Arrival cortisol measurement in veal calves and its association with body weight, protein fractions, animal health and performance.

Male dairy calves are exposed to an accumulation of transport, social and environmental stressors while transferred to fattening units. As a consequence, calves show high cortisol concentrations upon arrival at the veal facility. Whether cortisol levels as measured on arrival can be associated with animal health, welfare and production results is currently unknown. The first objective of this prospective cohort study was to determine possible associations of arrival serum cortisol concentration with health and production variables of veal calves and other arrival predictors like body weight and γ-globulin concentration. The second aim was to investigate potential clustering of arrival risk factors in veal calves for developing bovine respiratory disease (BRD) based on arrival body weight, serum cortisol concentration, total protein and protein fractions. In total, 105 male Holstein calves from two consecutive production cycles in a single, commercial white veal farm were blood sampled directly at arrival on the farm to determine serum cortisol, total protein and protein fractions. All calves were weighed the day after arrival and clinical signs, average daily weight gain (ADG) and carcass weight were collected. Also, all calves of both production cohorts were repeatedly examined by thoracic ultrasonography at the onset of group respiratory disease symptoms (2-3 weeks after arrival) and four weeks later. Linear and logistic mixed models together with k-means clustering were used for statistical analyses. Calves showed on average high, but individually variable serum cortisol concentrations (mean value = 96.6 ng/mL ± standard deviation (SD) = 48.8; Range (R) = 50.0-317 ng/mL). Arrival cortisol, body weight and γ-globulin content were not significantly associated. Serum cortisol and albumin concentrations at arrival were associated with chronic, unresponsive pneumonia. For each increase of serum cortisol concentration by 10 ng/mL, the odds for lung consolidation of ≥1 cm in depth at the second ultrasonography increased, odds ratio (OR) = 1.03 (95 % confidence interval (CI) = 1.01-1.06; P < 0.050). For every decrease in serum albumin concentration by 1g/L, the OR for developing pneumonia was 1.23 (95 % CI= 1.04-1.46; P < 0.015). Additionally, two clusters of calves were identified based upon arrival status: a low risk cluster with below average stress-induced cortisol values, above average body weight, no acute phase response (APR) and no failure of passive immune transfer (FPIT), and a high risk cluster with above average cortisol values, below average body weight, APR and FPIT. High risk calves had higher odds for developing clinical BRD (OR= 3.88 (95 % CI=1.20-12.53; P < 0.020)) and lung consolidation ≥1 cm in depth at week 6-7 after arrival (OR= 3.93 (95 % CI=1.34-11.53; P < 0.013)). Avoiding high cortisol levels upon arrival of calves is important for animal welfare, but also for reduction of the prevalence of chronic, unresponsive pneumonia and the associated need for (repeated) antimicrobial treatment and production losses.

Associations of barn air quality parameters with ultrasonographic lung lesions, airway inflammation and infection in group-housed calves.

