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

M. Keith Chaffin Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4475.

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Noah D. Cohen Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4475.

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Ronald J. Martens Equine Infectious Disease Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4475.

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Abstract

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.

Abstract

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.

Rhodococcus equi causes one of the most severe and devastating forms of pneumonia in foals. Rhodococcus equi is a gram-positive, soil-saprophytic, facultativeintracellular pathogen that has worldwide distribution.1–5 Pneumonia caused by R equi results in animal distress and economic losses to the equine breeding industry. Prevalence and case fatality rates are high.4 Diagnosis is challenging, particularly during the early stages of infection.2,5 Treatment is generally prolonged, expensive, associated with adverse effects, and not always successful.2,4,5 Pneumonia caused by R equi may negatively impact future athletic performance.4 Breeding farms reputed to have endemic pneumonia attributable to R equi are likely to lose clients.

Foals affected with pneumonia caused by R equi develop pyogranulomatous lesions in the lungs and mediastinal lymph nodes.2–5 Clinical signs include fever, cough, lethargy, nasal discharge, tachypnea, and progressive respiratory distress.5 Although pneumonia is the most common manifestation of infection, R equi can invade other organs and cause extrapulmonary disorders, such as osteomyelitis, abdominal lymphadenitis, and enterocolitis.6 Also, immune-mediated disorders, such as uveitis and polysynovitis, can result from R equi infections.6

Clinical signs of R equi–induced pneumonia do not typically become apparent until foals are 30 to 90 days old; however, there is a growing body of evidence that most, if not all, affected foals become infected early after birth during the neonatal period. Results of experiments indicate that foals < 2 weeks old are more susceptible to experimental infection with R equi, compared with the susceptibility of older foals.7 Epidemiologic data are consistent with the hypothesis that most foals with naturally developing disease become infected near the time of birth.8 Reasons for increased susceptibility of neonatal foals remain unknown. Foals from farms with endemic R equi infections that subsequently develop pneumonia caused by R equi have lower CD4+:CD8+ T-lymphocyte ratios during the first 2 weeks after birth than do foals that do not subsequently develop R equi–induced pneumonia.9 As foals increase in age, the immunophenotypic differences between affected and unaffected foals become smaller in magnitude until they are no longer apparent.9 Also, foals are born with an inherent inability to mount a Thelper-1–based cell-mediated immune response and to generate γ-interferon.10,11 These mechanisms represent critical components of the host protective immune response against R equi infections.12 Analysis of these findings suggests that affected foals probably have relatively ineffective or immature immune responses to R equi during the early neonatal period and are thereby less prepared to develop an adequate antimicrobial defense against R equi infections.9

We believe that R equi initially establishes a small nidus of infection in the lungs of certain susceptible foals during the first few days after birth, when their host immune defenses are immature. The infection insidiously increases in magnitude during the next several weeks or months until it becomes sufficiently severe to cause clinical signs of pneumonia. Thus, it appears that the first few days after birth represent a critical period during which R equi infections become established. Consequently, strategies to prevent infections during this critical period may be more effective than strategies designed to control infections in foals at several weeks to months of age, when most R equi infections are already established.

Currently, there are no vaccines available to prevent disease caused by R equi. The only proven effective immunoprophylactic strategy available is IV administration of R equi HIP to newborn foals.13 Although this is the best prophylactic program available, it is cost prohibitive for many horse owners, labor-intensive, and not universally effective.13–19

A novel strategy for prevention of pneumonia caused by R equi is the administration of effective antimicrobial agents to high-risk foals during the critical period when they are most likely to become infected with R equi. This strategy requires an antimicrobial that is safe, achieves therapeutic concentrations within tissues and cells at the primary site of infection, and maintains adequate concentrations throughout the critical period for infection. Therapeutic concentrations of an antimicrobial at the primary site of infection may result in bacterial death prior to establishment of a nidus of infection. Foals are primarily exposed to R equi from the environment via the respiratory route.1–3 Bronchoalveolar cells (alveolar macrophages and neutrophils) are the host defense cells that are initially exposed to the bacteria. Thus, an antimicrobial agent that achieves high and prolonged intracellular concentrations within these cells during the critical period for infection may effectively prevent infection. For this approach to be practical at equine breeding farms, it would be advantageous for the antimicrobial to have pharmacokinetic properties that allow oral administration and an infrequent dosing regimen.

Azithromycin is an antimicrobial that has been evaluated in various species for the treatment of animals infected with intracellular and pyogenic bacteria.20 Azithromycin has properties that are superior to erythromycin, including fewer adverse effects, convenience of once-daily oral administration, a substantial postantibiotic effect, better bioavailability, higher tissue concentrations, superior uptake into the intracellular environment, and prolonged tissue and intracellular concentrations.20 Azithromycin has gained considerable popularity as a treatment for R equi–infected foals.21–23 On the basis of its apparent safety, ease of administration, and pharmacokinetic and pharmacodynamic properties, azithromycin appears to be a suitable candidate to evaluate for use in antimicrobial chemoprophylaxis of R equi infections during the critical period when R equi infections most frequently develop.

We hypothesized that short-term oral administration of azithromycin to newborn foals would safely provide protection against naturally acquired infection with R equi, thereby reducing the prevalence of disease on farms where pneumonia caused by R equi is endemic. The primary objectives of the study reported here were to determine the effect of azithromycin chemoprophylaxis administered for the first 2 weeks after birth on the cumulative incidence of pneumonia attributable to R equi infections, age at onset of clinical signs of pneumonia, and mortality rate of foals affected with R equi– induced pneumonia for foals on equine breeding farms with endemic R equi infections. Secondary objectives of the study were to determine the effect of azithromycin chemoprophylaxis on the prevalence of azithromycinresistant R equi that caused pneumonia, susceptibility to azithromycin for R equi isolates cultured from the foals, and clinically apparent adverse effects of azithromycin among foals at R equi–endemic equine breeding farms.

Materials and Methods

The study was a controlled, randomized clinical trial performed during 2005 at equine breeding farms with a history of endemic R equi infections. The study protocol was approved by the Clinical Research Review Committee at the College of Veterinary Medicine & Biomedical Sciences, Texas A&M University.

Study population—Participating farms were selected from those that have participated in other studies9,24–27 conducted by our laboratory group to identify farmrelated, foal-related, hematologic, and immunophenotypic risk factors associated with development of pneumonia caused by R equi. Equine breeding farms were screened by one of the investigators (MKC), and farms had to meet the following criteria for inclusion in the study: anticipated that ≥ 25 foals would be born on the farm during 2005 and would remain on the farm for at least the first 150 days after birth, history of endemic pneumonia in foals attributed to R equi infection with morbidity rates ≥ 20% among foals for the preceding 2 years, ability and willingness to monitor all foals throughout the first 150 days after birth, availability of veterinary services for proper diagnosis and treatment of foals with R equi–induced pneumonia, availability of a veterinarian who was consistently willing to participate in the study and adhere to study protocols regarding diagnostic testing of foals with pneumonia, ability and willingness to provide required data for each enrolled foal, willingness to randomly assign foals to 2 treatment groups, ability and willingness to treat foals of 1 of the groups with azithromycin every 48 hours for the first 2 weeks after birth, and willingness to not treat foals of the other group (control foals) with azithromycin during the first 2 weeks after birth.

Criteria for enrollment of each foal into the study included that the foal must be born on the participating farm during 2005, the foal would remain on the farm for at least 150 days after birth, and the owner or owner's representative must provide consent for participation in the study and must consent to required diagnostic protocols should the foal develop clinical signs of pneumonia. Owners of foals were provided the opportunity to decline enrollment in the study and could withdraw from participation at any time and for any reason.

Assignment to groups and treatment of foals with azithromycin—Eligible foals at participating farms were assigned to 2 treatment groups. On each farm, foals were numbered chronologically on the basis of date of birth (ie, first foal born was foal No. 1, the second foal born was foal No. 2, and so on). Foals were then randomly assigned to 1 of 2 treatment groups by use of a computer-generated random sequence of the numbers 1 and 2. Group 1 foals served as control foals and were not treated with azithromycin during the first 2 weeks after birth; these foals did not receive a control substance. Group 2 foals were treated with azithromycina (10 mg/kg [4.5 mg/lb], PO, q 48 h) during the first 2 weeks after birth. The first treatment was administered on day 1 or 2 after birth (day of birth was designated as day 0), and subsequent treatments were administered on alternate days for a total of 7 treatments. Farm personnel were instructed to estimate the body weight of each foal at the time of each treatment and to administer azithromycin suspension in accordance with a provided dosage chart that contained itemized instructions for mixing the azithromycin suspension and determining the volume of suspension to administer to each foal (ie, amount of azithromycin was based on the estimated body weight).

Monitoring and diagnostic assessment of foals—Foals included in the study were housed at the participating farms. Standard housing, management, and preventative health care practices, as determined by the owners and managers of each farm, were used. Foals enrolled in the study were not deprived of any preventative or therapeutic practices that were typically provided at the farm. Farm personnel were requested to manage the foals from groups 1 and 2 similarly, and the only intentional difference in management of the foals was administration of azithromycin to the group 2 foals. Farm personnel were instructed to adhere to study protocols regarding diagnostic approaches for any enrolled foals with clinical signs of pneumonia. Foals were monitored from day 1 to day 150 after birth for clinical signs of pneumonia (eg, fever, cough, nasal discharge, tachypnea, or respiratory distress) or any adverse effects attributable to azithromycin (eg, diarrhea or hyperthermia).

