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International audience; AbstractThe aim of this study was to analyze bacterial profiles of bovine mastitic milk samples and samples from healthy quarters using Next Generation Sequencing of amplicons from 16S rRNA genes and to compare results with microbiological results by PCR assays of the same samples. A total of 49 samples were collected from one single dairy herd during the same day. The samples were divided in two sample sets, which were used in this study. The DNA extraction as well as the library preparation and sequencing of these two sets were performed separately, and results of the two datasets were then compared. The vast majority of genera detected appeared with low read numbers and/or in only a few samples. Results of PCR and microbiome analyses of samples infected with major pathogens Staphylococcus aureus or Streptococcus uberis were consistent as these genera also covered the majority of reads detected in the microbiome analysis. Analysis of alpha diversity revealed a much higher species richness in set 1 than in set 2. The dominating bacterial genera with the highest read numbers clearly differed between datasets, especially in PCR negative samples and samples positive for minor pathogens. In addition to this, linear discriminant analysis (LDA) was conducted between the two sets to identify significantly different genera/family level microbes. The genus Methylobacterium was much more common in set 2 compared to set 1, and genus Streptococcus more common in set 1. Our results indicate amplification of contaminating bacteria in excess in samples with no or minor amounts of pathogen DNA in dataset 2. There is a need for critical assessment of results of milk microbiome analyses.

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.

The aim of this study was to determine risk factors for bovine intramammary infection (IMI) associated with the most common bacterial species in Finland. Large databases of the Finnish milk-recording system and results of microbiological analyses of mastitic milk samples from Valio Ltd. (Helsinki, Finland) were analyzed. The study group comprised 29,969 cows with IMI from 4,173 dairy herds. A cow with a quarter milk sample in which DNA of target species was detected in the PathoProof Mastitis PCR Assay (Thermo Fisher Scientific, Waltham, MA) was determined to have IMI. Only cows with IMI caused by the 6 most common pathogens or groups of pathogens, coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Corynebacterium bovis, and Escherichia coli, were included. The control group comprised 160,176 IMI-free cows from the same herds as the study group. A multilevel logistic regression model was used to study herd- and cow-specific risk factors for incidence of IMI. Pathogen-specific results confirmed those of earlier studies, specifically that increasing parity increases prevalence of IMI regardless of causative pathogen. Holsteins were more susceptible to IMI than Nordic Reds except when the causative pathogen was CNS. Occurrence of IMI caused by C. bovis was not related to milk yield, in contrast to IMI caused by all other pathogens investigated. Organic milk production was associated with IMI only when the causative pathogen of IMI was Staph. aureus; Staph. aureus IMI was more likely to occur in conventional than in organic production. Cows in older freestall barns with parlor milking had an increased probability of contracting an IMI compared with cows in tiestall barns or in new freestall barns with automatic milking. This was the case for all IMI, except those caused by CNS, the prevalence of which was not associated with the milking system, and IMI caused by Staph. aureus, which was most common in cows housed in tiestall barns. A better breeding index for milk somatic cell count was associated with decreased occurrence of IMI, indicating that breeding for improved udder health has been successful in reducing the incidence of IMI caused by the most common pathogens in Finland. In the Finnish dairy sector, the importance of other measures to control IMI will increase as the Holstein breed progressively takes the place of the Nordic Red breed. Attention should be paid to hygiene and cleanliness, especially in old freestall barns. Based on our results, the increasing prevalence of automatic milking is not a reason for special concern.

Activity of lysosomal N-acetyl-β-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1–1·04 pmoles 4-MU/min/μl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

The aim of this study was to determine the intramammary dose of benzylpenicillin required to maintain a concentration in the milk above the MIC for the Gram-positive bacteria that cause mastitis. The product used in this study was a commercially available procaine benzylpenicillin in an oily suspension with micronized particles. Three dose levels were used: 200,000, 300,000, and 600,000IU. Concentrations of benzylpenicillin in cow milk and plasma were determined after a single intramammary dose was administered into one quarter of each of the five cows in each treatment group. Samples were analyzed using an HPLC-MS/MS method, which was validated during the study. Concentrations in the milk were well above the MIC for the target pathogens for all doses tested. There was a linear dose-dependent increase in the mean AUCs of benzylpenicillin concentrations in plasma and milk. At the first milking, 12hr after dosing, there was a significant difference between the mean milk benzylpenicillin concentrations in cows treated with a dose of 600,000IU, and those treated with 200,000 or 300,000IU. Although this study shows a linear relationship between the dose of procaine benzylpenicillin administered and the concentration in the milk in the healthy udder, it would be useful to conduct studies on cows with mastitis to define the optimum dose and duration of intramammary treatment with benzylpenicillin.

International audience; Since its introduction in the 1950s, colistin has been used mainly as a topical treatment in human medicine owing to its toxicity when given systemically. Sixty years later, colistin is being used as a last-resort drug to treat infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae (e.g., Escherichia coli, Klebsiella pneumoniae), for which mortality can be high. In veterinary medicine, colistin has been used for decades for the treatment and prevention of infectious diseases. Colistin has been administered frequently as a group treatment for animal gastrointestinal infections caused by Gram-negative bacteria within intensive husbandry systems. Given the ever-growing need to retain the efficacy of antimicrobials used to treat MDR infections in humans, the use of colistin in veterinary medicine is being re-evaluated. Despite extensive use in veterinary medicine, there is limited evidence for the development of resistance to colistin and no evidence has been found for the transmission of resistance in bacteria that have been spread from animals to humans. Since surveillance for colistin resistance in animals is limited and the potential for such transmission exists, there is a clear need to reinforce systematic monitoring of bacteria from food-producing animals for resistance to colistin (polymyxins). Furthermore, colistin should only be used for treatment of clinically affected animals and no longer for prophylaxis of diseases, in line with current principles of responsible use of antibiotics.

