Clinical manifestations, diagnosis, treatment, and prevention of Rhodococcus equi infections in foals

veterinary microbiology VeterinaryMicrobiology56 (1997)313-334

Clinical manifestations, diagnosis, treatment, and prevention of Rhodococcus equi infections in foals Steeve Gig&e Department

of Pathobiology,

Ontario

Veterinary

* , John F. Prescott College, 2WI

University

of Guelph.

Guelph,

Ont.,

Cam&

NIG

Abstract Since the 1986 Rhodococcus equi workshop, there have been major breakthroughs in understanding the epidemiology of, the virulence of, and the immune responseto, this intriguing pathogen. However, with the exception of the use of hyperimmune plasma for the prevention of the disease(Martens et al., 1989; Madigan et al., 1991) the clinical aspectsof R. equi infections have essentially remained unchanged. This article reviews the various clinical manifestations and summarizesrecent advancesin diagnosis, treatment and prevention of R. equi infections in foals. 0 1997 Elsevier ScienceB.V. Keywords:Rhodococcus

equi;

Clinicalmanifestation;Diagnosis;Treatment;Prevention;Foal

1. Introduction

Rhodococcus equi is one of the most important cause of disease in foals between 1 and 6 months of age, with most foals showing clinical signs before the age of 4 months. Although all horse farms are likely to be infected with R. equi, the clinical disease is enzootic and devastating on some farms, sporadic on others, and unrecognized on most. This probably reflects differences in environmental (temperature, dust, soil pH etc.) and management conditions as well as differences in virulence of isolates (Takai et al.. 1991). Even though R. equi may be isolated from areas never inhabited by horses, it is found in greater numbers where horses are present since the volatile fatty acids found in

* Corresponding author. Tel.: + l-519-8244120 ext. 4717; fax: + l-519-7670809; e-mail: [email protected]. 0378-l 135/97/$17.00 0 1997ElsevierScienceB.V. All rightsreserved. PII SO378-1135(97)00099-0

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manure enhance its growth (Hughes and Sulaiman, 1987). Intestinal carriage in adult horses is likely passive, representing only acquisition from contaminated soil, but the organism can replicate in the intestine of foals up to about three months of age (Takai et al., 1986a). Inhalation of contaminated dust particles is the most important route of pneumonic infection in foals.

2. clinical

manif~tatiOIls

2. I. Bronchopneumonia The most common manifestation of R. equi infections in foals is a chronic suppurative bronchopneumonia with extensive abscessation and associated suppurative lymphadenitis (Zink et al., 1986). The slow spread of the lung infection coupled with the remarkable ability of foals to compensate for the progressive loss of functional lung make early clinical diagnosis difficult. Early clinical signs may only include a slight increase in respiratory rate and a mild fever. These subtle clinical signs are either missed or ignored, allowing the condition to progress. Therefore, even with the chronic form of the disease, respiratory signs are often of apparently acute onset. The clinical signs of the ‘acute on chronic’ form of the disease may include decreased appetite, mild lethargy, fever (38.8-4O.O”C, up to 41.5”C), tachypnea, and increased effort of breathing characterized by nostril flaring and increased abdominal effort. Cough and bilateral nasal discharge are inconsistent findings. Most affected foals are in good body condition but weight loss may become apparent with chronicity in severely affected individuals. A smaller percentage of affected foals may present with a more devastating subacute form. These foals may be found dead or more commonly present in acute respiratory distress with a high fever and no previous history of clinical respiratory disease. These severely affected foals often die within several days despite aggressive treatment (Beech and Sweeney, 1991). On post mortem examination this subacute form of the disease is characterized by the presence of a diffuse miliary pyogranulomatous pneumonia and the maturity of the lesions suggest extensive lung involvement before apparition of the clinical signs (Martens et al., 1982). On auscultation, the lung sounds of foals affected by both the chronic or subacute form of the disease vary considerably and the findings can be confused by referred sounds from the upper airway. Inspiratory and/or expiratory wheezes or crackles may be audible over affected areas which are more commonly located cranioventrally. In more severely affected foals, auscultation may only reveal large airway sounds suggesting consolidation. Lung sounds are diminished over areas of severe consolidation, extensive peripheral abscess formation, or pleural effusion. However, pleural effusion is a rare finding in cases of R. equi pneumonia. The findings on auscultation do not necessarily correlate well with the severity of the pneumonia (Falcon et al., 1985). In some cases, severe disease may be present with few abnormal auscultatory findings. The sensitivity of thoracic auscultation can be significantly enhanced by inducing the foal to breathe deeply using a rebreathing bag or alternatively by cupping a hand over the

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nostrils for a few seconds. These two procedures should not be performed on foals in severe respiratory distress and care should be taken to avoid overstressing foals during physical examination. In addition to auscultation, thoracic percussion may be helpful to detect areas of consolidation or abscess formation, and may help to detect pleural effusion in rare cases with pleural involvement. 2.2. Intestinal manifestations Approximately 50% of pneumonic foals presented for necropsy also had intestinal manifestations (Zink et al., 1986). However, the vast majority of foals with R. equi pneumonia do not show clinical signs of intestinal disease. In the same study, only 4% of the foals had intestinal lesions without pneumonia. The intestinal form of R. equi infection is characterized by a multifocal ulcerative enterocolitis and typhlitis over the area of the Peyer’s patches with granulomatous or suppurative inflammation of the mesenteric and/or colonic lymph nodes (Zink et al., 1986). Occasionally, a single large abdominal abscess (usually in a mesenteric lymph node) often causing adhesion to the large or small bowel is the only finding (Zink et al., 1986). Peritonitis may result and the organism may be isolated from the peritoneal fluid (Baldwin et al., 1992). Clinical signs associated with the abdominal form of the disease may include fever, depression, anorexia, weight loss, colic, and diarrhea (Zink et al., 1986; Baldwin et al., 1992). Marked gastrointestinal lymphatic obstruction associated with increased protein concentration in the peritoneal fluid and hypoproteinemia may lead to ascites giving foals a pot-bellied appearance. Such foals have a poor prognosis because of the extensive granulomatous inflammation of the colonic mucosa and submucosa, and mesenteric lymph nodes. 2.3. Non-septic polysynovitis Immune-mediated polysynovitis, particularly of the tibiotarsal and stifle joints, is seen in approximately one-third of cases with R. equi pneumonia (Sweeney et al., 1987). Occasionally all joints are affected. The degree of joint effusion is variable and, in most cases, lameness is not apparent or limited to a stiff gait. Cytological examination of the synovial fluid usually reveals a non-septic mononuclear pleocytosis (Sweeney et al., 1987) and bacteriologic culture of the synovial fluid usually fails to yield an agent (Sweeney et al., 1987; Madison and Scarratt, 1988; Kenney et al., 1994). Histologic examination of the synovial membrane reveals lymphoplasmacytic synovitis (Madison and Scarratt, 1988; Kenney et al., 1994). Fluorescein-labeled anti-equine IgG staining of the synovial membrane of three affected foals revealed evidence of immunoglobulin within the synovial membrane (Madison and Scarratt, 1988). Rheumatoid factors (i.e. antibodies directed against autologous or heterologous Fc portion of immunoglobulin) were also identified in the synovial fluid of a foal with non-septic joint effusion and R. equi pneumonia (Kenney et al., 1994). Local therapy of the affected joints is not indicated as the effusion usually resolves without any apparent consequences as the pneumonia resolves. The presence of a non-septic polysynovitis in a foal between 1 and

