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Mastitis is a major production disease, causing significant economic losses for dairy farmers in South-Asian countries, as well as other parts of the world. Udder health control programs (UHCP) have been established in developed countries as an effective strategy for mastitis control but have not yet been introduced in South-Asian low-income countries like Bangladesh, India, Pakistan, and Sri Lanka. To launch UHCP successfully in dairy herds in South-Asia, it is important to know the current prevalence and risk factors for subclinical mastitis (SCM). Therefore, a narrative literature review was conducted with the aim to describe the dairy sector, the prevalence of SCM and its causal agents, risk factors for mastitis occurrence and the control measures suggested by different studies conducted in the selected countries. The literature revealed that India had the highest cattle population. Milking was mainly done by hand in all of the studied countries. Stall feeding was done in Bangladesh and Sri Lanka and limited access to grazing was also reported in some farms in India and Pakistan. There was substantial variation in the prevalence of SCM between studies in all 4 countries, ranging from about 20% to about 80%, but the average prevalence across all studies was high (50%). The most common causal agents for SCM were non-aureus staphylococci (NAS), Staphylococcus (S.) aureus, Streptococcus spp. and Escherichia (E.) coli. The management related risk factors reported for SCM were stall feeding of cows, a higher stock density, cracked floors, open drains, the presence of flies, poor drainage, peri-parturient diseases, infrequent dung removal and earth floors. The control measures suggested in these studies were to improve the hygiene and sanitation of cows, to improve the cleanliness of farms and milker's hands, to apply dry cow therapy, supplementing micronutrients and routine screening for SCM combined with taking intervention measures like isolation of cows or milking infected cows last, and proper treatment. Also, full hand milking, complete milking, machine milking, and providing feed and water immediately after milking have been recommended. Finally, we show that current literature often studies the same set of (non-manageable) risk factors, so more research is needed to obtain a comprehensive picture of the determinants of SCM. Randomized controlled trials are needed to truly quantify the effect of intervention under field conditions. Altogether, our work gives an overview of the udder health situation in South-Asia and provides the basis for the design of UHCP in this region.

Staphylococcus aureus (S. aureus) is a major mastitis pathogen with a detrimental impact on udder health in dairy cattle. Although S. aureus is generally classified as a contagious mastitis pathogen, control measures aimed at preventing contagious transmission are not always effective. Previous studies showed that various extramammary sites can be colonized with S. aureus and could be a reservoir for S. aureus intramammary infections (IMI). The aim of this study was to determine the prevalence of S. aureus extramammary site colonization in Dutch dairy cattle and to compare the spa genotypes of S. aureus isolates from IMI to spa genotypes of isolates from extramammary sites. Six herds were visited and from cows with a composite milk somatic cell count ≥200,000 cells/mL quarter milk samples and swabs from various extramammary sites (hock, groin, udder cleft, nares, and feces) were taken. Extramammary site samples were processed by a two-step high salt selective culture and presence of S. aureus was confirmed by femA PCR. S. aureus isolates from milk and extramammary sites were compared by spa typing. The cow level colonization varied from 0% to 73%, and the prevalence of IMI in the sampled cows varied from 0% to 61% between herds. The extramammary site with the highest prevalence of colonization was the hock (23%) and the lowest prevalence of colonization was found for the nares (5%) and feces (5%). Spa typing of S. aureus isolates from either extramammary sites or milk showed that in most herds there were one or two predominant S. aureus spa genotypes present. Different S. aureus spa genotypes could be categorized into three groups based on the distribution between milk or extramammary sites: i) predominantly milk associated, ii) associated with both milk and extramammary sites, and iii) associated with extramammary sites. In conclusion, we showed that the prevalence of extramammary site colonization differed significantly between herds and extramammary sites and that specific S. aureus spa genotypes were associated with milk (IMI) or extramammary site colonization. Extramammary S. aureus reservoirs could be a source for IMI that cannot be eradicated by intervention measures aimed at contagious mastitis pathogens.

Since 2013, selective dry cow treatment (SDCT) has been the standard approach in the Netherlands where farmers select cows for the use of antimicrobials at drying-off. Shortly after its introduction, antimicrobial usage decreased significantly, and no significant association was found between the level of SDCT and clinical mastitis (CM). Obviously, at that time long-term associations could not be evaluated. This study aimed to provide insight into the methods and level of implementation of SDCT on Dutch dairy farms with a conventional milking system (CMS) or an automatic milking system (AMS) in 2016 and 2017, several years after the implementation of SDCT. Udder health and antimicrobial use were also assessed. For this study, 262 farmers recorded dry cow treatments as well as all CM cases in the period from May 1, 2016, until April 30, 2017. Additionally, somatic cell count (SCC) data on cow and herd level, treatment data on herd level and questionnaire results on udder health management were collected. Data were analyzed using descriptive statistics with differences between milking systems being evaluated using nonparametric univariable statistics. In the study period, SDCT was applied on almost all (98.8%) of the participating dairy farms. The main reason for applying antimicrobials at drying-off was either the SCC history during the complete previous lactation or the SCC at the last milk recording before drying-off. The median percentage of cows treated with antimicrobials was 48.5%. The average incidence rate of CM was 27.3 cases per 100 cows per year. From all CM cases that were registered per herd, on average 32.8% were scored as mild, 42.2% as moderate, and 25.0% as severe CM. The mean bulk tank SCC of the herds was 168,989 cells/mL. A cow was considered to have subclinical mastitis (SCM) if individual SCC was ≥150,000 cells/mL for primiparous and ≥250,000 cells/mL for multiparous cows. Passing these threshold values after 2 earlier low SCC values was considered a new case of SCM. The mean incidence rate of SCM in these herds was 62.5 cases per 100 cows per year. Bulk tank SCC and the incidence rate of SCM on farms with a CMS were statistically lower than on farms with an AMS, whereas the incidence rate of CM did not significantly differ between both groups of farms. The AMS farms had more cows per herd treated with antimicrobials at drying-off and a larger proportion of severe CM cases than did CMS farms. It is unknown whether the differences are due to the milking system or to other differences between both types of farms. This study showed the level of adoption of SDCT, udder health, and antimicrobial usage parameters several years after the ban on the preventive use of antimicrobials in animal husbandry. It found that udder health parameters did not differ from those found in Dutch studies before and around the time of implementing SDCT, whereas SDCT was widely applied on Dutch dairy farms during the study period. Therefore, it was concluded that Dutch dairy farmers were able to handle the changed policy of antimicrobial use at drying-off while maintaining indicators of a good udder health.

In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely. info:eu-repo/semantics/publishedVersion

Due to the zoonotic origin of SARS-Coronavirus 2 (SARS-CoV-2), the potential for its transmission from humans back to animals and the possibility that it might establish ongoing infection pathways in other animal species has been discussed. Cats are highly susceptible to SARS-CoV-2 and were shown experimentally to transmit the virus to other cats. Infection of cats has been widely reported. Domestic cats in COVID-19-positive households could therefore be a part of a human to animal to human transmission pathway. Here, we report the results of a qualitative risk assessment focusing on the potential of cat to human transmission in such settings. The assessment was based on evidence available by October 2021. After the introduction of SARS-CoV-2 to a household by a human, cats may become infected and infected cats may pose an additional infection risk for other members of the household. In order to assess this additional risk qualitatively, expert opinion was elicited within the framework of a modified Delphi procedure. The conclusion was that the additional risk of infection of an additional person in a household associated with keeping a domestic cat is very low to negligible, depending on the intensity of cat-to-human interactions. The separation of cats from humans suffering from SARS-CoV-2 infection should contribute to preventing further transmission.