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85·7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83·0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97·0 and 95·8% compared with BC. For Staphylococcus spp., the corresponding figures were 86·7 and 75·4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37·0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Background: The Finnish dairy herd recording system maintains production and health records of cows and herds. Veterinarians and farmers register veterinary treatments in the system. Milk samples for microbiological analysis are routinely taken from mastitic cows. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes most samples using real-time PCR. This study addressed pathogen-specific microbiological data and treatment and culling records, in combination with cow and herd characteristics, from the Finnish dairy herd recording system during 2010-2012. Results: The data derived from 240,067 quarter milk samples from 93,529 dairy cows with mastitis; 238,235 cows from the same herds served as the control group. No target pathogen DNA was detected in 12% of the samples. In 49% of the positive samples, only one target species and in 19%, two species with one dominant species were present. The most common species in the samples with a single species only were coagulase-negative staphylococci (CNS) (43%), followed by Staphylococcus aureus (21%), Streptococcus uberis (9%), Streptococcus dysgalactiae (8%), Corynebacterium bovis (7%), and Escherichia coli (5%). On average, 36% of the study cows and 6% of the control cows had recorded mastitis treatments during lactation. The corresponding proportions were 16 and 6% at drying-off. For more than 75% of the treatments during lactation, diagnosis was acute clinical mastitis. In the milk samples from cows with a recorded mastitis treatment during lactation, CNS and S. aureus were most common, followed by streptococci. Altogether, 48% of the cows were culled during the study. Mastitis was reported as the most common reason to cull; 49% of study cows and 18% of control cows were culled because of mastitis. Culling was most likely if S. aureus was detected in the milk sample submitted during the culling year. Conclusions: The PCR test has proven to be an applicable method also for large-scale use in bacterial diagnostics. In the present study, microbiological diagnosis was unequivocal in the great majority of samples where a single species or two species with one dominating were detected. Coagulase-negative staphylococci and S. aureus were the most common species. S. aureus was also the most common pathogen among the culled cows, which emphasizes the importance of preventive measures.

Two methods of collecting milk samples from mastitic bovine mammary quarters were compared. Samples were taken in a consistent order in which standard aseptic technique sampling was done first, followed by insertion of a sterile cannula through the teat canal and collection of a second sample. Microbiological results of those two sampling techniques were compared. Milk samples were analysed using multiplex real-time polymerase chain reaction (PCR). The cannula technique produced a reduced number of microbial species or groups of species per sample compared with conventional sampling. Staphylococcus spp. were the most common species identified and were detected more often during conventional sampling than with cannula sampling. Staphylococcus spp. identified in milk samples could also have originated from the teat canal without being present in the milk. The number of samples positive for Trueperella pyogenes or yeasts in the conventional samples was twice as high as in the cannula samples, indicating that the presence of Trueperella pyogenes and yeast species should not necessarily be interpreted as being the causative agents of bovine intra-mammary infections (IMI).

Use of antimicrobials for veterinary indications related to reproduction in cattle and horses is reviewed. Antimicrobial compounds are widely used to treat and prevent infections of reproductive organs. Total amounts of antimicrobials for such purposes, estimated by weight, are low compared with major uses in food animals. The most common reproduction-related indication in cattle is mastitis. The number of intramammary products available for treatment of mastitis in the European Union is high. Metritis and endometritis also require antimicrobial treatment of cattle and specific products for intrauterine administration are available. The traditions and practices associated with the use of these products vary considerably among different countries. Parenteral antimicrobial treatment is used to treat acute clinical mastitis and puerperal metritis. Pharmacological characteristics of the antimicrobial administered parenterally are critical to achieve and maintain therapeutic concentrations in the target organs. In mares, the most common indications associated with reproduction are endometritis, retained placenta and placentitis. The number of authorized antimicrobial products for horses is limited. Horses are treated individually and off-label use of antimicrobials is very common. In veterinary indications related to reproduction, treatment practices exist that cannot be considered to be evidence-based or responsible use of antimicrobials. Not all products for local treatment have proven efficacy data. Examples of unnecessary uses are routine treatment of cows with retained placenta and use of post-breeding antibiotic treatments in mares.

Bovine mastitis caused by Escherichia coli can range from being a subclinical infection of the mammary gland to a severe systemic disease. Cow-dependent factors such as lactation stage and age affect the severity of coliform mastitis. Evidence for the efficacy of antimicrobial treatment for E. coli mastitis is very limited. Antimicrobial resistance is generally not a limiting factor for treatment, but it should be monitored to detect changes in resistance profiles. The only antimicrobials for which there is some scientific evidence of beneficial effects in the treatment for E. coli mastitis are fluoroquinolones and cephalosporins. Both are critically important drugs, the use of which in animals destined for food should be limited to specific indications and should be based on bacteriological diagnosis. The suggested routine protocol in dairy herds could target the primary antimicrobial treatment for mastitis, specifically infections caused by gram-positive bacteria. In E. coli mastitis with mild to moderate clinical signs, a non-antimicrobial approach (anti-inflammatory treatment, frequent milking and fluid therapy) should be the first option. In cases of severe E. coli mastitis, parenteral administration of fluoroquinolones, or third- or fourth-generation cephalosporins, is recommended due to the risk of unlimited growth of bacteria in the mammary gland and ensuing bacteremia. Evidence for the efficacy of intramammary-administered antimicrobial treatment for E. coli mastitis is so limited that it cannot be recommended. Nonsteroidal anti-inflammatory drugs have documented the efficacy in the treatment for E. coli mastitis and are recommended for supportive treatment for clinical mastitis.

Background The dairy industry has undergone substantial structural changes as intensive farming has developed during recent decades. Mastitis continues to be the most common production disease of dairy cows. Nationwide surveys of mastitis prevalence are useful in monitoring udder health of dairy herds and to study the impact of structural changes on the dairy industry. This survey on bovine subclinical mastitis was the first based on cow composite milk somatic cell count (SCC) data from the Finnish national health monitoring and milk recording database. A cow with composite milk SCC ≥200,000 cells/ml in at least one of the four test milkings during the year was considered to have subclinical mastitis and a cow with composite milk SCC ≥200,000 cells/ml in three or in all four test milkings during the year to have chronic subclinical mastitis. The aim of the study was to determine the prevalence of subclinical mastitis and chronic subclinical mastitis in Finland in 1991, 2001 and 2010 and to investigate cow and herd factors associated with elevated SCC. Results Prevalence of subclinical mastitis in Finland decreased over recent decades from 22.3% (1991) and 20.1% (2001) to 19.0% (2010). Prevalence of chronic subclinical mastitis was 20.4% in 1991, 15.5% in 2001 and 16.1% in 2010. The most significant cow and herd factors associated with subclinical mastitis or high milk SCC were increasing parity, Holstein breed, free-stalls with an automatic milking system and organic production. Milk SCC were highest from July to September. Main factors associated with chronic mastitis were increasing parity and Holstein breed. Conclusions Prevalence of subclinical mastitis in Finland decreased over recent decades, the greatest change taking place during the first decade of the study. Prevalence of chronic subclinical mastitis significantly decreased from 1991. The most significant factors associated with both types of mastitis were increasing parity and Holstein breed, and for subclinical mastitis also free-stalls with automatic milking. National surveys on mastitis prevalence should be carried out at regular intervals to monitor udder health of dairy cows and to study the impact of the ongoing structural changes in the dairy industry to enable interventions related to udder health to be made when needed. Electronic supplementary material The online version of this article (doi:10.1186/s13028-017-0288-x) contains supplementary material, which is available to authorized users.

