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.

Assessment of Leishmania infantum infection in equine populations in a canine visceral leishmaniosis transmission area.

BACKGROUND: Leishmaniosis, zoonosis that produces significant public health impacts, is caused by Leishmania infantum. Canines are the main domestic reservoir and, besides humans, other species of mammals could be infected when living in endemic areas. In this study, we detected equine Leishmania infantum infections in a canine visceral leishmaniosis transmission area and evaluated the clinical, haematological, biochemical and oxidative stress disorders. This study was conducted in Uruguaiana, Rio Grande do Sul, south of Brazil. Peripheral blood samples were collected from 124 animals (98 horses and 26 dogs) of both genders and several breeds after they underwent general and dermatologic examinations. RESULTS: Twenty five Leishmania infantum infected animals (20.16%), 14 horses and 11 dogs were detected by PCR (Polymerase Chain Reaction) amplification of kinetoplast DNA regions with 96% homology to Leishmania infantum (GenBank Accession No. L 19877.1). The clinical and haematological alterations of infected equines were skin lesions, nodules, lymphadenopathy, decreased levels in red blood cells and haematocrit (p < 0.05) and increase in urea serum concentration (p < 0.05), while CVL presented a decrease in red blood cells counts (p < 0.05), increase in lymphocytes (p < 0.05), and decrease in neutrophil-lymphocyte ratio (p < 0.05). Oxidative stress markers of plasma protein carbonyl and plasma lipid peroxidation were not statistically significant (p > 0.05) in both species. CONCLUSIONS: To our knowledge, this has been the first leishmaniosis equine survey performed in south of Brazil, caused by Leishmania infantum that were able to initially identify haematological and biochemical changes in the species, even in asymptomatic animals. We present evidence supporting those findings of haematological and biochemical changes could be related to infection. Surprisingly, the clinical manifestations of equine infection were similar to those found in canine visceral leishmaniosis. The equine population could be play an important role in the cycle of leishmaniosis in south Brazil and consequently indicates a great risk of public health. This evaluation of infected animals is important to establish the clinical and laboratory parameters involved in the disease progression.

Assessment of equine piroplasmids in the Nhecolândia sub-region of Brazilian Pantanal wetland using serological, parasitological, molecular, and hematological approaches.

Brazilian Pantanal is the world´s largest wetland ecosystem, where cattle's ranching is the most important economic activity. The objective of this study was to compile some epidemiological features on equine piroplasmids from the Nhecolândia sub-region of Pantanal wetland through the evaluation of the patterns of T. equi and B. caballi infections in different groups of horses; identification of the tick species that infest horses; and to study phylogenetic relationships among Theileria equi 18S rRNA gene sequences. During October 2015, blood and serum samples were collected from 170 horses in four different categories. Ticks, after identification, had their hemolymph and eggs examined for the presence of piroplasmid sporokinets. Also we searched parasites in the peripheral blood smears of the investigated horses. The number of red blood cells (RBCs) and the packed cell volume (PCV) ​​were determined to test for anemia in the infected animals, and exposure to B. caballi and T. equi was evaluated by enzyme-linked immunosorbent assay. "Catch all primers" based on 18S rRNA gene were used in polymerase chain reactions (PCR) to detect equine piroplasmids, followed by three nested PCRs for the phylogenetic analysis. The serological results showed that 61.8% and 52.9% of the horses sampled were exposed to T. equi and B. caballi, respectively. Piroplasmid DNA was detected in 43.5% of the horses analyzed. Our sequencing revealed 98-100% identity with some sequences previously published in GenBank for T. equi, and microheterogeneity among others. We found that 51.2% of the animals sampled were infested with Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus (Boophilus) microplus, singly or co-infested. Since positive and negative animals presented similar RBC and PCV values, and no sporokinets were found on blood smears, hemolymph and eggs of the ticks collected, we suggest that infected equines can act as asymptomatic carriers for piroplasmosis in the studied region. Our results together showed the enzootic characteristic of equine piroplasmids in Pantanal region highlighting the importance of using different methods for detection these parasites. Moreover, breeding mares and foals should be monitored since they displayed the greatest occurrences for molecular test (59.0% and 86.1% respectively) and tick infestations (87.2% and 63.9% respectively).

Assessment of Draxxin® (tulathromycin) as an inhibitor of in vitro growth of Babesia bovis, Babesia bigemina and Theileria equi.

Babesia bovis, Babesia bigemina and Theileria equi are worldwide tick-borne hemoprotozoan that cause diseases characterized by fever, anemia, weight loss and abortion. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the host for life, and becoming a reservoir for tick transmission. The live-attenuated vaccines for B. bovis and B. bigemina are not available for worldwide use due to legal restrictions and other concerns such as potential erythrocyte antigen and pathogen contamination, and a vaccine for T. equi is not available. The use of chemotherapeutics is essential to treat and control these diseases, but several studies have shown the development of drug-resistance by these parasites, and safe and effective alternative drugs are needed. Tulathromycin, a macrolide antibiotic, has proven to be effective against a vast range of bacteria and Plasmodium yoelli, a Babesia and Theileria related intra-erythrocytic apicomplexan. Draxxin® (tulathromycin) is currently licensed to treat infections that cause respiratory diseases in cattle in several countries. In this study, the activity of Draxxin® was tested in vitro on cultured B. bovis, B. bigemina and T. equi. Addition of the drug to in vitro cultures resulted in cessation of parasite replication of the three species tested, B. bovis, B. bigemina and T. equi, with estimated IC50 of 16.7 ±â€¯0.6 nM; 6.2 ±â€¯0.2 nM and 2.4 ±â€¯0.1 nM, respectively, at 72 h. Furthermore, neither parasites nor parasite DNA were detectable in cultures treated with IC100, suggesting Draxxin® is a highly effective anti-Babesia/Theileria drug. Importantly, the IC50 calculated for Draxxin® for the Babesia/Theileria parasites tested is lower that the IC50 calculated for some drugs currently in use to control these parasites. Collectively, the data strongly support in vivo testing of Draxxin® for the treatment of bovine babesiosis and equine piroplasmosis.

Prevalence of liver fluke infection in Irish horses and assessment of a serological test for diagnosis of equine fasciolosis.

BACKGROUND: There is little information on the prevalence of Fasciola hepatica infection in the horse population in Ireland or the potential impact of fluke infection on animal health. OBJECTIVES: To investigate F. hepatica infection in the Irish horse population and to assess the diagnostic potential of an indirect enzyme-linked immunosorbent assay (ELISA) based on the F. hepatica recombinant cathepsin L1 (CL1) antigen. STUDY DESIGN: Cross-sectional abattoir survey of horses for liver fluke status. METHODS: Animals (n = 200) were examined at an abattoir between May 2013 and April 2014. Horses were graded ante mortem for body condition score. Blood and faeces were collected and livers were examined post mortem by gross morphology. A cohort (n = 35) of livers were also examined histologically. Haematology and blood biochemistry, including serum liver enzyme activities, were measured and faeces were sedimented for egg counts. Serum was assayed by indirect ELISA using a recombinant CL1. RESULTS: The prevalence of liver fluke infection was 9.5%. There was no correlation between liver fluke status and time of year, breed classification, age group, sex, body condition score, ante mortem assessment, strongyle infection status, serum liver enzyme activities or CL1 concentration. A comparison of the CL1 ELISA in horse sera compared with a reference standard diagnosis showed high specificity of 95.6% (95% confidence interval [CI] 91.5-98.0%), but low sensitivity of 42.1% (95% CI 20.2-66.5%). MAIN LIMITATIONS: This study is limited by its nature as an abattoir study, the relatively small number of animals examined (n = 200), and the absence of a known negative group of horses. CONCLUSIONS: Blood biomarkers are not good indicators of liver fluke infection and the CL1 ELISA is not a sensitive tool for diagnosis of fluke infection in the horse. The prevalence of F. hepatica in horses indicates that further research is required to assess the potential impact of liver fluke on equine liver health.

A survey of UK prescribers' experience of, and opinions on, anthelmintic prescribing practices for livestock and equines.

The aim of this study was to determine practices, attitudes and experiences of UK prescribers of anthelmintics for horses and livestock. A questionnaire was sent by direct email to groups licenced to prescribe these medicines. These were veterinarians, Suitably Qualified Persons (SQPs, registered with the Animal Medicines Training Regulatory Authority) and veterinary pharmacists. The survey was also advertised through social media. It comprised questions relating to demographics, training experiences, current prescribing practices, as well as personal opinions on anthelmintic selection, diagnostics and anthelmintic resistance. A total of 193 veterinarians and 326 SQPs were included in final analysis. Pharmacists were excluded from detailed analysis due to the low numbers that responded (n=3). The results indicated that SQP participants were more likely to receive post-certification parasitology training than the veterinarians, and that both channels consulted similar sources for information about helminths and their control (paper articles in journals, online sources). The SQP participants stated a higher frequency of face-to-face interactions with clients/customers (96.1%) than the veterinarians (76.4%), who stated a higher frequency of telephone interactions (55.1% and 73.5%, respectively). Veterinarians were more likely to state that there were specific factors that limited interactions with their clients (54.1%) than SQPs (19.6%), such as competition from other suppliers. SQP participants considered a wider range of factors as important when deciding on which anthelmintic to recommend (i.e. knowledge of specific parasites, knowledge of specific anthelmintics, discussion of measures to avoid anthelmintic resistance and time to talk with clients/customers); however, the veterinarian participants were more likely to consider the results of diagnostic tests. While discussions about anthelmintic resistance were stated with similar frequency in both groups, less frequent were specific discussions about anthelmintic sensitivity testing. In-house faecal egg count analysis was more likely to be available from those that prescribed anthelmintics for equines alone, compared to prescribers who dispensed anthelmintics for livestock alone or livestock and equines. The SQP participants indicated that they felt a large number of organisations were responsible for ensuring that anthelmintics are used responsibly, whilst veterinarian participants were more likely to place responsibility on the prescribers alone. Taken together, these findings provide an insight into how prescribers of anthelmintics in the UK interact with their clients/customers before and at the point of sale and act as a unique source of information on how best practice advice pertaining to sustainable helminth control is disseminated by the various prescribing channels.

Assessment of Theileria equi and Babesia caballi infections in equine populations in Egypt by molecular, serological and hematological approaches.

BACKGROUND: Equine piroplasmosis (EP) caused by Theileria equi, Babesia caballi, or both, contributes to significant economic loss in the equine industry and remains uncontrolled in Egypt. This study focuses on surveying T. equi and B. caballi infections and hematological disorders in equine populations in Egypt. METHODS: Theileria equi and B. caballi infections were assessed in blood from 88 horses and 51 donkeys in Egypt using light microscopy, indirect immunofluorescent antibody test (IFAT), nested PCR (nPCR), and competitive-ELISA (cELISA) assays. PCR products were examined for specificity by DNA sequencing. Hematological alterations were evaluated using a standard cell counter. RESULTS: Microscopic analysis revealed EP infection in 11.4% and 17.8% of horses and donkeys respectively. IFAT detected 23.9% and 17.0% infection of T. equi and B. caballi, respectively, in horses, and 31.4% of T. equi and B. caballi in donkeys. T. equi cELISA detected 14.8% and 23.5% positive horses and donkeys, respectively, but the B. caballi RAP-1-based cELISA failed to detect any positives, a result hypothesized to be caused by sequence polymorphism found in the rap-1 genes. Nested-PCR analysis identified 36.4% and 43.1% positive horses and donkeys, respectively for T. equi and it also identified 19.3% and 15.7% positive horses and donkeys, respectively for B. caballi. The overall EP incidence found in the population under study was relatively high and comparable regardless of the diagnostic method used (56.8% using nPCR and 48.9% using IFAT). Hematologic analysis revealed macrocytic hypochromic anemia and thrombocytopenia in all piroplasma-infected horses. CONCLUSIONS: The data confirm relatively high levels of EP, likely causing hematological abnormalities in equines in Egypt, and also suggest the need for an improved serological test to diagnose B. caballi infection in this region.

Semi-quantitative differentiation of cyathostomin larval cultures by reverse line blot.

Cyathostomins are the most prevalent horse nematodes worldwide and over 50 species are described. The eggs and the infective larvae (L3) can easily be obtained or cultured from infected horses, but cannot be differentiated morphologically at species level. A reverse line blot (RLB) method based on the hybridization of a PCR fragment with a species specific probe, has previously been developed for the differentiation of individual eggs and/or L3s, but is too labor intensive for large scale studies. In the present study a RLB method on multiple pooled L3s for the semi-quantitative differentiation of cyathostomin larval cultures was developed and validated. First, the probability of the presence of a certain species within a pool was calculated as function of the frequency and the number of L3s within a pool. Ten L3s per pool were found to be optimal. Next, the probability, the chance of occurrence was calculated when 4 pools per culture were used. The probability distributions for 0, 1, 2, 3 or 4 positive pools were transformed into the corresponding median frequency of the cumulative probability: 0.014, 0.04, 0.08, 0.16 and 0.59, respectively. Based on these calculated probabilities, RLB on 10 L3s per pool and 4 pools per sample was validated by estimating the cross-hybridization, precision and accuracy in 3 groups of horses. First, absence of cross-hybridization was confirmed by differentiation of the same L3s (160 L3s from the 4 horses from group 1) in the RLB on individual as well as on pooled L3s. Cross-hybridization was excluded for 9 of the most common cyathostomins. Next, the precision and accuracy were determined by the differentiation of 10 replicates of 3 cultures from 3 horses from group 2 (1200 L3s). The coefficient of variation (CV) was between 0 and 0.90 and the accuracy was between 0.42 and 1.73. A Monte Carlo simulation based on the observed scores and associated probability distributions gave similar results as the use of a fixed median frequency. The LPGs obtained from 276 larval culture counts from a larger cohort (23 horses, group 3) were not significantly different from the LPGs obtained from summation of the LPG per species found by RLB on pooled L3s. The RLB on pooled L3s was found therefore an useful semi-quantitative method for the differentiation of the most common cyathostomin L3, with a workload of approximately one tenth of that of the RLB on individual L3s.

Attitudes towards implementation of surveillance-based parasite control on Kentucky Thoroughbred farms - Current strategies, awareness and willingness-to-pay.

REASONS FOR PERFORMING STUDY: Traditionally, equine parasite control has relied heavily on frequent routine anthelmintic treatments applied at regular intervals all year round. However, current recommendations aim to employ a more surveillance-based approach and it remains unknown to what extent these recommendations are being implemented on US horse farms. OBJECTIVES: To describe equine parasite control on Kentucky Thoroughbred farms and evaluate respondents' willingness to pay for various attributes of surveillance-based parasite control strategies. STUDY DESIGN: Questionnaire survey performed among the membership of the Kentucky Thoroughbred Farm Managers' Club. METHODS: The survey collected demographic data and information about current parasite control strategies. Further, respondents were asked to choose between hypothetical parasite control strategies described with a combination of different attributes: costs, time and effort needed, hypothetical disease-risk levels and hypothetical risks of anthelmintic resistance. Data were analysed with multivariable logistic analysis. RESULTS: About 26% responded to the survey (n = 112). Most respondents were concerned about anthelmintic resistance and incorporated veterinary advice in defining their deworming programme. However, almost 70% were following a traditional rotational deworming programme with little or no faecal surveillance. Respondents were willing to pay a premium for a product for which there is no known anthelmintic resistance and provided the highest possible decrease in health risks. The number of young horses on the farm, utilisation of veterinarian advice in developing a deworming programme, expressed concern about drug resistance in parasites and having documented drug resistance on the farm all associated significantly with the type of parasite control programme used. CONCLUSIONS: Traditional approaches for equine parasite control are still widely used in the Kentucky Thoroughbred industry. The data suggest that respondents were only willing to make these changes if they could be given assurance that the surveillance-based approach would prevent anthelmintic resistance and decrease health risks significantly for the horses.

Importance of the horse and financial impact of equine trypanosomiasis on cattle raising in Venezuela.

In Venezuela, horses are indispensable for extensive cattle raising, and extensive cattle raising prevails in all regions. This determines the numerical relationship between horses and cattle (r = 0.93) to be relatively constant nationwide. At regional level, the average extension of cattle ranches varies greatly. However, in relation to the area covered by pastures, the numbers of horses (r = 0.95) and cattle (r = 0.93) are relatively uniform nationwide. Water buffalo occupy small fractions of the territory; therefore, their numbers are related to the area of pastures less strongly (r = 0.56). There is no information on the numerical relationship between the numbers of horses and water buffalo. In the Llanos region of the country, equine trypanosomiasis is responsible for a high mortality in horses, causing considerable financial losses to cattle ranches. So far, such losses have not been assessed. For this region, in 2008, it can be calculated that: (1) with no treatment, losses owing to horse mortality caused by this hemoparasitosis would have amounted to US$7,486,000; (2) the diagnosis and treatment of affected horses would have required an investment of US$805,000; and (3) in terms of horses saved, this investment would have resulted in benefit of US$6,232,000. Therefore, for every monetary unit invested, there would be a benefit 7.75 times greater, this ratio being applicable to any year and all regions of the country. It follows that the profitability of investing in the diagnosis and treatment of equine trypanosomiasis is guaranteed.

Sustainable equine parasite control: perspectives and research needs.

Clinically important equine parasites are ubiquitous in managed horse populations. The traditional approach to parasite control is frequent administration of anthelmintics to all horses on a farm. However, increasing levels of anthelmintic resistance is forcing horse owners and veterinarians to shift this control paradigm. Treatment regimens involving routine deworming of all horses throughout the year are now being replaced by more sustainable approaches, which take in to account the importance of maintaining adequate parasite refugia. The selective therapy principle has been recommended for more than 15 years, but there is limited experience with this approach. The relative magnitude of the faecal egg count for an individual horse is a consistent trait, and this provides a reliable basis for selective therapy. But no studies have evaluated the consequences of selective therapy in the long-term, and such studies are strongly needed to validate this approach. Importantly, it remains unclear how selective therapy may affect the prevalence and intensity of other parasites of significant pathogenic potential (e.g. Strongylus vulgaris), which have become uncommon due to years of intensive chemotherapy. Consequently, a selective approach requires vigilant surveillance of the parasite fauna and intensity. This places a demands for reliable diagnostic tools. Also noteworthy is the fact that the majority of equine nematode parasites are more pathogenic during their larval stages, when they cannot be detected by traditional egg counting techniques. Consequently, parasite-specific diagnostic tools capable of assessing prepatent parasite burdens, and able to differentiate between strongyle species of different pathogenic potentials, would be of great value to the equine clinician. Tools for detecting infections with the tapeworm Anoplocephala perfoliata are laborious, difficult to interpret, and at present there is no established method to evaluate treatment efficacy. Thus, better diagnostic tools are needed for tapeworms as well. Biological control, especially the predacious fungi have demonstrated good potential as an adjunct for strongyle control and such a product could easily have a market in equine establishments. In summary, there is general agreement that the traditional treat-all at frequent interval approach should be abandoned, and that optimal parasite control can be maintained with far fewer anthelmintic treatments. But better diagnostic techniques and more evidence documenting the long-term consequences of selective therapy programs are needed to develop and validate systems for sustainable equine parasite control.

Pharmacological assessment of netobimin as a potential anthelmintic for use in horses: plasma disposition, faecal excretion and efficacy.

This study aimed to determine the plasma disposition and faecal excretion of netobimin (NTB) and its respective metabolites as well as the efficacy against strongyles in horses following oral administration. Netobimin (10mg/kg) was administered orally to 8 horses. Blood and faecal samples were collected from 1 to 120h post-treatment and analysed by high performance liquid chromatography (HPLC). Using a chiral phase-based HPLC, plasma disposition of ABZSO enantiomers produced was also determined. Faecal strongyle egg counts (EPG) were performed by a modified McMaster's technique before and after the treatment. Neither NTB nor ABZ were present and only albendazole sulphoxide (ABZSO) and sulphone metabolites (ABZSO(2)) were detected in the plasma samples. Maximum plasma concentration of ABZSO (0.53+/-0.14microg/ml) and ABZSO(2) (0.36+/-0.09microg/ml) were observed at (t(max)) 10.50 and 19.50h, respectively following administration of NTB. The area under the curve (AUC) of the two metabolites was similar to each other. Netobimin was not detected, and ABZ was predominant in faecal samples. The maximum plasma concentration (C(max)) of (-)ABZSO was significantly higher than (+)ABZSO, but the area under the curves (AUCs) of the enantiomer were not significantly different each other in plasma samples. The enantiomers of ABZSO were close to racemate in the faecal samples analyzed. Netobimin reduced the EPG by 100%, 100%, 77%, 80% and 75% 2, 4, 6, 8 and 10 weeks post-treatment, respectively. The specific behaviour of the two enantiomers probably reflects different enantioselectivity of the enzymatic systems of the liver which are responsible for sulphoxidation and sulphonation of ABZ. Considering the pharmacokinetic and efficacy parameters NTB could be used as an anthelmintic in horses.

Estimation of the transmission dynamics of Theileria equi and Babesia caballi in horses.

For the evaluation of the epidemiology of Theileria equi and Babesia caballi in a herd of 510 horses in SW Mongolia, several mathematical models of the transmission dynamics were constructed. Because the field data contain information on the presence of the parasite (determined by PCR) and the presence of antibodies (determined by IFAT), the models cater for maternal protection with antibodies, susceptible animals, infected animals and animals which have eliminated the parasite and also allow for age-dependent infection in susceptible animals. Maximum likelihood estimation procedures were used to estimate the model parameters and a Monte Carlo approach was applied to select the best fitting model. Overall, the results are in line with previous experimental work, and add evidence that the epidemiology of T. equi differs from that of Babesia spp. The presented modelling approach provides a useful tool for the investigation of some vector-borne diseases and the applied model selection procedure avoids asymptotical assumptions that may not be adequate for the analysis of epidemiological field data.

Assessment of serum protein electrophoresis for monitoring therapy of naturally acquired equine cyathostomin infections.

Serum protein electrophoresis (SPE) has previously been suggested as a means of assessing cyathostomin burdens in horses, although SPE used for that purpose is supported by little evidence. This clinical research report describes a study that objectively evaluated the use of SPE on a population of 38 horses following the administration of different anthelmintics. The population was subdivided into three groups, Groups F, M and P: 7.5 mg/kg bwt fenbendazole was administered to Group F on day -12; on day 0 0.4 mg/kg bwt moxidectin was administered to Group M and 19 mg/kg bwt pyrantel was administered to Group P. Faecal worm egg counts were obtained on days -14, 0 and 10. Groups M and P acted as controls for the Group F faecal egg count reduction test (FECRT) in which a high level of benzimidazole resistance was demonstrated. Group F was then used as a control group for the FECRT for both Groups P and M. A high anthelmintic efficacy of moxidectin and pyrantel was detected. SPE was performed on venous blood collected on days 0, 10, 30, 56 and 80. As the cyathostomins infecting the horses had been shown to be highly resistant to fenbendazole, Group F then served as a control group for comparison of any changes in protein fractions. Serum proteins did not vary significantly between groups on any of the sampling dates. No significant changes in serum proteins were observed in any group and no patterns were apparent on qualitative assessment of SPE profiles. SPE was therefore concluded to be an insensitive tool for the monitoring of cyathostomin treatment in horses in a clinical environment.

Application of the 51Cr-EDTA urinary recovery test for assessment of intestinal permeability in the horse.

Altered intestinal permeability is implicated in the pathogenesis of diverse equine medical conditions including alimentary laminitis and protein-losing enteropathies associated with parasitic infection. The aims of this study were to assess the feasibility of applying the 51Cr-EDTA absorption test for the assessment of intestinal permeability in the horse, and to apply this test in horses with experimentally induced alterations in gastrointestinal function. Four healthy ponies were administered 36 MBq of 51Cr-EDTA via naso-gastric tube, and urine samples were collected into polythene bags strapped to the pony's abdomen. Total urine voided every 6 h was collected during each test, and 1 ml samples were taken for measurement of gamma-radiation. Urinary recovery of 51Cr-EDTA was measured following intravenous atropine sulphate or bethanecol, and following 22 and 46 days of administration of 250,000 third-stage cyathostome larvae. There was no significant difference in urinary 51Cr-EDTA recovery following the control treatment, and following atropine or bethanecol administration, but significant increases were detected in the animals with experimental cyathostome infection consistent with increased permeability of the intestinal membrane. Motility modifying agents (bethanecol and atropine) did not affect absorption of 51Cr-EDTA, suggesting that subtle changes in motility might not affect the ability of this test to detect altered intestinal permeability. The finding of increased urinary recovery of 51Cr-EDTA in ponies with cyathostome infection suggests that 51Cr-EDTA may be a useful marker for assessment of intestinal permeability in the horse.

Changes in the EU legislation on Trichinella inspection--new challenges in the epidemiology.

The European Union (EU) countries are searching for new ways to certify meat free of Trichinella; however, with the expansion of the EU, the acceptance of a unilateral method is complicated by the variability of pig and human trichinellosis among EU countries, where significantly higher prevalence rates have been observed in the newly added eastern countries. Several attempts have been made to define Trichinella-free areas, but certification of Trichinella-free pig production farms appears to be the only feasible approach. The increasing prevalence of the non-encapsulating species, Trichinella pseudospiralis, in game, domestic pigs and humans has eliminated the compression technique from the new EU legislation to be enacted in 2006. Also, the observation that several species of Trichinella tolerate freezing in horse meat for up to 4 weeks has forced a change in legislation as well where freezing is no longer an option for certifying horse meat. Because current serological detection methods are not suited for meat inspection, classical direct detection methods and inactivation by freezing remain the methods of choice for pork. It has been proposed, therefore, to automate direct inspection methods as a cost effective alternative to certify pig farms free of Trichinella.

Assessment of Sarcocystis neurona sporocyst viability and differentiation between viable and nonviable sporocysts using propidium iodide stain.

Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of dead sporocysts. After live and dead sporocysts were mixed at various ratios, the number of unstained sporocysts detected after the staining procedure correlated significantly (r2 = 0.9978) with the expected numbers of live sporocysts. Sporocyst mixtures were also assayed for in vitro excystation and development in tissue cultures. The correlation between the percentage of plaques formed in tissue cultures and the percentage of expected infectious (live) sporocysts in each mixture was r2 = 0.6712. By analysis of variance, no statistically significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts (P = 0.3902) in each mixture. In addition, there was evidence of a relation between PI impermeability of sporocysts and animal infectivity. These results suggest that the PI dye-exclusion technique can be a useful tool in identifying viability and potential infectivity of S. neurona sporocysts and in differentiating between viable and nonviable sporocysts.

A non-lethal method for assessment of efficacy of antiparasitics against parasites in horses such as Anoplocephala perfoliata and Gasterophilus intestinalis.

Sourcing of horses naturally infected with parasites such as Anoplocephala perfoliata and Gasterophilus intestinalis for antiparasitic efficacy slaughter studies is often difficult, expensive, and usually excessive numbers of animals must be slaughtered before significant data can be obtained. To overcome this problem a "Modified Critical Treatment Method" was developed. This method does not require the horses to be sacrificed, but relies on the collection and processing of all faecal material containing expelled parasites from test animals. Efficacy is assessed by comparing, in the same horses, the numbers of parasites expelled with the test compound with the total numbers removed with test and reference endectocides. This, for the most part, means that farms that were previously unavailable, e.g. studs, can now be used for the collection of efficacy data.

Horse trichinellosis, an unresolved puzzle.

In spite of routine controls to detect Trichinella larvae in horse-meat, human infections due to horse-meat consumption continue to occur in France and Italy. The epidemiology of horse trichinellosis since its discovery in 1975 is outlined, addressing the possible modes of natural transmission to horses, the need to develop more sensitive methods for detecting Trichinella larvae in horses, and the economic impact of horse trichinellosis. Investigations of human outbreaks due to horse-meat consumption have implicated single cases of inadequate veterinary controls on horses imported from non-European Union countries. In particular, most cases of human infection have been attributed to horses imported from Eastern Europe, where pig trichinellosis is re-emerging and the main source of infection in horses.

Babesiosis in China.

The importance of babesiosis in livestock in China is discussed and mainly focused on bovine and equine babesiosis. Babesiosis is still one of the most important diseases affecting livestock and has caused great economic loss. Nine species of Babesia have been recognized in livestock: B. bigemina, B. bovis, B. major, B. motasi, B. ovis, B. perroncitoi, B. trautmanni, B. equi (Theileria equi), B. caballi. The distribution of Babesia follows the distribution of the tick vectors. The main vectors of bovine babesiosis are the one-host tick Boophilus microplus and the three-host ticks Rhipicephalus haemaphysaloides haemaphysaloides, Haemaphysalis punctata and Haemaphysalis longicornis. Bovine babesiosis has caused significant losses in milk and meat from cattle in most parts of China. The disease is also a barrier to improving productivity of local cattle by cross-breeding due to the high mortality of genetically superior but highly susceptible cattle, especially dairy cattle, imported from Babesia-free areas. Dermacentor nuttalli is the major vector of equine babesiosis and the tick is distributed in almost all parts of North China. Outbreaks of equine babesiosis have not been very common, but in some districts the disease has seriously affected horses, donkeys and mules.