After its first detection in 1996, the highly pathogenic avian influenza A(H5Nx) virus has spread extensively worldwide. HPAIv A(H5N1) was first detected in Indonesia in 2003 and has been endemic in poultry in this country ever since. However, Indonesia has limited information related to the phylodynamics of HPAIv A(H5N1) in poultry. The present study aimed to increase the understanding of the evolution and temporal dynamics of HPAIv H5N1 in Indonesian poultry between 2003 and 2016. To this end, HPAIv A(H5N1) hemagglutinin sequences of viruses collected from 2003 to 2016 were analyzed using Bayesian evolutionary analysis sampling trees. Results indicated that the common ancestor of Indonesian poultry HPAIv H5N1 arose approximately five years after the common ancestor worldwide of HPAI A(H5Nx). In addition, this study indicated that only two introductions of HPAIv A(H5N1) occurred, after which these viruses continued to evolve due to extensive spread among poultry. Furthermore, this study revealed the divergence of H5N1 clade 2.3.2.1c from H5N1 clade 2.3.2.1b. Both clades 2.3.2.1c and 2.3.2.1b share a common ancestor, clade 1, suggesting that clade 2.3.2.1 originated and diverged from China and other Asian countries. Since there was limited sequence and surveillance data for the HPAIv A(H5N1) from wild birds in Indonesia, the exact role of wild birds in the spread of HPAIv in Indonesia is currently unknown. The evolutionary dynamics of the Indonesian HPAIv A(H5N1) highlight the importance of continuing and improved genomic surveillance and adequate control measures in the different regions of both the poultry and wild birds. Spatial genomic surveillance is useful to take adequate control measures. Therefore, it will help to prevent the future evolution of HPAI A(H5N1) and pandemic threats.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are of clinical and economic importance for the global poultry industry. Many countries and integrations are involved in monitoring programmes to control both mycoplasma species. This review provides an extensive historic overview of the last seven decades on the development of the knowledge regarding the factors that influence the clinical expression of the disease, the epidemiology, and monitoring of both MG and MS. This includes the detection of new virulent strains, studies unravelling the transmission routes, survival characteristics, and the role of other avian hosts. Also the role of molecular typing tests in unravelling epidemiology and factors that complicate the interpretation of test results is discussed. The latter includes the presence of heterologous mycoplasma infections, the use of heterologous oil-emulsion vaccines, and the use of antibiotic treatments. Also the occurrence of MG and MS strains with low virulence and the use of live and/or inactivated MS and MS vaccines are discussed.

ObjectiveUnprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure.MethodsAir sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks’ housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR.ResultsInside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000–10 000 copies/m3). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks’ housing units and surfaces contained SARS-CoV-2 RNA.ConclusionsInfected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities.

Subclinical mastitis (SCM) in water buffalo is responsible for reduced milk yield and quality. This cross-sectional study was carried out to a) estimate the prevalence of SCM, b) identify risk factors associated with SCM, and c) identify farm-level risk factors associated with bulk milk somatic cell count (BMSCC). The buffalo farms included in this study represented five rearing systems: free-range, semi-free-range, household, semi-intensive, and intensive, providing a total of 3491 functional quarters of 880 lactating buffalo on 248 farms. The California mastitis test score was used to identify SCM. Bulk milk samples (n = 242) were used for farm-level BMSCC. Quarter and buffalo-level risk factors for SCM were measured using questionnaires and observations. The overall SCM prevalence was high at 27.9% at the quarter-level (25th and 75th percentiles: 8.3% and 41.7%) and 51.5% at buffalo-level (25th and 75th percentiles: 33.3% and 66.7%). The geometric mean BMSCC was 217,000 cells/mL of milk (ranging from 36,000-1,213,000 cells/mL), which is low on average, but some farms could improve substantially. The buffalo rearing system, udder location (left versus right), teat shape, udder asymmetry, number of milkers, and having a quarantine facility were associated with buffalo udder health. Our findings suggest that mainly using free-range rearing systems may help decrease the prevalence of SCM primarily by employing buffalo breeding and better farm biosecurity, and udder health control strategies can be designed based on our findings.

This study aimed to evaluate the effect of an extended voluntary waiting period (VWP) on SCC, SCC elevations and clinical mastitis incidence during the complete lactation and the first 6 weeks of the next lactation. Holstein-Friesian dairy cows (N = 154) were blocked for parity, expected milk yield, calving season and breeding value for persistency and were randomly distributed across 3 VWP (50, 125, or 200 d: VWP-50, VWP-125, VWP-200). Cows were monitored from calving until 6 weeks into the next lactation, or until culling. An elevation of SCC in milk was defined as SCC in milk ≥200 000 cells/ml after two previous weeks with SCC < 200 000 cells/ml. Over the complete lactation, extending the VWP did not affect SCC elevations and the occurrence of clinical mastitis per lactation or per cow per year. There was no clear effect of VWP length on SCC in the complete lactation, except that multiparous cows in VWP-125 had a higher SCC compared with multiparous cows in VWP-50. Dry-off antibiotic usage per cow per year was lower in VWP-200 compared with VWP-50 for multiparous cows. In the first 6 weeks of the next lactation, cows in VWP-200 had a higher SCC compared with cows in VWP-50, with no effect of VWP on the number of elevations of SCC or the occurrence of clinical mastitis. Extending the VWP may therefore be used to reduce the frequency of transition periods and the associated use of dry-cow antibiotics, with limited impact on udder health, and a similar occurrence of SCC elevations and clinical mastitis per year. This study aimed to evaluate the effect of an extended voluntary waiting period (VWP) on SCC, SCC elevations and clinical mastitis incidence during the complete lactation and the first 6 weeks of the next lactation. Holstein-Friesian dairy cows (N = 154) were blocked for parity, expected milk yield, calving season and breeding value for persistency and were randomly distributed across 3 VWP (50, 125, or 200 d: VWP-50, VWP-125, VWP-200). Cows were monitored from calving until 6 weeks into the next lactation, or until culling. An elevation of SCC in milk was defined as SCC in milk ≥200 000 cells/ml after two previous weeks with SCC < 200 000 cells/ml. Over the complete lactation, extending the VWP did not affect SCC elevations and the occurrence of clinical mastitis per lactation or per cow per year. There was no clear effect of VWP length on SCC in the complete lactation, except that multiparous cows in VWP-125 had a higher SCC compared with multiparous cows in VWP-50. Dry-off antibiotic usage per cow per year was lower in VWP-200 compared with VWP-50 for multiparous cows. In the first 6 weeks of the next lactation, cows in VWP-200 had a higher SCC compared with cows in VWP-50, with no effect of VWP on the number of elevations of SCC or the occurrence of clinical mastitis. Extending the VWP may therefore be used to reduce the frequency of transition periods and the associated use of dry-cow antibiotics, with limited impact on udder health, and a similar occurrence of SCC elevations and clinical mastitis per year.

Administering intramammary antimicrobials to all mammary quarters of dairy cows at drying-off [i.e., blanket dry cow therapy (BDCT)] has been a mainstay of mastitis prevention and control. However, as udder health has considerably improved over recent decades with reductions in intramammary infection prevalence at drying-off and the introduction of teat sealants, BDCT may no longer be necessary on all dairy farms, thereby supporting antimicrobial stewardship efforts. This narrative review summarizes available literature regarding current dry cow therapy practices and associ-ated impacts of selective dry cow therapy (SDCT) on udder health, milk production, economics, antimicro-bial use, and antimicrobial resistance. Various methods to identify infections at drying-off that could benefit from antimicrobial treatment are described for select-ing cows or mammary quarters for treatment, includ-ing utilizing somatic cell count thresholds, pathogen identification, previous clinical mastitis history, or a combination of criteria. Selection methods may be enacted at the herd, cow, or quarter levels. Producers' and veterinarians' motivations for antimicrobial use are discussed. Based on review findings, SDCT can be ad-opted without negative consequences for udder health and milk production, and concurrent teat sealant use is recommended, especially in udder quarters receiving no intramammary antimicrobials. Furthermore, herd selection should be considered for SDCT implementa-tion in addition to cow or quarter selection, as BDCT may still be temporarily necessary in some herds for optimal mastitis control. Costs and benefits of SDCT vary among herds, whereas impacts on antimicrobial resistance remain unclear. In summary, SDCT is a vi-able management option for maintaining udder health and milk production while improving antimicrobial stewardship in the dairy industry.

