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

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  • 1 Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.
  • | 2 Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.
  • | 3 Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.
  • | 4 6666 Ranch, 1102 Dash For Cash Rd, Guthrie, TX 79236.
  • | 5 6666 Ranch, 1102 Dash For Cash Rd, Guthrie, TX 79236.
  • | 6 Department of Animal Hygiene, School of Veterinary Medicine and Animal Sciences, Kitasoto University, Towada, Aomori 034-8628, Japan.

Abstract

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.

Abstract

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.

The gram-positive, facultative intracellular bacterium Rhodococcus equi is an important cause of disease and death in foals worldwide.1–3 Organisms that are virulent in foals carry an 80- to 90-kbp plasmid with a pathogenicity island that encodes at least 1 gene product necessary for causing disease in foals, namely virulence-associated protein A.1 Organisms that lack this plasmid are avirulent in foals but are commonly isolated from samples of air, feces, and soil at farms with horses.4–8 It is generally believed that R equi pneumonia in equids results from inhalation of aerosolized or airborne virulent bacteria.1–3 At the farm level, there is evidence that the cumulative incidence of R equi pneumonia at breeding farms is correlated with concentrations of virulent R equi in the air at those locations.8 Moreover, concentrations of airborne virulent R equi are generally higher in barns and stables than in paddocks.6 Evidence of exposure to airborne R equi and subsequent development of R equi pneumonia at the level of the individual foal is, to our knowledge, limited to a single report.9 Although a positive association between detection of virulent R equi in air samples collected from foals’ stalls at day 7 after birth and subsequent development of pneumonia was identified in that study,9 no similar association between detection of virulent R equi in air samples collected from foals’ stalls at birth or at 2 weeks of age and subsequent development of pneumonia was evident. Thus, data from that study were somewhat ambiguous. A major limitation of that report9 was that it included data from 47 foals, only 7 of which developed R equi pneumonia. The purpose of the study reported here was to determine whether concentrations of airborne virulent R equi in stalls housing foals during the first 2 weeks after birth are associated with subsequent development of R equi pneumonia in those foals. We elected to focus on foals during the first 2 weeks after birth because of clinical and epidemiological evidence of infection during this period10,11 and to be consistent with the aforementioned foal-level study9 of airborne R equi.

Materials and Methods

Farm and location of air sample collections—The farm at which the study was performed was selected because the farm veterinarian and general manager (GPB) were conducting an evaluation of screening tests for R equi pneumonia in foals, which was directed by one of the authors (MKC) during 2011, and because the cumulative incidence of R equi pneumonia in foals at that site during 2008, 2009, and 2010 was ≥ 15%. As part of that screening test evaluation, treatment was not initiated for any foal on the basis of screening test results and the veterinarians making decisions about diagnosis and treatment of R equi pneumonia were not informed of the results of testing. All foals born in or brought into a foaling stall within 18 hours after birth were eligible for inclusion in the study of this report. Foals born in or brought into foaling stalls on the day of birth were maintained in the foaling stall for 2 to 3 days and were moved to smaller holding pens adjacent to the foaling barn on day 3 or 4 after birth. The holding pens included a covered area (approx 3.66 × 3.66 m) and an uncovered area (approx 3.66 × 18.29 m). Foaling stalls were constructed of wood and were solid; they were bedded with barley or wheat straw. Holding pens were constructed of steel pipe rails (ie, not solid); they were bedded with sand. The farm owner's authorized agent (GPB) provided consent for collection of air samples from these locations for purposes of this project. The study period was January through May 2011. Collection of samples from foals for diagnostic testing and disease classification was approved by the Clinical Research Review Committee of the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

