Materials and Methods

This study was performed during 2008 on equine breeding farms with a history of recurrent foal pneumonia attributable to R equi infections. The study protocol was approved by the Clinical Research Review Committee at the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

Study population—Sample size calculations for the number of foals needed to detect significant differences between groups were performed on the basis of the following assumptions: the cumulative incidence of R equi pneumonia among control foals at participating farms would be 20%, and the cumulative incidence of R equi pneumonia among gallium-treated foals would be 7.5%. It was estimated that 268 foals were needed to detect a significant effect of gallium on the cumulative incidence of R equi pneumonia. It was estimated that 20% of enrolled foals would not complete the study (eg, because of death from other causes, errant compliance, voluntary withdrawal, or foals leaving the farm prior to 5 months of age). Therefore, we initially planned to enroll a minimum of 336 foals in the study to account for loss of numbers.

Selection of eligible farms—Several methods were used to identify prospective farms for inclusion in this study. First, veterinarians and personnel of R equi—endemic breeding farms who have collaborated with the investigators in previous studies were contacted and asked to consider participating in this study. Those previous studies^32–38 were designed to identify farm-related, foal-related, hematologic, and immunophenotypic risk factors associated with development of R equi pneumonia; to determine whether mares were a source of virulent R equi for their foals; to determine whether environmental concentrations of virulent R equi were associated with increased risk of R equi pneumonia; and to determine the prophylactic effects of azithromycin against R equi pneumonia. Second, numerous veterinarians who provide services for R equi-endemic breeding farms independently contacted the investigators and inquired about participating in the clinical trial. Third, a notice was posted on an electronic mailing list of equine veterinarians that provided basic information regarding the study and invited veterinarians who provide services for breeding farms with endemic R equi foal pneumonia to contact the investigators regarding participation in the clinical trial.

Prospective veterinarians were contacted via telephone, and the basic methods of the study were described. An introductory packet was sent to each veterinarian who expressed interest during the initial telephone discussion. The introductory packet described the study objectives and provided details of criteria for inclusion of farms, criteria for inclusion of individual foals, methods regarding administration of gallium and placebo formulations, methods regarding obtaining informed consent from foal owners, methods for monitoring foals, and methods for diagnosis in foals with clinical signs of pneumonia. The packet also detailed the study definition of an R equi—affected and —unaffected foal and included a copy of the informed consent form and data collection form. After reviewing the introductory packet, veterinarians were asked to visit with the owners and managers of their clients' farms, determine whether the farms met the criteria for inclusion, and assess the clients' level of interest in participating in this clinical trial.

A follow-up questionnaire regarding criteria for inclusion of the farm was sent 21 days later to each prospective veterinarian with instructions to complete the questionnaire and return it to the investigators via a preaddressed, postage-paid envelope. The information contained in this questionnaire was used to screen prospective veterinarians and farms for level of interest, capacity for continuing to follow the study protocol, and inclusion criteria. It was assumed that veterinarians who did not return the completed questionnaire were either not interested in further participation or were not suitable candidates for inclusion in the study.

Participating farms and veterinarians were further screened for inclusion by one of the investigators (MKC) on the basis of information gathered from questionnaires, telephone discussions, and, in some instances, visitation to the farms. Equine breeding farms were selected that met the following criteria for inclusion in the study: > 25 foals anticipated to be born on the farm during 2008, a history of recurrent foal pneumonia caused by R equi with cumulative incidence rates > 20% during the 2 previous years (2006 and 2007), ability and willingness to monitor all foals throughout the first 5 months after birth, availability of veterinary services for proper diagnosis and treatment of R equi pneumonia, ability and willingness to collect information and complete data collection forms for each foal enrolled in the study, willingness to allow randomized and double-masked assignment of foals to 2 treatment groups, willingness to treat foals assigned to group 2 with gallium formulation every 24 hours for the first 2 weeks after birth in a masked fashion, willingness to treat foals assigned to group 1 with a placebo formulation every 24 hours for the first 2 weeks after birth in a masked fashion, ability and willingness of the farm management and their veterinarians to follow study-defined protocols for the diagnosis of R equi pneumonia, and ability and willingness to obtain informed consent for participation from the owner of each enrolled foal. The authors have successfully used similar methods in previous studies^32–38 to select farms for investigation of endemic R equi pneumonia.

For each farm that was enrolled in the study, a primary contact person was identified, who was most frequently the broodmare or foal manager of the farm. Also, the veterinarian providing medical care to the foal population was identified. Notebooks containing explicit study instructions, log sheets for recording administration of medications, data collection forms, and informed consent forms were initially mailed prior to January 1, 2008. For enrolled farms that were located in Texas and Oklahoma, the study coordinator visited each farm and met with the veterinarian and broodmare or foal manager to provide detailed instructions for the study. Similar instructions were provided to more distant farms via conference telephone communications.

Selection of individual eligible foals—Criteria for enrollment of each foal into the study included that the foal must be born on the participating farm during 2008, the foal would remain on the farm for at least 150 days after birth, and the owner or owner's representative must provide informed consent for participation in the study, including consent to perform the 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 for any reason.

Preparation, storage, and shipment of medications—Gallium maltolate^a was compounded into a carboxymethylcellulose formulation in 4-L batches, with a final concentration of 100 mg of gallium maltolate/mL. Deionized water (2,800 mL) was added to a stainless steel bucket and placed on a heated mechanical stir plate. A stir bar was added, and the water was slowly heated and stirred until it was warm. Sixty grams of carboxymethylcellulose^b was slowly added to the warm water during stirring. Once all carboxymethylcellulose had dissolved (after approx 1 hour), the heat was removed and the carboxymethylcellulose solution was placed into a 6-L plastic container. The solution was allowed to cool to room temperature (21.1°C), and then simple syrup^c (800 mL) and benzyl alcohol^d (40 mL) were added and mixed thoroughly. Gallium maltolate^a (400 g) was then added to the solution during stirring. The solution was mechanically stirred and blended to liquefy any clumps of gallium. Deionized water was added to bring the solution to a total volume of 4 L.

