Pathogen-specific production losses in bovine mastitis.

Reduction in long-term milk yields represents a notable share of the economic losses caused by bovine mastitis. Efficient, economic, and safe measures to prevent these losses require knowledge of the causal agent of the disease. The aim of this study was to investigate pathogen-specific impacts of mastitis on milk production of dairy cows. The materials consisted of milk and health recording data and microbiological diagnoses of mastitic quarter milk samples of 20,234 Finnish dairy cows during 2010, 2011, and 2012. The 6 most common udder pathogens were included in the study: Staphylococcus aureus, non-aureus staphylococci (NAS), Escherichia coli, Corynebacterium bovis, Streptococcus uberis, and Streptococcus dysgalactiae. We used a 2-level multilevel model to estimate curves for lactations with and without mastitis. The data on lactation periods to be compared were collected from the same cow. To enable comparison among lactations representing diverse parities, the estimated lactation curves were adjusted to describe the cow's third lactation. Mastitis caused by each pathogen resulted in milk production loss. The extent of the reduction depended on the pathogen, the timing of mastitis during lactation, and the type of mastitis (clinical vs. subclinical). The 2 most commonly detected pathogens were NAS and Staph. aureus. Escherichia coli clinical mastitis diagnosed before peak lactation caused the largest loss, 10.6% of the 305-d milk yield (3.5 kg/d). The corresponding loss for Staph. aureus mastitis was 7.1% (2.3 kg/d). In Staph. aureus mastitis diagnosed between 54 and 120 d in milk, the loss was 4.3% (1.4 kg/d). The loss was almost equal in both clinical and subclinical mastitis caused by Staph. aureus. Mastitis caused by Strep. uberis and Strep. dysgalactiae resulted in losses ranging from 3.7% (1.2 kg/d) to 6.6% (2.1 kg/d) depending on type and timing of mastitis. Clinical mastitis caused by the minor pathogens C. bovis and NAS also had a negative effect on milk production: 7.4% (2.4 kg/d) in C. bovis and 5.7% (1.8 kg/d) in NAS when both were diagnosed before peak lactation. In conclusion, minor pathogens should not be underestimated as a cause of milk yield reduction. On single dairy farms, control of E. coli mastitis would bring about a significant increase in milk production. Reducing Staph. aureus mastitis is the greatest challenge for the Finnish dairy sector.

The effect of a cannula milk sampling technique on the microbiological diagnosis of bovine mastitis.

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).

Factors associated with intramammary infection in dairy cows caused by coagulase-negative staphylococci, Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Corynebacterium bovis, or Escherichia coli.

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.

Use of antimicrobials in the treatment of reproductive diseases in cattle and horses.

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.

Efficacy of 5-day parenteral versus intramammary benzylpenicillin for treatment of clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro.

The efficacy of parenteral (intramuscular) or intramammary (IMM) benzylpenicillin treatment for clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro was investigated. Cows with clinical mastitis in 1 udder quarter were randomly placed into 2 treatment groups. The preliminary bacteriological diagnosis of intramammary infection (IMI) was based on on-farm culturing, and the bacteriological diagnoses were later confirmed by a quantitative PCR assay. Clinical mastitis caused by gram-positive bacteria susceptible to benzylpenicillin was treated with penicillin via either the parenteral route (20mg/kg) or IMM route (600mg) once per day for 5d. The outcome of the treatment was evaluated 3 to 4wk after the onset of the treatment. The affected quarter was examined to assess the clinical cure, and milk samples were collected from the affected quarter to determine the bacteriological cure and milk N-acetyl-ß-d-glucosaminidase activity. The survival and the composite milk somatic cell counts of the treated cows were followed up for 6 and 3mo after treatment, respectively. A total of 140 cows with clinical mastitis were included in the study, 61 being treated with benzylpenicillin parenterally and 79 via the IMM route. From all quarters treated, 108 of 140 (77.1%) were cured clinically and 77 of 140 (55.0%) were cured bacteriologically. The route of treatment did not significantly affect the outcome of the treatment; 80.3% of the quarters with parenteral treatment and 74.7% of the quarters with IMM treatment showed a clinical cure, and 54.1 and 55.7% a bacteriological cure, respectively. The milk N-acetyl-ß-d-glucosaminidase activity was significantly lower in the quarters with a clinical or bacteriological cure than in the quarters with no cure. The 6-mo survival and the proportion of cows with composite milk somatic cell counts <200,000/mL among the treated cows during the 3-mo follow-up period did not significantly differ between the treatment groups. In conclusion, the outcome of either parenteral or IMM benzylpenicillin treatment of clinical mastitis caused by penicillin-susceptible bacteria was similar.

