Identifying and addressing barriers and opportunities for bovine respiratory disease complex vaccination: a consensus paper on practical recommendations for best practise vaccination.

Many questions are raised, and challenges faced in the new era of (intranasal) bovine respiratory disease complex vaccination. An increase in vaccination rate is expected, due to its positive impact on cattle health, reduction of antimicrobial use and economic factors. However, engagement of farmers and veterinarians with regard to vaccination is often affected by limitations, resulting in the development of barriers to vaccination, but also opportunities to overcome these. The objective of the report is to provide practical recommendations and a consensus on best practises for BRDC vaccination, addressing barriers faced by veterinarians and farmers. The report combines an evidence review with expert opinions and includes discussions on different vaccination approaches, such as intranasal and systemic protocols. As result of the discussions, several barriers to BRDC vaccination were identified, including a lack of knowledge or visibility of the disease's impact, the preference for blanket antibiotic use over vaccination, resistance to change, the need for visible success, uncertainty about the best time to vaccinate, and concerns about adverse reactions and vaccine efficacy in the presence of maternal antibodies. While these barriers seem substantial, they provide opportunities for the veterinary sector. Indeed, veterinarians are encouraged to use the argumentation presented, along with local case studies and diagnostic testing to highlight the impact of disease, while conducting calf health audits, ensuring expectations are managed to achieve visible success. Overall, this consensus paper aims to provide practical recommendations and support for veterinarians and farmers to overcome barriers and increase BRDC vaccination rates in cattle.

Bayesian evaluation of sensitivity and specificity of blood culture media and hypoglycemia in sepsis-suspected calves.

BACKGROUND: Sepsis is a life-threatening condition for which critically important antimicrobials are often indicated. The value of blood culture for sepsis is indisputable, but appropriate guidelines on sampling and interpretation are currently lacking in cattle. OBJECTIVE: Compare the diagnostic accuracy of 2 blood culture media (pediatric plus [PP] and plus aerobic [PA]) and hypoglycemia for bacteremia detection. Estimate the contamination risk of blood cultures in critically ill calves. ANIMALS: One hundred twenty-six critically ill calves, 0 to 114 days. METHODS: Retrospective cross-sectional study in which the performance of PP, PA and hypoglycemia to diagnose sepsis was assessed using a Bayesian latent class model. A Cox proportional hazards model was used to compare time to positivity (TTP). Potential contamination was descriptively analyzed. Isolates were considered relevant when they were; member of the Enterobacterales, isolated from both blood cultures vials, or well-known, significant bovine pathogens. RESULTS: The sensitivities for PP, PA, and hypoglycemia were higher when excluding assumed contaminants; 68.7% (95% credibility interval = 30.5%-93.7%), 87.5% (47.0%-99.5%), and 61.3% (49.7%-72.4%), respectively. Specificity was estimated at 95.1% (82.2%-99.7%), 94.2% (80.7%-99.7%), and 72.4% (64.6%-79.6%), respectively. Out of 121 interpretable samples, 14.9% grew a presumed contaminant in PA, PP, or both. There was no significant difference in the TTP between PA and PP. CONCLUSIONS AND CLINICAL IMPORTANCE: PA and PP appear to outperform hypoglycemia as diagnostic tests for sepsis. PA seems most sensitive, but a larger sample size is required to verify this. Accuracy increased greatly after excluding assumed contaminants. The type of culture did not influence TTP or the contamination rate.

Diagnostic performance of Mycoplasmopsis bovis antibody ELISA tests on bulk tank milk from dairy herds.

BACKGROUND: Testing of bulk tank milk (BTM) for Mycoplasmopsis bovis (previously Mycoplasma bovis) antibodies is increasingly popular. However the performance of some commercially available tests is unknown, and cutoff values possibly need to be adjusted in light of the purpose. Therefore, the aim of this study was to compare the diagnostic performance of three commercially available M. bovis antibody ELISAs on BTM, and to explore optimal cutoff values for screening purposes. A prospective diagnostic test accuracy study was performed on 156 BTM samples from Belgian and Swiss dairy farms using Bayesian Latent Class Analysis. Samples were initially classified using manufacturer cutoff values, followed by generated values. RESULTS: Following the manufacturer's guidelines, sensitivity of 91.4%, 25.6%, 69.2%, and specificity of 67.2%, 96.8%, 85.8% were observed for ID-screen, Bio K432, and Bio K302, respectively. Optimization of cutoffs resulted in a sensitivity of 89.0%, 82.0%, and 85.5%, and a specificity of 83.4%, 75.1%, 77.2%, respectively. CONCLUSIONS: The ID-screen showed the highest diagnostic performance after optimization of cutoff values, and could be useful for screening. Both Bio-X tests may be of value for diagnostic or confirmation purposes due to their high specificity.