Participating veterinarians examined foals that had clinical signs of pneumonia or were identified as potentially having adverse effects attributable to azithromycin. For foals with clinical signs of pneumonia, veterinarians were instructed to perform a complete physical examination, thoracic auscultation, and at least 1 additional diagnostic procedure (thoracic radiography, thoracic ultrasonography, or tracheobronchial aspiration with microbiologic culture and cytologic examination).

Participating veterinarians classified each foal as affected or unaffected with pneumonia caused by R equi. An affected foal was defined as a 20- to 150-day-old foal that had clinical signs of pneumonia (eg, tachypnea, fever, nasal discharge, or cough) and from which R equi was isolated from a tracheobronchial aspirate or postmortem lung specimen or that had clinical signs of pneumonia and radiographically visible multifocal pulmonary opacities, sonographically visible peripheral pulmonary abscesses or consolidation, or cytologically visible gram-positive coccobacilli in tracheobronchial aspirate specimens.9,24–27 Foals that developed clinical signs of pneumonia but for which results of the required diagnostic tests did not meet the case definition for pneumonia caused by R equi were classified as unaffected foals. Foals that did not develop clinical signs of pneumonia during the first 150 days after birth also were classified as unaffected foals. Foals that developed signs of pneumonia but for which the required diagnostic procedures were not conducted were omitted from the data analysis.

Additional data collection—Data were collected from each foal regarding group assignment, breed, sex, date of birth, and whether the foal received R equi HIP. For foals that received HIP, data were collected regarding the commercial source of HIP, number of HIP transfusions, age at time of each transfusion, and volume of HIP administered for each transfusion. Data were collected from each foal regarding whether the foal left the farm prior to 150 days of age, age at which the foal left the farm, and total number of days the foal was on the breeding farm during the first 150 days after birth.

For foals affected with pneumonia attributable to R equi, data were collected regarding clinical signs; age at onset of clinical signs; diagnostic methods used for classifying the foal as affected with R equi–induced pneumonia; results of diagnostic tests; treatments administered; and whether the foal died, was euthanatized, or recovered from the disease. For foals that died or were euthanatized, data were collected regarding age at time of death and results of necropsy.

Data were collected for group 2 foals regarding estimated weight of each foal at the time of each treatment, volume of azithromycin suspension administered, and date of each treatment. Participating veterinarians recorded data from each group 2 foal regarding potential adverse effects of azithromycin treatment, age at onset, clinical signs, methods of treatment, and outcome. To further assess potential adverse effects of diarrhea associated with azithromycin administration, data were recorded for each day during the first 28 days after birth for clinical signs of diarrhea. From these data, the total number of days that a foal had diarrhea during the first 14 and 28 days after birth was calculated.

Determination of MICs of R equi isolates—To assess whether isolates of R equi were resistant to azithromycin as a result of azithromycin chemoprophylaxis, we determined MICs of azithromycin against R equi isolates obtained from fecal specimens of study foals and from clinical specimens from clinically affected foals. Fresh fecal samples were collected from enrolled foals at 3 (days 17 through 24) and 4 (days 25 through 32) weeks of age. Fecal specimens were collected from the top of a fecal pile observed to have been freshly voided; specimens were placed into labeled plastic bags. Fecal specimens were frozen at −20°C until shipped to our laboratory in insulated containers with ice packs. Once received, they were frozen at −80°C until microbiological culture was performed.

Microbiological culture of fecal specimens was performed in duplicate by use of a modified NANAT R equi–selective agar medium, as described elsewhere28; both positive and negative control samples were used. Use of the modified NANAT agar medium effectively minimized concomitant growth of bacterial and fungal contaminants without inhibiting growth of R equi.28 Culture plates were incubated at 37°C for 36 hours. One to 5 colonies of R equi were harvested and streaked onto blood agar media plates and incubated at 37°C for 48 hours. Isolated colonies were confirmed as R equi during morphologic examination by use of Gram stain and with the synergistic hemolysis test.29 A colorimeter was used, and bacteria were suspended in saline (0.45% NaCl) solution to obtain a McFarland 1 turbidity reading. This solution was spread smoothly onto Mueller-Hinton agar, and azithromycin test stripsb were placed on the plates.30 Plates were incubated at 37°C for 24 hours. The MIC was determined at 24 hours, in accordance with the manufacturer's recommended protocol.b Reference organisms with known MICs were used for quality-control purposes, as recommended by the manufacturer of the azithromycin test strips. Mean MIC of the duplicate cultures was recorded.

In addition, clinical laboratories used by participating veterinarians provided R equi isolates recovered from enrolled affected foals (ie, cultured from tracheobronchial aspirates or postmortem specimens) to our laboratory. Pure colonies of R equi were prepared on beads and frozen at −80°C and then shipped via overnight mail in insulated containers with ice packs. On receipt by our laboratory, such isolates were grown on modified NANAT R equi–selective media, and MICs were determined by use of the azithromycin test strips, as described for isolates of R equi obtained from fecal specimens.

Quantitation of R equi in fecal samples—At approximately the midpoint of the study, we noticed that the fecal samples from group 2 foals appeared to have fewer R equi colonies than did the fecal samples from group 1 foals. Subsequently, we arbitrarily selected a small group of fecal samples from 3-week-old foals (30 group 1 foals and 33 group 2 foals). In addition, we arbitrarily selected a small group of fecal samples from 4-week-old foals (30 group 1 foals and 35 group 2 foals). The fecal concentrations of R equi in these samples were determined as described elsewhere.28

Data analysis—Data were analyzed by use of descriptive and inferential methods. Distributions of data that were continuous or discrete (eg, MIC values) were summarily described by use of median and interquartile ranges (ie, range of the 25th to 75th percentiles). Categoric data were summarized by use of contingency tables. For inferential analyses, the following approaches were used. Comparisons between groups (eg, azithromycin-treated and control groups) for continuous data were made with Wilcoxon rank sum tests31 because these data often had a non-Gaussian distribution. Categoric data were compared by use of χ2 or Fisher exact tests.32 The association between development of pneumonia attributable to R equi and treatment group was assessed with logistic regression analysis33; ORs were determined by exponentiating coefficients, and 95% CIs for the OR were derived by use of maximum-likelihood estimators.33 Because standard logistic regression did not account for the correlation of observations obtained from foals from the same farm, mixed-effects logistic regression34 was used to estimate the OR (and 95% CI) for the association of treatment effect on the odds of developing pneumonia attributable to R equi, wherein farm was included as a random effect and treatment group was a fixed effect.35 For all analyses, values of P ≤ 0.05 were considered significant.

The rationale for administration of azithromycin was to prevent disease, similar to use of a vaccine. Thus, the efficacy of azithromycin for preventing disease also was estimated by use of the following equation:

article image
where the OR was that for the association of treatment with disease estimated from logistic regression analysis. Estimates of efficacy were made for both the point estimate of the OR and the lower and upper bounds of the 95% CI for each OR.

Results

Participating farms—Twenty-five equine breeding farms were screened by one of the investigators (MKC) for eligibility to participate in the study. Fourteen farms met the criteria for eligibility, and 12 farms and their representative veterinarians initially agreed to participate in the study. One farm voluntarily withdrew from the study because of the time and effort associated with adhering to study protocols. Data from another farm (including 18 group 1 and 17 group 2 foals) were excluded from data analysis because all group 2 foals at that farm received improper dosages of azithromycin (< 3 mg/kg [< 1.4 mg/lb], PO, q 48 h). The remaining 10 participating farms were distributed among 4 states (5 farms in Texas, 3 farms in Florida, 1 farm in Kentucky, and 1 farm in Oklahoma).

Participating foals—For the 10 participating farms, 465 foals were eligible for inclusion in the study. Fortyseven foals were excluded from enrollment because of lack of owner consent (n = 28), severe illness unrelated to R equi during the neonatal period (7), and various reasons that were not reported to the investigators (12). Of the 418 foals that were initially enrolled, 35 were voluntarily withdrawn from the study (2 foals from group 2 were withdrawn because several azithromycin treatments were not administered, 13 control and 11 azithromycin-treated foals were withdrawn because the foals left the farm prior to 150 days after birth and were lost to follow-up monitoring, 3 control and 3 azithromycin-treated foals were withdrawn because of death as a result of illness or injury unrelated to R equi or respiratory tract disease prior to 150 days of age, and 1 control and 2 azithromycin-treated foals were withdrawn for unreported reasons).

Data were provided for 348 foals. The investigators elected to exclude an additional 10 foals from the analyses for various reasons. One control and 1 azithromycin-treated foal were omitted because of inadequate data, 1 control and 1 azithromycin-treated foal were omitted because they developed pneumonia and the required diagnostic procedures were not performed to adequately classify the foals as affected or unaffected, 1 control foal and 2 azithromycin-treated foals left the farm prior to 150 days of age and were lost to follow-up monitoring, and 2 control foals and 1 treatment foal were omitted because of death prior to 150 days of age as a result of illness or injury unrelated to R equi or respiratory tract disease.