Air sample collection—For each foal, an air sample was collected from the foaling stall on day 1 after birth and from holding pens on days 4, 7, and 14 after birth. A portable air sampling devicea was used for collecting air samples. Culture plates (100 mm in diameter) containing a modified NANAT medium (selective for R equi and commonly used for epidemiological studies of R equi4,5,7,9) were used during sample collection, as previously described.9 To approximate methods of air sample collection used in previous studies,6–9 the air sampler was placed on the ground to collect air at a height of approximately 10 cm above the stall floor or paddock ground. For each sample collection, 500 L of air was aspirated at a rate of 100 L/min onto a modified NANAT culture plate (1 plate/air sample). Before each sample was collected, the sieve of the air sampler was disinfected with an isopropanol wipe.12 On each day of collection, air samples were collected between 6 am and 6 pm; most samples were collected between 6 am and 12 pm. During each air sample collection, the individual mare and foal pair for which the sample was being collected were present in the stall or pen.

Microbiological culture and modified colony immunoblot assay—The methods for microbiological culture and the modified colony immunoblot have been reported previously.4,5,7,9 Culture plates were transported chilled and with icepacks in insulated containers to the Equine Infectious Disease Laboratory at Texas A&M University. Upon arrival, plates were incubated at 37°C for 48 hours and then analyzed for the presence of R equi. Rhodococcus equi was identified on the basis of morphological characteristics: semitransparent, salmon-colored, smooth, glistening, mucoid colonies with irregularly round or teardrop shape, which may or may not coalesce. For each air sample, a total (ie, virulent plus avirulent) concentration of R equi was determined by counting the number of CFUs identified as R equi per plate (ie, per air sample [500 L of air collected at a rate of 100 L/min]) and recorded, and plates that were identified as having R equi present were then tested for virulent R equi by immunoblotting.

The concentration of virulent R equi in each air sample was determined by use of a modified immunoblotting technique for detection of virulence-associated protein A7,9 that allows for the quantification of virulent R equi within a background of bacterial and fungal contamination. For each set of colony immunoblots performed, a positive and a negative control specimen were included. Nitrocellulose membranesb were placed onto the modified NANAT culture plates that contained at least 1 CFU of R equi after incubation. Once the nitrocellulose membranes were completely saturated, they were removed and air-dried for 30 minutes at room temperature (approx 22°C) and then baked in a hybridization ovenc at 100°C for 1 minute. Membranes were then incubated in a 5% nonfat dry milkd solution diluted in TBSSe at 37°C for 1 hour to block unbound sites. The nitrocellulose membranes were then washed 3 times (10 min/wash) in TBSS with 0.05% Tween 20.f Membranes were incubated (approx 18 hours) at 4°C on a rocking platformg with the monoclonal antibody (provided by ST) diluted 1:10,000 in 5% nonfat dry milk. Membranes underwent 3 washes (10 min/wash) with fresh TBSS at 37°C. Horseradish peroxidase–conjugated goat IgG fraction against mouse IgGh (diluted 1:2,000 in 5% nonfat dry milk) was added to the membranes, which were then incubated for 1 hour at 37°C. Membranes underwent 3 washes (10 min/wash) with fresh TBSS at 37°C, 1 wash with citrate-EDTA buffer (10mM sodium citratei and 10mM EDTAj [pH, 5.0]) for 5 minutes at 37°C, 1 wash with citrate-EDTA buffer and 1% dextran sulfatek for 10 minutes at 37°C, and 3 washes (5 min/wash) with citrate-EDTA buffer at 37°C. Following addition of the substrates 3,3′,5,5′-tetramethylbenzidinel and hydrogen peroxide, the membranes were then incubated at 37°C for 30 minutes or until the positive control membrane was sufficiently developed. The substrate was discarded, and distilled water was added to terminate development. On visual examination, virulent R equi colonies were determined as those that appeared blue; colonies of avirulent R equi and contaminant bacteria remained colorless. Air sample concentrations of virulent R equi were expressed as the number of CFUs per plate (ie, per air sample [500 L of air collected at a rate of 100 L/min]).

