Effects of a mastitis treatment strategy with or without on-farm testing.

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

Development and evaluation of 4 loop-mediated isothermal amplification assays to detect mastitis-causing bacteria in bovine milk samples.

Farmers prefer fast, sensitive, and on-site tests for treatment decisions on mastitis. Due to the time to results of the currently available diagnostic tools, these are rarely used for that purpose. Genotypic tests that do not require a growth step may be suitable for on-site testing, for example loop-mediated isothermal amplification (LAMP), which has been described as a sensitive test that can be used on-site. Therefore, this study aimed to develop and evaluate LAMP assays for the detection of a subset of mastitis-causing pathogens, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus spp., in milk from cows with clinical mastitis. Furthermore, a generic nucleic acid lateral flow immunoassay (NALFIA) was evaluated as a potential on-site readout of the LAMP assays. For each assay of LAMP and NALFIA, the limit of detection and analytical specificity were determined using isolates, and the diagnostic specificity was determined using selected samples with known etiology. In addition, the diagnostic specificity of LAMP was determined using field samples with unknown etiology at testing. Bacteriological culture with identification by mass spectrometry was used as a reference method. The 4 assays had a kappa ≥0.73 with the reference method when testing the selected samples, but ≥0.47 when testing field samples. After correcting for prevalence, kappa was ≥0.80 for the E. coli, K. pneumoniae, and Staph. aureus assays. The Streptococcus spp. assay had a kappa of 0.47 (0.48 after correction) with the reference method, probably caused by the assay broadly targeting a genus instead of a particular species. The NALFIA readout was found to have kappa ≥0.81 for the E. coli, Staph. aureus, and Streptococcus spp. assays at a generic runtime, but for the K. pneumoniae assay a shorter runtime could be used. In conclusion, LAMP is a promising method for fast on-site tests for mastitis-causing pathogens if the current elaborate method for sample preparation is replaced by a simplified protocol. The NALFIA is an easy and reliable readout for on-site use, with the observation that for the current assay designs a generic runtime is not yet possible for the chosen set of pathogens. If associated with a simple and fast sample preparation protocol, the combination of LAMP and NALFIA has the potential to enable fast and reliable on-site testing of clinical mastitis milk samples.

Agreement between four commercial diagnostic tests and routine bacteriological culture of milk to determine the udder infection status of dairy cows.

Mastitis is usually treated based on clinical signs or somatic cell count information rather than on results of bacteriological culture of milk. In many countries an optimal mastitis treatment is considered important from the perspective of therapy efficacy, prudent antimicrobial use and farm economics. Farmers can optimize their mastitis treatment decisions if they know whether and which mastitis pathogen is involved. Information on the mastitis pathogen involved can be acquired from diagnostic mastitis tests such as culture-based tests. This study aimed to determine the agreement of four commercial culture-based mastitis tests with routine bacteriological culture of milk to determine the intramammary infection status of a quarter or cow. The commercial culture-based tests evaluated in this study were CHROMagar Mastitis (CHROMagar, France), Hardy Diagnostics Mastitis Triplate (Hardy Diagnostics, USA), Minnesota Easy Culture System II Tri-plate (University of Minnesota, USA), and VétoRapid (Vetoquinol, the Netherlands). We used 866 prospectively collected milk samples, routinely submitted to the bacteriological laboratory of GD Animal Health for routine bacteriological culture of milk from April to June 2016. Samples were cultured on routine bacteriological culture of milk and on the commercial culture-based tests. We calculated the agreement beyond chance of each commercial culture-based test result with the result of routine bacteriological culture using 2x2 contingency tables. Furthermore, inter-reader agreement was determined for 597 samples read by two masked readers. The agreement of the four commercial culture-based mastitis tests with routine bacteriological culture of milk for Gram-positive bacteria ranged from 0.14 (95% CI 0.11-0.16) using Hardy Diagnostics Mastitis Triplate to 0.25 (95% CI 0.22-0.28) using Minnesota Easy Culture System II Tri-plate. The agreement for Gram-negative bacteria was approximately 0.70 (95% CI 0.66-0.74) for all four commercial culture-based tests. The agreement for no growth ranged from 0.22 (95% CI 0.19-0.25) using Hardy Diagnostics Mastitis Triplate to 0.34 (95% CI 0.31-0.38) using VétoRapid. This category was affected by prevalence and bias as the prevalence adjusted and bias adjusted kappa ranged from 0.63 (95% CI 0.56-0.69) using CHROMagar Mastitis to 0.68 (95% CI 0.62-0.74) using Hardy Diagnostic Mastitis Triplate. Agreement between readers was almost perfect. Although only for Gram-negative bacteria a good agreement was found between commercial culture-based tests and routine bacteriological culture of milk, and further on-farm evaluations are needed to determine the effect of these findings on udder health, commercial culture-based tests are of added value to support decisions whether and how to treat cows with mastitis.