Data for 338 foals from 10 farms were analyzed. Randomization resulted in 168 foals (49.7%) assigned to group 1 and 170 (50.3%) assigned to group 2 (Table 1). There was no significant difference between groups in the distribution of foals among the participating farms. All 338 foals were born between January 1 and May 31, 2005 (Table 2). There was no significant difference between groups in the distribution of foals born during each month. There were no significant differences between groups in the distribution of foals among breeds (Table 3). Of the control foals, 89 (53.0%) were male and 79 (47.0%) were female. Of the group 2 foals, 85 (50.0%) were male and 85 (50.0%) were female. There were no significant differences between groups with regard to sex. All 338 foals remained on the farms and were monitored for signs of pneumonia until 150 days of age.

Table 1—

Distribution of 338 foals among 10 equine breeding farms in a study conducted to determine the effect of azithromycin administered to foals during the first 2 weeks after birth on the cumulative incidence of pneumonia caused by Rhodococcus equi infection.

FarmTotal No. of foalsUntreated foalsAzithromycin-treated foals
No.%No.%
11015352.54847.5
2371951.41848.6
3251352.01248.0
4241250.01250.0
5271244.41555.6
6321546.91753.1
713753.8646.2
8401947.52152.5
9221045.51254.5
1017847.1952.9
Table 2—

Month of birth for 338 foals in a study conducted to determine the effect of azithromycin administered to foals during the first 2 weeks after birth on the cumulative incidence of pneumonia caused by R equi infection.

MonthTotal No. of foalsUntreated foalsAzithromycin-treated foals
No.%*No.%*
January492514.92414.1
February854225.04325.3
March1075532.75230.6
April693219.03721.8
May28148.3148.2

Represents the percentage based on the cumulative number of untreated or azithromycin-treated foals.

Values in column do not total to 100% because of rounding.

Table 3—

Distribution of breed for 338 foals in a study conducted to determine the effect of azithromycin administered to foals during the first 2 weeks after birth on the prevalence of pneumonia caused by R equi infection.

BreedTotal No. of foalsUntreated foalsAzithromycin-treated foals
No.%*No.%*
Thoroughbred18810461.98449.4
Quarter Horse984325.65532.4
Arabian1784.895.3
Paint621.242.3
Appaloosa26116.5158.8
Mixed30031.8

See Table 2 for key.

Table 4—

Proportion of foals affected by pneumonia caused by R equi infection at 10 equine breeding farms.

FarmTotal No. of foalsUntreated foalsAzithromycin-treated foals
No. with pneumonia/No. of foals%No. with pneumonia/No. of foals%
11013/535.70/480
2374/1921.01/185.6
3250/1300/120
4244/1233.31/128.3
5273/1225.00/150
6321/156.70/170
7130/700/60
84010/1952.63/2114.3
9224/1040.01/128.3
10176/875.03/933.3

Of the 338 foals, 283 (83.7%) received IV transfusions of R equi HIP. Of the control foals, 140 (83.3%) received HIP, and of the azithromycin-treated foals, 143 (84.1%) received HIP. There were no significant differences between groups regarding the proportion of foals that received HIP. When these data were examined on the basis of farm, there were still no significant differences between groups. Of the control foals that received HIP, 122 (87.1%) received HIP obtained from 1 commercial source,c and 18 (12.9%) received HIP obtained from another commercial source.d Of the 143 group 2 foals that received HIP, 122 (85.3%) received HIP from 1 commercial source,c and 21 (14.7%) received HIP from another commercial source.d There were no significant differences between groups in the proportion of foals that received HIP from either commercial source.

Of the foals that received HIP, the median number of transfusions with HIP was 1 (range, 1 to 3) and 2 (range, 1 to 2) for control and azithromycin-treated foals, respectively. There were no significant differences between groups in the number of transfusions with HIP. When these data were analyzed categorically (< 2 transfusions vs ≥ 2 transfusions), there were no significant differences between groups.

Distribution of age of foals at the time of the first transfusion with HIP was not significantly different between transfused foals in group 1 (median, 1 day; range, 1 to 7 days) and transfused foals in group 2 (median, 1 day; range, 1 to 21 days). There were 68 (40.5%) foals in group 1 and 74 (43.5%) foals in group 2 that received a second transfusion with HIP. The distribution of ages at the time of the second transfusion with HIP was not significantly different between transfused foals in group 1 (median, 21 days; range, 2 to 21 days) and transfused foals in group 2 (median, 21 days; range, 14 to 21 days). Two control foals received a third transfusion with HIP (1 foal at 42 days and 1 foal at 64 days); none of the azithromycin-treated foals received a third HIP transfusion. The proportion of foals that received a third transfusion was not significantly different between control (2/168 [1.2%]) and azithromycin-treated foals (0/170 [0%]). Among the transfused foals, the volume of HIP administered for each transfusion was not significantly different between foals in group 1 (median, 1,000 mL; range, 700 to 1,000 mL) and foals in group 2 (median, 1,000 mL; range, 700 to 1,000 mL).

Cumulative incidence of pneumonia attributable to R equi—Overall, 44 (13.0%) foals were classified as affected with R equi–induced pneumonia, and 294 (87.0%) foals were classified as unaffected. The proportion of affected foals was significantly (P < 0.001) higher for control foals (35/168 [20.8%]) than for azithromycin-treated foals (9/170 [5.3%]). Control foals were significantly more likely (OR, 4.7; 95% CI, 2.2 to 10.1) to be affected with pneumonia attributable to R equi than were azithromycin-treated foals.

The proportion of affected foals at each farm ranged from 0% to 52.9% (Table 4). Two farms had no affected foals during the study. For each of the 8 farms that had at least 1 affected foal, the proportion of affected foals was higher for group 1 foals than for group 2 foals. When data were analyzed to omit the 2 farms with no affected foals, the proportion of affected foals was significantly (P < 0.001) higher for control foals (35/148 [23.6%]) than for azithromycin-treated foals (9/152 [5.9%]). Control foals were significantly more likely (OR, 4.9; 95% CI, 2.3 to 10.6) to be affected with pneumonia attributable to R equi than were azithromycin-treated foals.

In addition to the 2 farms with no affected foals, there was 1 farm that had only 1 affected foal. When data were analyzed to omit all 3 farms with ≤ 1 affected foal, the proportion of affected foals was significantly (P < 0.001) higher for control foals (34/133 [25.6%]) than for azithromycin-treated foals (9/135 [6.7%]). Control foals were significantly more likely (OR, 4.8; 95% CI, 3.2 to 7.2) to be affected with pneumonia attributable to R equi than were azithromycin-treated foals.

The aforementioned results ignored effects of farm. Because of the variation in prevalence of disease among farms and the expectation that foals from a particular farm were more similar to each other than they were to foals from other farms, the data were analyzed by use of mixed-effects logistic regression in which farm was treated as a random effect. When data from all 10 farms were included in mixed-effects modeling, control foals were significantly more likely (OR, 6.9; 95% CI, 3.4 to 14.2) to be affected with pneumonia attributable to R equi than were azithromycin-treated foals. When data from the 2 farms with no affected foals were omitted, control foals were significantly more likely (OR, 6.9; 95% CI, 3.3 to 14.5) to be affected with pneumonia attributable to R equi than were azithromycin-treated foals. When data from the 3 farms with ≤ 1 affected foal were omitted, control foals were significantly more likely (OR, 6.7; 95% CI, 3.2 to 14.4) to be affected with pneumonia attributable to R equi than were azithromycin-treated foals. In all 3 mixed-effects models, the CI for the SD of the random effect for farm did not include 0, which indicated that there was not a significant effect of farm.

Age of affected foals at the time of onset of clinical signs of pneumonia attributable to R equi was plotted (Figure 1). There was no significant difference in age at onset of clinical signs between control foals (median, 61 days; range, 28 to 142 days) and azithromycintreated foals (median, 63 days; range, 44 to 128 days). When these data were analyzed by use of mixed-effects models to account for effects of farm, the age at onset was still not significantly different between groups.

Figure 1—
Figure 1—

Box-and-whiskers plots of the age at onset of clinical signs of pneumonia for foals affected with pneumonia attributable to Rhodococcus equi. Group 1 foals were 168 untreated control foals, and group 2 foals were 170 foals treated with azithromycin at 48-hour intervals during the first 2 weeks after birth. Boxes represent the interquartile range (25th to 75th percentiles). Within each box, the horizontal line with a triangle represents the median value. Bars above and below the boxes extend to the 95th and 5th percentiles, respectively. The horizontal line with a circle represents an outlier.

Citation: Journal of the American Veterinary Medical Association 232, 7; 10.2460/javma.232.7.1035

Efficacy of azithromycin chemoprophylaxis— When data for all 10 farms were included, the estimated efficacy of azithromycin chemoprophylaxis was 78.7% (95% CI, 54.5% to 90.1%). When the 2 farms with no affected foals were omitted, the estimated efficacy was 79.6% (95% CI, 56.5% to 90.6%). When the 3 farms with ≤ 1 affected foal were omitted, the estimated efficacy was 79.2% (95% CI, 68.8% to 86.1%).