Accuracy and inter-rater agreement among practitioners using quick thoracic ultrasonography to diagnose calf pneumonia.

BACKGROUND: Thoracic ultrasonography (TUS) is a commonly used tool for on-farm detection of pneumonia in calves. Different scanning methods have been described, but the performance of novice practitioners after training has not been documented. METHODS: In this study, 38 practitioners performed quick TUS (qTUS) on 18-23 calves each. Pneumonia was defined as lung consolidation 1 cm or more in depth. Diagnostic parameters (accuracy [Acc], sensitivity [Se] and specificity [Sp]) were compared to those of an experienced operator. Cohen's kappa and Krippendorff's alpha (Kalpha) were determined. The potential effects of training and exam sessions on performance were evaluated. RESULTS: The average relative Se and Sp were 0.66 (standard deviation [SD] = 0.26; minimum [Min.]-Maximum [Max.] = 0-1) and 0.71 (SD = 0.19; Min.-Max. = 0.25-1), respectively. The average relative Acc was 0.73 (SD = 0.11; Min.-Max. = 0.52-0.96). Over all sessions, Cohen's kappa averaged 0.40 (SD = 0.24; Min.-Max. = 0.014-0.90) and Kalpha was 0.24 (95% confidence interval [CI]: 0.20-0.27), indicating 'fair' agreement. Calf age and housing influenced Se and Sp. Supervised practical training improved Se by 17.5% (95% CI: 0.01-0.34). LIMITATIONS: The separate effects of calf age and housing could not be determined. CONCLUSION: This study showed that qTUS, like any other clinical skill, has a learning curve, and variability in performance can be substantial. Adequate training and certification of one's skill are recommended to assure good diagnostic accuracy.

Abomasitis associated with halofuginone intoxication in pre-weaned calves.

BACKGROUND: In this case series abomasitis as a consequence of halofuginone intoxication is suspected. CASE PRESENTATION: Seven Belgian-Blue calves with complaints of anorexia and weight loss were presented to an university clinic. Ultrasonography showed thickening and edema of the abomasal wall in all cases, suggesting abomasitis. Abomasitis was confirmed on necropsy in three cases. Retrospective analysis clarified the uptake of an overdose of halofuginone lactate (348-421 µg/kg/day). Four animals fully recovered after removal of halofuginone lactate administration, therapy for comorbidities (pneumonia, diarrhoea) and supportive therapy. CONCLUSION: To the authors' knowledge, this case series is the first report associating halofuginone lactate use with abomasitis. This was suspected after clinical improvement of four of the presented animals after terminating the administration of a high dose of halofuginone lactate, and exclusion of other possible causes. Underlying mechanisms are still unclear.

Associations of serostatus upon arrival with clinical respiratory disease, lung consolidation, and growth in veal calves.

