A multidisciplinary approach to reduce and refine antimicrobial drugs use for diarrhoea in dairy calves.

This study evaluated the impact of a multidisciplinary intervention to reduce and refine antimicrobial drug use for treatment of diarrhoea in dairy calves. The intervention consisted of modification of management practices for disease prevention, health evaluation training of farm staff, implementation of an algorithm directed at antimicrobial therapy for diarrhoeic calves, and monthly farm visits. A combination of retrospective (before intervention period [BP], 1 year) and prospective (immediately after intervention period [AP], 1 year) cohorts were used. Health outcomes measured included incidence of diarrhoea and overall mortality. Antimicrobial treatment rates for diarrhoea and total mass of antimicrobial drugs used at the calf and farm level were also evaluated. Outcomes were assessed using the χ2 or Fisher's exact test, and odds ratios and 95% confidence intervals (95% CI) were calculated. Records of 2049 and 2251 calves from 10 farms were available in the BP and AP, respectively. Reduced antimicrobial treatment rates occurred on seven farms (P < 0.01), but not on three farms in the AP. A total of 85% (1303/1573) and 18% (310/1698) of diarrhoeic calves were treated with antimicrobial drugs in the BP and AP (P < 0.001), respectively. There were no differences in the incidence of diarrhoea or mortality between periods. In the AP, the use sulfamethazine and trimethoprim decreased on eight farms, one farm discontinued use of lincomycin and spectinomycin, while two farms discontinued use and one reduced use of cephalosporins. This multidisciplinary approach was effective in reducing antimicrobial drug use for calf diarrhoea on dairy farms without negative impacts on calf health.

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.

Finite element analysis of wall stress in the equine pulmonary artery.

REASONS FOR PERFORMING STUDY: Arterial calcification is found frequently in the pulmonary artery of racehorses, but the aetiology is unknown. Calcification might be associated with increased wall stress due to arterial geometry (shape) and exercise-induced hypertension. HYPOTHESIS: High wall stress levels are found in the regions associated with calcified lesion formation, exacerbated as transluminal pressure increases to levels associated with exercise. METHODS: The pulmonary arteries of 5 horses, unaffected by calcification, were dissected and pressurised to resting and exercising physiological transluminal pressures and scanned with MRI. Arterial geometries were reconstructed to form 3D computer models and finite element analyses performed. Wall stress levels were measured in 4 regions of interest: the arterial trunk and bifurcation, the wall ipsilateral and contralateral to the bifurcation. Measurements were made for arterial transluminal pressures of 25, 50 and 100 mmHg. RESULTS: High wall stress levels were consistently found at the pulmonary artery bifurcation and wall ipsilateral to the bifurcation, where calcified lesions typically form. Lower wall stress levels were found along the trunk and the wall contralateral to the bifurcation where lesions are less frequently found. Wall stress levels increased 5-fold over a 4-fold increase in pressure. The wall stress levels ranged 10 kPa in the wall of the branch contralateral to the bifurcation at 25 mmHg to 400 kPa in the bifurcation at 100 mmHg. CONCLUSIONS: Wall stress from arterial geometry and increased pulmonary artery transluminal pressure are factors that may be associated with calcification of the equine pulmonary artery. POTENTIAL RELEVANCE: Arterial calcification may increase the risk of arterial wall failure in racing horses.

Clara cell secretory protein is reduced in equine recurrent airway obstruction.

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

Histamine bronchoprovocation does not affect bronchoalveolar lavage fluid cytology, gene expression and protein concentrations of IL-4, IL-8 and IFN-gamma.

In diagnosing inflammatory airway disease (IAD) in performance horses, a histamine bronchoprovocation (HBP) test is often performed. In previously published studies, HBP is usually undertaken prior to cytological examination of the bronchoalveolar lavage (BAL) cells. The purpose of this study was to determine if HBP alters (1) the total nucleated cell numbers and distribution in BAL fluid (BALF) and (2) the mRNA and protein concentrations of selected cytokines in BAL cells and BALF, respectively. BALF was initially collected endoscopically from the right middle or diaphragmatic lung lobe in eight healthy young Standardbred horses. Five to six days later, HBP was performed by aerosolization of histamine (8mg) over a 2min period. BALF was again collected within 2-4h of the HBP from the left middle or diaphragmatic lung lobe. In both samples, total and differential WBC counts were obtained. The gene expressions of interleukin-4 (IL-4), IL-8, interferon-gamma (IFN-gamma) and beta-actin in BAL cells were measured using real-time RT-PCR. The cytokine protein concentrations were measured in the BALF using ELISA. HBP was not associated with either a change in the total BAL cell number or in the distribution of the BAL cells. BAL cell expression of IL-4, IL-8 and IFN-gamma, detected in all samples with the exception of IL-4 in one horse (post-HBP), was not altered as a result of HBP. HBP was not associated with a significant change in IL-8 or IFN-gamma concentrations in the BALF. IL-4 protein was undetectable in BALF either prior to or following HBP. We conclude that HBP can precede BALF collection performed within 2-4h of the former without affecting selected parameters analysed in the BAL cells or BALF.

Characterization of the Fecal Bacterial Microbiota of Healthy and Diarrheic Dairy Calves.

BACKGROUND: Neonatal diarrhea accounts for more than 50% of total deaths in dairy calves. Few population-based studies of cattle have investigated how the microbiota is impacted during diarrhea. OBJECTIVES: To characterize the fecal microbiota and predict the functional potential of the microbial communities in healthy and diarrheic calves. METHODS: Fifteen diarrheic calves between the ages of 1 and 30 days and 15 age-matched healthy control calves were enrolled from 2 dairy farms. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene (Illumina, San Diego, CA). RESULTS: Significant differences in community membership and structure were identified among healthy calves from different farms. Differences in community membership and structure also were identified between healthy and diarrheic calves within each farm. Based on linear discriminant analysis effect size (LEfSe), the genera Bifidobacterium, Megamonas, and a genus of the family Bifidobacteriaceae were associated with health at farm 1, whereas Lachnospiraceae incertae sedis, Dietzia and an unclassified genus of the family Veillonellaceae were significantly associated with health at farm 2. The Phylogenetic Investigation of Communities Reconstruction of Unobserved States (PICRUSt) analysis indicated that diarrheic calves had decreased abundances of genes responsible for metabolism of various vitamins, amino acids, and carbohydrate. CLINICAL RELEVANCE: The fecal microbiota of healthy dairy calves appeared to be farm specific as were the changes observed during diarrhea. The differences in microbiota structure and membership between healthy and diarrheic calves suggest that dysbiosis can occur in diarrheic calves and it is associated with changes in predictive metagenomic function.

Detection of Bovine Coronavirus in Healthy and Diarrheic Dairy Calves.

BACKGROUND: BCoV is identified in both healthy and diarrheic calves, complicating its assessment as a primary pathogen. OBJECTIVES: To investigate the detection rates of bovine coronavirus (BCoV) in feces of healthy and diarrheic calves and to describe the usefulness of a pancoronavirus reverse transcriptase (RT) PCR (PanCoV-RT-PCR) assay to identify BCoV in samples of diarrheic calves. ANIMALS: Two hundred and eighty-six calves <21 days. Calves with liquid or semiliquid feces, temperature >39.5°C, and inappetence were considered as cases, and those that had pasty or firm feces and normal physical examination were designated as controls. METHODS: Prospective case-control study. A specific BCoV-RT-PCR assay was used to detect BCoV in fecal samples. Association between BCoV and health status was evaluated by exact and random effect logistic regression. Fecal (n = 28) and nasal (n = 8) samples from diarrheic calves were tested for the presence of BCoV by both the PanCoV-RT-PCR and a specific BCoV-RT-PCR assays. A Kappa coefficient test was used to assess the level of agreement of both assays. RESULTS: BCoV was detected in 55% (157/286) of calves; 46% (66/143), and 64% (91/143) of healthy and diarrheic calves, respectively. Diarrheic calves had higher odds of BCoV presence than healthy calves (OR: 2.16, 95% CI: 1.26 to 3.83, P = 0.004). A good agreement between PanCoV-RT-PCR and BCoV-RT-PCR to detect BCoV was identified (κ = 0.68, 95% CI: 0.392 to 0.967; P < 0.001). CONCLUSIONS AND CLINICAL IMPORTANCE: BCoV was more likely to be detected in diarrheic than healthy calves. The PanCoV-RT-PCR assay can be a useful tool to detect CoV samples from diarrheic calves.

Frequency dependence of forced oscillatory respiratory mechanics in horses with heaves.

The effect of measurement frequency on respiratory mechanics was investigated in six horses with reversible allergic airway disease. Total respiratory impedance was measured at 1.5, 2.0, 3.0, and 5.0 Hz by using the forced oscillation technique with the horses in remission, after acute antigenic challenge producing clinical heaves, and with heaves but after the administration of 2 mg fenoterol by inhalation. The slopes of the magnitude (magnitude of Zrs) and real part (R) of total respiratory impedance over the frequency range 1.5-3 Hz changed significantly after antigenic challenge and fenoterol. The ratio of R at 2 Hz to R at 3 Hz, however, discriminated better among the three conditions. Compliance and resonant frequency (calculated by using a three-element model) changed significantly after antigenic challenge and fenoterol, but inertance did not. We concluded that horses with heaves showed frequency dependence of R and (magnitude of Zrs) at frequencies up to 3 Hz and that parameters derived from a three-element model were useful indicators of small airway obstruction in the horse.

Effect of exercise on the cardiovascular and respiratory systems of heavy turkeys and relevance to sudden death syndrome.

The cardiorespiratory response to walking exercise was studied in 14-to-18-week-old male and female heavy turkeys. Blood pressure, electrocardiograms, respiratory rate, body temperature, blood gases, and electrolytes were measured at rest and after exercise at two different room temperatures. After exercise, systolic, diastolic, and mean blood pressures decreased, and heart rate and body temperature increased, while a severe lactic acidosis developed, in both sexes and at both room temperatures. Fifty-four percent of the turkeys developed arrhythmias, mostly a transient post-exercise sinus arrhythmia of no clinical significance. Changes in electrolytes were clinically insignificant. Blood gases remained within normal limits, demonstrating adequate gas-exchange and pulmonary capability. Within the limits of this study, the cardiac capacity of the heavy turkey in response to exercise appeared very limited. The pathogenesis of sudden death syndrome of turkeys is still unknown, but cardiovascular changes occurring after exercise could be severe enough to cause death.

Effect of exercise on cardiac output and other cardiovascular parameters of heavy turkeys and relevance to the sudden death syndrome.

The cardiovascular response to treadmill exercise was studied in 14-to-18-week-old male and female heavy turkeys. Arterial blood pressure, cardiac output, body temperature, and heart rate were measured before and during each period of exercise. Total peripheral resistance, stroke volume, and stroke index were calculated from those parameters. Systolic, diastolic, and mean blood pressures significantly decreased during exercise. Cardiac output increased during exercise in males and females by 64% and 23%, respectively, while total peripheral resistance fell by approximately 40% in both sexes. Exercise had no significant effect on stroke volume and stroke index. The cardiac functional capacity of the heavy turkey in response to mild exercise appears very limited. This failure of the cardiovascular system to adequately respond to increased metabolic needs suggests that hemodynamic failure is a major component in the pathogenesis of sudden death syndrome of turkeys.

Use of a virulence-associated protein based enzyme-linked immunosorbent assay for Rhodococcus equi serology in horses.

An enzyme-linked immunosorbent assay (ELISA) was developed against Rhodococcus equi using Triton X-114 detergent extracted whole cell material, in which the virulence associated protein (VapA) predominated. Enzymelinked immunosorbent assay titres corresponded to antibody reacting with VapA on Western blots. There was considerable variation in antibody titres of nonimmunised mares and in the time when the colostrally derived antibody of their foals had declined to low or undetectable titres. In general, antibodies in foals declined to their lowest levels at age 4-8 weeks. Seroconversion occurred in foals age 8-10 weeks, but the precise time depended on maternal titre and the month in which the foal was born. Foals reaching age 8 weeks in late summer showed more marked seroconversion than foals born earlier. The ELISA was used to follow the response to immunisation with the same Triton X-114 extracted material. Six mares immunised before parturition with the antigen in aluminium hydroxide adjuvant developed high titres, up to > 102,400 and transferred them to their foals through colostrum. Their foals responded to immunisation with 0.5-1.0 mg antigen 3, 5, 7 and 9 weeks after birth. Antibody titres following immunisation with similar dosage reached up to > 102,400 in a separate group of foals of nonimmunised mares. Nonvaccinated control foals seroconverted at age 6-8 weeks. The VapA based ELISA is useful to follow the course of natural infection with R. equi or immunisation with VapA based antigen.

Pulmonary eosinophilia associated with increased airway responsiveness in young racing horses.

Horses are known to acquire small airway disease (SAD), an allergen-induced naturally occurring syndrome of reversible obstructive lung disease accompanied by airway hyperresponsiveness and increased inflammatory cell numbers on bronchoalveolar lavage (BAL). This disorder has received scant attention in young racehorses. The purpose of the present report was to examine the effect of BAL eosinophilia in young racehorses on clinical examination, BAL, hematology, airway responsiveness, and on pulmonary function at rest and after a standardized exercise challenge. Five (3 males, 2 females; age 2.6 +/- 0.9 years) with a history of respiratory compromise and BAL eosinophil differential count > 5% and 6 controls (4 males, 2 females; age 3.5 +/- 1.0 years) training and performing to expectation with normal BAL cell differential (eosinophils < 1%) were studied. Respiratory system clinical examination was performed and expressed as a clinical score. Arterial blood gas measurements, CBC, and pulmonary function testing were performed at rest. Pulmonary mechanics measurements were repeated 1 hour and 20 hours after a standardized treadmill exercise challenge. Incremental histamine inhalation challenge was performed and the concentration of histamine effecting a 35% decrease in dynamic compliance (PC35CDyn) was determined. Significant differences were noted between and controls with regard to clinical score (P = .01), blood eosinophils (P = .04), BAL cell count (P = .04), BAL macrophage differential (P = .04), PC35CDyn (P = .008), and tidal volume and respiratory rate at 20 hours following exercise challenge (P = .05). We conclude that pulmonary eosinophilia and airway hyperresponsiveness are manifest in some young horses without overt airway obstruction at rest. We speculate that these may be early events in the natural progression of heaves.

An evaluation of the multiple-breath nitrogen washout as a pulmonary function test in horses.

Multiple-breath nitrogen washouts (MBNW) were performed with 29 light horses. Seven normal horses were used to examine the reproducibility, and 22, ranging from normal to severely diseased, were used to examine the changes in chronic obstructive pulmonary disease (COPD) and the effect of a bronchodilator, salbutamol, on the distribution of ventilation. The MBNW were analyzed using the functional residual capacity (FRC), end-tidal N2 concentration of the final breath of the MBNW (FETN2,fb), end-tidal N2 concentration when the cumulative expired volume was equal to body weight (FETN2,bw), lung clearance index (LCI), Becklake index (BI), mixing ratio (MR), index of distribution of inspiration (IDI), pulmonary N2 clearance delay (PCD) and ventilatory efficiency (EFF). The LCI, MR, IDI, PCD and EFF were calculated at end-tidal N2 concentrations (FETN2), equal to 3%, 2% and FETN2,fb. The EFF was also calculated at FETN2 = 8%, as was BI. The within day coefficient of variation for most indices of the MBNW was 10-15%. None of the indices varied significantly from day-to-day. The FETN2,fb, FETN2,bw, MR, IDI, PCD, and EFFfb, EFF3% and EFF2% differed between the horses with COPD and the normal horses, and all of the indices, except FRC, were correlated with a histopathological score of the small airways. The FETN2,bw appeared to be the most sensitive index in detecting the horses with COPD. In the horses with COPD, salbutamol caused a decrease in FRC and FETN2,fb indicating increased ventilation of the lung. However, LCI increased suggesting a less efficient distribution of ventilation.

Evaluation of a guarded bronchoscopic method for microbial sampling of the lower airways in foals.

A novel method to reduce contamination of the bronchoscope during microbial sampling of the lower airways of foals was evaluated. Methylene blue (MB) was used as a nasopharyngeal dye marker to assess the relative contamination from the upper airways of bronchoalveolar lavage (BAL) specimens obtained by standard bronchoscopy (SB) and a "guarded" bronchoscopic method (GB). For GB, a clear sterile cellulose sheath was fitted over the bronchoscope in an effort to protect the endoscope tip and channel from contamination. Methylene blue was detected visually in seven of eight BAL samples from foals following SB, but in none of the samples recovered by GB (p less than 0.001). Significantly less MB was detected in BAL by spectrophotometry in the GB group as well (p less than 0.02). The GB was next employed to study the microbial flora in the lower airways of healthy weaned foals (n = 30). Bacteria were isolated from 29 of 30 (97%) BAL samples, and in moderate or large numbers from 26 of 30 (87%) of the foals. Potential pathogens, including Bordetella bronchiseptica, Streptococcus zooepidemicus, Staphylococcus aureus, Mycoplasma felis and Streptococcus pneumoniae, were cultured from the lower airways of foals. In conclusion, the bronchoscope and bronchoalveolar lavage specimens were readily contaminated by a dye marker placed in the nasopharynx of foals, and the degree of contamination was significantly reduced by sheathing the endoscope. This contamination during bronchoscopy may obscure the interpretation of isolates from BAL specimens from foals, which may possess a bacterial flora in the lower airways without cytological evidence of inflammation.

