Karin Persson Waller

Staphylococcus chromogenes and Staphylococcus simulans are commonly found in intramammary infections (IMI) associated with bovine subclinical mastitis, but little is known about genotypic variation and relatedness within species. This includes knowledge about genes encoding antimicrobial resistance (AMR) and potential virulence factors (pVF). The aim of this study was therefore to investigate these aspects by whole-genome sequencing of milk isolates from Swedish dairy cows with subclinical mastitis in an observational study. We also wanted to study if specific genotypes were associated with persistent IMI and the inflammatory response at udder quarter level. In total, 105 and 118 isolates of S. chromogenes and S. simulans, respectively, were included. Isolates were characterized using a 7-locus multilocus sequence typing (7-MLST), core genome analysis and in-silico analysis of AMR and pVF genes. Forty-seven sequence types (ST) and 7 core genome clusters of S. chromogenes were identified, and the most common ST were ST-6 and ST-109, both belonging to cluster VII. A 7-locus MLST scheme for S. simulans was not available, but 3 core genome clusters and 5 subclusters were described. Overall, substantial variation in ST and clusters among cows and herds were found in both species. Some ST of S. chromogenes were found in several herds, indicating spread between herds. Moreover, within-herd spread of the same genotype was observed for both species. Only a few AMR genes (blaZ, str_pS194, vga(A)) were detected in a limited number of isolates, with the exception of blaZ coding for β-lactamase, which was identified in 22% of the isolates of S. chromogenes with ST-19, ST-102, and ST-103 more commonly carrying this gene compared with other STs. However, the blaZ gene was not identified in S. simulans. The average total number of pVF detected per isolate was similar in S. chromogenes (n = 30) and S. simulans (n = 33), but some variation in total numbers and presence of specific pVF or functional groups of pVF, was shown between ST/clusters within species. Differences in inflammatory response and potentially in persistent IMI at udder quarter level were found between S. chromogenes subtypes but not between S. simulans subtypes. In conclusion, the results from the present study generates new insight into the epidemiology of bovine S. chromogenes and S. simulans IMI, which can have implications for future prevention and antimicrobial treatment of infections related to these species.

Subclinical mastitis can be common among freshly calved heifers (FCH), but the prevalence differs between herds possibly due to variation in risk factors. The aims of this observational study were to identify differences in occurrence of IMI in FCH between herds with documented good or not so good first parity udder health based on cow SCC (CSCC) in early lactation, and to study herd differences in animal factors important for udder health such as udder and hock skin lesions and animal cleanliness. Three groups of herds were included, those with high proportion of FCH with low (≤75,000 cells/ml) CSCC at the first 2 milk recordings after calving (LL), herds with high proportion of FCH with high (>100,000 cells/ml) CSCC at the first and low CSCC at the second recording (HL), and herds with high proportion of FCH with high CSCC at both recordings (HH). Thirty-one herds (13 LL, 11 HL, 15 HH) were visited 3 times during a 12-mo period for observation of cleanliness and hock lesions, and sampling of udder/teat skin using swab cloths of milk-fed calves, early pregnant heifers and late pregnant heifers. In 25 (9 LL, 9 HL, 7 HH) udder quarter samples from colostrum and milk on d 3-4 after calving were taken by the farmers from FCH during one year. The farmers also provided information on calving (individual or group), use of restraint and oxytocin at milking, and presence of teat and udder skin lesions. Bacterial growth in swab samples and quarter samples was investigated by culturing, and a selection of isolates was genotyped using whole genome sequencing (WGS). Cleanliness, hock and udder skin lesions other than udder-thigh dermatitis, and growth of bacteria in swab samples did not differ between herd groups. It was more common that FCH from LL herds, compared with FCH in HH and HL herds, calved in a group of animals. Use of restraint at milking was more common in LL herds than in HH herds while presence of udder-thigh dermatitis was lowest in LL herds. Specific infection was found in 14% of 5,593 quarter samples from 722 FCH. The most common IMI was S. chromogenes. Growth of S. simulans was more common in HH than in LL and HL herds. In colostrum samples, S. haemolyticus was more common in HL and HH than in LL herds. The proportion of quarters with the same specific infection at both samplings was higher in HH than in LL herds and tended to be higher in HH than in HL herds. The proportion of quarters with S. chromogenes IMI at both samplings tended to differ between herd groups and was highest in HH herds. WGS found the same sequence type of S. chromogenes and S. aureus in both samples in almost all quarters with the same infection at both samplings. The differences in IMI between herd groups were in line with the higher SCC in HH herds. The reasons for the predominance of S. chromogenes IMI in FCH need further studies.