The positive and negative controls included in each set of colony immunoblots consisted of pure cultures of virulent (ATCC strain 33701) and avirulent (ATCC strain 33703) R equi grown on modified NANAT culture plates. The pure culture strains had been previously grown in R equi minimal medium,13 and 10-fold serial dilutions were performed with PBS solution.m One hundred microliters of the dilution containing approximately 104 CFUs of R equi/mL was plated for each strain. Colony immunoblot control plates were incubated under the same conditions as the air sample plates.

Classification of disease status—All foals were monitored daily by farm personnel for clinical signs of pneumonia until 20 weeks after birth. Clinical signs suggestive of pneumonia included fever, lethargy, signs of depression, cough, nasal discharge, polysynovitis, tachypnea, increased respiratory effort, respiratory distress, and detection of a tracheal rattle or pulmonary crackles or wheezes via auscultation. For each foal that developed clinical signs of pneumonia, thoracic ultrasonography and collection of a transendoscopic TBA sample with a commercially available triple-guarded cathetern were performed. Between uses, the endoscope was disinfected with a 3.4% glutaraldehyde solutiono following a standard protocol used in our laboratory and known to be microbicidal against R equi. Each sample of TBA fluid was submitted for microbiological culture and cytologic evaluation to the Texas Veterinary Medical Diagnostic Laboratory in College Station, Tex. A foal was classified as having R equi pneumonia when it had clinical signs of pneumonia at 3 to 20 weeks after birth and assessments yielded at least 2 of the following findings: R equi detected in TBA fluid via microbiological culture, cytologic evidence of gram-positive intracellular coccobacilli in the TBA sample, and ultrasonographic evidence of peripheral pulmonary consolidation or abscesses at the time of examination for clinical signs of pneumonia.

Data analysis—Data were tabulated for descriptive purposes; however, these summary tables did not account for repeated observations on individual foals. The air sample concentrations (the number of CFUs per plate [ie, per air sample {500 L of air collected at a rate of 100 L/min}]) of virulent R equi (primary aim) and total R equi (virulent and avirulent organisms; secondary aim) were analyzed as counts that had values of 0 and non-0 positive integers. A generalized linear mixed model with a Poisson link was used for analysis of the these count data for association with pneumonia, age at time of air sample collection, and month of sample collection; individual foal was modeled as a random effect to account for repeated measures (random-effects Poisson regression analysis). Confidence intervals (95%) were calculated via random-effects Poisson regression analysis with maximum likelihood methods. Posthoc random-effects logistic regression analysis also was conducted by use of a generalized linear mixed model with a logit link for the binary outcome of R equi (virulent or total) counts dichotomized as either < 2 or ≥ 2 (representing < 2 CFUs/500 L of air or ≥ 2 CFUs/500 L of air). Results of logistic regression models were summarized as ORs and CIs that were estimated via maximum likelihood methods. The correlation structure of measures used for random-effects models was that of compound symmetry. All analyses were performed with statistical software,p and a value of P < 0.05 was considered significant for all inferential analyses. Model fit was assessed visually by plots of standardized residuals and random-effects residuals versus quantiles of a standard normal distribution.

Results

One hundred twenty-one foals were included in the study. Of the 121 foals, 24 (20%) developed R equi pneumonia. These 24 foals each developed clinical signs of pneumonia and had ultrasonographic evidence of areas of pulmonary consolidation or abscess formation; microbiological culture of the sample of TBA fluid obtained from each foal with pneumonia yielded growth of R equi. At the time of detection of pneumonia, the median age of the 24 affected foals was 58 days (range, 36 to 92 days). The other 97 foals did not develop clinical signs of pneumonia.

On the basis of the study design, an air sample was to be collected from the foaling stall on day 1 after birth and from holding pens on days 4, 7, and 14 after birth for each foal. Of the 484 total samples of air that could have been collected (4 samples for each of the 121 foals), 471 (97%) were collected. Samples were not available for analysis often because of lack of available staff to perform the collections; missed sample collection from stalls or pens occurred at random for various foals at each time point.

