Epidemiology of antimicrobial resistance in commensal E. coli from healthy dairy cattle on a Mediterranean pasture-based system of Australia: A cross-sectional study.

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 - 72.7) and calf feces (67.1%; 95% CI = 62.0 - 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 - 49.4) and 24.6% (95% CI = 20.2 - 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 - 10.3); calf: 30.0% (95% CI = 25.2 - 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 - 8.7); calf: 40.3% (95% CI = 35.1 - 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 - 4.3); calf: 22.0% (95% CI = 17.7 - 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC ß-lactamase and aminoglycoside phosphotransferase.

Bacteriological outcomes following random allocation to quarter-level selection based on California Mastitis Test score or cow-level allocation based on somatic cell count for dry cow therapy.

Intramammary infusion of antimicrobials at the end of lactation (dry cow therapy) has been a cornerstone of mastitis management for many years. However, as only a proportion of cows are infected at this time, treating only those cows likely to be infected is an important strategy to reduce antimicrobial usage and minimize risk of emergence of antimicrobial resistance. Such an approach requires the ability to discriminate between cows and quarters likely to be infected and uninfected. This study compared assignment of cows or quarters to antimicrobial treatment at the end of lactation based on cow composite somatic cell count (SCC; i.e., all quarters of cows with a maximum SCC across lactation >200,000 cells/mL received an antimicrobial; n = 891 cows, SCC-group) or assignment to quarter-level treatment based on a quarter level California Mastitis Test (CMT) score ≥ trace (n = 884 cows; CMT-group) performed immediately before drying off. All quarters of all cows also received an infusion of a bismuth-based internal teat sealant. Milk samples were collected for microbiology following the last milking, and again within 4 d of calving. Clinical mastitis records from dry off to 30 d into the subsequent lactation were collected. Multilevel, multivariable models were used to assess the effect of assignment to antimicrobial treatment. At drying off, a total of 575 (8.8%) and 147 (2.3%) of the 6,528 quarters had a minor, and a major intramammary infection (IMI), respectively. At drying off, 2089/3270 (63.9%) and 883/3311 (26.7%) of quarters were treated with dry cow therapy in the CMT and SCC-groups, respectively. Apparent bacteriological cure proportion for any IMI was higher in quarters assigned to the CMT than the SCC-group (349/368 (0.95, 95% CI 0.92-0.97) versus 313/346 (0.90, 95% CI 0.87-0.93)). New IMI proportion was lower among quarters assigned to the CMT than SCC-group [101/3,212 (0.032, 95% CI 0.025-0.038) versus 142/3,232 (0.044, 95% CI 0.036-0.051)]. The prevalence of any IMI postcalving was lower in quarters assigned to the CMT than SCC-group [119/3,243 (0.037, 95% CI: 0.030-0.044) versus 173/3,265 (0.054, 95% CI: 0.045-0.062)]. There was no difference in incidence of clinical mastitis between treatment groups. The total mass of antimicrobials used was 63% higher in the CMT-group than in the SCC-group (3.47 versus 2.12 mg/kg of liveweight). Selection of quarters for antimicrobial treatment at the end of lactation based on CMT resulted in greater proportion undergoing bacteriological cure, reduced risk of any new IMI and reduced post calving prevalence of any IMI compared with selection of cows based on SCC. However, CMT-based selection resulted in higher antimicrobial use compared with SCC-based selection, and further research is required to analyze the cost benefit and impact on risk of antimicrobial resistance of these 2 strategies.

Development of an intrauterine infection model in the postpartum dairy cow.

AIMS: To develop an intrauterine infection model for Trueperella pyogenes in postpartum dairy cows and to assess the effect of this infection on the degree of intrauterine inflammation and concentrations of progesterone in serum. METHODS: The oestrous cycles of 36 healthy, non-pregnant, postpartum dairy cows were synchronised. They were then treated by intrauterine infusion of 0.5 g cephapirin before being blocked by age and randomly assigned to treatment with intrauterine infusion of saline (n = 18), 107 (n = 9) or 109 (n = 9) cfu of T. pyogenes, approximately 4 days after the expected time of ovulation (Day 0). Prior to intrauterine infusion on Day 0 and again on Days 3, 7, 10, and 15, cytobrush samples were collected from the uterus of each cow for microbiology and assessment of the percentage of polymorphonuclear neutrophils (PMN%). Blood samples were collected on the same days for measurement of concentrations of progesterone in serum, and uterine lumen diameter was assessed daily using transrectal ultrasonography. RESULTS: Trueperella pyogenes was isolated from 5/18 (28%), 7/9 (78%) and 8/9 (89%) cows infused with saline, 107 or 109 cfu of T. pyogenes, respectively (p < 0.001). Mean PMN% in the control cows did not change over time (p > 0.05), whereas it was higher on Days 7 and 10 than Day 0 in the 107 cfu group, and higher on Days 3 and 10 than Day 0 in the 109 cfu group (p < 0.05). The percentage of observations with uterine lumen diameters >2 mm was higher in cows infused with 107 (29.3 (95% CI = 14.5-44.2)%) or 109 cfu (19.2 (95% CI = 7.0-31.5)%) than in control cows (3.1 (95% CI = 0.1-6.0)%) (p < 0.001). Mean concentrations of progesterone in serum were higher in cows infused with 107 cfu (2.01 (SE 0.19) ng/mL) than cows infused with 109 cfu (1.01 (SE 0.27) ng/mL), with the control group intermediate (1.41 (SE 0.19) ng/mL) (p = 0.03). CONCLUSIONS: Infusion of 107 or 109 cfu of T. pyogenes resulted in the establishment of intrauterine infection in 83% of cows. Infection resulted in increased uterine lumen diameter, and an inflammatory response, i.e. elevated PMN%. CLINICAL RELEVANCE: This intrauterine infection model may be useful for future research on, for example, the pathogenesis of intrauterine infection in postpartum dairy cows.

