Use of inline measures of l-lactate dehydrogenase for classification of posttreatment mammary Staphylococcus aureus infection status in dairy cows.

An automated method for determining whether dairy cows with subclinical mammary infections recover after antibiotic treatment would be a useful tool in dairy production. For that purpose, inline l-lactate dehydrogenase (LDH) measurements was modeled using a dynamic linear model; the variance parameters were estimated using the expectation-maximization algorithm. The method used to classify cows as infected or uninfected was based on a multiprocess Kalman filter. Two learning data sets were created: infected and uninfected. The infected data set consisted of records from 48 cows with subclinical Staphylococcus aureus infection from 4 herds collected in 2010. The uninfected data set came from 35 uninfected cows collected during 2013 from 2 herds. Bacteriological culturing was used as gold standard. To test the model, we collected data from the 48 infected cows 50 d after antibiotic treatment. As a result of the treatment, this test data set consisted of 25 cows that still had a subclinical infection and 23 cows that were recovered. Model sensitivity was 36.0% and specificity was 82.6%. To a large extent, l-lactate dehydrogenase reflected the cow's immune response to the presence of pathogens in the udder. However, cows that were classified correctly before treatment had a better chance of correct classification after treatment. This indicated a variation between cows in immune response to subclinical mammary infection that may complicate the detection of subclinically infected cows and determination of recovery.

Changes in milk yield, lactate dehydrogenase, milking frequency, and interquarter yield ratio persist for up to 8 weeks after antibiotic treatment of mastitis.

Within the dairy industry, the appearance of milk and withdrawal time due to antibiotic residuals in the milk are used to determine recovery status after cases of treated mastitis. However, both milk production and dairy cow behavior have been shown to be affected after the normalization of milk appearance, indicating that animals may not have fully recovered. The aim of the present study was to describe the changes in milk yield, lactate dehydrogenase activity, milking frequency, and interquarter yield ratio (defined as the coefficient of variation between the active quarters) after cases of naturally occurring mastitis with special focus on the recovery period after antibiotic treatment. A second aim was to examine whether these changes were affected by the pathogens present at the time of mastitis diagnosis. This retrospective study was based on a cohort data set including 1,032 lactations from 795 dairy cows kept on 2 Danish farms and milked by an automatic milking system. A total of 174 treated mastitis cases were compared with nontreated control cows from 5 wk before treatment and until 8 wk after. Treated mastitis resulted in reduced milk yield, elevated lactate dehydrogenase activity, lower milking frequency, and elevated interquarter yield ratio. Within these measures, deviations from baseline levels and from the control cows were found as early as 1 to 3 wk before the antibiotic treatment and peaked around the days of treatment. In some cases, the mastitic cows returned to premastitis levels, whereas in others they remained affected throughout the rest of the observation period. To correctly estimate the effects of treated mastitis and the recovery status of cows, it is important to take the individual cow into account and not only compare with herd levels, as this might mask the true degree of the changes. The effects on each outcome variable depended on the involved pathogen and differences were found between primiparous cows and older animals. However, in general, the changes in milk production, lactate dehydrogenase activity, and interquarter yield ratio showed parallels, suggesting that the recovery period continued for weeks after antibiotic treatment. These results call for further investigation into management of mastitic dairy cows to optimize recovery, limit milk loss, and ensure animal welfare during the period after mastitis.

Behavioral changes in freestall-housed dairy cows with naturally occurring clinical mastitis.

Dairy cows exhibit classic signs of sickness behavior during mastitis. However, knowledge about the consequences of naturally occurring mastitis in freestall-housed dairy cows, milked in automatic milking systems, is lacking. The aim of the present study was to describe the behavior of dairy cows after diagnosis and antibiotic treatment of mastitis. In the days before and after antibiotic treatment, the milking behavior, feeding, and activity were examined in 30 mastitic and 30 control Danish Holstein-Friesian cows kept in freestalls and milked by an automatic milking system. Sickness behavior was evident in the mastitic dairy cows and local clinical signs in the udder as well as behavioral changes persisted beyond the 3 d of antibiotic treatment. In the days before diagnosis and treatment, feed intake was reduced compared with the control animals. Although reduced by the antibiotic treatment, this difference persisted until at least 10 d after diagnosis. Sick cows spent less time lying in the initial days after treatment, reversing to the level of the control cows within the 10 d posttreatment period. In the 48 h before antibiotic treatment, the mastitic cows showed increased restlessness during milking, as seen by a higher frequency of tripping and kicking. Mastitic cows continued to show increased kicking during milking even after the antibiotic treatment period. These results show that the behavioral changes induced by naturally occurring mastitis persisted beyond the days of antibiotic treatment, thereby calling for further investigation into management of mastitic dairy cows to optimize recovery and ensure animal welfare during the recovery period after clinical mastitis.