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6 months of age is highly suggestive of R. equi infection investigation (see section on diagnosis).

313-334

and deserves further

2.4. Septic arthritis and osteomyelitis Bacteremic spread of the organism from the lungs or gastrointestinal tract may occasionally result in septic arthritis and/or osteomyelitis. However, foals can occasionally develop R. equi septic arthritis or osteomyelitis without apparent lung or other source of infection. The degree of lameness of foals with septic arthritis distinguishes them from foals with immune-mediated polysynovitis. In equivocal cases, bacterial culture and cytological examination of the synovial fluid should be performed. Foals with R. equi osteomyelitis usually show signs of lameness and swelling over the affected area. In most cases the clinical and pathological findings bear all the hallmark of primary metaphysitis (P type osteomyelitis) (Collates et al., 1990; Desjardins and Vachon, 1990; Firth et al., 1993). The extension of bone infection through the metaphyseal cortex and into surrounding tissue may lead to cellulitis and sometimes septic arthritis. In addition to appropriate antimicrobial therapy (see section on treatment), foals with R. equi septic arthritis and osteomyelitis often require aggressive local therapy and the prognosis is guarded. R. equi vertebral osteomyelitis, although recognized for the first time by Rooney (19661, has been described more frequently recently (Gig&e and Lavoie, 1994; Olchowy, 1994; Chaffin et al., 1995). Early signs of vertebral osteomyelitis are frequently limited to fever, lethargy, stiff gait, reluctance to move, and resistance to palpation (Mayhew, 1989). However, the diagnosis is rarely made until the infection extends to the epidural space causing signs of spinal cord or nerve compression. Such signs may include paresis, ataxia, paralysis, or cauda equina syndrome depending on the severity and site of compression. Radiography is important in making a diagnosis of vertebral osteomyelitis. However, in 4 of the 6 cases of R. equi vertebral osteomyelitis recently reported the radiographs were normal despite extensive lesions seen on post-mortem examination (Gig&e and Lavoie, 1994; Olchowy, 1994; Chaffin et al., 1995). Radiographic changes in the vertebral body may not be evident until 2-8 weeks after the onset of clinical signs (Digby and Kersley, 1979). When available, nuclear scintigraphy (Markel et al., 1986) or computed tomography (Markel et al., 1988) should be used to assist localization of the lesion(s) in early cases of vertebral osteomyelitis. Any foals between 1 and 6 months of age presented for lameness or neurologic signs along with leukocytosis or hyperfibrinogenemia should be evaluated for R. equi infection. 2.5. Other manifestations in foals Ulcerative lymphangitis, cellulitis, and subcutaneous abscesses have also been reported (Dewes, 1972; Smith and Jang, 1980; Zink et al., 1986; Perdrizet and Scott, 1987). The larvae of Strongyloides westen’ have been proposed as the primary insult to the skin in a few cases (Dewes, 1972, 1989) but their exact role remain to be determined. Other rare manifestations of R. equi infections in foals include uveitis

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(Beech and Sweeney, 19911, panophthalmitis (Blogg et al., 19831, nephritis, and hepatic and renal abscessation (Ellenberger and Genetzki, 1986). 2.6. Manifestations in adult horses Disease due to R. equi is rare in adult horses. There are a few reports of sporadically occurring illness similar to that observed in foals involving primarily lungs or colon and related lymph nodes, or rarely as a wound infection (Roberts et al., 1980; Ellenberger and Genetzki, 1986; Zink et al., 1986). In one adult horse, acquired combined immunodeficiency of unknown origin led to R. equi septicemia and lung abscessation (Freestone et al., 1987). Rarely, the organism has also been isolated from infertile mares and aborted fetuses (Zink et al., 1986; Fitzgerald and Yamini, 1995).

3. Diagnosis The distinction between lower respiratory tract infections caused by R. equi and that caused by other pathogens is problematic especially on farms with no previous history of R. equi infections. Many diagnostic tests including a complete blood count (CBC), fibrinogen level, radiographs, and serology may help distinguish R. equi pneumonia from that caused by other pathogens. However, bacteriologic culture combined with cytological examination of tracheobronchial exudate are still the ‘gold standards’ used to arrive at a definitive diagnosis. 3.1. Clinical laboratory tests A CBC including plasma protein and fibrinogen concentration should be performed in all cases suspected of having R. equi pneumonia. Hyperlibrinogenemia is the most consistent laboratory finding, although rare cases may have normal fibrinogen concentrations. Neutrophilic leukocytosis with or without monocytosis is also common (Falcon et al., 1985; Sweeney et al., 1987). One study showed significantly higher fibrinogen concentrations and white blood cell counts (WBC) in non-survivors than in survivors (Falcon et al., 1985) whereas another study showed no difference between the 2 groups (Sweeney et al., 1987). In a review of 40 cases of lung abscesses there was a trend toward higher fibrinogen concentrations and WBC for the foals from which R. equi was isolated from the tracheal wash as opposed to foals from which another pathogen was isolated (Lavoie et al., 1994). However, in all these studies there was a considerable overlap in ranges, which preclude the use of fibrinogen concentrations and WBC as diagnostic tests or prognostic indicators for an individual animal. A CBC also permits evaluation of the hydration status and a crude evaluation of the immune system. Persistent lymphopenia ( < 1.0 X 109/1), especially in Arabian foals, is suggestive of combined immunodeficiency and deserve a more thorough evaluation of the immune function. Workers in Ireland have consistently noted thrombocytosis in foals infected with R. equi (Leadon et al., 1988). This finding has not been repeated by workers elsewhere. Finally, arterial blood gas analysis is useful for monitoring the