Risk factors for clinical or subclinical mastitis following infusion of internal teat sealant alone at the end of lactation in cows with low somatic cell counts.

AIMS: To identify risk factors for subclinical and clinical mastitis in cows with low somatic cell counts (SCC) following infusion with internal teat sealant (ITS) at drying off. METHODS: Cows with no history of clinical mastitis and a maximum SCC <250,000 cells/mL at any herd test in the lactation before drying off were randomly selected from 36 herds. In the final week of lactation, quarter milk samples were collected aseptically from each selected cow for microbiology, and each quarter was then infused with ITS. Clinical mastitis records from 22 herds and herd test data from all herds were collated to determine potential herd- or cow-level explanatory variables for clinical mastitis over the dry period or in the first 60 days of the subsequent lactation, and subclinical mastitis (SCC >200,000 cells/mL; SCM) at the first herd test after calving. Multivariable, multilevel, logistic regression analyses were used to model the data. RESULTS: At drying off, 44/1,514 (2.8%) cows were infected with a major pathogen. Two of 1,001 (0.2%) cows were diagnosed with clinical mastitis over the dry period. There were 128/1,514 (8.5%) cows with SCM at the first herd test after calving. The odds of SCM were greater for cows with a major pathogen present at drying off than those without (OR = 4.7 (95% CI = 2.29-9.65); p < 0.001), and for third or greater lactation than second lactation cows (OR = 3.16 (95% CI = 1.70-5.88); p < 0.001). For every 1L increase in milk yield at the last herd test before drying off the OR for SCM was 1.07 (95% CI = 1.01-1.13); (p = 0.02), and for each 1 unit increase in ln maximum SCC in the lactation before drying off the OR for SCM was 1.54 (95% CI = 1.13-2.10); (p = 0.01). There were 30/976 (3.1%) cows diagnosed with clinical mastitis in the first 60 days after calving. The odds of clinical mastitis were greater for cows producing >15 L/day at the last herd test of the preceding lactation than cows producing <10 L/day (OR = 4.79 (95% CI = 1.48-15.46); p = 0.009); for each 1 unit increase in ln maximum SCC in the previous lactation the OR for clinical mastitis was 1.96 (95% CI = 1.09-3.54); (p = 0.03), and the odds increased with increasing herd-level cow-case lactational incidence of clinical mastitis in the preceding lactation (p = 0.003). CONCLUSIONS AND CLINICAL RELEVANCE: Selection of cows with low SCC for ITS infusion should consider cow milk yield and maximum cow SCC in the preceding lactation.

Antimicrobial susceptibilities in dairy herds that differ in dry cow therapy usage.

Intramammary infusion of antimicrobials at the end of lactation (dry cow therapy; DCT) is a central part of mastitis control programs and is one of the major indications for antimicrobial use in dairy cows. However, with increasing focus on prudent use of antimicrobials and concerns about emergence of antimicrobial resistance, the practice of treating every cow at the end of lactation with DCT is in question. This cross-sectional, observational study determined the minimum inhibitory concentrations (MIC) of 10 antimicrobials for coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis isolates from milk samples from dairy cows with somatic cell counts >200,000 cells/mL in herds that had been organic for >3 yr (n = 7), or had used either ampicillin-cloxacillin DCT (n = 11) or cephalonium DCT (n = 8) in the preceding 3 yr. The organic herds were certified under the United States Department of Agriculture National Organic Program, meaning that there was no blanket DCT, and minimal use of antimicrobials in general, with a loss of organic status of the animal if treated with antimicrobials. Breakpoints (where available) were used to categorize isolates as resistant, intermediate, or susceptible to antimicrobials. The MIC distributions of isolates from different herd types were compared using binomial or multinomial logistic regression. Of 240 CNS isolates, 12.9, 0.8, 7.1, 32.6, and 1.2%, were intermediate or resistant to ampicillin, cephalothin, erythromycin, penicillin, and tetracycline, respectively. Of 320 Staph. aureus isolates, 29.0, 2.5, 1.2, and 34.9% were intermediately resistant or resistant to ampicillin, penicillin, erythromycin, and oxacillin, respectively. Of 184 Strep. uberis isolates, 1.1, 25.0, 1.6, and 1.6% were intermediately resistant or resistant to erythromycin, penicillin, pirlimycin, and tetracycline, respectively. Generally, the MIC of CNS and streptococcal isolates from organic herds were lower than isolates from herds using DCT. However, the differences in MIC distributions occurred at MIC below clinical breakpoints, so that the bacteriological cure rates may not differ between isolates of differing MIC. Bimodal distributions of MIC for ampicillin and penicillin were found in Staph. aureus isolates from organic herds, suggesting that isolates with a higher MIC are a natural part of the bacterial population of the bovine mammary gland, or that isolates with higher MIC have persisted within these organic herds from a time when antimicrobials had been used. Given these observations, further work is required to determine if exposure to DCT is causally associated with the risk of elevated MIC, and whether reduction or removal of DCT from herds would reduce the risk of elevated MIC of mastitis pathogens.

Detecting intramammary infection at the end of lactation in dairy cows.

