Ronald J. Martens

Temporal changes in cytokine expression of foals during the first month of life

Foals are uniquely susceptible to a wide variety of opportunistic infections normally associated with immunodeficiencies. Little is understood about the immune system of foals during the neonatal period. An apparent age-related susceptibility predisposes neonatal foals to infectious diseases and hinders therapeutic and preventative interventions for these diseases. Cytokine expression is correlated with the type of immune response as well as the severity of a disease. In this study, we measured foal peripheral blood mononuclear cell (PBMC)-specific mRNA cytokine expression from 72 foals from three different farms during the first 4 weeks of life. Interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) were cloned and transcribed in vitro to generate antisense probes for ribonuclease protection assays. Using linear mixed-effect models, we determined that IFN-gamma, TGF-beta1, and IL-1alpha increased significantly (P<0.05) with age.

Determination of the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in Rhodococcus equi isolates and treatment outcome in foals infected with antimicrobial-resistant isolates of R equi

OBJECTIVE To determine the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in Rhodococcus equi isolates and to describe treatment outcome in foals infected with antimicrobial-resistant isolates of R equi. DESIGN Cross-sectional study. SAMPLE POPULATION 38 isolates classified as resistant to macrolide antimicrobials or rifampin received from 9 veterinary diagnostic laboratories between January 1997 and December 2008. PROCEDURES For each isolate, the minimum inhibitory concentration of macrolide antimicrobials (ie, azithromycin, erythromycin, and clarithromycin) and rifampin was determined by use of a concentration-gradient test. Prevalence of R equi isolates from Florida and Texas resistant to macrolide antimicrobials or rifampin was determined. Outcome of antimicrobial treatment in foals infected with antimicrobial-resistant isolates of R equi was determined. RESULTS Only 24 of 38 (63.2%) isolates were resistant to >or= 1 antimicrobial. Two isolates were resistant only to rifampin, whereas 22 isolates were resistant to azithromycin, erythromycin, clarithromycin, and rifampin. The overall prevalence of antimicrobial-resistant isolates in submissions received from Florida and Texas was 3.7% (12/328). The survival proportion of foals infected with resistant R equi isolates (2/8 [25.0%]) was significantly less, compared with the survival proportion in foals that received the same antimicrobial treatment from which antimicrobial-susceptible isolates were cultured (55/79 [69.6%]). Odds of nonsurvival for foals infected with resistant R equi isolates were 6.9 (95% confidence interval, 1.3 to 37) times the odds for foals infected with susceptible isolates. CONCLUSIONS AND CLINICAL RELEVANCE Interpretation of the results emphasized the importance of microbiological culture and antimicrobial susceptibility testing in foals with pneumonia caused by R equi.

Chemoprophylactic effects of azithromycin against Rhodococcus equi-induced pneumonia among foals at equine breeding farms with endemic infections.

OBJECTIVE To determine the effect of azithromycin chemoprophylaxis on the cumulative incidence of pneumonia caused by Rhodococcus equi, age at onset of pneumonia, and minimum inhibitory concentration (MIC) of azithromycin for R equi isolates cultured from fecal and clinical samples. DESIGN Controlled, randomized clinical trial. ANIMALS 338 foals born and raised at 10 equine breeding farms; each farm had a history of endemic R equi infections. PROCEDURES Group 1 foals were control foals, and group 2 foals were treated with azithromycin (10 mg/kg [4.5 mg/lb], PO, q 48 h) during the first 2 weeks after birth. Foals were monitored for development of pneumonia attributable to R equi infection and for adverse effects of azithromycin. Isolates of R equi were tested for susceptibility to azithromycin. RESULTS The proportion of R equi-affected foals was significantly higher for control foals (20.8%) than for azithromycin-treated foals (5.3%). Adverse effects of azithromycin treatment were not detected, and there were no significant differences between groups for the MICs of azithromycin for R equi isolates cultured from fecal or clinical samples. CONCLUSIONS AND CLINICAL RELEVANCE Azithromycin chemoprophylaxis effectively reduced the cumulative incidence of pneumonia attributable to R equi among foals at breeding farms with endemic R equi infections. There was no evidence of resistance to azithromycin. Nonetheless, caution must be used because it is possible that resistance could develop with widespread use of azithromycin as a preventative treatment. Further investigation is needed before azithromycin chemoprophylaxis can be recommended for control of R equi infections.

Association of soil concentrations of Rhodococcus equi and incidence of pneumonia attributable to Rhodococcus equi in foals on farms in central Kentucky

OBJECTIVE To determine whether soil concentrations of total or virulent Rhodococcus equi differed among breeding farms with and without foals with pneumonia caused by R equi. SAMPLE POPULATION 37 farms in central Kentucky. Procedures-During January, March, and July 2006, the total concentration of R equi and concentration of virulent R equi were determined by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively, in soil specimens obtained from farms. Differences in concentrations and proportion of virulent isolates within and among time points were compared among farms. RESULTS Soil concentrations of total or virulent R equi did not vary among farms at any time point. Virulent R equi were identified in soil samples from all farms. Greater density of mares and foals was significantly associated with farms having foals with pneumonia attributable to R equi. Among farms with affected foals, there was a significant association of increased incidence of pneumonia attributable to R equi with an increase in the proportion of virulent bacteria between samples collected in March and July. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that virulent R equi were commonly recovered from soil of horse breeding farms in central Kentucky, regardless of the status of foals with pneumonia attributable to R equi on each farm. The incidence of foals with pneumonia attributable to R equi can be expected to be higher at farms with a greater density of mares and foals.

Protective Role of Neutrophils in Mice Experimentally Infected with Rhodococcus equi

ABSTRACT Neutrophils are important in controlling early infections with the intracellular bacterium Rhodococcus equi. Antineutrophil monoclonal antibody (RB6-8C5)-induced neutrophil deficiency during the first week after experimental infection of mice with R. equi resulted in more severe disease and significantly increased tissue concentrations of R. equi.

Prevalence of Virulent Rhodococcus Equi in Soil from Five R. Equi-Endemic Horse-Breeding Farms and Restriction Fragment Length Polymorphisms of Virulence Plasmids in Isolates from Soil and Infected Foals in Texas

Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R. equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15–17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims. Rhodococcus equi were isolated from soil of all 5 farms; however, virulent R. equi were only isolated from 3 of the 5 farms and represented 18.8% (87 of 462) of total isolates. Of the 87 virulent soil isolates, 56 (64.5%) contained an 85-kb type I plasmid, 23 (26.4%) an 87-kb type I plasmid, 7 (8%) a newly defined 85-kb type III plasmid (Tx 43), and 1 (1.1%) a newly defined 85-kb type IV plasmid (Tx 47). Of the 100 isolates from infected foals, 96 were virulent. Of the 96 virulent isolates, 51 (53.1%) contained an 85-kb type I plasmid, 39 (40.6%) an 87-kb type I plasmid, 4 (4.2%) an 85-kb type III plasmid (Tx 43), and 2 (2.1%) an 85-kb type IV plasmid (Tx 47). There are at least 4 different R. equi virulence-associated plasmids in Texas, 2 of which have not previously been described. Based upon virulence plasmid typing, there is geographic diversity among isolates of R. equi from clinical and environmental samples on horse-breeding farms in Texas. There is not a strong correlation between the presence of virulent R. equi in farm soils and the R. equi disease status of those farms.

Basal and stimulus-induced cytokine expression is selectively impaired in peripheral blood mononuclear cells of newborn foals.

Neonates are thought to be generally deficient in production of Th-1-associated cytokines at birth, and thereby more susceptible to bacterial infections. Using neonatal foals as a model, this study examined the age-dependent maturation of both basal and stimulus-induced immune responses, as reflected by the expression of a panel of Th-1-associated and pro-inflammatory cytokines. Results showed that although the basal production of IFN-gamma and IL-6 was impaired (P<0.05) in PBMCs of neonatal foals at birth, the basal production of IL-8, IL-12(p35/p40) and IL-23(p19/p40) were either in excess of or comparable to that of older foals. In response to Rhodococcus equi and CpG-ODN stimulation in vitro, PBMCs of neonatal foals showed increased (P<0.05) expression of IFN-gamma and IL-6, and preferentially increased expression of either IL-23(p19/p40) with R. equi stimulation or IL-12(p35/p40) with CpG-ODN stimulation. The magnitude of these stimulus-induced responses (except for IL-23p19), were significantly (P<0.05) less for newborn foals than for older foals. The selective impairment of age-dependent basal and stimulus-induced cytokine expression by newborn foals may reflect the different functional state of various TLR pathways in newborns, and be directly associated with their age-dependent susceptibility to infection. Our results indicate that CpG-ODNs can selectively stimulate deficient cytokines (P<0.05) from PBMCs in newborn foals in vitro, suggesting immunoprophylactic or therapeutic potential of CpG-ODNs.

Virulence of Rhodococcus equi Isolated from Cats and Dogs

ABSTRACT Nine cat isolates and nine dog isolates of Rhodococcus equi from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent R. equi strains and contained no virulence plasmids.

Study of serum amyloid A concentrations as a means of achieving early diagnosis of Rhodococcus equi pneumonia

REASONS FOR PERFORMING STUDY Prognosis of Rhodococcus equi pneumonia can be challenging because the course of the disease is often insidious and overt clinical signs are subtle. Early diagnosis is considered desirable because it may offer the chance of more successful implementation of treatment and, thereby, improved outcome. Serological tests have previously failed to be accurate for early detection or diagnosis. Measurement of serum amyloid A (SAA) prior to and at the time of clinical signs was therefore chosen in order to assess its potential clinical use. OBJECTIVE To determine whether SAA concentrations differentiate foals affected with R. equi pneumonia from unaffected foals, either prior to the onset of disease or at the time of onset of clinical signs. HYPOTHESIS SAA concentrations are significantly higher among foals that develop R. equi pneumonia than in foals from the same environment that remain clinically unaffected. METHODS Serum samples were obtained from 212 foals 7-14 days and 196 foals 21-28 days post partum, and from affected foals and age-matched controls at the time of onset of signs of pneumonia. SAA concentration was determined for each sample. RESULTS There were no significant differences between SAA concentrations of foals with R. equi and clinically unaffected foals during the 2 periods of examination or at the time of onset of clinical signs of R. equi pneumonia. CONCLUSIONS Concentrations of SAA are variable among foals with R. equi pneumonia and cannot be used reliably either as an ancillary diagnostic tool or to screen for early detection of disease during the first month post partum. POTENTIAL RELEVANCE Bimonthly monitoring concentration of SAA is not useful as a screening test for early detection of R. equi pneumonia and does not facilitate diagnosis of this disease when used according to the protocol of this study.

Age-related changes in cytokine expression by neutrophils of foals stimulated with virulent Rhodococcus equi in vitro

Although evidence exists that neutrophils play a vital role in resistance to infection with Rhodococcus equi, the means by which neutrophils exert their effects have not been clearly defined. In the present study we evaluated differences in cytokine expression by unstimulated and R. equi-stimulated neutrophils obtained from newborn foals and subsequently at 2-, 4-, and 8-weeks of age. Stimulation with virulent R. equi induced significantly (P<0.05) greater expression of IFNgamma, TNFalpha, IL-6, IL-8, IL-12p40, IL-12p35, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, and there were significant effects of age on expression of IL-6, IL-8, IL-12p40 and IL-23p19. Neutrophil expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. Expression of IL-12p40 by R. equi-stimulated neutrophils from newborn and 2-week-old foals did not differ from that of unstimulated neutrophils; however, expression of IL-12p40 by neutrophils from 4- and 8-week-old foals was significantly greater when stimulated by R. equi than without stimulation. These results demonstrate that foal neutrophils increase mRNA expression of many pro-inflammatory cytokines, including IFNgamma, in response to in vitro stimulation with R. equi, and that the magnitude of this expression with respect to IL-6, IL-8, IL-12p40 and IL-23p19 is influenced by age. The clinical importance of the age-related difference in R. equi-induced expression of IL-12p40 to susceptibility to R. equi pneumonia remains to be determined.