Use of Serial Quantitative PCR of the vapA Gene of Rhodococcus equi in Feces for Early Detection of R. equi Pneumonia in Foals.

BACKGROUND: Current screening tests for Rhodococcus equi pneumonia in foals lack adequate accuracy for clinical use. Real-time, quantitative PCR (qPCR) for virulent R. equi in feces has not been systematically evaluated as a screening test. OBJECTIVE: The objective of this study was to evaluate the accuracy of qPCR for vapA in serially collected fecal samples as a screening test for R. equi pneumonia in foals. ANIMALS: One hundred and twenty-five foals born in 2011 at a ranch in Texas. METHODS: Fecal samples were collected concurrently with thoracic ultrasonography (TUS) screening examinations at ages 3, 5, and 7 weeks. Affected (pneumonic) foals (n = 25) were matched by age and date-of-birth to unaffected (n = 25) and subclinical (ie, having thoracic TUS lesions but no clinical signs of pneumonia) foals (n = 75). DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. RESULTS: Subsequently affected foals had significantly greater concentrations of vapA in feces than foals that did not develop pneumonia (unaffected and subclinical foals) at 5 and 7 weeks of age. Accuracy of fecal qPCR, however, was poor as a screening test to differentiate foals that would develop clinical signs of pneumonia from those that would remain free of clinical signs (including foals with subclinical pulmonary lesions attributed to R. equi) using receiver operating characteristic (ROC) methods. CONCLUSIONS AND CLINICAL IMPORTANCE: In the population studied, serial qPCR on feces lacked adequate accuracy as a screening test for clinical R. equi foal pneumonia.

Estimating the Sensitivity and Specificity of Real-Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals.

BACKGROUND: Real-time, quantitative PCR (qPCR) methods for detecting Rhodococcus equi in feces have been developed as a noninvasive, rapid diagnostic test for R. equi pneumonia, but have not been evaluated in a large population of foals. OBJECTIVE: The objective of this study was to evaluate the clinical utility of fecal PCR as a diagnostic test for R. equi pneumonia in foals using receiver operating characteristic (ROC) methods. ANIMALS: 186 foals born in 2011 at an R. equi-endemic ranch in Texas. METHODS: Fecal samples were collected at the time of onset of clinical signs for pneumonic foals (n = 31). Foals with pneumonia were matched by age and birth date to healthy (n = 31) and subclinical (n = 124) control foals; fecal samples were collected from these controls. DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. RESULTS: Concentration of R. equi in feces differed significantly (P < .05) among groups. The area under the ROC curve for fecal qPCR for diagnosis of R. equi pneumonia was 89% (95% CI, 83-99), with a sensitivity of 94% and specificity of 72%. CONCLUSIONS AND CLINICAL IMPORTANCE: qPCR of feces can be useful as an alternative to tracheobronchial aspiration for the diagnosis of R. equi in foals with clinical signs of pneumonia. Caution should be used in extrapolating results of this study to other populations because fecal concentration of R. equi might vary by geographic location or management practices.

Concurrent ivermectin and Solanum spp. toxicosis in a herd of horses.

BACKGROUND: Representatives from a herd of horses with acute onset of neurologic signs after administration of ivermectin presented for evaluation and treatment. OBJECTIVES: Describe clinical signs of horses intoxicated by ingestion of Solanum sp. and administered ivermectin. ANIMALS: Six of 11 affected unrelated horses presented for evaluation and treatment. The remaining 5 affected horses were treated at the farm. Four additional horses, housed separately, were unaffected. METHODS: Case series is presented. Serum ivermectin concentrations were evaluated in the 6 hospitalized horses. The remnants of the tubes of ivermectin paste were analyzed for ivermectin concentration. The hay fed to the affected horses was analyzed for the presence of toxic plants. RESULTS: Serum ivermectin concentrations were higher than expected, given the dosage of ivermectin administered. The ivermectin concentration remaining in the administration tubes did not exceed specifications. The hay was heavily contaminated by 2 Solanum species. All horses returned to normal neurologic function with supportive care. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses might exhibit signs of ivermectin toxicity after appropriate dosing of the drug if they concurrently consume toxic plants of the Solanum family.

Natural Heterobilharzia americana infection in horses in Texas.

