Postnatal changes in epigenetic modifications of neutrophils of foals are associated with increased ROS function and regulation of neutrophil function.

Neonates of all species, including foals, are highly susceptible to infection, and neutrophils play a crucial role in innate immunity to infection. Evidence exists that neutrophils of neonatal foals are functionally deficient during the first weeks of life, including expression of cytokine genes such as IFNG. We hypothesized that postnatal epigenetic changes were likely to regulate the observed age-related changes in foal neutrophils. Using ChIP-Seq, we identified significant differences in trimethylated histone H3 lysine 4, an epigenetic modification associated with active promoters and enhancers, in neutrophils in foals at 30 days of age relative to 1 day of age. These chromatin changes were associated with genes implicated in immune responses and were consistent with age-related changes in neutrophil functional responses including ROS generation and IFN expression. Postnatal changes in epigenetic modifications suggest that environmentally-mediated cues help to promote maturation of neutrophil functional responses. Elucidating the environmental triggers and their signaling pathways could provide a means for improving innate immune responses of neonates to improve their ability to combat infectious diseases.

Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals.

Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and Rhodococcus equi-stimulated mRNA expression of the cytokines interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, and IL-8 using real-time PCR, degranulation by quantifying the amount of ß-D glucuronidase activity, and reactive oxygen species (ROS) generation using flow cytometry. In vivo administration of the CpG-ODN formulation on days 1 and 7 resulted in significantly (P<0.05) increased IFN-γ mRNA expression by foal neutrophils on days 3, 9, and 14. Degranulation was significantly (P<0.05) lower for foals in the CpG-ODN-treated group than the control group at days 3 and 14, but not at other days. No effect of treatment on ROS generation was detected. These results indicate that CpG-ODN administration to foals might improve innate and adaptive immune responses that could protect foals against infectious diseases and possibly improve responses to vaccination.

Macrolide- and rifampin-resistant Rhodococcus equi on a horse breeding farm, Kentucky, USA.

Macrolide and rifampin resistance developed on a horse breeding farm after widespread use was instituted for treatment of subclinical pulmonary lesions in foals. Resistance occurred in 6 (24%) of 25 pretreatment and 8 (62%) of 13 (62%) posttreatment isolates from affected foals. Drug-resistant isolates formed 2 distinct genotypic clusters.

Association of perinatal exposure to airborne Rhodococcus equi with risk of pneumonia caused by R equi in foals.

OBJECTIVE: To determine whether the concentrations of airborne virulent Rhodococcus equi in stalls housing foals during the first 2 weeks after birth are associated with subsequent development of R equi pneumonia in those foals. SAMPLE: Air samples collected from foaling stalls and holding pens in which foals were housed during the first 2 weeks after birth. PROCEDURES: At a breeding farm in Texas, air samples (500 L each) were collected (January through May 2011) from stalls and pens in which 121 foals were housed on day 1 and on days 4, 7, and 14 after birth. For each sample, the concentration of airborne virulent R equi was determined with an immunoblot technique. The association between development of pneumonia and airborne R equi concentration was evaluated via random-effects Poisson regression analysis. RESULTS: Some air samples were not available for analysis. Of the 471 air samples collected from stalls that housed 121 foals, 90 (19%) contained virulent R equi. Twenty-four of 121 (20%) foals developed R equi pneumonia. Concentrations of virulent R equi in air samples from stalls housing foals that developed R equi pneumonia were significantly higher than those in samples from stalls housing foals that did not develop pneumonia. Accounting for disease effects, air sample concentrations of virulent R equi did not differ significantly by day after birth or by month of birth. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of foals to airborne virulent R equi during the first 2 weeks after birth was significantly (and likely causally) associated with development of R equi pneumonia.

Association of airborne concentration of virulent Rhodococcus equi with location (stall versus paddock) and month (January through June) on 30 horse breeding farms in central Kentucky.

OBJECTIVE: To determine whether the concentration of airborne virulent Rhodococcus equi varied by location (stall vs paddock) and month on horse farms. SAMPLE: Air samples from stalls and paddocks used to house mares and foals on 30 horse breeding farms in central Kentucky. PROCEDURES: Air samples from 1 stall and 1 paddock were obtained monthly from each farm from January through June 2009. Concentrations of airborne virulent R equi were determined via a modified colony immunoblot assay. Random-effects logistic regression was used to determine the association of the presence of airborne virulent R equi with location from which air samples were obtained and month during which samples were collected. RESULTS: Of 180 air samples, virulent R equi was identified in 49 (27%) and 13 (7%) obtained from stalls and paddocks, respectively. The OR of detecting virulent R equi in air samples from stalls versus paddocks was 5.2 (95% confidence interval, 2.1 to 13.1). Of 60 air samples, virulent R equi was identified in 25 (42%), 18 (30%), and 6 (10%) obtained from stalls during January and February, March and April, and May and June, respectively. The OR of detecting virulent R equi from stall air samples collected during May and June versus January and February was 0.22 (95% confidence interval, 0.08 to 0.63). CONCLUSIONS AND CLINICAL RELEVANCE: Foals were more likely to be exposed to airborne virulent R equi when housed in stalls versus paddocks and earlier (January and February) versus later (May and June) during the foaling season.

Effects of location for collection of air samples on a farm and time of day of sample collection on airborne concentrations of virulent Rhodococcus equi at two horse breeding farms.

OBJECTIVE: To determine whether airborne concentrations of virulent Rhodococcus equi at 2 horse breeding farms varied on the basis of location, time of day, and month. SAMPLE POPULATION: 2 farms in central Kentucky with recurrent R equi-induced pneumonia in foals. PROCEDURES: From February through July 2008, air samples were collected hourly for a 24-hour period each month from stalls and paddocks used to house mares and their foals. Concentrations of airborne virulent R equi were determined via a modified colony immunoblot technique. Differences were compared by use of zero-inflated negative binomial methods to determine effects of location, time, and month. RESULTS: Whether mares and foals were housed predominantly in stalls or paddocks significantly affected results for location of sample collection (stall vs paddock) by increasing airborne concentrations of virulent R equi at the site where horses were predominantly housed. Airborne concentrations of virulent R equi were significantly higher from 6:00 pm through 11:59 pm than for the period from midnight through 5:59 am. Airborne concentrations of virulent R equi did not differ significantly between farms or among months. CONCLUSIONS AND CLINICAL RELEVANCE: Airborne concentrations of virulent R equi were significantly increased when horses were predominantly housed at the site for collection of air samples (ie, higher in stalls when horses were predominantly housed in stalls and higher in paddocks when horses were predominantly housed in paddocks). Concentrations of virulent R equi among air samples collected between the hours of 6:00 am and midnight appeared similar.

Antimicrobial activity of tulathromycin and 14 other antimicrobials against virulent Rhodococcus equi in vitro.

This study determined the antimicrobial activity of tulathromycin against Rhodococcus equi in vitro. Ninety-eight virulent isolates of R. equi from equine clinical cases were examined, of which 20 isolates were macrolide resistant. A custom 96-well antimicrobial susceptibility testing plate was used, allowing 14 additional antimicrobials to be tested against R. equi. Isolates were cultured with various concentrations of antimicrobials, and minimal inhibitory concentration (MIC) values were determined. Tulathromycin was found to have poor activity in vitro against R. equi isolates susceptible or resistant to macrolides, with MIC50 and MIC90 values >64 ug/mL for all isolates. MIC values for other macrolides tested were similar to previously published data.

Detection of strain variation in isolates of Rhodococcus equi from an affected foal using repetitive sequence-based polymerase chain reaction.

Rhodococcus equi is an important pathogen of foals aged 1-6 months. Evidence exists that foals are exposed to a wide diversity of R. equi strains in their environment. However, limited data are available regarding the extent to which genotypic variation exists among isolates infecting individual foals. Therefore, electrophoresis of repetitive sequence-based polymerase chain reaction (rep-PCR) amplicons in an automated microfluidics chip format was used to genotype 9 virulent R. equi isolates obtained from distinct anatomic locations in a single foal. Four of the isolates were obtained from different regions of the lung, and 5 were from abscessed intra-abdominal lymph nodes (LNs). Six distinct genotypes were identified among the 9 isolates. None of the pulmonary isolates was identical; however, a pulmonary isolate was found to be identical to an isolate recovered from a small intestinal LN, and another pulmonary isolate was identical to an isolate from a mesenteric LN. These results indicate that foals can be infected with multiple strains of virulent R. equi. Furthermore, identical strains can be found in multiple, remote anatomic locations in an infected foal, and this can occur for >1 strain in the same foal. The automated system used in the current study provided a rapid, reproducible, and discriminating method for typing R. equi isolates.

In vitro antimicrobial activity of gallium maltolate against virulent Rhodococcus equi.

The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48 h. Both the MIC(50) and the MIC(90) after 24h of growth were 558 ng/mL (8 µM) and after 48 h of growth were 2230 ng/mL (32 µM). There were no apparent differences between MICs of macrolide-resistant and macrolide-susceptible isolates.

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.