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oxygenation and acid base status during therapy of pneumonic foals showing signs of respiratory distress or cyanosis. 3.2. Imaging techniques Thoracic radiography is useful in evaluating the severity of pneumonia and in assessing response to therapy. A prominent alveolar pattern characterized by ill defined regional consolidation is the most common radiographic abnormality (Falcon et al., 1985). The consolidated lesions are often seen as more discrete nodular and cavitary lesions compatible with pulmonary abscessation. Abscesses may contain gas which can be seen radiographically. Radiographic evidence of tracheobronchial lymphadenopathy characterized by nodular densities displacing the trachea dorsally was reported to be useful prognostically as it was observed more often in foals that did not recover (Falcon et al., 1985). Although in one study non-survivors tended to have more severe radiographic lesions than survivors (Falcon et al., 1985) many survivors have very severe radiographic lesions and radiographs should not be used as the sole criterion for prognostication and euthanasia (Sweeney et al., 1987; Beech and Sweeney, 1991). In foals less than 3 months of age radiographic evidence of nodular lung lesions and lymphadenopathy are highly suggestive of R. equi infections. However, in foals of 3 months and older S. zooepidemicus is another common cause of lung abscesses(Lavoie et al., 1994). In some cases of R. equi pneumonia, a mild to severe bronchointerstitial pattern is the only radiographic finding. In foals presented with acute respiratory distress and having a prominent bronchointerstitial pattern on radiographic evaluation, the major differential diagnosis is sporadic severe bronchointerstitial pneumonia. The cause of this syndrome is unknown although several etiologies have been proposed (Buergelt et al., 1986; Prescott et al., 1991; Wilson, 1992a; Lakritz et al., 1993). R. equi can be isolated from some of these cases but its exact role in the pathophysiology, if any, remains to be determined. In some cases of respiratory distress in foals Pneumocystis carinii may be present with R. equi or may cause respiratory disease by itself (Ainsworth et al., 1993a; Ewing et al., 1994a). In one study 3 out of 5 foals with combined R. equi-P. carinii infections had a distinct reticulonodular (miliary) pattern radiographically, which may aid in the antemortem diagnosis of P. carinii infections (Ainsworth et al., 1993a). Ultrasonography is a helpful diagnostic tool when lung involvement includes peripheral areas but may not be as useful as radiography to evaluate the extent of lung lesions since abscesses with overlying aerated lung will not be detected. However, in most horses and foals with pulmonary abscessation the periphery of the lung is affected enabling the ultrasonographer to successfully image some of the abscesses(Reef, 1991). To the authors’ knowledge, comparison between ultrasonography and radiography has not been critically evaluated in foals with R. equi pneumonia. Nevertheless, ultrasonography may prove to be very useful in evaluating the severity of pneumonia and in assessing response to therapy especially for equine practitioners who do not have access to thoracic radiography. A sector scanner equipped with a 7.5 or 5.0 MHz transducer is preferred but the linear scanner commonly used in equine reproduction can also be used. Scintigraphic perfusion imaging showed major pulmonary perfusion defects characterized by a decrease or absence of radioisotope activity in affected areas of the lungs in

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4 foals experimentally infected with R. equi (Martens et al., 1989). The perfusion scans correlated well with radiographic and necropsy assessments of functional lung tissue. Further studies are necessary to evaluate the value of scintigraphy compared to radiology in foals with pneumonia. 3.3. Serologic tests A number of workers have investigated serological approaches to diagnose R. equi infections in foals. These tests divide into 3 major types: agar gel immunodiffusion, synergistic hemolysis inhibition, and enzyme-linked immunosorbent assay. The serologic diagnosis of R. equi infections is problematic because the widespread exposure of foals to this organism at a young age leads to antibody production without necessarily producing clinical disease. In addition, maternally-derived antibody may cause positive reactions in some serological assays which further confound the interpretation of the test. Finally, serologic tests of proven sensitivity and specificity are not commercially available. 3.3.1. Agar gel immunodiffusion (AGID) Virtually all strains of R. equi produce membranolytic exoenzymes originally called ‘equi factors’ (Prescott et al., 1982). These exoenzymes consist of a cholesterol oxidase and phospholipases C. The AGID test detects mainly precipitating antibodies against these exoenzymes but the identity of other antigens sometimes detected is unknown. The vast majority of adult horses are negative for precipitating antibody on AGID, consequently, there are no false positives as a result of maternally-derived antibody (Gaskin et al., 1990). In the same study, the earliest detection of such antibody in foals was at 26 days of age. There are contradicting data regarding the sensitivity and specificity of this test making it difficult to interpret (Table 1). After seroconversion, clearance of

Table 1 Agar gel immunodiffusion

(AGID)

Author

Nakazawa Hietala

Hoffman

Skalka

in the diagnosis

Culture-confirmed R. equi pneumonia et al., 1987

100% (18/l@ necropsy

et al., 1985

0% (O/54)

50% (11/22) TBA

et at., 1993

and Svastova,

of R. equi pneumonia Non-R. equi infections a

60% (3/5) TBA 1985

-

31% (16/52)

in foals

Clinically healthy foals

Sensitivity

Specificity

18% (9/51)

100%

100%

-

50%

-

60%

65% (31/48)

b

69%

-

a Foals that were negative for R. equi on culture of a bronchial lavage (Hoffman et al., 1993) or on culture of the affected tissues at necropsy (Nakazawa et al., 1987). b Clinically healthy foals were not considered in calculation of the specificity since bacterial culture was not attempted.

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precipitating antibodies in sick foals receiving appropriate antimicrobial been reported to take 2-6 weeks (Gaskin et al., 1990)

therapy has

3.3.2. Synergistic hemolysis inhibition (SHI) The exoenzymes previously described can interact with a phospholipase D produced by Corynebacterium pseudotuberculosis, or Staphylococcus aureus beta toxin to collectively destroy mammalian erythrocyte membranes causing hemolysis (Linder and Bemheimer, 1982). Presence of antibodies against these exoenzymes will result in inhibition of this synergistic hemolysis. No study has defined the sensitivity and specificity of the SIB assay in relation to culture of a tracheobronchial aspirate. In one study, a SIB assay distinguished foals with naturally occurring pneumonia from healthy foals but failed to identify foals in the early stages of experimentally induced disease (Prescott et al., 1984b). Using a more sensitive SHI assay, Skalka and Svastova (1985) and Skalka (1987) demonstrated the widespread nature of subclinical infection in foals. As opposed to AGID, the SIB assay is quantitative but perhaps too sensitive for diagnostic purposes, detecting more subclinical infections than AGID (Gaskin et al., 1990). The SIB assay is also more difficult to perform in the laboratory. 3.3.3. Enzyme-linked immunosorbent assay (ELISA) Four different enzyme-linked immunosorbent assays (ELISA) have been described for measuring the humoral immune response to R. equi. All these ELISA were developed using different antigens and, in most cases, the antigens are poorly defined. ELISA studies using supematant antigens prepared in different ways showed that antibodies to R. equi are widespread in the horse population and that there was no apparent correlation between ELISA titers and clinical disease (Ellenberger et al., 1984; Hietala et al., 1985). In another study, Tween 20 detergent extract from the type strain ATCC 6939 gave the broadest crossreactivity among the strains tested and was found superior to supematant of autoclaved or homogenized cells (Takai et al., 1985). Using this ELISA, the mean values of anti-R. equi IgG antibodies in foals known or suspected to be ill with R. equi pneumonia were higher than in normal foals in a limited number of animal tested (Takai et al., 1985, 1986b). This ELISA was more sensitive than AGID (Takai et al., 1985). The sensitivity and specificity of this ELISA in relation to bacterial culture of a tracheobronchial aspirate are however unknown. A radial immunodiffusion enzyme assay that combine the principles of radial immunodiffusion and ELISA without the need for serum titration or spectrophotometer to interpret the results has also been applied to the serologic diagnosis of R. equi infections and found to correlate well with Tween 20 ELISA (Takai et al., 1990). Recently, Prescott et al. (1996) developed an ELISA based on Triton X-l 14 detergent extracted whole cell material in which the virulence-associated protein (VapA) predominates. This ELISA also demonstrated seroconversion in clinically healthy foals between 8 and 10 weeks of age. Foals experimentally infected with a virulent strain of R. equi seroconverted two weeks post challenge whereas foals experimentally infected with a non-virulent strain did not (Gigdre and Prescott, unpublished data), which suggests that a diagnostic advantage of this ELISA might be the failure to detect exposure to non-virulent strains. The sensitivity and specificity of this ELISA have not yet been evaluated in a clinical setting.

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In summary, there is a lack of sensitivity and specificity data on which to assess the significance of most serological assays. Serologic tests (AGID, SIB, ELISA) may be more useful at the farm level to detect overall exposure (see section on prevention) than at the individual level to diagnose clinical infection with R. equi. Simple reliance on serology as a diagnostic test for R. equi infections will result in overdiagnosis of this disease and in missing infections in the early stages. Reliable criteria for serological diagnosis of R. equi pneumonia are required. 3.4. Cytology and culture Because of the lack of field studies comparing the sensitivity and specificity of serologic tests, bacteriologic culture combined with cytological examination of a tracheobronchial aspirate (TBA) is still the only acceptable way to arrive at a definitive diagnosis of R. equi pneumonia. TBA can easily be obtained by percutaneous transtracheal aspiration or, alternatively, with a sterile polyethylene tube passed through the biopsy channel of a flexible fiber optic endoscope. Endoscopy has the advantage of allowing visualization and selective aspiration of exudate when it is present (which may therefore enhance recovery of R. equi). However, bacterial contamination of nasal or pharyngeal origin commonly occurs with endoscopy. The use of a sterile cellulose sheath over the endoscope (Hoffman et al., 19911, or a guarded endoscopy swab or aspiration catheter passed through the biopsy channel of the endoscope (Sweeney et al., 1989; Darien et al., 1990) significantly reduces but may not completely eliminate upper airway contamination. TBA should be avoided in foals with severe respiratory distress. In one study only 7 (62%) of the 11 foals with positive R. equi culture at necropsy and 57 (64%) of the 89 foals with radiographic evidence of lung abscessation yielded R. equi on culture of a TBA (Hillidge, 1987). Martens et al. (1982) also reported that bacterial culture of a TBA was not consistent in detecting the presence of R. equi in experimentally infected foals. However, in 2 other studies all 17 foals, in which R. equi was isolated from the lung parenchyma at necropsy, were previously found to yield the organism on culture of a TBA suggesting that culture of a TBA is a consistent and reliable method of diagnosing R. equi pneumonia (Muller and Madigan, 1992; Lavoie et al., 1994). Larger case series are required to determine the exact sensitivity of TBA for the diagnosis of R. equi pneumonia in foals. R. equi can usually be isolated within 48 h of culture of fresh specimens obtained prior to initiation of antimicrobial therapy but cultures should be incubated for 72 h before discarding, as some colonies may not be evident before this, especially if other bacteria are present. In foals that have recently received antimicrobials the culture may take several days to grow or may only be isolated anaerobically (Prescott and Hoffman, 1993). Multiple other pathogens may be isolated along with R. equi (Falcon et al., 1985; Sweeney et al., 1987). Foals without clinical disease exposed to contaminated environments may have R. equi in their tracheas as a result of inhalation of contaminated dust. In one study conducted on a farm with enzootic R. equi pneumonia 77 (35%) of 216 foals sampled had positive TBA cultures but no signs of respiratory disease (Ardans et al., 1986). For this reason, bacteriologic culture of a TBA should be interpreted in the context of cytological evaluation, physical examination and laboratory results. A light growth of R. equi from

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a foal with no clinical signs of respiratory disease, normal fibrinogen and WBC, and no cytological abnormalities may be an incidental finding. The use of polymerase chain reaction (PCR) based on the VapA gene sequence would be a more rapid and perhaps more sensitive mean of identifying R. equi in TBA samples than isolation especially if the foal sampled is being treated with antimicrobial agents. In the study of diagnostic methods reported by Anzai et al. in this issue, culture of a TBA was found to be highly reliable and, interestingly, more sensitive than either PCR or indirect immunofluorescence staining of TBA cells. A TBA is also the sample of choice for cytological examination. Cytologically, intracellular Gram-positive pleomorphic rods could be identified in 22 (61%) of 48 foals in which R. equi was cultured from a TBA. In 8 (22%) foals, however, no bacteria were seen on Gram stain and in 6 (17%) foals, only other bacterial types could be seen (Sweeney et al., 1987). Bronchoalveolar lavage (BAL) fluid is not a suitable sample for cytological diagnosis of pneumonia since approximately 50% of the horses with confirmed pneumonia have normal BAL fluid analysis despite evidence of acute inflammation on cytological examination of a TBA (Rossier et al., 1991). However, if P. carinii is suspected (with or without R. equi), BAL fluid would be the sample of choice for an antemortem diagnosis (Ewing et al., 1994a). Blood culture are not routinely performed in cases of R. equi pneumonia. Leadon et al. (1988) however reported positive blood cultures in 6 of 10 foals with R. equi pneumonia and foals experimentally infected with virulent R. equi are often septicemic (Gig&e and Prescott, unpublished data). Blood culture is also the most sensitive means of diagnosis in human patients infected with this organism (Donisi et al., 1996). Whether blood culture would be a useful diagnostic tool or prognostic index in foals remains to be determined. Positive R. equi culture from nasal or fecal swabs cannot be taken as evidence of infection. R. equi can be cultured from the feces of normal horses even if they live in farms with no history of R. equi pneumonia (Nakazawa et al., 1983; Takai et al., 1986a,c). The quantitative culture of the feces of foals at weekly intervals was advocated as an aid in early diagnosis of R. equi enteritis in foals because the bacterial count per gram of feces increased at the same time as clinical signs appeared (Takai et al., 1986~). However, a single fecal sample from a foal has no diagnostic value because of individual as well as farm to farm variation in the number of R. equi in the feces (Woolcock et al., 1980; Nakazawa et al., 1983; Takai et al., 1986a,c). Furthermore, a negative fecal culture may not be helpful in ruling out R. equi infection since, in one study, only 5 (17%) of 30 foals with confirmed R. equi pneumonia had positive fecal cultures (Ardans et al., 1986).

4. Treatment

of R. equi pneumonia

A wide variety of antimicrobial agents are effective against R. equi in vitro (Table 2). However, because R. equi is an intracellular pathogen surviving and replicating in macrophages (Hondalus and Mosser, 1994) and it causes granulomatous lesions with thick caseous material, many of these drugs are ineffective in vivo. For example, in one

S. Gigukre, Table 2 In vitro susceptibility

of 60 R. equi isolates

Antibiotics Aminoglycosides: amikacin gentamicin neomycin Beta-lactams: ampicillin amoxicillin/clavulanic ceftiofur cephalothin penicillin ticarcillin Fluoroquinolones: ciprofloxacin enrofloxacin Macrolides erythromycin tihnicosin Miscellaneous chloramphenicol rifampin tetracycline trimethoprim/sulfa

J.F. Prescott/

Number isolates

acid

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Microbiology

a of equine

origin

of tested

Sensitive (%)

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to selected

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antimicrobial Intermediate (%I

323

agents Resistant (%I

23 60 34

82.6 96.7 88.2

0 3.3 5.9

17.4 0 5.9

60 24 22 5.5 59 22

18.3 62.5 50 14.5 10.2 18.2

28.3 12.5 22.7 9.1 13.6 63.6

53.3 25 27.3 76.4 76.3 18.2

20 22

95.0 22.1

0 63.6

5.0 13.6

60 22

86.7 13.6

11.7 13.6

1.6 12.7

31 18 54 60

67.7 83.3 53.7 68.3

19.4 0 24.1 3.3

12.9 16.7 22.2 28.3

a The isolates were obtained between 1985 and 1996 from the Clinical Veterinary College and from the Ontario Ministry of Agriculture, Laboratory Services Branch.

Bacteriology Laboratory of the Ontario Food and Rural Affairs, Veterinary

study all 17 foals with R. equi pneumonia treated with the combination of penicillin and gentamicin died despite the fact that all isolates were sensitive to gentamicin (Sweeney et al., 1987). The combination of erythromycin and rifampin has become the treatment of choice for R. equi infections in foals and has dramatically reduced foal mortality since its introduction (Hillidge, 1987; Sweeney et al., 1987). The two drugs are bacteriostatic against R. equi (Nordmann and Ronco, 1992a) but highly effective in vitro (Table 2). Their combination is synergistic both in vitro and in vivo (Prescott and Nicholson, 1984a; Nordmann et al., 1992b3) and use in combination reduces the likelihood of resistance to either drug (Nordmann and Ronco, 1992a). Rifampin and, to a lesser extent, erythromycin are lipid soluble, allowing them to penetrate caseous material. Rifampin is concentrated approximately 2-fold in phagocytic cells and is effective against many intracellular bacteria (Maurin and Raoult, 1993). Erythromycin is concentrated lo- to 20-fold in granulocytes and alveolar macrophages by an active mechanism but, despite high intracellular concentrations, its intracellular antibacterial activity at best equals that against extracellular bacteria (Maurin and Raoult, 1993; van den Broek, 1993). The concentration of. erytbromycin within lysosomes may explain the poor

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efficacy of the drug against intracellular pathogens since many of them, like R. equi, can prevent phagosome-lysosome fusion. Alternatively, acidity within the phagolysosome may reduce the efficacy of erythromycin (Maurin and Raoult, 1993). The recommended dosage regimen for rifampin is 5 mg/kg every 12 h or 10 mg/kg every 24 h orally (Hillidge, 1987; Castro et al., 1986; Burrows et al., 1992). Rifampin may causes urine and other secretions (tears, saliva, etc.) to turn red-orange but is usually well tolerated when administered orally for long period of time. Intravenous administration of rifampin may result in severe effects including weakness, unsteadiness, profuse sweating and distress (Burrows et al., 1992). Recommended dosage of the acid soluble estolate or ethylsuccinate esters of erythromycin is 25 mg/kg every 8 or 12 h (Prescott et al., 1983; Hillidge, 1987). A recent pharmacokinetic study indicated that administration of the salt preparations erythromycin stearate and erythromycin phosphate could be substituted for erythromycin estolate or erythromycin ethylsuccinate in the treatment of R. equi infections in foals (Ewing et al., 1994b). The authors suggested that twice daily administration of a higher dose (37.5 mg/kg) could be substituted for the standard regimen but the clinical efficacy and safety of this dosing regimen remains to be shown. A third antimicrobial agent may be necessary if another pathogen resistant to erythromycin or rifampin is isolated in significant numbers along with R. equi. Resolution of clinical signs, normalization of plasma fibrinogen and radiographic resolution of lung lesions are commonly used to guide the duration of therapy which generally ranges between 4 and 9 weeks (Hillidge, 1987). Return of plasma fibrinogen values to normal for at least one week prior to discontinuation of therapy is the best criterion. Long term therapy is expensive but fundamental since relapses may occur if therapy is discontinued early. A positive clinical response within 7 days associated with a decrease in plasma fibrinogen concentration and severity of lung lesions on radiographs suggests a favorable prognosis. In one study, 11 of 13 foals that failed to respond to therapy died within the first week (Hillidge, 1987). Although well tolerated by most foals, the combination of erythromycin and rifampin commonly causes fecal consistency to soften. Most of the time this effect is self limiting and does not necessitate cessation of therapy but these foals should be monitored carefully because some may develop depression and severe diarrhea, leading to dehydration and electrolyte loss that necessitate intensive fluid therapy and cessation of oral erythromycin. If the R. equi infection being treated is severe when the foal develop watery diarrhea, oral erythromycin can be replaced by intravenous (IV) erythromycin lactobionate (5 mg/kg diluted in saline and given as a slow infusion every 6 h). Although IV administered erythromycin is excreted in the bile, administration of IV erythromycin in association with oral rifampin and supportive treatment has resulted in cessation of the diarrhea in many foals that had developed severe diarrhea when treated orally. Administration of rifampin is rarely associated with diarrhea in horses and even in foals with severe diarrhea its administration can usually be continued, although its absorption may be reduced. If the R. equi infection being treated is not severe or is under resolution, or if IV administration of erythromycin is too costly or cannot be administered on the farm, alternative antimicrobial agents may be used (see below). Foals that develop severe diarrhea when treated with oral erythromycin may develop Gram-negative bacteremia with Sulmonella spp. or other enteric organisms. Therefore,

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additional Gram-negative coverage is recommended. The combination of gentamicin or amikacin with erythromycin or rifampin in vitro gave significant antagonistic activity against R. equi compared with either drug alone (Prescott and Nicholson, 1984a; Nordmann and Ronco, 1992a). Therefore, we do not recommend concurrent administration of an aminoglycoside with either erythromycin or rifampin for the treatment of R. equi infections. There seems to be a geographic and farm to farm variation in the incidence and severity of erythromycin induced diarrhea. Another complication associated with the initiation of treatment with erythromycin and rifampin is partial anorexia, mild colic, and bruxism as seen with gastric ulcerations. The signs usually resolve after temporary cessation (1 or 2 dosages) (Prescott and Hoffman, 1993). During surges of very hot weather an idiosyncratic reaction characterized by severe hyperthermia and tachypnea, and increased liver enzymes concentrations has been described in foals treated with erythromycin (Wilson, 1992a; Prescott and Hoffman, 1993). Administration of antipyretic drugs and placing the foal in a cold environment will treat this problem. Diarrhea has also been observed occasionally in the dams of nursing foals while the foals are being treated with oral erythromycin presumably because coprophagic behavior leads to ingestion of sufficient active erythromycin to perturb the intestinal flora of the mare (Wilson, 1992a). Although Wilson (1992a) reported that erythromycin alone has been used successfully to treat R. equi pneumonia we do not recommend this practice because (1) erythromycin alone was ineffective in a mouse model of R. equi pneumonia despite excellent activity in vitro (Nordmamr et al., 1992b); (2) erythromycin is poorly effective in the intracellular environment and against intracellular pathogens in general (Maurin and Raoult, 1993); and (3) long term use of erythromycin alone is often associated with development of resistance (Prescott and Sweeney, 1985). Similarly, rifampin should not be used in monotherapy because this increases the chance of resistance developing (Nordmann and Ronco, 1992a). Even though the vast majority of R. equi isolates from foals are sensitive to erythromycin (MIC I 0.25 pg/ml) and rifampin (MIC I 0.06 pg/rnl) resistant strains to either drugs have been encountered. Kenney et al. (1994) also reported progressive development of resistance to both erythromycin and rifampin during treatment. Therapy of foals infected with resistant isolates is problematic because of the limited range of alternative effective drugs. 4.1. Alternative antimicrobial

agents

High doses of the trimethoprim-sulfonamide (TMS) combination (30 mg/kg of combination every 8 or 12 h) have proved effective in foals with mild or early R. equi pneumonia without marked evidence of pulmonary abscessation or for continued therapy in foals responding well to other antimicrobials (Wilson, 1992b). However, TMS is unlikely to be as effective as erythromycin and rifampin for the treatment of severe R. equi pneumonia with extensive abscessation because of its poor activity in caseous material and against most intracellular pathogens. Enrofloxacin, alone or in combination with other antibiotics (ceftiofur or rifampin), has recently been used successfully to treat a limited number of foals with culture-con-

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fiied R. equi pneumonia where conventional therapy was not possible because of bacterial resistance, cost, or erythromycin induced diarrhea (B. Bentz and W. Russell, personal communication). Using a dose of 5.0 mg/kg orally every 24 h for up to 5 weeks, no side effects were noted but a few foals receiving higher doses developed joint effusion and lameness that resolved following discontinuation of therapy. Experimentally, oral administration of enrofloxacin to foals has resulted in lameness, joint effusion and cartilage lesions with doses as low as 5 mg/kg/day (lower dose tested) but the severity of these effects was dose dependent (D. Copeland, Bayer Corporation, personal communication). Most R. equi isolates are only of intermediate sensitivity to enrofloxacin (Table 2). However, fluoroquinolones are concentrated approximately 6-7fold in granulocytes and 3-fold in macrophages, and are effective in the intracellular environment (Maurin and Raoult, 1993). Interestingly, ciprofloxacin, a better antimicrobial based on in vitro susceptibility testing, was ineffective in a mouse model of R. equi pneumonia (Nordmann et al., 1992b). Further studies on the pharmacokinetics, toxicity, and clinical efficacy of enrofloxacin in foals are however required before we can recommend its use for the treatment of R. equi pneumonia. If one elects to use enrofloxacin as an alternative antimicrobial agent for the treatment of susceptible R. equi isolates in foals, the owner should be made aware of the potential side effects and that the product is not approved for use in horses. In an experimental model of R. equi infection in mice the most effective drugs in monotherapy were found to be, in order of effectiveness, vancomycin, imipenem, and rifampin (Nordmann et al., 1992b). Amikacin, erythromycin, ciprofloxacin, or minocycline in monotherapy did not lead to a significant decrease in bacterial counts. The most active drugs in combination were those including vancomycin or rifampin but these combinations were not different from vancomycin monotherapy. Many antimicrobial agents have been used with inconsistent results in human patients infected with R. equi. The proposed optimum antimicrobial regimen for R. equi infections in humans is combination of vancomycin with imipenem (IV) for at least three weeks followed by an oral antimicrobial combination such as erythromycin and rifampin or minocycline and rifampin (Nordmann, 1995). There are no reports on the administration of vancomycin or imipenem, alone or in combination for the treatment of R. equi infections in foals or on their pharmacokinetic behaviors in foals. These drugs are cost prohibitive for most horses. Incorporation of antimicrobial agents with poor intracellular activity (e.g. aminoglycosides and beta-la&u& into liposomes or other colloidal carriers has been shown to significantly increase intracellular activity of many drugs against various bacterial pathogens (Fattal et al., 1993). It would be interesting to explore the efficacy of liposome-containing antibiotics for the treatment of R. equi pneumonia. 4.2. Additional therapy

Nursing care, provision of adequate nutrition and hydration, and maintaining the foal in a cool and well ventilated environment are important. Oxygen therapy using humidified oxygen by pharyngeal insufflation in moderately hypoxemic foals, or by percutaneous transtracheal oxygenation in severely hypoxemic animals (Hoffman and Viel,

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1992) is indicated in foals with respiratory distress, persistent hypoxemia or cyanosis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are of value in reducing fever and improving attitude and appetite in febrile depressed anorectic foals. NSAIDs should be used judiciously because they may lead to gastrointestinal ulceration and nephrotoxicity especially if the foal is hypovolemic. Nebulization may be helpful in selected cases with tenacious secretions and non-productive cough. However, most cases of R. equi pneumonia do not benefit from nebulization and the procedure is stressful to some foals. Similarly, it is our experience and that of others (Beech and Sweeney, 1991) that bronchodilators such as aminophylline and beta-2-adrenergic agents (e.g. clenbuterol, albuterol) are rarely helpful clinically. Furthermore, aminophylline should be used cautiously in foals concurrently treated with erythromycin because the latter delays clearance and elevates blood levels of aminophylline, thus potentiating toxicity. Specific hyperimmune plasma is effective in the prevention of the disease (see section on prevention) but was of no value once the infection had established in an experimental model of R. equi pneumonia (Chaffin et al., 1991). It would be useful to explore the efficacy of cytokines (IL-2, INFg, IL-12) in treatment of R. equi pneumonia when these become available.

5. Prognosis Prior to the introduction of the combination erythromycin-rifampin as the recommended treatment, the prognosis of R. equi pneumonia was poor with reported mortality rate as high as 80% (Elissade et al., 1980). Using erythromycin and rifampin Hillidge (1987) reported a successful outcome (as assessed by survival) in 50 (88%) of 57 foals with confirmed R. equi pneumonia. However, until recently there was no information on the impact of R. equi infections on future athletic performance. Ainsworth et al. (1993b) performed radiography, hematological and BAL analysis, and pulmonary function tests in 5 horses (6-18 months) with prior R. equi pneumonia and compared the results to age-matched controls with no history of respiratory disease. There was no significant differences between the two groups. Bernard et al. (1991) evaluated the influence of R. equi pneumonia on future racing performance. Thirty horses that previously had R. equi pneumonia were compared to the affected foals’ dams’ progeny. There were no significant differences in total earnings, average earning index, and age at the first race between horses that previously had R. equi pneumonia and controls or the North American average. These results suggest that horses with previous R. equi pneumonia are likely to perform as well as expected if they race but, since only foals registered with the Jockey Club were considered, it does not indicate if they are as likely to race as a control population. Christley and Hodgson (1994) examined the case histories and race records of 11 horses previously affected with R. equi pneumonia. Seven of them eventually raced at least once, four of the seven won races, and 5 placed. However, the small number of horses available prevented statistical analysis and full determination of the effect of previous R. equi pneumonia. Recently, a large collaborative study involving several major veterinary hospitals was conducted in order to definitively assess the influence of prior R. equi pneumonia on racing performance (D.

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Ainsworth, personal communication). The records of 115 foals (49 thoroughbreds [TB] and 66 standardbreds [SB]) that had chest radiographs and were diagnosed with R. equi pneumonia based on culture of a TBA were reviewed. All cases were treated with erythromycin and rifampin between 1984 and 1994. The survival rate was significantly higher in SB (80%) than in TB (61%). Death was more likely in foals presented with respiratory distress and the non-survivors had a higher radiologic score on admission than survivors. Of the survivors 54% (for both SB and TB) had at least one racing start as opposed to 65% for the control population suggesting that horses contracting R. equi pneumonia as foals are slightly less likely to race. However, as also shown by Bernard et al. (1991), those foals that raced performed as well as expected.

6. Prevention 6.1. Decreasing the size of infective challenge There is a progressive build up of infection on horse farms with prolonged use so that enzootic farms are likely to be those used for breeding horses for many years, those with heavy concentrations of mares and foals, and those located where summer temperatures are high, where the soil type is sandy, and where dust is extensive. Although the total number of R. equi in the environment may be similar, farms with enzootic disease appear to be more heavily infected with virulent (VapA positive) organisms than those where the disease is not present (Takai et al., 1991). Large numbers of foals kept on bare, dusty, manure containing paddocks will result in heavy challenge, with clinical disease maintaining virulent bacteria. Although environmental conditions are major contributing factors, they are often impossible to alter. It is important to house foals in well ventilated, dust free areas, and avoid dirt paddocks and crowding. Pasture must be rotated to decrease dust formation and by consequent inhalation of R. equi. Any sandy or dirt areas should ideally be planted with grass and made ‘off limits’ to foals or, alternatively, irrigation may be useful in decreasing dust formation. Manure should be removed from paddocks and composted. Because mares and foals tend to congregate around water sources and shade in hot summers, reduction in the size of mare-foal bands may help reducing the destruction of grass and exposure to barren soil. Breeding practices that results in mares foaling in winter months may decrease the incidence of the disease as environmental challenge will be minimized during the foal’s most susceptible age period. 6.2. Earlier recognition of the disease Early recognition of R. equi cases with isolation and treatment of infected foals will reduce losses, prevent the spread of virulent organism and limit the cost of therapy in farms enzootically infected. In one study on a large breeding farm with enzootic R. equi problems, twice weekly complete physical examination with auscultation of the lungs was successful in promoting early diagnosis and preventing mortality (Prescott et al., 1989). However, twice weekly or more frequent examination by a veterinarian is

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expensive and not possible on most farms. Another approach recently found to be helpful in early diagnosis of R. equi infections is testing foals from birth to 5 months of age at 2-week intervals with the AGID assay (Gaskin et al., 1990; Wilkins et al., 1993). Higuchi et al. (1997) also suggest serological surveillance of R. equi antibody in foals at 30 and 45 days of age using Tween 20 ELISA. Seroconversion only indicates exposure and many seropositive foals will not develop a clinical R. equi infection. Foals should not be treated based only on a positive serological result. However, positive foals should be monitored carefully (daily temperature, frequent physical examination, frequent plasma fibrinogen evaluation and, when possible, ultrasonographic evaluation) so that they can be subjected to appropriate diagnostic procedures and treated as soon as they show clinical, hematological or ultrasonographic evidence of disease. Foals seropositive on AGID or ELISA may develop pneumonic disease due to causes other than R. equi and when possible a definitive diagnosis should be made by TBA. A few foals may also develop clinical R. equi pneumonia without seroconversion (Gaskin et al., 1990; Hoffman et al., 1993). For this reason a combination of careful daily observation and recording of temperature values as well as serological surveillance and measurement of plasma fibrinogen concentrations at 2-week intervals, although labor intensive, may prove to be the best approach to early diagnosis of R. equi infections on enzootic farms. 6.3. Passive Immunization Martens et al. (1989) were the first to show the immunoprophylactic capacity of specific hyperimmune plasma in an experimental model of R. equi pneumonia in foals. Subsequently, it was shown that administration of 1 L of hyperimmune plasma obtained from donors vaccinated with an autogenous bacterin was also highly effective in significantly reducing the incidence and mortality due to R. equi pneumonia when compared to non treated controls foals raised on the same farm (Madigan et al., 1991: Muller and Madigan, 1992). In the same study and in experimental model of infection. vaccination of mares did not provide protection against R. equi pneumonia despite a significant increase in colostral specific R. equi antibody and partial passive transfer to foals (Madigan et al., 1991; Martens et al., 1991). How hyperimmune plasma protects against R. equi pneumonia remains to be determined but it is likely through opsonizing antibodies. However, other factors such as fibronectin, complement and cytokines may also play a role in protection. Passive immunization of foals has proved highly beneficial and cost effective in preventing the disease on many farms. However, failures have also been reported. In a recent study, administration of hyperimmune plasma with high antibody levels within 48 h of birth failed to protect against R. equi pneumonia since 9 (26%) of 34 transfused foals and 12 (21%) of 57 non-treated controls developed pneumonia (Hurley and Begg, 1995). The reasons for the failure of hyperimmune plasma to protect foals on this farm are unknown but may relate to exposure to R. equi after the decline of passively-transferred antibodies to a non-protective level. The ideal timing for administration of hyperimmune plasma to foals on enzootic farms is not clear. In the first field studies described above the plasma was administered to most foals in the first 30 days of age but based on their experience the authors state that I L of plasma should be administered during the first l-2 weeks of life in foals

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born during the R. equi season, or at the beginning of the warm season for foals born early in the year (Muller and Madigan, 1992). However, decline of passively transferred antibodies to non-protective levels at the time when natural R. equi exposure is still high is a concern if plasma is administered shortly after birth. Further studies are necessary to evaluate the best time for administration of hyperimmune plasma but administration within the first week of life followed by a second administration at approximately 25-30 days of age, although expensive, may prove to be the best approach. R. equi specific hyperimmune plasma obtained from donors vaccinated with a bacterin is commercially available (Polymune R, Veterinary Dynamics Inc., Templeton, CA). Work reported in this issue by Becu et al. suggests that, in Argentina, R. equi has been controlled in enzootic farms by a program of mare vaccination and monitoring the presence of maternally-derived anti-R. equi antibodies in foals at 2 days of age by AGID. Foals with few antibodies can then be transfused with hyperimmune plasma obtained from donors vaccinated with the same antigen. All foals are transfused at 25 days of age, irrespective of antibody level. Recently, a concentrated equine serum product (Seramune oral equine IgG, Sera Inc., Shawnee Mission, Kansas) marketed for the prevention of failure of passive transfer of immunity was also shown to slightly increase specific R. equi antibodies titers in treated foals as assessed by ELISA. The product was also shown to delay but not prevent seroconversion on the AGID assay for a period of 2-3 weeks beyond the untreated control group (Davis et al., 1995). However, until studies demonstrating efficacy in preventing the development of R. equi pneumonia are available, we cannot recommend its use in the prophylaxis of R. equi infections. 6.4. Active immunization

It would be considerably more convenient to control R. equi pneumonia on enzootitally infected farms by the active immunization of mares and their foals with a protective antigen rather than by intravenous administration of large volume of plasma. Further work is needed to examine active immunization based on a better understanding of the antigens of importance and the method of adjuvant required to produce the effective Thl immune response required for protection against R. equi pneumonia (immunity to R. equi is discussed elsewhere in this issue). It has long been suggested by European workers that equine herpesvirus type 2 (EHV-2) has the capacity of initiating a two phase respiratory syndrome in foals. The first (viral) stage is characterized by occasional appearance of nasal discharge and mild fever. The foals apparently recover but 2-3 weeks later develop severe pulmonary abscessation caused by R. equi suggesting that EHV-2 may act as a trigger mechanism for the development of R. equi pneumonia (P&i et al., 1978; Belt& et al., 1980). Recently, active immunization of foals with immunostimulating complexes (ISCOMs) containing selected envelope components of EHV-2 was attempted in a farm with enzootic EHV-2-R. equi infections. Eighteen of 19 foals immunized twice remained clinically healthy and one had mild transient respiratory signs. Three of the 5 non-vaccinated controls died of R. equi pneumonia and one developed severe respiratory disease but survived suggesting that, in some farms, EHV-2 may predispose to R. equi

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pneumonia (Nordengrahn et al., 1996). However, the incidence of such predisposition, if it occurs, remains to be determined.

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