When random effects logistic regression was used to account for farm effects, estimated efficacy was 85.5% (95% CI, 70.6% to 93.0%) when all 10 farms were included. When the 2 farms with no affected foals were omitted, estimated efficacy was 85.5% (95% CI, 69.7% to 93.1%). When the 3 farms with ≤ 1 affected foal were omitted, estimated efficacy was 85.1% (95% CI, 68.8% to 93.1%).

Clinical signs of affected foals—All 44 affected foals reportedly had clinical signs of pneumonia. Overall, 31 (70.4%) affected foals had a fever. Affected control foals had a higher prevalence of fever (27/35 [77.1%]) than did affected azithromycin-treated foals (4/9 foals [44.4%]); however, these prevalences did not differ significantly (P = 0.055). Overall, 20 (45.4%) affected foals had signs of lethargy. There was no difference in the prevalence of lethargy between affected control foals (16/35 [45.7%]) and affected azithromycin-treated foals (4/9 [44.4%]). Overall, 41 (93.2%) affected foals had a cough. There was no difference in the prevalence of cough between affected control foals (32/35 [91.4%]) and affected group 2 foals (9/9 [100%]). Overall, 21 (47.7%) affected foals had nasal discharge. There was no difference in the prevalence of nasal discharge between affected control foals (16/35 [45.7%]) and affected group 2 foals (5/9 [55.6%]). Overall, 19 (43.2%) affected foals had tachypnea. There was no significant difference in the prevalence of tachypnea between affected control foals (13/35 [37.1%]) and affected group 2 foals (6/9 [66.7%]). Overall, 18 (40.9%) affected foals had respiratory distress. There was no significant difference in the prevalence of respiratory distress between affected control foals (12/35 [34.3%]) and affected group 2 foals (6/9 [66.7%]).

Diagnostic testing of affected foals—All 44 affected foals were classified on the basis of clinical signs of pneumonia and at least one of the required diagnostic criteria. Of the affected foals, 10 (22.7%) had a tracheobronchial aspirate obtained, and R equi was isolated from the specimen. There was no difference in the proportion of affected foals that had isolation of R equi from a tracheobronchial aspirate between group 1 (9/35 [25.7%]) and group 2 (1/9 [11.1%]) foals. Cytologic examination was performed on 4 (40.0%) of the tracheobronchial aspirates from affected group 1 foals, and all 4 had cytologic evidence of gram-positive coccobacilli.

Of the affected foals, 11 (25.0%) had radiographic evidence of multifocal pulmonary opacities. There was no significant difference in the proportion of affected foals that had radiographic evidence of multifocal pulmonary opacities between group 1 (9/35 [25.7%]) and group 2 (2/9 [22.2%]) foals.

Ultrasonographic evidence of peripheral pulmonary abscesses or consolidation was the most frequently used diagnostic modality among foals in this study. Thirty-two (72.7%) affected foals had ultrasonographic evidence of peripheral abscesses or consolidation. There was no significant difference in the proportion of affected foals that had ultrasonographic evidence of peripheral pulmonary abscesses or consolidation between group 1 (24/35 [68.6%]) and group 2 (8/9 [88.9%] foals).

Two (4.5%) affected foals had radiographic and ultrasonographic evidence of pneumonia attributable to R equi. Two (4.5%) affected foals had radiographic evidence of pneumonia attributable to R equi and isolation of R equi from a tracheobronchial aspirate. Five (11.4%) affected foals had ultrasonographic evidence of peripheral abscesses or consolidation and isolation of R equi from a tracheobronchial aspirate.

Outcome of affected foals—All 44 affected foals were treated with macrolides (with or without rifampin) appropriate for R equi–induced pneumonia. Forty-three (97.7%) foals made a complete recovery. One group 2 foal failed to respond to treatment and died at 98 days of age as a result of pneumonia attributable to R equi. Necropsy of this foal revealed severe pyogranulomatous pneumonia, and R equi was isolated from necropsy specimens. The difference in survival between group 1 (35/35 [100%]) and group 2 (8/9 [88.9%]) foals was not significant.

Adverse effects of azithromycin—Adverse effects attributable to azithromycin were not reported by farm personnel. One veterinarian reported that diarrhea in 2 foals at 1 farm could have been associated with administration of azithromycin. Hyperthermia was not detected as an adverse effect in any azithromycin-treated foals. Overall, there were 115 (34.0%) foals that had diarrhea during the period 1 to 14 days after birth. There was no difference in the proportion of foals that had diarrhea during this age period between group 1 (54/168 [32.1%]) and group 2 (61/170 [35.9%]) foals. Of the 115 foals with diarrhea, there was no difference in the number of days with diarrhea during the period 1 to 14 days after birth between group 1 foals (median, 3 days; range, 1 to 12 days) and group 2 foals (median, 3 days; range, 1 to 11 days).

Overall, there were 136 (40.2%) foals that had diarrhea during the period 1 to 28 days after birth. There was no difference in the proportion of foals that had diarrhea during the period 1 to 28 days after birth between group 1 (65/168 [38.7%]) and group 2 (71/170 [41.8%]) foals. Of the 136 foals with diarrhea, there was no difference in the number of days with diarrhea during the period 1 to 28 days after birth between group 1 (median, 4 days; range, 1 to 15 days) and group 2 (median, 4 days; range, 1 to 13 days) foals.

MICs against R equi isolates from fecal samples— Fecal samples at 3 weeks of age were obtained from 99 (58.9%) group 1 and 105 (61.8%) group 2 foals. Selective media resulted in culture of R equi from 92 (92.9%) fecal samples of group 1 foals and 96 (91.4%) fecal samples of group 2 foals. There were no significant differences between groups regarding whether R equi organisms were cultured from feces. There was no significant difference in the number of R equi colonies used for MIC testing of isolates from fecal samples of group 1 (median, 3 colonies; range, 2 to 5 colonies) and group 2 (median, 3 colonies; range, 2 to 4 colonies) foals. There were no significant differences in the distribution of azithromycin MICs for isolates from fecal samples of group 1 (median, 1 μg/mL; range, 0.32 to 1.5 μg/mL) and group 2 (median, 0.87 μg/mL; range, 0.5 to 1.5 μg/mL) foals.

Fecal samples at 4 weeks of age were obtained from 97 (57.7%) group 1 foals and 105 (61.8%) group 2 foals. Selective media resulted in culture of R equi from 93 (95.9%) fecal samples of group 1 foals and 94 (89.5%) fecal samples of group 2 foals. There were no significant differences between groups regarding whether R equi was cultured from feces. There was no significant difference in the number of R equi colonies used for MIC testing of fecal isolates from group 1 (median, 3 colonies; range, 1 to 5 colonies) and group 2 (median, 3 colonies; range, 1 to 4 colonies) foals. There were no significant differences in the distribution of azithromycin MICs for isolates from fecal samples of group 1 (median, 1 μg/mL; range, 0.25 to 1.5 μg/mL) and group 2 (median, 1 μg/mL; range, 0.38 to 1.5 μg/mL) foals.

MICs against R equi isolates from clinical samples—Only 5 isolates of R equi from clinical samples of affected foals were obtained for MIC testing. Four isolates were from tracheobronchial aspirates of affected foals, and 1 isolate was from a lung abscess found during necropsy. Three isolates were from affected group 1 foals, and 2 isolates were from affected group 2 foals, including 1 isolate obtained during necropsy of the single foal that died. There were no significant differences in the azithromycin MICs of isolates from affected group 1 (median, 0.75 μg/mL; range, 0.5 to 1.0 μg/mL) and group 2 (median, 0.63 μg/mL; range, 0.5 to 0.75 μg/mL) foals.

Quantitative R equi culture of fecal samples— Quantitative R equi cultures of fecal samples were performed for fecal samples of 3-week-old foals (30 group 1 foals and 33 group 2 foals). At 3 weeks of age, group 2 foals had significantly (P < 0.001) lower concentrations of R equi (median, 3,000 CFUs/g; range, 600 to 10,000 CFUs/g) in fecal samples than did group 1 foals (median, 6,000 CFUs/g; range, 750 to 37,500 CFUs/g; Figure 2). Quantitative cultures were performed for fecal samples of 4-week-old foals (30 group 1 foals and 35 group 2 foals). At 4 weeks of age, group 2 foals had significantly (P < 0.001) lower concentrations of R equi (median, 3,000 CFUs/g; range, 800 to 8,000 CFUs/g) in fecal samples than did group 1 foals (median, 7,000 CFUs/g; range, 2,000 to 13,500 CFUs/g; Figure 3). When a mixed-effects model was used to include terms for farm (random effect) and disease (R equi– induced pneumonia or no pneumonia; fixed effect), concentrations of R equi in fecal samples remained significantly (P < 0.001) lower in azithromycintreated than in untreated control foals, which indicated that this difference was not attributable to farm or disease effects.

Figure 2—
Figure 2—

Box-and-whiskers plots of distributions of concentrations of R equi in fecal samples collected from foals at 3 weeks of age. Quantitative R equi cultures were performed on fecal samples from 30 control foals (group 1) and 32 azithromycin-treated foals (group 2). CFU = Colony-forming unit. See Figure 1 for remainder of key.