Associations of udder-health indicators with cow factors and with intramammary infection in dairy cows.

The objective of this study was to investigate if and how cow factors and intramammary infection (IMI) are associated with 4 different udder-health indicators in dairy cows as a first step in investigating whether the diagnostic performance of these indicators can be improved. The investigated indicators were somatic cell count (SCC), lactate dehydrogenase (LDH), N-acetyl-ß-d-glucosaminidase (NAGase), and alkaline phosphatase (AP) measured in milk. In this cross-sectional study, approximately 1,000 cows from 25 dairy herds were sampled for bacteriology (quarter milk samples) during 3 consecutive days: the day before test milking, at the day of test milking, and at the day after test milking. The whole-udder test milking sample was analyzed for milk composition, SCC, LDH, NAGase, and AP. Cow data (parity, breed, milk yield, percentage of milk fat and protein, milk urea concentration, and days in milk from the sampled test milking) were collected from the Swedish milk-recording scheme. Of the sampled cows 485 were considered IMI negative and were used in multivariable mixed-effect linear regression models to investigate associations between cow factors and the udder-health indicators. A second modeling including all cows, both IMI negative and IMI positive (256 cows), was also performed. The results showed that all udder-health indicators were affected by cow factors but that different cow factors were associated with different indicators. Intramammary-infection status was significantly associated with all udder-health indicators except AP. Parity and milk urea concentration were the only cow factors associated with all indicators in all models. The significant cow factors explained 23% of the variation in SCC and >30% of the variation in LDH, NAGase, and AP in IMI-negative cows, showing that LDH, NAGase, and AP are more affected than SCC by cow factors. The IMI status explained 23% of the variation in SCC in the model with all cows but only 7% of the variation in LDH and 2% of the variation in NAGase, indicating that SCC has the best potential as a diagnostic tool in finding cows with IMI. However, further studies are needed to investigate whether the diagnostic properties of these udder-health indicators will improve with adjustment according to their associations with different cow factors when used as a diagnostic tool for finding cows with IMI.

Short communication: Changes in gait symmetry in healthy and lame dairy cows based on 3-dimensional ground reaction force curves following claw trimming.

Lameness is a frequent health problem in dairy cows. This preliminary study aimed to detect gait differences between healthy and lame walking cows using 3-dimensional force plates. We examined left-right leg symmetry changes of healthy and lame Holstein dairy cows following claw trimming. Gait scoring (GS) was performed on d -5, 0, 1, and 7 relative to claw trimming. Before the experiment, 5 cows walked normally (initial GS=1) and 4 cows limped moderately on a hind leg (initial GS=3). Gait was measured on d -2, -1, 0, 1, and 7 relative to trimming by obtaining ground reaction forces as cows walked repeatedly across 2 parallel 3-dimensional force plates. From the ground reaction forces, stance phase data were derived using computerized procedures. Left-right leg symmetries of entire curves in the 3 force directions were calculated. Effects of lameness and trimming were analyzed in a mixed model, using a low lameness threshold (GS>1). One week after claw trimming, only one cow was mildly lame. In addition, the symmetries of all 3 dimensions were significantly improved shortly after trimming. Importantly, lameness significantly worsened vertical symmetry. Lame cows walked significantly more slowly than healthy cows. In conclusion, all force symmetries seemed capable of detecting gait responses to claw trimming. Although our results are based on a small number of animals, vertical leg symmetry was affected by lameness.

Gram-typing of mastitis bacteria in milk samples using flow cytometry.

Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive identification of bacterial pathogens, although shipment of samples to diagnostic laboratories delays treatment decisions. Due to the lack of fast on-site tests that can identify the causative pathogens, antibiotic treatments are often initiated before bacterial identification. The present study describes a flow cytometry-based method, which can detect and distinguish gram-negative and gram-positive bacteria in mastitis milk samples. The differentiation was based on bacterial fluorescence intensities upon labeling with biotin-conjugated wheat germ agglutinin and acridine orange. Initially 19 in-house bacterial cultures (4 gram-negative and 15 gram-positive strains) were analyzed, and biotin-conjugated wheat germ agglutinin and acridine orange florescence intensities were determined for gram-negative and gram-positive bacteria, respectively. Fluorescence cut-off values were established based on receiver operating characteristic curves for the 19 bacterial cultures. The method was then tested on 53 selected mastitis cases obtained from the department biobank (milk samples from 6 gram-negative and 47 gram-positive mastitis cases). Gram-negative bacteria in milk samples were detected with a sensitivity of 1 and a specificity of 0.74, when classification was based on the previously established cut-off values. However, when receiver operating characteristic curves were constructed for the 53 mastitis cases, results indicate that a sensitivity and specificity of 1 could be reached if cut-off values were reduced. This flow cytometry-based technique could potentially provide dairy farmers and attending veterinarians with on-site information on bacterial gram-type and prevent ineffective antimicrobial treatment in mastitis cases caused by gram-negative bacteria.