Barn climate is believed to play a major role in the bovine respiratory disease complex. However, the exact air quality parameters associated with (sub)clinical pneumonia or airway inflammation in calves are currently unknown. The objective of this cross-sectional study was to assess associations of air quality parameters with clinical signs, lung consolidation, pulmonary inflammation and infection in group-housed calves. In total, 60 beef and dairy farms were visited from January to April 2017 and 428 calves sampled. Measured air quality parameters included continuous 24-h measurements of ammonia concentration, relative humidity and temperature and punctual measurements of air velocity, ammonia, CO2 and bacterial air load. Calf sampling consisted of clinical examination, thoracic ultrasonography and broncho-alveolar lavage sampling for bacteriological and cytological analysis of broncho-alveolar lavage fluid (BALf). Average air temperature was 14.2 °C (standard deviation (SD) 4.4, range 5.5-23.9) and relative humidity 68.8 % (SD 8.9, range 52.2-91.6). Average ammonia concentration was 1.7 ppm (SD 0.9, range 0-10.0). Lung consolidations of ≥1 cm, ≥3 cm and ≥6 cm in depth were present in 41.1 % (176/428), 27.1 % (116/428) and 16.1 % (69/428) of the calves, respectively. Average pen temperature was positively associated with consolidations of ≥1 cm (P = 0.005), ≥3 cm (P = 0.002) and ≥6 cm (P < 0.01). Ammonia exposure, in hours>4 ppm, was associated with lung consolidation ≥1 cm (odds ratio (OR) = 1.73; confidence interval (CI) = 1.02-3.07; P = 0.04). Ammonia concentration was positively associated with BALf epithelial cell percentage (P = 0.01). Air velocity >0.8 m/s was associated with increased odds of lung consolidation of ≥3 cm (OR = 6.8; CI = 1.2-38.5; P = 0.04) and ≥6 cm (OR = 15.9; CI = 1.2-200.0; P = 0.03). The prevalence of lung consolidations ≥1 cm was higher in the draught (81.8 %; P = 0.0092) and warm, dry and ammonia accumulation clusters (54.2 %; P = 0.02) compared to the presumably normal cluster (31.6 %). In addition, in the warm, dry and ammonia cluster the prevalence of lung consolidations ≥3 cm (38.1 %; P = 0.04) and ≥6 cm (31.4 %; P = 0.01) in depth were higher compared to the presumably normal climate cluster (18.2 % and 9.1 %, respectively). Of all frequently measured indoor air quality parameters, only average temperature, ammonia concentration and air velocity were associated with pneumonia and might therefore be preferable for cost-effective evaluation of calf barn climate.

Effects of omega-3 fatty acids on immune, health and growth variables in veal calves.

Under the present intensive rearing conditions, calves face a series of stressors and multiple pathogens often necessitating antimicrobial use. Multiple feed additives are currently explored for their ability to prevent disease and limit the use of antimicrobials. Supplementation of the polyunsaturated long chain n-3 fatty acids eicosapentaenoic (EPA) and docohexaenoic (DHA) from marine origin has been proposed as a strategy to improve immune function and prevent excessive inflammation reactions. The aim of this randomized clinical trial was to explore the effects of n-3 fatty acids (PUFAs) used as feed supplement on health, production and immune variables in a veal calf setting. One hundred-seventy calves were randomly assigned to 3 treatment groups: microalgae (MA, n = 57, 2.5 g DHA/animal/day), fish oil (FO, n = 57, 2.5 g EPA + DHA/animal/day)] and a control group (CON, n = 56). Average daily gain (ADG), bodyweight at 12 weeks on feed and slaughter weight were determined. Health monitoring consisted of recording of clinical signs and repeated thoracic ultrasonography. After 5, 8 and 11 weeks of supplementation, the function of neutrophils, monocytes and peripheral blood mononuclear cells (PBMCs) was evaluated ex vivo by measuring reactive oxygen species (ROS) production by neutrophils and monocytes and proliferation of and cytokine release by PBMCs. Under the field conditions of this study, dietary supplementation of MA and FO showed very limited immunomodulatory effects. Feeding MA led to increased ROS production by neutrophils, Estimate (E) = 0.38, Standard Error (SE) = 0.14; P < 0.05, compared to the control calves after 5 weeks of in-feed supplementation. FO reduced IL-6 secretion E= -0.29, SE= 0.11; P < 0.05 compared to MA treated animals after 11 weeks on feed. Health and production variables were unaffected by treatments. The doses of EPA and DHA used in this study did not cause immunomodulatory changes in highly stressed calves to such an extent that this led to better health or growth of animals.

Rapid Identification of Mycoplasma bovis Strains from Bovine Bronchoalveolar Lavage Fluid with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry after Enrichment Procedure.

Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.

The presence of Mycoplasma bovis in colostrum.