To reduce antimicrobial use, infusion of antimicrobials into only infected cows at the end of lactation (selective dry cow therapy) is preferable to infusion of every cow with antimicrobials. Use of selective dry cow antimicrobial therapy requires differentiation of probably infected from uninfected cows to enable treatment allocation. Milk somatic cell count (SCC) has been used to distinguish between cows with and without intramammary infection (IMI). However, SCC may be influenced by milk yield, stage of lactation, breed, and herd-level variables such as prevalence of infection. Cut points for SCC, to distinguish between cows with and without an IMI, may need to differ between cow age groups and breeds, or among herds. This study evaluated associations between SCC and major pathogen IMI in one or more quarters of 2,606 cows from 36 herds in 4 regions of New Zealand. In the last week of lactation, cows selected at random had milk samples collected from each quarter, and the teat-end condition and hygiene of the udder were scored. Herd- and cow-level data including age, breed, milk volume, and SCC at each production were recorded, and bulk tank milk SCC and volume of milk shipped were collated. At cow level, the association between average, maximum, and last cow-composite SCC, and presence of a major pathogen IMI in one or more quarters of cows, was examined using receiver operator curves. Predictive logistic regression models were then developed that included potential effect modifiers such as age, milk yield, and bulk tank milk SCC. The population average prevalence of major pathogen IMI was 7.2% of cows (95% confidence interval = 5.9-8.6), and this varied significantly between herds. The average, maximum, and last cow-composite SCC of lactation were all predictive of presence of a major pathogen IMI and did not differ in their ability to discriminate infected from uninfected cows. However, the optimal cut points for the last SCC, the maximum SCC, and average SCC were 108, 152, and 105 × 1,000 cells/mL, respectively. Inclusion of age, bulk tank SCC, and history of clinical mastitis improved overall model fit. However, inclusion of these variables did not improve the discriminatory power of maximum cow-composite SCC used alone. We conclude that cow-composite SCC on its own resulted in sensitivities and specificities of between 0.76 and 0.86, and 0.71 to 0.80, respectively, for determination of presence of major pattern IMI, and the predictive value was not improved by addition of other predictor variables.

Evaluation of fully oxidised ß-carotene as a feed ingredient to reduce bacterial infection and somatic cell counts in pasture-fed cows with subclinical mastitis.

AIMS: To assess the effect of oral supplementation with fully oxidised ß-carotene (OxBC) on bacteriological cure, the incidence of clinical mastitis, and somatic cell counts (SCC) in pasture-fed cows with subclinical intramammary infection. METHODS: Cows from four dairy herds were enrolled in early lactation if they had quarter-level SCC >200,000 cells/mL and they had a recognised bacterial intramammary pathogen in one or more quarters. They were randomly assigned to be individually fed from Day 0, for a mean of 40 days, with 0.5 kg of a cereal-based supplementary feed that either contained 300 mg of OxBC (treatment; n = 129 quarters) or did not (control; n = 135 quarters). Quarter-milk samples were collected on Days 21 and 42 for microbiology and SCC assessment. Bacteriological cure was defined as having occurred when the bacteria present on Day 0 were not isolated from samples collected on Days 21 or 42. A new intramammary infection was defined as a bacterial species isolated either on Day 21 or 42 differing from that isolated on Day 0. Clinical mastitis was diagnosed and recorded by herdowners up to Day 42. RESULTS: The bacteriological cure rate was greater for quarters from cows in the treatment group (13.9 (95% CI = 4.1-23.7)%) than for cows in the control group (6.9 (95% CI = 4.8-9.1)%; p = 0.02). The percentage of quarters that developed a new intramammary infection at Day 21 or 42 was greater for cows in the treatment group (17.9 (95% CI = 6.7-29.1)%) than for cows in the control group (13.0 (95% CI = 4.3-21.8)%; p < 0.01). The prevalence of quarters that were infected on Day 42 was less in cows in the treatment group (79.9 (95% CI = 62.3-97.6)%) than the control group (88.2 (95% CI = 78.4-97.9)%; p = 0.009). The incidence of quarters diagnosed with clinical mastitis by Day 42 was lower in cows from the treatment group (1/129 (0.78 (95% CI = 0.02-4.24)%)) than in cows from the control group (6/135 (4.44 (95% CI = 1.65-9.42)%; p = 0.03)). Mean quarter-level SCC was not statistically different between treatment groups (p = 0.34). CONCLUSIONS AND CLINICAL RELEVANCE: Feeding 300 mg/cow/day of OxBC resulted in a higher bacteriological cure rate, a lower prevalence of intramammary infection, and a lower incidence of clinical mastitis compared to untreated controls. However new intramammary infections increased in treated cows, and the magnitude of the increased bacteriological cure was low, resulting in 80% of cows remaining infected at Day 42. Therefore treatment with OxBC should be considered as an adjunct to other mastitis control measures.

Cow-level risk factors for clinical mastitis in the dry period in cows treated with an internal teat sealant alone at the end of lactation.

