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.

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.

Hemorrhagic bowel syndrome in dairy cattle: Gross, histological, and microbiological characterization.

Hemorrhagic bowel syndrome (HBS) is a sporadic and fatal disease of predominantly lactating dairy cattle, characterized by segmental hemorrhage and luminal clot formation in the small intestine. Although, Clostridium perfringens and Aspergillus fumigatus have been associated with HBS, the pathogenesis and cause are currently unknown. In this study, 18 naturally occurring cases of HBS (7 necropsied immediately following euthanasia, 11 with 12-48 hour postmortem intervals) were investigated to characterize the pathology and the intestinal microbiome. Hemorrhagic bowel syndrome was characterized by a single small-intestinal, intramucosal hematoma with dissection of the lamina muscularis mucosae. In most cases necropsied immediately after euthanasia (4/7), the intestinal mucosa proximal to the hematoma contained 9 to 14, dispersed, solitary or clustered, erosions or lacerations measuring 4 to 45 mm. In 77% (37/48) of these mucosal lesions, microscopic splitting of the lamina muscularis mucosae comparable to the hematoma was present. These findings suggest the intramucosal hematoma to originate from small mucosal erosions through dissecting hemorrhage within the lamina muscularis mucosae. No invasive fungal growth was observed in any tissue. Bacteriological cultivation and nanopore sequencing showed a polymicrobial population at the hematoma and unaffected intestine, with mostly mild presence of C perfringens at selective culture. Gross and microscopic lesions, as well as the culture and sequencing results, were not in support of involvement of C perfringens or A fumigatus in the pathogenesis of HBS.

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 in vitro effect of lactose on Clostridium perfringens alpha toxin production and the implications of lactose consumption for in vivo anti-alpha toxin antibody production.

Necro-hemorrhagic enteritis in calves, caused by Clostridium perfringens type A, is a fatal disease, mostly affecting calves in intensive rearing systems. The lack of development of active immunity against α toxin, an essential virulence factor in the pathogenesis, has been proposed as a main trigger. In this experimental study, the effect of a set of milk replacer components on α toxin production, and the effect of lactose on in vivo antibody production, were investigated. For the latter, Holstein-Friesian bull calves (n = 18) were fed an all liquid diet that contained either a milk replacer with high-lactose content (45% DM) or the same milk replacer that was lactase treated, resulting in a lactose-free equivalent. Antibody levels against α toxin were monitored from 2 to 12 wk of age. In the in vitro part of the study, a concentration-dependent inhibitory effect of lactose on in vitro C. perfringens α toxin activity was observed, whereas protein did not influence α toxin activity. The in vivo experiment then showed from the age of 10 wk onwards, that anti-α toxin antibody levels of high-lactose animals declined, whereas antibody levels of the animals consuming lactose-free milk replacer remained the same throughout the trial. This points to a natural decline in maternal immunity of lactose-consuming animals, that is not compensated by the development of an active immunity, resulting in inferior protection. This study suggests that dietary lactose reduces C. perfringens α toxin production in vivo, which may lead to a decreased antigen presentation and thus lower serum antibody levels against the toxin. Consequently, any event causing massive α toxin production puts lactose-consuming calves at higher risk of developing necro-hemorrhagic enteritis.

Effect of on-arrival bovine respiratory disease vaccination on ultrasound-confirmed pneumonia and production parameters in male dairy calves: A randomized clinical trial.