Respiratory tract infections remain a major problem during calf rearing, especially among milk (formula)-fed veal. Preconditioning of calves through appropriate colostrum management and vaccination could be helpful to address this issue. The objective of this study was to investigate whether the presence of serum antibodies against major respiratory tract pathogens (bovine respiratory syncytial virus, parainfluenza 3 virus, bovine coronavirus, Mycoplasmopsis bovis, Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica) and total serum IgG concentration in calves upon arrival at the veal facility were associated with the occurrence of clinical bovine respiratory disease (BRD) or lung consolidation in the first 3 wk, as assessed by both the Wisconsin BRD scorecard (based on 5 clinical signs: cough, rectal temperature, ear position, and nasal and ocular discharge) and by quick thoracic ultrasound scanning. Additionally, the association between calves' serostatus production parameters were explored. A prospective cohort study was conducted among 442 male dairy calves on a large veal calf facility in Belgium. Both clinical scoring and quick thoracic ultrasound scanning were performed on all calves at 4 key moments in the production cycle: arrival at the facility, initiation of first metaphylactic antimicrobial treatment at peak incidence of BRD (wk 1), end of the first metaphylactic treatment (short-term evaluation) and at wk 10 (long-term evaluation). Mixed effects logit regression models were fitted to quantify relationships. The outcomes of interest were clinical respiratory disease (Wisconsin BRD scorecard positive), lung consolidation (≥1 cm or ≥ 3 cm), average daily weight gain, and cold carcass weight. In the first week of production, incidence of lung consolidation (≥1 cm) quickly increased from 14.9% upon arrival to 43.0% at the peak of the BRD incidence, while clinical BRD increased from 3.6% to 16.1%. The main finding of this study was that calves who were seropositive for bovine respiratory syncytial virus and bovine coronavirus at arrival had reduced odds of developing lung consolidation at the peak of the outbreak, 0.58 odds ratio (95% CI: 0.38-0.89) and 0.37 odds ratio (95% CI: 0.20-0.69), respectively. No relationships between serum IgG concentration at arrival and the development of lung consolidations or clinical respiratory disease were found. Nevertheless, on average, throughout the first 10 wk of the fattening cycle, calves with failed transfer of passive immunity (serum IgG < 7.5 g/L) gained 40 g/d (95% CI: 10-70 g/d) less weight (average daily gain). Hence, ensuring that calves have a positive serostatus for these respiratory tract pathogens before entering the facility may help lower the incidence of lung consolidations, subsequently reducing treatment incidence and the adverse effects on primary economic outcomes.

Differentiating airway inflammation in calves based on cluster analysis of bronchoalveolar lavage fluid cytology.

BACKGROUND: Nonbronchoscopic bronchoalveolar lavage (nBAL) is routinely performed in calves, and airway cytology has great potential in airway disease diagnostics. A good reference framework for nBAL cytology is lacking. OBJECTIVES: To distinguish different cytological profiles in nBAL from grouped housed calves using cluster analysis, and characterize these profiles on individual and herd levels. ANIMALS: Three hundred thirty-eight group-housed calves from 60 herds (mainly dairy and beef ). METHODS: Cross-sectional study. Differential counts of white blood cells were determined on nBAL fluid, followed by differentiation of cytological profiles by K-means-based cluster analysis. These profiles were characterized by reference values, decision tree analysis, and associations with clinical, ultrasonographic, bacteriological, and cytological features. RESULTS: A normal (55.9%), a neutrophilic (41.1%), and an eosinophilic profile (3.0%) were identified. The normal profile was characterized by reference values of 2.3% to 47.4% neutrophils, 35.1% to 95.1% macrophages, 0.4 to 22.9% lymphocytes, and 0.0% to 0.9% eosinophils. The neutrophilic profile was characterized by ≥44.5% neutrophils, <1.6% eosinophils, and <11.5% lymphocytes. This profile was associated with the isolation of Pasteurella multocida, the presence of neutrophils with toxic granulation, and the presence of phagocytosed bacteria in neutrophils. The eosinophilic profile was characterized by eosinophils ≥1.6% (neutrophilia present) or ≥2.4% (neutrophilia absent), and associated with the presence of mast cells. On herd level, the neutrophilic and eosinophilic profiles were present in 85.0% and 15.0% of the herds, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides a first step in the development of cytological guidelines, aiding the assessment of airway health and inflammation in calves through nBAL fluid cytology.

Sepsis and survival in critically ill calves: Risk factors and antimicrobial use.

BACKGROUND: Sepsis is a life-threatening disease for which critically important antimicrobials (CIA) frequently are used. Diagnostic and therapeutic guidelines for sepsis and critically ill calves are largely lacking. OBJECTIVES: Identify factors associated with mortality in critically ill calves and describe bacteria obtained from blood cultures of critically ill calves with sepsis and their antimicrobial resistance. ANIMALS: Two-hundred thirty critically ill calves, mainly Belgian Blue beef cattle. METHODS: Retrospective cohort study. Logistic regression, survival analysis, and decision tree analysis were used to determine factors associated with mortality. RESULTS: Of the critically ill calves, 34.3% had sepsis and 61.3% died. The final survival model indicated that calves with sepsis (hazard risk [HR]: 1.6; 95% confidence interval [CI]: 1.0-2.5; P = .05), abnormal behavior (HR: 2.3; 95% CI: 1.3-4.0; P = .005), and hypothermia (HR: 0.82; 95% CI: 0.72-0.95; P = .01) had a significantly higher mortality risk. In a second survival model, hypothermia (HR: 0.87; 95% CI: 0.78-0.96; P = .004) and hypoglycemia (HR: 2.2; 95% CI: 1.5-3.3; P < .001) were risk factors for mortality. Decision tree analysis emphasized the importance of behavior, hypochloremia, hypoglycemia, hyperkalemia, and lung ultrasonography for mortality risk. Escherichia coli (30.6%) was most frequently isolated from blood cultures, of which 90.9% were multidrug resistant. Inappropriate use of antimicrobials was frequent for penicillin, amoxicillin, and sulfamethoxazole/trimethoprim, but less for CIA. CONCLUSIONS AND CLINICAL IMPORTANCE: Many critically ill calves have sepsis, which increases mortality risk. Bacteria involved are often resistant to first-intention antimicrobials but less resistant to CIA. The other identified risk factors for mortality can support therapeutic decision-making.

