Exploring herd-level perinatal calf mortality risk factors in eastern Canadian dairy farms.

This closed cohort study aimed to identify the associations between dairy calf management practices and herd-level perinatal calf mortality risk. From February 2020 to June 2021, predominantly Holstein dairy farms in Québec (n = 1832) and New Brunswick (n = 52), Canada, registered in the dairy herd improvement program, were visited once. A questionnaire covering all aspects of precalving, calving, and colostrum management was administered. Data regarding perinatal mortality were retrieved from the dairy herd improvement program database for each farm for 2021. Perinatal mortality was calculated for each farm as the proportion of calves dead at birth or dying within 24 h of life. A multivariable negative binomial model was used to assess herd-level factors associated with the risk of perinatal mortality. The final model included the lying surface in the calving area, the typical time to first colostrum intake, typical cow-calf contact time, the proportion of males born, the proportion of assisted calvings, and herd size. Herd-level perinatal mortality risk ranged from 0 to 38.1% (mean (SE) = 7.6% ± 0.1%). A greater proportion of males born, proportion of assisted calvings, and delayed colostrum feeding were associated with increased herd-level perinatal mortality. Factors associated with a decreased herd-level perinatal mortality risk were having a typical cow-calf contact time between 7 and 12 h after calving compared with reduced cow-calf contact time, soft lying surfaces in the calving area compared with concrete and mat-lying surfaces, and an increased number of calvings per year. Our results show that while some of the significant risk factors are not well understood (i.e., calving area lying surface, typical cow-calf contact time); Canadian farmers could focus on the factors under their control (i.e., time to first colostrum feeding, proportion of difficult calvings, males born, and calvings per year) to reduce the risk of perinatal mortality. Future work should focus on qualitative research to understand the dairy farmer motivations and limitations to implementing practices identified in this and other studies to reduce perinatal mortality.

Thoracic Ultrasonography and Bronchoalveolar Lavage Fluid Analysis in Holstein Calves with Subclinical Lung Lesions.

BACKGROUND: Thoracic ultrasonography (US) and bronchoalveolar lavage fluid (BALF) analysis are antemortem methods used to identify the lung lesions associated with bovine respiratory disease (BRD). Accuracy of US and the cell distributions in BALF have not been characterized in calves with subclinical disease. OBJECTIVES: To evaluate the accuracy of US and BALF and describe BALF characteristics in calves with subclinical lung lesions. ANIMALS: Twenty-five Holstein calves, 1-12 weeks old. METHODS: In this prospective study, calves with low respiratory scores underwent US, BALF and postmortem examination (normal US, n = 5; comet-tails, n = 5; consolidation, n = 15). Bronchoalveolar lavage fluid was collected and analyzed for total and differential cell counts. Lung lesions were assessed by gross and histopathologic examination. Data were analyzed using nonparametric methods and relative risk analysis. The accuracy of US and BALF were estimated relative to postmortem examination. RESULTS: The sensitivity and specificity of US for detecting lung lesions was 94% (95% CI, 69-100%) and 100% (95% CI, 64-100%), respectively. A cut-point of ≥4% BALF neutrophils was associated with the highest BALF sensitivity and specificity, 81% (95% CI, 56-94%) and 75% (95% CI, 36-95%). The presence of consolidation on US increased the risk of having a BALF neutrophil proportion ≥4% (RR, 3.9; 95% CI, 1.13-13.45; P = .003). CONCLUSIONS AND CLINICAL IMPORTANCE: Ultrasonography accurately detects lung lesions in calves with subclinical disease. Clinicians should use a cut-point of ≥4% BALF neutrophils to diagnose subclinical respiratory disease.

Nonculture molecular techniques for diagnosis of bacterial disease in animals: a diagnostic laboratory perspective.

The past decade has seen remarkable technical advances in infectious disease diagnosis, and the pace of innovation is likely to continue. Many of these techniques are well suited to pathogen identification directly from pathologic or clinical samples, which is the focus of this review. Polymerase chain reaction (PCR) and gene sequencing are now routinely performed on frozen or fixed tissues for diagnosis of bacterial infections of animals. These assays are most useful for pathogens that are difficult to culture or identify phenotypically, when propagation poses a biosafety hazard, or when suitable fresh tissue is not available. Multiplex PCR assays, DNA microarrays, in situ hybridization, massive parallel DNA sequencing, microbiome profiling, molecular typing of pathogens, identification of antimicrobial resistance genes, and mass spectrometry are additional emerging technologies for the diagnosis of bacterial infections from pathologic and clinical samples in animals. These technical advances come, however, with 2 caveats. First, in the age of molecular diagnosis, quality control has become more important than ever to identify and control for the presence of inhibitors, cross-contamination, inadequate templates from diagnostic specimens, and other causes of erroneous microbial identifications. Second, the attraction of these technologic advances can obscure the reality that medical diagnoses cannot be made on the basis of molecular testing alone but instead through integrated consideration of clinical, pathologic, and laboratory findings. Proper validation of the method is required. It is critical that veterinary diagnosticians understand not only the value but also the limitations of these technical advances for routine diagnosis of infectious disease.

Failure of respiratory defenses in the pathogenesis of bacterial pneumonia of cattle.

The respiratory system is well defended against inhaled bacteria by a dynamic system of interacting layers, including mucociliary clearance, host defense factors including antimicrobial peptides in the epithelial lining fluid, proinflammatory responses of the respiratory epithelium, resident alveolar macrophages, and recruited neutrophils and monocytes. Nevertheless, these manifold defenses are susceptible to failure as a result of stress, glucocorticoids, viral infections, abrupt exposure to cold air, and poor air quality. When some of these defenses fail, the lung can be colonized by bacterial pathogens that are equipped to evade the remaining defenses, resulting in the development of pneumonia. This review considers the mechanisms by which these predisposing factors compromise the defenses of the lung, with a focus on the development of bacterial pneumonia in cattle and supplemented with advances based on mouse models and the study of human disease. Deepening our understanding of how the respiratory defenses fail is expected to lead to interventions that restore these dynamic immune responses and prevent disease.

Hydrogen peroxide production and free radical-mediated cell stress in Mycoplasma bovis pneumonia.

Mycoplasma bovis causes chronic pneumonia and polyarthritis in feedlot cattle. M. bovis infects the lungs of most feedlot cattle, but the majority of calves never develop disease. Competing explanations are that some strains of M. bovis are more virulent than others or, alternatively, that calves require some other abnormality to be present in order for M. bovis to cause disease. We hypothesize that H2O2 production is an important virulence factor of M. bovis, causing oxidative injury to lung tissue. A second hypothesis is that isolates associated with caseonecrotic bronchopneumonia have an increased capacity for H2O2 production. Immunohistochemical markers of oxidative stress (4-hydroxynonenal, HN) and nitrative stress (3-nitrotyrosine, NT) were compared in lungs of calves with caseonecrotic bronchopneumonia characteristic of M. bovis infection, with other forms of bronchopneumonia or with non-inflamed lungs. HN and NT were identified in M. bovis pneumonia, mainly in foci of caseous necrosis. HN was not observed in inflamed non-necrotic tissue in lesions typical of pneumonic pasteurellosis. H2O2 production by M. bovis was identified, but the levels did not differ in isolates from calves with caseonecrotic bronchopneumonia compared with those with non-inflamed lungs or other forms of pneumonia. These findings provide evidence that oxidative and nitrative injury contribute to the formation of the caseonecrotic lesions that are characteristic of M. bovis pneumonia and that production of H2O2 by M. bovis may contribute to this oxidative injury.

Localization and functional characterization of pulmonary bovine odorant-binding protein.

Bovine odorant-binding protein (OBP) may function in olfaction and defense against oxidative injury, but its role in inflammation and defense against bacterial infection has not been investigated. Expression of OBP was discovered in the bovine lung and found to undergo changes in abundance during glucocorticoid administration and stress. OBP was localized to nasal, tracheal, and bronchial mucosal glands with immunohistochemistry, with faint expression in airway surface epithelium and none in bronchioles or alveoli. Two isoforms of OBP were identified, appearing to be differentially regulated during lipopolysaccharide-induced pulmonary inflammation, but differences between these isoforms were not revealed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Functional studies showed no effect of OBP on in vitro growth of Escherichia coli or Mannheimia haemolytica under iron-replete or iron-depleted conditions, nor did OBP opsonize bacteria for an enhanced neutrophil oxidative burst. However, OBP did reduce the ability of supernatants from lipopolysaccharide-stimulated macrophages to induce neutrophil chemotaxis. These findings indicate that OBP may inhibit neutrophil recruitment by inflammatory mediators, and they suggest an ability to bind macrophage-derived inflammatory mediators within the airways.