International audience; Antimicrobials are important tools for the therapy of infectious bacterial diseases in companion animals. Loss of efficacy of antimicrobial substances can seriously compromise animal health and welfare. A need for the development of new antimicrobials for the therapy of multiresistant infections, particularly those caused by Gram-negative bacteria, has been acknowledged in human medicine and a future corresponding need in veterinary medicine is expected. A unique aspect related to antimicrobial resistance and risk of resistance transfer in companion animals is their close contact with humans. This creates opportunities for interspecies transmission of resistant bacteria. Yet, the current knowledge of this field is limited and no risk assessment is performed when approving new veterinary antimicrobials. The objective of this review is to summarize the current knowledge on the use and indications for antimicrobials in companion animals, drug-resistant bacteria of concern among companion animals, risk factors for colonization of companion animals with resistant bacteria and transmission of antimicrobial resistance (bacteria and/or resistance determinants) between animals and humans. The major antimicrobial resistance microbiological hazards originating from companion animals that directly or indirectly may cause adverse health effects in humans are MRSA, methicillin-resistant Staphylococcus pseudintermedius, VRE, ESBL- or carbapenemase-producing Enterobacteriaceae and Gram-negative bacteria. In the face of the previously recognized microbiological hazards, a risk assessment tool could be applied in applications for marketing authorization for medicinal products for companion animals. This would allow the approval of new veterinary medicinal antimicrobials for which risk levels are estimated as acceptable for public health.

Bovine mastitis is an economic and a welfare problem on dairy farms. The objective of this study was to estimate the costs of clinical mastitis (CM), having a special focus on the cost variation related to culling decisions. A dynamic optimization model was developed to determine an optimal replacement time of a mastitic cow and to estimate the costs of CM, taking into account the risk of premature culling and the uncertainty in CM prevalence. Six lactations were analyzed at monthly periods for Ayrshire and Holstein-Friesian breeds. The estimates reflect Finnish production conditions where mastitis is treated only by veterinarians. Biological parameters of the model were adapted from the literature and the Finnish dairy herd health recording system. Field data were used to produce the risk parameters of culling due to mastitis on commercial dairy farms. The model recommended treating the cows with CM and keeping them in most cases until their fifth lactation. A cheaper (-20%) heifer transferred the optimum to the previous lactation and a more expensive (+20%) heifer to the following lactation. Conditional on optimal replacements, the average cost of CM of an Ayrshire (Holstein-Friesian costs in parentheses) cow was €485 (€458), varying from €209 (€112) to €1,006 (€946). The costs were at the highest when the occurrence of CM was at a top yield phase. In the scenario where the risk of culling due to mastitis was included in the model, the average cost of CM was €596 (€623). Disposing of a young cow at the end of her first lactation month caused the highest costs. The costs converted to figures per cow-year were €121 (€147) with optimal cullings and €155 (€191) in the current Finnish conditions. Thus, the increase in the costs of CM due to premature cullings was 28% (30%.) The main cost sources were long-term production losses regardless of the culling decisions. Premature culling formed 20% (23%) of the total costs. To decrease the costs of CM, more emphasis should be given to hidden costs, especially the high cost of premature culling should be underlined.

The aim of the study was to evaluate the effect of sampling technique on the microbiological results of bovine milk samples using multiplex real-time PCR. Comparison was made between a technique where the milk sample was taken directly from the udder cistern of the udder quarter using a needle and vacuum tube and conventional sampling. The effect of different cycle threshold (Ct) cutoff limits on the results was also tested to estimate the amount of amplified DNA in the samples. A total of 113 quarters from 53 cows were tested pairwise using both techniques, and each sample was studied with real-time PCR. Sampling from the udder cistern reduced the number of species per sample compared with conventional sampling. In conventional samples, the number of positive Staphylococcus spp. results was over twice that of samples taken with the needle technique, indicating that most of the Staphylococcus spp. originated from the teat or environmental sources. The Ct values also showed that Staphylococcus spp. were present in most samples only in low numbers. Routine use of multiplex real-time PCR in mastitis diagnostics could benefit from critical evaluation of positive Staphylococcus spp. results with Ct values between 34.0 and 37.0. Our results emphasize the importance of a careful aseptic milk sampling technique and a microbiologically positive result for a milk sample should not be automatically interpreted as an intramammary infection or mastitis.

Acute mastitis is one of the most common diseases of high-producing dairy cows. However, there is still lack of knowledge on which precise behaviours change first at the beginning of acute mastitis, and whether behavioural changes might serve as a tool for early detection of clinical mastitis. In addition, mastitis can cause motivational conflict in the behavioural priorities of a cow, and thus blur the classical patterns of sickness behaviour: cows’ increased lying behaviour which may be uncomfortable or painful due to the sore udder. To study this, we monitored the behaviour of six cows after induction of acute endotoxin mastitis. Cows served as their own controls. Their behaviour was filmed from 1 day before the induction (control day) to 24 h after the induction (induction day). To follow-up on the inflammation, milk samples were taken and the cows’ health status was checked at regular intervals. Mean daily durations were determined for all behaviours, except stepping and body care, for which mean daily frequencies were counted. For standing and lying behaviours, also mean bout durations were analyzed. To examine the effects of time since the induction of mastitis, data were accordingly divided in 2-h periods. We analyzed the effect of induction on overall behaviour and behavioural rhythms with linear mixed models, taking repeated observations into account. Overall cows spent less time lying on the induction day than on the day before, and less on the side of the inflamed udder quarter. Cows also spent longer time overall for eating silage during the induction day, and they also stepped more than during the previous day. Cows spent less time lying and ruminated and drank less when the udder was severely swollen and when they had high fever. We concluded that unlike in typical sickness behaviour, cows did not increase their time spent lying, but instead stood more, and avoided lying on the side of the inflamed udder quarter. We suggest that pain experienced in the udder overrides the motivational the state of the cows’ sickness behaviour. In the future, novel mastitis detection tools should therefore be able automatically detect and combine changes on both physiological parameters (i.e. fever) and complete behavioural patterns consisting of resting, standing and eating behaviours.