Dynamics of subclinical pneumonia in male dairy calves in relation to antimicrobial therapy and production outcomes.

Quick thoracic ultrasonography (qTUS) is increasingly used as an on-farm method to diagnose clinical and subclinical pneumonia in dairy calves. The primary objective of this prospective cohort study was to describe dynamics of lung consolidation in a purchase-dependent production system for male dairy calves in relation to antimicrobial therapy and respiratory diagnostics. In addition, we studied the association of cured and uncured pneumonia with average daily gain (ADG) and cold carcass weight (CCW). The third objective was to determine the effects of arriving with lung consolidation on the probability of developing chronic unresponsive pneumonia and reduced performance. A total of 295 male dairy calves were intensively followed by qTUS and clinical scoring on 7 strategic occasions (wk 1, 2, 3, 4, 6, 8, and 12) during the production cycle. Of the calves, 17.6% (52/295) arrived with a lung consolidation ≥1 cm. At the first outbreak of respiratory disease (wk 1 after arrival), this incidence had risen to 30.8%. Initial therapy with tulathromycin and subsequently doxycycline appeared ineffective, resulting in a increase to 43.8% of calves having pneumonia in wk 4. At the start of the first outbreak (wk 1), the majority (86.8%) of the pneumonia cases were subclinical. At wk 4, the outbreak became more clinical, and treatment with amoxicillin resulted in a cure risk of 52.7%. Culture and nanopore sequencing diagnostics on nonendoscopic broncho-alveolar lavage (nBAL) samples identified bovine respiratory syncytial virus and Mycoplasma bovis as the dominant agents in the first outbreak. The isolated M. bovis strain showed mutations associated with macrolide resistance. The second outbreak was characterized by a Pasteurella multocida superinfection and isolation of multiple M. bovis strains from nBAL diagnostic testing. Evaluated over the complete observation period, 83.4% of the calves developed consolidations ≥1 cm on qTUS. Of these calves, 53.9% (135/246) were cured by antimicrobial therapy. Chronic pneumonia (≥30 subsequent days of pneumonia) was seen in 13.9% of the animals (n = 41). Calves with uncured or chronic pneumonia had a lower ADG (992 ± 174 g/d and 930 ± 146 g/d, respectively) compared with calves that never developed pneumonia (ADG = 1,103 ± 156 g/d). In contrast, calves that did fully cure trended toward a lower ADG than calves that never developed pneumonia, but differences were no longer significant. Also, the effect of uncured pneumonia was no longer significant for CCW. Calves with lung consolidation upon arrival had a lower ADG (981 ± 159 g/d vs. 1,045 ± 159 g/d) and were more likely to develop chronic pneumonia [odds ratio = 4.2; 95% confidence interval = 2.1-8.6] compared with calves without consolidation upon arrival. Animals with chronic pneumonia, in turn, had a lower CCW than animals without chronic pneumonia (10.3 ± 4.4 kg; 95% confidence interval: 1.6-19.1 kg). This study documents the consequences of subclinical pneumonia upon arrival and pneumonia developed later in the production cycle on production outcomes in a veal calf setting. Both qTUS and nBAL diagnostics provide important information, offering potential for better control and prevention of bovine respiratory disease in dairy calves.

The DECIDE project: from surveillance data to decision-support for farmers and veterinarians.