Indoor Air Quality (IAQ) is strongly associated with animal health and wellbeing. To identify possible problems of the indoor environment of macaques (Macaca spp.), we assessed the IAQ. The temperature (°C), relative humidity (%) and concentrations of inhalable dust (mg/m3), endotoxins (EU/m3), ammonia (ppm) and fungal aerosols were measured at stationary fixed locations in indoor enclosures of group-housed rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). In addition, the personal exposure of caretakers to inhalable dust and endotoxins was measured and evaluated. Furthermore, the air circulation was assessed with non-toxic smoke, and the number of times the macaques sneezed was recorded. The indoor temperature and relative humidity for both species were within comfortable ranges. The geometric mean (GM) ammonia, dust and endotoxin concentrations were 1.84 and 0.58 ppm, 0.07 and 0.07 mg/m3, and 24.8 and 6.44 EU/m3 in the rhesus and cynomolgus macaque units, respectively. The GM dust concentrations were significantly higher during the daytime than during the nighttime. Airborne fungi ranged between 425 and 1877 CFU/m3. Personal measurements on the caretakers showed GM dust and endotoxin concentrations of 4.2 mg/m3 and 439.0 EU/m3, respectively. The number of sneezes and the IAQ parameters were not correlated. The smoke test revealed a suboptimal air flow pattern. Although the dust, endotoxins and ammonia were revealed to be within accepted human threshold limit values (TLV), caretakers were exposed to dust and endotoxin levels exceeding existing occupational reference values.

In the Dutch national surveillance system, an increasing number of reports were received in the summer of 2017 from farmers about unusual behavior of their cows. The cows were grouping during the day in summer in one part of the barn and did not move for several hours, which, according to the farmers, led to reduced food and water intake and lying time and resulted in decreased milk production and increased risk of lameness. Many farmers perceived magnetic fields from, for instance, high-voltage lines, automated milking systems, or solar panels as possible causes for the behavior of their cows. Our aim for the study was to study potential factors such as magnetic fields and other factors such as barn climate and insect burden for adverse grouping behavior of dairy cows in the barn. For each case herd, 2 control herds were selected in the same postal area code. A case was a herd in which cattle grouped at least on 7 occasions in a month for several hours. In a control herd, the cows were in the barn during the same time period as in the matching case herd but did not show adverse grouping behavior. A questionnaire was administered by telephone in 31 case herds and 62 control herds. The questionnaire gathered information on behavior of the cows and potential risk factors. In addition, data on the distance of the herd to high-voltage lines was obtained. From a total of 74 variables, all variables with a P-value ≤0.10 were included in full multivariable logistic regression model. Backward selection was carried out at P ≤ 0.10. The grouping behavior of the cows started in most herds in June, was seen only during the day, and lasted mostly 6 to 8 h, with cows often grouped in the northern part of the barn. Identified risk factors appeared to be recently constructed barns, measured stray voltage in barns, and presence of fans in barns. Given the cross-sectional design of the case-control study, causality for these risk factors leading to adverse behavior of the cows could not be proven. Dissemination of the results to farmers hopefully results in measures that can prevent the unusual grouping behavior of cows.

Background Hepatitis E virus (HEV) genotype 3 and 4 is a zoonosis that causes hepatitis in humans. Humans can become infected by consumption of pork or contact with pigs. Pigs are the main reservoir of the virus worldwide and the virus is present on most pig farms. Main body Though HEV is present on most farms, the proportion of infected pigs at slaughter and thus the level of exposure to consumers differs between farms and countries. Understanding the cause of that difference is necessary to install effective measures to lower HEV in pigs at slaughter. Here, HEV studies are reviewed that include infection dynamics of HEV in pigs and on farms, risk factors for HEV farm prevalence, and that describe mechanisms and sources that could generate persistence on farms. Most pigs become infected after maternal immunity has waned, at the end of the nursing or beginning of the fattening phase. Risk factors increasing the likelihood of a high farm prevalence or proportion of actively infected slaughter pigs comprise of factors such as farm demographics, internal and external biosecurity and immunomodulating coinfections. On-farm persistence of HEV is plausible, because of a high transmission rate and a constant influx of susceptible pigs. Environmental sources of HEV that enhance persistence are contaminated manure storages, water and fomites. Conclusion As HEV is persistently present on most pig farms, current risk mitigation should focus on lowering transmission within farms, especially between farm compartments. Yet, one should be aware of the paradox of increasing the proportion of actively infected pigs at slaughter by reducing transmission insufficiently. Vaccination of pigs may aid HEV control in the future.

In Europe, monitoring of breeding stock for Salmonella Pullorum (SP) or Salmonella Gallinarum (SG) infections is compulsory at the point of lay. Vaccinations against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are increasingly administered in Europe. These vaccines might induce cross-reactions in the rapid plate agglutination (RPA) SP/SG test due to shared O-antigens, possibly resulting in a lower test specificity. The extent to which the specificity of SP/SG serological tests is influenced by SE and/or ST vaccinations in the field has not been reported. In this paper, we report the diagnostic and flock specificity of the commercially available RPA SP/SG test using 1:2-1:16 serum dilutions on four panels of sera: SPF sera, field sera from flocks of varying age and SE/ST vaccination status, and reference sera from an international proficiency testing scheme. The results showed that the use of live SE/ST vaccines did not influence the specificity of the RPA SP/SG test. Inactivated vaccines showed a drop of the diagnostic specificity to 96.54% and a flock specificity of 34.1% when the 1:2 serum dilution was used. The 1:8 serum dilution showed a diagnostic specificity of 99.41% and a flock specificity of 86.4%. In conclusion, the use of SE/ST vaccines has either no effect or a modest effect on the specificity of the RPA SP/SG test used to monitor flocks. The main factors are the type of vaccine, and the serum dilution used for testing and a cut-off.

Bovine tuberculosis (bTB) is a chronic, infectious and zoonotic disease of domestic and wild animals caused mainly by Mycobacterium bovis. This study investigated farm management factors associated with recurrent bTB herd breakdowns (n = 2935) disclosed in the period 23 May 2016 to 21 May 2018 and is a follow-up to our 2020 paper which looked at long duration bTB herd breakdowns. A case control study design was used to construct an explanatory set of farm-level management factors associated with recurrent bTB herd breakdowns. In Northern Ireland, a Department of Agriculture Environment and Rural Affairs (DAERA) Veterinarian investigates bTB herd breakdowns using standardised guidelines to allocate a disease source. In this study, source was strongly linked to carryover of infection, suggesting that the diagnostic tests had failed to clear herd infection during the breakdown period. Other results from this study associated with recurrent bTB herd breakdowns were herd size and type (dairy herds 43% of cases), with both these variables intrinsically linked. Other associated risk factors were time of application of slurry, badger access to silage clamps, badger setts in the locality, cattle grazing silage fields immediately post-harvest, number of parcels of land the farmer associated with bTB, number of land parcels used for grazing and region of the country.

OHEJP Project: BIOPIGEE Pigs are a reservoir of hepatitis E virus (HEV), which causes hepatitis in humans. To study the epidemiology of HEV in pig farms, sampling methods are currently used that cause discomfort to pigs, such as rectal sampling. In line with the 3Rs principle, we aimed to evaluate non-invasive methods to detect pens with HEV-shedding pigs. Twenty-eight pens of one farm were sampled cross-sectionally. Individual rectal swabs (IRS) were collected to determine prevalence within pens. Four pen-level samples were compared: a pool of IRS per pen (P), boot socks (BS), oral fluid (OF) and pooled faecal droppings (FD). Each sample was tested by RT-PCR and the sensitivity and specificity of each method was determined by Bayesian latent class analysis. According to IRS, 19/28 pens were HEV positive. BS had a sensitivity of 95% and detected HEV in pens with 10% of pigs shedding; however, specificity was below 30%. FD were comparably accurate to P, with a sensitivity and specificity of 94% and 86%, respectively. BS sampling is thus advised to detect early shedding of HEV or pen contamination, and FD to determine the duration of shedding. This study demonstrates that non-invasive sampling can replace rectal swabs in research on HEV in pigs.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens. Supplementary Information The online version contains supplementary material available at 10.1186/s13567-022-01026-z.

