Comparison of loop-mediated isothermal amplification (LAMP) and PCR for the diagnosis of infection with Trypanosoma brucei ssp. in equids in The Gambia.

INTRODUCTION: Infection of equids with Trypanosoma brucei (T. brucei) ssp. is of socioeconomic importance across sub-Saharan Africa as the disease often progresses to cause fatal meningoencephalitis. Loop-mediated isothermal amplification (LAMP) has been developed as a cost-effective molecular diagnostic test and is potentially applicable for use in field-based laboratories. PART I: Threshold levels for T. brucei ssp. detection by LAMP were determined using whole equine blood specimens spiked with known concentrations of parasites. Results were compared to OIE antemortem gold standard of T. brucei-PCR (TBR-PCR). RESULTS I: Threshold for detection of T. brucei ssp. on extracted DNA from whole blood was 1 parasite/ml blood for LAMP and TBR-PCR, and there was excellent agreement (14/15) between tests at high (1 x 103/ml) concentrations of parasites. Detection threshold was 100 parasites/ml using LAMP on whole blood (LWB). Threshold for LWB improved to 10 parasites/ml with detergent included. Performance was excellent for LAMP at high (1 x 103/ml) concentrations of parasites (15/15, 100%) but was variable at lower concentrations. Agreement between tests was weak to moderate, with the highest for TBR-PCR and LAMP on DNA extracted from whole blood (Cohen's kappa 0.95, 95% CI 0.64-1.00). PART II: A prospective cross-sectional study of working equids meeting clinical criteria for trypanosomiasis was undertaken in The Gambia. LAMP was evaluated against subsequent TBR-PCR. RESULTS II: Whole blood samples from 321 equids in The Gambia were processed under field conditions. There was weak agreement between LWB and TBR-PCR (Cohen's kappa 0.34, 95% CI 0.19-0.49) but excellent agreement when testing CSF (100% agreement on 6 samples). CONCLUSIONS: Findings support that LAMP is comparable to PCR when used on CSF samples in the field, an important tool for clinical decision making. Results suggest repeatability is low in animals with low parasitaemia. Negative samples should be interpreted in the context of clinical presentation.

Variation in disease phenotype is marked in equine trypanosomiasis.

BACKGROUND: Equine trypanosomiasis is a severe and prevalent disease that has the greatest impact globally upon working equids due to its distribution across lower income countries. Morbidity and mortality rates are high; disease management strategies in endemic regions are ineffective and cost prohibitive. Individual variation in disease phenotype in other species suggests host factors could reveal novel treatment and control targets but has not been investigated in equids. METHODS: A prospective clinical evaluation of equines presenting for a free veterinary examination was performed in hyperendemic villages in The Gambia. Age, body condition score and body weight were estimated by validated methods, and haematocrit and total protein concentration measured. Animals fulfilling 2 out of 5 clinical inclusion criteria (anaemia, poor body condition, pyrexia, history of abortion, oedema) for a diagnosis of trypanosomiasis received trypanocidal treatment with follow-up at 1 and 2 weeks. Blood samples underwent PCR analysis with specific Trypanosoma spp. primers and results were compared to the subject's clinical and clinicopathological features. A mixed effects generalised linear model was generated to evaluate the association of infection status with degree of pyrexia and anaemia. RESULTS: Morbidity was high within examined (n = 641) and selected (n = 247) study populations. PCR status was not associated with a defined disease phenotype; there was intra- and inter-species variability. Donkeys were more frequently Trypanosoma spp.-positive (P < 0.001) and febrile (P < 0.001) than horses, but infected horses were more anaemic (P < 0.001), and in poorer body condition (P < 0.001) than donkeys. Sex was correlated to disease phenotype: males were more anaemic (P = 0.03) and febrile (P < 0.001). Haemoparasite co-infections were more common than a single infection. CONCLUSIONS: There was evidence of diversity in trypanosomiasis clinical signs plus variable disease phenotypes within equid subpopulations that warrant further investigation. The complex co-infection profile of field cases requires greater consideration to optimise disease management.

Analysis of Theileria equi diversity in The Gambia using a novel genotyping method.

Theileria equi, one of the primary pathogens causing equine piroplasmosis, has previously been sub-classified into a number of clades on the basis of 18S SSU rRNA gene sequence diversity. This partitioning of the parasite population has potential implications for host immunity, treatment and vaccine development. To detect and identify different clade genotypes among and within individual equine blood samples, a novel PCR-based technique was designed and optimized. Theileria equi has only recently been described in The Gambia, and the developed genotyping technique was used to analyse blood samples taken from 42 piroplasmosis-positive horses and donkeys within the country. Three different T. equi genotypes were detected within the population, including the same genotype as the recently described Theileria haneyi, with 61.9% of individuals found to be infected with more than one genotype. Overall, there was a trend that males were more likely to have a multiple genotype infection. Thus, the novel genotyping technique has been shown to be effective in analysis of field populations and offers researchers a rapid method of identifying multiple T. equi genotypes both within individuals and equine populations in epidemiological studies.

Safety and efficacy of three trypanocides in confirmed field cases of trypanosomiasis in working equines in The Gambia: a prospective, randomised, non-inferiority trial.

BACKGROUND: Globally, working equines have a continued and growing socioeconomic role in supporting the livelihoods of between 300-600 million people in low income countries which is rarely recognised at a national or international level. Infectious diseases have significant impact on welfare and productivity in this population and equine trypanosomiasis is a priority disease due to its severity and prevalence. Strategies are required to improve the prevention, diagnosis, management and treatment of trypanosomiasis in equines and more data are required on the efficacy and safety of current trypanocidal drugs. METHODS: A prospective randomised, open-label non-inferiority trial was performed in The Gambia on horses and donkeys that fulfilled 2/5 clinical inclusion criteria (anaemia, poor body condition, pyrexia, history of abortion, oedema). Following randomised trypanocidal treatment (diminazene diaceturate, melarsomine dihydrochloride or isometamidium chloride), animals were observed for immediate adverse drug reactions and follow-up assessment was performed at 1 and 2 weeks. Blood samples underwent PCR analysis with specific Trypanosoma sp. primers. Treatment efficacy was assessed by measuring changes in clinical parameters, clinicopathological results and PCR-status post-treatment after evaluating for bias. Using PCR status as the outcome variable, non-inferiority of isometamidium treatment was determined if the upper bound limit of a 2-sided 95% CI was less than 10%. RESULTS: There was a significant beneficial effect upon the Trypanosoma sp. PCR positive population following trypanocidal treatment for all groups. The findings of clinical evaluation and PCR status supported a superior treatment effect for isometamidium. Melarsomine dihydrochloride efficacy was inferior to isometamidium. There were immediate, self-limiting side effects to isometamidium in donkeys (26%). Diminazene had the longest duration of action as judged by PCR status. CONCLUSIONS: The data support the continued use of isometamidium following careful dose titration in donkeys and diminazene for trypanosomiasis in equines using the doses and routes of administration reported.

Molecular confirmation of Sarcocystis fayeri in a donkey.

Sarcocystis fayeri is a canine protozoan parasite with an equine intermediate host. Historically classified as an incidental pathogen, recent literature has described the toxic effects of Sarcocystis fayeri in human food poisoning, and highlighted potential involvement in equine neuromuscular disease. Until now, horses were believed to be the exclusive intermediate host. This study reports the first molecular confirmation of S. fayeri in a donkey, and gives rise to the consideration of donkeys being a potential reservoir for the parasite. This finding is of particular importance in understanding the epidemiology of this disease.

Erratum to: Arrhythmogenic right ventricular cardiomyopathy secondary to adipose infiltration as a cause of episodic collapse in a horse.

[This corrects the article DOI: 10.1186/s13620-015-0052-3.].

Perioperative trends in plasma colloid osmotic pressure in horses undergoing surgery.

OBJECTIVE: To compare perioperative trends in plasma colloid osmotic pressure (COP) between horses undergoing orthopedic and colic surgery. DESIGN: Prospective clinical study September 2009-January 2011. SETTING: Veterinary university teaching hospital. ANIMALS: Thirty-three healthy, client-owned horses presenting for orthopedic surgery (non-GI) and 85 client-owned horses presenting for emergency exploratory celiotomy (GI, gastrointestinal). INTERVENTIONS: None. MEASUREMENTS: Data relating to the horse's parameters on presentation, surgical lesion, post-operative management and survival were extracted from computerized clinical records. Heparinized blood samples were taken on presentation (PreOp, pre-operative), on recovery from anesthesia (T0), at 12 (T12) and 24 (T24) hours post recovery. COP was measured within 4 hours of collection. RESULTS: There was no significant difference in PreOp or T0 COP between groups. Both groups had a significant decrease in COP during anesthesia. When compared to their respective pre-operative values, horses in the non-GI group had significantly increased COP at T12, whereas those in the GI group had significantly reduced COP. This trend was continued at T24. Horses in the GI group placed on intravenous crystalloid isotonic fluids post-operatively had a significantly lower COP at T12 and T24. Horses in the GI group that did not survive had significantly lower post-operative COP values at T24. CONCLUSIONS: Horses undergoing exploratory celiotomy had significantly lower COP post-operatively than those horses undergoing orthopedic surgery. This difference was more marked in those horses receiving isotonic crystalloid intravenous fluid therapy post-operatively and in those that did not survive to discharge. In the non-GI group an increase in COP post-operatively was common.

Arrhythmogenic right ventricular cardiomyopathy secondary to adipose infiltration as a cause of episodic collapse in a horse.

A 15-year-old Clydesdale cross gelding was investigated and managed over a 2-year period for intermittent collapse. The horse presented initially after an observed episode of collapse at rest, and had a resting tachycardia, elevated Cardiac Troponin I and polycythaemia. Multiple dysrhythmias were detected on telemetric electrocardiography. Vital parameters, cardiac rhythm and red cell count returned to reference range with prolonged rest but further resting syncopal episodes were observed, and due to safety concerns and limited treatment options the horse was euthanased. Post mortem evaluation identified extensive infiltration and replacement of right and left ventricular myocardial fibres with adipose and fibrous tissue, consistent with arrhythmogenic right ventricular cardiomyopathy. This report provides further information regarding the clinical and pathological features of this rarely reported condition.

The prevalence and nature of cardiac arrhythmias in horses following general anaesthesia and surgery.

BACKGROUND: The prevalence and nature of arrhythmias in horses following general anaesthesia and surgery is poorly documented. It has been proposed that horses undergoing emergency surgery for gastrointestinal disorders may be at particular risk of developing arrhythmias. Our primary objective was to determine the prevalence and nature of arrhythmias in horses following anaesthesia in a clinical setting and to establish if there was a difference in the prevalence of arrhythmias between horses with and without gastrointestinal disease undergoing surgery. Our secondary objective was to assess selected available risk factors for association with the development of arrhythmias following anaesthesia and surgery. METHODS: Horses with evidence of gastrointestinal disease undergoing an exploratory laparotomy and horses with no evidence of gastrointestinal disease undergoing orthopaedic surgery between September 2009 and January 2011 were recruited prospectively. A telemetric electrocardiogram (ECG) was fitted to each horse following recovery from anaesthesia and left in place for 24 hours. Selected electrolytes were measured before, during and after surgery and data was extracted from clinical records for analysis. Recorded ECGs were analysed and the arrhythmias characterised. Multivariable logistic regression was used to identify risk factors associated with the development of arrhythmias. RESULTS: Sixty-seven horses with gastrointestinal disease and 37 without gastrointestinal disease were recruited. Arrhythmias were very common during the post-operative period in both groups of horses. Supra-ventricular and bradyarrhythmias predominated in both groups. There were no significant differences in prevalence of any type of arrhythmias between the horses with or without gastrointestinal disease. Post-operative tachycardia and sodium derangements were associated with the development of any type of arrhythmia. CONCLUSIONS: This is the first study to report the prevalence of arrhythmias in horses during the post-operative period in a clinical setting. This study shows that arrhythmias are very common in horses following surgery. It showed no differences between those horses with or without gastrointestinal disease. Arrhythmias occurring in horses during the post-anaesthetic period require further investigation.