Citation: Journal of the American Veterinary Medical Association 232, 7; 10.2460/javma.232.7.1035

Figure 3—
Figure 3—

Box-and-whiskers plots of distributions of concentrations of R equi in fecal samples collected from foals at 4 weeks of age. Quantitative R equi cultures were performed on fecal samples from 30 control foals (group 1) and 35 azithromycin-treated foals (group 2). See Figures 1 and 2 for remainder of key.

Citation: Journal of the American Veterinary Medical Association 232, 7; 10.2460/javma.232.7.1035

Discussion

Prophylactic strategies to control or prevent pneumonia attributable to R equi in foals at equine breeding farms on which the infection is endemic are limited. The primary objective of the study reported here was to determine the chemoprophylactic effects of azithromycin administered orally every 48 hours for the first 2 weeks after birth on the cumulative incidence of pneumonia attributable to R equi on equine breeding farms with endemic R equi infections. Analysis of results of this study revealed that azithromycin chemoprophylaxis was an effective method for reducing the cumulative incidence of R equi–induced pneumonia on these equine breeding farms. Control foals had an overall cumulative incidence of 20.8%, and azithromycin-treated foals had an overall cumulative incidence of 5.3%; these values resulted in an estimated protective efficacy of 78.7% without adjusting for farm effects and > 85.5% after adjusting for farm effects. Control foals were approximately 6.9 times as likely to develop pneumonia attributable to R equi as were azithromycin-treated foals. Thus, there was a significant difference between groups, regardless of the methods used for analysis.

Azithromycin was chosen for the study reported here for a number of reasons, including its apparent safety, ease of administration, and pharmacokinetic and pharmacodynamic properties, which are likely to result in adequate and prolonged concentrations within the bronchoalveolar cells at the time of initial exposure to R equi. Pharmacokinetic properties of azithromycin have been reported for foals 6 to 14 weeks old.21–23 Azithromycin is absorbed well in foals after oral administration and achieves high tissue and intracellular concentrations.21,22 Azithromycin achieves extremely high concentrations within the bronchoalveolar cells (15- to 170-fold higher than plasma concentrations) and pulmonary epithelial lining fluid (up to 16-fold higher than plasma concentrations),21–23 which are likely the sites where foals are initially exposed to aerosolized R equi from the environment. In addition, azithromycin achieves intracellular concentrations in polymorphonuclear leukocytes that are approximately 37 times the plasma concentrations.22 The half-life of azithromycin in polymorphonuclear leukocytes is 49.2 hours,22 and the half-life in bronchoalveolar cells is 54.4 hours.23 Following an intragastric dose of 10 mg/kg, azithromycin concentrations in bronchoalveolar cells exceeded the MIC90 for R equi for at least 120 hours, and azithromycin concentrations in pulmonary epithelial lining fluid exceeded the MIC90 for at least 48 hours.23 Thus, azithromycin achieves extremely high (above the MIC90 for R equi) and prolonged intracellular concentrations in bronchoalveolar cells, pulmonary epithelial lining fluid, and neutrophils.21,23 The persistence of high concentrations of azithromycin in bronchoalveolar cells and pulmonary epithelial lining fluid 48 hours after administration suggests that administration at 48-hour intervals may be adequate for treatment of foals with R equi infections.21 For treatment of clinically affected foals with pneumonia attributable to R equi, it has been recommended to administer azithromycin at 10 mg/kg, PO, every 24 to 48 hours.21,22 On the basis of reported data,21–23 azithromycin administered at 10 mg/kg, PO, every 48 hours likely maintains adequate concentrations of azithromycin at the sites where R equi initially colonizes and begins to multiply.

Antimicrobial chemoprophylaxis has been used for numerous conditions in human and veterinary medicine. One major concern with antimicrobial chemoprophylaxis is the potential for adverse effects from the antimicrobial being administered.36 Azithromycin has not been associated with adverse effects in studies21–23 performed in foals, and clinical experience indicates that azithromycin is reasonably safe in foals; however, to our knowledge, large-scale safety studies in foals have not been reported. Potential adverse effects of macrolides in foals include hyperthermia, tachypnea, and colitis and enterocolitis in the dam of treated foals.37,38 In the study reported here, we considered it prudent to monitor foals closely and to record any potential adverse effects of azithromycin, particularly clinical signs related to diarrhea or hyperthermia. No adverse effects of azithromycin were reported, other than by 1 veterinarian who reported that mild diarrhea in 2 foals may have been related to azithromycin administration. Diarrhea is a common disorder in neonatal foals, and many causes exist.39 Among a farm population of foals, the exact cause of diarrhea is frequently undetermined, and thus it can be difficult or impossible to determine whether diarrhea in a foal was caused by azithromycin administration or by numerous other potential factors. To attempt to account for this, we collected data from all enrolled foals regarding whether they had diarrhea during the first 14 and 28 days after birth and the number of days with diarrhea during these time periods. We detected no differences between control and azithromycin-treated foals in the proportion of foals with diarrhea during the first 14 and 28 days after birth. Also, we detected no difference between control and azithromycin-treated foals in the number of days with diarrhea during the first 14 and 28 days after birth. From these data, we concluded that adverse effects of azithromycin chemoprophylaxis did not represent a substantial problem. Nonetheless, veterinarians should be aware of the potential for adverse effects and should monitor foals appropriately. Additional investigations are needed to better understand the clinical characteristics and frequency of adverse effects associated with azithromycin chemoprophylaxis.

Another major concern associated with antimicrobial chemoprophylaxis is the potential for development of bacterial resistance.36 Resistance to antimicrobials can be acquired through genetic mutation or acquisition of genetic elements.36 Because of this concern, we considered it critical in the study reported here to evaluate development of azithromycin resistance among isolates of R equi from fecal samples and tracheobronchial aspirates. When the azithromycin MICs against R equi isolates from fecal samples were compared among the foals in each group, we found no evidence of azithromycin resistance at either the 3- or 4-week age period. Also, we attempted to obtain isolates from clinical samples of affected foals to further evaluate azithromycin resistance; however, we only obtained isolates from clinical samples of 5 affected foals, only 2 of which were azithromycin-treated foals. Although this number of isolates was smaller than we had hoped and was insufficient to make any strong conclusions, we did not detect azithromycin resistance in the 2 isolates from azithromycin-treated foals, including the 1 azithromycin-treated foal that died.

Another concern associated with antimicrobial chemoprophylaxis is the potential for nontarget bacteria, including non-R equi pathogens and commensal and transient microbial flora, to be exposed to an administered antimicrobial and develop resistance.36 It was beyond the scope of the study reported here to monitor azithromycin resistance of nontarget microbes other than R equi, and additional investigations are certainly needed to better understand the impact of azithromycin chemoprophylaxis on the emergence of resistant microbes. Veterinarians should critically evaluate prophylactic use of antimicrobials to assure that the health and well-being of domestic animals and humans are not adversely affected.36 Although the results of our study revealed significant beneficial effects of azithromycin chemoprophylaxis for foals with R equi infections and did not indicate emergence of azithromycin-resistant R equi, the investigators do not advocate prophylactic use of azithromycin on a widespread basis because of the potential for development of antimicrobial resistance. For veterinarians who choose to use azithromycin chemoprophylaxis at breeding farms for the prevention of pneumonia attributable to R equi, it is recommended that caution be exercised because of the potential for azithromycin resistance and that appropriate surveillance programs36 be implemented to detect and characterize antimicrobial resistance that may develop. Additional investigation of alternative preventative strategies in foals during the first 2 weeks after birth is indicated. Such alternatives may include minimizing exposure to virulent R equi during the first 2 weeks after birth or use of preventative medications that are not relied on to treat microbial infections in humans or domestic animals.40,41

Although it was not initially a major objective of the study, we noticed that microbial cultures of fecal samples from azithromycin-treated foals appeared to grow fewer R equi colonies on R equi–selective media than did microbial cultures of fecal samples from control foals. Thus, we elected to further evaluate this finding by selecting a population of fecal samples from control and azithromycin-treated foals to perform quantitative R equi microbiological cultures.28 Analysis of the results revealed that 3- and 4-week-old foals treated with azithromycin at 48-hour intervals during the first 2 weeks after birth had significantly lower concentrations of R equi in fecal samples than did control foals, which indicated that azithromycin chemoprophylaxis may reduce the concentration of total R equi in foal feces. Although we did not determine the concentration of virulent R equi in these fecal samples,28 this finding could have important ramifications for fecal shedding of this organism. The environmental burden of R equi likely depends on many factors, including fecal shedding from adult horses, affected foals, and unaffected foals.42–44 If azithromycin chemoprophylaxis reduces fecal shedding of R equi, then it may impact the prevalence of R equi in treated foals as well as the environmental burden of the bacterium. This finding deserves further investigation to determine the impact of azithromycin chemoprophylaxis on fecal contamination of the environment.

The azithromycin product used in the study reported here is approved for use in humans and is commercially available as a powder that is sold in 1-g packets.a The powder was mixed with tap water to create a suspension and administered shortly after preparation of the suspension. Farm personnel reported that the formulation was easy to mix and simple to administer to the foals. We did not use any generic or compounded azithromycin products in this study. Caution should be exercised regarding extrapolating the results of this study to any generic or compounded azithromycin products.