Most air samples (381/471 [81%]) yielded virulent R equi counts of 0 (ie, 0 CFUs/air sample; Table 1); counts of virulent R equi ranged from 0 to 4/culture plate across which a sample of 500 L of air had been aspirated. Random-effects Poisson regression analysis revealed no significant association of virulent R equi counts with either age (1 [reference category], 4, 7, or 14 days) or month of birth (January [reference category], February, March, April, or May); data were tabulated for descriptive purposes. Although air sample concentrations of virulent R equi appeared to be highest for foals born during the month of March, compared with findings of foals born during the other months, this difference was not significant (P = 0.099). Concentrations of virulent R equi in air samples collected for foals that subsequently developed R equi pneumonia were significantly (P = 0.05) higher than those in air samples collected for foals that did not subsequently develop R equi pneumonia. Across all time points (ie, days after birth of foal), virulent R equi counts in air samples from stalls or pens of foals that developed disease were higher by a mean value of 0.5 (95% CI, 0.1 to 0.9 counts), compared with findings for foals that did not develop disease. Adjusting for age via multivariable random-effects Poisson regression analysis, pneumonia remained significantly associated with higher counts of airborne virulent R equi (Tables 2 and 3); results also remained significant for association of disease with higher airborne virulent R equi concentration after adjusting for effects of age and month of birth.

Table 1—

Distribution of concentrations of virulent Rhodococcus equi in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals during the 2-week period after birth (4 samples/foal) with data categorized on the basis of day after each foal's birth, month of foal's birth, and whether foals did (n = 24) or did not (97) develop pneumonia caused by R equi.

VariableVirulent R equi concentration (CFUs/500 L of air)
01234Total
Day after birth of foal
   19517710120
   410113510120
   79414611116
   149116512115
   Total381602343471
Month of foal's birth
   January60521169
   February10311300117
   March105251321146
   April8517311107
   May28220032
   Total381602343471
Development of R equi pneumonia in foal
   No309501332377
   Yes7210101094
   Total381602343471

Data are reported as number of air samples.

A portable air sampler was used to collect 500 L of air (at approx 10 cm above the stall floor or ground) at a rate of 100 L/min onto a culture plate (1 plate/air sample) at days 1, 4, 7, and 14 after each foal's birth. Air sample concentrations of virulent R equi were expressed as the number of CFUs per plate (ie, per air sample [500 L of air collected at a rate of 100 L/min]) and recorded as counts. Some samples were not available for analysis, often because of lack of available staff to perform the collections; missed sample collection from stalls or pens occurred at random for various foals at each time point.

Table 2—

Distribution of concentrations of virulent R equi in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals on days 1, 4, 7, and 14 after each foal's birth with data categorized on the basis of whether foals did (n = 24) or did not (97) develop pneumonia caused by R equi.

Day after birth of foalDevelopment of R equi pneumonia in foalVirulent R equi concentration (CFUs/500 L of air)
01234Total
1No781540097
Yes17231023
Total9517710120
4No821031096
Yes19320024
Total10113510120
7No761141193
Yes18320023
Total9414611116
14No731421191
Yes18230124
Total9116512115

Data are reported as number of air samples.

See Table 1 for remainder of key.

Table 3—

Results of multivariable random-effects Poisson regression analysis for the association of concentrations of virulent R equi in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals, including 24 foals that developed R equi pneumonia, during the 2-week period after birth (4 samples/foal) with development of pneumonia and age of foal, or development of pneumonia, age of foal, and month of birth of foal.