AIMS: To assess potential cow-level risk factors for clinical mastitis during the dry period in cows infused with internal teat sealant (ITS) alone at drying off, and associations with elevated somatic cell counts (SCC) at the first herd test, or clinical mastitis in the subsequent lactation. METHODS: Ten herds reporting an incidence of clinical mastitis during the dry period of >3% amongst cows infused with ITS alone at the end of the 2018/2019 lactation were enrolled. Cow data collected included age, breed, milk yield and SCC at the last herd test before drying off, drying off date, clinical mastitis records during the dry period and in the subsequent lactation, and SCC at the first herd test following calving. RESULTS: Overall, 2,401 cows were infused with ITS alone and 196/2,401 (8.2%) cows were diagnosed with clinical mastitis during the dry period. In the final multivariable model for risk of clinical mastitis there was an interaction between age and milk yield at the last herd test (p < 0.001), with cows aged >4 years and producing ≥10 L/cow/day having a higher risk of clinical mastitis than younger cows or those producing <10 L/cow/day. The risk of clinical mastitis was higher for cows dried off in the last week of lactation than for cows dried off >1 week before the end of lactation for the herd (p = 0.033). The percentage of cows with elevated SCC (>150,000 cells/mL) at the first herd test in the following lactation was higher amongst those diagnosed with clinical mastitis over the dry period compared with those that were not (p = 0.048). The hazard of clinical mastitis in the first 60 days after calving was also higher in cows diagnosed with clinical mastitis in the dry period than those that were not (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: The risk of clinical mastitis in the dry period following infusion of ITS alone at the end of lactation was associated with cow age, milk yield before drying off and timing of drying off. Cows diagnosed with clinical mastitis over the dry period had a higher risk of clinical mastitis and of elevated SCC in the subsequent lactation. This was an observational study so no causal inferences can be made, however herdowners and veterinarians should follow current recommendations regarding management of milk yield before drying off, and selection of cows for treatment with ITS alone.

Increasing conception rate by addition of a second prostaglandin injection in an Ovsynch and progesterone treatment programme for dairy cows not detected in oestrus before the start of the seasonal mating period.

AIMS: To assess the effect on conception rate to first service, 3- and 6-week in-calf rates, final pregnancy rate and the hazard of pregnancy, of addition of a second prostaglandin (PG) injection 24 hours after the first, in a synchrony programme combining Ovsynch and progesterone in pasture-based dairy cows that had not been detected in oestrus before the start of the seasonal mating period. METHODS: This prospective, negative-controlled study was conducted using anoestrous cows (n = 1,411) from nine spring-calving dairy herds in the Waikato region of New Zealand. Ovaries of cows not detected in oestrus were examined by transrectal ultrasonography and the presence of a corpus luteum determined. All cows were then treated with an intravaginal progesterone-releasing device and injected I/M with gonadorelin (Day -9). Cows were injected I/M with cloprostenol at device removal (Day -2) and on Day -1 were injected with either water (single PG; n = 720), or cloprostenol (double PG; n = 692). On Day 0 cows were injected again with gonadorelin, unless previously detected in oestrus, and were inseminated on Day 1. Based on dated pregnancy diagnosis by transrectal ultrasonography, cows were categorised as conceiving to first insemination, becoming pregnant in the first 3 or 6 weeks, or becoming pregnant by the end of the mating period. RESULTS: Cows in the double PG group had increased conception rate to first service (45.3 (95% CI = 45.1-45.4)% vs. 41.1 (95% CI = 41.0-41.3)%; p < 0.001), 3-week in-calf rate (53.4 (95% CI = 53.6-54.0)% vs. 49.2 (95% CI = 49.0-49.4)%; p < 0.001), and 6-week in-calf rate (67.2 (95% CI = 65.7-68.8)% vs. 63.5 (95% CI = 62.0-65.0)%; p = 0.014) compared to cows in the single PG group. Final pregnancy rate and hazard of pregnancy did not differ between treatment groups (p >0.2). CONCLUSIONS: Addition of a second PG injection 24 hours after the first, increased conception rate to first service and the proportion of cows pregnant by 3 and 6 weeks after commencement of the mating period. CLINICAL RELEVANCE: Improved outcomes can be achieved when treating those cows not detected in oestrus by the start of the seasonal mating period by addition of a second PG injection to the currently recommended treatment protocol for these animals.

Prevalence of endometritis diagnosed by vaginal discharge scoring or uterine cytology in dairy cows and herds.

Endometritis, diagnosed either by assessing the proportion of nucleated cells that are neutrophils (PMN%) following cytology of the endometrium or by assessing the degree of purulent material within the vagina (purulent vaginal discharge or PVD score), is prevalent among dairy cows. However, limited data exist as to the degree of variation among herds in the prevalence of endometritis diagnosed by these 2 methods. Thus, we undertook a study involving uterine cytological and vaginal sampling at a median of 41 d in milk of 1,807 cows from 100 seasonally breeding dairy herds in New Zealand. The optimal cut-point for PMN% was determined by receiver operator characteristic (ROC) curve analysis using conception to first artificial insemination (AI) as the outcome variable. The prevalence of disease was then calculated at the cow and herd levels, and an estimate of the effect of clustering of cow within a herd was calculated. Reproductive outcomes were collated and associations between endometritis and reproductive outcomes assessed using multivariable models. The optimal PMN% cut-point was ≥2%. The correlation of results for cows within a herd (the intraclass correlation) was 0.03, which was significant; hence, subsequent modeling accounted for this clustering. The cow-level prevalence of PMN% ≥2% was 27.0% [95% confidence interval (CI): 25.0 to 29.1%], whereas the mean within-herd prevalence of PMN% ≥2% was 27.1% (95% CI: 24.7 to 29.6%; range: 5.0 to 63.6%), and the prevalence among herds varied significantly. An elevated PMN% (≥2%) was significantly associated with a reduction in the proportion of cows conceiving to first AI (45.8 vs. 54.5%), a reduced proportion of cows submitted for AI in the first 3 wk of the seasonal breeding program (83.7 vs. 89.3%), and a lower proportion pregnant in the first 3 (44.4 vs. 55.4%) and 6 wk (67.5 vs. 76.4%) of the breeding program relative to cows with a low PMN% (i.e., <2%). A total of 24.6% of cows had a PVD score ≥2. The herd mean prevalence of PVD score ≥2 was 25.1% (95% CI: 22.5 to 27.7%; range: 5.0 to 65.0%) and varied significantly among herds. The level of agreement (kappa) between the PVD score and PMN% was low (16.8%) and nonsignificant. The effects of PVD score and PMN% on reproductive outcomes were independent. The within-herd median prevalence of endometritis based on combining both diagnostic tests and using a Bayesian latent class model was 22.9% (Bayesian 95% CI: 10.4 to 40.1%). We conclude that more than one-fifth of dairy cows have endometritis diagnosed either by PMN% or PVD in seasonal breeding herds when assessed at an average of 41 DIM, which was, on average, 30 d before the start of the seasonal breeding program. There is large and unexplained variation in prevalence of endometritis among herds. The 2 diagnostic methods were both associated with reproductive outcomes but have low levels of agreement between them and their effects appear to be independent.

