield was not affected by walking distance, but one of the groups experienced a lower milk yield when time away from the paddock was increased. This result suggests that, for some cows, lying times may be shorter when experiencing a longer time away from the paddock, which may also affect milk yield. Overall, this study indicates that paddock behaviours are associated with walking distance to the milking parlour and time spent away from the paddock. Efforts to reduce walking distance and time spent away from the paddock are likely to provide cows with greater opportunity to engage in daily behaviours in the paddock that meet their needs and maintain their milk yield.