In herds with Mycoplasma bovis circulation, colostrum is often considered infectious. However, in contrast to milk, the presence of M. bovis in colostrum was not previously evidenced. In this survey, the presence of M. bovis DNA was determined with real-time PCR in 368 colostrum samples from 17 herds, recently infected with M. bovis. Only 1.9% of the samples tested positive, with 13 herds having no positive samples and an overall within-herd prevalence of 3.2% (SD: 4.9%; Range: 0-30.0%). These results show that in infected herds M. bovis DNA can be retrieved in colostrum. To what extend colostrum is infectious remains to be determined.

Effects of glycerol-esters of saturated short- and medium chain fatty acids on immune, health and growth variables in veal calves.

In veal and dairy beef production systems, Holstein bull calves experience many stressors and excessive pathogen exposure, necessitating the use of antimicrobials for welfare and production reasons. The aim of this randomized clinical trial was to explore the effects of esterified fatty acids used as feed supplement on health, production and immune variables in veal calves. Different glycerol-esters of fatty acids were used: short chain fatty acid (SCFA)-based glycerol-mono- (C4) and tributyrate (C4), and medium chain fatty acid (MCFA)-based glycerol-monocaprylate/monocaprinate (C8/C10) and glycerol-monolaurate (C12) in two different doses. One hundred sixty eight calves (2-to 4-week-old) were randomly assigned to 6 treatment groups; tributyrate (0.5 g/animal/day); monobutyrate (1 g/animal/day); low C8/C10 (7 g/animal/day) and high C8/C10 (10 g/animal/day); low C12 (4 g/animal/day) and high C12 (6 g/animal/day) and a control group (CON). Duration of in-feed supplementation was 14 weeks. Average daily gain, bodyweight at 14 weeks on feed and slaughter weight were determined. Health monitoring consisted of clinical signs and repeated thoracic ultrasonography. After 4, 8 and 12 weeks of supplementation, the function of neutrophils, monocytes and peripheral blood mononuclear cells (PBMCs) was evaluated ex vivo by measuring reactive oxygen species (ROS) production by neutrophils and monocytes, proliferation of and cytokine release by PBMCs. Study power was based upon ROS production by neutrophils and treatment groups were too limited to detect significant differences in growth and health variables. Glycerol-ester supplementation resulted in different effects on immune cell function, depending on the type and dose of the glycerol-ester as well as duration of supplementation. Our main findings were increased secretion of interleukin IL-17A by PBMCs at 4 weeks of feed supplementation in high C8/C10 (P< 0.01), low C12 (P < 0.01) and monobutyrate (P< 0.01) groups, combined with decreased ROS production in neutrophils (P < 0.001) and monocytes (P < 0.05) in the high C8/C10 and monocytes (P < 0.05) in low C12 groups compared to the control animals. After 12 weeks on feed, ROS production by neutrophils (P < 0.001) and monocytes (P < 0.01) of monobutyrate and by monocytes (P < 0.01) of tributyrate groups was decreased compared to control calves. In summary, supplementation of glycerol-esters of MCFAs resulted in immune-modulatory effects, which did not manifest themselves in improved health and growth of calves under the conditions and limitations of this study. Especially doses of high C8/C10 and low C12 show potential to promote an early, robust pro-inflammatory response with diminished ROS production. This might be beneficial for clearance of pathogens in young calves in periods of stress and high pathogen load.

Non-specific, agar medium-related peaks can result in false positive Mycoplasma alkalescens and Mycoplasma arginini identification by MALDI-TOF MS.

