M. Cameron

Evaluation of selective dry cow treatment following on-farm culture: Risk of postcalving intramammary infection and clinical mastitis in the subsequent lactation

The objective of the study was to evaluate the utility of a Petrifilm-based on-farm culture system when used to make selective antimicrobial treatment decisions on low somatic cell count cows (<200,000 cells/mL) at drying off. A total of 729 cows from 16 commercial dairy herds with a low bulk tank somatic cell count (<250,000 cells/mL) were randomly assigned to receive either blanket dry cow therapy (DCT) or Petrifilm-based selective DCT. Cows belonging to the blanket DCT group were infused with a commercial dry cow antimicrobial product and an internal teat sealant (ITS) at drying off. Using composite milk samples collected on the day before drying off, cows in the selective DCT group were treated at drying off based on the results obtained by the Petrifilm on-farm culture system with DCT + ITS (Petrifilm culture positive), or ITS alone (Petrifilm culture negative). Quarters of all cows were sampled for standard laboratory bacteriology on the day before drying off, at 3 to 4d in milk (DIM), at 5 to 18 DIM, and from the first case of clinical mastitis occurring within 120 DIM. Multilevel logistic regression was used to assess the effect of study group (blanket or selective DCT) and resulting dry cow treatment (DCT + ITS, or ITS alone) on the risk of intramammary infection (IMI) at calving and the risk of a first case of clinical mastitis between calving and 120 DIM. According to univariable analysis, no difference was observed between study groups with respect to quarter-level cure risk and new IMI risk over the dry period. Likewise, the risk of IMI at calving and the risk of clinical mastitis in the first 120 DIM was not different between quarters belonging to cows in the blanket DCT group and quarters belonging to cows in the selective DCT group. The results of this study indicate that selective DCT based on results obtained by the Petrifilm on-farm culture system achieved the same level of success with respect to treatment and prevention of IMI over the dry period as blanket DCT and did not affect the risk of clinical mastitis in the first 120 d of the subsequent lactation.

Identification of bovine-associated coagulase-negative staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol

This study evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) for the identification of bovine-associated coagulase-negative staphylococci (CNS), a heterogeneous group of different species. Additionally, we aimed to expand the MALDI-ToF MS database with new reference spectra as required to fill the gaps within the existing commercial spectral library. A total of 258 isolates of CNS were used in the study, covering 16 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 219), and the remainder were identified by sequencing of 16S rRNA, hsp60, or both rpoB and hsp60. The genotypic identification was considered the gold standard identification. All MALDI-ToF MS identifications were carried out using the direct transfer method. In a preliminary evaluation (n = 32 isolates; 2 of each species) with the existing commercial database, MALDI-ToF MS showed a typeability of 81% (26/32) and an accuracy of 96% (25/26). In the main evaluation (n = 226 isolates), MALDI-ToF MS with the existing commercial Biotyper (Bruker Daltonics Inc., Billerica, MA) database achieved a typeability of 92.0% (208/226) and an accuracy of 99.5% (207/208). Based on the assessment of the existing commercial database and prior knowledge of the species, a total of 13 custom reference spectra, covering 8 species, were created and added to the commercial database. Using the custom reference spectra expanded database, isolates were identified by MALDI-ToF MS with 100% typeability and 100% accuracy. Whereas the MALDI-ToF MS manufacturers cutoff for species-level identification is 2.000, the reduction of the species level cutpoint to ≥1.700 improved the species-level identification rates (from 64 to 92% for the existing commercial database) when classifying CNS isolates. Overall, MALDI-ToF MS using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.

Evaluation of a 3M Petrifilm on-farm culture system for the detection of intramammary infection at the end of lactation.