Farmers, veterinarians and other animal health managers in the livestock sector are currently missing sufficient information on prevalence and burden of contagious endemic animal diseases. They need adequate tools for risk assessment and prioritization of control measures for these diseases. The DECIDE project develops data-driven decision-support tools, which present (i) robust and early signals of disease emergence and options for diagnostic confirmation; and (ii) options for controlling the disease along with their implications in terms of disease spread, economic burden and animal welfare. DECIDE focuses on respiratory and gastro-intestinal syndromes in the three most important terrestrial livestock species (pigs, poultry, cattle) and on reduced growth and mortality in two of the most important aquaculture species (salmon and trout). For each of these, we (i) identify the stakeholder needs; (ii) determine the burden of disease and costs of control measures; (iii) develop data sharing frameworks based on federated data access and meta-information sharing; (iv) build multivariate and multi-level models for creating early warning systems; and (v) rank interventions based on multiple criteria. Together, all of this forms decision-support tools to be integrated in existing farm management systems wherever possible and to be evaluated in several pilot implementations in farms across Europe. The results of DECIDE lead to improved use of surveillance data and evidence-based decisions on disease control. Improved disease control is essential for a sustainable food chain in Europe with increased animal health and welfare and that protects human health.

Comparison and optimisation of screening cutoff values for Mycoplasma bovis antibody ELISAs using serum from youngstock.

BACKGROUND: Mycoplasma bovis-associated disease can cause tremendous production losses, welfare issues and high antimicrobial use. Therefore, screening cattle for M. bovis antibodies before entering the herd is a popular and possibly cost-efficient way to reduce disease introduction. However, interpretation of results can be challenging due to variable accuracy between tests and populations. This study's objectives were to compare the diagnostic test accuracy of three commercially available M. bovis antibody ELISAs (ID-screen, Bio K302 and Bio K432) and to explore optimal cutoff values for screening purposes. METHODS: A prospective diagnostic test accuracy study was performed on 170 serum samples from youngstock using Bayesian latent class modelling. Samples were categorised using manufacturer and generated cutoff values. RESULTS: Using the manufacturers' guidelines, ID-screen, Bio K432 and Bio K302 showed 97.6%, 67.4% and 33.6% sensitivity, and 78.8%, 97.6% and 99.1% specificity, respectively. Optimised cutoffs resulted in 94.8%, 82.6% and 78.3% sensitivity, and 94.2%, 92.5% and 79.4% specificity, respectively. CONCLUSIONS: The highest diagnostic accuracy for detecting M. bovis antibodies was obtained by ID-screen (≥110%). However, by adjusting cutoff values, the sensitivity of Bio-X tests could be markedly increased, making these tests also applicable as screening tools. LIMITATIONS: Interpretation needs to be careful as antibodies may be linked to both infectious and non-infectious status.

Randomized field trial comparing the efficacy of florfenicol and oxytetracycline in a natural outbreak of calf pneumonia using lung reaeration as a cure criterion.

BACKGROUND: Respiratory infections are the main indication for antimicrobial use in calves. Optimal treatment duration currently is unknown, but shorter duration would likely decrease selection for antimicrobial resistance. HYPOTHESIS/OBJECTIVES: Determine differences in cure rate and healing time between animals treated with florfenicol and oxytetracycline in a natural outbreak of respiratory disease using reaeration observed on thoracic ultrasound examination as healing criterion. ANIMALS: Commercial farm housing 130, 3 to 9 month old Belgian blue beef calves. METHODS: Randomized clinical trial during an outbreak of respiratory disease. Metaphylactic treatment was initiated, randomly treating animals with either florfenicol or oxytetracycline. Ultrasonographic follow-up was done the first day and every other day for a 14-day period. At the individual animal level, treatment was discontinued when reaeration of the lungs occurred. Differences in cure rate and healing time were determined. RESULTS: Of the 130 animals studied, 67.7% developed a lung consolidation ≥0.5 cm. The mean ultrasonographic healing time was 2.5 days in the florfenicol group compared to 3.1 days in the oxytetracycline group (P = .04). After single treatment, 80.6% and 60.3% had no consolidations in the florfenicol and oxytetracycline groups, respectively (P = .01). A Mycoplasma bovis strain was genetically and phenotypically determined to be susceptible to both antimicrobials. CONCLUSIONS AND CLINICAL IMPORTANCE: Ultrasonographic lung reaeration shows potential as a cure criterion to rationalize antimicrobial use for outbreaks of pneumonia. In our study, florfenicol resulted in a faster cure and higher reduction in antimicrobial usage than did oxytetracycline.