The study reported here has numerous limitations, including potential for biases by the farm personnel and veterinarians. Farm personnel were aware as to which foals were assigned to group 1 or 2 (whether or not they received azithromycin). Also, veterinarians who ultimately classified each foal as affected or unaffected with pneumonia attributable to R equi were also aware of which foals were assigned to group 1 or 2. We considered and would have preferred a study design whereby farm personnel and participating veterinarians would have remained unaware of foal assignments; however, several logistic issues made this impractical. Most participating farms had a resident veterinarian who was intimately involved with day-to-day management and treatment of foals, so to keep veterinarians unaware of group assignments, we would have needed an indistinguishable control treatment for group 1 foals. Providing control treatments to all group 1 foals would have made participation more labor-intensive by doubling the therapeutic interventions required at each participating farm. Also, it would have required that treatment suspensions (with and without azithromycin) be prepared in advance. Unfortunately, the azithromycin suspension we used was designed for preparation immediately prior to use, and it was difficult to maintain homogenously in suspension for prolonged periods. Thus, we were concerned that preprepared suspensions would result in erroneous dosages of azithromycin being administered to group 2 foals. Despite the lack of a blinded study design, we believe that our objective and clearly defined criteria for affected and unaffected foals minimized potential biases on the part of participating veterinarians. Furthermore, given the severity of R equi–induced pneumonia, it is unlikely that affected group 2 foals would have gone undetected, even had the veterinarians been biased.

Another limitation of the study was the impact that farm- and year-related effects can have on the cumulative incidence of pneumonia attributable to R equi, even in control foals. Our proposed inclusion criteria for farms were designed to provide some homogeneity among farms; nevertheless, a significant effect of farm was detected in our study. Also, the cumulative incidence of R equi–induced pneumonia may vary from year to year, even on farms on which the bacterium is endemic. Other studies9,24–27 have revealed significant farm and year effects on the cumulative incidence of pneumonia attributable to R equi. In the study reported here, 2 farms had no affected foals during 2005, and a third farm had only 1 affected foal, despite the fact that in the preceding 2 years, all 3 of these farms had cumulative incidence rates > 20%. Because of the low prevalence of disease on these 3 farms, we elected to analyze the data with and without inclusion of those farms. The results were similar when data from those farms were excluded from analyses.

Potential for bias associated with attrition at the farm and individual foal level existed, but we believe appropriate precautions were taken to prevent such biases from inadvertently altering the results. One farm voluntarily withdrew from the study, and data from another farm were excluded from analyses because of administration of inadequate dosages of azithromycin. In addition, 45 enrolled foals were withdrawn or excluded from data analyses (35 by farm personnel and 10 by the investigators) for various reasons. We examined data for these excluded foals and the reasons for exclusion closely to ensure that exclusion would not result in significant bias on the prevalence of affected foals. Of the 45 excluded foals, only 2 had clinical signs of pneumonia (1 control and 1 azithromycin-treated foal); inadequate diagnostic testing prevented us from accurately classifying those foals as affected or unaffected. Even if those foals had been affected by pneumonia attributable to R equi, inclusion would not have significantly altered our findings.

Additional limitations of the study reported here included the potential for a few inaccurate doses of azithromycin administered to foals assigned to group 2 because body weights were estimated. Also, foals may have failed to swallow the entire orally administered dose of azithromycin. Another potential limitation to this study was compliance at specific farms; however, our impression was that personnel at the farms adhered to the study protocols in a thorough and complete manner and that major inadequacies were discovered and the data appropriately excluded from analyses.

We certainly recognize the possibility for misclassification of foals (affected vs unaffected); however, we believe that the study was designed in a manner that accurately categorized foals as affected or unaffected with pneumonia attributable to R equi yet was still within the reasonable confines of a farm-based clinical trial. Multiple veterinarians representing multiple breeding farms were needed to participate in this study; thus, it was essential to choose diagnostic criteria for R equi–induced pneumonia that were suitable for the variations in equipment, diagnostic preferences (ie, radiographic, ultrasonographic, and clinicopathologic), and clinical experiences of numerous participating veterinarians and farm owners. Certainly some veterinarians were more experienced at interpreting thoracic radiographs or ultrasonograms than were other veterinarians, and some variability likely existed regarding the quality of images and accuracy of interpretations. The diagnostic criteria used for diagnosis of pneumonia attributable to R equi varied among foals but most frequently consisted of ultrasonographic45 detection of peripheral lung lesions consistent with R equi–induced pneumonia combined with clinical signs of pneumonia. Because microbiological confirmation of diagnosis was frequently not performed, some foals classified as affected may have had pneumonia caused by other agents. We believe that the impact of this misclassification was likely small. Participating veterinarians each had extensive experience with diagnosis of R equi infections and treatment of foals with pneumonia attributable to R equi. In addition, sensitivity and specificity of microbiological culture of tracheobronchial aspirates for diagnosis of R equi are not perfect. Sensitivity ranges from 57% to 100%.46–53 We have used similar diagnostic criteria for classification of affected and unaffected foals in other studies9,24–27 of pneumonia attributable to R equi, and we believe that misclassification is infrequent.

In the study reported here, we were not able to determine the effect of azithromycin on the prevalence of subclinical R equi infections. Clinically normal foals of this study were not serially tested by evaluation of thoracic radiographs, thoracic ultrasonographs, or tracheobronchial aspirates. Such standardized testing of all foals may have been preferable but was not practical or economically feasible for this farm-based clinical trial. The definition we used for an affected foal required that affected foals have clinical signs of respiratory tract disease; thus, foals affected only with subclinical infections would have been classified as unaffected foals. Additional studies are needed to determine the impact of azithromycin chemoprophylaxis on foals with subclinical R equi infections.

Caution should be used when extrapolating these data to other populations of foals. Our study population was limited to foals that were born on farms with endemic R equi infections and that remained on the farms until 150 days of age. Our study population did not include foals that arrived on the farms after birth or foals that left the farms prior to 150 days of age. The effects of azithromycin chemoprophylaxis for such foals will require further investigation. Also, our study population consisted primarily of foals that were also treated prophylactically with HIP. The impact of azithromycin chemoprophylaxis for foals that did not receive HIP could not be determined; however, a significant effect of azithromycin was detected among foals receiving HIP, which suggested that azithromycin chemoprophylaxis alone may be expected to be superior to HIP prophylaxis alone.

Epidemiologic evidence suggests that most, if not all, foals naturally affected with pneumonia attributable to R equi become infected soon after birth,8 and another study7 revealed that foals < 2 weeks old are more susceptible to experimentally induced infection. Immunologic studies9–12 have revealed that neonatal foals have immature defense mechanisms for preventing infection with R equi and are thus likely more susceptible to R equi infection during the first few weeks after birth. The data from our study, which were collected from foals at farms on which pneumonia attributable to R equi was endemic, provide additional support for the concept that many naturally affected foals acquire R equi infections during the first 2 weeks after birth.

Analysis of results of the study reported here revealed that azithromycin chemoprophylaxis was an effective strategy for reducing the cumulative incidence of pneumonia attributable to R equi among foals at equine breeding farms with endemic R equi infections. Adverse effects of azithromycin chemoprophylaxis were not detected. We did not detect an effect on azithromycin MICs for R equi isolates cultured from fecal or clinical specimens; however, these data must be interpreted cautiously in light of the limited duration of the study and limited number of foals included. In addition, these data provide evidence that azithromycin chemoprophylaxis may have minimized the impact of fecal shedding on environmental burdens of R equi. Additional studies are needed to better elucidate the effects of antimicrobial chemoprophylaxis on environmental burdens of R equi and emergence of antimicrobial-resistant microbes.

ABBREVIATIONS

HIP

Hyperimmune plasma

MIC

Minimum inhibitory concentration

NANAT

Nalidixic acid-novobiocin-actidione-potassium tellurite

OR

Odds ratio

CI

Confidence interval

MIC90

Minimum inhibitory concentration for 90% of the isolates

a.

Zithromax, azithromycin for oral suspension, 1-g packets, Pfizer Labs, New York, NY.

b.

Azithromycin Etest for minimum inhibitory concentration determination, AB Biodisk, Solna, Sweden.

c.

Rhodococcus equi antibody, hyperimmune plasma, MG Biologics, Ames, Iowa.

d.

Plasvacc, Polymune REA, Templeton, Calif.

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    • Export Citation
  • 16.

    Becu T, Polledo G, Gaskin JM. Immunoprophylaxis of Rhodococcus equi pneumonia in foals. Vet Microbiol 1997;56:193204.

  • 17.

    Higuchi T, Arakawa T, Hashikura S, et al. Effect of prophylactic administration of hyperimmune plasma to prevent Rhodococcus equi infections on foals from endemically affected farms. Zentralbl Veterinarmed [B] 1999;46:641648.

    • Search Google Scholar
    • Export Citation
  • 18.

    Giguere S, Gaskin JM, Miller C, et al. Evaluation of a commercially available hyperimmune plasma product for prevention of naturally acquired pneumonia caused by Rhodococcus equi in foals. J Am Vet Med Assoc 2002;220:5963.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Caston SS, McClure SR, Martens RJ, et al. Effect of hyperimmune plasma on the severity of pneumonia caused by Rhodococcus equi in experimentally infected foals. Vet Ther 2006;7:361375.

    • Search Google Scholar
    • Export Citation
  • 20.

    Peters DH, Friedel HA, McTavish D. Azithromycin. A review of its antimicrobial activity, antimicrobial activity, pharmacokinetic properties and clinical efficacy. Drugs 1992;44:750799.

    • Search Google Scholar
    • Export Citation
  • 21.

    Jacks S, Giguere S, Gronwall RR, et al. Pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals. Am J Vet Res 2001;62:18701875.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Davis JL, Gardner SY, Jones SL, et al. Pharmacokinetics of azithromycin in foals after IV and oral dose and disposition into phagocytes. J Vet Pharmacol Ther 2002;25:99104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Suarez-Mier G, Giguère S, Lee EA. Pulmonary disposition of erythromycin, azithromycin, and clarithromycin in foals. J Vet Pharmacol Ther 2007;30:109115.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Chaffin MK, Cohen ND, Martens RJ. Evaluation of equine breeding farm characteristics as risk factors for development of Rhodococcus equi pneumonia in foals. J Am Vet Med Assoc 2003;222:467475.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Chaffin MK, Cohen ND, Martens RJ. Evaluation of equine breeding farm management and preventative health practices as risk factors for development of Rhodococcus equi pneumonia in foals. J Am Vet Med Assoc 2003;222:476485.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Chaffin MK, Cohen ND, Edwards RF, et al. Foal-related risk factors associated with development of Rhodococcus equi pneumonia on farms with endemic infection. J Am Vet Med Assoc 2003;223:17911799.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Cohen ND, O'Conor MS, Chaffin MK, et al. Farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals. J Am Vet Med Assoc 2005;226:404413.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Grimm MB, Cohen ND, Slovis NM, et al. Evaluation of fecal samples from mares as a source of Rhodococcus equi for their foals by use of quantitative bacteriologic culture and colony immunoblot analyses. Am J Vet Res 2007;68:6371.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Prescott JF, Lastra M, Barksdale L. Equi factors in the identification of Corynebacterium equi Magnusson. J Clin Microbiol 1982;16:988990.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Brown DF, Brown L. Evaluation of the E test, a novel method of quantifying antimicrobial activity. J Antimicrob Chemother 1991;27:185190.

  • 31.

    Rosner BA. Nonparametric methods. In: Fundamentals of biostatistics. 2nd ed. Boston: Duxbury Press, 1986;278293.

  • 32.

    Rosner BA. Hypothesis testing: categorical data. Nonparametric methods. In: Fundamentals of biostatistics. 2nd ed. Boston: Duxbury Press, 1986;302357.

    • Search Google Scholar
    • Export Citation
  • 33.

    Hosmer DW, Lemeshow S. Applied logistic regression. New York: John Wiley & Sons, 1989;187215.

  • 34.

    Breslow N, Clayton DG. Approximate inference in generalized linear mixed models. J Am Stat Assoc 1993;88:925.

  • 35.

    Pinheiro JC, Chao EC. Efficient Laplacian and adaptive Gaussian quadrature algorithms for multilevel generalized linear mixed models. J Comput Graph Stat 2006;15:5881.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Morley PS, Apley MD, Besser E, et al. Antimicrobial drug use in veterinary medicine. J Vet Intern Med 2005;19:617629.

  • 37.

    Stratton-Phelps M, Wilson WD, Gardner IA. Risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: 143 cases (1986–1996). J Am Vet Med Assoc 2000;217:6873.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Lakritz J, Wilson WD. Erythromycin and other macrolide antibiotics for treating Rhodococcus equi pneumonia in foals. Compend Contin Educ Pract Vet 2002;3:256261.

    • Search Google Scholar
    • Export Citation
  • 39.

    Magdesian KG. Neonatal foal diarrhea. Vet Clin North Am Equine Pract 2005;21:295312.

  • 40.

    Martens JR, Harrington JR, Cohen ND, et al. Gallium therapy: a novel metal-based antimicrobial strategy for potential control of Rhodococcus equi foal pneumonia, in Proceedings. 52nd Annu Meet Am Assoc Equine Pract 2006;219221.

    • Search Google Scholar
    • Export Citation
  • 41.

    Harrington JR, Martens RJ, Cohen ND, et al. Antimicrobial activity of gallium against virulent Rhodococcus equi in vitro and in vivo. J Vet Pharmacol Ther 2006;29:121127.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Takai S, Ohbushi S, Koike K, et al. Prevalence of virulent Rhodococcus equi in isolates from soil and feces of horses from horse-breeding farms with and without endemic infections. J Clin Microbiol 1991;29:28872889.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Takai S, Takahagi J, Sato Y, et al. Molecular epidemiology of virulent Rhodococcus equi in horses and their environment. In: Nakajima H, Rossdale PD, eds. Equine infectious diseases VII. Newmarket, England: R & W Publications Ltd, 1997;183187.

    • Search Google Scholar
    • Export Citation
  • 44.

    Takai S, Anzai T, Yamaguchi K, et al. Prevalence of virulence plasmids in environmental isolates of Rhodococcus equi from horse-breeding farms in Hokkaido. J Equine Sci 1994;5:2125.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Slovis NM, McCracken JL, Mundy G. How to use thoracic ultrasound to screen foals for Rhodococcus equi at affected farms, in Proceedings. 51st Annu Meet Am Assoc Equine Pract 2005;274278.

    • Search Google Scholar
    • Export Citation
  • 46.

    Lavoie JP, Fiset L, Laverty S. Review of 40 cases of lung abscesses in foals and adult horses. Equine Vet J 1994;26:348352.

  • 47.

    Higuchi T, Hashikura S, Hagiwara S, et al. Isolation of virulent Rhodococcus equi from transtracheal aspirates of foals serodiagnosed by enzyme-linked immunosorbent assay. J Vet Med Sci 1997;59:10971101.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Hillidge CJ. Use of erythromycin-rifampin combination in treatment of Rhodococcus equi pneumonia. Vet Microbiol 1987;14:337342.

  • 49.

    Mueller NS, Madigan JE. Methods of implementation of an immunoprophylaxis program for the prevention of Rhodococcus equi pneumonia: results of a 5-year field study, in Proceedings. 39th Annu Meet Am Assoc Equine Pract 1992;193201.

    • Search Google Scholar
    • Export Citation
  • 50.

    Anzai T, Wada R, Nakanishi A, et al. Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals. Vet Microbiol 1997;56:335345.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Ardans AA, Hietala SK, Spensley MS, et al. Studies of naturally occurring and experimental Rhodococcus equi, in Proceedings. 32nd Annu Meet Am Assoc Equine Pract 1986;129144.

    • Search Google Scholar
    • Export Citation
  • 52.

    Sellon DC, Besser TE, Vivrette SL, et al. Comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of Rhodococcus equi pneumonia in foals. J Clin Microbiol 2001;39:12891293.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 53.

    Martens RJ, Fiske RA, Renshaw HW. Experimental subacute foal pneumonia induced by aerosol administration of Corynebacterium equi. Equine Vet J 1982;14:111116.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Box-and-whiskers plots of the age at onset of clinical signs of pneumonia for foals affected with pneumonia attributable to Rhodococcus equi. Group 1 foals were 168 untreated control foals, and group 2 foals were 170 foals treated with azithromycin at 48-hour intervals during the first 2 weeks after birth. Boxes represent the interquartile range (25th to 75th percentiles). Within each box, the horizontal line with a triangle represents the median value. Bars above and below the boxes extend to the 95th and 5th percentiles, respectively. The horizontal line with a circle represents an outlier.

  • Figure 2—

    Box-and-whiskers plots of distributions of concentrations of R equi in fecal samples collected from foals at 3 weeks of age. Quantitative R equi cultures were performed on fecal samples from 30 control foals (group 1) and 32 azithromycin-treated foals (group 2). CFU = Colony-forming unit. See Figure 1 for remainder of key.

  • Figure 3—

    Box-and-whiskers plots of distributions of concentrations of R equi in fecal samples collected from foals at 4 weeks of age. Quantitative R equi cultures were performed on fecal samples from 30 control foals (group 1) and 35 azithromycin-treated foals (group 2). See Figures 1 and 2 for remainder of key.

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    Cohen ND, Chaffin MK, Martens RJ. Control and prevention of pneumonia in foals caused by Rhodococcus equi. Compend Contin Educ Pract Vet 2000;22:10621070.

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    Chaffin MK, Martens RJ. Extrapulmonary disorders associated with Rhodococcus equi pneumonia in foals: retrospective study of 61 cases (1988–1996), in Proceedings. 43rd Annu Meet Am Assoc Equine Pract 1997;79:80.

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    Martens RJ, Martens JG, Fiske RA. Rhodococcus equi foal pneumonia: pathogenesis and immunoprophylaxis, in Proceedings. 35th Annu Meet Am Assoc Equine Pract 1989;199213.

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    Horowitz ML, Cohen ND, Takai S, et al. Application of Sartwell's model (logarithmic-normal distribution of incubation periods) to age at onset and age at death of foals with Rhodococcus equi pneumonia as evidence of perinatal infection. J Vet Intern Med 2001;15:171175.

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    Chaffin MK, Cohen ND, Martens RJ, et al. Hematologic and immunophenotypic factors associated with the development of Rhodococcus equi pneumonia of foals at equine breeding farms with endemic infection. Vet Immunol Immunopathol 2004;100:3348.