The schistosome Heterobilharzia americana infects dogs, raccoons, and other mammals in the southeastern United States. Migration of eggs into the liver results in parasitic granulomas with varying degrees of fibrosis and inflammation. Recently, hepatic parasitic granulomas in horses were shown to be caused by H. americana infection. In the present study, samples of liver from 11 of 12 horses with hepatic granulomas identified at necropsy (n = 11) or surgical biopsy (n = 1) were used for DNA extraction, polymerase chain reaction amplification and sequencing using primers specific for a portion of the H. americana small subunit ribosomal RNA gene. A polymerase chain reaction amplicon of the correct size was produced from the extracted DNA in 8 of the 11 horses. Amplicons from 5 of the 8 positive horses were sequenced and had 100% identity with H. americana. In all but 2 of the 12 horses, Heterobilharzia was not responsible for the primary clinical disease, and the hepatic granulomas were considered an incidental finding.

Equine rhabdomyolysis.

A 1.5-year-old Quarter Horse gelding with a history of chronic nasal discharge and leukocytosis presented with signs of increased lethargy and muscular pain. The horse quickly became recumbent and unable to rise and was euthanized due to a poor prognosis. At necropsy, severe bilateral guttural pouch empyema was observed, as well as numerous well-demarcated areas of pallor within the skeletal muscles of all major muscle groups. Polymerase chain reaction testing of the guttural pouch exudate confirmed an infection with Streptococcus equi subsp. equi, and an S. equi-associated immune-mediated rhabdomyolysis was initially considered to be the most likely diagnosis. This report briefly discusses the various etiologies that should be considered in cases of equine myopathy, and it demonstrates the complexity of these poorly understood muscular disorders.

Pharmacokinetics of an orally administered methylcellulose formulation of gallium maltolate in neonatal foals.

Gallium is a trivalent semi-metal with anti-microbial effects because of its incorporation into crucial iron-dependent reproductive enzyme systems. Gallium maltolate (GaM) provides significant gallium bioavailability to people and mice following oral administration and to neonatal foals following intragastric administration. To study the prophylactic and therapeutic effects of GaM against Rhodococcus equi pneumonia in foals, we developed a methylcellulose formulation of GaM (GaM-MCF) for oral administration to neonatal foals. Normal neonatal foals were studied. Six foals received 20 mg/kg and another six foals received 40 mg/kg of GaM-MCF orally. Serial serum samples were collected and serum gallium concentrations were determined using inductively coupled plasma mass spectroscopy. Gallium was rapidly absorbed (T(max) of 4 h), and a mean C(max) of 0.90 or 1.8 microg/mL was achieved in foals receiving 20 or 40 mg/kg respectively. Marked variability existed in C(max) among foals: only half of the foals receiving 20 mg/kg attained serum concentrations of >0.7 microg/mL, a level suggested to be therapeutic against R. equi by previous studies. Mean elimination half-life was 32.8 or 32.4 h for foals receiving 20 or 40 mg/kg respectively. The results of this study suggest that at least 30 mg/kg orally every 24 h should be considered in future pharmacodynamic and efficacy studies.

Massive pulmonary thromboembolism in six horses.

This report involves 6 cases in which medical records and post mortem findings were reviewed leading to the diagnosis of massive pulmonary thromboembolism (MPTE). All horses were mature and MPTE has not been recognised previously as a sequel to generalised systemic illness in mature horses. The clinical data and pathological findings of the cases are reported and the authors conclude that MPTE is an uncommon but important complication of medical and surgical disorders in mature horses. In 3 of the cases, the condition was nonfatal suggesting that some horses having developed PTE survive and the condition may not be recognised in such cases. The incidence of the condition might be higher than supposed.

Study of serum amyloid A concentrations as a means of achieving early diagnosis of Rhodococcus equi pneumonia.

REASONS FOR PERFORMING STUDY: Prognosis of Rhodococcus equi pneumonia can be challenging because the course of the disease is often insidious and overt clinical signs are subtle. Early diagnosis is considered desirable because it may offer the chance of more successful implementation of treatment and, thereby, improved outcome. Serological tests have previously failed to be accurate for early detection or diagnosis. Measurement of serum amyloid A (SAA) prior to and at the time of clinical signs was therefore chosen in order to assess its potential clinical use. OBJECTIVE: To determine whether SAA concentrations differentiate foals affected with R. equi pneumonia from unaffected foals, either prior to the onset of disease or at the time of onset of clinical signs. HYPOTHESIS: SAA concentrations are significantly higher among foals that develop R. equi pneumonia than in foals from the same environment that remain clinically unaffected. METHODS: Serum samples were obtained from 212 foals 7-14 days and 196 foals 21-28 days post partum, and from affected foals and age-matched controls at the time of onset of signs of pneumonia. SAA concentration was determined for each sample. RESULTS: There were no significant differences between SAA concentrations of foals with R. equi and clinically unaffected foals during the 2 periods of examination or at the time of onset of clinical signs of R. equi pneumonia. CONCLUSIONS: Concentrations of SAA are variable among foals with R. equi pneumonia and cannot be used reliably either as an ancillary diagnostic tool or to screen for early detection of disease during the first month post partum. POTENTIAL RELEVANCE: Bimonthly monitoring concentration of SAA is not useful as a screening test for early detection of R. equi pneumonia and does not facilitate diagnosis of this disease when used according to the protocol of this study.