MALDI-TOF MS is a fast and accurate tool to identify Mycoplasma species in liquid media. However, when trying to identify presumptive Mycoplasma bovis (M. bovis) colonies from solid medium (the "direct transfer method") a surprisingly high occurrence of M. arginini and M. alkalescens identification was observed. It was hypothesized that agar medium components are associated with false positive identification with Mycoplasma spp., as M. bovis colonies are very small and grow into the agar. The objective of this study was to determine whether complete modified pleuropneumonia-like organism (PPLO) agar (supplemented with horse serum, sodium pyruvate, technical yeast extract, ampicillin sodium salt and colistin) and the separate components, result in false identification as Mycoplasma spp. by MALDI-TOF MS. A total of 100 samples were examined, of which 33% of the modified PPLO agar spots were identified as M. alkalescens (16%) and M. arginini (17%)), albeit with relatively low score values (< 1.85). No false identification of M. bovis was obtained. Several medium components (unsupplemented PPLO agar, horse serum and colistin) resulted in spectra with peaks showing close matches with peaks present in the M. alkalescens and M. arginini database spectra. This study shows that the direct transfer method should be interpreted with caution, and one should strive to pick as little as possible agar when sampling Mycoplasma-like colonies from solid medium containing PPLO agar, horse serum and/or colistin.

Isolation of Drug-Resistant Gallibacterium anatis from Calves with Unresponsive Bronchopneumonia, Belgium.

Gallibacterium anatis is an opportunistic pathogen, previously associated with deaths in poultry, domestic birds, and occasionally humans. We obtained G. anatis isolates from bronchoalveolar lavage samples of 10 calves with bronchopneumonia unresponsive to antimicrobial therapy. Collected isolates were multidrug-resistant to extensively drug-resistant, exhibiting resistance against 5-7 classes of antimicrobial drugs. Whole-genome sequencing revealed 24 different antimicrobial-resistance determinants, including genes not previously described in the Gallibacterium genus or even the Pasteurellaceae family, such as aadA23, blaCARB-8, tet(Y), and qnrD1. Some resistance genes were closely linked in resistance gene cassettes with either transposases in close proximity or situated on putative mobile elements or predicted plasmids. Single-nucleotide polymorphism genotyping revealed large genetic variation between the G. anatis isolates, including isolates retrieved from the same farm. G. anatis might play a hitherto unrecognized role as a respiratory pathogen and resistance gene reservoir in cattle and has unknown zoonotic potential.

Comparison of bronchoalveolar lavage fluid bacteriology and cytology in calves classified based on combined clinical scoring and lung ultrasonography.

Respiratory tract infections are the leading cause of antimicrobial use in calves. Combining clinical examination and lung ultrasonography allows on-farm classification of calves as healthy or suffering from an upper respiratory tract infection (URTI), subclinical or clinical pneumonia. This might help to improve targeted antimicrobial therapy, restricting treatment to pneumonic cases. However, to what extent these diagnostic categories coincide with expected bacteriological and cytological bronchoalveolar lavage fluid (BALf) characteristics is currently unknown. The objective of this study was therefore to compare BALf bacteriology and cytology between healthy calves and calves with URTI, subclinical and clinical pneumonia. The hypothesis was that calves with subclinical and clinical pneumonia would have higher quantitative bacterial counts, bacterial isolation rates and neutrophil counts than URTIs or healthy animals. A cross-sectional study was performed on 305 indoor group-housed dairy and beef calves, from 62 farms. Calves were classified by combining clinical examination and lung ultrasonography. Clinical respiratory disease was defined using the Wisconsin score card and the Healthy Criterion (HC). The HC classified calves as clinically ill if at least one clinical sign was present. Ultrasonographic lung consolidation with a depth of ≥1 cm was considered indicative for pneumonia. Cytology and bacteriology were performed on BALf sampled by non-endoscopic bronchoalveolar lavage. Calves with clinical pneumonia were further subdivided based on culture result and presence of neutrophils phagocytosing bacteria. Combined lung ultrasonography and clinical examination (HC) classified 25.9 % (79/305) of the calves as healthy, 33.1 % (101/305) as URTI, 10.2 % (31/305) as subclinical and 30.8 % (94/305) as clinical pneumonia. Bacterial isolation rates and quantitative BALf culture results did not differ between groups. Calves with clinical pneumonia and neutrophil phagocytosis showed a significantly higher BALf neutrophil percentage compared to healthy calves (59.0 % vs. 37.7 % in healthy calves, P =.03). Inversely, lymphocyte percentage was lower in these calves (1.8 % vs. 5.3 % in healthy calves, P = .003). Classification of calves using lung ultrasonography and clinical scoring did not correspond with BALf bacteriology and cytology findings, as extrapolated from human and companion animal medicine. Under the current housing conditions of this study high rates of non-infectious airway inflammation or airway colonization by opportunistic pathogens, rather than infection might explain this. Isolation of respiratory pathogens from calves with various signs of respiratory disease or ultrasonographic lesions should be interpreted carefully. Of all cytological features, phagocytosis by neutrophils in BALf might be a useful criterion supporting the diagnosis of bacterial respiratory tract infection.