Understanding under which conditions conjugative plasmids encoding antibiotic resistance can invade bacterial communities in the gut is of particular interest to combat the spread of antibiotic resistance within and between animals and humans. We extended a one-compartment model of conjugation to a two-compartment model, to analyse how differences in plasmid dynamics in the gut lumen and at the gut wall affect the invasion of plasmids. We compared scenarios with one and two compartments, different migration rates between the lumen and wall compartments, and different population dynamics. We focused on the effect of attachment and detachment rates on plasmid dynamics, explicitly describing pair formation followed by plasmid transfer in the pairs. The parameter space allowing plasmid invasion in the one-compartment model is affected by plasmid costs and intrinsic conjugation rates of the transconjugant, but not by these characteristics of the donor. The parameter space allowing plasmid invasion in the two-compartment model is affected by attachment and detachment rates in the lumen and wall compartment, and by the bacterial density at the wall. The one- and two-compartment models predict the same parameter space for plasmid invasion if the conditions in both compartments are equal to the conditions in the one-compartment model. In contrast, the addition of the wall compartment widens the parameter space allowing invasion compared with the one-compartment model, if the density at the wall is higher than in the lumen, or if the attachment rate at the wall is high and the detachment rate at the wall is low. We also compared the pair-formation models with bulk-conjugation models that describe conjugation by instantaneous transfer of the plasmid at contact between cells, without explicitly describing pair formation. Our results show that pair-formation and bulk-conjugation models predict the same parameter space for plasmid invasion. From our simulations, we conclude that conditions at the gut wall should be taken into account to describe plasmid dynamics in the gut and that transconjugant characteristics rather than donor characteristics should be used to parameterize the models.

Staphylococcus pseudintermedius can be transmitted between dogs and their owners and can cause opportunistic infections in humans. Whole genome sequencing was applied to identify the relatedness between isolates from human infections and isolates from dogs in the same households. Genome SNP diversity and distribution of plasmids and antimicrobial resistance genes identified related and unrelated isolates in both households. Our study shows that within-host bacterial diversity is present in S. pseudintermedius, demonstrating that multiple isolates from each host should preferably be sequenced to study transmission dynamics.

There is great interest in identifying gut microbiota development patterns and underlying assembly rules that can inform strategies to improve broiler health and performance. Microbiota stratification using community types helps to simplify complex and dynamic ecosystem principles of the intestinal microbiota. This study aimed to identify community types to increase insight in intestinal microbiota variation between broilers and to identify factors that explain this variation. A total of 10 well-performing poultry flocks on four farms were followed. From each flock, the cecal content of nine broilers was collected at 7, 14, and 35 days posthatch. A total of two robust community types were observed using different clustering methods, one of which was dominated by 7-day-old broilers, and one by 35-day-old broilers. Broilers, 14-day-old, were divided across both community types. This is the first study that showed conserved cecal microbiota development trajectories in commercial broiler flocks. In addition to the temporal development with age, the cecal microbiota variation between broilers was explained by the flock, body weight, and the different feed components. Our data support a conserved development of cecal microbiota, despite strong influence of environmental factors. Further investigation of mechanisms underlying microbiota development and function is required to facilitate intestinal health promoting management, diagnostics, and nutritional interventions.

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2 positive cats and dogs in ten infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of ten farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all ten farms. This study shows evidence of SARS-CoV-2 infection in twelve feral cats and two dogs. Eleven cats (19%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10% to 18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2.

Humans can become infected with hepatitis E virus (HEV) by consumption of undercooked pork. To reduce the burden of HEV in humans, mitigation on pig farms is needed. HEV is found on most pig farms globally, yet within-farm seroprevalence estimates vary considerably. Understanding of the underlying variation in infection dynamics within and between farms currently lacks. Therefore, we investigated HEV infection dynamics by sampling 1711 batches of slaughter pigs from 208 Dutch farms over an 8-month period. Four farm types, conventional, organic, and two types with strict focus on biosecurity, were included. Sera were tested individually with an anti-HEV antibody ELISA and pooled per batch with PCR. All farms delivered seropositive pigs to slaughter, yet batches (resembling farm compartments) had varying results. By combining PCR and ELISA results, infection moment and extent per batch could be classified as low transmission, early, intermediate or late. Cluster analysis of batch infection moments per farm resulted in four clusters with distinct infection patterns. Cluster 1 farms delivered almost exclusively PCR negative, ELISA positive batches to slaughter (PCR−ELISA+), indicating relatively early age of HEV infection. Cluster 2 and 3 farms delivered 0.3 and 0.7 of batches with intermediate infection moment (PCR+ELISA+) respectively and only few batches with early infection. Cluster 4 farms delivered low transmission (PCR−ELISA−) and late infection (PCR+ELISA−) batches, demonstrating that those farms can prevent or delay HEV transmission to farm compartments. Farm type partly coincided with cluster assignment, indicating that biosecurity and management are related to age of HEV infection.

BackgroundThe FAIR (Findable, Accessible, Interoperable, Reusable) principles were proposed in 2016 to set a path towards reusability of research datasets. In this systematic review, we assessed the FAIRness of datasets associated with peer-reviewed articles in veterinary epidemiology research published since 2017, specifically looking at salmonids and dairy cattle. We considered the differences in practices between molecular epidemiology, the branch of epidemiology using genetic sequences of pathogens and hosts to describe disease patterns, and non-molecular epidemiology.ResultsA total of 152 articles were included in the assessment. Consistent with previous assessments conducted in other disciplines, our results showed that most datasets used in non-molecular epidemiological studies were not available (i.e., neither findable nor accessible). Data availability was much higher for molecular epidemiology papers, in line with a strong repository base available to scientists in this discipline. The available data objects generally scored favourably for Findable, Accessible and Reusable indicators, but Interoperability was more problematic.ConclusionsNone of the datasets assessed in this study met all the requirements set by the FAIR principles. Interoperability, in particular, requires specific skills in data management which may not yet be broadly available in the epidemiology community. In the discussion, we present recommendations on how veterinary research could move towards greater reusability according to FAIR principles. Overall, although many initiatives to improve data access have been started in the research community, their impact on the availability of datasets underlying published articles remains unclear to date.

Plasmid-mediated antimicrobial resistance is a major contributor to the spread of resistance genes within bacterial communities. Successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission. While these key parameters are readily quantified in vitro , the influence of interactions with other microbiome members is largely unknown. Here, we investigated the influence of three genera of lactic acid bacteria (LAB) derived from the chicken gastrointestinal microbiome on the spread of an epidemic narrow-range ESBL resistance plasmid, IncI1 carrying bla CTX-M-1 , in mixed cultures of isogenic Escherichia coli strains. Secreted products of LAB decreased E. coli growth rates in a genus-specific manner but did not affect plasmid transfer rates. Importantly, we quantified plasmid transfer rates by controlling for density-dependent mating opportunities. Parametrization of a mathematical model with our in vitro estimates illustrated that small fitness costs of plasmid carriage may tip the balance towards plasmid loss under growth conditions in the gastrointestinal tract. This work shows that microbial interactions can influence plasmid success and provides an experimental-theoretical framework for further study of plasmid transfer in a microbiome context.

We report a case of rabies in a sand cat, Felis margarita, from Saudi Arabia. This incident suggests hitherto undocumented spillover infection in this species. Our report highlights the shortcomings of passive reporting, necessity of wildlife surveillance, and the need for a comprehensive One Health approach to disease prevention and control.

Online somatic cell count (SCC) measurement is widely used in dairy herds milked with automatic milking systems (AMS) and gives the opportunity to closely monitor individual cow udder health. Using automated SCC data, we observed cows displaying a remarkably regularly fluctuating SCC (rfSCC) pattern, which is described in this study. We aimed to (1) estimate the prevalence of rfSCC in cows milked by AMS, (2) characterize the rfSCC pattern, and (3) identify factors potentially associated with the rfSCC pattern. We analyzed 30-d episodes of composite SCC recordings of 1,000 cows from 55 dairy herds from 6 countries using an AMS with automated SCC measurement, and we identified the rfSCC pattern in 4.7% (95% CI: 3.5-6.2%) of these episodes. The rfSCC episodes had a median SCC of 701 × 1,000 cells/mL (2.5-97.5% quantile: 539-1,162), a median amplitude of 552 × 1,000 cells/mL (2.5-97.5% quantile: 409-886), and a median cycle length of 4.1 d (2.5-97.5% quantile: 3.7-4.9). Bacteriological culture data from quarter-milk samples collected every 2 wk in 1 Dutch AMS herd were analyzed, yielding no clear association between pathogen species and the rfSCC pattern found in that herd. Altogether, we described an intriguing phenomenon, present in almost 5% of the cows during a 1-mo study period. Further work is needed to quantify its importance in terms of udder health, but also to elucidate the mechanism behind this remarkable SCC pattern.