A placebo formulation was also compounded that was similar in appearance, color, smell, and consistency to the gallium formulation. The placebo formulation was prepared in 5-L batches. A solution of carboxymethylcellulose was prepared, similar to that described for the gallium formulation, except that 3,000 mL of deionized water was used instead of 2,800 mL. A separate mixture (660 g of coffee creamer^e [used to adjust color and consistency], 39 mL of benzyl alcohol,^d and 780 mL of simple syrup^c) was prepared in a 7-L plastic container and mixed thoroughly. The carboxymethylcellulose solution was then added to this mixture and blended thoroughly. Once the formulation was thoroughly blended into a homogeneous mixture, food coloring^f (750 μL of yellow coloring followed by 125 μL of red coloring) was added to match the color of the gallium formulation. The formulation was mixed and blended thoroughly to produce a formulation homogeneous in consistency and color.

Gallium maltolate and placebo formulations were prepared and packaged on different days to minimize the risk of accidentally mistaking one formulation for the other. Catheter-tip syringes^g (volume, 35 mL) were loaded with either gallium or placebo formulations. For each formulation, approximately of half the syringes were loaded with 18 mL and the other half were loaded with 21 mL of formulation. The syringe tips were sealed with black rubber caps.^h Loaded syringes were stored in labeled boxes and stored in a refrigerated room until packaging for shipment to the farms.

Formulations of medications were sent to each farm on a monthly basis, starting January 1, 2008, on the basis of communications with the broodmare or foal manager regarding the projected number of eligible foals expected to be born during the upcoming month. Based on the randomized treatment chart for each farm, 14 syringes of the assigned treatment for each foal were packaged and sealed in a plastic bag.ⁱ Each syringe was labeled with farm name, foal identification number, and the day of treatment. Syringes intended for the first 7 days after birth contained 18 mL of the assigned treatment, and syringes intended for days 8 to 14 contained 21 mL. Packages of bagged syringes were placed into labeled cardboard boxes with ice packs and shipped via overnight mail to the individual farms. Farm personnel were instructed to refrigerate the medications until time of administration to enrolled foals.

Randomized assignment of foals to treatment groups—On each farm, enrolled foals were assigned a study identification number on the basis of their chronologically ordered date of birth. Prior to initiation of the study, foals on each farm were randomly assigned to 1 of 2 treatment groups on the basis of their assigned identification numbers by use of a computer-generated random sequence of the numbers 1 and 2. A treatment chart was prepared by study personnel for each farm, detailing which foal identification numbers were assigned to which treatment group. The investigators, farm personnel, and participating veterinarians were not, at any time, aware of which foals were assigned to which treatment group. The treatment chart was used by study personnel for packaging and labeling syringes of medications.

Administration of medications to foals—Foals assigned to group 1 served as control foals and were treated with 18 mL of placebo formulation orally once daily for the first 7 days after birth, followed by 21 mL of placebo formulation orally once daily for days 8 to 14. Foals assigned to group 2 served as principal foals and were treated with 18 mL of gallium formulation orally once daily for the first 7 days after birth, followed by 21 mL of gallium formulation orally once daily from days 8 to 14.

Farm personnel were instructed to administer the medications strictly on the basis of the labeled information provided on the syringes (farm and foal identification numbers and day of treatment). The dosage of gallium administered to each foal was calculated to approximate 30 mg/kg administered orally once daily. On the basis of information contained in a weight-analysis report prepared by a consulting service at a prospective participating breeding farm, the mean body weight of foals was 55.5, 60, 64.5, 70.5, and 75 kg, respectively, on days 1, 4, 7, 11, and 14 after birth. Dosages for the first week of treatment were determined on the basis of the mean body weight at day 4 (60 kg), and dosages for the second week of treatment were determined on the basis of the mean body weight on day 11 (70.5 kg).

Monitoring and diagnostic assessment of foals—Foals enrolled in the study were housed at the participating farms. Standard housing, management, and preventative health-care practices, as determined by the owners, managers, and veterinarians of each farm, were used. Foals were not deprived of any preventative or therapeutic practices that were normally provided. Farm personnel and veterinarians were instructed to adhere to study protocols regarding diagnostic approaches for enrolled foals with clinical signs of pneumonia. Foals were monitored from days 1 to 150 after birth for clinical signs of pneumonia (eg, fever, cough, nasal discharge, tachypnea, respiratory distress, or abnormal lung sounds) or any adverse effects attributable to administration of study medications.

Participating veterinarians were instructed to examine foals that developed clinical signs of pneumonia or were identified as potentially having adverse effects attributable to study medications. For foals with clinical signs of pneumonia, veterinarians were instructed to perform a complete physical examination, thoracic auscultation, and at least one of the following diagnostic procedures: thoracic radiography, thoracic ultrasonography, or tracheobronchial aspiration with microbiological culture and cytologic examination.