Treatment for bovine Escherichia coli mastitis - an evidence-based approach.

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.

Milk haptoglobin, milk amyloid A, and N-acetyl-ß-D-glucosaminidase activity in bovines with naturally occurring clinical mastitis diagnosed with a quantitative PCR test.

The associations between quantitative bacteriological results from a real-time PCR test and concentrations of acute-phase proteins (APP) and N-acetyl-ß-d-glucosaminidase (NAGase) activity in milk in naturally occurring clinical mastitis were investigated. Milk APP concentrations and NAGase activity in clinical mastitis caused by different udder pathogens were studied. The associations between the severity of the clinical signs and concentrations of APP and NAGase activity were estimated. Milk samples from 281 cases of clinical mastitis were collected from 3 Estonian dairy farms and analyzed by PCR to identify pathogens. Twenty-seven samples out of 281 (9.6%) were PCR negative. Milk samples containing 4 or more bacterial species (n=28) were considered possibly contaminated and excluded from all further analyses. In total, 443 bacterial identifications were made from the remaining 226 milk samples. A single bacterial species was detected in 68 samples (30.1%), 2 species were detected in 99 samples (43.8%), and 3 species were detected in 59 (26.1%) samples. To determine the inflammatory response in the udder, the concentrations of milk amyloid A (MAA) and haptoglobin (Hp) and NAGase activity in the milk were analyzed. A significant positive association was found between the severity of the clinical signs and inflammatory markers in the milk. Milk amyloid A and Hp concentrations and NAGase activity were significantly higher in samples with large quantities of bacterial DNA from Escherichia coli or Streptococcus dysgalactiae compared with milk samples not containing those species. Large quantities of bacterial DNA from Trueperella pyogenes or Streptococcus uberis in the milk were associated with elevated concentrations of Hp and high NAGase activity, but not with increased MAA concentrations. Milk samples containing Corynebacterium bovis and coagulase-negative staphylococci had significantly lower concentrations of MAA and Hp and lower NAGase activity compared with samples where these species were not detected. It can be concluded that concentrations of APP and NAGase activity in the milk were associated with the quantity of bacterial DNA in the milk samples.

Is the biofilm formation and slime producing ability of coagulase-negative staphylococci associated with the persistence and severity of intramammary infection?

Biofilm and slime formation assists bacteria in avoiding the host immune defence and antimicrobial therapy. It is suspected to affect the severity or persistence of mastitis caused by coagulase-negative staphylococci (CNS), which are a common cause of bovine mastitis. The phenotypic biofilm formation ability of 244 CNS isolates (199 isolates from bovine mastitis and 52 type and reference strains) was investigated with a tissue culture plate (TCP) assay and fluorescent in situ hybridization (FISH). Slime production of the strains was assessed using Congo red agar (CRA) plates. Additionally, genes encoding the adhesion proteins MSCRAMM (microbial surface components recognizing adhesive matrix molecules) and biofilm-associated proteins (bap) were detected. The severity of intramammary infection (IMI) in mastitis from which the isolates originated was measured with milk N-acetyl-ß-D-glucosaminidase (NAGase) activity. One-third of isolates from mastitis produced biofilm when analysed with TCP or FISH. The kappa test value, measuring the agreement between two tests, differed between CNS species. Slime production was less frequent for isolates of the common mastitis species Staphylococcus chromogenes (0.2% of isolates produced slime) and Staphylococcus simulans (3.5%) compared to Staphylococcus epidermidis (40%). No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was rare in isolates originating from IMI. Only 12.7% of isolates from persistent IMI and 1.8% of isolates from spontaneously eliminated IMI produced slime. The eno gene encoding laminin-binding protein was most frequently detected among the isolates from mastitis, 75% of them having this gene. Only a few other MSCRAMM genes were detected.

Costs of clinical mastitis with special reference to premature culling.

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.

Acute phase proteins in milk in naturally acquired bovine mastitis caused by different pathogens.