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    Breathnach CC, Sturgill-Wright T, Stiltner JL, et al. Foals are interferon gamma–deficient at birth. Vet Immunol Immunopathol 2006;112:199209.

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    Boyd NK, Cohen ND, Lim WS, et al. Temporal changes in cytokine expression of foals during the first month of life. Vet Immunol Immunopathol 2003;92:7585.

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    Hines SA, Stone DM, Hines MT, et al. Clearance of virulent but not avirulent Rhodococcus equi from the lungs of adult horses is associated with intracytoplasmic gamma interferon production by CD4+ and CD8+ T lymphocytes. Clin Diagn Lab Immunol 2003;10:208215.

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    Martens RJ, Martens JG, Fiske RA. Rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals. Equine Vet J 1989;21:249255.

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    Madigan JE, Hietala S, Muller N. Protection against naturally acquired Rhodococcus equi pneumonia in foals by administration of hyperimmune plasma. J Reprod Fertil Suppl 1991;44:571578.

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    Hurley JR, Begg AP. Failure of hyperimmune plasma to prevent pneumonia caused by Rhodococcus equi in foals. Aust Vet J 1995;72:418420.

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  • 16.

    Becu T, Polledo G, Gaskin JM. Immunoprophylaxis of Rhodococcus equi pneumonia in foals. Vet Microbiol 1997;56:193204.

  • 17.

    Higuchi T, Arakawa T, Hashikura S, et al. Effect of prophylactic administration of hyperimmune plasma to prevent Rhodococcus equi infections on foals from endemically affected farms. Zentralbl Veterinarmed [B] 1999;46:641648.

    • Search Google Scholar
    • Export Citation
  • 18.

    Giguere S, Gaskin JM, Miller C, et al. Evaluation of a commercially available hyperimmune plasma product for prevention of naturally acquired pneumonia caused by Rhodococcus equi in foals. J Am Vet Med Assoc 2002;220:5963.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Caston SS, McClure SR, Martens RJ, et al. Effect of hyperimmune plasma on the severity of pneumonia caused by Rhodococcus equi in experimentally infected foals. Vet Ther 2006;7:361375.

    • Search Google Scholar
    • Export Citation
  • 20.

    Peters DH, Friedel HA, McTavish D. Azithromycin. A review of its antimicrobial activity, antimicrobial activity, pharmacokinetic properties and clinical efficacy. Drugs 1992;44:750799.

    • Search Google Scholar
    • Export Citation
  • 21.

    Jacks S, Giguere S, Gronwall RR, et al. Pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals. Am J Vet Res 2001;62:18701875.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Davis JL, Gardner SY, Jones SL, et al. Pharmacokinetics of azithromycin in foals after IV and oral dose and disposition into phagocytes. J Vet Pharmacol Ther 2002;25:99104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Suarez-Mier G, Giguère S, Lee EA. Pulmonary disposition of erythromycin, azithromycin, and clarithromycin in foals. J Vet Pharmacol Ther 2007;30:109115.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Chaffin MK, Cohen ND, Martens RJ. Evaluation of equine breeding farm characteristics as risk factors for development of Rhodococcus equi pneumonia in foals. J Am Vet Med Assoc 2003;222:467475.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Chaffin MK, Cohen ND, Martens RJ. Evaluation of equine breeding farm management and preventative health practices as risk factors for development of Rhodococcus equi pneumonia in foals. J Am Vet Med Assoc 2003;222:476485.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Chaffin MK, Cohen ND, Edwards RF, et al. Foal-related risk factors associated with development of Rhodococcus equi pneumonia on farms with endemic infection. J Am Vet Med Assoc 2003;223:17911799.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Cohen ND, O'Conor MS, Chaffin MK, et al. Farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals. J Am Vet Med Assoc 2005;226:404413.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Grimm MB, Cohen ND, Slovis NM, et al. Evaluation of fecal samples from mares as a source of Rhodococcus equi for their foals by use of quantitative bacteriologic culture and colony immunoblot analyses. Am J Vet Res 2007;68:6371.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Prescott JF, Lastra M, Barksdale L. Equi factors in the identification of Corynebacterium equi Magnusson. J Clin Microbiol 1982;16:988990.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Brown DF, Brown L. Evaluation of the E test, a novel method of quantifying antimicrobial activity. J Antimicrob Chemother 1991;27:185190.

  • 31.

    Rosner BA. Nonparametric methods. In: Fundamentals of biostatistics. 2nd ed. Boston: Duxbury Press, 1986;278293.

  • 32.

    Rosner BA. Hypothesis testing: categorical data. Nonparametric methods. In: Fundamentals of biostatistics. 2nd ed. Boston: Duxbury Press, 1986;302357.

    • Search Google Scholar
    • Export Citation
  • 33.

    Hosmer DW, Lemeshow S. Applied logistic regression. New York: John Wiley & Sons, 1989;187215.

  • 34.

    Breslow N, Clayton DG. Approximate inference in generalized linear mixed models. J Am Stat Assoc 1993;88:925.

  • 35.

    Pinheiro JC, Chao EC. Efficient Laplacian and adaptive Gaussian quadrature algorithms for multilevel generalized linear mixed models. J Comput Graph Stat 2006;15:5881.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Morley PS, Apley MD, Besser E, et al. Antimicrobial drug use in veterinary medicine. J Vet Intern Med 2005;19:617629.

  • 37.

    Stratton-Phelps M, Wilson WD, Gardner IA. Risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: 143 cases (1986–1996). J Am Vet Med Assoc 2000;217:6873.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Lakritz J, Wilson WD. Erythromycin and other macrolide antibiotics for treating Rhodococcus equi pneumonia in foals. Compend Contin Educ Pract Vet 2002;3:256261.

    • Search Google Scholar
    • Export Citation
  • 39.

    Magdesian KG. Neonatal foal diarrhea. Vet Clin North Am Equine Pract 2005;21:295312.

  • 40.

    Martens JR, Harrington JR, Cohen ND, et al. Gallium therapy: a novel metal-based antimicrobial strategy for potential control of Rhodococcus equi foal pneumonia, in Proceedings. 52nd Annu Meet Am Assoc Equine Pract 2006;219221.

    • Search Google Scholar
    • Export Citation
  • 41.

    Harrington JR, Martens RJ, Cohen ND, et al. Antimicrobial activity of gallium against virulent Rhodococcus equi in vitro and in vivo. J Vet Pharmacol Ther 2006;29:121127.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Takai S, Ohbushi S, Koike K, et al. Prevalence of virulent Rhodococcus equi in isolates from soil and feces of horses from horse-breeding farms with and without endemic infections. J Clin Microbiol 1991;29:28872889.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Takai S, Takahagi J, Sato Y, et al. Molecular epidemiology of virulent Rhodococcus equi in horses and their environment. In: Nakajima H, Rossdale PD, eds. Equine infectious diseases VII. Newmarket, England: R & W Publications Ltd, 1997;183187.

    • Search Google Scholar
    • Export Citation
  • 44.

    Takai S, Anzai T, Yamaguchi K, et al. Prevalence of virulence plasmids in environmental isolates of Rhodococcus equi from horse-breeding farms in Hokkaido. J Equine Sci 1994;5:2125.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Slovis NM, McCracken JL, Mundy G. How to use thoracic ultrasound to screen foals for Rhodococcus equi at affected farms, in Proceedings. 51st Annu Meet Am Assoc Equine Pract 2005;274278.

    • Search Google Scholar
    • Export Citation
  • 46.

    Lavoie JP, Fiset L, Laverty S. Review of 40 cases of lung abscesses in foals and adult horses. Equine Vet J 1994;26:348352.

  • 47.

    Higuchi T, Hashikura S, Hagiwara S, et al. Isolation of virulent Rhodococcus equi from transtracheal aspirates of foals serodiagnosed by enzyme-linked immunosorbent assay. J Vet Med Sci 1997;59:10971101.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Hillidge CJ. Use of erythromycin-rifampin combination in treatment of Rhodococcus equi pneumonia. Vet Microbiol 1987;14:337342.

  • 49.

    Mueller NS, Madigan JE. Methods of implementation of an immunoprophylaxis program for the prevention of Rhodococcus equi pneumonia: results of a 5-year field study, in Proceedings. 39th Annu Meet Am Assoc Equine Pract 1992;193201.

    • Search Google Scholar
    • Export Citation
  • 50.

    Anzai T, Wada R, Nakanishi A, et al. Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals. Vet Microbiol 1997;56:335345.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Ardans AA, Hietala SK, Spensley MS, et al. Studies of naturally occurring and experimental Rhodococcus equi, in Proceedings. 32nd Annu Meet Am Assoc Equine Pract 1986;129144.

    • Search Google Scholar
    • Export Citation
  • 52.

    Sellon DC, Besser TE, Vivrette SL, et al. Comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of Rhodococcus equi pneumonia in foals. J Clin Microbiol 2001;39:12891293.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 53.

    Martens RJ, Fiske RA, Renshaw HW. Experimental subacute foal pneumonia induced by aerosol administration of Corynebacterium equi. Equine Vet J 1982;14:111116.

    • Crossref
    • Search Google Scholar
    • Export Citation

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