Classification of foals as affected or unaffected—Participating veterinarians classified each foal as affected or unaffected with pneumonia attributable to R equi, according to study-defined criteria. An affected foal was defined as a 15- to 150-day-old foal that had clinical signs of pneumonia and from which R equi was isolated from a tracheobronchial aspirate or lung specimen at postmortem examination 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.²¹ 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 for each foal regarding foal identification number, owner's name, dam's name, breed, sex, date of birth, and whether the foal received hyperimmune plasma. For foals that received hyperimmune plasma, data were collected regarding the commercial source of hyperimmune plasma, the number of transfusions, age at the time of each transfusion, volume administered for each transfusion, and route of administration. Data were collected from each foal regarding whether the foal left the farm prior to 150 days of age, age at the time the foal left the farm, and the total number of days the foal was on the breeding farm during the first 150 days after birth.

Data were collected for each foal regarding each of the first 14 days after birth as to whether study medications were administered and the labeled information on the syringes that were administered. From these data, the following variables were determined: whether the appropriate syringe was administered on each day, the total number of treatments administered, whether all treatments were administered correctly and on the correct day, and the number of treatments administered incorrectly.

For foals affected with pneumonia attributable to R equi, data were collected regarding the presence of individual clinical signs (fever, lethargy, cough, nasal discharge, tachypnea, respiratory distress, or other), age at onset of clinical signs, diagnostic methods used for classifying the foal as affected with R equi—induced pneumonia, results of each diagnostic test, antimicrobial treatments administered to the foal, duration of antimicrobial treatment, and whether the foal died, was euthanized, or recovered from the disease. For foals that died or were euthanized, data were collected regarding age at the time of death and results of necropsy.

Participating veterinarians recorded data from each foal regarding potential adverse effects of study medications, including age at onset, clinical signs, methods of treatment, and outcome. To further assess whether diarrhea was associated with study medications, data were recorded as to whether the foal had diarrhea during each of the first 21 days after birth. From these data, the following variables were calculated: whether the foal developed diarrhea during the first 3 weeks after birth, age at onset of diarrhea, whether the foal had diarrhea during the first week after birth, the number of days of diarrhea during the first week after birth, whether the foal had diarrhea during the second week after birth, the number of days of diarrhea during the second week after birth, whether the foal had diarrhea during the third week after birth, the number of days of diarrhea during the third week after birth, and the total number of days that the foal had diarrhea during the first 3 weeks after birth.

Statistical analysis—Data were analyzed by use of descriptive and inferential methods. Distributions of data that were continuous or discrete (eg, age at onset of pneumonia) were summarily described by use of median and interquartile ranges (ie, 25th to 75th percentiles). Categorical data were summarized by use of contingency tables. For inferential analyses, comparisons between groups (eg, control and gallium-treated groups) for continuous data were made with Wilcoxon rank sum tests³⁹ because these data often had a non-Gaussian distribution. Categorical data were compared by use of χ² or Fisher exact tests.⁴⁰ The association between development of pneumonia attributable to R equi and treatment group was assessed with logistic regression analysis⁴¹; ORs were determined by exponentiation of coefficients, and 95% CIs for the OR were derived by use of maximum-likelihood estimators.⁴² Because standard logistic regression did not account for the correlation of observations obtained from foals on the same farm, mixed-effects logistic regression⁴³ 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 modeled as a random effect and treatment group was a fixed effect.⁴³ For all analyses, values of P < 0.05 were considered significant. All investigators, participating veterinarians, and farm personnel remained masked to the treatment group assignment of foals until after data analysis was complete. Values of P ≤ 0.05 were considered significant.

Results

Participating farms—Forty-eight veterinarians from 14 states in the United States and 2 other countries (Germany and Argentina), representing 62 equine breeding farms, were initially considered and screened for eligibility for participation in the study. On the basis of the results of screening, 20 farms met the criteria for eligibility, and of those, 19 farms and their representative veterinarians initially agreed to participate in the study. Five farms voluntarily withdrew from the study in January when at least 1 of the first 3 enrolled foals developed diarrhea during the first week of treatment. A sixth farm, which had initially enrolled 2 foals each in group 1 and group 2, voluntarily withdrew from the study because of the time and effort associated with adhering to study protocols. A seventh farm voluntarily withdrew from the study because of unexpected changes in their horse population, resulting in a severe reduction in the number of foals available for inclusion in the study. The remaining 12 participating farms enrolled in the study were distributed among 4 states (7 farms in Texas, 3 in Oklahoma, 1 in Illinois, and 1 in Iowa).

Participating foals—For the 12 participating farms, 602 foals were eligible for inclusion in the study. Farm personnel excluded 56 foals from enrollment because of lack of owner consent (n = 36 foals), illness unrelated to R equi infection during the neonatal period (10), and various elective reasons that were not reported to the investigators (10). Of the 546 foals that were enrolled in the study, the investigators elected to exclude an additional 58 foals from the data analysis for various reasons. There was no known evidence of pneumonia in 56 excluded foals. Eighteen control foals and 21 gallium-treated foals were omitted because they left the farm prior to 100 days of age and were lost to follow-up monitoring for onset of respiratory disease. One control and 1 gallium-treated foal were omitted because they left the farm between 100 and 150 days of age and were lost to follow-up monitoring. Four control foals and 7 gallium-treated foals were omitted because they died or were euthanized prior to 150 days of age because of an injury or disease unrelated to R equi infection or respiratory tract disease. One control and 2 gallium-treated foals were omitted because the farms did not submit data forms for these foals. An additional control and gallium-treated foal were omitted because they developed clinical signs of pneumonia, and the required diagnostic procedures were not performed to adequately classify the foals as R equi affected or unaffected.

Data for 483 foals from 12 farms were analyzed. Randomization resulted in 245 (51%) foals being assigned to group 1 and 238 (49%) foals being assigned to group 2 (Table 1). There was no significant difference between groups in the distribution of foals among the participating farms. All 483 foals were born between January 1 and September 1, 2008 (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 placebo-treated foals, 121 (49.0%) were male and 124 (51.0%) were female. Of the gallium-treated foals, 132 (55.0%) were male and 106 (45.0%) were female. There were no significant differences between groups with regard to sex. All 483 foals remained on the farms and were monitored for signs of pneumonia until 150 days of age.

Most (99.0%; 240 placebo-treated and 236 gallium-treated) foals received all 14 scheduled treatments of medication. One placebo-treated foal and 1 gallium-treated foal missed 2 of the 14 scheduled treatments. Four placebo-treated foals and 1 gallium-treated foal missed one of the scheduled treatments. There was no difference between treatment groups in the distribution of foals that received 14, 13, or 12 of the scheduled treatments.

None of the 483 foals received a treatment other than the randomly assigned scheduled treatments (ie, no foals assigned to group 1 received gallium formulation, and no foals assigned to group 2 received placebo formulation). Most (97.5%; 238 placebo-treated and 233 gallium-treated) foals received all 14 treatments on the appropriate days. Seven (2.9%) placebo-treated foals and 5 (2.1%) gallium-treated foals did not receive all 14 treatments on the appropriate day. There was no difference between groups in the distribution of foals that received all 14 treatments on the appropriate day. Most (238 placebo-treated and 233 gallium-treated) foals had no treatments that were administered incorrectly (ie, incorrect day or not administered). Six (2.4%) placebo-treated and 2 (0.8%) gallium-treated foals received 1 incorrect treatment. One (0.4%) placebo-treated and 3 (1.3%) gallium-treated foals received 2 incorrect treatments. There was no difference between groups in the distribution of foals that received 0, 1, or 2 incorrect treatments.

Of the 483 foals, 355 (73%) received IV transfusions of hyperimmune plasma. Of the placebo-treated foals, 182 (74%) received hyperimmune plasma, and of the gallium-treated foals, 173 (73%) received hyperimmune plasma. There were no significant differences between groups regarding the proportion of foals that received hyperimmune plasma. Of the placebo-treated foals that received hyperimmune plasma, 89 (49%) received hyperimmune plasma obtained from 1 commercial source,^j 62 (34%) received hyperimmune plasma obtained from a second commercial source,^k 19 (10%) received hyperimmune plasma from a third commercial source,^l and 12 (6.6%) received hyperimmune plasma produced from a local herd of donor horses. Of the gallium-treated foals that received hyperimmune plasma, 96 (55%) received hyperimmune plasma from 1 commercial source,^j 51 (29%) received hyperimmune plasma from a second commercial source,^k 12 (6.9%) received hyperimmune plasma from a third commercial source,^l and 14 (8.1%) received hyperimmune plasma from a local herd of donor horses. There were no significant differences between groups in the proportion of foals that received hyperimmune plasma from each source.

There was no difference between groups in the number of transfusions with hyperimmune plasma. Of the 355 foals that received hyperimmune plasma, 50 of 182 (27%) placebo-treated and 35 of 173 (20%) gallium-treated foals received 1 transfusion. Distribution of age of foals at the time of the first transfusion with hyperimmune plasma was not significantly different between transfused foals in group 1 (median, 1 day; range, 1 to 3 days) and transfused foals in group 2 (median, 1 day; range, 1 to 4 days). There were 130 (71%) foals in group 1 and 137 (79%) in group 2 that received a second transfusion with hyperimmune plasma. The distribution of ages at the time of the second transfusion with hyperimmune plasma was not significantly different between transfused foals in group 1 (median, 14 days; range, 2 to 30 days) and group 2 (median, 14 days; range, 2 to 32 days). Two placebo-treated foals (at 30 days) and 1 gallium-treated foal (at 40 days) received a third transfusion of hyperimmune plasma. The proportion of foals that received a third transfusion was not significantly different between group 1 (2/245 [0.8%]) and group 2 (1/238 [0.4%]). Among the transfused foals, the volume of hyperimmune plasma administered for each transfusion was not significantly different between foals in group 1 (median, 1,000 mL; range, 300 to 2,200 mL) and group 2 (median, 1,000 mL; range, 300 to 2,200 mL).

Cumulative incidence of pneumonia—Of the 483 foals enrolled in the study, 182 (38%) developed clinical signs of pneumonia. Of the 245 placebo-treated foals, 84 (34%) developed pneumonia, and of the 238 gallium-treated foals, 98 (41%) developed pneumonia. There was no significant difference between groups in the proportion of foals that developed pneumonia.

Cumulative incidence of pneumonia attributable to R equi—Overall, 161 (33%) foals were classified as affected with R equi pneumonia and 322 (67%) were classified as unaffected. Of 245 placebo-treated foals, 78 (32%) were classified as affected with R equi pneumonia (Table 4). Of 238 gallium-treated foals, 83 (35%) were classified as affected with R equi pneumonia. There was no significant difference between groups in the proportion of foals that developed R equi pneumonia.

The results did not account for 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 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 12 farms were included in mixed-effects modeling, there was no significant difference in the odds of R equi pneumonia between placebo-treated and gallium-treated foals (OR, 1.2; 95% CI, 0.8 to 1.8). In mixed-effects models, the 95% CI for the SD of the random effect for farm did not include 0, indicating that the random effect of farm was significant.

The overall proportion of affected foals at each farm ranged from 13% to 76%. The proportion of affected placebo-treated foals at each farm ranged from 11% to 77% (Table 4). The proportion of affected gallium-treated foals at each farm ranged from 16% to 75%. There was no significant difference between groups in the proportion of affected foals at any farm; thus, the absence of gallium effect on cumulative incidence was consistent among farms. At 7 (58%) farms, the cumulative incidence of R equi pneumonia was higher for gallium-treated foals, whereas at 5 (42%) farms, the cumulative incidence was higher for placebo-treated foals.

The median age of affected foals at the time of onset of clinical signs of pneumonia attributable to R equi was 52 days (range, 15 to 144 days) for placebo-treated foals and 52 days (range, 16 to 136 days) for gallium-treated foals. There was no significant difference between groups in the distribution of age at onset of clinical signs of pneumonia attributable to R equi.

Of 355 foals that received hyperimmune plasma, 103 (29%) developed R equi pneumonia. Of 128 foals that did not receive hyperimmune plasma, 58 (45%) developed R equi pneumonia. The odds of developing R equi pneumonia were significantly (P = 0.001) lower (OR, 0.49; 95% CI, 0.32 to 0.75) among foals that received hyperimmune plasma, compared with those for foals that did not receive hyperimmune plasma.

Outcome of affected foals—Of the 161 affected foals, 15 foals were euthanized or died from R equi pneumonia. The overall case fatality rate was 9.3%. Of 78 affected placebo-treated foals, 5 (6.4%) were euthanized or died from R equi pneumonia. Of the 83 affected gallium-treated foals, 10 (12.0%) were euthanized or died from R equi pneumonia. The difference in survival rate between placebo- and gallium-treated foals was not significant. Necropsy examination was performed for 4 placebo-treated and 8 gallium-treated foals that died. Necropsy results for all 12 foals revealed severe pyogranulomatous pneumonia, and R equi was isolated from necropsy specimens of all 12 foals. Median age of death was 51 days (range, 33 to 140 days) for 5 placebo-treated foals and 57 days (range, 42 to 65 days) for 10 gallium-treated foals, and this difference was not significant. Of the 161 affected foals, 146 (90.7%) made a complete recovery. Of 78 affected placebo-treated foals, 73 (94%) made a complete recovery. Of 83 affected gallium-treated foals, 73 (88%) made a complete recovery.

Clinical signs of affected foals—All 161 affected foals reportedly had clinical signs of pneumonia. Overall, 68% (109/161) of affected foals had a fever. There was no significant difference in the prevalence of fever among affected group 1 (52/78 [67%]) and group 2 (57/83 [69%]) foals. Overall, 53% (85/161) of affected foals reportedly were lethargic. There was no significant difference in the prevalence of lethargy between affected group 1 (38/78 [49%]) and group 2 (47/83 [57%]) foals. Overall, 71% (115/161) of affected foals had a cough. There was no significant difference in the prevalence of cough between affected group 1 (59/78 [76%]) and group 2 (56/83 [67%]) foals. Overall, 31% (50/161) of affected foals had nasal discharge. There was no significant difference in the prevalence of nasal discharge between affected group 1 (26/78 [33%]) and group 2 (24/83 [29%]) foals. Overall, 31% (50/161) of affected foals had tachypnea. There was no significant difference in the prevalence of tachypnea between affected group 1 (24/78 [31%]) and group 2 (26/83 [31%]) foals. Overall, 43% (70/161) affected foals had respiratory distress. There was no significant difference in the prevalence of respiratory distress between affected group 1 (29/78 [37%]) and group 2 (41/83 [49%]) foals. Overall, 47% (75/161) affected foals had abnormal lung sounds detected during thoracic auscultation. There was no significant difference in the prevalence of abnormal lung sounds between affected group 1 (37/78 [47%]) and group 2 (38/83 [46%] foals). Three of 78 (3.8%) group 1 foals were reported to have an audible tracheal rattle. One of 83 (1.2%) affected group 2 foals was reported to have polysynovitis. Overall, 3 of 161 (2%) affected foals had neurologic signs attributable to R equi infection; 1 of these foals was in group 1, and 2 were in group 2.

Diagnostic testing of affected foals—All 161 affected foals were classified on the basis of clinical signs of pneumonia and at least one of the required diagnostic criteria. There were no significant differences between groups in the proportion of affected foals that underwent each diagnostic procedure. There were no significant differences between groups in the proportion of affected foals that had a positive test result for any of the diagnostic procedures. Ultrasonographic evidence of peripheral pulmonary abscesses or consolidation was the most frequently used diagnostic modality among affected foals in this study. Thoracic ultrasonography was performed for 66 (85%) affected group 1 foals and 63 (76%) affected group 2 foals. Of those, peripheral pulmonary consolidation or abscessation was visible in 66 (100%) group 1 foals and 61 (97%) group 2 foals. Thoracic radiography was performed for 6 (8%) affected group 1 foals and 7 (8%) affected group 2 foals. Of those, 5 group 1 foals and 7 group 2 foals had focal or multifocal pulmonary opacities visible on radiographs. A tracheobronchial aspirate was performed on 22 (28%) affected group 1 foals and 27 (33%) affected group 2 foals. Microbiological culture was performed for all 49 affected foals for which tracheobronchial aspirates were performed. Of those 49 foals, R equi was isolated from 16 (73%) group 1 foals and 21 (78%) group 2 foals. Cytologic examination of tracheobronchial aspirates was performed for 19 (24%) affected group 1 foals and 22 (27%) affected group 2 foals. Of those, gram-positive pleomorphic coccobacilli were identified cytologically for 15 group 1 foals and 18 group 2 foals.

Treatment of affected foals—All 161 affected foals were treated with either erythromycin, azithromycin, or clarithromycin. The proportion of foals in each group that were treated with each antimicrobial agent was summarized (Table 5). There were no significant differences between groups in the proportion of affected foals that were treated with each antimicrobial agent.

Median duration of antimicrobial treatment was 26 days (range, 2 to 90 days) for affected group 1 foals and 28 days (range, 1 to 90 days) for affected group 2 foals. There was no significant difference between groups.

Adverse effects of gallium and placebo medications—Adverse effects attributable to gallium were limited to diarrhea, which 6 of 12 veterinarians reported as a possible adverse effect of medications administered. Data regarding diarrhea for each foal at those 6 farms were recorded, representing 228 foals. Of these 228 foals, 69 (30%) developed diarrhea during the first 3 weeks after birth. Of 114 placebo-treated foals, 38 (33%) developed diarrhea during the first 3 weeks after birth. Of 114 gallium-treated foals, 31 (27%) developed diarrhea during the first 3 weeks after birth. There was no significant difference between groups in the proportion of foals that developed diarrhea during the first 3 weeks after birth.

Of the 69 foals with diarrhea, the age at onset of diarrhea was significantly (P = 0.005) younger for gallium-treated foals (median, 6 days; range, 1 to 21 days) than for placebo-treated foals (median, 12 days; range, 3 to 20 days). Overall, 25 of 228 (11%) foals developed diarrhea during the period from 1 to 7 days after birth. There was a significant (P < 0.001) difference in the proportion of foals that had diarrhea during the first week after birth between group 1 (6/114 [5.3%]) and group 2 (19/114 [16.7%]). Overall, 38 of 228 (17%) foals developed diarrhea during the period from 8 to 14 days after birth. The proportion of foals that developed diarrhea during the period from 8 to 14 days was significantly (P = 0.026) greater for placebo-treated foals (26/114 [22.8%]) than for gallium-treated foals (12/114 [10.5%]). Overall, 30 of 228 (13%) foals developed diarrhea during the period from 15 to 21 days after birth. There was no significant difference in the proportion of foals that had diarrhea during the third week after birth between group 1 (18/114 [16%]) and group 2 (12/114 [11%]).

Of the 69 foals with diarrhea, gallium-treated foals had significantly (P < 0.001) more days with diarrhea (median, 1 day; range, 0 to 5 days) during the period from 1 to 7 days after birth than did placebo-treated foals (median, 0 days; range, 0 to 3 days). Of the 69 foals with diarrhea, gallium-treated foals had significantly (P = 0.006) fewer days with diarrhea (median, 0 days; range, 0 to 3 days) during the period from 8 to 15 days after birth than did placebo-treated foals (median, 0 days; range, 0 to 5 days). For the 69 foals with diarrhea, there were no differences in the number of days with diarrhea during the period from 15 to 21 days after birth between gallium-treated (median, 0 days; range, 0 to 4 days) and placebo-treated foals (median, 0 days; range, 0 to 3 days). For the 69 foals with diarrhea, there was no difference in the total number of days with diarrhea during the period from 1 to 21 days after birth between group 1 (median, 2.5 days; range, 1 to 6 days) and group 2 (median, 2 days; range, 1 to 8 days).

All foals with diarrhea were treated empirically at the farms on the basis of the recommendations of the participating veterinarian. Details of those treatments were not available to the investigators but generally consisted of gastrointestinal protectants, antiulcer medications, and, rarely, IV fluid treatment. All foals with diarrhea recovered without apparent complications.

Discussion

There are limited prophylactic strategies available to control or prevent pneumonia attributable to R equi in foals on equine breeding farms where such infections are prevalent or recurrent. Results of the study reported here indicated that gallium chemoprophylaxis did not reduce the cumulative incidence of R equi-induced pneumonia on equine breeding farms with endemic infections. Gallium-treated foals had an overall cumulative disease incidence of 35%, which was not significantly different from the incidence of control foals (32%). Foals treated with gallium were equally likely to develop pneumonia attributable to R equi as were placebo-treated foals. In addition, chemoprophylaxis with gallium did not affect the age at onset of pneumonia, case fatality rate, or duration of antimicrobial treatment of affected foals.

Gallium (Ga³⁺), a trivalent semimetal that shares many chemical similarities with ferric iron (Fe³⁺),^{22–24,44,45} enters mammalian cells, including macrophages, and competes with ferric iron for uptake by intracellular bacteria, resulting in bacterial acquisition of gallium instead of ferric iron. The antimicrobial effects of gallium are attributable to its incorporation into Fe-dependent metabolic and reproductive enzyme systems and its inability to undergo redox cycling (unlike ferric iron), which leads to bacterial death.²⁴ Gallium maltolate provides high gallium bioavailability following oral administration in humans and a variety of laboratory animals and is not associated with toxicoses in those species.^22–24 Recent studies^26,27,30 reveal that gallium effectively inhibits growth of R equi in vitro, extracellularly, and within macrophages. Also, gallium is readily absorbed when administered orally to mice and effectively reduces tissue burdens of R equi in experimentally infected mice.²⁶ When administered intragastrically to neonatal foals, gallium is safe and readily absorbed and high serum concentrations are achieved.^29,31 In a previous study,²⁸ gallium was readily absorbed in a dose-dependent manner when gallium was compounded into a carboxymethycellulose formulation and administered orally to neonatal foals. There was marked variability among foals in maximum serum gallium concentrations; thus, it was recommended in that report²⁸ to administer at least 30 mg/kg orally every 24 hours to consistently achieve adequate serum concentrations of gallium. On the basis of those results, we chose a dosage of 30 mg/kg administered orally every 24 hours for the study reported here.

The reasons for the lack of prophylactic effects associated with administration of gallium were unknown, but several possibilities exist. Variable absorption of gallium among treated foals was expected on the basis of previous pharmacokinetic reports^28,31; however, daily dosages of 30 mg/kg should have resulted in most foals achieving serum concentrations > 0.7 μg/mL, a concentration thought to be effective at inhibiting growth of R equi. One possible explanation is that higher serum concentrations of gallium are actually needed to effectively kill or inhibit growth of R equi. Minimum inhibitory concentrations of gallium against R equi isolates have not been reported, and further investigation to determine such values is warranted. Another possible explanation for the lack of efficacy is that gallium does not adequately reach effective concentrations within the pulmonary tissues or the intracellular environment of macrophages, where R equi infection initially develops. Limited pharmacokinetic studies^28,31 have been reported for neonatal foals, and there are no reports of pharmacodynamic properties of gallium in foals. Investigations into the pharmacodynamic properties of gallium are needed to better elucidate the ability of gallium to distribute to the sites of infection.

Another possible explanation for the lack of prophylactic effect of gallium is inadequate stability of the carboxymethylcellulose formulation of gallium. In our study, syringes of gallium formulation were stored in a refrigerated environment prior to and after shipment to the participating farms and, during shipment via overnight delivery, were stored with ice packs. Because new shipments were sent out monthly, it is unlikely that any syringes were stored for > 5 weeks prior to administration. Nonetheless, there have been no studies reported that determine the stability of this compounded formulation under various storage conditions, and such studies are needed.

Another possible explanation for the lack of prophylactic effect is that chemoprophylaxis during the first 2 weeks after birth is inadequate to reduce the incidence of clinical disease. Although a previous study²¹ revealed that azithromycin, when administered daily for the first 14 days after birth, was effective at reducing the cumulative incidence of R equi pneumonia, another study^m from a large breeding farm in Germany reported contradictory findings. In that study,^m prophylactic effects were not apparent when azithromycin was administered during the first month after birth to foals.

The only adverse effect of gallium reported by veterinarians in the present study was diarrhea. Diarrhea is a common disorder in neonatal foals, and numerous etiologies exist.⁴⁶ Among the foals in the present study, the exact cause of diarrhea was frequently undetermined, making it difficult or impossible to determine whether diarrhea in an individual foal was caused by gallium administration or by numerous other potential factors. We attempted to determine the effect of gallium treatment on the incidence of diarrhea at the 6 farms that reported diarrhea as a potential adverse effect of medications. Data were collected from all enrolled foals at those farms regarding whether they had diarrhea during the first, second, and third weeks after birth and the number of days with diarrhea during each of those weeks. The incidence of diarrhea for gallium-treated foals was compared with that of the placebo-treated foals. Unfortunately, data were not obtained regarding the incidence of diarrhea in foals at each farm that were not enrolled in the study (ie, did not receive gallium or placebo treatments); thus, it was difficult to make conclusions regarding the effects of gallium. Over the first 3 weeks after birth, data did not reveal differences between treatment groups in the proportion of foals that developed diarrhea or the total number of days with diarrhea. However, data did reveal a temporal effect of gallium on the incidence of diarrhea and the number of days with diarrhea. A significantly higher proportion (16.7%) of foals treated with gallium developed diarrhea during the first week after birth than did placebo-treated foals (5.3%). In addition, gallium-treated foals had significantly more days of diarrhea during the first week after birth than did placebo-treated foals. For the second week after birth, a higher proportion of placebo-treated foals developed diarrhea and had more days of diarrhea than did the gallium-treated foals. For the third week after birth, after study medications had been discontinued, there were no differences between the treatment groups regarding the incidence of diarrhea or the number of days with diarrhea. From these data, it was concluded that the overall incidence of diarrhea was similar for gallium- and placebo-treated foals but that gallium may result in an increased incidence of diarrhea in foals < 7 days of age. This temporal effect on the incidence of diarrhea was not recognized in previous studies.²⁹ Whether gallium alters the intestinal flora of foals < 7 days of age is unknown; the mechanism by which gallium may result in diarrhea in foals is unknown, and we cannot exclude the possibility that this temporal effect on the incidence of diarrhea could have been the result of chance alone. Nonetheless, veterinarians should be aware of the potential for diarrhea in foals treated with gallium, particularly during the first week after birth. Additional investigations are needed to better understand the clinical characteristics and frequency of adverse effects associated with gallium treatment in neonatal foals.

Many clinical trials are limited by biases that may be introduced from a nonmasked study design; however, to minimize such biases among farm personnel, participating veterinarians, and investigators, a double-masked study design was used. None of the farm personnel, veterinarians, or investigators were aware of which foals received gallium until data analyses were completed. We believe that this study design effectively minimized bias; thus, such biases were unlikely to have affected the results. Nevertheless, the study reported here had limitations.

One limitation of the study was the effect that farm- and year-related factors can have on the cumulative incidence of pneumonia attributable to R equi, even in control foals. The proposed inclusion criteria for farms were designed to provide some homogeneity among farms; however, the cumulative incidence of R equi pneumonia was variable from farm to farm. Also, the cumulative incidence of R equi—induced pneumonia may vary from year to year, even on farms where the bacterium is endemic. Other studies^10,32–35 have revealed significant effects of farm and year on the cumulative incidence of pneumonia attributable to R equi. In the study reported here, the overall cumulative incidence of R equi pneumonia was 33%, which was considerably higher than that in many other studies of R equi pneumonia. This was not unexpected inasmuch as farms were selected for high cumulative incidence. Because of the high incidence of clinical disease in this study, we do not believe that year variability posed major limitations to the study. There was marked variability among farms in the incidence of R equi pneumonia; however, it is our opinion that this was likely equally distributed among the 2 treatment groups.

Potential for bias associated with withdrawal of farms and loss of numbers of foals existed, but we believe appropriate precautions were taken to prevent such biases from inadvertently altering the results. Seven farms voluntarily withdrew from the study for various reasons. All of these farms withdrew early in the study; thus, few foals were enrolled. Although the loss of these farms could have resulted in an inadequate number of foals to complete the study, we had planned for such loss of numbers, and even without these farms, adequate numbers of foals were enrolled in the study. Nonetheless, the withdrawal of these farms might have introduced bias, but we were not able to avoid any such bias. In addition, data from 58 enrolled foals were excluded from analyses for a number of reasons. The authors closely examined data for these excluded foals and the reasons for exclusion to ensure that exclusion would not result in important bias regarding the prevalence of affected foals. Only 2 of the excluded foals had clinical signs of pneumonia, and even if both had been affected by pneumonia attributable to R equi, their inclusion would not have substantially altered the findings.

Another limitation of the study reported here was the potential for inaccurate dosages of gallium administered to foals assigned to group 2. The double-masked study design required that syringes of medications be prepared in advance, so that farm personnel would not know which foals received which medications. Such a design made it logistically difficult or impossible to administer exact dosages of gallium formulation. Instead, we projected that the mean body weight of foals on days 4 and 11 after birth would be approximately 60 and 70 kg, respectively. To avoid logistic problems associated with providing different volumes of medications for each day to account for daily weight gain, we elected to treat all foals for the first week after birth on the basis of a projected body weight of 60 kg and to treat all foals for the second week after birth at a projected body weight of 70.5 kg. Syringes of medications were prepared in advance to provide 30 mg of gallium/kg/d for each foal assigned to group 2, and similar volumes of placebo were prepared for foals assigned to group 1. Some enrolled foals weighed less than the mean projected body weights, and likewise, other enrolled foals weighed more than the mean projected body weights. Despite the approximated dosages of gallium administered to group 2 foals, we believe that the study design likely provided approximately 30 mg of gallium/kg/d to foals assigned to group 2. Some foals may have failed to swallow the entire orally administered dose of gallium.

Another potential limitation of this study was compliance at specific farms; however, the authors' impression was that personnel at the farms and the participating veterinarians adhered to the study protocols in an exceptionally thorough and complete manner. Of 483 foals, 97.5% received all 14 assigned medications on the appropriate day and no foals received the wrong medication. Of 182 foals with clinical signs of pneumonia, veterinarians appropriately followed study protocols regarding diagnostic testing for all foals except 2, and data from those 2 foals were omitted from analyses.

As with any study of disease, the potential for mis-classification of disease outcome (ie, affected vs unaffected with R equi pneumonia) existed; 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 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. 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 ultrasonographic⁴⁷ detection of peripheral lung lesions consistent with R equi—induced pneumonia combined with clinical signs of pneumonia. Because microbiological confirmation of diagnosis was not frequently performed, some foals classified as affected may have had pneumonia caused by agents other than R equi. The authors believe the effect of this misclassification was likely small and equally distributed among foals in each treatment group. Participating veterinarians each had extensive experience with diagnosis and treatment of pneumonia attributable to R equi in foals. In addition, sensitivity and specificity of microbiological culture of tracheobronchial aspirates for diagnosis of R equi are not perfect; reported sensitivity ranges from 57% to 100%.^48–55 Furthermore, the randomization and double-masked methods used in this study were likely to have minimized any misclassification bias.

In the study reported here, it was not possible to determine the prophylactic effect of gallium on the incidence of subclinical R equi infections. Certainly, some foals develop R equi—associated lung abscesses that spontaneously resolve without developing clinically apparent signs of disease. Clinically normal foals of this study were not serially screened by use of thoracic radiography or thoracic ultrasonography. The primary objective was to determine the effect of gallium on the incidence of clinical disease rather than subclinical disease. An alternative approach would have been to study subclinical disease and use serial ultrasonographic testing of all foals to detect subclinical disease; however, such screening methods would have likely resulted in foals that had positive test results being treated with antimicrobials prior to onset of clinical signs. The authors considered this approach when designing this study but believed it was most important to design the study to detect chemoprophylactic effects on clinically apparent disease rather than subclinical disease. The definition used for an affected foal required that affected foals had 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 effect of gallium chemoprophylaxis on foals with subclinical infections of R equi.

Caution should be used when extrapolating these data to other populations of foals. The 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. The study population did not include foals that arrived at the farms after birth or foals that left the farms prior to 150 days of age. Also, the study population consisted primarily of foals that were also treated prophylactically with hyperimmune plasma. The effect of gallium chemoprophylaxis in foals that do not receive hyperimmune plasma could not be determined.

Although it was not a primary aim of this investigation, results indicated that foals that received hyperimmune plasma were significantly less likely to develop R equi pneumonia than were foals that did not receive hyperimmune plasma. Only 29% of foals treated prophylactically with hyperimmune plasma developed R equi pneumonia, as opposed to 45% of foals that were not treated with hyperimmune plasma. Whether hyperimmune plasma was administered to foals and the frequency of administration varied widely from farm to farm, but varied little among foals on the same farm; thus, it was not possible to separate the effect of hyperimmune plasma from the effect of farm. Nonetheless, we believe these data provide some evidence to support the concept that hyperimmune plasma has protective effects against R equi pneumonia. Previous studies^14–20 provide conflicting evidence regarding the significance of prophylactic effects of hyperimmune plasma on farms with endemic infections.