Amy L. Johnson

Type A botulism in horses in the United States: a review of the past ten years (1998-2008).

The objective of the current retrospective study was to describe naturally occurring type A botulism in horses in the United States. In the past 10 years, the Botulism Laboratory at the University of Pennsylvanias School of Veterinary Medicine has identified 3 isolated cases and 8 outbreaks of type A botulism in horses via samples positive for Clostridium botulinum type A toxin or spores using the mouse bioassay test. Additional information was obtained by review of submission forms and by telephone or email interviews. Almost all type A cases and outbreaks occurred in the western United States, with Oregon and Idaho overrepresented. Type A toxin was identified in only 1 outbreak; all other identified cases and outbreaks were positive for spores but not preformed toxin. Reported clinical signs included progressive muscle weakness, recumbency, decreased tail and/or tongue tone, dysphagia, respiratory distress, and death. Isolated cases involved foals ≤1 month of age; outbreaks involved horses ≥11 months. One hundred and nineteen horses were potentially exposed to the toxin source; 54 out of 119 showed signs of botulism, and 49 out of 54 affected horses were confirmed dead. The number of horses affected per outbreak ranged from 2 to 24. The source of infection was confirmed to be hay or silage in 6 out of 8 outbreaks and was unknown in 2 out of 8 outbreaks. The present report is the first description of outbreaks of type A botulism in horses and has important implications for prevention and treatment. Based on these findings, type A botulism should be considered in suspect cases of equine botulism in the western United States.

Validation of an In-Clinic Enzyme-Linked Immunosorbent Assay Kit for Diagnosis of Borrelia Burgdorferi Infection in Horses

Confirmation of Borrelia burgdorferi infection in horses has required enzyme-linked immunosorbent assay (ELISA) or Western blot tests performed by reference laboratories. An in-clinic C6 ELISA SNAP kit has been marketed for dogs. This canine kit was evaluated for horses using serum from experimentally infected ponies. Serum samples originated from 2 previous studies. In the first study, 7 ponies were exposed to B. burgdorferi–infected ticks; 4 ponies served as uninfected controls. Serum samples were obtained bimonthly for 9 months. In the second study, 16 ponies were exposed to B. burgdorferi–infected ticks. After confirmation of infection by skin culture, polymerase chain reaction (PCR), and serology, the ponies were allocated to 4 groups that received tetracycline, doxycycline, ceftiofur, or no treatment. Serum samples were obtained monthly, both before and after antibiotic treatments, for 11 months. For the current study, selected samples (n = 220) from both studies were tested with IDEXX SNAP Heartworm Ab/Borrelia burgdorferi Ab/Ehrlichia canis Ab Test Kits. Tested samples included samples taken before infection, from various times postinfection, and after antibiotic treatments. Results from confirmed positive or negative samples were used to determine sensitivity and specificity of the assay. Results indicate that the test kits have fair sensitivity (63%) and very high specificity (100%) for horses recently infected with B. burgdorferi. Validation of this test provides equine practitioners with an inexpensive, in-clinic method to confirm infection, although its moderate sensitivity may result in a moderate chance of a false negative test.

Evaluation of EEG based determination of unconsciousness vs. loss of posture in broilers.

Evaluation of the loss of consciousness in poultry is an essential component in evaluating bird welfare under a variety of situations and applications. Many current approaches to evaluating loss of consciousness are qualitative and require observation of the bird. This study outlines a quantitative method for determining the point at which a bird loses consciousness. In this study, commercial broilers were individually anesthetized and the brain activity recorded as the bird became unconscious. A wireless EEG transmitter was surgically implanted and the bird anesthetized after a 24-48 h recovery. Each bird was monitored during treatment with isoflurane anesthesia and EEG data was evaluated using a frequency based approach. The alpha/delta (A/D) ratio and loss of posture (LOP) were used to determine the point at which the birds went unconscious. There was no statistically significant difference between time to unconsciousness as measured by A/D ratio or LOP.