Streptococcus uberis is an environmental udder pathogen that infects cattle and can cause persistent intramammary infection (IMI), despite the fact that isolates are mainly susceptible to antibiotics. As biofilm growth can cause persistent infection, the ability of ten S. uberis isolates from clinical and subclinical IMIs to form biofilms on the polystyrene surface of a conventional 96-microplates model was examined. Biofilm formation was judged by different staining methods (crystal violet and resazurin) and by atomic force and fluorescence microscopy. These analyses revealed that two out of ten S. uberis strains tested were able to form biofilms. Upon treatment with Proteinase K, biofilms of S. uberis were completely disintegrated, which indicates that biofilm formation is protein-mediated in these strains. Addition of trace amounts of milk, the natural growth medium of S. uberis, significantly increased biofilm formation by most of the strains initially classified as non-biofilm producers. Alpha-casein and β-casein were the primary inducers of biofilm growth, and casein degradation by serine protease activity was required to achieve maximal biofilm production. These results suggest that the extracellular proteolytic activity of S. uberis contributes to an increased biofilm formation. Such a mode of growth induced by host proteins might help to explain the persistence of IMIs caused by this pathogen.

Automatic milking (AM) is increasing in modern dairy farming, and over 8,000 farms worldwide currently use this technology. Automatic milking system is designed to replace conventional milking managed by a milker in a milking parlor or in tie stalls. Cows are generally milked more frequently in AM than in conventional milking, and milking is quarter-based instead of udder-based. Despite improvements in the milking process and often building of a new barn before the introduction of AM, udder health of the cows has not improved; on the contrary, problems may appear following conversion from conventional milking to AM. This review focuses on udder health of dairy cows in AM, and we discuss several aspects of cow and milking management in AM associated with udder health. Finally, adequate management methods in AM are suggested. According to several studies comparing udder health between automatic and conventional milking or comparing udder health before and after the introduction of automatic milking in the same herds, udder health has deteriorated during the first year or more after the introduction of AM. Automatic detection of subclinical and clinical mastitis and cleaning the teats before milking are challenges of AM. Failures in mastitis detection and milking hygiene pose a risk for udder health. These risk factors can partly be controlled by management actions taken by the farmer, but AM also needs further technical development. To maintain good udder health in AM, it is imperative that the barn is properly designed to keep the cows clean and the cow traffic flowing. Milking frequency must be maintained for every cow according to its stage of lactation and milk production. Careful observation of the cows and knowledge of how to use all data gathered from the system are also important. "Automatic" does not mean that the role of a competent herdsman is in any way diminished.

The aim of this study was to identify specific phylogeny groups, virulence genes or antimicrobial resistance traits of Escherichia coli isolated in bovine mastitis associated to clinical signs, persistence of intramammary infection in the quarter and recovery from mastitis. A total of 154 E. coli isolates from bovine clinical mastitis, 144 from the acute stage and 10 from follow-up samples 3 weeks later, originating from 144 cows in 65 dairy herds in Southern Finland were investigated. Phylogeny groups and virulence genes of the isolates were determined using polymerase chain reaction, and antimicrobial susceptibility using the VetMIC™ microdilution method. In ten cows (11.8%), infection persisted, confirmed by re-isolation of the same pulsed-field gel electrophoresis type from the affected quarter at 3 weeks post-treatment. The majority of isolates, 119 (82.6%), belonged to phylogeny group A, which mainly consisted of commensal strains. Altogether 56 isolates (38.9%) had at least one virulence gene detected. Most common virulence genes detected were irp2, iucD, papC iss; genes svg, stx1, stx2, cnf1 and hlyA were not found. Combinations of virulence genes varied greatly. Forty (27.8%) of the 144 E. coli isolates showed resistance to at least one antimicrobial tested. None of the studied phylogeny groups, virulence factors or antimicrobial resistance traits was associated with clinical signs, persistence of intramammary infection or clinical recovery from mastitis. The results support the conclusion that mastitis-causing E. coli bacteria are typical commensals.

Udder health and milk production were monitored in cows transferred from tie stalls or loose housing with conventional milking to loose housing with either automatic or conventional milking. Data were collected from 182 Finnish farms from September 1999 to February 2006. Data from the first year before and first year after the changes were compared. A total of 88 herds changed from conventional milking (CM herds) to automatic milking (AM herds), 29 of which were housed in tie stalls and 59 of which were housed in a loose housing barn before the change. Additionally, 94 CM herds milked in loose housing barns that had been housed in tie stalls before the change were included. Milk record data consisted of annual herd size, parity, breed, calving dates, test day data [date, milk yield, and cow somatic cell count (SCC)] and records for treatments of clinical mastitis. Calculations were made for energy-corrected milk yield and logarithmic SCC (logSCC), proportion of cows at risk that experienced an SCC >200,000 cells/mL for the first time (highSCC), and number of treatments of clinical mastitis within a herd. Cows in tie stalls had higher milk yield (28.5 +/- 0.29 vs. 26.5 +/- 0.46 kg/d) and a lower logSCC (4.86 +/- 0.01 vs. 4.95 +/- 0.02) than cows in loose housing barns before the change. After the change, CM herds had slightly better udder health than AM herds because the proportion of cows at risk for highSCC was larger in AM herds (3.3 vs. 2.1%). The change in milking and housing systems caused a decline of 0.8 +/- 0.25 kg/d per cow in energy-corrected milk yield, a slight increase in cow logSCC (from 4.88 +/- 0.01 to 4.93 +/- 0.01), and an increase of 0.6% in the proportion of cows having highSCC (from 2.5 to 3.1). The impact was clearer on herds that began automatic milking. Based on the results, the increase in bulk milk SCC of herds milked automatically in Finland was probably due to reduced separation of mastitic milk in AM herds.

Staphylococcus aureus isolates collected from sites of intramammary infection during a 10-month period and from extramammary sites (dairy cow teat skin, teat canals, and skin lesions; milking liners; and hands and nostrils of milking personnel) at two separately managed Finnish dairy herd establishments were analyzed to study the sources and reservoirs of bovine S. aureus intramammary infection. Selected isolates were subjected to pulsed-field gel electrophoresis (PFGE) typing and PCR analysis for genes encoding hemolysins ( hla to hlg ), leukocidins ( lukED and lukM ), superantigens ( sea , sec , sed , seg to seo , seu , and tst ), adhesins ( fnbA and fnbB ), and penicillin and methicillin resistance ( blaZ and mecA ). S. aureus was found throughout the herds in 94% of the cows. Nine PFGE types were found, with the herds each having their own predominant type and sharing one type. The degree of diversity of PFGE types in herd II, which integrated foreign heifers, was higher than that in herd I. For both herds, the majority of the PFGE-typed isolates both from milk and from extramammary sites represented the predominant PFGE types. In isolates from herd I, the most prevalent genes were hla - hlg , lukED , and fnbA ; in those from herd II, they were hla , hld , hlg , lukED , and fnbA . The other genes were pulsotype linked within the herds. The predominant PFGE types carried both fnbA and fnbB ; only fnbA was detected in the other PFGE types. No connection between specific virulence genes and the origins of isolates was found. The results suggest that for the two herds, most S. aureus isolates from extramammary sites were indistinguishable from the isolates infecting the mammary gland and that those sites can thus act as origins and reservoirs of intramammary infections. However, contamination in the opposite direction cannot be excluded.