The purpose of this study was to evaluate a 3M Petrifilm-based on-farm culture system for the detection of intramammary infection (IMI) in low somatic cell count (SCC) cows (<200,000 cells/mL) at drying off. The main objectives were to determine the test characteristics and the predictive values of the Petrifilm on-farm culture system. The ability of dairy producers to correctly classify cows as infected or uninfected based on Petrifilm culture and a set colony count threshold was also assessed. A total of 360 cows originating from 16 low bulk tank SCC (<250,000 cells/mL) dairy herds were enrolled at drying off. Enrolled cows had an expected dry period of 30-90 days, a SCC<200,000 cells/mL on the last 3 tests prior to drying off, no clinical mastitis in the same time period, and no antibiotic treatment in the last 14 days. Quarter milk samples were collected on the day prior to drying off, and a composite milk sample was created by combining 5 mL of milk from each quarter sample. Composite milk samples were cultured on-farm using the Petrifilm culture system, which provided results within 24h. Quarter milk samples were cultured in a reference laboratory, and the results were aggregated to the cow level. On the day of drying off, the Petrifilm was read by the producer and cows were classified as positive if ≥5 colonies (equivalent to 50 colony forming units/mL) were present. When read by the producer, 47.8% of the cows cultured negative on Petrifilm and were infused with only an internal teat sealant at drying off. The test characteristics of the Petrifilm on-farm culture system were calculated by comparing the producer-derived Petrifilm results to those obtained by standard laboratory culture. The sensitivity and specificity of the Petrifilm on-farm culture system were 85.2% (78.5-90.5) and 73.2% (66.4-79.3), respectively. The negative predictive value of the Petrifilm test system was high (86.6%) when estimated using the prevalence of IMI in this data set, and the positive predictive value was moderate (70.9%). An automated 3M Petrifilm reader was used to obtain accurate colony counts. The agreement between Petrifilm results obtained by the producer and those obtained by the automated Petrifilm reader was high, with a kappa value of 0.82 (0.75-0.89).

Evaluation of selective dry cow treatment following on-farm culture: Milk yield and somatic cell count in the subsequent lactation

Compared with blanket dry cow therapy (DCT), the selective antimicrobial treatment of cows based upon on-farm culture results has the potential to reduce the amount of antimicrobials used in dairy production. The objective of the current study was to determine the effect of a Petrifilm (3M Canada, London, Ontario) on-farm culture-based selective DCT program on milk yield and somatic cell count (SCC) in the following lactation. A total of 729 low-SCC (<200,000 cells/mL) cows from 16 commercial dairy herds with a low bulk tank SCC (<250,000 cells/mL) were randomly assigned to receive either blanket DCT or Petrifilm-based selective DCT. Cows belonging to the blanket DCT group were infused with a commercial DCT product and an internal teat sealant (ITS) at drying off. Using composite milk samples collected on the day before drying off, cows in the selective DCT group were treated at drying off based on the results obtained by the Petrifilm on-farm culture system with DCT and ITS (Petrifilm culture positive) or ITS alone (Petrifilm culture negative). Milk test-day records for the following lactation were obtained from Dairy Herd Improvement for all cows enrolled in the trial. Repeated measures linear mixed models were used to assess the effect of study group (blanket or selective DCT) on test-day milk production and natural logarithm of SCC over the first 180 d of the subsequent lactation. According to the final multivariable models, when low-SCC cows were selectively treated with DCT at drying off based on results obtained using the Petrifilm on-farm culture system, no effect on milk production (least squares means for blanket DCT = 39.3 kg vs. selective DCT = 39.0 kg) or natural logarithm of SCC (least squares means for blanket DCT = 3.95 vs. selective DCT = 3.97) was observed in the subsequent lactation when compared with cows receiving blanket DCT. The results of this study indicate that selective DCT based on results obtained by the Petrifilm on-farm culture system enabled a reduction in the use of DCT without negatively affecting milk production and milk quality.

Short communication: Evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci

This study evaluated MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci (CNS). A total of 861 CNS isolates were used in the study, covering 21 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 804) and the remainder were identified by sequencing of hsp60 (n = 56) and tuf (n = 1). The genotypic identification was considered the gold standard identification. Using a direct transfer protocol and the existing commercial database, MALDI-TOF mass spectrometry showed a typeability of 96.5% (831/861) and an accuracy of 99.2% (824/831). Using a custom reference spectra expanded database, which included an additional 13 in-house created reference spectra, isolates were identified by MALDI-TOF mass spectrometry with 99.2% (854/861) typeability and 99.4% (849/854) accuracy. Overall, MALDI-TOF mass spectrometry using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.