Dutch dairy farmers' need for microbiological mastitis diagnostics.

Although several microbiological mastitis diagnostic tools are currently available, dairy farmers rarely use them to base treatment decisions on. In this study, we conducted a telephone interview among 195 randomly selected Dutch dairy farmers to determine their current use of and their need for microbiological diagnostics for clinical mastitis (CM), subclinical mastitis (SCM), and dry-cow treatment (DCT), followed by the test characteristics they consider important. A structured questionnaire was used, based on face-to-face interviews previously held with other farmers. The answers were registered in a database and analyzed using descriptive statistics and univariable and multivariable models. Antimicrobial treatment decisions for CM, SCM, and DCT were mainly based on clinical signs and somatic cell count. In case of CM, 34% of farmers indicated that they currently submit milk samples for bacteriological culture (BC). This would increase to 71% if an on-farm test resulting in treatment advice within 12 h were available. For SCM, use would increase from 22 to 55%, and for DCT, from 7 to 34%, if the same 12-h test were available. For CM and DCT, the preferred test outcome was advice on which antibiotic to use, according to 58 and 15% of the farmers, respectively. For SCM, the preferred test outcome was the causative bacterium for 38% of the farmers. Farmers who currently submit CM milk samples for BC were 13.1 times more likely to indicate, as the preferred test outcome, advice on which antibiotic to use, compared with farmers who do not currently submit CM milk samples for BC. Fourteen percent of the farmers indicated not being interested at all in microbiological mastitis diagnostics for CM. For SCM and DCT, 27 and 55%, respectively, were not interested in microbiological mastitis diagnostics. Regarding test characteristics that farmers considered important, reliability was most often indicated (44-51% of the farmers). Additionally, a preferred time-to-result of ≤8 h for CM and ≤20 to 24 h for SCM and DCT and ≤7% false test outcomes were indicated as desired characteristics of microbiological mastitis diagnostics. Overall, a need seems to exist for microbiological mastitis diagnostic tests among Dutch dairy farmers, specifically for CM, and resulting in a treatment advice. The availability of a reliable diagnostic test, with a suitable time-to-result, will likely increase the use of microbiological mastitis diagnostics and eventually optimize antibiotic usage.

Culicoides species associated with sheep in the Netherlands and the effect of a permethrin insecticide.

Culicoides spp. act as vectors for a number of viral diseases of animals including bluetongue in sheep. The aims of this study were to determine: (1) which Culicoides spp. are associated with sheep in The Netherlands; (2) the time of the day when they are most active; and (3) the effect of treatment of animals with a permethrin insecticide. Two pairs of sheep were each housed within mosquito tents of either one or two layers of netting and all trapped Culicoides spp. were identified microscopically. For the permethrin insecticide study, one of two pairs of sheep was treated with 3.6% permethrin and all animals were housed in tents of similar design. Of the 6210 midges captured, 54.1% were identified as C. chiopterus and 42.7% as C. obsoletus. C. imicola was not identified. The average insect feeding rate was 35-40% and midge activity was greatest around sunset. Permethrin treatment reduced the number of midges captured by 50% and also resulted in a decrease in the percentage of midges that had fed. The findings provide useful information on the behaviour and distribution of Culicoides spp. that will facilitate the development of appropriate control strategies to minimise the risk of insect-vector borne virus diseases such as bluetongue.