The concentrations of haptoglobin (Hp) and serum amyloid A (SAA) and the activity of N-acetyl-ß-D-glucosaminidase (NAGase) in milk from 234 cows with spontaneous mastitis caused by different pathogens were measured to assess whether they corresponded with the clinical signs of mastitis and whether there were any differences between pathogens. Ninety-eight of the cows had clinical mastitis and 136 had subclinical mastitis. There were statistically significant positive correlations between the concentrations of SAA and Hp and the activity of NAGase. Significant differences in the concentrations of acute phase proteins and NAGase activity were found in milk from cows with mastitis caused by different pathogens. The highest concentrations of Hp and NAGase were found in cases of mastitis caused by Escherichia coli and Arcanobacterium pyogenes, and the lowest concentrations were from cases of mastitis caused by coagulase-negative staphylococci. Very low SAA concentrations were found in milk from the cases caused by A pyogenes, in contrast to cases caused by other major mastitis pathogens. The median concentration of SAA was over 10 times higher in cases of mastitis caused by E coli than in mastitis caused by other pathogens. There were significant differences in the mean Hp concentration and NAGase activity between clinical and subclinical mastitis. In approximately one-third of the samples, the Hp concentration was below the detection limit, potentially compromising the use of Hp as a mastitis marker.

Invited review: udder health of dairy cows in automatic milking.

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.

Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria.

Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4h were compared with those of conventional bacterial culture (48h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Efficacy of enrofloxacin in the treatment of naturally occurring acute clinical Escherichia coli mastitis.

The efficacy of the combination of systemic enrofloxacin (5mg/kg twice with a 24-h interval, first dose i.v., second dose s.c.) and the nonsteroidal antiinflammatory agent ketoprofen (3mg/kg i.m. or 4 mg/kg per os daily for 1 to 3 d) treatment was compared with antiinflammatory treatment only in dairy cows with naturally occurring acute clinical Escherichia coli mastitis. A total of 132 cows with acute clinical mastitis and with confirmed growth of E. coli in a pretreatment milk sample were randomly allocated to 1 of 2 treatment groups. Response to treatment was evaluated clinically and by bacteriological culturing and determination of N-acetyl-beta-d-glucosaminidase (NAGase) activity on d 2 and 21 posttreatment. Enrofloxacin treatment did not increase bacteriological (90.5% of treated vs. 86.8% of nontreated cured) or clinical cure (46.7% of treated vs. 57.1% of nontreated cured), cow survival (95.3% of treated vs. 92.7% of nontreated), or quarter milk production assessed 21 d posttreatment (21.8 vs. 29.3% return to preinfection level for nontreated cows), nor did it decrease mammary gland tissue damage estimated using determination of milk NAGase activity (24.0+/-0.3 vs. 18.3+/-1.3 pmol of 4-methylumbelliferone per min per microL for nontreated cows). Treatment did not influence the number of study cows remaining in the herd after 6 mo (71.9% of treated vs. 80.6% of nontreated). The only significant effects of enrofloxacin were enhancing the bacteriological cure (odds ratio=3.32 for treated cows) and decreasing the clinical cure (odds ratio=0.05 for treated cows) on d 2 posttreatment. Our results did not support the use of enrofloxacin to treat acute clinical E. coli mastitis.

Reflection paper on MRSA in food-producing and companion animals: epidemiology and control options for human and animal health.

The scope of this reflection paper was to review the latest research on the risk of MRSA infection and colonization in animals. Attention focused on occurrence, risk factors for colonization and infection, and human contact hazard for livestock, horses, and companion animals. Whereas the clonal relationship between MRSA strains of CC398 is straightforward in livestock this is less obvious in horses. Small companion animals typically share MRSA strains that seem to exchange with a human reservoir. Management and therapeutic options have been suggested for livestock, horses, companion animals, as well as instructions on safety measures for persons in contact with animals. Conclusions were drawn with emphasis on future research activities, especially to confirm the apparent evolution of the organism and to demonstrate efficiency of control strategies.

Udder health of cows changing from tie stalls or free stalls with conventional milking to free stalls with either conventional or automatic milking.

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.

Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing.

In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The "no-growth" samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10(3) to 10(7) bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-beta-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.

Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens.

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes.

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.

Treatment of mastitis during lactation.

Treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account. In acute mastitis, where bacteriological diagnosis is not available, treatment should be initiated based on herd data and personal experience. Rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial. Treating subclinical mastitis with antimicrobials during lactation is seldom economical, because of high treatment costs and generally poor efficacy. All mastitis treatment should be evidence-based, i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. Use of on-farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials.