Rhodococcus equi (Proceedings)

Pneumonia in foals, caused by Rhodococcus equi (R. equi) is a well-known worldwide problem. Other less common clinical manifestations of R. equi infections in foals include ulcerative enterocolitis, colonic/mesenteric lymphadenopathy, immune mediated synovitis and uveitis, osteomyelitis, pyogranulomatous dermatitis, brain abscess, immune mediated anemia and septic arthritis. Inhalation of contaminated dust particles is thought to be an important route for pneumonic infection of foals. Ingestion of the organisms is a significant route of exposure and immunization but may not lead to hematogenous pneumonia unless the foal has multiple exposures to very large number of bacteria. Recent epidemiologic evidence indicates that foals that develop R. equi pneumonia are most commonly infected during the first few days of life, but clinical signs do not develop until foals are 30 to 60 days of age or older.

PATHOGENESIS

R. equi is a facultative intracellular pathogen and its infectivity is limited to cells of the monocyte-macrophage lineage. The virulence mechanisms of R. equi are associated with the virulence plasmid. These 80-90 kb plasmids encoding a family of seven closely related virulence-associated proteins designated VapA and VapC to Vap H, are responsible for the ability of R. equi to persist in, and eventually destroy alveolar macrophages. Plasmid-cured derivatives of virulent R. equi strains lose their ability to replicate and survive in macrophages and fail to induce pneumonia in foals, confirming the importance of these plasmids for the virulence of R. equi.

Foal pneumonia caused by R. equi is endemic on some farms, intermittent on others, and absent on most farms. Anecdotally, some mares have reportedly had multiple affected foals, while foals of other mares from the same environment are consistently unaffected. The source of infection for foals remains unknown. Results of previous studies indicate that the feces of mares is a potential source of R. equi for the environment and possibly a direct source of infection for foals. A study looking at 171 mares in central Kentucky looked at the association between R. equi pneumonia status of the foal and shedding of virulent R. equi by its dam. Shedding of virulent R. equi was observed in at least 1 sampling period for every mare examined, and >33% were culture-positive during all sampling periods. However, significant differences were not observed in either the fecal concentrations of total or virulent R. equi from dams of affected foals compared to dams of unaffected foals. In conclusion dams of affected foals do not shed more R. equi in feces than do dams of unaffected foals, indicating that heavier shedding by particular mares does not explain infection in their foals. However, the finding that virulent R. equi in the feces of all sampled mares during at least 1 sampling period indicates that mares are likely an important source of R. equi for their surrounding environment.

It remains unknown as to what host factors determine the outcome of exposure, however it is clear that cell-mediated immunity plays a critical role in resistance to R. equi. The clearance of virulent strains of R. equi from the lungs of adult horses is associated with the production of interferon-gamma (IFN-γ) by CD4+ and CD8+ T cells. In a study of experimental R. equi infection of foals, CD4+ T cells collected from those foals infected with the virulent strain failed to produce IFN-γ mRNA and instead produced significantly elevated amounts of interleukin (IL)-4 mRNA, indicating a Th2-type immune response. . Recent data indicate that decreased numbers of peripheral CD4+ T cells in foals may contribute to their increased susceptibility to infection, though the precise contribution of these cells to resistance was not determined

CLINICAL MANIFESTATIONS

The most common manifestation of R. equi in foals is a suppurative bronchopneumonia with extensive abscessation and suppurative lymphadenitis. The slow spread of the lung infection coupled with the remarkable ability of foals to compensate for the progressive loss of functional lung makes early diagnosis difficult. Early clinical signs may only include a slight increase in respiratory rate and mild fever. These clinical signs are often missed, allowing the disease to progress. Therefore the respiratory signs are often apparently acute in onset. A smaller percentage of these foals may be found dead or more commonly present in acute respiratory distress with high fevers (105-106 F) and no previous history of clinical respiratory disease. Approximately 50% of R. equi pneumonic foals presented to necropsy also had intestinal manifestations characterized by granulomatous or suppurative inflammation of the Peyer's patches and the mesenteric and/or colonic lymph nodes. Interestingly, the majority of the foals with R. equi pneumonia do not show clinical signs of the intestinal disease. It has been speculated however, that foals with subclinical intestinal manifestations may not gain body weight as readily as they should have. In the same study, only 4% of the foals with intestinal R. equi lesions did not have pneumonia. Immune-mediated polysynovitis, particularly the tibiotarsal and stifle joints can be seen in 30% of cases with R. equi pneumonia.

DIAGNOSIS

The insidious course of infection makes early diagnosis difficult. Recognition of foals with R. equi pneumonia prior to the development of clinical signs would likely reduce losses and limit costs associated with long-term treatment of affected foals. Many diagnostic test including complete blood cell count, fibrinogen level, thoracic ultrasound, radiographs and serology have all been used to help distinguish R. equi pneumonia from that caused by other pathogens. However, bacteriologic culture or PCR amplification combined with cytological examination of a tracheobronchial aspirate (TBA) are still the "gold standards" used to arrive at a definitive diagnosis. A recent study suggested that serological assays, whether performed on single or paired samples, cannot be used to reliably establish, confirm or exclude a diagnosis of R. equi pneumonia in foals. These serological test are problematic because of the wide spread exposure of foals to this organism at a young age which would initiate appropriate antibody production. Measurements of white blood cell count (WBC) or fibrinogen concentrations are non-specific indicators of infection or inflammation. A recent study revealed that although both measurement of fibrinogen concentrations and WBC concentration were useful for early identification of R. equi-infected foals, WBC was significantly better than measurement of fibrinogen concentrations. On the basis of their data a farm where the prevalence of the disease is high, foals with a WBC concentration of < 13,000 cells/µL are unlikely infected with a negative predicative value of 95.1% at a prevalence of 40%. On an endemic farm results of a WBC > 13,000 cells/µL or noting a foal with a fever would warrant a careful examination by the veterinarian. Foals with a WBC > 14,000 cells/µL with no clinical signs of disease and normal lung sounds should be considered for additional diagnostic tests such as thoracic ultrasonography. Ultrasonography may reveal abnormalities of the peripheral pulmonary parenchyma. If these abnormalities are detected then a TBA and/or antibiotic treatment should be initiated. Farms with endemic R. equi that have suffered significant morbidity and/or mortality rates should be monitoring rectal temperatures 2x daily, with febrile foals selected for further testing (thoracic ultrasonography) or treatment. In my experience performing twice monthly thoracic ultrasonography (starting at 3-4 weeks of age until 4 months of age) has demonstrated to be a very effective for early recognition and reduction of mortality attributed to R. equi pneumonia on several endemic farms. Diagnostic thoracic ultrasonography has been shown to be an accurate alternative imaging modality for detection of pulmonary pathology attributed to R. equi pneumonia in foals when thoracic radiography is not available. Pulmonary lesions were assigned a grade according to the severity (Table 1). The grading scale ranged from 0 (Normal) to 10 (The entire lung surface is affected). A grading scale was implemented to aid in the documentation of lesions, determine if treatment was successful and help with the communication and description of pneumonia. The foal's grade was determined not by the total number of lesions that were visualized but by the highest grade visualized. For exampled a foal with multiple one's of the left hemithorax as well as one grade 3 would be identified as a grade 3 in the left thorax. The rationale for this early screening is the belief that earlier initiation of specific treatment will not only improve the prognosis for recovery but also reduce the treatment period (2 weeks rather than 4-8 weeks of antibiotic treatments). Treatment was discontinued when the ultrasound score achieved a grade of zero. The implementation of thoracic ultrasonography on endemic farms appears to be highly sensitive because no foals which were diagnosed as a grade 0 developed clinical disease. The farms which implemented thoracic ultrasonography had no mortalities and a marked reduction of clinical disease associated with R. equi. Thoracic ultrasonography is a practical, quick, accurate and useful diagnostic modality when screening for R. equi. In addition the pulmonary lesion grades should be routinely employed when monitoring treatments and communicating the description of the lesions.

TREATMENT

Although control trials to evaluate optimal treatment are lacking, erythromycin 25mg/kg PO TID and rifampin 5mg/kg PO BID are considered standard treatment. This protocol is considered costly, labor intensive (3 times a day treatment) and can result in adverse reactions in treated foals (including diarrhea and hyperthermia). Other macrolides such as azithromycin and clarithromycin have been shown to have greater bioavailability, to be more chemically stable, and achieve high tissue as well an intracellular concentrations, . The pharmacokinetics of azithromycin have shown that 10mg/kg PO q 24 h for the first 5 days of treatment and then every other day achieve appropriate minimal inhibitory concentrations (MIC) in bronchoalveolar cells , pulmonary epithelial lining fluid and in polymorphonuclear leukocytes. . On the basis of pharmacokinetic values and minimum inhibitory concentrations of R. equi isolates, clarithromycin at a dose of 7.5 mg/kg PO q 12h can keep serum levels above the MIC90. Azithromycin or clarithromycin are commonly used in combination with rifampin because of their synergistic properties against R. equi. In one retrospective study evaluating 81 foals with naturally acquired R. equi pneumonia the combination clarithromycinrifampin was superior to azithromycinrifampin or erythromycinrifampin for the treatment of pneumonia caused by R. equi in foals in a referral population. The study evaluated shortterm (discharge from the hospital) and longterm (apparently healthy as a yearling) success rates, days of hospitalization, days with fever, days with tachypnea, and percentage of radiographic improvement were compared among groups.

Recent pharmacokinetic data of doxycycline in 4 to 8 week old foals have shown that oral administration at a dosage of 10 mg/kg every 12 h would maintain serum, pulmonary epithelial lining fluid, and BAL cell activity above the minimum inhibitory concentrations of R. equi. Therefore doxycycline may be an option for foals that have adverse reactions to the macrolides.

The overall prognosis for successful outcome (survival) has been reported to be approximately 80%. Foals that recover from R. equi pneumonia and make it to the race track have been shown to perform as well as expected.

PROPHYLAXIS

Passive Immunity

The only method proven to decrease the incidence of R. equi pneumonia is transfusion of hyperimmune plasma. The amount of plasma that should be administered and the time(s) of administration for optimal protection are unknown. It is recommended that 1L of hyperimmune (HI) plasma should be administered during the 1st 24 hours of life and again 25 days later. The rationale for this approach is that it is reported that exposure and infection occur early in life and that younger foals are more susceptible to infection than older foals. The basis of this belief include the finding that experimental infection resulted in more severe disease in foals infected at < 2 weeks of age than in foals infected at > 2 weeks of age. In another study evaluating HI plasma, the HI treated foals compared to the untreated group had lung lesions that were less severe and bacterial numbers which were significantly lower in the transfused foals. Studies do however emphasize that the protection conferred by HI plasma is not complete (decrease incidence of disease by 30-40%). For optimal control of the disease on endemic farms administration of hyperimmune plasma should be combined with other control strategies. The two commercially North American USDA approved anti-R. equi HI plasma have high anti-VapA antibody titers (Lake Immunogenics , Inc., Ontario, N.Y.and Plasvacc USA, Inc. Templeton, CA).

Chemoprophylaxis

Chemoprophylaxis may reduce the prevalence of R. equi on endemic farms. The general principles for chemoprophylaxis is that the target population must have a high risk of infection (endemic farm) with a known time period of susceptibility (first week of life) coupled with the identity of the target organism (R. equi). The use of chemoprophylaxis is not a new principle and is currently being used in equine medicine today for example pre and post operative antimicrobial administration in orthopedic surgeries and abdominal surgeries. Some of the concerns with the use of chemoprophylaxis are the potential development of superinfections, the development of resistance and the development of antibiotic induced colitis.

AZITHROMYCIN

The pharmacokinetics of Azithromycin (10mg/kg PO) in new born foals revealed an oral bioavailability of 89% with a prolonged half life when compared to the older foal. No adverse reactions were noted during the 150 day follow up period. On the basis of the pharmacokinetic data a dosage of 10mg/kg PO every 48 hours would maintain levels above the MIC90 for R. equi. . In a randomized prospective clinical trial at 10 equine breeding farms with endemic infection, foals were either administered azithromycin 10mg/kg PO EOD for 7 doses starting at either day 1 or day 2 of age or a placebo. A total of 338 foals participated in the study. 84% of these foals also received HI plasma. The prevalence of disease was 21% in the control group compared to 5% in the treatment group. The conclusion of this study was that the use of azithromycin for R. equi chemoprophylaxis may be of benefit.

GALLIUM MALTOLATE

Gallium maltolate (GaM), a trivalent semi-metal, is approved by the FDA for use in humans to treat hypercalcemia of malignancy and is chemically similar to ferric iron (Fe3+ ). Gallium functions as an iron mimic which has been used to control various microorganisms (Example: Mycobacterium tuberculosis) by exploiting their iron dependency. Gallium when given orally readily binds to the iron transport proteins transferrin and lactoferrin, which subsequently concentrates in macrophages, the target cell of R. equi. This results in inhibition of several iron dependent enzyme systems that are crucial to bacterial survival and multiplication. Gallium has recently been shown to inhibit the growth of R. equi in a highly dose dependent nature in test tube cultures. In a murine study GaM was orally absorbed in a dose dependent manner. The control mice and treatment groups (Low dose GaM 10mg/kg PO SID for 10 days or High dose GaM 50mg/kg PO SID for 10 days) were inoculated on day 4 with virulent R. equi by intraperitoneal injection. On day 10 all mice were killed and the lungs, liver and spleen where harvested for bacterial cultures and GaM concentrations. Concentrations of R. equi in the lung, liver and spleen tissues of mice treated with both high and low doses of GaM were similar. The median concentrations of R. equi in the spleens, lungs and livers of untreated control mice were ~ 12-, 16- and 42 fold greater, retrospectively, than those of mice treated with GaM. Bioavailability and safety studies in newborn foals have shown that 20mg/kg PO resulted in serum concentrations (> 700 ng/ml) of Ga considered sufficient to suppress growth (95%) of R. equi. Current studies are being conducted in Kentucky giving foals on endemic farms 20mg/kg GaM PO SID for the first month of life and assessing if foals given GaM would have a decreased incidence of R. equi pneumonia. Currently no foals in the study group have had any adverse reactions or growth retardation compared to the controls. Gallium seems to have great potential for the prevention and control of disease in foals caused by R. equi. Short term therapy (1 month) may provide additional time for maturation of innate and adaptive functions and could substantially reduce the incidence of disease on R. equi endemic farms.