Pulmonary structure and function in adult dairy cows with an expanded lung field.

Pulmonary function tests were performed on seven adult dairy cows with an expanded lung field (ExLF) and the results were compared to the values from seven cows with normal lung fields. The cows with ExLF had an increased functional residual capacity (FRC) and end-tidal N2 concentration of the final breath of the multiple-breath N2 washout (FETN2,fb), and an abnormal distribution of ventilation. The measurements of ventilation and gas exchange and pulmonary mechanics did not differ between the two groups. Postmortem examination of the lungs of five of the cows in each group indicated the cows with ExLF had a bronchiolitis with varying degrees of goblet cell hyperplasia, epithelial cell hyperplasia and metaplasia, smooth muscle hypertrophy, inflammatory cell infiltrate, vascular thickening, peribronchiolar fibrosis, and a mild overinflation of the lung. A histopathological score, based on the preceding indices and bronchus-associated lymphoid tissue, was correlated with FRC, FETN2,fb, the lung clearance index and pulmonary resistance. There were no correlations between the histopathological score and any of the measures of ventilation and gas exchange.

Hemostatic studies in racing standardbred horses with exercise-induced pulmonary hemorrhage. Hemostatic parameters at rest and after moderate exercise.

The purpose of this study was to determine whether a defect in hemostasis might be a factor in the etiology of exercise-induced pulmonary hemorrhage (EIPH). Hemostatic parameters were evaluated in 22 EIPH-positive and ten EIPH-negative racing horses while in a rested state. Nineteen EIPH-positive and ten EIPH-negative horses were further evaluated just before and immediately after a 15 min exercise period on a 260 m oval track. When EIPH-positive and EIPH-negative horses were compared at rest, there was no significant difference in any of the coagulation and fibrinolytic parameters studied. There was however, a significant difference in platelet function as assessed by aggregometry. The platelets from affected horses were significantly less responsive than those from nonaffected horses when exposed in vitro to the platelet agonists adenosine diphosphate, collagen and platelet activating factor. Exercise tended to increase the packed cell volume and factor VIII/von Willebrand factor and to decrease platelet aggregation responses to low concentrations of adenosine diphosphate. These effects of exercise however were quantitatively similar in both EIPH-positive and EIPH-negative horses. Reduced platelet function may therefore be a contributing factor in the bleeding characteristic of horses with EIPH.

Changes in the bacterial flora of the upper and lower respiratory tracts and bronchoalveolar lavage differential cell counts in feedlot calves treated for respiratory diseases.

Serial nasopharyngeal swab and bronchoalveolar lavage cultures were used to estimate changes in the bacterial flora of the respiratory tracts of calves during the first month after arrival in the feedlot. Bronchoalveolar lavage (BAL) differential cell counts served to evaluate pulmonary inflammatory changes during this period. Two groups of calves were studied, one consisting of clinically normal controls (n = 60), the other, of cases (n = 59) which received treatment for respiratory disease (penicillin +/- trimethoprimsulfadoxine). A variety of organisms, including Pasteurella multocida, Pasteurella haemolytica, Haemophilus somnus, Mycoplasma bovis and Mycoplasma bovirhinis, were present in the upper and lower airways of both groups during the postarrival period. With the exception of M. bovis, an overall decline in the prevalence of these organisms was observed during the course of the study. In cases, there was a marked decrease in the number of Pasteurella spp. and H. somnus isolates immediately following treatment. For the Pasteurella spp., however, this effect was shortlived as they often appeared to recolonize the respiratory tract within eight days of terminating antimicrobial therapy. Treatment did not appear to affect the frequency of isolating M. bovis. Its prevalence, in both groups of calves, increased to levels approaching 100% during the course of the study. All Pasteurella spp. isolates were tested for susceptibility to several commonly used antimicrobials. Resistance was only evident among P. haemolytica isolated from cases and in every instance this was to a combination of penicillin, ampicillin and tetracycline. Significantly more isolates were resistant after treatment than before. There were BAL differential cell count abnormalities indicative of inflammation in both cases and controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of the duration and distribution of equine influenza virus subtype 2 (H3N8) antigens in experimentally infected ponies in vivo.