Genome-Wide Association Study Reveals Genetic Markers for Antimicrobial Resistance in Mycoplasma bovis.

Mycoplasma bovis causes many health and welfare problems in cattle. Due to the absence of clear insights regarding transmission dynamics and the lack of a registered vaccine in Europe, control of an outbreak depends mainly on antimicrobial therapy. Unfortunately, antimicrobial susceptibility testing (AST) is usually not performed, because it is time-consuming and no standard protocol or clinical breakpoints are available. Fast identification of genetic markers associated with acquired resistance may at least partly resolve former issues. Therefore, the aims of this study were to implement a first genome-wide association study (GWAS) approach to identify genetic markers linked to antimicrobial resistance (AMR) in M. bovis using rapid long-read sequencing and to evaluate different epidemiological cutoff (ECOFF) thresholds. High-quality genomes of 100 M. bovis isolates were generated by Nanopore sequencing, and isolates were categorized as wild-type or non-wild-type isolates based on MIC testing results. Subsequently, a k-mer-based GWAS analysis was performed to link genotypes with phenotypes based on different ECOFF thresholds. This resulted in potential genetic markers for macrolides (gamithromycin and tylosin) (23S rRNA gene and 50S ribosomal unit) and enrofloxacin (GyrA and ParC). Also, for tilmicosin and the tetracyclines, previously described mutations in both 23S rRNA alleles and in one or both 16S rRNA alleles were observed. In addition, two new 16S rRNA mutations were possibly associated with gentamicin resistance. In conclusion, this study shows the potential of quick high-quality Nanopore sequencing and GWAS analysis in the evaluation of phenotypic ECOFF thresholds and the rapid identification of M. bovis strains with acquired resistance. IMPORTANCE Mycoplasma bovis is a leading cause of pneumonia but also causes other clinical signs in cattle. Since no effective vaccine is available, current M. bovis outbreak treatment relies primarily on the use of antimicrobials. However, M. bovis is naturally resistant to different antimicrobials, and acquired resistance against macrolides and fluoroquinolones is frequently described. Therefore, AST is important to provide appropriate and rapid antimicrobial treatment in the framework of AMR and to prevent the disease from spreading and/or becoming chronic. Unfortunately, phenotypic AST is time-consuming and, due to the lack of clinical breakpoints, the interpretation of AST in M. bovis is limited to the use of ECOFF values. Therefore, the objective of this study was to identify known and potentially new genetic markers linked to AMR phenotypes of M. bovis isolates, exploiting the power of a GWAS approach. For this, we used high-quality and complete Nanopore-sequenced M. bovis genomes of 100 isolates.

Evaluation of Nanopore Sequencing as a Diagnostic Tool for the Rapid Identification of Mycoplasma bovis from Individual and Pooled Respiratory Tract Samples.

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, Nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study Nanopore sequencing was compared to rapid identification of M. bovis with matrix-assisted laser desorption ionization-time of flight mass spectrometry (RIMM) and a triplex real-time PCR assay in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) samples obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis. Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results for the pooled samples were compared with those for the individual samples to determine sensitivity and specificity. The BLCM showed good sensitivity (77.3% [95% credible interval, 57.8 to 92.8%]) and high specificity (97.4% [91.5 to 99.7%]) for Nanopore sequencing, compared to RIMM (sensitivity, 93.0% [76.8 to 99.5%]; specificity, 91.3% [82.5 to 97.0%]) and real-time PCR (sensitivity, 94.6% [89.7 to 97.7%]; specificity, 86.0% [76.1 to 93.6%]). Sensitivity and specificity of pooled analysis for M. bovis were 85.7% (95% confidence interval, 59.8 to 111.6%) and 90.0% (71.4 to 108.6%%), respectively, for Nanopore sequencing and 100% (100% to 100%) and 88.9% (68.4 to 109.4%) for RIMM. In conclusion, Nanopore sequencing is a rapid, reliable tool for the identification of M. bovis. To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for Nanopore sequencing and RIMM is possible.

Case Report: Multidrug Resistant Raoultella ornithinolytica in a Septicemic Calf.