Randomized, controlled, superiority study of extended duration of therapy with an intramammary antibiotic for treatment of clinical mastitis.

The objectives of this study were to evaluate the efficacy of therapy of clinical mastitis following intramammary infusion of 2 durations of intramammary antibiotic therapy and to analyze clinical and microbiology data at enrollment or within 4 d of commencement of treatment as potential predictors of subsequent clinical and bacteriological cure. A total of 304 glands were randomly assigned to intramammary treatment with a combination of amoxicillin, clavulanic acid, and prednisolone either 3 times at 12-h intervals (3×12, n = 156) or 5 times at 12-h intervals (5×12 hourly, n = 148). At enrollment (d 0), assessments were made of the cow's demeanor, rectal temperature, teat-end hyperkeratosis, presence of heat or swelling of the gland, presence of clots in the milk, and California Mastitis Test (CMT) score. Cows were revisited on d 4, 14, and 21 and assessed for rectal temperature, the presence of clots in milk, heat or swelling of the gland, and CMT score. Milk samples were collected from enrolled glands for bacteriology (d 0, 4, 14, and 21) and for somatic cell count (d 14 and 21). Data were analyzed using generalized linear models. Treatment group was the key independent variable, but herd was included in the models. Other potentially confounding variables were included where associated at the bivariate level. A second series of analyses were undertaken to predict likelihood of clinical and bacteriological cure using the clinical signs cow age and days in milk, as well as bacteriological results at d 0 and 4. Although we found a higher clinical failure rate in the 3×12 than 5×12 hourly treatment group (28.2 ± 5.0 vs. 13.4 ± 3.6%, respectively), we noted no difference in bacteriological cure percentage (73.3 ± 7.8 vs. 72.0 ± 7.4%, respectively). The presence of heat or swelling at d 4, days in milk at initiation of treatment, age of the cow, and change in CMT score between d 0 and 4 were predictive of clinical and bacteriological cure. We concluded that increasing the duration of treatment resulted in significantly fewer clinical failures, but had no effect on cure proportion, somatic cell count, or new infection rate. Knowledge of cow age and days in milk and clinical signs were predictive of clinical and bacteriological cure proportion and may provide guidance for producers, under veterinary direction, as to when to extend or recommence therapy.

Risk factors for subclinical hypocalcaemia, and associations between subclinical hypocalcaemia and reproductive performance, in pasture-based dairy herds in New Zealand.

AIMS To estimate the herd-level prevalence of subclinical hypocalcaemia within 3 days of calving, to determine cow and herd-level risk factors for this condition, and associations with reproductive performance, in pasture-based cows in New Zealand. METHODS Between 10 and 15 clinically healthy cows ≥3-years-old (n=1,051) were enrolled from 76 spring-calving dairy herds. Blood samples were collected from all cows on 1 or 2 days within 3 days of calving, and assayed for total concentrations of Ca in serum. Subclinical hypocalcaemia was defined as concentrations of Ca in serum ≤2.14 mmol/L, and herd-level prevalence was the percentage of sampled cows with subclinical hypocalcaemia. Breeding and pregnancy diagnosis data were obtained for each cow to calculate reproductive outcomes. Herd-level data about management practices were collected from farmers at enrolment. Associations between cow-level variables, and reproductive outcomes, and subclinical hypocalcaemia were examined using multivariable logistic regression mixed models. Associations between herd-level variables and prevalence of subclinical hypocalcaemia were examined using a multivariable linear regression model. RESULTS Mean herd-level prevalence of subclinical hypocalcaemia was 52 (95% CI=49-55)%. Risk of subclinical hypocalcaemia at the cow-level was associated with an interaction between age of cow and interval from calving to sampling (p=0.03). Increasing age of cow was associated with increased risk of subclinical hypocalcaemia, and cows sampled on the day of calving had an increased risk compared with cows sampled 1 or 2 days after calving. Increased herd-level prevalence of subclinical hypocalcaemia was associated with feeding grass silage (p=0.06) or maize silage (p=0.004), and feeding increasing amounts of elemental Mg in the form of magnesium oxide (p=0.02). The mean farmer-reported herd-level prevalence of clinical hypocalcaemia was 2.9 (95% CI=2.1-3.8)%. Subclinical hypocalcaemia was not associated with any of the reproductive outcomes measured. CONCLUSIONS AND CLINICAL RELEVANCE There was a high prevalence of subclinical hypocalcaemia in the pasture-fed, spring-calving dairy herds sampled and a large between-herd variation in prevalence. Subclinical hypocalcaemia increased with increasing age, and declined with time postpartum. Herd-level prevalence was associated with feeding grass silage and maize silage to pre-calving cows and with increasing amounts of supplemented magnesium oxide. Subclinical hypocalcaemia was not associated with reproductive outcomes.