Accuracy of transcranial magnetic stimulation and a Bayesian latent class model for diagnosis of spinal cord dysfunction in horses.

BACKGROUND: Spinal cord dysfunction/compression and ataxia are common in horses. Presumptive diagnosis is most commonly based on neurological examination and cervical radiography, but the interest into the diagnostic value of transcranial magnetic stimulation (TMS) with recording of magnetic motor evoked potentials has increased. The problem for the evaluation of diagnostic tests for spinal cord dysfunction is the absence of a gold standard in the living animal. OBJECTIVES: To compare diagnostic accuracy of TMS, cervical radiography, and neurological examination. ANIMALS: One hundred seventy-four horses admitted at the clinic for neurological examination. METHODS: Retrospective comparison of neurological examination, cervical radiography, and different TMS criteria, using Bayesian latent class modeling to account for the absence of a gold standard. RESULTS: The Bayesian estimate of the prevalence (95% CI) of spinal cord dysfunction was 58.1 (48.3%-68.3%). Sensitivity and specificity of neurological examination were 97.6 (91.4%-99.9%) and 74.7 (61.0%-96.3%), for radiography they were 43.0 (32.3%-54.6%) and 77.3 (67.1%-86.1%), respectively. Transcranial magnetic stimulation reached a sensitivity and specificity of 87.5 (68.2%-99.2%) and 97.4 (90.4%-99.9%). For TMS, the highest accuracy was obtained using the minimum latency time for the pelvic limbs (Youden's index = 0.85). In all evaluated models, cervical radiography performed poorest. CLINICAL RELEVANCE: Transcranial magnetic stimulation-magnetic motor evoked potential (TMS-MMEP) was the best test to diagnose spinal cord disease, the neurological examination was the second best, but the accuracy of cervical radiography was low. Selecting animals based on neurological examination (highest sensitivity) and confirming disease by TMS-MMEP (highest specificity) would currently be the optimal diagnostic strategy.

Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS.

Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing ≥2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.

Determination of magnetic motor evoked potential latency time cutoff values for detection of spinal cord dysfunction in horses.

BACKGROUND: Transcranial magnetic stimulation (TMS) and recording of magnetic motor evoked potentials (MMEP) can detect neurological dysfunction in horses but cutoff values based on confirmed spinal cord dysfunction are lacking. OBJECTIVES: To determine latency time cutoff for neurological dysfunction. ANIMALS: Five control horses and 17 horses with proprioceptive ataxia. METHODS: Case-control study with receiver operating characteristic curve analysis, based on diagnostic imaging, TMS, and histopathological findings. Horses were included if all 3 examinations were performed. RESULTS: Diagnostic imaging and histopathology did not show abnormalities in the control group but confirmed spinal cord compression in 14 of 17 ataxic horses. In the remaining 3 horses, histopathological lesions were mild to severe, but diagnostic imaging did not confirm spinal cord compression. In control horses, latency time values of thoracic and pelvic limbs were significantly lower than in ataxic horses (20 ± 1 vs 34 ± 16 milliseconds, P = .05; and 39 ± 1 vs 78 ± 26 milliseconds, P = .004). Optimal cutoff values to detect spinal cord dysfunction were 22 milliseconds (sensitivity [95% CI interval], 88% [73%-100%]; specificity, 100% [100%-100%]) in thoracic and 40 milliseconds (sensitivity, 94% [83%-100%]; specificity, 100% [100%-100%]) in pelvic limbs. To detect spinal cord dysfunction caused by compression, the optimal cutoff for thoracic limbs remained 22 milliseconds, while it increased to 43 milliseconds in pelvic limbs (sensitivity, 100% [100%-100%]; specificity, 100% [100%-100%] for thoracic and pelvic limbs). CONCLUSIONS AND CLINICAL IMPORTANCE: Magnetic motor evoked potential analysis using these cutoff values is a promising diagnostic tool for spinal cord dysfunction diagnosis in horses.