An outbreak of disease in a Swedish beef cattle herd initiated an in-depth study to investigate the presence of bacteria and viruses in the blood of clinically healthy (n = 10) and clinically diseased cattle (n = 20) using whole-genome shotgun sequencing (WGSS). The occurrence of infectious agents was also investigated in ticks found attached to healthy cattle (n = 61) and wild deer (n = 23), and in spleen samples from wild deer (n = 30) and wild boars (n = 10). Moreover, blood samples from 84 clinically healthy young stock were analysed for antibodies against Anaplasma phagocytophilum and Babesia divergens. The WGSS revealed the presence of at least three distinct Mycoplasma variants that were most closely related to Mycoplasma wenyonii. Two of these were very similar to a divergent M. wenyonii variant previously only detected in Mexico. These variants tended to be more common in the diseased cattle than in the healthy cattle but were not detected in the ticks or wild animals. The DNA of A. phagocytophilum was detected in similar proportions in diseased (33%) and healthy (40%) cattle, while 70% of the deer, 8% of ticks collected from the cattle and 19% of the ticks collected from deer were positive. Almost all the isolates from the cattle, deer and ticks belonged to Ecotype 1. Based on sequencing of the groEL-gene, most isolates of A. phagocytophilum from cattle were similar and belonged to a different cluster than the isolates from wild deer. Antibodies against A. phagocytophilum were detected in all the analysed samples. In conclusion, uncommon variants of Mycoplasma were detected, probably associated with the disease outbreak in combination with immune suppression due to granulocytic anaplasmosis. Moreover, A. phagocytophilum was found to be circulating within this cattle population, while circulation between cattle and deer occurred infrequently.

Bovine mastitis at calving or early lactation is often associated with intra-mammary bacterial infections (IMI) at drying-off (DO) or during the dry period (DP). The IMI risk is associated with management routines at the herd, but knowledge on how farmers and veterinarians comply with national recommendations is scarce, as is their attitudes to the importance of such routines. Therefore, the main aims of this study were to collect information on farmer routines and attitudes, and on veterinary advice and attitudes to DO and DP. Associations between routines and advice, and demographic herd and veterinary variables were also studied. Web-based questionnaires were sent to 2472 dairy farmers and 517 veterinarians. The answers were summarized descriptively, and associations with demographics were evaluated using univariable regression models. The response rate was 14% for farmers and 25% for veterinarians. Routines and advice were in line with recommendations at the time of the study in many, but not all, areas of questioning. Significant associations between herd routines or veterinary advice and demographic variables were also found. Milking system and post-graduate training were the variables associated with the largest number of farmer and veterinary answers, respectively. In conclusion, the results indicate a need for more education on good routines during DO and DP. It was also clear that the national recommendations valid at the time of the study were in need of revision.