Immunohistochemical identification of collagen in the equine lung.

Recurrent airway obstruction (RAO) in the horse is a disease characterized by reversible bronchoconstriction and by mucus and neutrophil accumulation in the airways. It has been hypothesized that in horses with RAO, remodeling changes occur that are similar to those described in humans with asthma. Although collagen fibrils are present surrounding normal airways, they are a prominent feature of airway remodeling in human asthma with evidence of enhanced collagen III and I fibril deposition. An immunolabeling method was developed to identify collagen I and III in equine lung and to describe the collagen fiber type and distribution within the walls of the noncartilagenous bronchioles. The health status of 14 horses was characterized by clinical respiratory exam, bronchoalveolar lavage cytology, and pulmonary function tests. Following postmortem examination and histological assessment, horses were divided into RAO-affected (n = 4) and nonaffected (n = 10) groups. Eight sections per horse from all lung regions were evaluated histologically. Results of the study showed that collagens I and III were present in the lamina propria and adventitial area of the noncartilaginous bronchioles. There was clear staining differentiation between collagen I or III, airway smooth muscle, and the airway epithelium. Collagen I and III were present in the lamina propria and adventitial areas of the noncartilaginous bronchioles of horses, and there was no significant difference in the relative amount of collagen I and III between this group of RAO-affected and nonaffected horses.

Cardiac myxosarcoma with adrenal adenoma and pituitary hyperplasia resembling Carney complex in a dog.

A 9-year-old female Golden Retriever was presented with an acute onset of progressive respiratory distress. Echocardiography revealed a left atrial mass that limited blood flow from the pulmonary veins. The pathological evaluation revealed a left atrial ossifying myxosarcoma, bilateral adrenocortical adenomas, multifocal pituitary hyperplasia with expression of adrenocorticotrophic hormone, and multiple pituitary Rathke's cleft cysts. These pathologic findings are similar to those described in Carney complex, a familial human syndrome characterized by cardiac myxoma and extracardiac tumors associated with mutations in the protein kinase A regulator gene PRKAR1A. Mutations were not detected in PRKAR1A exons in the present case.

Laboratory findings, histopathology, and immunophenotype of lymphoma in domestic ferrets.

Lymphoma is a common tumor in ferrets, but anatomic distribution, histomorphology, immunophenotype, laboratory abnormalities, and response to chemotherapy are incompletely defined. In this study, lymphoma was diagnosed by histopathology of tumor tissue in 29 ferrets ranging in age from 0.8 to 8.5 years, including 12 males and 17 females. Tumors involved the viscera of the abdominal cavity (n = 11), thoracic cavity (n = 1), or abdominal and thoracic cavities (n = 7); the skin (n = 2); or the viscera of both body cavities plus other sites (n = 8). Microscopically, all tumors had diffuse architecture. Assessment by histomorphology and immunophenotype classified tumors as peripheral T-cell lymphoma (n = 17), anaplastic large T-cell lymphoma (n = 5), anaplastic large B-cell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 1), and Hodgkin-like lymphoma (n = 2). Cytologic evaluation of tumor tissue was diagnostic in 11 of 13 cases. Twenty-two of 27 ferrets had anemia, 2 had leukemia, and 5 were neutropenic. Common comorbid disorders were adrenal disease (n = 27) and insulinoma (n = 6). Tumors most frequently involved mesenteric lymph nodes, while enlargement of peripheral lymph nodes was uncommon (n = 3). Ferrets with Hodgkin-like lymphoma had massive enlargement of single lymph nodes. Mean survival of ferrets not immediately euthanized was 5.0 months (T-cell lymphoma) and 8.4 months (B-cell lymphoma). Ferrets treated with chemotherapy survived an average of 4.3 months (T-cell lymphoma, n = 9) or 8.8 months (B-cell lymphoma, n = 4). Results indicate that lymphomas in ferrets most commonly affect abdominal viscera, may be amenable to cytologic diagnosis, are frequently associated with anemia and, in some cases, may be chemosensitive, resulting in relatively long survival times.

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant (p<0.01), and these effects were significantly (p<0.02, p<0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant (p<0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Effect of bovine granulocyte colony-stimulating factor on the development of pneumonia caused by Mannheimia haemolytica.