The high degree of commingling and accumulation of stressors during and after transport makes prevention of bovine respiratory disease (BRD) extremely challenging in the veal and dairy beef industry. Upon arrival, vaccination for agents involved in BRD is practically most achievable, but its efficacy under such conditions in dairy veal calves is unknown. Given the high prevalence of subclinical pneumonia in these settings, the primary objective of the present study was to determine the effect of 2 vaccination protocols administered upon arrival against bovine respiratory syncytial virus (BRSV), bovine parainfluenza type 3 virus (BPI-3), and Mannheimia haemolytica on clinical BRD and lung ultrasonographic findings in dairy veal calves. In addition, the effects of vaccination on average daily live weight gain and cold carcass weight were determined. In this randomized clinical trial, 443 male dairy calves were assigned to one of 3 groups: a negative, placebo-controlled group (n = 151), a vaccination group with 2 subcutaneous injections 4 wk apart with an inactivated vaccine containing BRSV, BPI-3, and M. haemolytica (parenteral [PE] group; n = 149) and a second vaccination group receiving an intranasal live-attenuated vaccine containing BRSV and BPI-3 and 2 subcutaneous vaccinations with the same inactivated vaccine as the PE vaccination group (intranasal-parenteral [IN-PE] group; n = 143). Clinical scoring and quick thoracic ultrasonography (qTUS) were performed on all calves on arrival (wk 0), at the peak of respiratory disease (outbreak; wk 1), at the end of the first antimicrobial group treatment (wk 3), and at a long-term evaluation point (wk 10). Culture and nanopore sequencing on nonendoscopic bronchoalveolar lavage (nBAL) samples were used to identify pathogens involved in the outbreak. Upon arrival, 15.1% of the calves had lung consolidation ≥1cm and incidence quickly rose to 42.8% during the outbreak. In both the PE and IN-PE group, the odds of pneumonia in wk 10 were reduced by 62% (odds ratio [OR] = 0.38; 95% confidence interval [CI] = 0.23-0.64) and 41% (OR = 0.59; 95% CI = 0.37-0.96), respectively. Short-term cure rate (50.3%), as determined immediately after the first group antimicrobial treatment, was not influenced by vaccination. In contrast, long-term cure rate, determined at wk 10, was affected by vaccination with higher cure in the PE group compared with the control group (69.4% vs. 51.2%; OR = 2.2; 95% CI = 1.1-5.0). Average daily gain in the first 10 wk of production was not affected by vaccination. Vaccination resulted in an increase in cold carcass weight of 3.5 and 4.3 kg in the PE (95% CI = -0.9-7.9) and IN-PE group (95% CI = -0.17-8.7), respectively. In conclusion, under the conditions of the present study, vaccination upon arrival resulted in a reduced prevalence of pneumonia at wk 10 of production, likely caused both by an improved cure rate of secondary infections and a reduced incidence of new cases between outbreak and long-term evaluation. The present protocol, using qTUS for pneumonia detection and nBAL diagnostics for pathogen identification adds a new dimension to randomized clinical trials on respiratory disease in calves.

Detection of Chlamydia and Chlamydia-like organisms in bovine placental tissue.

This study aimed to investigate the presence of Chlamydia spp. and Parachlamydia acanthamoebae in bovine placental tissue originating from abortion and non-abortion cases in Belgium. Placentas of 164 late term bovine abortions (last trimester of gestation) and 41 non-abortion (collected after calving) cases were analysed by PCR for Chlamydia spp., Chlamydia abortus, C. psittaci and P. acanthamoebae. Additionally, a subset of 101 (75 abortion and 26 non-abortion cases) of these placenta samples were also analysed by histopathology to detect possible Chlamydia-induced lesions. In 5.4% (11/205) of the cases, Chlamydia spp. were detected, and three of those cases were positive for C. psittaci. Parachlamydia acanthamoebae was detected in 36% (75/205) of the cases, being 44% (n = 72) in abortions and 7.3% (n = 3) in non-abortions cases (p < .001). None of the cases was positive for C. abortus. Purulent and/or necrotizing placentitis with or without vasculitis was observed in 18.8% (19/101) of the histopathologically analysed placenta samples. In 5.9% (6/101) of the cases, placentitis was observed along with vasculitis. In the abortion cases, 24% (18/75) of the samples showed purulent and/or necrotizing placentitis, while purulent and/or necrotizing placentitis was visible in 3.9% (1/26) of the non-abortion cases. Placental lesions of inflammation and/or necrosis were present in 44% (15/34) of the cases where P. acanthamoebae was detected, while inflammation and/or necrosis was present in 20.9% (14/67) of the negative cases (p < .05). The detection of Chlamydia spp. and especially P. acanthamoebae, in combination with correlated histological lesions such as purulent and/or necrotizing placentitis and/or vasculitis in placental tissue following abortion, suggests a potential role of this pathogen in cases of bovine abortion in Belgium. Further in-depth studies are necessary to unravel the role of these species as abortifacient agents in cattle and to include them in bovine abortion monitoring programmes.

Clinical insights into the three-dimensional anatomy of cheek teeth in alpacas based on micro-computed tomography - Part 2: Maxillary cheek teeth.