AssociationVariableCoefficient95% CIP value
Airborne virulent R equi counts with pneumonia and agePneumonia0.5< 0.1 to 0.90.050
Day after birth (relative to day 1)
   Day 4–0.3–0.8 to 0.30.317
   Day 70.0–0.5 to 0.50.978
   Day 140.1–0.4 to 0.60.622
Airborne virulent R equi counts with pneumonia, age, and month of birthPneumonia0.5< 0.1 to 0.90.045
Day after birth (relative to day 1)
   Day 4–0.3–0.8 to 0.30.369
   Day 70.0–0.5 to 0.50.990
   Day 140.1–0.5 to 0.70.685
Month of birth (relative to January)
   February–0.5–1.3 to 0.30.249
   March0.6–0.1 to 1.20.099
   April0.2–0.5 to 0.90.596
   May–0.3–1.5 to 0.80.577

See Table 1 for key.

Examination of the data suggested that the difference between affected and unaffected foals appeared to be associated primarily with counts of 2 virulent R equi colonies/500 L of air in the foals’ environment. Thus, we created a binary variable for virulent R equi counts < 2 CFUs/500 L of air or ≥ 2 CFUs/500 L of air. Via random-effects logistic regression analysis (ie, a generalized linear model with a logit link), this binary outcome was significantly (P = 0.009) associated with increased odds of disease: for foals for which samples yielded ≥ 2 CFUs of virulent R equi/500 L of air, there was a 3.9-fold (95% CI, 1.4-to 10.6-fold) increase in the odds of disease, relative to foals for which samples yielded < 2 CFUs of virulent R equi/500 L of air. This association remained significant even after adjusting for effects of age or age and month of birth (Table 4).

Table 4—

Results of multivariable random-effects logistic regression analysis for the association of concentrations of virulent R equi ≥ 2 CFUs (vs < 2 CFUs) in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals, including 24 foals that developed R equi pneumonia, during the 2-week period after birth (4 samples/foal) with development of pneumonia and age of foal, or development of pneumonia, age of foal, and month of birth of foal.

AssociationVariableOR95% CIP value
Airborne virulent R equi counts ≥ 2 CFUs with pneumonia and agePneumonia3.91.4 to 10.60.009
Day after birth (relative to day 1)
   Day 40.70.3 to 1.30.228
   Day 71.10.6 to 2.10.720
   Day 141.10.6 to 2.10.721
Airborne virulent R equi counts ≥ 2 CFUs with pneumonia, age, and month of birthPneumonia4.21.4 to 11.90.009
Day after birth (relative to day 1)
   Day 40.70.3 to 1.30.239
   Day 71.10.6 to 2.00.776
   Day 141.10.6 to 2.00.755
Month of birth (relative to January)
   February0.40.1 to 2.00.240
   March2.30.6 to 9.50.250
   April0.70.1 to 3.80.715
   May0.90.1 to 7.40.897

See Table 1 for key.

Results of analysis of total R equi counts in the collected air samples were similar to those for virulent organisms, although concentrations of total R equi counts were generally higher. Of the 471 air samples, 297 (63%) yielded a total R equi count of 0 CFUs/500 L of air; counts of R equi colonies ranged from 0 to 14 CFUs/500L of air (Table 5). Random-effects Poisson regression analysis revealed that, after adjusting for effects of age or age and month of birth, total R equi counts in air samples collected from pens and stalls housing foals that developed R equi pneumonia were significantly higher than total R equi counts in air samples collected from pens and stalls housing foals that did not develop R equi pneumonia (Table 6). By use of the binary variable of total R equi counts < 2 CFUs/500 L of air or total R equi counts ≥ 2 CFUs/500 L of air and random-effects logistic regression analysis, the odds of disease (OR, 2.1; 95% CI, 1.1 to 3.9) was significantly (P = 0.017) higher for foals for which air samples had 2 CFUs of R equi, compared with findings for foals for which air samples had < 2 CFUs of R equi, after adjusting for effects of age or effects of age and month of birth (Table 7).

Table 5—

Distribution of concentrations of total (virulent and avirulent) R equi in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals during the 2-week period after birth (4 samples/foal) with data categorized on the basis of whether foals did (n = 24) or did not (97) develop pneumonia caused by R equi.