Aims: To screen putative virulence genes in Staphylococcus aureus causing persistent and nonpersistent bovine intramammary infections (IMI) with different clinical characteristics. To examine, whether a possible relationship exists between genetic profile and infection persistence, clinical signs of infection, clonal type determined by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance. Methods and Results: One-hundred and sixty-one S. aureus isolates derived from bovine IMI, consisting of 17 different PFGE types, were screened by conventional and multiplex-polymerase chain reaction (PCR) for 24 virulence genes for haemolysins (hla-hlg), leukocidins (lukED, lukM), exfoliative toxins (eta, etb), enterotoxins (sea-seo, seu), toxic-shock syndrome toxin (tst), and genes encoding penicillin (blaZ) and methicillin resistance (mecA). The majority of S. aureus isolated at the onset of mastitis carried haemolysin genes (76·7–97·4%), lukED (96·6%), and at least one gene for pyrogenic toxin superantigen (PTSAg) (69·0%). Strains carrying PTSAg-encoding genes were more common among predominant PFGE types and in persistent IMI. Strains concomitantly possessing sed, sej, and blaZ, putatively plasmid-encoded, were typically found in connection with persistent IMI. Conclusions: Our results suggest that certain genetic elements are over-representative in S. aureus isolates especially from persistent bovine mastitis. This phenomenon seems to be in connection with clonal type and is often concomitant with penicillin resistance. Significance and Impact of the Study: This is the first study to investigate associations between a large number of bacterial factors and outcome of S. aureus mastitis. The finding that widespread clonal types of S. aureus causing bovine mastitis of low treatment response may harbour characteristic genes could be improved for strain-specific diagnostic purposes.

A database containing pathogen-specific information on mastitis has been established in Finland. The data consist of the results from routine milk samples collected from clinical and subclinical mastitis and submitted to laboratories all over the country. In the database, bacteriological information has been recorded on an individual cow basis. A total of 77,051 pathogen records submitted to the database from 1 January 2004 to 1 January 2006 were retrieved for this preliminary report of pathogen distribution. Staphylococcus aureus (18.26% and 17.73%) and coagulase negative staphylococci (CNS) (17.57% and 23.51%) were the most frequently isolated pathogens from the milk samples. Seasonal and regional differences in pathogen distribution were also found. The pathogen information is primarily used for targeting the antimicrobial treatment of the cow, but also for designing specific mastitis control strategies for the herds. Pathogen data can also be useful for national mastitis control programmes an...

Eighty-four calves with respiratory disease from 18 herds in different parts of Finland were chosen for a study evaluating the capacity of different respiratory pathogens to cause changes in different acute phase protein concentrations, white blood cell (WBC) count and clinical signs. The selected acute phase proteins were fibrinogen, haptoglobin, serum amyloid-A, lipopolysaccharide binding protein and alpha1-acid glycoprotein. From each calf, a paired blood sample was obtained for serological studies of bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine coronavirus, bovine adenovirus-3 and bovine adenovirus-7. Tracheobronchial lavage was performed to detect bacteria and mycoplasma. Isolation of Pasteurella multocida was associated with increased concentrations of all tested acute phase proteins. For other pathogens, no significant relationships were observed. No association was present between viral or bacterial findings and WBC count.

Background Coagulase-negative staphylococci (CoNS) are the most common bovine mastitis causing bacteria in many countries. It is known that resistance for antimicrobials is in general more common in CoNS than in Staphylococcus aureus but little is known about the antimicrobial resistance of specific CoNS species. In this study, 400 CoNS isolates from bovine mastitic milk samples were identified to species level using ribotyping and MALDI-TOF MS, and their antimicrobial susceptibility was determined using a commercially available microdilution system. The results were interpreted according to the epidemiological cut-off values by the European Committee on Antimicrobial Susceptibility testing. Results The most common CoNS species were S. simulans, S. epidermidis, S. chromogenes and S. haemolyticus. Penicillin resistance was the most common type of antimicrobial resistance. Staphylococcus epidermidis was the most resistant among the four major species. Almost one-third of our S. epidermidis isolates were resistant to >2 antimicrobials and close to 7 % were multidrug resistant. The majority of S. epidermidis isolates were resistant to benzylpenicillin. On the contrary, only few S. simulans isolates were penicillin-resistant. Phenotypic oxacillin resistance was found in all four main species, and 34 % of the isolates were oxacillin resistant. However, only 21 isolates (5 %) were positive for the mecA gene. Of these, 20 were S. epidermidis and one S. sciuri. mecC positive isolates were not found. Conclusion Staphylococcus epidermidis differed from the three other major CoNS species as resistance to the tested antimicrobials was common, several isolates were multidrug resistant, and 19 % of the isolates carried the mecA gene encoding methicillin resistance. Electronic supplementary material The online version of this article (doi:10.1186/s13028-016-0193-8) contains supplementary material, which is available to authorized users.

Detection of mastitis and reliability of alarms were evaluated in eight herds with automatic milking. Herds were visited once to gather milk samples and the farmers followed up alarms for 15 to 30 days. Sensitivity of detecting quarters with SCC over the threshold value of 200,000 or 1,000,000 cells/ml was 18.0 to 42.9% in Group A, based on electrical conductivity (EC) and 5.5 to13.0% in Group B, based on EC and milk colour. Specificity was over 97.0%. In Group A, 6/17 of the EC alarms were false positives. None of the seven EC alarms in Group B was false positive. On the day that the farmer observed clinical mastitis, more than half of the quarters received alarms, but only milk from some quarters was automatically diverted. In total 58.4% (Group A) and 26.8% (Group B) of cows had alarms based on EC, and 84.1% (

The coagulase-negative staphylococcal species causing mastitis in lactating cattle were identified and possible differences in the clinical characteristics or persistence of mastitis caused by different CNS were evaluated. The effect of antimicrobial treatment was also assessed. In addition, AFLP-typing of CNS was compared with the phenotypic identification. A total of 133 clinical or subclinical quarter cases of intramammary infection caused by CNS from the practice area of the Ambulatory Clinic of the University of Helsinki were studied. Bacteriological diagnosis was based on biochemical (API) testing. Staphylococcus simulans (43.6%) followed by S. chromogenes (23.3%) were the most common CNS species isolated from the milk samples. Ninety-nine isolates were genotyped using AFLP-analysis. Only 75.0% of S. chromogenes and S. simulans isolates identified with API test were clustered with the type strains of these species. Approximately half of the mastitis cases were clinical, and in the majority clinical signs were mild. The severity and persistence of intramammary infection were unaffected by CNS species. Fifty-nine percent of the quarter cases were treated with antimicrobials, and the rest were left without treatment. Mastitis due to beta-lactamase-negative CNS was treated with penicillin G and that due to beta-lactamase-positive CNS with cloxacillin. Nineteen percent of the isolates were beta-lactamase-positive. The bacterial cure rate for quarters treated with antimicrobials was high, 85.9%, as opposed to only 45.5% for untreated quarters. Bacterial cure rates for the most common CNS species or AFLP clusters were not statistically different. Further studies on identification of CNS species are needed.