Temporal changes in cytokine expression of foals during the first month of life.

Foals are uniquely susceptible to a wide variety of opportunistic infections normally associated with immunodeficiencies. Little is understood about the immune system of foals during the neonatal period. An apparent age-related susceptibility predisposes neonatal foals to infectious diseases and hinders therapeutic and preventative interventions for these diseases. Cytokine expression is correlated with the type of immune response as well as the severity of a disease. In this study, we measured foal peripheral blood mononuclear cell (PBMC)-specific mRNA cytokine expression from 72 foals from three different farms during the first 4 weeks of life. Interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) were cloned and transcribed in vitro to generate antisense probes for ribonuclease protection assays. Using linear mixed-effect models, we determined that IFN-gamma, TGF-beta1, and IL-1alpha increased significantly (P<0.05) with age.

Prevalence of virulent Rhodococcus equi in soil from five R. equi-endemic horse-breeding farms and restriction fragment length polymorphisms of virulence plasmids in isolates from soil and infected foals in Texas.

Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R. equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims. Rhodococcus equi were isolated from soil of all 5 farms; however, virulent R. equi were only isolated from 3 of the 5 farms and represented 18.8% (87 of 462) of total isolates. Of the 87 virulent soil isolates, 56 (64.5%) contained an 85-kb type I plasmid, 23 (26.4%) an 87-kb type I plasmid, 7 (8%) a newly defined 85-kb type III plasmid (Tx 43), and 1 (1.1%) a newly defined 85-kb type IV plasmid (Tx 47). Of the 100 isolates from infected foals, 96 were virulent. Of the 96 virulent isolates, 51 (53.1%) contained an 85-kb type I plasmid, 39 (40.6%) an 87-kb type I plasmid, 4 (4.2%) an 85-kb type III plasmid (Tx 43), and 2 (2.1%) an 85-kb type IV plasmid (Tx 47). There are at least 4 different R. equi virulence-associated plasmids in Texas, 2 of which have not previously been described. Based upon virulence plasmid typing, there is geographic diversity among isolates of R. equi from clinical and environmental samples on horse-breeding farms in Texas. There is not a strong correlation between the presence of virulent R. equi in farm soils and the R. equi disease status of those farms.

Voluntary limb-load distribution in horses with acute and chronic laminitis.

OBJECTIVES: To compare limb-load distribution between horses with and without acute or chronic laminitis. ANIMALS: 10 horses with carbohydrate-induced acute laminitis, 20 horses with naturally occurring chronic laminitis, and 20 horses without foot abnormalities (controls). PROCEDURES: Limb-load distribution was determined, using a custom-designed system that allowed simultaneous quantification of the mean percentage of body weight voluntarily placed on each limb (ie, mean limb load) and the SD of the mean load over a 5-minute period (ie, load distribution profile [LDP]). Load distribution profile was used as an index of frequency of load redistribution. RESULTS: Mean loads on fore- and hind limbs in control horses were 58 and 42%, respectively, and loads were equally and normally distributed between left and right limbs. In addition, forelimb LDP was greater, compared with hind limbs, and was affected by head and neck movement. In comparison, limb-load distribution in horses with chronic laminitis was characterized by an increase in the preferential loading of a forelimb, a decrease in total forelimb load, and an increase in LDP that was correlated with severity of lameness. In horses with carbohydrate-induced acute laminitis, mean limb loads after onset of lameness were not different from those prior to lameness; however, LDP was significantly decreased after onset of lameness. CONCLUSION AND CLINICAL RELEVANCE: Quantification of limb-load distribution may be an applicable screening method for detecting acute laminitis, grading severity of lameness, and monitoring rehabilitation of horses with chronic laminitis.

Application of Sartwell's model (lognormal distribution of incubation periods) to age at onset and age at death of foals with Rhodococcus equi pneumonia as evidence of perinatal infection.