BACKGROUND: Scientific knowledge regarding alpaca dentistry is relatively limited despite its clinical implications. The present gap in available supportive data leads to limited treatment options for dental pathology in alpacas in comparison to other species. The main goal of this study was to gain novel insights into the general and pulp morphology of maxillary cheek teeth to allow development of more advanced treatment strategies in the future. Also, the risk of causing pulp exposure when floating maxillary cheek teeth was of particular interest. Concurent research focusing on the anatomy of mandibular cheek teeth has been performed accordingly. The results obtained in mandibular teeth are expected to be non-extrapolatable because of the structural differences between mandibular and maxillary teeth. RESULTS: Pulp morphology of maxillary cheek teeth showed great variation. A common pulp chamber was identified in 46/83 (55.4%) teeth with a mean dental age of 2 years and 7 months (± 2 years and 5 months). Pulpal segmentation was more commonly observed in teeth of increasing age. Full columnar segmentation was seen in 33/69 teeth (47.8%), whereas within-column segmentation was observed in 36/83 teeth (43.4%). Age and degree of segmentation of the pulpal tissue varied greatly according to Triadan position. Physical contact between roots of adjacent teeth was found in the majority of examined molars (range 82-94%) which resulted in morphological adaptations at the level of the root tips. The measured sub-occlusal dentinal thickness was as low as 0.46 mm above pulp horn 2 in a 14 years and 11 months old Triadan 09, emphasizing the risk of pulp exposure attributed to dental floating. CONCLUSION: This study offers an objective description of age-dependent maxillary cheek teeth pulp morphology in alpacas. Current findings are of great value to provide a basis for the development of tooth-saving techniques as a treatment for dental disease in this species. Observed physical contact between the roots of different examined molars may be a facilitating factor in the spread of apical infection in chronically diseased cases. Finally, a conservative approach regarding dental floating is recommended in order to avoid iatrogenic damage to pulp tissue.

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.

Dermal immune responses against Psoroptes ovis in two cattle breeds and effects of anti-inflammatory dexamethasone treatment on the development of psoroptic mange.

Psoroptic mange is a common disease of livestock, caused by Psoroptes ovis. Compared to Holstein-Friesian (HF) cattle, the Belgian Blue (BB) cattle breed is highly susceptible to the infestation. However, the mechanism for this difference is still unclear. To determine the factors responsible for this breed susceptibility, the immune response to P. ovis was studied in experimentally infested BB and HF cattle, using clinical signs, histology, immunohistochemical profiling and gene expression analysis of skin biopsies. The mite numbers and lesion area of BB cattle were greater than in HF during the whole study period. Significant influxes of eosinophils in the epidermis and dermis were detected in comparison with the pre-infestation samples in both breeds, with significantly higher eosinophils in BB at 6 weeks post infestation (wpi). Mast cell numbers were unaffected at all stages of infestation in HF, but were significantly elevated relative to pre-infestation in BB cattle at 2 and 6 wpi. The more pronounced cutaneous eosinophilia and higher IL-4 levels at 6 wpi in BB cattle suggest that a Th2-type immune response is underlying the higher susceptibility of the BB breed. In naturally infested BB cattle, development of the psoroptic mange lesions and eosinophils and CD3+ T cell areas were severely depressed after anti-inflammatory treatment with dexamethasone. Together, these results suggest that a stronger Th2-type immune response to P. ovis causes the skin lesions in psoroptic mange in BB cattle and that local anti-inflammatory treatment could potentially be an alternative to control the pathology caused by this parasite.

Clinical insights into the three-dimensional anatomy of cheek teeth in alpacas based on micro-computed tomography. Part 1: mandibular cheek teeth.

BACKGROUND: Despite dental disease being a common health concern in alpacas, important dental pathology including apical infection, remains poorly understood. Treatment options are limited compared to veterinary dentistry techniques in other species. The primary goal of this study was to increase understanding of the external and internal anatomy of mandibular cheek teeth to enable the development of tooth sparing techniques in this species. Also, an objective evaluation of the sub-occlusal dentinal thickness in normal mandibular cheek teeth is warranted to understand the risks associated with reduction of overgrown teeth. RESULTS: Overall pulp anatomy was variably characterized by the presence of a common pulp chamber in younger teeth, and segmentation of pulp cavities into multiple separate pulp entities within the same tooth with increasing age. A common pulp chamber was identified in 55.3% (26/47) of teeth with a mean dental age of 1 year and 11 months (± 1 year and 8 months). Columnar segmentation was recorded in the remaining teeth with a mean dental age of 6 years and 5 months (± 3 years and 11 months). Age of segmentation of the common pulp chamber into multiple separate pulp entities shows wide variation and is dependent of the specific Triadan position. The present study illustrates the presence of disto-mesial root contacts between adjacent tooth roots, often leading to morphological adaptations, most frequently observed between Triadan 09-10s (80%) and 10-11s (67%). The measured sub-occlusal dentinal thickness was as low as 1.11 mm over some pulp horns. The sub-occlusal dentinal thickness was lower than 2, 3, and 4 mm in 13.1, 38.1 and 61.4% of performed measurements, respectively. CONCLUSION: This study provides detailed information on age-dependent mandibular cheek teeth anatomy in alpacas, which may support the use and development of advanced dental treatments in this species such as endodontics and tooth sectioning techniques. Apical morphological adaptations caused by disto-mesial root contact between adjacent mandibular cheek teeth are clearly illustrated. The limited amount of sub-occlusal secondary dentin warrants a cautious approach with regards to dental floating in alpacas.