Quality of bulk tank milk samples from Danish dairy herds based on real-time polymerase chain reaction identification of mastitis pathogens.

Results of a commercial real-time PCR analysis for 11 mastitis pathogens from bulk tank milk (BTM) samples from all 4,258 Danish dairy herds in November 2009 to January 2010 were compared with somatic cell count (SCC) and total bacteria count (TBC) estimates in BTM. For Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis, a low real-time PCR cycle threshold (Ct) value (corresponding to high bacterial DNA quantity) was correlated with higher SCC and higher TBC. For Staphylococcus aureus, low Ct values were correlated only with higher SCC. For the environmental mastitis pathogens Klebsiella spp., Enterococcus spp., and Escherichia coli, low Ct values had a correlation with higher TBC. Staphylococcus spp. were found in the BTM from all herds, Strep. uberis in 95%, Staph. aureus in 91%, and Strep. dysgalactiae in 86%, whereas E. coli, Klebsiella, and Strep. agalactiae were found in 61, 13, and 7% of the herds. It is concluded that the real-time PCR used provides results that are related to the milk quality in the herds. Real-time PCR can be used in the same way as culture for monitoring BTM samples, and is especially useful for bacteria with low prevalence (e.g., Strep. agalactiae).

Danish stable schools for experiential common learning in groups of organic dairy farmers.

The farmer field school (FFS) is a concept for farmers' learning, knowledge exchange, and empowerment that has been developed and used in developing countries. In Denmark, a research project focusing on explicit non-antibiotic strategies involves farmers who have actively expressed an interest in phasing out antibiotics from their herds through promotion of animal health. One way of reaching this goal was to form participatory focused farmer groups in an FFS approach, which was adapted to Danish conditions and named "stable schools." Four stable schools were established and went through a 1-yr cycle with 2 visits at each of the 5 or 6 farms connected to each group. A facilitator was connected to each group whose role was to write the meeting agenda together with the host farmer, direct the meeting, and write the minutes to send to the group members after the meeting. Through group focus interviews and individual semistructured qualitative interviews of all participants, the approach of the farmers' goal-directed work toward a common goal was judged to be very valuable and fruitful and based on a common learning process. Complex farming situations were the focus of all groups and in this context, problems were identified and solutions proposed based on each farmer's individual goals. In this article, we describe the experiences of 4 stable school groups (each comprising farmers and a facilitator), and the common process of building a concept that is suitable for Danish organic dairy farming.

Development and daily management of an explicit strategy of nonuse of antimicrobial drugs in twelve Danish organic dairy herds.

Promotion of animal health and well-being at the individual animal and herd level is an important goal in organic farming. At the same time, chemical products affecting the natural balance among living organisms are prohibited in all areas of the organic farm. From an animal welfare point of view, however, no animal must suffer. Therefore, veterinary drugs are allowed under the European Union's regulations for organic farming, despite the fact that they are powerful cell toxins affecting both pathogenic and necessary bacteria, and as such in organic terminology, are regarded as "chemical" or "artificial" products. In this article, we present and discuss interviews with 12 Danish organic dairy producers who claim that minimized use or nonuse of antimicrobial drugs is an explicit goal. The dairy producers were at different levels with regard to reduced antimicrobial treatment. An explicit strategy of no antimicrobial treatments is based primarily on a long-term effort to improve herd health, and secondarily, on finding alternative treatments for diseased animals. Improved hygiene, outdoor access, use of nursing cows, and blinding of chronic mastitis quarters were the main techniques in developing a strategy of not using antimicrobial treatments in the herd by dairy producers. Producers' perception of disease changed from something unavoidable to a disturbing break in the daily rhythm that often could have been avoided. Change toward a nonantimicrobial strategy was gradual and stepwise. All dairy producers in this study desired to preserve the possibility of using antimicrobial drugs in emergencies.

A stochastic model simulating pathogen-specific mastitis control in a dairy herd.