Sepsis is a frequent life-threatening condition in young calves, requiring rapid broad spectrum and bactericidal therapy to maximize survival chances. Few studies have identified and characterized bacteria involved in sepsis in calves. This report demonstrates the involvement of a multidrug resistant Raoultella ornithinolytica, an emerging pathogen in human medicine, in a calf with suspected sepsis. R. ornithinolytica was identified by MALDI-TOF MS from blood cultures of a critically ill calf. Susceptibility testing showed phenotypic resistance against ampicillin, gentamicin, potentiated sulphonamides, streptomycin, tetracyclines and intermediate susceptibility for enrofloxacin. Whole genome sequencing confirmed identification as R. ornithinolytica and the multidrug resistant character of the isolate. Antimicrobial resistance genes acting against aminoglycosides, beta-lactam antibiotics, fosfomycin, quinolones, sulphonamides, trimethoprim and tetracyclines were found. The calf recovered after empirical parenteral therapy with enrofloxacin and sodium penicillin for seven days. Ancillary therapy consisted of fluid therapy, ketoprofen and doxapram hydrochloride. To the authors' knowledge, this is the first report characterizing a multidrug resistant R. ornithinolytica isolate from blood culture in cattle. It is currently unknown whether animals and farms may act as reservoirs for multidrug resistant R. ornithinolytica strains.

Antimicrobial Susceptibility of Mycoplasma bovis Isolates from Veal, Dairy and Beef Herds.

Mycoplasma bovis is an important pathogen causing mostly pneumonia in calves and mastitis in dairy cattle. In the absence of an effective vaccine, antimicrobial therapy remains the main control measure. Antimicrobial use in veal calves is substantially higher than in conventional herds, but whether veal calves also harbor more resistant M. bovis strains is currently unknown. Therefore, we compared antimicrobial susceptibility test results of M. bovis isolates from different cattle sectors and genomic clusters. The minimum inhibitory concentration of nine antimicrobials was determined for 141 Belgian M. bovis isolates (29 dairy, 69 beef, 12 mixed, 31 veal farms), and was used to estimate the epidemiological cut-off. Acquired resistance was frequently observed for the macrolides, while no acquired resistance to oxytetracycline and doxycycline, minimal acquired resistance to florfenicol and tiamulin, and a limited acquired resistance to enrofloxacin was seen. M. bovis isolates from beef cattle or genomic cluster III had higher odds of being gamithromycin-resistant than those from dairy cattle or genomic clusters IV and V. In this study, no cattle industry could be identified as source of resistant M. bovis strains. A single guideline for antimicrobial use for M. bovis infections, with a small remark for gamithromycin, is likely sufficient.

High quality genome assemblies of Mycoplasma bovis using a taxon-specific Bonito basecaller for MinION and Flongle long-read nanopore sequencing.

BACKGROUND: Implementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1-2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained. RESULTS: Here, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness). CONCLUSIONS: These results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.

Phylogenomic analysis of Mycoplasma bovis from Belgian veal, dairy and beef herds.

M. bovis is one of the leading causes of respiratory disease and antimicrobial use in cattle. The pathogen is widespread in different cattle industries worldwide, but highest prevalence is found in the veal industry. Knowledge on M. bovis strain distribution over the dairy, beef and veal industries is crucial for the design of effective control and prevention programs, but currently undocumented. Therefore, the present study evaluated the molecular epidemiology and genetic relatedness of M. bovis isolates obtained from Belgian beef, dairy and veal farms, and how these relate to M. bovis strains obtained worldwide. Full genomes of one hundred Belgian M. bovis isolates collected over a 5-year period (2014-2019), obtained from 27 dairy, 38 beef and 29 veal farms, were sequenced by long-read nanopore sequencing. Consensus sequences were used to generate a phylogenetic tree in order to associate genetic clusters with cattle sector, geographical area and year of isolation. The phylogenetic analysis of the Belgian M. bovis isolates resulted in 5 major clusters and 1 outlier. No sector-specific M. bovis clustering was identified. On a world scale, Belgian isolates clustered with Israeli, European and American strains. Different M. bovis clusters circulated for at least 1.5 consecutive years throughout the country, affecting all observed industries. Therefore, the high prevalence in the veal industry is more likely the consequence of frequent purchase from the dairy and beef industry, than that a reservoir of veal specific strains on farm would exist. These results emphasize the importance of biosecurity in M. bovis control and prevention.

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.

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.