Accuracy and inter-rater reliability of lung auscultation by bovine practitioners when compared with ultrasonographic findings.

In practice, veterinary surgeons frequently rely on lung auscultation as a confirmation test for pneumonia. To what extent diagnostic accuracy of lung auscultation varies between different practitioners is currently unknown. In this diagnostic test study, 49 Dutch veterinarians each auscultated between 8 and 10 calves, and communicated whether they would decide to treat the animal with antimicrobials or not. They were not allowed to perform any other aspect of the clinical examination. Their decisions were compared with lung ultrasonography findings. The average sensitivity and specificity of lung auscultation were 0.63 (sd=0.2; range=0.2-1.0) and 0.46 (sd=0.3; range=0.0-1.0), respectively. Of the participants, 8.2 per cent were 100 per cent sensitive, 16.3 per cent were 100 per cent specific, and only 4.0 per cent were perfect. The Krippendorff's alpha was 0.18 (95 per cent confidence interval: -0.01 to 0.38), signifying poor reliability between multiple raters. Regardless of the poor diagnostic accuracy in this study, especially the large variation in a confirmation test between different practitioners could potentially cause professional damage as well as misuse of antimicrobials. This study could be seen as a gentle stimulus to regularly evaluate one's diagnostic skills. Both complementary training and the use of more accurate techniques with less inter-rater variation could improve the situation.

Optimizing identification of Mycoplasma bovis by MALDI-TOF MS.

Fast and accurate identification of Mycoplasma bovis in cattle samples is of great importance for rational treatment and control of pneumonia, arthritis and mastitis. However, which growth conditions will allow the fastest identification of M. bovis with MALDI-TOF MS remains unclear. Therefore, growth conditions and incubation time were investigated to optimize identification of M. bovis with MALDI-TOF MS and an in-house library was constructed. Nine different M. bovis strains were inoculated in triplicate in three liquid media (B1-3). Basic broth (B1) consisted of pleuropneumonia-like organism broth, enriched with 25% horse serum and 0.7% yeast extract. B2 and B3 were additionally supplemented with 0.5% pyruvate or 520 µg/mL ampicillin, respectively. Protein extraction was performed after 24, 48, 72, 96 and 120 h of incubation (37 °C, 5% CO2) and processed with Autoflex III smartbeam. Identification scores ≥1.7 were interpreted as reliable. The present study showed reliable identification of M. bovis with MALDI-TOF MS as early as 24 h after inoculation, and in broth supplemented with pyruvate, up to 120 h after inoculation. Serial dilutions showed improved survival of M. bovis in broth with pyruvate. The addition of ampicillin to prevent contamination, did not impair identification of M. bovis and state-of-the-art in-house libraries contributed to higher identification scores for M. bovis with MALDI-TOF MS.

Factors associated with lung cytology as obtained by non-endoscopic broncho-alveolar lavage in group-housed calves.