Common aims of animal health surveillance systems are the timely detection of emerging diseases and health status monitoring. This study aimed to evaluate the coverage and representativeness of passive surveillance components for cattle and swine in the Netherlands from 2015–2019. The passive surveillance components consisted of a telephone helpdesk for veterinary advice and diagnostic and postmortem facilities. Spatial analysis showed heterogeneity (range in RR = 0.26–5.37) of participation across the Netherlands. Generalized linear mixed models showed that distance to the diagnostic facility and farm density were associated with the number of contacts of farmers with the helpdesk and postmortem examination. The contact rate of veterinary practices was associated with their number of clients, ranging in RR from 0.39 to 1.59. We concluded that the evaluation indicated differences in coverage of the passive surveillance components across regions, farms and veterinary practices. Due to the absence of emerging infections in the study period, we were unable to estimate the consequences of the observed differences for the early detection of disease. Nevertheless, regions and veterinary practices with low participation in passive surveillance might be a risk for early detection, and consequently, further understanding of the motivation to participate in passive surveillance components is needed.

Bovine viral diarrhea (BVD) is controlled in many countries by detection and culling of persistently infected (PI) animals. The most important risk factor for BVDV introduction is purchase. An introduced cow can be PI and transmit the virus to other cattle in the herd. If she is not PI but is pregnant, there is still a risk because the subsequently born calf may be PI, when she encountered the virus in early pregnancy. To control this risk, all cows > 1 year from non-BVDV-free herds that are introduced in herds that participate in the Dutch BVDV control program are tested for virus and antibodies. Depending on the results, subsequent measures such as suspension of the BVDV-free status, removing the animals from the herd, or testing the off-spring of the cow for virus, are undertaken. The aim of this study was to evaluate the results of this risk mitigating measure. Data on cattle movements, calving's, herd-level BVDV status, and animal-level test data were available from all dairy herds that participated in the national BVDV control program (>14,000 dairy herds) for the year 2019. The data were combined and parameters of interest were calculated, i.e., (i) the number of purchased BVD virus positive cattle and (ii) the number of BVD virus positive calves born from purchased cows within 9 months after introduction. In 2019, 217,301 cattle were introduced in Dutch dairy herds that participated in the BVDV control program. Of these, 49,820 were tested for presence of BVD virus and 27 (0.05%) cows introduced in 21 different herds tested BVD virus positive. Out of 46,727 cattle that were tested for antibodies, 20.5% tested positive. The seropositive cows produced 4,341 viable calves, of which 3,062 were tested for virus and subsequently, 40 (1.3%) were found BVD virus positive. These 40 BVD virus positive calves were born in 23 herds. The risk mitigating measure led to detection of 67 BVD virus positive animals in 44 unique herds in 2019. This study makes plausible that the probability and impact of re-introduction of BVDV can be minimized by testing introduced cattle and their subsequently born calves.

Salmonella entericaserotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+and CD8+T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes signifcant economic losses to the swine industry worldwide. Little is known regarding the epidemiology of this infection in tropical countries. To address this problem in Costa Rica, a seroepidemiological study was carried out in two phases. In the frst phase, a pilot study was conducted in nine farms with the clinical diagnosis of PRRSV. In total, 265 pig serum samples were collected from animals ranging in age from 1 to 15 weeks of age. This study aimed to establish the duration of maternal immunity in piglets, to identify the period of viremia, and to determine when seroconversion occurs. In the second phase, a cross-sectional serology study was performed on a representative sample of the Costa Rican national herds in the second phase. The twenty-fve selected farms represent all provinces and were classifed according to herd size (100 to 2000 sows). In each farm, pigs aged 8, 10, and 12 weeks were sampled, as well as gilts based on the pilot study. In total 1281 pigs were sampled across all 25 farms. The aim of the cross-sectional study was to quantify the seroprevalence of PRRSV in Costa Rican pig farms and to describe its geographical distribution in this tropical country. The prevalence of positive farms was 44% (11/25), and these farms were located in six of the seven provinces of Costa Rica. Overall, 58% (344/596) of the pigs were seropositive to PRRSV. The age of the pigs and the ecozone where farms were located were signifcantly related with PRRSV seroprevalence in animals and herds, respectively. El virus del síndrome reproductivo y respiratorio porcino (PRRSV) causa importantes pérdidas económicas a la industria porcina en todo el mundo. Se sabe poco sobre la epidemiología de esta infección en los países tropicales. Para abordar este problema para abordar este problema en Costa Rica, se llevó a cabo un estudio seroepidemiológico en dos fases. En la primera fase, se realizó un estudio piloto en nueve granjas con diagnóstico clínico de PRRSV. En total, se recogieron 265 muestras de suero de cerdos de edades comprendidas entre de 1 a 15 semanas de edad. Este estudio tenía como objetivo establecer la duración de la inmunidad materna en los lechones, identificar el período de viremia y determinar cuándo se produce la seroconversión. En la segunda fase, se realizó un estudio transversal de serología realizado en una muestra representativa de las piaras nacionales de Costa Rica en la segunda fase. Las veinticinco granjas seleccionadas representan todas las provincias y se clasificaron según el tamaño de la piara (100 a 2000 cerdas). En cada granja se muestrearon cerdos de 8, 10 y 12 semanas, así como cerdas jóvenes, según el estudio piloto. En total se muestrearon 1281 cerdos en las 25 explotaciones. El objetivo del estudio transversal era cuantificar la seroprevalencia del PRRSV en las explotaciones porcinas costarricenses y describir su distribución geográfica en este país tropical. La prevalencia de explotaciones positivas fue del 44% (11/25), y estas explotaciones estaban localizadas en seis de las siete provincias de Costa Rica. En general, el 58% (344/596) de los cerdos eran seropositivos al PRRSV. La edad de los cerdos y la ecozona en la que se encontraban las explotaciones estaban significativamente relacionadas con la seroprevalencia del PRRSV en los animales y las piaras, respectivamente. Funding RM was a recipient of a scholarship from the Ministry of Science and Technology and Telecommunications (MICITT) PND018–15-2 from PINN POSGRADOS of Costa Rica to conduct doctoral studies. This work was supported partially by the Department of Farm Animal Health, University of Utrecht. Financial contribution to this study also came from Regional Research Center for Sustainable Animal Production (CRIPAS), Universidad Nacional, Costa Rica. Escuela de Medicina Veterinaria

Meat inspection records of one large cattle slaughterhouse were analyzed to evaluate the added value of slaughterhouse data for cattle health surveillance in the Netherlands. Data were available from January 2015 to September 2018, consisting of 467,361 meat inspection records. Analyses included (1) an assessment of the representativeness of the cattle herds in the slaughterhouse data in relation to the cattle herd population in the Netherlands, and (2) multivariable analyses to quantify associations between meat inspection findings and farm of origin characteristics, and the trends in time of the findings in slaughtered cattle. Ninety percent of the meat inspection records originated from dairy cattle therefore this paper only presents the results of dairy herds (N = 422,194 cattle). The dairy herds in the slaughterhouse data seemed representative for the Dutch dairy population although their regional coverage differed from the distribution of dairy herds in the Netherlands. Non-dairy herds were underrepresented in the slaughterhouse data which stresses the importance of the inclusion of data from other slaughterhouses that may be more specialized in slaughtering beef cattle. Inspection records were categorized into 15 indicators related to ante-mortem and post-mortem findings. Following multivariable analyses, seven indicators were deemed of added value to existing cattle health surveillance components, as they provided either new information or information regarding specific health problems.