Technical success and effectiveness of teat cleaning and the management factors associated with them were evaluated in 9 automatic milking herds. In total, 616 teats cleaned with a cleaning cup and 716 teats cleaned with rotating brushes were included. Technical success and the effectiveness of teat cleaning, including the location and nature of the dirt, were evaluated visually. On average, 79.9% of teat cleanings with a cleaning cup, and 85.0% of those cleaned with brushes succeeded technically; that is, the teat was correctly positioned in the cleaning device throughout the whole cleaning process. The difference between use of teat cups and brushes was significant. However, because technical success of teat cleaning is strongly dependent on herd characteristics, these results should be interpreted with caution. Factors associated with the technical success of teat cleaning with a cleaning cup were herd, days in milk, behavior of the cow, teat color, and teat location. For rotating brushes, behavior of the cow, teat location, udder and teat structure, and days in milk were associated with technical success. Excessive udder hair and technical failure of the automatic milking machine also caused a few technically unsuccessful teat cleanings with a cleaning cup. Teats with technically successful teat cleanings were evaluated for the effectiveness of teat cleaning. From originally dirty teats, the cleaning cup had a significant advantage over the brushes in the percentage of teats that became clean or almost clean during the cleaning process (79.8 vs. 72.9%). Teat orifices were least effectively cleaned compared with the teat barrel and apex. Bedding material (peat, sawdust, or straw) on the teat was cleaned almost completely. Factors associated with the effectiveness of teat cleaning were teat cleanliness before cleaning, herd, teat cleaning method, and teat condition. The variation among herds indicates the likelihood that herd management factors can be adjusted to improve milking hygiene. There is also a need to improve the precision and effectiveness of the teat cleaning mechanisms of automatic milking systems.

A nationwide survey was conducted in Finland to estimate prevalence of bovine mastitis, distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens. In total, 12,661 quarter milk samples were collected from 3282 dairy cows at 216 farms. These were randomly selected from a database covering all Finnish dairy farms. Quarter milk samples collected by the dairy advisors were submitted for somatic cell counting, bacteriological examination, and testing for antimicrobial susceptibility. If the milk SCC of a cow or of a quarter exceeded 300,000/mL, the cow was defined as having mastitis. The results were compared with those of a previous survey done in 1995. The prevalence of mastitis continued to decrease from 38% in 1995 to 31% in 2001. Compared with the study from 1995, the number of quarters with bacterial growth in 2001 increased significantly from 21.0 to 33.5%. This mainly resulted from increased prevalence of Corynebacterium bovis. Coagulase-negative staphylococci remained the most common bacterial group, comprising almost one-half of the pathogens isolated, whereas the relative number of Staphylococcus aureus isolations decreased from the time of the previous study. According to in vitro antimicrobial susceptibility testing, the enterococci demonstrated the highest level of resistance. Compared with the other Nordic countries, penicillin resistance among the staphylococci was still at a relatively high level in Finland (52.1 and 32.0% for Staphylococcus aureus and coagulase-negative staphylococci, respectively). Streptococci isolated from mastitis were very susceptible to beta-lactam antibiotics, as also found in the previous survey in 1995.

The effect of bovine lactoferrin (Lf) was studied in experimental Escherichia coli mastitis, using enrofloxacin as a comparator. Mastitis was induced in six clinically healthy primiparous dairy cows by infusing 1500 colony-forming units of E. coli into a single udder quarter. The challenge was repeated into a contralateral quarter of the same cows 3 weeks later. At the first challenge, three cows were treated with 1.5 g of bovine lactoferrin intramammarily three times (12, 20 and 36 h postchallenge, PC), and the other three cows received 5 mg/kg of enrofloxacin (Baytril®) parenterally (12, 36 and 60 h PC). Flunixin meglumine (2.2 mg/kg) was administered to all cows twice at 24-h intervals. During the second challenge, the treatments for the two groups were reversed. Intramammary challenge with E. coli produced clinical mastitis in all cows, but the severity of the disease varied markedly. No statistically significant differences between treatment groups were observed in clinical signs such as rectal temperature, rumen motility and general attitude. Milk somatic cell count, daily milk yield and bacterial counts in cows treated with Lf and those receiving enrofloxacin also did not differ significantly. However, a trend for a more rapid elimination of bacteria was seen in the cows treated with enrofloxacin. Milk NAGase activity also decreased significantly faster in the group treated with enrofloxacin. The concentration of lipopolysaccharide in milk compared with the number of bacteria was significantly lower in Lf than in enrofloxacin-treated cows (20 h PC).

Minimal inhibition concentration (MIC) values of 100 Finnish and 100 Israeli Escherichia coli isolated from clinical bovine mastitis were determined for ampicillin, cephalexin, ceftazidime, dihydrostreptomycin, gentamicin, tetracycline, trimethoprim-sulfadiazine, and ciprofloxacin by an agar dilution method. The in vitro antimicrobial susceptibility of the E. coli isolates was high; only 27% showed resistance to one or more tested antimicrobial agents. Fifteen percent of the Israeli isolates and 14% of the Finnish isolates were resistant to tetracycline, 3 and 16% to cephalexin, 10 and 7% to ampicillin, 13 and 9% to dihydrostreptomycin, and 4 and 2% to trimethoprim-sulfadiazine. No gentamicin–, ceftazidime-, or ciprofloxacin-resistant isolates were detected. Eleven percent of all the isolates were resistant to two or more antimicrobial agents. Tetracycline was most often associated with multiresistant patterns. Most of the multiresistant isolates had very high MIC values, whereas most of those that were resistant to only one tested antibiotic had MIC values close to the susceptibility breakpoint. Antimicrobial resistance appeared to pose no problem in E. coli isolated from mastitic milk of both countries. This is probably due to the controlled use of antimicrobial agents in the treatment of dairy herds. Some differences were present in the resistance patterns, which may reflect the different use of antimicrobial agents in these two countries.