Airborne virulent R equi concentration (CFUs/500 L of air)Development of pneumoniaTotal
NoYes
0246 (65)51 (54)297 (63)
161 (16)13 (14)74 (16)
234 (9)15 (16)49 (10)
317 (4)6 (6)23 (5)
47 (2)2 (2)9 (2)
54 (1)0 (0)4 (1)
65 (1)3 (3)8 (2)
71 (< 1)1 (1)2 (< 1)
81 (< 1)1 (1)2 (< 1)
90 (0)0 (0)0 (0)
100 (0)1 (1)1 (< 1)
110 (0)1 (1)1 (< 1)
120 (0)0 (0)0 (0)
130 (0)0 (0)0 (0)
141 (< 1)0 (0)1 (< 1)
Total377 (100)94 (100)471 (100)

Data are reported as number of air samples (%).

See Table 1 for remainder of key.

Table 6—

Results of multivariable random-effects Poisson regression analysis for the association of concentrations of total (virulent and avirulent) R equi in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals, including 24 foals that developed R equi pneumonia, during the 2-week period after birth (4 samples/foal) with development of pneumonia and age of foal, or development of pneumonia, age of foal, and month of birth of foal.

AssociationVariableCoefficient95% CIP value
Airborne total R equi counts with pneumonia and agePneumonia0.5< 0.1 to 0.90.012
Day after birth (relative to day 1)
   Day 4–0.2–0.6 to 0.20.332
   Day 70.1–0.3 to 0.40.806
   Day 140.1–0.3 to 0.40.649
Airborne total R equi counts with pneumonia, age, and month of birthPneumonia0.60.1 to < 1.00.009
Day after birth (relative to day 1)
   Day 4–0.2–0.6 to 0.20.323
   Day 70.1–0.3 to 0.40.812
   Day 140.1–0.3 to 0.40.660
Month of birth (relative to January)
   February0.0–0.6 to 0.60.927
   March0.60.0 to 1.10.060
   April0.2–0.4 to 0.80.497
   May–0.1–0.9 to 0.80.921

See Table 1 for key.

Table 7—

Results of multivariable random-effects logistic regression analysis for the association of concentrations of total (virulent and avirulent) R equi ≥ 2 CFUs (vs < 2 CFUs) in 471 samples of air (500 L each) collected at a breeding farm from foaling stalls and mare-foal pens used to house 121 foals, including 24 foals that developed R equi pneumonia, during the 2-week period after birth (4 samples/foal) with development of pneumonia and age of foal, or development of pneumonia, age of foal, and month of birth of foal.

AssociationVariableOR95% CIP value
Airborne total R equi counts ≥ 2 CFUs with pneumonia and agePneumonia2.11.1 to 3.90.017
Day after birth (relative to day 1)
   Day 41.00.5 to 1.80.967
   Day 71.20.7 to 2.20.462
   Day 141.40.8 to 2.50.249
Airborne total R equi counts ≥ 2 CFUs with pneumonia, age, and month of birthPneumonia2.21.2 to 4.00.014
Day after birth (relative to day 1)
   Day 41.00.5 to 1.80.949
   Day 71.20.7 to 2.20.470
   Day 141.40.8 to 2.50.263
Month of birth (relative to January)
   February1.10.4 to 2.80.875
   March2.91.2 to 7.10.017
   April2.10.8 to 5.50.115
   May1.70.5 to 1.70.378

See Table 1 for key.