The distributions of the incubation periods for infectious and neoplastic diseases originating from point-source exposures, and for genetic diseases, follow a lognormal distribution (Sartwell's model). Conversely, incubation periods in propagated outbreaks and diseases with strong environmental components do not follow a lognormal distribution. In this study Sartwell's model was applied to the age at onset and age at death of foals with Rhodococcus equi pneumonia. The age at onset of clinical signs and age at death were compiled for 107 foals that had been diagnosed with R. equi pneumonia at breeding farms in Argentina and Japan. For each outcome (disease and death), these data followed a lognormal distribution. A group of 115 foals with colic from the University of California were used as a comparison group. The age at onset of clinical signs for these foals did not follow a lognormal distribution. These results were consistent with the hypothesis that foals are infected with R. equi during the 1st several days of life, similar to a point-source exposure.

Association of disease with isolation and virulence of Rhodococcus equi from farm soil and foals with pneumonia.

OBJECTIVE: To determine whether isolation and virulence of Rhodococcus equi from soil and infected foals are associated with clinical disease. DESIGN: Cross-sectional and case-control study. SAMPLE POPULATION: R equi isolates from 50 foals with pneumonia and soil samples from 33 farms with and 33 farms without a history of R equi infection (affected and control, respectively). PROCEDURE: R equi was selectively isolated from soil samples. Soil and clinical isolates were evaluated for virulence-associated protein antigen plasmids (VapA-P) and resistance to the beta-lactam antibiotics penicillin G and cephalothin. Microbiologic cultures and VapA-P assays were performed at 2 independent laboratories. RESULTS: VapA-P was detected in 49 of 50 (98%) clinical isolates; there was complete agreement between laboratories. Rhodococcus equi was isolated from soil on 28 of 33 (84.8%) affected farms and 24 of 33 (72.7%) control farms, but there was poor agreement between laboratories. Virulence-associated protein antigen plasmids were detected on 14 of 66 (21.2%) farms by either laboratory, but results agreed for only 1 of the 14 VapA-P-positive farms. We did not detect significant associations between disease status and isolation of R equi from soil, detection of VapA-P in soil isolates, or resistance of soil isolates to beta-lactam antibiotics. No association between beta-lactam antibiotic resistance and presence of VapA-P was detected. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of soil microbiologic culture and VapA-P assay results, it is not possible to determine whether foals on a given farm are at increased risk of developing disease caused by R equi.

Empyema of the guttural pouch (auditory tube diverticulum) in horses: 91 cases (1977-1997).

OBJECTIVE: To identify features of guttural pouch (auditory tube diverticulum) empyema in horses and compare findings of uncomplicated guttural pouch empyema with guttural pouch empyema complicated by chondroids. DESIGN: Retrospective study. ANIMALS: 91 horses with guttural pouch empyema. PROCEDURE: Medical records of horses with guttural pouch empyema were reviewed. RESULTS: The most common owner complaint and abnormal finding was persistent nasal discharge. Chondroids were detected in 21% (19/91) of affected horses. Streptococcus equi was isolated from the guttural pouch in 14 of 44 horses; for Streptococcus spp, in vitro resistance to sulfadimethoxine and trimethoprim-sulfamethoxazole was detected. Retropharyngeal swelling and pharyngeal narrowing were significantly more prevalent in horses with chondroids, compared with horses with uncomplicated empyema. Ninety-three percent of affected horses were discharged from the hospital; at time of discharge, 66% had complete resolution of disease, 19% had improvement without resolution, and 15% did not have improvement. CONCLUSIONS AND CLINICAL RELEVANCE: Horses with persistent nasal discharge should be examined endoscopically for guttural pouch empyema. Treatment with lavage offers a good prognosis for resolution of uncomplicated guttural pouch empyema. Aggressive treatment with lavage and endoscopic snare removal of chondroids offers a good prognosis and may make surgical intervention unnecessary.

Randomized controlled trial of effects of Escherichia coli antiserum on serum immunoglobulin G concentrations and morbidity and mortality rates in foals.

OBJECTIVE: To determine whether administration of commercially available Escherichia coli antiserum to neonatal foals would affect serum IgG concentration or morbidity and mortality rates during the first 60 days of life. DESIGN: Randomized controlled trial. ANIMALS: 271 neonatal foals on 4 well-managed farms. PROCEDURE: Foals were randomly assigned to a treatment or control group. All foals were allowed to suckle colostrum normally. In addition, treatment-group foals were given E coli antiserum (10 micromilligrams) orally between 0 and 8 hours after birth. Serum samples were obtained between 18 and 36 hours after birth, and serum IgG concentration was determined. Foals were monitored for the first 60 days after birth, and causes of disease or death were recorded. RESULTS: Groups did not differ significantly in regard to breed, sex, month of birth, season of birth, age of dams, parity of dams, duration of gestation, or specific gravity of colostrum before suckling. In addition, groups did not differ significantly in regard to mean serum IgG concentration, prevalence of complete or partial failure of passive transfer of immunity, frequency or causes of disease, or frequency of death from infectious causes. CLINICAL IMPLICATIONS: In this group of foals on well-managed farms, administration of E coli antiserum did not alter serum IgG concentrations or morbidity and mortality rates during the first 60 days of life.

Factors associated with survival in septicemic foals: 65 cases (1988-1995).

In order to identify variables obtained at admission that could be used to predict survival in septicemic foals, medical records of 65 foals diagnosed with septicemia were reviewed. Initially, variables were analyzed independently (univariate analysis) for association with survival. Of the physical examination and historical data examined using univariate analysis, the ability to stand at admission, respiratory rate > or = 60 breaths per minute (bpm), and normal-appearing mucous membranes were significantly associated with survival. Foals with history of induced parturition were significantly less likely to survive. The following hematologic and serum biochemical variables determined at admission were significantly associated with survival: white blood cell count > or = 6,000 cells/microL, neutrophil count > 4,000 cells/microL, serum albumin concentration > 2.2 g/dL, serum glucose concentration > 120 mg/dL, blood pH > or = 7.35, and positive base excess. The administration of plasma at admission was significantly associated with survival. Stepwise multiple logistic regression analysis was performed to evaluate the association between survival and variables identified as significantly associated with survival in bivariate analysis. The final multivariate model selected included the variables standing, duration of clinical signs (24-hour intervals) prior to admission, respiratory rate > or = 60 bpm, neutropenia (< or = 4,000 cells/ microL), and neonatal age category. The probability of survival was significantly increased for foals that were standing, had a respiratory rate > or = 60 bpm, and that had a neutrophil count > 4,000 cells/microL at admission. Probability of survival was significantly decreased for foals that had a longer duration of clinical signs prior to admission. For each 24 hours of duration, the estimated risk of death was increased by 5.8-fold.

Effects of postoperative peritoneal lavage on pharmacokinetics of gentamicin in horses after celiotomy.

OBJECTIVE: To evaluate the effect of peritoneal lavage on pharmacokinetics of gentamicin sulfate in healthy horses after experimental celiotomy. ANIMALS: 13 clinically normal horses. PROCEDURE: Horses were randomly assigned to control or experimental groups. All horses received gentamicin (6.6 mg/kg of body weight, IV, q 24 h) before surgery, underwent experimental abdominal surgery, and had abdominal drains placed percutaneously. Horses of the experimental group received postoperative peritoneal lavage; horses of the control group did not receive peritoneal lavage. The day after surgery, 24 hours after the preoperative dose of gentamicin, a second dose of gentamicin was administered. Three and 15 hours after this second dose of gentamicin, horses of the experimental group received peritoneal lavage. Venous blood was obtained, for determination of concentration of gentamicin, immediately before and at specified intervals during the 24-hour period after the second dose of gentamicin. RESULTS: There were no differences in any of the pharmacokinetic values of gentamicin between horses of the control and experimental groups. CONCLUSIONS: Peritoneal lavage had no effect on pharmacokinetics of gentamicin in healthy horses after abdominal surgery, in which localized nonseptic peritonitis was induced. CLINICAL RELEVANCE: Peritoneal lavage in horses with localized nonseptic peritonitis or for the prevention of intra-abdominal adhesions should not necessitate alteration of the dosage of gentamicin to maintain predictable serum concentrations.

Magnetic resonance imaging of the brain of normal neonatal foals.

Magnetic resonance imaging (MRI) was performed on the brain of 5 normal, anesthetized, neonatal (age 3-to-6 days) Quarter Horse foals. The objectives of the study were to develop a technique for imaging the brain of neonatal foals, and to ascertain their normal brain anatomy. Intravenous propofol was administered for induction and maintenance of general anesthesia. Using spin echo MR techniques, T1 weighted sagittal and transverse views, and spin density and T2 weighted transverse views were successfully made of each foal. MR images provided excellent visualization of many anatomic structures of the brain and head. MRI of the brain is feasible for selected neonatal equine patients.