International audience; Mastitis affects the quality of milk and is a potential health risk for the other cows. In a well managed dairy herd, in addition to clinical mastitis, subclinical mastitis should be efficiently detected. Bacteriological sampling is not feasible as a routine test to identify subclinical mastitis, and indirect tests of mastitis are more suitable for selecting cows with intramammary infections for subsequent bacteriological sampling. Mastitis affects the composition of milk, and the degree of changes depends on the infecting agent and the inflammatory response. Indicators of inflammation in the milk which can be determined using rapid, reliable and easy routine techniques, can be used for the early detection of mastitis. The measuring of the somatic cell count in milk is the standard method, but the analysis technique is problematic for routine use in herds. The most promising parameters for monitoring subclinical mastitis are milk N-acetyl-$\beta$-D-glucosaminidase activity, lactose, and electrical conductivity along with some other indicators such as optical and milk flow measurements, preferably with an inter-quarter evaluation included in the test. Acute phase proteins, haptoglobin and serum amyloid A, are also potential candidates for mastitis monitoring. New mastitis detection systems which can be adapted into on-line use are urgently needed, since dairy units are growing bigger and automatic milking systems are being taken into use.

Quarter milk samples were taken from 48 clinically healthy, pregnant Finnish Ayrshire and Friesian dairy cows at the last milking before drying-off, and 2 and 6 days later to determine the lactoferrin and citrate concentrations in the udder secretion. The mean lactoferrin concentration in the milk increased from 5.29 mg ml(-1) on the last day of drying-off process to 8.09 and 11.26 mg ml(-1), 2 and 6 days later, respectively. Citrate concentration decreased from 1.85 mg ml(-1) to 1.54 and 1.09 mg ml(-1), respectively. Median molar ratio (citrate to native-Lf) decreased from 153 to 86 and further to 44. Lf and citrate concentrations in milk varied greatly among cows. Sampling time had a statistically significant effect on lactoferrin and citrate concentrations in milk while the breed and the parity of the cows had no effect on either of these variables.

We investigated the differences in the effect of intramammarily infused endotoxin for cows in early (EL) and late (LL) lactation. In this crossover study, nine cows were challenged twice with 100 microg of intramammarily infused Escherichia coli 0111:B4 LPS, each cow serving its own control. Systemic and local signs were recorded throughout the experiment to assess the severity of each cow's response. Daily milk yield and indicators of inflammation in blood and milk were also recorded. The response was significantly more severe in the EL period. Before the challenge, milk yield and serum free fatty acid concentration were higher and serum urea concentration lower in EL compared with LL cows. No significant differences were present in other parameters. After the challenge, milk SCC and leukocyte function, measured as chemiluminescence, increased, more markedly in EL. Blood neutrophil content varied from neutropenia to neutrophilia. Simultaneously with neutropenia, the function of neutrophils increased in LL but decreased in EL. Serum cortisol peaked 4 h after challenge in both groups. In conclusion, endotoxin challenge resulted in more severe response in EL. During that time cows also had impaired neutrophil function postchallenge.

The efficacy of intramammary treatments containing procaine penicillin G alone (treatment A) or a combination of procaine penicillin G and neomycin (treatment B) was compared in treating clinical bovine mastitis caused by gram-positive bacteria susceptible in vitro to penicillin G. Both treatments were supplemented with a single intramuscular injection of procaine penicillin G on the first day of treatment. The study was carried out using a double blind design on commercial dairy farms in Southern Finland. A total of 56 quarters were treated with treatment A and 61 with treatment B. The cure rates for both treatments were equal, which suggests that the use of the penicillin G-aminoglycoside combination does not increase the efficacy of the treatment over that achieved by using penicillin G alone in bovine clinical mastitis caused by penicillin-susceptible, gram-positive bacteria.

The objective of this study was to determine the effect of an anionic diet on mineral metabolism, acid-base status and udder oedema of dairy cows fed grass silage based diets during the dry period. Eighteen pregnant, non-lactating Friesian cows were divided randomly into two groups according to their expected calving date. Dietary cation-anion balance (DCAB), calculated as mill equivalents [(Na + + K + ) - (Cl - + S 2- )], for high DCAB (control) and low DCAB treatments were +254 and -41 mEq kg -1 dry matter (DM), respectively. Anionic salts were ammonium chloride (NH 4 Cl), magnesium chloride (MgCl 2 ) and magnesium sulphate (MgSO 4 ). Cows received grass silage (5.2 kg DM), hay (0.9 kg DM) and a concentrate mixture (2.7 kg DM) until calving. Blood and urine samples were collected 4, 3, 2 and 1 week before the expected calving date, at calving, 1 day and 1 week after calving. Udder oedema was evaluated by a quantitative and a subjective method. Acidification resulted in a marked decrease in urinary pH, increased urinary Ca excretion and a change in blood acidbase balance. Blood Ca 2+ and plasma Ca tot concentrations were more stable at parturition for the anionic group, although such differences were not statistically significant. Anionic salts (DCAB -41 mEq kg -1 DM) did not cause udder oedema in experimental cows., Kokeen tarkoituksena oli selvittää anionisten suolojenvaikutuksia ummessa olevien lehmien veren ja virtsan kivennäisaineenvaihduntaan, happo-emästasapainoon sekä erityisesti utarepöhöön. Kationi-anioni -tasapaino laskettiin [(Na + + K + ) -(Cl - + S 2- )] mEq/kg kuiva-ainetta (ka), ja se oli joko +254 tai -41 mEq/kg ka. Lehmät saivat säilörehua 5,2 kg ka, heinää 0,9 kg ka ja täysrehua 2,7 kg ka nel-jä viikkoa ennen odotettua poikimista poikimispäivään saakka. Veri- ja virtsanäytteet otettiin 4, 3, 2 ja 1 viikkoa ennen odotettua poikimista sekä poikimispäivänä, 1 vrk ja 1 viikko poikimisen jälkeen. Utare-pöhö mitattiin ja arvioitiin silmämääräisesti. Anioniset suolat alensivat virtsan pH:ta, lisäsivät kalsiumin erittymistä virtsaan ja muuttivat veren happo- emästasapainoa. Veren ionisoitunut kalsiumpitoisuus ja plasman kalsiumpitoisuus pysyivät anionisia suoloja saaneilla lehmillä poikimisen aikaan vakaampina kuin lehmillä, jotka eivät olleet saaneet anionisia suoloja. Anionisten suolojen syöttäminen (-40 mEq/kg ka) ennen poikimista ei aiheuttanut lehmille utarepöhöä.