The objective of this study was to develop a model simulating mastitis control in dairy herds and to investigate how sensitive the model is when varying the effect parameters according to the uncertainty. The model simulates 9 pathogen-specific mastitis types, each of which can be subclinical or clinical. The clinical cases can be 1 of 4 severities defined according to the effect of the mastitis case: mild, moderate, severe, and permanent effect. The risk factors include lactation stage, parity, yield level, previous diseases, season, and contagious spread of the infection from herd mates. Occurrence of mastitis is modeled to have direct effects on feed intake, body weight, milk yield, somatic cell count in the milk, subsequent mastitis cases within the cow and in herd mates, voluntary and involuntary culling, mortality, and milk withdrawal. Thirty-five scenarios were simulated to study model behavior and model sensitivity. The consequences per cow/yr of mastitis in the default simulated herd included 0.42 clinical mastitis occurrences, 0.56 subclinical mastitis occurrences, loss of 385-kg milk yield, a 1.3% reduced feed intake, 61-kg milk withdrawal and 146 in reduced economic net return. Based on scenarios demonstrating model behavior and sensitivity analysis, the model appears to produce valid consequences of mastitis control strategies. Representation of the effect of subclinical mastitis and of variation in mastitis severity was concluded in this study to be important when modeling mastitis economics in a dairy herd. The model offers the opportunity to study the long-term herd specific effects of a wide range of control strategies against mastitis.

Comparison of antimicrobial susceptibility of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy herds in the midwestern United States and Denmark.

An observational study was conducted to compare the antimicrobial susceptibility patterns of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy farms in Wisconsin, United States, and southern Jutland, Denmark. Bulk tank milk samples and data regarding management and production were collected from 30 organic and 30 conventional dairy farms in Wisconsin and 20 organic and 20 conventional dairy farms in Denmark. S. aureus isolates were tested for resistance against 15 antimicrobial agents by semiautomatic microbroth dilution methods in each country. Of the 118 bulk tank milk samples in Wisconsin, 71 samples (60%) yielded at least one S. aureus isolate, and a total of 331 isolates were collected. Of the 40 bulk tank milk samples from Denmark, 27 samples (55%) yielded at least one S. aureus isolate, and a total of 152 isolates were collected. Significant differences between organic and conventional dairies were detected only to ciprofloxacin in Wisconsin and avilamycin in Denmark. Significant differences (P < 0.05) between the two countries were detected in nine antimicrobials. Denmark had a higher probability of having reduced susceptibility to ciprofloxacin and streptomycin (P = 0.015 and 0.003, respectively). Wisconsin isolates had a higher probability of having reduced susceptibility to seven other antimicrobial agents (bacitracin, gentamicin, kanamycin, penicillin, sulphamethoxazole, tetracycline, and trimethoprim). We found small differences between organic and conventional farm types in each country and larger differences between the two national agricultural systems.

Reduced medication in organic farming with emphasis on organic dairy production.

Medication is an important focus area in organic animal husbandry. The combination of goals relating to improved animal welfare and reduced use of chemicals in general creates a common wish to reduce medication. Based on data from current Danish research projects in organic dairy farming, one specific organic medication pattern or policy cannot be described. The disease treatment pattern is influenced by many factors, e.g. the interaction with colleagues, veterinarians and agricultural advisors. No significant difference could be found with regard to incidence of mastitis treatments or somatic cell counts in 27 organic and 57 conventional herds. A marked tendency to shorter treatment periods in relation to mastitis treatments was described for organic farms in comparison with conventional farms (1.9 days versus 3.2 days (5 organic and 7 conventional herds)). In a study of development of health advisory service in organic herds, the dialogue between farmer, veterinarian and agricultural cattle advisor changed the treatment pattern markedly during a period of 6 months. Among important future challenges for veterinarians in organic farming is pointed at the constructive, open, and critical interaction with the single organic farmer as well as the organic animal husbandry system in general.

Effect of mastitis treatment and somatic cell counts on milk yield in Danish organic dairy cows.

Production and disease data from 17,488 lactations in 48 Danish organic dairy herds from 1997 to 2001 were analyzed to obtain estimates on the effect of somatic cell counts (SCC) and mastitis treatment on milk production. A multilevel three-parameter piecewise random coefficients linear model with energy-corrected milk (ECM) as dependent variable and herd, lactation, and test days as levels, was used to model the lactation curve. Covariates related to production, SCC, veterinary treatments, and reproductive performance in the previous lactation as well as information on other diseases in the current lactation were included to describe the production capacity of the individual cow. The average daily milk production at herd level was 20.8, 24.2, and 25.8 kg of ECM/d in first, second, and third or later lactation. The estimates for production losses were on average 0.2, 0.3, and 0.4 kg of ECM/d in first, second, and third or later lactation with each twofold increase in SCC between 100,000 and 1,500,000 cells/ml. The effect varied with the stage of lactation and was nonsignificant around 60 d postpartum and highest at the end of the lactation. The production losses in cows treated for mastitis varied with parity and stage of lactation and were modified by the SCC after treatment. For a cow in third lactation with a SCC below 100,000 cells/ ml before treatment at days in milk = 15, the predicted loss was 435 kg of ECM, including a loss of 135 kg of ECM because of higher SCC compared with the level before treatment. Most of the variation in production related to SCC and mastitis was at the lactation level, and no significant differences were found between herds grouped according to milk production level, SCC, or prevalence of mastitis treatment.