The purpose of this experiment was to study the duration and distribution of equine influenza virus in actively infected ponies over a 3 wk period. Pony foals (6-8 mo old) were infected experimentally by nebulizing equine influenza subtype-2 virus ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals (n = 6) had a febrile response, a deep hacking cough and mucopurulent nasal discharge for 7 to 10 d. The virus was isolated from nasopharyngeal swabs of all the ponies 3 and 5 d after infection and all the ponies seroconverted to the virus. Samples were taken from the nasopharynx, mid-trachea and the mainstem bronchus with cytology brushes through an endoscope as well as from bronchoalveolar lavage fluid. On days 3 to 7 post-infection, ciliacytophtorea (the presence of cilia and ciliated plates separated from columnar epithelial cells) was recognized on routine cytological stain. Indirect immunoperoxidase staining utilizing polyclonal antibodies demonstrated viral antigen in intact and fragmented ciliated epithelial cells and in fragments of ciliated plates. The infected cells and cell fragments were particularly evident on days 3 and 5 post-infection in the nasopharynx, mid-trachea and mainstem bronchus and on days 3 to 7 post-infection in the bronchoalveolar lavage samples. On days 7 and 21 post-infection, viral antigen was identified in vacuoles of alveolar macrophage-like cells collected by bronchoalveolar lavage. It can be concluded from this study that equine influenza virus can infect not only the upper airways but also the bronchial epithelium and that viral antigen can persist up to 21 d post-infection.

Pathogenesis and clinical signs of equine herpesvirus-1 in experimentally infected ponies in vivo.

Equine herpesvirus-1 (EHV-1) causes respiratory disease, neonatal death, abortion and neurologic disease. The main purpose of this study was to identify viral antigen in respiratory tract samples by immunoperoxidase staining. Six pony foals were selected on the basis of demonstrating seronegativity to EHV-1 by virus neutralization and housed in isolation. They were infected experimentally by administering EHV-1 nebulized ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals had febrile responses, nasal discharge, and enlarged submandibular lymph nodes. Sporadic coughing was also heard. EHV-1 was isolated from nasopharyngeal swabs of 4/6 ponies and seroconversion was demonstrated in all foals. Bronchoscopic examination of the large airways revealed hyperemia. The incidence of recovery of Actinobacillus suis from nasopharyngeal swabs increased initially, with recovery of Streptococcus zooepidemicus isolates predominating at 3 wk post-infection. Cytology brushes were used to sequentially sample the respiratory tract of the infected ponies at the nasopharynx, mid-trachea and the mainstem bronchus. Bronchoalveolar lavage provided lung cells. Immunocytochemistry techniques were applied to both types of samples to locate EHV-1 antigen. Indirect immunoperoxidase staining of samples utilizing monoclonal antibodies specific for EHV-1 demonstrated viral antigen associated with cellular debris, primarily in the nasopharyngeal samples on days 3-9 post-infection.

Evaluation of an in vitro degranulation challenge procedure for equine pulmonary mast cells.

Pulmonary mast cells (PMC) are important components of the inflammatory process in equine allergic lung diseases such as heaves. Very little, however, is known of the degranulation kinetics of these cells and thus, their pathophysiologic role remains largely speculative. The purpose of this study was to develop a repeatable protocol for in vitro equine PMC degranulation. Five mature horses (sex: 2 M, 3 F; age: 8.8 +/- 6.5 y), historically free of pulmonary disease and normal on clinical respiratory examination, arterial blood gas analysis, pulmonary mechanics testing and histamine inhalation challenge, were studied. Bronchoalveolar lavage was performed on 4 separate occasions, at least 2 d apart, in a different lung lobe on each occasion. The lavage fluid was concentrated by centrifugation. Cells were resuspended in modified HEPES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC concentration determined. Cell inocula containing 30,000 PMC were incubated with 10(-8) to 6 x 10(-5) M A23187. Cells were then separated by centrifugation and histamine release (HR) was determined by fluorometric assay. The procedure was readily performed and yielded sufficient PMC for 30 to 60 inocula per lavage. Maximal HR (34.4 +/- 16.1%) was obtained with 10(-5) M A23187. The degranulation process was largely complete by 20 min but cell lysis was negligible. The challenge was repeatable within horse and produced a mean coefficient of variability of 23.0% following 20 min incubation with 10(-5) M A23187. We conclude that equine PMC degranulation can be repeatably performed in vitro and speculate that this protocol may be useful in further studies on the pathophysiology and treatment of equine allergic lung diseases.