Extensive studies have shown the polymorphonuclear leukocytes (PMN) dysfunction inextricably links to parturition. To investigate the effect of parity on PMN function, phorbol 12-myristate 13-acetate (PMA) stimulated luminol-amplified chemiluminescence (CL) and viability. of blood and milk PMN were investigated in primiparous and pluriparous dairy cows during periparturient period. The CL kinetics of blood and milk PMN and hematological profiles were also, assessed. Milk PMN CL was always lower than blood PMN CL. Blood and milk PMN CL and milk PMN viability were significantly higher in primiparous cows throughout the study. Blood PMN CL in pluriparous cows showed a,sharper decrease. Both in pluriparous and in primiparous cows, minimal blood PMN CL appeared at periparturient day (PPD) 2. After PPD 7, blood PMN CL recovery rate was faster in primiparous cows. Milk PMN CL was minimal at PPD 2. in both groups. Whereas no changes were observed in blood PMN viability, the viability of milk PMN in. primiparous cows was substantially higher than in pluriparous cows. The number of circulating eosinophils and immature neutrophils was substantially higher in primiparous cows throughout the study. The CL kinetics of blood PMN at PPD -2 and 2 and of milk PMN at PPD 2 exhibited different responses to PMA, with higher intensity and durability, peaking and subsiding more slowly in primiparous dairy cows. The pronounced. reduction in PMN CL and viability in milk PMN of pluriparous cows may be involved in the underlying mechanisms that make these animals more susceptible to periparturient infectious diseases.

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half-life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty-eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.

Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors. The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E. coli, clinical pictures of coliform mastitis, environmental conditions and herd management. The genes of nine virulence factors were detected by polymerase chain reaction. Presence of K1 and K5 capsules was assessed by use of specific bacteriophages. Serum resistance was tested by a turbidimetric assay. Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E. Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17. Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants. Genes for F17a, CS31A, Afa7D and Afa7E were not detected. Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates. Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113). K1 and K5 capsules were not detected.

The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated fdr marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains.

Pleuromutilins (tiamulin and valnemulin) are antimicrobial agents that are used mainly in veterinary medicine, especially for swine and to a lesser extent for poultry and rabbits. In pigs, tiamulin and valnemulin are used to treat swine dysentery, spirochaete-associated diarrhoea, porcine proliferative enteropathy, enzootic pneumonia and other infections where Mycoplasma is involved. There are concerns about the reported increases in the MICs of tiamulin and valnemulin for porcine Brachyspira hyodysenteriae isolates from different European countries, as only a limited number of antimicrobials are available for the treatment of swine dysentery where resistance to these antimicrobials is already common and widespread. The loss of pleuromutilins as effective tools to treat swine dysentery because of further increases in resistance or as a consequence of restrictions would present a considerable threat to pig health, welfare and productivity. In humans, only one product containing pleuromutilins (retapamulin) is authorized currently for topical use; however, products for oral and intravenous administration to humans with serious multidrug-resistant skin infections and respiratory infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA), are being developed. The objective of this review is to summarize the current knowledge on the usage of pleuromutilins, resistance development and the potential impact of this resistance on animal and human health.

Macrolides and lincosamides are important antibacterials for the treatment of many common infections in cattle and pigs. Products for in-feed medication with these compounds in combination with other antimicrobials are commonly used in Europe. Most recently approved injectable macrolides have very long elimination half-lives in both pigs and cattle, which allows once-only dosing regimens. Both in-feed medication and use of long-acting injections result in low concentrations of the active substance for prolonged periods, which causes concerns related to development of antimicrobial resistance.Acquired resistance to macrolides and lincosamides among food animal pathogens, including some zoonotic bacteria, has now emerged. A comparison of studies on the prevalence of resistance is difficult, since for many micro-organisms no agreed standards for susceptibility testing are available. With animal pathogens, the most dramatic increase in resistance has been seen in the genus Brachyspira. Resistance towards macrolides and lincosamides has also been detected in staphylococci isolated from pigs and streptococci from cattle. This article reviews the use of macrolides and lincosamides in cattle and pigs, as well as the development of resistance in target and some zoonotic pathogens. The focus of the review is on European conditions.

Beta (beta) and delta (delta)-hemolysin of Staphylococcus aureus strains were cultured in vitro in milk lactoserum (whey) prepared from both healthy and mastitis bovine milk. Production of beta- and delta-hemolysins were detected in 12 out of 50 strains studied. The association between N-acetyl-beta-D-glucosaminidase (NAGase) activity, plasmin activity (PL) and trypsin inhibitory capacity (TIC), known as inflammatory indicators for mastitis, and hemolytic activity were also studied. Mastitic milk decreased directly the lytic effect of both beta-and delta-hemolysins of S. aureus on hemolytical blood agar plates. S. aureus in healthy milk samples produced more beta-hemolysin (3 times) and delta-hemolysin (2 times) when compared to S. aureus supernatants in milk from infected quarters. Furthermore, beta- and delta-hemolysis correlated negatively with TIC and NAGase and PL activities. Addition of reduced glutathione (GSH) or beta-mercaptoethanol into the artificial medium enhanced hemolysins activity.

Five Finnish Ayrshire cows in mid or end-lactation were treated with 40 mg sulphadiazine/kg and 8 mg trimethoprim/kg using intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) routes. Elimination of sulphadiazine was not affected by the route of administration (median t(½)4.4–5.0 h) while elimination of trimethoprim was strongly limited by slow absorption from the injection site after s.c. and i.m. administration (median for apparent t(½). 21–25 h) compared to that after i.v. administration (median t(½) 1.2 h; p

Six Finnish Ayrshire cows were challenged intramammarily with 1500 CFU of Escherichia coli (E. coli) into single udder quarters, and the challenge was repeated into contralateral quarters 3 weeks later. All cows received flunixine meglumine once, and 3 of them were also treated with enrofloxacin. At the 2nd challenge, treatments were changed vice versa. The development of mastitis was followed by monitoring of systemic and local clinical signs, and with serial milk and serum samples. Intramammary challenge with E. coli produced clinical mastitis in all cows, the severity of the disease varying greatly between the animals. No significant changes between the 2 treatment regimens or sequent challenges were found for any of the clinical parameters. The response of each cow followed the same pattern after both challenges; three of the cows became mildly and the other 3 either moderately or severely affected. Two severely affected cows had to be euthanized because of severe mastitis. Serum haptoglobin and amyloid-A concentrations peaked 2-3 days after bacterial challenge. Serum haptoglobin did not correlate with the severity of the disease. Serum amyloid-A rose gradually in the severely affected cows, and significant differences were found between severely versus moderately or mildly affected cows at day 4. Serum tumor necrosis factor alpha concentrations increased only in the severely affected cows. Serum cortisol response was prolonged in the severely diseased animals, and was significantly lower after the second challenge. Serum nitrite/nitrate concentration increased in the severely affected cows. This indicated excess nitric oxide production during acute E. coli mastitis. Strongly decreased milk production, and high bacterial growth in the infected quarters were best predictors for the outcome from acute E. coli mastitis.