Comparing Occurrence of Bovine Respiratory Pathogens Detected by High-Throughput Real-Time PCR in Nasal Swabs and Non-Endoscopic Bronchoalveolar Lavage Samples from Dairy and Veal Calves.

This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Mycoplasma species, Pasteurella multocida, and Truepurella pyogenes). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of Mannheimia haemolytica in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.

Acute phase protein concentration in pharyngeal swabs from clinically healthy commercial dairy calves.

BACKGROUND: Early diagnosis of disease in calves is crucial for fast recovery and prudent use of antibiotics. The serum concentration of acute phase proteins (APPs) is up- or downregulated in response to tissue injury and has been studied widely in human medicine. There is growing interest in using APPs as biomarkers for different diseases and as a tool to initiate and monitor treatment in veterinary medicine as well. The concentration of APPs in saliva in healthy calves has not been established and the use of pharyngeal swabs offers a non-invasive alternative to blood sampling. Pharyngeal swabs, tracheal aspirate (TA) and blood samples were collected from 84 clinically healthy commercial dairy calves and analyzed for the APPs serum amyloid A (SAA), haptoglobin (Hp) and lipopolysaccharide binding protein (LBP). RESULTS: We found detectable concentrations of SAA, Hp and LBP in pharyngeal swabs from calves, as well as in TA and serum. There were no biologically interesting correlations between the SAA concentrations in serum and TA or pharyngeal swabs. This also applied to Hp and LBP concentrations in serum and TA or pharyngeal swabs. CONCLUSIONS: SAA, Hp and LBP can be measured in saliva and TA from calves, but there was no correlation between the specific APP concentration in serum and pharyngeal swab or TA. There was a considerable technical variation in the sampling method for both pharyngeal swab and TA, and further validation of the methods is needed.

Field Experience of Antibody Testing against Mycoplasma bovis in Adult Cows in Commercial Danish Dairy Cattle Herds.

Mycoplasma bovis in cattle is difficult to diagnose. Recently, the ID screen® mycoplasma bovis indirect ELISA (ID screen) was commercially released by IDVet. The objectives of this study were to: (1) gain and share experience of using the ID screen in adult dairy cows under field conditions; (2) determine the correlation between antibody levels in milk and serum and (3) compare the ID screen results with those of the Bio K 302 (BioX 302) ELISA from BioX Diagnostics. Paired serum and milk samples were collected from 270 cows from 12 Danish dairy herds with three categories of M. bovis disease history. The ID screen tested nearly all cows positive in all, but the three non-infected herds, while the BioX 302 tested very few cows positive. The ID screen is therefore a much more sensitive test than the BioX 302. However, cows in five exposed herds without signs of ongoing infection and two herds with no history of M. bovis infection also tested ID screen positive. Therefore, the performance and interpretation of the test must be investigated under field conditions in best practice test evaluation setups. A concordance correlation coefficient of 0.66 (95% CI: 0.59-0.72) between the ID screen serum and milk results indicates that milk samples can replace serum samples for the ID screen diagnosis of M. bovis in adult cows.

Increased incidence rate of undesired early heifer departure in Mycoplasma bovis-antibody positive Danish dairy cattle herds.

Mycoplasma bovis infections cause disease and production losses in cattle worldwide. The long-term consequences are not well described despite being important for management decisions during and after disease outbreaks. We investigated the association between M. bovis antibody-positivity and undesired early departure (UED, i.e. death, euthanasia or slaughter) before first calving in a cohort of 636 heifers from 36 Danish dairy herds with and without a history of M. bovis-associated disease. The herds were visited 4 times at 3-month intervals and blood samples from young stock and milk samples from lactating cows were collected. Poisson regression was performed to examine the association with UED as outcome, logarithmic transformation of risk time as offset and herd as a random effect. Individual antibody measurements and group-level variables representing the infection level among young stock and cows, age and mortality variables were included in the model. The incidence rate ratio of UED increased by 1.23 times for every 10% increase in M. bovis young stock seroprevalence, while the effect of individual antibody level was modified by age and influenced UED less. In conclusion, UED in heifers was associated with M. bovis antibody-positivity in young stock and should be controlled in dairy herds to reduce losses.

Mycoplasma bovis antibody dynamics in naturally exposed dairy calves according to two diagnostic tests.

BACKGROUND: Inexpensive and convenient diagnostic tests for use in clinical work and for the surveillance of infection with Mycoplasma bovis are in demand. The objective of this longitudinal field study was to gain knowledge about the dynamics of antibodies against M. bovis in sera from naturally exposed calves with and without different clinical signs, measured by two different ELISA tests. RESULTS: A total of 83 calves were subject to between one and five blood samples and clinical examinations using a standard protocol during five herd visits to each of four outbreak dairy herds. The blood samples were analysed for the presence of antibodies against M. bovis using the commercial IgG ELISA test BioX K302 (BioX) and an in-house indirect IgG ELISA test (MilA ELISA). Linear mixed models were used to describe and compare the antibody dynamics as measured by the two tests in relation to the disease status and age of the animals. The BioX ELISA response was below the recommended cut-off (37 ODC%) for the entire study period in many of the calves. The estimated mean ODC% increased slowly but did not reach the recommended individual animal cut-off in three of the four herds. The highest estimated ODC% was not reached until the calf was 110-130 days old. The MilA ELISA response rose above the recommended cut-off (135 antibody units (AU)) in almost all calves, and in two herds, the estimated mean was above the individual animal cut-off shortly after the birth of the calf. The highest estimated antibody concentration was reached when the calf was approximately 60 days old. Disease status of the calf was not significantly associated with the results of either test. CONCLUSIONS: We conclude that the BioX ELISA cannot be recommended for use in calves below 3 months of age. The MilA ELISA was able to detect antibodies shortly after birth (i.e. from approximately 3 weeks of age and onwards) and is therefore a more sensitive test for M. bovis exposure in young calves. Neither ELISA seemed able to differentiate between calves with arthritis and/or otitis media, and respiratory disease.

Latent class analysis of bulk tank milk PCR and ELISA testing for herd level diagnosis of Mycoplasma bovis.

The bacterium Mycoplasma bovis causes disease in cattle of all ages. An apparent increase in the occurrence of M. bovis associated outbreaks among Danish dairy cattle herds since 2011 has prompted a need for knowledge regarding herd-level diagnostic performance. Therefore, the objective of this study was to evaluate the herd-level diagnostic performance of an indirect ELISA test by comparison to a real-time PCR test when diagnosing M. bovis in cattle herds of bulk tank milk. Bulk tank milk samples from Danish dairy herds (N=3437) were analysed with both the antibody detecting BIO K 302 M. bovis ELISA kit and the antigen detecting PathoProof Mastitis Major-3 kit. As none of these are considered a gold standard test for herd-level diagnostics we applied a series of Bayesian latent class analyses for a range of ELISA cut-off values. The negative and positive predictive values were calculated for hypothetical true national prevalences (1, 5, 10, 15 and 20%) of infected herds. We estimated that the ELISA test had a median sensitivity and specificity of 60.4 [37.5-96.2 95% Posterior Credibility Interval] and 97.3 [94.0-99.8 95% PCI] at the currently recommended cut-off (37% Optical density Coefficient). These changed to 43.5 [21.1-92.5 95% PCI] and 99.6 [98.8-100 95% PCI] if the cut-off was increased to 50 ODC%. In addition, herd-level diagnosis by ELISA would result in fewer false positives at a cut-off value of 50 ODC% compared to 37 ODC% without compromising the negative predictive value.