Diagnosis of respiratory disease in preweaned dairy calves using sequential thoracic ultrasonography and clinical respiratory scoring: Temporal transitions and association with growth rates.

Bovine respiratory disease (BRD) in dairy calves is a multifactorial condition, involving environmental, host, and pathogen factors. Thoracic ultrasound scoring (TUS) has recently been validated as an accurate method of detecting BRD-related lung pathology in dairy calves. Previous studies investigating the use of TUS in preweaned dairy calves have largely been based on cross-sectional data from all-year production systems. The objectives of this longitudinal observational study were to characterize the temporal transitions in TUS scores in dairy calves from pasture-based, seasonal-calving herds using sequential examinations during the preweaning period, and to investigate the relationship between the presence and temporal pattern of BRD, diagnosed by TUS or clinical respiratory scoring (CRS), and average daily gain (ADG). In spring of 2019, 317 preweaned calves from 7 commercial dairy farms were recruited at less than 4 wk old (ranging from 1-27 d of age). Each farm was examined on at least 3 occasions at 20- to 28-d intervals and housed indoors in group or individual pens. At each visit TUS scores, CRS scores based on the University of Wisconsin Calf Respiratory Score Chart (https://www.vetmed.wisc.edu/fapm/wp-content/uploads/2020/01/calf_respiratory_scoring_chart.pdf), and live weight using a dairy breed-specific weigh band were recorded. All data were recorded by the same 2 veterinarians over the course of the study. The final data set consisted of 966 TUS and CRS scores collected from 317 calves over a period of approximately 6 wk from 7 farms. The data were analyzed in multivariable, mixed effects, linear regression models, with separate models constructed for TUS and CRS scores. Random effects (intercepts) were included for calf, farm, and visit week. Additionally, a random slope was included for age at sampling by farm. Median farm TUS score ranged from 0 to 2.5 over the 3 visits (possible range: 0-5). The percentage of calves with a TUS score ≥3 (consolidation of the full thickness of 1 lung lobe), on each farm ranged from 0 to 50%. The median CRS in calves on individual farms ranged from 1 to 3 over the 3 visits (possible range: 0-12). The percentage of calves on each farm with a CRS score ≥5 (possible range: 0-12) ranged from 0 to 26%. The TUS and CRS scores were weakly correlated. The TUS was associated with reduced ADG. Calves with TUS scores ≥3 grew at 126 g/d less than unaffected calves over the 3-wk period before examination. The predicted effect on ADG was dependent on the age and duration over which the animal was affected. Calves affected later (i.e., between visits 2 and 3) had lower predicted weights at 63 d compared with calves with increased TUS scores earlier in the study period. Calves with a TUS score ≥3 at each of the 3 sampling points had the lowest weight at 63 d of age. There was no association of CRS with ADG. This study showed that in contrast to CRS, higher TUS scores are associated with lower ADG, with weight loss being more pronounced in chronic cases.

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.

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.

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.

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.

Storage time and temperature affect the isolation rate of Mannheimia haemolytica and Pasteurella multocida from bovine bronchoalveolar lavage samples.

BACKGROUND: A microbiological diagnosis is essential to better target antimicrobial treatment, control and prevention of respiratory tract infections in cattle. Under field conditions, non-endoscopic broncho-alveolar lavage (nBAL) samples are increasingly collected. To what extent the highly variable turnaround time and storage temperatures between sampling and cultivation affect the isolation rate of bacterial pathogens is unknown. Therefore, the objective of this experimental study was to determine the effect of different storage temperatures (0 °C, 8 °C, 23 °C and 36 °C) and times (0,2,4,6,8,24,48 h) on the isolation rate and concentration of Pasteurellaceae in nBAL samples from clinically affected animals. RESULTS: At a storage temperature temperature of 36 °C isolation rates of Mannheimia haemolytica and Pasteurella multocida were significantly reduced 6 h and 48 h after sampling, respectively. At room temperature (23 °C), a decrease in M. haemolytica and P. multocida isolation rate was noticed, starting at 24 and 48 h after sampling, respectively, but only significant for P. multocida at 48 h. The presence of microbial contamination negatively affected the isolation of P. multocida in clinical nBAL samples, but not of M. haemolytica. CONCLUSION: Optimal M. haemolytica and P. multocida isolation rates from clinical nBAL samples are obtained after storage at 0 °C or 8 °C, provided that the sample is cultivated within 24 h after sampling. The maximum period a sample can be stored without an effect on the M. haemolytica and P. multocida isolation success varies and is dependent on the storage temperature and the degree of microbial contamination.

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.