Rapid assessment of bovine colostrum quality: How reliable are transmission infrared spectroscopy and digital and optical refractometers?

The objectives of this study were to evaluate the performance of the transmission infrared (IR) spectroscopic method and digital and optical Brix refractometers for measurement of colostral IgG concentration and assessment of colostrum quality of dairy cows. Colostrum samples (n = 258) were collected from Holstein cows on 30 commercial dairy farms in Nova Scotia and Newfoundland, Canada. Colostral IgG concentrations of 255 samples were measured by the reference radial immunodiffusion (RID) assay and IR spectroscopy. The Brix scores were determined on 240 of these samples using both the digital and optical Brix refractometers. Approximately half (48%) of the colostrum samples had RID IgG concentrations <50 g/L, which was the cut-point for poor quality. The correlation between RID and IR IgG concentrations was 0.88. The correlations between RID IgG concentration and Brix scores, as determined by the digital and optical refractometers, were 0.72 and 0.71, respectively. The optimal cutoff levels for distinguishing good- and poor-quality colostrum using IR spectroscopy, and digital and optical Brix refractometers were at 35 g/L and 23% Brix, respectively. The IR spectroscopy showed higher sensitivity (90%) and specificity (86%) than the digital (74 and 80%, respectively) and optical (73 and 80%, respectively) Brix refractometers for assessment of colostrum quality, as compared with RID. In conclusion, the transmission-IR spectroscopy is a rapid and accurate method for assessing colostrum quality, but is a laboratory-based method, whereas Brix refractometers were less accurate but could be used on-farm.

Whole-genome sequence analysis of antimicrobial resistance genes in Streptococcus uberis and Streptococcus dysgalactiae isolates from Canadian dairy herds

The objectives of this study are to determine the occurrence of antimicrobial resistance (AMR) genes using whole-genome sequence (WGS) of Streptococcus uberis (S. uberis) and Streptococcus dysgalactiae (S. dysgalactiae) isolates, recovered from dairy cows in the Canadian Maritime Provinces. A secondary objective included the exploration of the association between phenotypic AMR and the genomic characteristics (genome size, guanine–cytosine content, and occurrence of unique gene sequences). Initially, 91 isolates were sequenced, and of these isolates, 89 were assembled. Furthermore, 16 isolates were excluded due to larger than expected genomic sizes (>2.3 bp × 1,000 bp). In the final analysis, 73 were used with complete WGS and minimum inhibitory concentration records, which were part of the previous phenotypic AMR study, representing 18 dairy herds from the Maritime region of Canada (1). A total of 23 unique AMR gene sequences were found in the bacterial genomes, with a mean number of 8.1 (minimum: 5; maximum: 13) per genome. Overall, there were 10 AMR genes [ANT(6), TEM-127, TEM-163, TEM-89, TEM-95, Linb, Lnub, Ermb, Ermc, and TetS] present only in S. uberis genomes and 2 genes unique (EF-TU and TEM-71) to the S. dysgalactiae genomes; 11 AMR genes [APH(3′), TEM-1, TEM-136, TEM-157, TEM-47, TetM, bl2b, gyrA, parE, phoP, and rpoB] were found in both bacterial species. Two-way tabulations showed association between the phenotypic susceptibility to lincosamides and the presence of linB (P = 0.002) and lnuB (P < 0.001) genes and the between the presence of tetM (P = 0.015) and tetS (P = 0.064) genes and phenotypic resistance to tetracyclines only for the S. uberis isolates. The logistic model showed that the odds of resistance (to any of the phenotypically tested antimicrobials) was 4.35 times higher when there were >11 AMR genes present in the genome, compared with <7 AMR genes (P < 0.001). The odds of resistance was lower for S. dysgalactiae than S. uberis (P = 0.031). When the within-herd somatic cell count was >250,000 cells/mL, a trend toward higher odds of resistance compared with the baseline category of <150,000 cells/mL was observed. When the isolate corresponded to a post-mastitis sample, there were lower odds of resistance when compared with non-clinical isolates (P = 0.01). The results of this study showed the strength of associations between phenotypic AMR resistance of both mastitis pathogens and their genotypic resistome and other epidemiological characteristics.