Dairy cattle are typically disbudded or dehorned. Little is known, however, about the biological function and role of horns during thermoregulatory processes in cattle, and thus about the potential physiological consequences of horn removal. Anecdotal evidence suggests that dairy cow horns increase in temperature during rumination, and few studies on other bovid species indicate that horns aid thermoregulation. The objective of this study was, therefore, to elucidate a possible thermoregulatory function of the horns in dairy cattle. Using non-invasive infrared thermography, we measured the superficial temperature of the horns, eyes, and ears of 18 focal cows on three different farms in a temperate climate zone under various environmental circumstances. Observations of social and non-social behaviours were conducted as well. Based on environmental temperature, humidity, and wind speed, the heat load index (HLI) was calculated as a measure of the heat load experienced by a cow. The temperature of the horns increased by 0.18 °C per unit HLI, indicating that horns serve the dissipation of heat. Dehorned cows had higher eye temperatures than horned cows, though this result should be interpreted with caution as the low sample size and experimental setup prevent casual conclusions. We did not, however, find changes in horn temperature during rumination, nor with any other behaviours. Our study thus supports a role of horns in thermoregulation, but not related to rumination. These results should be considered when assessing the potential consequences of horn removal, a painful procedure.

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

A reduction in antimicrobial use (AMU) is needed to curb the increase in antimicrobial resistance in broiler production. Improvements in biosecurity can contribute to a lower incidence of disease and thereby lower the need for AMU. However, veterinary advice related to AMU reduction or biosecurity is often not complied with, and this has been linked to the attitudes of farmers. Behavior change promoted by coaching may facilitate uptake and compliance regarding veterinary advice. Thirty broiler farms in Belgium and the Netherlands with high AMU were included in this study for 13 months. For each farmer, the attitude towards AMU reduction was quantified using an adjusted Awareness, Desire, Knowledge, Ability, and Reinforcement (ADKAR®) change management model, and farm biosecurity was assessed with the Biocheck.UGent™ tool. Subsequently, farmers were coached to improve disease prevention and antimicrobial stewardship. After the individual coaching of farmers, there was a change in their attitudes regarding AMU, reflected by an increase in ADKAR® scores. Biosecurity levels improved by around 6% on average, and AMU was reduced by 7% on average without negative effects on performance parameters. Despite these improvements, no significant association could be found between higher ADKAR® scores and lower AMU. Further investigation into sociological models is needed as a tool to reduce AMU in livestock production.

Major histocompatibility complex (MHC) peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein antigens to elicit functional T cell responses. Liquid chromatography-mass spectrometry (LC-MS) analysis of MHC eluted ligands (EL data) has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of antigen presentation have reached a high level of accuracy for both MHC class I and class II. Here, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte antigen class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by EL data derived from cell lines expressing a range of DRB3 alleles prevalent in Holstein-Friesian populations. The model generated (NetBoLAIIpan - available as a web-server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced MS peptidomics with immunoinformatics for characterization of the BoLA-DR antigen presentation system and provide a novel tool that can be utilised to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

Mastitis is an important problem in meat-producing sheep, but few studies have investigated the transmission dynamics of mastitis pathogens in these animals. The objective of this study was to describe the pathogens causing intramammary infections (IMI) in suckler ewes, their effect on somatic cell count, and the dynamics of these IMI in early lactation. We enrolled 15 sheep flocks early after lambing and selected ewes in each flock that were sampled twice with a 3-wk interval. Milk samples from both glands of each ewe were bacteriologically cultured, and somatic cell count was measured. Non-aureus Staphylococcus spp. were the most prevalent culture results. Somatic cell counts were most strongly increased in ewes infected with Mannheimia haemolytica, whereas staphylococci, including Staphylococcus aureus, were associated with a moderate increase in somatic cell count. The proportion of udder halves that remained culture-positive with Staphylococcus spp. during the 3-wk sampling interval was moderate, but M. haemolytica infections were stable during this time period. A substantial number of new infections were seen in the early lactation study period for non-aureus Staphylococcus spp., Staph. aureus, and Corynebacterium spp., but not for M. haemolytica or Streptococcus spp. The number of new IMI of Staph. aureus was associated with the number of Staph. aureus-infected udder halves in the flock at the first sampling moment, indicative of contagious transmission. Altogether, we show that substantial transmission happens in early lactation in suckler ewes, but that the dynamics differ between pathogen species. More research is needed to further describe transmission in different stages of lactation and to identify transmission routes, to develop effective interventions to control mastitis.

Simple Summary Silvopastoral Systems (SPS) are agroforestry arrangements that combine forage grasses with shrubs and trees for animal nutrition and complementary uses. Their contribution to sustainable livestock production and animal welfare is well recognized, mainly with Zebu cattle in tropical regions. In order to obtain information in temperate climate zones, 130 beef cattle of European breeds were selected from four commercial herds and randomly allocated to two contiguous plots: SPS and Open Pastures Systems (OPS). The trees in the SPS were Eucalyptus globulus globulus for paper pulp production. They were planted with two meters between each tree over native, diverse grasses. Over the course of one year, individual body weights and animal welfare indicators were collected every 45 days. There were no differences observed in weight gain between the two systems. No sign of impaired welfare, such as lameness, integument alterations, coughing, nasal/ocular discharge, or hampered respiration, was observed in either system. Silvopastoral systems offer animals a sustainable and richer environment, which will improve their welfare. The additional income provided by the wood production allows the farmers to maintain their traditional cattle farming lifestyle. Abstract The potential benefits of keeping Zebu cattle in silvopastoral systems are well described in tropical regions. In order to obtain information on European breeds of beef cattle (Bos taurus taurus) in temperate climate zones, individual records of body weight and welfare indicators were obtained from 130 beef cattle. These belonged to four herds and were randomly allocated to two contiguous plots: Silvopastoral Systems (SPS) and Open Pastures Systems (OPS). The SPS in this study were areas with exotic trees of Eucalyptus globulus globulus for paper pulp production planted in a 2 × 2 design (two meters between each tree) over diverse, native grasses. The OPS were large open areas with a great diversity of native grasses, herbs, and small plots of trees where the animals could rest and shelter from extreme weather conditions. Over the course of one year, individual body weights and a number of specific animal welfare indicators were measured every 45 days. After a descriptive analysis, a generalized linear mixed model (GLMM) with a Gaussian distribution, with time and system (OPS or SPS) fitted as fixed effects and individuals nested by herd as random intercepts, was used. The results showed that weight gain did not differ between the two systems. None of the animals showed any sign of impaired welfare in either system over the study period. Silvopastoral systems offer animals a sustainable and richer environment that will improves their welfare. The additional income provided by the wood production allows the farmers to maintain their traditional cattle farming lifestyle.

The etiology of mastitis is crucial information to use antimicrobials prudently for control and treatment. This study aimed to evaluate the effects of mastitis diagnosis and treatment strategies with on-farm testing, on cure, new intramammary infections (IMI), somatic cell count (SCC), and antimicrobial use, compared with farmers' current diagnosis and treatment strategies. The on-farm tests used, CHROMagar Mastitis (CHROMagar, Paris, France) and Minnesota Easy Culture System II Tri-plate (University of Minnesota, St. Paul, MN), both had etiological groups of IMI as result, being gram-positive growth, gram-negative growth, or culture negative. Two randomized controlled trials were conducted on 15 herds: trial 1 prospectively enrolled 155 cows with clinical mastitis, and trial 2 cross-sectionally included 78 cows with subclinical mastitis. In both trials, cows were randomly distributed over 3 equal-sized groups: a test group using CHROMagar, a test group using Minnesota, and a control group not using on-farm tests. Farmers decided whether or not to treat, and which antimicrobial treatment would be applied, using information available on the day of enrollment (control group), complemented with the on-farm test result 1 d after enrollment (both test groups). For clinical mastitis, an antimicrobial treatment was given in 58% of cases that used CHROMagar, in 80% that used Minnesota, and in 86% of the controls. For subclinical mastitis, an antimicrobial treatment was given in 50% of cases that used CHROMagar, in 54% that used Minnesota, and in 4% of the controls. Bacteriological cure rate of clinical mastitis was lowest in the CHROMagar group [odds ratio 0.18 (95%CI 0.03-0.99)] compared with the controls. Using the Minnesota on-farm test for subclinical mastitis diagnosis and treatments resulted in fewer new IMI on d 21 [odds ratio 0.06 (95%CI 0.00-0.74)] compared with the controls. Clinical cure rate, percentage of new IMI, and SCC on d 21 of clinical mastitis were comparable among the groups. Using on-farm tests in farmers' decision-making process resulted in more treatments in accordance with the etiology of mastitis than without on-farm testing. A diagnosis and treatment strategy with on-farm testing is advised in cows with clinical mastitis to enhance prudent antimicrobial use. For subclinical mastitis, however, on-farm testing may lead to an unacceptable increase in use of antimicrobials and thus should not be advised as the common approach.