The role of recruited neutrophils in Mannheimia haemolytica infection is controversial. We hypothesized that the neutrophilia induced by recombinant bovine granulocyte colony-stimulating factor (GCSF) would lead to rapid bacterial clearance and less severe lesions after infection with M. haemolytica. Two experiments (A and B) were conducted in which four calves per experiment were treated daily with 5 microg/kg GCSF and four calves per experiment were treated with saline. All 16 calves were challenged with 5 x 10(9) colony-forming units (cfu)/ml (experiment A) or 4.5 x 10(8) cfu/ml (experiment B) of M. haemolytica bacteria, into the right bronchus by bronchoscope-placed catheter. The mean maximal blood neutrophil counts in non-GCSF-treated and GCSF-treated calves before bacterial challenge were 5.6 +/- 0.7 x 10(9)/liter and 25.4 +/- 2.7 x 10(9)/liter, respectively. Two untreated calves became neutropenic and were euthanatized 2 days after infection because of severe respiratory distress. GCSF-treated calves had a 37% reduction in lung lesions compared with nontreated calves, and this difference was significant (P=0.04) when the effect of previous antibody titre to leukotoxin was considered. The effect of GCSF treatment on the severity of clinical signs seemed to be influenced by the antibody titre to M. haemolytica leukotoxin, although this effect could not be conclusively addressed. In conclusion, GCSF induced neutrophilia and partially protected calves against experimental infection with M. haemolytica. These results imply that increased numbers of neutrophils may, under some circumstances, protect against severe pneumonia caused by M. haemolytica.

The importance of interleukin-8 as a neutrophil chemoattractant in the lungs of cattle with pneumonic pasteurellosis.

Interleukin-8 (IL-8), an in vitro and in vivo neutrophil chemoattractant, is expressed at high levels in the lesions observed in bovine pneumonic pasteurellosis. Because of the role of neutrophils in the pathogenesis of pneumonic pasteurellosis, we investigated the relative importance of IL-8 as a neutrophil chemoattractant in this disease. Bronchoalveolar lavage (BAL) fluid was harvested from calves experimentally infected with bovine herpesvirus-1 and challenged with Mannheimia haemolytica. Neutrophil chemotactic activity was measured in pneumonic BAL fluid samples treated with a neutralizing monoclonal antibody to ovine IL-8, and compared to the activity in samples treated with an isotype-matched control antibody. Bronchoalveolar lavage fluid was analyzed at a dilution which induced a half-maximal response, and the concentrations of antibody were optimized in a preliminary experiment. Following incubation of replicate samples of diluted pneumonic bovine BAL fluid with 70 microg/mL of IL-8-neutralizing antibody or control antibody, the neutrophil chemotactic activities of the samples were determined using an in vitro microchemotaxis assay. Overall, pretreatment of BAL fluid samples with neutralizing anti-IL-8 antibody reduced neutrophil chemotactic activity by 15% to 60%, compared to pretreatment with control antibody. This effect was highly significant (P < 0.001), and was present in 5 of 5 samples. These data indicate that IL-8 is an important neutrophil chemoattractant in calves with pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present in the lesions.

Intravascular lymphoma presenting as bilateral panophthalmitis and retinal detachment in a dog.

A seven-year-old, spayed female boxer was presented for sudden blindness and red eyes of one week's duration. Ophthalmic examination revealed bilateral uveitis with complete bullous retinal detachments. Initial diagnostic testing failed to reveal evidence of systemic disease. Cytopathology of subretinal aspirates confirmed the presence of macrophages with intracytoplasmic retinal pigment, epithelial and choroidal pigment, and monocytes. Tentative diagnoses were idiopathic uveitis and retinal detachment. The dog was treated with immunosuppressive doses of prednisone. The retinas reattached and vision returned. By two months, the dog became blind and systemically ill. Postmortem and histopathological examinations revealed intravascular lymphoma in multiple organs, including the eyes.

Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant.

Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species. In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein. The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin. The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis. Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8. Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry. Injection of 3.3 ng to 3.3 microg of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils. Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection. Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils. These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo.

Expression of the neutrophil chemoattractant interleukin-8 in the lesions of bovine pneumonic pasteurellosis.

