A MULTIMODAL CLINICAL AND DIAGNOSTIC APPROACH TO BOVINE RESPIRATORY DISEASE COMPLEX (BRDC) IN DAIRY CALVES

Calf bronchopneumonia (BP), or Bovine Respiratory Disease Complex (BRDC), to date is still difficult to diagnose due to the multifactorial nature of its etiopathogenesis and aspecificity of clinical symptomatology. In this research project, four studies were conducted to investigate various aspects of BRDC, the objectives were: 1) To study the prevalence of BRDC-related pathogens in the territory of northern Italy; 2) To develop an accurate ultrasound diagnosis method that can be used under field conditions; 3) To standardize lung auscultation in order to increase its diagnostic accuracy; 4) To analyze hemogas-analytical and metabolic changes during BRDC by correlating them with clinical and ultrasonographic diagnosis. For this purpose, pre-weaning Italian Friesian calves from herds in northern Italy were included, and observations were performed under field conditions. In our first study, an epidemiological investigation of pathogens in the lower airways of calves that showed respiratory symptoms for less than three days and had not received antibiotic treatment in the previous two weeks was conducted. Ten farms in northern Italy were included, and trans-tracheal washes (TTW) were performed on 6 calves from each farm. Culture and molecular tests were performed on the samples, the results were analyzed obtaining the total and farm prevalence, and the prevalence of coinfections were evaluated. Pasteurella multocida and Mannheimia haemolytica were found in 37% and 12% of the samples, respectively, with a prevalence of 70% and 40% of the farms examined. The presence of Mycoplasma bovis was detected in 20% of the farms with a prevalence of 13% of the tested population. The viral pathogens detected were Bovine Coronavirus and Bovine Respiratory Syncytial Virus in 15% and 7% of cases, respectively. Only coinfections determined by two pathogens were evidenced: for P. multocida in 36% of the isolations, M. haemolytica (86%), M. bovis (50%), BCoV (56%), BRSV and others in 100% of the isolations. We observed that the prevalence of Mycoplasma bovis, although low in the population, reaches very high values at the farm level. In our population, the etiological agents with higher occurrence were Pasteurella multocida and BCoV. In order to reinforce the prudent use of antimicrobials, diagnoses should also be investigated from the etiological aspect so as to set up treatment choices in the most effective way. In the second study, the accuracy of a rapid ultrasound technique: Focused Lung Ultrasound (FLUS) was evaluated by comparing it with systematic Thoracic Ultrasonography (TUS) nowadays considered an imperfect gold standard for the diagnosis of BRDC-associated lung lesions. For this study 135 animals were subjected to both ultrasound investigations, and the McNemar test was used to evaluate statistical differences and agreement was assessed by weighted Kappa. A total of 76 calves out of 135 had a TUS score ≥ 2 (focal lung lesion > 1 cm) and were therefore considered to be affected by BRDC. The FLUS had sensitivity of 81.6% (95% CI = 71.0-89.5%), specificity = 100% (95% CI = 93.9-100%), positive predictive value of 100%, negative predictive value of 96.6% (95% CI = 94.7-97.9%) and accuracy of 97% (95% CI = 92.6-99.2%). McNemar's test showed a difference of 10.3% between FLUS and TUS. The agreement between TUS and FLUS was substantial (weighted kappa test 0.78). This study shows that the focused method could be used as an additional tool for evaluating consolidation, especially when examining a large number of postweaned dairy calves. In our third study, we investigated the accuracy of lung auscultation. This method suffers from non-unique definition of lung sounds, resulting in poor/moderate accuracy. To evaluate if standardization of nomenclature and method could increase accuracy in diagnosis, 330 calves