Lameness, one of the most important disorders in the dairy industry, is related to postpartum diseases and has an effect on dairy cow welfare, leading to changes in cows' daily behavioral variables. This study quantified the effect of lameness on the daily time budget of dairy cows in the transition period. In total, 784 multiparous dairy cows from 8 commercial Dutch dairy farms were visually scored on their locomotion (score of 1-5) and body condition (score of 1-5). Each cow was scored in the early and late dry period as well as in wk 4 and 8 postpartum. Cows with locomotion scores 1 and 2 were grouped together as nonlame, cows with score 3 were considered moderately lame, and cows with scores 4 and 5 were grouped together as severely lame. Cows were equipped with 2 types of sensors that measured behavioral parameters. The leg sensor provided number of steps, number of stand-ups (moving from lying to standing), lying time, number of lying bouts, and lying bout length. The neck sensor provided eating time, number of eating bouts, eating bout length, rumination time, number of rumination bouts, and rumination bout length. Sensor data for each behavioral parameter were averaged between 2 d before and 2 d after locomotion scoring. The percentage of nonlame cows decreased from 63% in the early dry period to 46% at 8 wk in lactation; this decrease was more severe for cows with higher parity. Cows that calved in autumn had the highest odds for lameness. Body condition score loss of >0.75 point in early lactation was associated with lameness in wk 4 postpartum. Moderately lame cows had a reduction of daily eating time of around 20 min, whereas severely lame cows had a reduction of almost 40 min. Similarly, moderately and severely lame dry cows showed a reduction of 200 steps/d, and severely lame cows in lactation showed a reduction of 600 steps/d. Daily lying time increased by 26 min and lying bout length increased by 8 min in severely lame cows compared with nonlame cows. These results indicate a high prevalence of lameness on Dutch dairy farms, with an increase in higher locomotion scores from the dry period into early lactation. Time budgets for multiparous dairy cows differed between the dry period and the lactating period, with a higher locomotion score (increased lameness) having an effect on cows' complete behavioral profile. Body condition score loss in early lactation was associated with poor locomotion postpartum, whereas lameness resulted in less eating time in the dry period and early lactation, creating a harmful cycle.

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups.

Highly pathogenic avian influenza viruses’ (HPAIVs) transmission from wild birds to poultry occurs globally, threatening animal and public health. To predict the HPAI outbreak risk in relation to wild bird densities and land cover variables, we performed a case-control study of 26 HPAI outbreaks (cases) on Dutch poultry farms, each matched with four comparable controls. We trained machine learning classifiers to predict outbreak risk with predictors analyzed at different spatial scales. Of the 20 best explaining predictors, 17 consisted of densities of water-associated bird species, 2 of birds of prey, and 1 represented the surrounding landscape, i.e., agricultural cover. The spatial distribution of mallard (Anas platyrhynchos) contributed most to risk prediction, followed by mute swan (Cygnus olor), common kestrel (Falco tinnunculus) and brant goose (Branta bernicla). The model successfully distinguished cases from controls, with an area under the receiver operating characteristic curve of 0.92, indicating accurate prediction of HPAI outbreak risk despite the limited numbers of cases. Different classification algorithms led to similar predictions, demonstrating robustness of the risk maps. These analyses and risk maps facilitate insights into the role of wild bird species and support prioritization of areas for surveillance, biosecurity measures and establishments of new poultry farms to reduce HPAI outbreak risks.

AIMS: The aims of this study were to determine antimicrobial prescription patterns and the factors affecting antimicrobial selection amongst Australian dairy veterinarians. METHODS: A structured questionnaire was administered to Australian dairy cattle veterinarians using the Qualtrics online survey platform. Questions focused on their (1) demographics; (2) opinions surrounding antimicrobial use, resistance, and stewardship; (3) decision-making drivers of both prescription and selection of commonly prescribed antimicrobials; (4) awareness on the guidelines for antimicrobial usage and sources of information concerning antimicrobials. KEY RESULTS: A total of 135 responses (14.1% response rate) from all eight dairying regions in Australia were received. The attitudes, perceptions, and concerns of dairy veterinarians towards antimicrobials indicated a high agreement regarding label indications (96%), consequences of off-label prescription (95%), and the presence of an antimicrobial resistance (AMR) risk (73%), when prescribing antibiotics. A four-dimensional categorical principal components analysis (CATPCA) model indicated most of the variation in opinion was due to AMR risk, trade-offs, prescription concerns and active substance concerns. The first active substance most dairy veterinarians chose for a scenario involving mastitis and dry cow therapy (DCT) treatment was cloxacillin. Decision-making drivers for antimicrobial choice when providing advice regarding the supply of antimicrobials for mastitis and DCT treatment were predominately clinical factors; however, diagnostics were rarely used in determining antimicrobial choice due to cost of implementation, diagnostic accuracy (sensitivity, specificity), and benefit issues. Non-clinical decision-making drivers included the perception of practicality for Australian Veterinary Association (AVA) prescription guidelines, opinions surrounding AMR risk and prescription concerns, consideration of Expert Advisory Group on Antimicrobial Resistance (EAGAR) scores, number of years worked with dairy farms, and the number of dairy farms they regularly consult for. When available at the practice, prescription policies were considered to impact on animal welfare outcomes and on the probability of AMR emergence. The major information sources influencing decision making on antimicrobial prescription for the Australian dairy veterinarians were clinical experience (93%) and product labels (81%). CONCLUSIONS: Australian dairy veterinarians are generally aware of the risk of resistance to antimicrobials and the need for stewardship, with clinical factors having the most impact on antimicrobial prescription. However, non-clinical factors incorporating awareness of guidelines and their attitudes on antimicrobial resistance risk and prescription concerns impact on the choice and prescription of antimicrobials. IMPLICATIONS: The development of prescription policy and guidelines, alongside effective communicative extension programs to increase veterinarian uptake, provides an avenue to mitigate AMR risk in Australian dairy cattle.

Colostrum intake is one of the most important factors in neonatal health in ruminants, mainly because of its unique immunological properties. Both in practice as well as in research, the attention of lactogenic immunity is focused on the importance of colostral antibodies and less attention is given to the functional role of maternal cells in colostrum. Here we study the transfer of maternal leukocytes via colostrum and the functionality in goat kids. In experiment 1, twenty twin pairs of goat kids from dams previously immunized with an inactivated Mycobacterium avium subsp. paratuberculosis (MAP) vaccine were fed maternal colostrum from their dam (kid 1) or pasteurized and frozen/thawed bovine colostrum (kid 2). The presence of cell mediated immune response (CMIR) against Mycobacterium avium antigens in the kids was assessed using intradermal skin testing with PPD-A tuberculin. Linear mixed effect models showed an increase in skin thickness in response to intradermal PPD-A injection in maternal colostrum fed kids compared to bovine colostrum fed kids. After intradermal PPD-A application, serum concentration of MAP specific antibodies increased in kids fed maternal colostrum, indicating antigen specific activation of the adaptive immune system. We did not detect a similar increase in antibodies in the kids fed bovine colostrum. In experiment 2, a more reductionistic approach was applied to specifically study the effects of the transfer of maternal colostral leukocytes on CMIR in goat kids. Similar to experiment 1, twin kids from MAP immunized dams were randomly divided over two groups. The experimental group received colostrum replacer supplemented with fluorescently labelled colostral cells of the dam and the control group received colostrum replacer only. No difference in skin response following intradermal PPD-A injection was observed between both groups of kids. Histologic examination of the skin at the intradermal injection site did not show fluorescently labelled cells. In conclusion, in our initial experiment we observed an antigen specific CMIR in goat kids fed fresh colostrum with colostral leukocytes from vaccinated dams. The lack of a DTH response in kids fed colostrum replacer supplemented with maternal colostrum derived leukocytes indicated that the complete colostral matrix is probably required for colostrum leukocytes to transfer across the intestinal epithelial barrier and modulate the neonatal immune response. In line with earlier studies, our results indicate that caprine maternal leukocytes present in colostrum can functionally contribute to the newborns’ early adaptive immune responses adding to the importance of colostrum feeding in ruminant neonates.