Effect of treatment with oral Ca boluses following calving on concentrations of Ca in serum in pasture-based dairy cows.

AIMS To assess the effect of the administration of two oral Ca boluses on concentrations of total Ca, ß-hydoxybutyrate (BHB) and non-esterified fatty acids (NEFA) in serum, and urine pH, in recently calved pasture-fed dairy cows. METHODS Friesian or Friesian cross Jersey cows from one dairy farm were blocked by age and randomly assigned to no treatment (control; n=14), or treatment (n=13) with two oral Ca boluses administered approximately 12 hours apart, with the first bolus being given within 14 hours of calving. Each bolus weighed 198 g and contained 43 g of Ca; 31 g of Ca from calcium chloride and 12 g of Ca from calcium sulfate. Cows were enrolled over three calendar days, and all cows were managed in one group during the 24-hour study period. Blood samples were collected at 0, 1, 2, 4, 8, 12, 13, 14, 16, 20 and 24 hours after the initial treatment. Serum from each time point was analysed for concentrations of total Ca, and from 0, 12, and 24 hours for NEFA and BHB. Urine was collected at 0, 12 and 24 hours for pH measurement and pH was categorised as <7 or ≥7. The effect of treatment on percentage change in concentrations of Ca in serum relative to 0 hours, and concentrations of NEFA, BHB and urine pH, was examined using multivariable repeated measures mixed models with cow as a random effect. RESULTS In the final multivariable model for percentage change in concentrations of Ca, there was an interaction between time and treatment (p=0.004), with the percentage increase being higher in treatment than control cows at 1, 2, 4, 8 and 13 hours. At 12 hours, 5/13 (41%) treated cows had a urine pH <7compared to 0/12 (0%) control cows (p<0.001), and at 24 hours 13/13 (100%) treated cows had urine pH <7 compared to 0/12 (0%) control cows (p<0.001). Over the 24-hour period, mean concentrations of NEFA or BHB in serum were similar in treated and control cows (p>0.3). CONCLUSIONS AND CLINICAL RELEVANCE Oral treatment with two Ca boluses increased concentrations of total Ca in serum and decreased urine pH in pasture-fed cows. This bolus has the potential to reduce the prevalence and duration of subclinical hypocalcaemia in recently calved cows.

Viscerosensory input drives angiotensin II type 1A receptor-expressing neurons in the solitary tract nucleus.

Homeostatic regulation of visceral organ function requires integrated processing of neural and neurohormonal sensory signals. The nucleus of the solitary tract (NTS) is the primary sensory nucleus for cranial visceral sensory afferents. Angiotensin II (ANG II) is known to modulate peripheral visceral reflexes, in part, by activating ANG II type 1A receptors (AT1AR) in the NTS. AT1AR-expressing NTS neurons occur throughout the NTS with a defined subnuclear distribution, and most of these neurons are depolarized by ANG II. In this study we determined whether AT1AR-expressing NTS neurons receive direct visceral sensory input, and whether this input is modulated by ANG II. Using AT1AR-GFP mice to make targeted whole cell recordings from AT1AR-expressing NTS neurons, we demonstrate that two-thirds (37 of 56) of AT1AR-expressing neurons receive direct excitatory, visceral sensory input. In half of the neurons tested (4 of 8) the excitatory visceral sensory input was significantly reduced by application of the transient receptor potential vallinoid type 1 receptor agonist, capsaicin, indicating AT1AR-expressing neurons can receive either C- or A-fiber-mediated input. Application of ANG II to a subset of second-order AT1AR-expressing neurons did not affect spontaneous, evoked, or asynchronous glutamate release from visceral sensory afferents. Thus it is unlikely that AT1AR-expressing viscerosensory neurons terminate on AT1AR-expressing NTS neurons. Our data suggest that ANG II is likely to modulate multiple visceral sensory modalities by altering the excitability of second-order AT1AR-expressing NTS neurons.

Pegbovigrastim treatment affects gene expression in neutrophils of pasture-fed, periparturient cows.

Treatment with granulocyte colony-stimulating factor has been reported to increase circulating neutrophil count and enhance neutrophil function in the periparturient cow. It was hypothesized that a commercially available recombinant bovine granulocyte colony-stimulating factor product (pegbovigrastim) affects gene expression profiles of neutrophils and supports neutrophil function in periparturient cows. Hence this study was undertaken to analyze expression of genes involved in neutrophil functions, including migration, interaction with pathogens, and cell survival. It also assessed the hypothesis that gene expression profiles in neutrophils are modulated by negative energy balance in the peripartum period. Holstein-Friesian, Jersey, and mixed-breed cows on pasture were blocked by expected calving date and body condition score and randomly assigned in a 2 × 2 factorial design. Cows were fed to exceed energy requirements prepartum (122%) or restricted to approximately 85% of prepartum energy requirements. At approximately 7 d before expected calving date, half the cows in each feed group were randomly assigned to be injected with pegbovigrastim or saline. Treatments were repeated within 24 h after calving. Blood samples were collected pretreatment approximately 7 d before calving (d -7). Blood, uterine flush, and milk samples were collected at 4 (d 4) and 7 d in milk (d 7) to measure the expression of a panel of 20 genes representing cell adhesion, pattern recognition, inflammation and cytokine response, antimicrobial capacity, and apoptosis functions in neutrophils using NanoString technology (NanoString Technologies Inc., Seattle, WA) to quantify RNA copy numbers. No effects were observed of prepartum feeding group or a feeding group × treatment interaction for any of the investigated genes. An effect was observed of time on expression of several genes in blood neutrophils. After calving, expression of 2 of 4 cell adhesion-related genes, 3 of 4 pattern recognition receptors, 2 of 4 inflammatory genes, 2 antimicrobial genes, and 2 of 4 cell survival genes was significantly greater at d 4 or 7 or both compared with before calving (d -7). Expression of ICAM1, TLR2, and PTGS2 was significantly higher in blood neutrophils from animals treated with pegbovigrastim compared with untreated controls, suggesting greater migration, pattern recognition, and inflammatory response ability. Pegbovigrastim also affected RNA expression in uterine cells with ICAM1, NOD1, CLEC6A, PTGS2, MPO, DEFB5, and CATHL6 being expressed at higher levels and SELL, ITGB8, IL8RB, and IL10 at lower levels. Milk somatic cells showed a similar pattern but with fewer significant changes. In contrast to the reported decline in neutrophil function in the transition period, neutrophil gene expression was increased for many of the genes studied, an apparent attempt to compensate for reduced neutrophil function. Treatment with pegbovigrastim further increased expression of several genes involved in these processes in blood neutrophils and changed uterine cells to a phenotype with increased antimicrobial capacity, typical for neutrophils that have migrated into their target tissue.