Discussion

Although virulent R equi are necessary for the development of pneumonia attributed to R equi in foals, the role of the environmental exposure as a component cause of the disease remains uncertain. Evidence exists that the presence or concentration of virulent R equi in feces of dams or soil is not associated with increased odds of R equi pneumonia in foals at breeding farms.4,5,8 In contrast, concentrations of airborne virulent R equi have been reported to correlate with cumulative incidence of R equi at Thoroughbred breeding farms in Australia.8 Although informative at the level of farm, these data do not provide evidence of exposure at the level of individual foals. Because the cumulative incidence of R equi pneumonia among foals at affected farms is typically < 30%,4,5,8,11,14,15 investigation of exposure at the level of the individual foal is warranted to elucidate the role of environmental exposure in the risk of disease.

Results of the present study indicated that concentrations of airborne virulent R equi (and total [virulent and avirulent] R equi) in air samples from stalls and holding pens used to house foals (and their dams) during the first 2 weeks after birth were significantly higher for foals that subsequently developed R equi pneumonia, compared with findings for foals that did not subsequently develop R equi pneumonia. These findings are consistent with results of a smaller scale investigation by our group at a farm in Kentucky, which indicated that detection of virulent R equi in air samples from stalls housing 7-day-old foals that later developed pneumonia attributed to R equi was significantly more likely than detection of virulent R equi in air samples from stalls housing 7-day-old foals that remained unaffected.9 However, unlike that investigation, there were no significant differences among CFU counts of virulent R equi in air samples on days 1, 4, 7, and 14 after birth in the present study. It is unclear whether this discrepancy in results between the 2 studies reflects differences between the farms’ management and environmental conditions, or chance alone.

Although not significant, there was weak evidence in the present study that concentrations of virulent and total R equi in air samples from foaling stalls and pens were significantly higher during the birth month of March, compared with findings for the birth months of January, February, April, or May. Reasons for this finding are unclear. In Kentucky, it was observed that concentrations of airborne virulent R equi were highest during the 2-month period of January and February, followed by the 2-month period of March and April, and then of May and June.16 In Australia, airborne concentrations have been found to be higher during warmer months, but the evaluated air samples were collected from outdoor settings (ie, lanes and paddocks).8 Because the greatest number of foals were born during the month of March in the present study, it is possible that the higher density of mares and foals in barns and pens or a greater level of activity of horses or people might have contributed to an increase in airborne concentrations of R equi.7,9

The present study had a number of important limitations. The magnitudes of observed effects were relatively modest. Consistent with results of other studies,6–9,16 air samples in the present study were collected approximately 10 cm above the ground surface of stalls or pens. Although neonatal foals spend a large proportion of their time in recumbency,13,17 the sample collection site we used may have been a poor surrogate for concentrations of virulent R equi to which foals were exposed at the level of their nares, where inhalation of infectious agents is presumed to occur. Moreover, the 5-minute sample collection period may have yielded poor indication of the cumulative exposure of foals during a 24-hour period. Developing techniques for continuous air sample collection at the level of the foals’ nares over longer periods (eg, 24 hours) would better reflect exposures of individual foals, and might lead to larger magnitudes of estimated effects. The sample collection technique would need to accommodate a large number of foals because only a proportion of foals (typically < 30%) will develop R equi pneumonia on farms in which the organism is endemic.

In the present study, air sample collection was restricted to 4 time points during the first 2 weeks after birth of the foals. Our rationale for collecting samples during the first 2 weeks after birth was based on the premise that causes should precede effects: pneumonia caused by R equi generally is not evident in foals ≤ 2 weeks of age. Clinical and epidemiological data indicate that many foals become infected during the early postnatal period.10,11 Thus, higher concentrations of virulent R equi in the air in stalls of ≤ 2-week-old foals that subsequently develop pneumonia caused by this bacterium would be more likely to be causally associated with disease than samples collected when foals were older because the early sample collection should have preceded disease (ie, causes must precede effects). Nevertheless, it is possible that concentrations of airborne virulent R equi in stalls and pens (or paddocks where foals were later housed) to which foals are exposed at older ages also may be associated with pneumonia caused by the bacterium.