Simple Summary To determine the level of welfare on a dairy farm is a complex task. There is no protocol available that can serve as a ‘gold standard’. The Welfare Quality protocol is the most extensive one, but it takes about a full day to perform. We, therefore, examined if it would be possible to replace the time-consuming parts, like lengthy behavioural observations, with simple measurements in the environment. This resulted in a new Welfare Monitor that can be executed in 1.5 h on a farm with 100 cows. Welfare assessment was appreciated by the farmers, and they responded to advice for improvements on their farm. Besides promoting better welfare, this approach also led to a better financial result for the farms. Abstract The Welfare Quality® assessment protocol (WQ) is the most extensive way to measure animal welfare. This study was set up to determine if resource-based welfare indicators, that are easier and faster to measure, could replace the more time consuming, animal-based measurements of the WQ. The WQ was applied on 60 dairy farms in the Netherlands, with good, moderate and poor welfare. The WQ protocol classified most farms (87%) as ‘acceptable’. Several of the animal-based measures of WQ correlated well with measures in the environment. Using these correlations, an alternative welfare assessment protocol (new Welfare Monitor) was designed, which takes approximately 1.5 h for a farm with 100 dairy cows. Because the opinion of farmers about welfare assessment is important if one wants to improve conditions for the cows at a farm, another objective of this study was to evaluate the usefulness of the new Welfare Monitor for the farmer. Over two years, the farms were visited, and advice was given to improve the conditions at the farm. After the first welfare assessment and advice, farmers improved the conditions for their cows substantially. Farms where the category score had increased made more improvements on average than those that did not upgrade.

Many protocols have been developed to assess farm animal welfare. However, the validity of these protocols is still subject to debate. The present study aimed to compare nine welfare assessment protocols, namely: (1) Welfare Quality© (WQ), (2) a modified version of Welfare Quality (WQ Mod), which has a better discriminative power, (3) WelzijnsWijzer (Welfare Indicator, WW), (4) a new Welfare Monitor (WM), (5) Continue Welzijns Monitor (Continuous Welfare Monitor, CWM), (6) KoeKompas (Cow Compass, KK), (7) Cow Comfort Scoring System (CCSS), (8) Stall Standing Index (SSI) and (9) a Welfare Index (WI Tuyttens). In addition, a simple welfare estimation by veterinarians (Estimate vets, EV) was added. Rank correlation coefficients were calculated between each of the welfare assessment protocol scores and mean hair cortisol concentrations from 10 cows at 58 dairy farms spread over the Netherlands. Because it has been suggested that the hair cortisol level is related to stress, experienced over a long period of time, we expected a negative correlation between cortisol and the result of the welfare protocol scores. Only the simple welfare estimation by veterinarians (EV) (ρ = −0.28) had a poor, but significant, negative correlation with hair cortisol. This correlations, however, failed to reach significance after correction of p-values for multiple correlations. Most of the results of the different welfare assessment protocols had a poor, fair or strong positive correlation with each other, supporting the notion that they measure something similar. Additional analyses revealed that the modified Welfare Quality protocol parameters housing (ρ = −0.30), the new Welfare Monitor (WM) parameter health (ρ = −0.33), and milk yield (ρ = −0.33) showed negative correlations with cortisol. We conclude that because only five out of all the parameter scores from the welfare assessment protocols showed a negative, albeit weak, correlation with cortisol, hair cortisol levels may not provide a long term indicator for stress in dairy cattle, or alternatively, that the protocols might not yield valid indices for cow welfare.

Foot-and-mouth disease (FDM) is a disease of cloven-hoofed animals with high costs in animal welfare and animal production. Up to now, transmission between farms in FMD-endemic areas has been given little attention. Between farm transmission can be quantified by distance independent transmission parameters and a spatial transmission kernel indicating the rate of transmission of an infected farm to susceptible farms depending on the distance. The spatial transmission kernel and distance-independent transmission parameters were estimated from data of an FMD outbreak in Lamphaya Klang subdistrict in Thailand between 2016 and 2017. The spatial between-farm transmission rate in Lamphaya Klang subdistrict was higher compared with the spatial between-farm transmission rate from FMDV in epidemic areas. The result can be explained by the larger size of the within-farm outbreak in the endemic area due to no culling. The inclusion of distance-independent transmission parameters improved the model fit, which suggests the presence of transmission sources from outside the area and spread within the area independent of the distance between farms. The remaining distance-dependent transmission was mainly local and could be due to over-the-fence transmission or other forms of contact between nearby farms. Farm size on the kernel positively affects the transmission rate, by increasing both infectivity and susceptibility with increasing farm size. The results showed that both distance-dependent transmission and distance-independent transmission were contributed to FMDV transmission in Lamphaya Klang outbreak. These transmission parameters help to gain knowledge about FMD transmission dynamic in the endemic area.

Platelet and leukocyte count reference intervals (RIs) for cattle differ by age and while adult RIs are known, RIs for calves are studied less. The aims of this observational study are to evaluate variation of platelet counts of Holstein Friesian calves over the first 14 days of life and to propose RIs for platelet and leukocyte counts of Holstein Friesian calves aged 0&ndash, 60 days. In a longitudinal study, 19 calves were blood sampled 17 times, in the first 14 days of their lives. Blood was collected in a citrate blood tube and platelet counts were determined. We assessed the course of platelet counts. In a field study, 457 healthy calves were blood sampled once. Blood was collected in an EDTA blood tube and platelet and leukocyte counts were determined. The RIs were calculated by the 2.5 and 97.5 percentiles. Platelet counts started to increase 24 h after birth (mean platelet count 381 &times, 109/L &plusmn, 138 &times, 109/L) and stabilized after five days (mean platelet count 642 &times, 265 &times, 109/L). In calves up to six days of age, platelet counts were lower than in calves older than five days. In conclusion, the RIs of platelet and leukocyte counts in calves were wider in range than the RIs for adult cattle, therefore, calf specific RIs for platelet and leukocyte counts should be used. From 6 until 60 days of age, we propose an RI for platelet counts of 287&ndash, 1372 &times, 109/L and for the first 60 days of life an RI for leukocyte counts of 4.0&ndash, 18.9 &times, 109/L.

Two-way transmission on mink farms Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic virus—one that spilled over from another species to infect and transmit among humans. We know that humans can infect other animals with SARS-CoV-2, such as domestic cats and even tigers in zoos. Oude Munnink et al. used whole-genome sequencing to show that SARS-CoV-2 infections were rife among mink farms in the southeastern Netherlands, all of which are destined to be closed by March 2021 (see the Perspective by Zhou and Shi). Toward the end of June 2020, 68% of mink farm workers tested positive for the virus or had antibodies to SARS-CoV-2. These large clusters of infection were initiated by human COVID-19 cases with viruses that bear the D614G mutation. Sequencing has subsequently shown that mink-to-human transmission also occurred. More work must be done to understand whether there is a risk that mustelids may become a reservoir for SARS-CoV-2. Science, this issue p. 172; see also p. 120, Genetic evidence indicates that SARS-CoV-2 transmission occurred from humans to mink as well as from mink to humans on farms in the Netherlands., Animal experiments have shown that nonhuman primates, cats, ferrets, hamsters, rabbits, and bats can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, SARS-CoV-2 RNA has been detected in felids, mink, and dogs in the field. Here, we describe an in-depth investigation using whole-genome sequencing of outbreaks on 16 mink farms and the humans living or working on these farms. We conclude that the virus was initially introduced by humans and has since evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period, several weeks before detection. Despite enhanced biosecurity, early warning surveillance, and immediate culling of animals in affected farms, transmission occurred between mink farms in three large transmission clusters with unknown modes of transmission. Of the tested mink farm residents, employees, and/or individuals with whom they had been in contact, 68% had evidence of SARS-CoV-2 infection. Individuals for which whole genomes were available were shown to have been infected with strains with an animal sequence signature, providing evidence of animal-to-human transmission of SARS-CoV-2 within mink farms.