We investigated the expression of interleukin-8 (IL-8) in pneumonic pasteurellosis of cattle because neutrophils are important mediators of tissue injury in this disease and because IL-8 is a major neutrophil chemoattractant in other species. We also compared IL-8 expression in bacterial and viral pneumonia, since the latter lacks the severe neutrophil exudation typical of pneumonic pasteurellosis. IL-8 expression was assessed by northern analysis, in situ hybridization, enzyme-linked immunosorbent assay, and in vivo bioassay. IL-8 mRNA expression was elevated dramatically in lesions of pneumonic pasteurellosis compared to unaffected lung from the same calves. In situ hybridization revealed intense expression of IL-8 mRNA in alveolar macrophages and neutrophils and milder expression in bronchiolar and alveolar epithelium, interstitial cells, and pleural mesothelium. Bronchoalveolar lavage fluid from lesional lung contained 16.06+/-4.00 ng/ml IL-8, whereas those from nonlesional and normal lung contained 0.34+/-0.11 and 0.01+/-0.002 ng/ml, respectively. We detected IL-8 expression at only minimal levels in bovine respiratory syncytial viral pneumonia. Lung extracts from lesions of pneumonic pasteurellosis induced vigorous neutrophil infiltration following injection into bovine skin, and 89% depletion of IL-8 from the extract reduced this neutrophil influx by 60%. These results demonstrate consistent upregulation of IL-8 expression in lesions of pneumonic pasteurellosis, implying a role for IL-8 in the ongoing recruitment of neutrophils to established lesions of pneumonic pasteurellosis. Because neutrophil-mediated tissue injury is critical to the pathogenesis of pneumonic pasteurellosis, these data suggest that neutralization of IL-8 activity could ameliorate the severe clinical signs and lesions of this disease.

Multiple aneurysmal bone cysts in a foal.

Multiple aneurysmal bone cysts (ABCs) are previously unreported in horses. An ABC was diagnosed in the left 3rd metacarpal of a Thoroughbred foal, which partially resolved following surgical curettage. A 2nd ABC developed in the left tibia, 7 wk postoperatively, and the foal was euthanized.

A prospective study of the immunophenotype and temporal changes in the histologic lesions of canine demodicosis.

Mural folliculitis is a consistent histologic lesion of canine demodicosis. The objective of this study was to describe the immunophenotype and to evaluate temporal changes in histologic lesions of demodicosis during the course of therapy. Five dogs with demodicosis were examined and biopsied biweekly for up to 14 weeks; three dogs were evaluated once only. Lymphocyte subsets infiltrating the lesions were quantified using immunohistochemistry to detect CD3, CD21, CD4, and CD8 antigens. Lymphocyte subsets in blood were analyzed from four dogs using flow cytometry. Mural folliculitis was always present during clinically active disease. In contrast, following resolution of clinical lesions, perifolliculitis and/or perifollicular granulomas were present but mural folliculitis was absent. Most lymphocytes infiltrating the follicular epithelium in lesions of mural folliculitis were CD3+ and CD8+; the ratio of CD4+ :CD8+ cells in this epithelium was 0.032. In contrast, the perifollicular dermis contained approximately equal numbers of CD4+ cells and CD8+ cells, with slightly fewer CD21+B cells. In peripheral blood, the ratio of CD4+:CD8+ lymphocytes was reduced and the percentage of CD8+ cells was increased in three of four dogs. These results indicate that mural folliculitis is a consistent lesion of clinically active canine demodicosis and is characterized by infiltration of the follicular epithelium by CD3+ CD8+ T lymphocytes. These lymphocytes are cytotoxic T cells, which may mediate the injury to the follicular epithelium in demodicosis. Alternatively, CD8+ T cells may play a role in resistance to Demodex canis infection or may represent a deleterious immune response in dogs that develop demodicosis.

Establishment of Demodex canis on canine skin engrafted onto scid-beige mice.

A small animal model of canine demodicosis is described. Normal canine skin was engrafted onto scid (severe combined immunodeficient)-beige mice, which lack functional B and T lymphocytes and have reduced natural killer cell activity. The xenografts were later infected with Demodex canis collected from a dog with demodicosis. At 30-112 days following infection, mites were seen histologically in the canine hair follicles of the engrafted skin. Demodex canis adults, nymphs, larvae, and eggs were present in samples macerated in sodium hydroxide. Mite infestations could not be demonstrated in the mouse skin, nor were mites passed from the infected graft to uninfected skin grafts on in-contact mice. This model may be utilized to assess the efficacy of miticidal treatments, to evaluate the importance of specific components of the immune response, and to study the biology of D. canis.