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention

Equine protozoal myeloencephalitis (EPM) remains an important neurologic disease of horses. There are no pathognomonic clinical signs for the disease. Affected horses can have focal or multifocal central nervous system (CNS) disease. EPM can be difficult to diagnose antemortem. It is caused by either of 2 parasites, Sarcocystis neurona and Neospora hughesi, with much less known about N. hughesi. Although risk factors such as transport stress and breed and age correlations have been identified, biologic factors such as genetic predispositions of individual animals, and parasite‐specific factors such as strain differences in virulence, remain largely undetermined. This consensus statement update presents current published knowledge of the parasite biology, host immune response, disease pathogenesis, epidemiology, and risk factors. Importantly, the statement provides recommendations for EPM diagnosis, treatment, and prevention.

Accurate Antemortem Diagnosis of Equine Protozoal Myeloencephalitis (EPM) Based on Detecting Intrathecal Antibodies against Sarcocystis neurona Using the SnSAG2 and SnSAG4/3 ELISAs

BACKGROUND Recent work demonstrated the value of antigen-specific antibody indices (AI and C-value) to detect intrathecal antibody production against Sarcocystis neurona for antemortem diagnosis of equine protozoal myeloencephalitis (EPM). OBJECTIVES The study was conducted to assess whether the antigen-specific antibody indices can be reduced to a simple serum : cerebrospinal fluid (CSF) titer ratio to achieve accurate EPM diagnosis. ANIMALS Paired serum and CSF samples from 128 horses diagnosed by postmortem examination. The sample set included 44 EPM cases, 35 cervical-vertebral malformation (CVM) cases, 39 neurologic cases other than EPM or CVM, and 10 non-neurologic cases. METHODS Antibodies against S. neurona were measured in serum and CSF pairs using the SnSAG2 and SnSAG4/3 (SnSAG2, 4/3) ELISAs, and the ratio of each respective serum titer to CSF titer was determined. Likelihood ratios and diagnostic sensitivity and specificity were calculated based on serum titers, CSF titers, and serum : CSF titer ratios. RESULTS Excellent diagnostic sensitivity and specificity was obtained from the SnSAG2, 4/3 serum : CSF titer ratio. Sensitivity and specificity of 93.2 and 81.1%, respectively, were achieved using a ratio cutoff of ≤100, whereas sensitivity and specificity were 86.4 and 95.9%, respectively, if a more rigorous cutoff of ≤50 was used. Antibody titers in CSF also provided good diagnostic accuracy. Serum antibody titers alone yielded much lower sensitivity and specificity. CONCLUSIONS AND CLINICAL IMPORTANCE The study confirms the value of detecting intrathecal antibody production for antemortem diagnosis of EPM, and they further show that the antigen-specific antibody indices can be reduced in practice to a simple serum : CSF titer ratio.

Indirect fluorescent antibody test and surface antigen ELISAs for antemortem diagnosis of equine protozoal myeloencephalitis.

BACKGROUND Recent research suggests that serum : CSF titer ratios could provide the most accurate antemortem diagnosis of equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona. OBJECTIVES The purpose of this study was to assess the utility of two commercially available tests, the indirect fluorescent antibody test (IFAT) and the surface antigen 2, 4/3 ELISA (SAG2, 4/3 ELISA), using archived paired serum and CSF samples. ANIMALS Samples were obtained from 4 types of clinical patients. Confirmed positive cases (n = 9 horses; 11 sample sets) had neurologic deficits and postmortem lesions consistent with EPM. Confirmed negative cases (n = 28) had variable clinical signs and postmortem lesions consistent with another disease. Suspected positive cases (n = 6) had neurologic deficits consistent with EPM, marked improvement after treatment, and exclusion of other diseases. Suspected negative cases (n = 14) had variable signs with a strong presumptive diagnosis of another disease. METHODS For each test, descriptive statistics were calculated using serum results alone, CSF results alone, and a serum : CSF titer ratio. RESULTS Overall accuracy was highest for SAG2, 4/3 ELISA titer ratio at 0.97 (95% CI 0.88-0.99) with sensitivity = 0.88 (95% CI 0.66-0.97) and specificity = 1 (95% CI 0.92-1). IFAT CSF and titer ratio results also showed high accuracy at 0.88 (95% CI 0.77-0.94), but lower sensitivity = 0.65 (95% CI 0.41-0.83). CONCLUSIONS AND CLINICAL IMPORTANCE Using serum results alone was least accurate for both test types. The more accurate methods, such as the SAG2, 4/3 ELISA serum : CSF titer ratio, should be utilized.

Utility of 2 Immunological Tests for Antemortem Diagnosis of Equine Protozoal Myeloencephalitis (Sarcocystis neurona Infection) in Naturally Occurring Cases

BACKGROUND Antemortem diagnosis of equine protozoal myeloencephalitis (EPM) is challenging. Limited information is available regarding a commercial test (surface antigen 1 [SAG-1] ELISA). Performance of another commercial test (indirect fluorescent antibody test [IFAT]) using samples from an independent group has not been well described. HYPOTHESIS/OBJECTIVES The primary goal was to evaluate the SAG-1 ELISA and IFAT using naturally occurring EPM cases. A secondary goal was to obtain more information regarding clinical presentation. ANIMALS Hospital cases were admitted over 20 months and classified into 4 groups. Confirmed positive cases (n = 9) had asymmetric or multifocal neurologic deficits or both and postmortem lesions consistent with EPM. Confirmed negative cases (n = 17) had variable clinical signs and postmortem lesions consistent with another neurologic disease (or no lesions). Suspected positive cases (n = 10) had asymmetric or multifocal deficits or both, marked improvement after treatment for EPM, and other likely diseases excluded. Suspected negative cases (n = 29) had orthopedic disease and no neurologic deficits. METHODS Results of immunological testing (SAG-1 ELISA and IFAT on serum or cerebrospinal fluid [CSF] or both), neurologic examinations, CSF analyses, and postmortem examinations were analyzed retrospectively. RESULTS SAG-1 ELISA sensitivity was 12.5% (95% CI, 1.6-38.4) and specificity was 97.1% (95% CI, 84.7-99.9) using serum. IFAT sensitivity was 94.4% (95% CI, 72.7-99.9) and specificity was 85.2% (95% CI, 66.3-95.8) using serum; sensitivity was 92.3% (95% CI, 64.0-99.8) and specificity was 89.7% (95% CI, 72.7-97.8) using CSF. CONCLUSIONS AND CLINICAL IMPORTANCE Low sensitivity of the SAG-1 ELISA limited its usefulness for antemortem diagnosis of EPM in this patient population.

A Multicenter Retrospective Study of 151 Renal Biopsies in Horses

BACKGROUND Renal biopsies are uncommonly performed in horses and little is known about their diagnostic utility and associated complication rate. OBJECTIVE To describe the techniques, the complication rate, risk factors, and histopathology results; as well as evaluate the safety and diagnostic utility of renal biopsy in the horse. ANIMALS One hundred and forty-six horses from which 151 renal biopsies were obtained. Animals ranged in age from 48 hours to 30 years. METHODS Multicenter retrospective study, with participation of 14 institutions (1983-2009). RESULTS Renal biopsy in horses was associated with a similar rate of complications (11.3%) to that occurring in humans and companion animals. Complications were generally associated with hemorrhage or signs of colic, and required treatment in 3% of cases. Fatality rate was low (1/151; 0.7%). Biopsy specimens yielded sufficient tissue for a histopathologic diagnosis in most cases (94%) but diagnoses had only fair (72%) agreement with postmortem findings. Risk factors for complications included biopsy specimens of the left kidney (P = .030), a diagnosis of neoplasia (P = .004), and low urine specific gravity (P = .030). No association with complications was found for age, sex, breed, institution, presenting complaint, other initial clinicopathologic data, biopsy instrument, needle size, or use of ultrasonographic guidance. CONCLUSIONS AND CLINICAL IMPORTANCE Renal biopsy in horses has low morbidity and results in a morphological histopathologic diagnosis in 94% of cases. However, this procedure might result in serious complications and should only be used when information obtained would be likely to impact decisions regarding patient management and prognosis.

Hepatic and metabolic changes in surgical colic patients: a pilot study

Objective – To determine: (1) changes in blood ammonia, bile acid (BA), bilirubin, triglyceride, and glucose concentrations and liver enzyme activities in perioperative colic patients and (2) the association between these laboratory findings and short-term survival. Design – Prospective observational clinical study. Animals – Thirty-two adult horses undergoing exploratory celiotomy for colic. Interventions – None. Measurements and Main Results – Blood samples were collected preoperatively and at 24–36 and 72–84 hours postoperatively and analyzed for blood ammonia, BA, bilirubin, triglyceride, and glucose concentrations and sorbitol dehydrogenase (SDH) and gamma glutamyl transferase (GGT) activities. Short-term survival was defined as survival to hospital discharge. Data were analyzed using a Fishers exact test and analysis of variance. Mildly increased blood ammonia concentrations were present in 2 horses at admission. Postoperative blood ammonia concentrations were within reference intervals in all horses. There were increases in liver enzyme activities as well as in BA, triglyceride, and total bilirubin concentrations. Horses with markedly increased admission BA concentrations and SDH activities did not survive. BA concentrations and SDH activities decreased postoperatively. There was no association between GGT activity and survival; GGT activity remained increased postoperatively. Blood triglyceride concentration was increased in almost all horses postoperatively; horses that did not survive had higher triglyceride concentrations at 24–36 hours postoperatively than horses that survived. Conclusion – Alterations in metabolism and hepatobiliary function are common in colic patients. The results of this study provide further prognostic indices for colic patients and highlight areas for improvement in patient management.OBJECTIVE To determine: (1) changes in blood ammonia, bile acid (BA), bilirubin, triglyceride, and glucose concentrations and liver enzyme activities in perioperative colic patients and (2) the association between these laboratory findings and short-term survival. DESIGN Prospective observational clinical study. ANIMALS Thirty-two adult horses undergoing exploratory celiotomy for colic. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Blood samples were collected preoperatively and at 24-36 and 72-84 hours postoperatively and analyzed for blood ammonia, BA, bilirubin, triglyceride, and glucose concentrations and sorbitol dehydrogenase (SDH) and gamma glutamyl transferase (GGT) activities. Short-term survival was defined as survival to hospital discharge. Data were analyzed using a Fishers exact test and analysis of variance. Mildly increased blood ammonia concentrations were present in 2 horses at admission. Postoperative blood ammonia concentrations were within reference intervals in all horses. There were increases in liver enzyme activities as well as in BA, triglyceride, and total bilirubin concentrations. Horses with markedly increased admission BA concentrations and SDH activities did not survive. BA concentrations and SDH activities decreased postoperatively. There was no association between GGT activity and survival; GGT activity remained increased postoperatively. Blood triglyceride concentration was increased in almost all horses postoperatively; horses that did not survive had higher triglyceride concentrations at 24-36 hours postoperatively than horses that survived. CONCLUSION Alterations in metabolism and hepatobiliary function are common in colic patients. The results of this study provide further prognostic indices for colic patients and highlight areas for improvement in patient management.

Quantitative real-time PCR for detection of the neurotoxin gene of Clostridium botulinum type B in equine and bovine samples

Clostridium botulinum type B is estimated to cause more than 85% of cases of equine botulism in the United States, as well as many outbreaks in cattle. In this study, a quantitative real-time polymerase chain reaction for detection of the neurotoxin gene of C. botulinum type B was compared to the mouse bioassay using 45 positive and 43 negative samples of equine, bovine or associated environmental origin. The sensitivity of the qPCR assay was 96%, whereas the sensitivity of the mouse bioassay was 84%. The specificity of the qPCR assay was 95% and the specificity of the mouse bioassay was 100%.