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Interferon Gamma Regulation in the Foal
Department of Veterinary Sciences
Respiratory diseases remain one of the major impediments to the equine industry. Young foals, in particular, suffer frequently and severely from viral and bacterial infections of the respiratory tract. Rhodococcus equi, in particular, remains the most common cause of subacute or chronic abscessating bronchopneumonia in young foals. Epidemiological and experimental data indicate foals less than 1 month of age are at greatest risk for infection.
The underlying hypothesis of this research project is that the decreased production of interferon-gamma by young foals contributes to their susceptibility to infection with Rhodococcus equi and other intracellular pathogens. We have already demonstrated that young foals are most deficient at interferon-gamma production at the time when R. equi exposure likely occurs. Here we propose to analyze cytokine mRNA expression in a large population of foals from multiple farms that have a high incidence of R. equi infections. We also will investigate possible approaches to enhance interferon-gamma production in the foal and thereby increase resistance to infection.
2010 Project Description
A challenge model for Rhodococcus equi infection was developed. A total of 24 foals (average age 4 +/-3 days) were used in this challenge model and two additional foals served as uninfected controls. Foals were challenged with an intrabronchial instillation of R. equi (UKVDL isolate provided by M. Donahue). The isolate was confirmed to contain the virulence-associate plasmid. Bacterial cultures were grown on agar plates overnight prior to inoculation. After sedation, a flexible fiberoptic endoscope was used to deliver the bacteria suspended in 35 ml of sterile PBS into both main bronchi. A high (108) and low (107) dose was administered to 6 and 18 foals, respectively. The foals were returned to their stalls and dams and monitored daily for changes in rectal temperature, respiration, and pulse as determined by physical exam and auscultation of the lungs. Ultrasound scanning was performed weekly beginning one week after the inoculation and continuing throughout the time the foal remained on the study. Blood was obtained via jugular venipuncture on the day of each ultrasound scan for fibrinogen analysis. Clinical signs recorded included lung auscultations, overall attitude, appetite, resting respiratory rate, pulse and fever. Suspect lung lesions (>2 cm) were identified by ultrasound.
A group of 7 foals that developed lesions in their lungs, as identified by ultrasound scanning, or other clinical signs were treated with antibiotics; clarithromycin (7.5 mg/kg) and rifampin (5 mg/kg) given per os twice daily. All foals were euthanized 21 days post challenge. At necropsy, both lungs from each animal were weighed and scored for pneumonia based on the total % of lung field affected. Seven sections of lung were histopathologically evaluated and graded on the distribution, degree of pyogranulomatous inflammation, and alveolar necrosis. A pneumonia score was generated individually for each of the 7 sections and for the whole animal. Sections of abscess wall, pneumonic lung (if abscesses were not evident), or left cranial lung lobe (if neither abscesses nor pneumonia were present) were obtained to analyze fibrosis. Fibrosis scores were generated by staining paraffin embedded tissue sections with trichrome and visually measuring the thickness of the pleura, interstitium, peribronchiolar region, and abscess wall. Lung tissue samples were obtained for bacteriological culturing and mRNA analyses.
Infection with Rhodococcus equi led to subacute or chronic abscessating bronchopneumonia in the foals. All foals developed clinical signs of rhodococcal pneumonia within 21 days post challenge. There was a significant rise in fibrinogen levels post-infection peaking on day 12. At necropsy, all foals exhibited multiple microgranulomas and abscesses in their lungs.
There was a significant effect of dosage on clinical score and bacterial recovery from the lung tissues. There was no effect of dosage on pathological findings at necropsy. Microarray analysis of lung sections indicated increased expression of inflammatory cytokine, chemokine and various proteases in samples collected adjacent to lesions. Expression of these genes decreased in samples collected further from the lesions, though overall expression of the targeted genes remained elevated in these samples when compared to samples from uninfected control lung tissue.
Treatment of the foals with antibiotics significantly reduced bacterial numbers in the lungs. While overall lung pathology and mRNA expression was reduced by antibiotic treatment, this was not statistically significant. We observed a dose-dependent relationship between the number of bacteria cultured from the lung and the dose of R. equi administered. Antibiotic treatment did significantly reduce the bacterial load in the lung and lessened clinical signs, as expected.
Together, these results indicate that a successful challenge model of acute Rhodococcal pneumonia in the foals was employed. However, it should be noted that the lesions were not typical of field cases which present as a chronic abscessating bronchopneumonia. While we did note abcessation in some of the infected foals, this was not the predominant lesion. Had the dose been lower and the incubation period longer, we might have expected to see more typical lesions.
Dawson, T.R.M.Y., Horohov, D.W., Meijer, W.G., Muscatello, G., 2010, Current understanding of the equine immune response to Rhodococcus equi. An immunological review of R. equi pneumonia. Vet Immunol Immunop 135, 1-11.
Sturgill, T.L., Horohov, D.W., 2010, Vaccination Response of Young Foals to Keyhole Limpet Hemocyanin: Evidence of Effective Priming in the Presence of Maternal Antibodies. J Equine Vet Sci 30, 359-364.