Another limitation of the present study was that it is possible that the significant association between higher concentrations of airborne virulent R equi with subsequent pneumonia caused by this bacterium in foals is not directly causal. For example, it is possible that the higher concentrations of virulent R equi in the air samples reflected higher concentrations of other particulates or poorer ventilation at the time that the air samples were collected. Conceivably, poorer ventilation and inhalation of particulates that are damaging to the lungs could predispose foals to subsequent development of R equi pneumonia, such that the concentration of virulent (or total) R equi is simply a surrogate marker of the causal exposure.

Variation in the air sample concentration and cumulative incidence of R equi pneumonia among farms has been reported,7,8,14–16 but the present study provided data from a single farm only. This limitation was somewhat diminished by the consistency of association between the presence of airborne virulent R equi and subsequent development of pneumonia attributed to R equi at a farm in Kentucky.9 In previous studies,7,9,16 we recorded R equi concentrations in air samples as CFUs of R equi/m3 rather than as counts (CFUs of R equi/500 L of air). The count (concentration) data in the present study can be converted easily to units of CFUs/m3 by doubling: for example, 1 CFU of R equi/500 L of air collected over 5 minutes = 2 CFUs of R equi/m3. We elected to use the number of colony counts directly observed on plates because the data were easily interpretable. Qualitative results were unchanged by conversion to units of CFU/m3, as one could predict from multiplying all results by a constant (ie, factor of 2).

Results of the present study have provided evidence that concentrations of airborne virulent R equi during the first 2 weeks after birth are associated with the risk of subsequent development of pneumonia in foals caused by this bacterium at the farm involved in the investigation. Thus, it is plausible that strategies to reduce concentrations of airborne virulent R equi in areas where foals are housed might reduce the risk of disease. Investigation of the role of concentrations of airborne virulent R equi on disease development would be greatly enhanced by methods for better characterization of foal-level exposure to airborne R equi.

ABBREVIATIONS

CI

Confidence interval

NANAT

Nalidixic acid-novobiocin-tellurite

TBA

Tracheobronchial aspirate

TBSS

Tris-buffered saline solution

a.

MAS-100 Eco, Merck Inc, Whitehouse Station, NJ.

b.

Nitrocellulose membrane, pore size 0.45 μm, Bio-Rad Laboratories, Hercules, Calif.

c.

Hybridization oven, VWR International, West Chester, Pa.

d.

Nonfat dry milk, Bio-Rad Laboratories, Hercules, Calif.

e.

Tris-buffered saline solution, Bio-Rad Laboratories, Hercules, Calif.

f.

Tween 20 solution, Bio-Rad Laboratories, Hercules, Calif.

g.

Rocking platform, VWR International, West Chester, Pa.

h.

Horseradish peroxidase-conjugated goat IgG fraction against mouse IgG, MP Biomedicals Inc, Aurora, Ohio.

i.

Sodium citrate, Sigma Chemical Co, St Louis, Mo.

j.

EDTA, Sigma Chemical Co, St Louis, Mo.

k.

Dextran sulfate, Sigma Chemical Co, St Louis, Mo.

l.

3,3′,5,5′-tetramethylbenzidine, Sigma Chemical Co, St Louis, Mo.

m.

Dulbecco PBS solution, Mediatech Inc, Manassas, Va.

n.

Triple stage tracheal wash catheter, MILA International Inc, Erlanger, Ky.

o.

CIDEX-PLUS, Advanced Sterilization Products, Irvine, Calif.

p.

S-PLUS, version 8.2, Tibco Inc, Seattle, Wash.

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Contributor Notes

Supported by the American Quarter Horse Foundation and the Link Equine Research Endowment, Texas A&M University.

The air sampler used for this project was purchased with a grant from the Grayson-Jockey Club Research Foundation.

The authors thank Stephanie Buntain, Stephanie Standridge, Grant Wicks, and Kyle Wicks for technical assistance.

Address correspondence to Dr. Cohen (ncohen@cvm.tamu.edu).