were examined. Each calf received: clinical evaluation with Wisconsin Score (sick if > 4), TUS ultrasound (sick if ≥ 2) and lung auscultation. Thoracic auscultation was classified as follows: AUSC1 (all pathological sounds), AUSC2 in which hisses, crackles, increased bronchial sounds and pleural rubs were considered, and AUSC3 when only increased bronchial sounds or pleural rubs were evaluated. The accuracy of AUSC categorizations was determined using three imperfect diagnostic tests by a Bayesian latent class model and sensitivity analysis (informative vs. weakly informative vs. non-informative priors and with vs. without covariance between ultrasound and clinical scoring). Based on the priors used, sensitivity (95¼I) of AUSC1 ranged from 0.89 (0.80-0.97) to 0.95 (0.86-0.99), with specificity (95¼I) from 0.54 (0.45-0.71) to 0.60 (0.47-0.94). Removing the increased breath sound from the AUSC categorizations results in a pronounced increase in specificity (for AUSC3, specificity ranged from 0.97 [0.93-0.99] to 0.98 [0.94-0.99]), but at the cost of a decrease in sensitivity (from 0.66 [0.54-0.78] to 0.81 [0.65-0.97]). The impact of priors on posterior densities of AUSC was limited. We could conclude that a standardized definition of lung sounds improved the accuracy of AUSC for BP diagnosis in calves. The last study in this research project aimed to determine what is the best diagnostic approach to identify metabolic abnormalities in sick calves. A total of 231 animals were enrolled in which were performed: clinical and ultrasonographic evaluation, blood gas examination and serum titration of paraoxonase-1 (PON-1) as a biomarker of inflammation. Calves were grouped as "healthy" or "calves with BP" based on clinical scores (Wisconsin [WISC5], California [CALIF], an alternative Wisconsin approach [WISC2points]) and two TUS cut-offs (≥ 1 cm; ≥ 3 cm); and as "healthy," "upper respiratory tract infection," "subclinical BP," and "clinical BP" based on the combination of clinical and TUS scores. Abnormalities were more extensive in calves diagnosed with TUS. Glucose concentrations were significantly lower in "calves with BP" (median TUS1cm = 5.2 mmol/L, 25-75% interquartile range [IQR] 5.5-6.6; median TUS3cm = 5.1 mmol/L, IQR 4.4-6.1) than in "healthy" (median TUS1cm = 5.8 mmol/L, 5.5-6.6; median TUS3cm = 5.7 mmol/L, IQR 5.2-6.3; P < 0.001). The concentration of unmeasured strong ions was significantly lower in "BP calves" diagnosed by TUS1cm (median = 0.8 mmol/L, IQR -0.9-3) than in "healthy" (median = 2.8, IQR 1.1-4.9; P < 0.001). PON-1 was significantly lower in "BP calves" (median TUS1cm = 64.1 U/mL, IQR 40.8-78; median TUS3cm = 59.2 U/mL, IQR 37.7-72.4) than in "healthy" (median TUS1cm = 77.3 U/mL, IQR 61.9-96.5; median TUS3cm = 72.9 U/mL, IQR 53.4-95.4; P < 0.001). None of the parameters showed clear distinctions between calves with clinical and subclinical BP. In conclusion, calves diagnosed with TUS identified more significant metabolic abnormalities. The present study provided new knowledge on BRDC, and in particular on aspects related to epidemiology, the tools we possess to improve the diagnosis of BP in the field, and the evaluation of metabolic changes in animals affected by respiratory disease. Thanks to the results obtained, we can assert that to date, thoracic ultrasonography remains the best in vivo method for identifying sick animals, and related to clinical scores, it discriminates with greater accuracy those animals manifesting metabolic and hemogasanalytic alterations. In addition, the development of rapid ultrasound techniques such as FLUS are shown to be very applicable under field conditions. Diagnostic methods considered inaccurate, such as auscultation, can be a valuable tool by providing unambiguous definition and interpretation of sounds. Finally, in a One Health perspective, it is necessary to perform etiologic diagnosis aimed at setting up a proper vaccination and treatment plan.