Dry-cow therapy with antibiotics (DCT) and treatment with internal teat sealants (ITS) are often used to control mastitis in dairy cows. However, the knowledge on farmer and veterinary compliance with recommendations for DCT and ITS is scarce. Thus, the main aim was to collect information on farmer routines and veterinary advice for such treatments. Associations with herd and veterinary variables were also studied. Web-based questionnaires including questions on demographics and the use of DCT and ITS were sent to 2472 farmers and 517 veterinarians in Sweden. The answers were summarized descriptively, and associations with demographics were evaluated using univariable regression models. The response rate was 14% for farmers and 25% for veterinarians. Among the farmers, 81% used selective DCT (SDCT), 3% used blanket DCT (BDCT), and 16% did not use DCT. Almost all (93%) veterinarians prescribed DCT and among those most recommended SDCT while 8% recommended BDCT. Eighty-two percent of the farmers did not use ITS and 45% of the veterinarians never prescribed ITS. Milking system and milk production, and post-graduate training and number of mastitis cases per month were associated with the largest numbers of farmer and veterinary answers, respectively. In conclusion, many farmer routines and veterinary advice complied with the recommendations available at the time, but a clear need for more education was also identified. The results also indicated that an up-date of the national recommendations was warranted.

Bovine clinical mastitis is an important problem for the dairy industry, and Staphylococcus aureus is a common mastitis-causing pathogen in many countries. Detailed knowledge on genetic variation of Staph. aureus strains within the bovine population, including changes over time, can be useful for mastitis control programs, because severity of disease and effects on milk production are at least partly strain-associated. Therefore, the major aim of this study was to compare sequence types of Staph. aureus isolated from cases of bovine clinical mastitis from 2002 to 2003 with sequence types of a more recent set of isolates collected from 2013 to 2018, using core genome multi-locus sequence typing (cgMLST). We also wanted to compare antibiotic resistance genes of isolates from the 2 sets, to identify changes that may have occurred over time in the Staph. aureus population. A total of 157 isolates of Staph. aureus, almost equally distributed between the 2 time periods, were subjected to high-throughput sequencing and cgMLST. The results showed that the most prevalent sequence types found among the 2002 to 2003 isolates belonged to the clonal complexes CC97, CC133, and CC151, and that those complexes still dominated among the isolates from 2013 to 2018. However, a population shift from CC133 to CC97 and CC151 over time was observed. Likewise, no important differences in prevalence of antibiotic resistance genes were found between the 2 sets of isolates. As expected, genes belonging to the major facilitator superfamily of transporter proteins, and multidrug and toxic compound extrusion transporters, were very common. Moreover, several genes and mutations conferring resistance to fosfomycin were present, but not in CC97 isolates. The β-lactamase gene blaZ was found in only 3 out of 81 isolates from 2002 to 2003 and 1 out of 76 isolates in 2013 to 2018. In conclusion, the results indicate that mastitis-associated Staph. aureus strains circulating among dairy cows in Sweden exhibit a remarkable genotypic persistence over a time frame of close to 15 yr.

Group B Streptococcus (GBS; Streptococcus agalactiae ) is a major neonatal and opportunistic bacterial pathogen of humans and an important cause of mastitis in dairy cattle with significant impacts on food security. Following the introduction of mastitis control programmes in the 1950s, GBS was nearly eradicated from the dairy industry in northern Europe, followed by re-emergence in the 21st century. Here, we sought to explain this re-emergence based on short and long read sequencing of historical (1953–1978; n=44) and contemporary (1997–2012; n=76) bovine GBS isolates. Our data show that a globally distributed bovine-associated lineage of GBS was commonly detected among historical isolates but never among contemporary isolates. By contrast, tetracycline resistance, which is present in all major GBS clones adapted to humans, was commonly and uniquely detected in contemporary bovine isolates. These observations provide evidence for strain replacement and suggest a human origin of newly emerged strains. Three novel GBS plasmids were identified, including two showing >98 % sequence similarity with plasmids from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis , which co-exist with GBS in the human oropharynx. Our findings support introduction of GBS into the dairy population due to human-to-cattle jumps on multiple occasions and demonstrate that reverse zoonotic transmission can erase successes of animal disease control campaigns.