Effect of altering the type of dietary carbohydrate early postpartum on reproductive performance and milk production in pasture-grazed dairy cows.

The objective of this study was to assess the effect of increasing dietary starch for approximately 30 d postpartum on reproduction outcomes in pasture-grazed, seasonal-calving dairy cows. Cows (n = 948) from 3 commercial herds were blocked by age (2, 3, and >3 yr), breed, and expected calving date and randomly assigned to 1 of 2 postpartum treatment groups: high starch (34.7 ± 1.9% nonstructural carbohydrate; mean ± SD) or low starch (22.5 ± 0.4% nonstructural carbohydrate). The high-starch group in all 3 farms received 4.0 to 4.5 kg/d of a 75:25 cracked corn:barley grain mixture in the dairy parlor, split evenly between the morning and afternoon milkings. The low-starch cows received 5.0 to 5.5 kg/d of a 50:50 mixture of palm kernel meal:soy hulls (herds 1 and 3) fed in the parlor; low-starch cows in the remaining herd (herd 2) did not receive a concentrate feed. Cows were cograzed on ryegrass-white clover dominant pastures and were offered corn silage (herds 1 and 3) and canola, corn distillers grain, and palm kernel meal (herd 1) throughout the study. At 1 mo before the start of the seasonal breeding period, the high-starch supplement was removed, and within each herd treatment groups were managed similarly through breeding. Presence of purulent vaginal discharge was assessed at 28 DIM, and tail paint was assessed weekly from 2 to 6 wk postpartum for signs of estrus. The interval to first observed estrus was unaffected by treatment (32.7 vs. 33.5 ± 2 d for high and low starch, respectively), but there were tendencies for a herd × treatment interaction for proportion of cows pregnant to first service and for pregnancy within 6 wk. This interaction was significant for the proportion of cows finally pregnant; a lower proportion of high-starch cows were pregnant to first service, pregnant by 6 wk, and pregnant by the end of the seasonal breeding period in herd 1, but diet did not affect these outcomes in the other herds. Our results do not support a positive effect on reproduction from increasing dietary starch in seasonally bred grazing dairy cows. However, the interactions indicate variability in the herd response to dietary starch early postpartum and imply that pregnancy rate could potentially be compromised through the provision of starch to grazing dairy cows in early lactation (i.e., prebreeding). The experiment was not designed to define the reasons for these interactions, but differences should be considered in future research on the subject.

Predicting intramammary infection status at drying off using indirect testing of milk samples.

AIMS To evaluate the Rapid Mastitis test (RMT, or California Mastitis test) and electrical conductivity (EC) at drying off when used alone or in combination with herd test data (maximum or last herd test somatic cell counts (SCC) before drying off), to define cows or quarters with intramammary infection, using microbiological culture as the gold standard. METHODS Quarter-level milk samples (n=609) from clinically healthy cows (n=153), in three herds in the Waikato region of New Zealand, were tested at drying off using the RMT and EC, and were collected for microbiological culture. The maximum SCC and the SCC at the last herd test of the preceding lactation were determined for each cow. The sensitivity, specificity, and positive and negative predictive values for each test were calculated for different cut-points, using microbiological culture as the gold standard. Receiver operating characteristic curves were generated and the area under the curve (AUC) was calculated for each test. The same parameters were calculated for combinations of two tests in parallel or in series. RESULTS Infection with any pathogen was detected in 62/153 (40.5%) cows and 99/609 (16.3%) quarters, and with major pathogens in 7/153 (4.6%) cows and 8/609 (1.3%) quarters. When predicting infection with any pathogen at the cow-level, the coefficient of agreement was highest for SCC (<0.32) and RMT (<0.28) and lowest for EC (<0.12); the AUC for RMT and EC when used singly ranged from 0.57-0.69, and in combination with SCC ranged from 0.68-0.75. AUC were similar for tests that used either the last or the maximum SCC. CONCLUSIONS AND CLINICAL RELEVANCE When evaluated singly, RMT and EC had only low to moderate diagnostic utility compared to bacteriological culture. When they were combined with SCC and interpreted in parallel, the results were improved, but only moderately. For herds that conduct herd testing, a single herd test late in lactation was as predictive of intramammary infection at drying off as multiple herd tests through the lactation. For herds that do not conduct herd testing, RMT has greater utility than EC.