BACKGROUND: Respiratory infections are the main indication for antimicrobial use in calves. As in humans and horses, studying inflammation of the deep airways by lung cytology raises the possibility of preventing respiratory disease and targeting its treatment in the future. Whether lung cytology findings coincide with clinical signs and lung ultrasonographic findings is currently unknown. Therefore, the objective of the present study was to determine the association of lung cytology with clinical signs, lung consolidation and broncho-alveolar lavage fluid (BALf) characteristics (including bacteriology). A total of 352 indoor group-housed calves aged between 1 and 6 months from 62 conveniently selected commercial herds were included in this cross-sectional study. Clinical examination, thoracic ultrasound and bacteriology and cytology on non-endoscopic broncho-alveolar lavage (nBAL) samples were performed. RESULTS: Pneumonia, defined as presence of ultrasonographic lung consolidations ≥1 cm in depth, affected 42.4% of the calves. Mean BALf neutrophil percentage was 36.6% (SD 23.8; R 0-97.4) and only a positive induced tracheal cough reflex (P = 0.04), standing posture (P = 0.03) increased breathing rate (P = 0.02) and isolation of Pasteurella multocida (P = 0.005), were associated with increased neutrophil percentage. No significant associations between lung ultrasonographic findings and cytology results were present, except for presence of basophils in BALf and consolidation of > 3 cm in depth (OR = 2.6; CI = 1.2-5.6; P = 0.01). Abnormal lung sounds were associated with detection of eosinophils in BALf (OR = 2.8; CI = 1.0-8.1; P = 0.05). Total nucleated cell count (TNCC) (P <  0.001) was positively and macrophage percentage (P = 0.02) negatively associated with volume of lavage fluid recovered. Macroscopic blood staining of BALf increased TNCC (P = 0.002) and lymphocyte percentage (P = 0.001). CONCLUSIONS: Only a limited number of clinical signs and ultrasonographic findings were associated with nBAL cytology. BALf cytology offers additional and distinct information in calves aiding in detection and prevention of respiratory conditions. In this population, selected from herds not reporting any recent respiratory illness, a high number of calves had ultrasonographic lung consolidation and high neutrophil percentage in BALf, suggesting that subclinical disease presentations frequently occur.

Randomized field trial on the effects of body weight and short transport on stress and immune variables in 2- to 4-week-old dairy calves.

BACKGROUND: Whether underweight calves respond differently to transport stress, enhancing their disease risk, is currently unknown. OBJECTIVE: To determine the effects of low body weight and transport stress on immune variables. ANIMALS: Twenty-one 2- to 4-week-old male Holstein calves, housed on a commercial farm. METHODS: Randomized clinical trial. Full factorial design with 4 treatment groups: low body weight (≤46 kg)/no transport (LOWCON); low body weight/transport (LOWTRANS); normal body weight (>46 kg)/no transport (NORMCON), and normal body weight/transport (NORMTRANS). Transport duration was 2 hours. RESULTS: Transport significantly increased serum cortisol concentration (77.8 µg/mL; 95% confidence interval [CI], 37.8-131.6; P < .001), interleukin (IL)-17A (344.9 pg/mL; 95% CI, 32.2-556.5; P = .04), and tumor necrosis factor-α (TNF-α) (218.2 pg/mL; 95% CI, 32.5-368.3; P = .03) production after lipopolysaccharide (LPS) stimulation. Body weight did not affect any of the studied variables. However, the interaction of transport and body weight was significant. LOWTRANS calves showed increased monocyte count (2.0 × 109 /L; 95% CI, 0.6-4.2; P < .05) and interleukin IL-17A production (106.0 pg/mL; 95% CI, 4.2-306.9; P = .03) compared to normal weight calves and increased TNF-α production (275.6 pg/mL; 95% CI, 2.6-463.0; P = .02) compared to LOWCON calves in unstimulated peripheral blood mononuclear cells (PBMCs) after transport. CONCLUSION AND CLINICAL IMPORTANCE: These findings contribute to our understanding of increased disease susceptibility of underweight calves when transported. Gamma globulin concentration was identified as important interfering factor in studies on immune variables in neonatal calves.

The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics.

A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.

Magnetic motor evoked potentials of cervical muscles in horses.

BACKGROUND: When surgical treatment of cervical vertebral malformation is considered, precise localization of compression sites is essential, but remains challenging. Magnetic motor evoked potentials (mMEP) from paravertebral muscles are useful in localizing spinal cord lesions, but no information about cervical muscle mMEP in horses is available yet. Therefore, the aim of this study was to determine the possibility, normal values, inter- and intra-observer agreement and factors that have an effect on cervical mMEP in healthy horses. METHODS: Transcranial magnetic stimulation was performed on 50 normal horses and 4 (2 left, 2 right) muscle responses were recorded at the middle of each cervical vertebra (C1-C7) and additionally just caudal to C7 to evaluate cervical nerves (Cn) Cn1 to Cn8. Latency time and amplitude of the recorded mMEP were defined by both an experienced and an unexperienced operator. RESULTS: Latency increased gradually from 14.2 ± 1.38 ms for Cn3 to 17.7 ± 1.36 ms for Cn8, was significantly influenced by cervical nerve (P < 0.01), gender (P = 0.02) and height (P = 0.03) and had a good intra-observer agreement. The smallest mean amplitude (4.35 ± 2.37 mV) was found at Cn2, the largest (5.99 ± 2.53 mV) at Cn3. Amplitude was only significantly influenced by cervical nerve (P < 0.01) and had a low intra-observer agreement. No significant effect of observer on latency (P = 0.88) or amplitude (P = 0.99) measurements was found. CONCLUSION: mMEP of cervical muscles in normal horses are easy to collect and to evaluate with limited intra- and inter-observer variation concerning amplitude and should be investigated in future studies in ataxic horses to evaluate its clinical value.

Rapid detection of tetracycline resistance in bovine Pasteurella multocida isolates by MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA).

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8-101.5) and 100% (95% CI = 100-100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 93.5% (95% CI = 86.3-100.6) and 96.3% (95% CI = 91.3-101.3) respectively, classifying 95% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

Use of a national identification database to determine the lifetime prognosis in cattle with necrotic laryngitis and the predictive value of venous pCO2.

BACKGROUND: Necrotic laryngitis, caused by Fusobacterium necrophorum, frequently requires surgical intervention (laryngostomy) in the chronic stage. HYPOTHESIS/OBJECTIVES: To determine survival until slaughter of cattle surgically treated for necrotic laryngitis and to identify predictors of mortality. ANIMALS: A total of 221 cattle diagnosed with necrotic laryngitis by laryngoscopy and surgically treated METHODS: Retrospective cohort study. Clinical records were matched with the national cattle identification, registration, and movement database. Information on possible predictors including clinical examination, biochemistry, and surgery was collected. A multivariable Cox proportional hazard model was used to identify predictors of mortality. RESULTS: The overall survival rate was 65.2% and 58.6% of the animals with a completed life cycle could be slaughtered. Animals <6 months old experienced significantly higher mortality risk (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.1-3.5). The venous partial pressure of carbon dioxide (pCO2 ; HR, 2.4; 95% CI, 1.4-4.2) at a 64.5 mm Hg cut-off was most significantly associated with mortality. Sensitivity and specificity of the final model consisting of age and pCO2 were 49.1 and 86.4%, respectively. Instead of pCO2 , total carbon dioxide (TCO2 ) could also be used, with similar diagnostic accuracy. CONCLUSIONS AND CLINICAL RELEVANCE: The lifetime prognosis for chronic necrotic laryngitis in cattle with surgical intervention appears fair. Age, venous pCO2 and TCO2 are easily accessible predictors of survival to support owners and veterinarians in their decision process of whether or not to operate and to identify high risk animals that require more intensive follow-up.