Antimicrobial Susceptibility Patterns of Environmental Streptococci Recovered from Bovine Milk Samples in the Maritime Provinces of Canada

Determination of antimicrobial susceptibility of bovine mastitis pathogens is important for guiding antimicrobial treatment decisions and for the detection of emerging resistance. Environmental streptococci are ubiquitous in the farm environment and are a frequent cause of mastitis in dairy cows. The aim of the study was to determine patterns of antimicrobial susceptibility among species of environmental streptococci isolated from dairy cows in the Maritime Provinces of Canada. The collection consisted of 192 isolates identified in milk samples collected from 177 cows originating from 18 dairy herds. Results were aggregated into: (1) Streptococcus uberis (n = 70), (2) Streptococcus dysgalactiae (n = 28), (3) other Streptococci spp. (n = 35), (4), Lactococcus spp. (n = 32), and (5) Enterococcus spp. (n = 27). Minimum inhibitory concentrations (MICs) were determined using the Sensititre microdilution system and mastitis plate format. Multilevel logistic regression models were used to analyze the data, with antimicrobial susceptibility as the outcome. The proportion of susceptible S. uberis ranged from 23% (for penicillin) to 99% (for penicillin/novobiocin), with a median of 82%. All S. dysgalactiae were susceptible to all antimicrobials except for penicillin (93% susceptible) and tetracycline (18% susceptible). The range of susceptibility for other Streptococcus spp. was 43% (for tetracycline) to 100%, with a median percent susceptibility of 92%. Lactococcus spp. isolates displayed percent susceptibilities ranging from 0% (for penicillin) to 97% (for erythromycin), median 75%. For the antimicrobials tested, the minimum inhibitory concentrations were higher for Enterococcus spp. than for the other species. According to the multilevel models, there was a significant interaction between antimicrobial and bacterial species, indicating that susceptibility against a particular antimicrobial varied among the species of environmental streptococci and vice versa. Generally, susceptibility decreased with increasing within-herd average somatic cell count, isolates recovered in mid-lactation were more susceptible than isolates recovered in early lactation, and isolates recovered in samples collected post-clinical mastitis were more susceptible than isolates recovered from non-clinical lactating quarters. The results of this research support continued susceptibility of environmental streptococci to beta-lactam antimicrobials. A departure from the expected susceptibility to beta-lactams was the apparent reduced susceptibility of S. uberis to penicillin.

Investigation of within- and between-herd variability of bovine leukaemia virus bulk tank milk antibody levels over different sampling intervals in the Canadian Maritimes

Bulk tank milk (BTM) samples are used to determine the infection status and estimate dairy herd prevalence for bovine leukaemia virus (BLV) using an antibody ELISA assay. BLV ELISA variability between samples from the same herd or from different herds has not been investigated over long time periods. The main objective of this study was to determine the within-herd and between-herd variability of a BTM BLV ELISA assay over 1-month, 3-month, and 3-year sampling intervals. All of the Canadian Maritime region dairy herds (n = 523) that were active in 2013 and 2016 were included (83.9% and 86.9% of total herds in 2013 and 2016, respectively). BLV antibody levels were measured in three BTM samples collected at 1-month intervals in early 2013 as well as two BTM samples collected over a 3-month interval in early 2016. Random-effects models, with fixed effects for sample replicate and province and random effects for herd, were used to estimate the variability between BTM samples from the same herd and between herds for 1-month, 3-month, and 3-year sampling intervals. The majority of variability of BTM BLV ELISA results was seen between herds (1-month, 6.792 ± 0.533; 3-month, 7.806 ± 0.652; 3-year, 6.222 ± 0.528). Unexplained variance between samples from the same herd, on square-root scale, was greatest for the 3-year (0.976 ± 0.104), followed by the 1-month (0.611 ± 0.035) then the 3-month (0.557 ± 0.071) intervals. Variability of BTM antibody levels within the same herd was present but was much smaller than the variability between herds, and was greatest for the 3-year sampling interval. The 3-month sampling interval resulted in the least variability and is appropriate to use for estimating the baseline level of within-herd prevalence for BLV control programs. Knowledge of the baseline variability and within-herd prevalence can help to determine effectiveness of control programs when BTM sampling is repeated at longer intervals.