Antimicrobial usage and risk of retreatment for mild to moderate clinical mastitis cases on dairy farms following on-farm bacterial culture and selective therapy.

AIMS: To assess antimicrobial usage for treatment of mild to moderate clinical mastitis, and risk of retreatment, following implementation of an on-farm bacterial culture system and selective therapy based on culture results, and to assess compliance with treatment decision tree protocols and the level of agreement between results from on-farm culture and laboratory-based microbiology methods. MATERIALS AND METHODS: Herdowners from seven dairy herds were asked to collect milk samples from cases of mild to moderate clinical mastitis between July 2015 and May 2016. All samples were cultured on-farm using a commercially available selective media and were also submitted for laboratory-based culture. Within sequential pairs of cows with mastitis, half were assigned to be treated without regard to culture results (Blanket group), and half were treated based on the on-farm culture results (Selective group) according to decision tree diagrams provided to the farmers. Culture results, treatments, and retreatments for clinical mastitis were recorded. The sum of the daily doses of antimicrobials used per cow, the number of retreatments and interval to first retreatment were compared between treatment groups. RESULTS: The geometric mean sum of daily doses for quarters assigned to the Selective (1.72 (95% CI=1.55-1.90)) group was lower than for the Blanket (2.38 (95% CI=2.17-2.60)) group (p=0.005). The percentage of cows retreated for clinical mastitis did not differ between the Selective (21.7 (95% CI=10.5-25.9)%) and Blanket (26.1 (95% CI=20.9-31.3)%) groups (p=0.13), and there was no difference between groups in the hazard that cows would be retreated within 60 days of enrolment (hazard ratio=0.82 (95% CI=0.39-1.69); p=0.59). Compliance with the treatment protocols was higher amongst quarters assigned to the Selective (199/233; 85.4%) compared with the Blanket (171/249; 68.7%) group (p<0.001), and varied between farms from 64-94%. The overall agreement between results from on-farm and laboratory culture was 188/331 (56.9%; kappa=0.31; p<0.001), but varied between farms from 44.7-88.2% (p<0.001). CONCLUSIONS: Use of on-farm culture with selective antimicrobial therapy resulted in approximately 25% lower antimicrobial usage, but was not associated with an increase in the proportion of cows retreated for clinical mastitis. CLINICAL RELEVANCE: This study has demonstrated that on-farm culture and selective therapy based on culture results can be implemented on-farm. However, farms varied in their implementation of both the treatment protocols and microbiology procedures. Where such systems are to be used on-farm, specific training and on-going monitoring is required.

Factors associated with colostrum quality in individual cows from dairy herds in the Waikato region of New Zealand.

AIMS: To examine associations between various cow-level factors and quality of first-milking colostrum (measured as Brix), and to evaluate herd-level associations between vaccination against calf diarrhoea and colostrum quality, in cows from dairy herds in the Waikato region of New Zealand. METHODS: A single colostrum sample was collected, by complete udder evacuation, from each of 20 cows from 29 dairy herds in the Waikato region of New Zealand during the 2016 spring calving period. Vaccination pre-partum with a calf diarrhoea vaccine was used in 15 herds. Each colostrum sample was tested using a digital Brix refractometer. The body condition score of each cow was recorded at the time of sample collection and farmers provided records of clinical mastitis and facial eczema from the previous 12 months, as well as the age and breed of cows. Associations between cow-level variables in non-vaccinated herds and Brix were examined using a multivariable linear mixed model and estimated marginal means obtained for different categories. RESULTS: Mean Brix of 281 samples from cows in non-vaccinated herds was 18.7 (SD 0.26)%; 63/281 (22.4%) samples had Brix ≥22% and 152/281 (54.1%) had Brix ≥18%. Mean Brix of colostrum samples from cows aged ≥6 years (20.2 (95% CI=19.1-21.2)%) was higher than for samples from 2-year-old cows (18.6 (95% CI=17.3-19.9)%) (p=0.005). Colostrum that was collected at the first milking on the day of calving had higher Brix (20.0 (95% CI=19.1-20.9)%) than colostrum collected from cows that calved the previous day (17.5 (95% CI=16.5-18.4)%) (p<0.001). Mean Brix of colostrum samples from cows which produced ≥8 L (18.2 (95% CI=17.1-19.2)%) tended to be lower than from cows which produced <8 L first-milking colostrum (19.1 (95% CI=18.3-20.0)%) (p=0.08). Among vaccinating herds, 9/15 (60%) had ≥60% colostrum samples with Brix ≥18% compared with 4/14 (29%) of non-vaccinating herds (p=0.04). CONCLUSIONS AND CLINICAL RELEVANCE: Colostrum quality, as measured by Brix, was associated with the total volume of first-milking colostrum, interval from calving to colostrum collection and cow age. Vaccination against calf diarrhoea was associated with a higher proportion of colostrum samples with adequate Brix. Careful selection of colostrum donor cows should ensure newborn calves are fed adequate quality colostrum which should be beneficial in preventing failure of passive transfer of IgG. Testing of colostrum from individual cows with a Brix refractometer is advocated for the selection of colostrum for feeding newborn calves.

Functional and neurochemical characterization of angiotensin type 1A receptor-expressing neurons in the nucleus of the solitary tract of the mouse.

Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT1AR) most widely expressed in adult neurons. Activation of the AT1R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT1R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT1AR-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT1AR promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT1AR-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT1AR-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT1AR-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT1AR-GFP neurons and TH-GFP neurons showed similar AT1AR-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT1AR-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing.