Impact of pH modification of the empirically used tobramycin ophthalmic solution on MIC90 concentration in tears and aqueous humor of donkeys (Equus asinus).

BACKGROUND: Commercial tobramycin ophthalmic solution is frequently used empirically to treat ocular disorders in equines, despite being primarily formulated for use in humans. It has been noted that tobramycin MIC90 concentration (minimal inhibitory concentration to 90% of microbial growth) rapidly declined following topical administration. It is hypothesized that adjustment of the pH of the empirically used tobramycin ophthalmic solution -prepared for human use- with the pH of the tears of donkeys, could increase the bioavailability of the drug and subsequently improve its penetration to the aqueous humor. Therefore, this study aimed to evaluate the impact of pH adjustment of the empirically used tobramycin ophthalmic solution on MIC90 concentration in tears and aqueous humor of donkeys (Equus asinus). The study was conducted on six (n = 6) clinically healthy donkeys. In each donkey, one eye was randomly selected to receive 210 µg tobramycin of the commercial tobramycin (CT) and used as a positive control (C group, n = 6). The other eye (treated eye) received 210 µg of the modified tobramycin ophthalmic solution (MT) (T group, n = 6). Tears and aqueous humor samples were collected 5-, 10-, 15-, 30- min, and 1-, 2-, 4-, and 6 h post-instillation. RESULTS: Modifying the pH of the empirically used commercial tobramycin ophthalmic solution in donkeys at a pH of 8.26 enhanced the drug's bioavailability. The MIC90 of the most hazardous bacteria isolated from equines' eyes such as Pseudomonas aeruginosa (MIC90 = 128 µg/ml) and Staphylococcus aureus (MIC90 = 256 µg/ml) was covered early (5 min post-instillation) and over a longer period in donkey tears (239-342 min) and aqueous humor (238-330 min) with the modified tobramycin solution. CONCLUSIONS: Adjustment of the pH of the commercial tobramycin ophthalmic solution, empirically used by veterinarians to treat donkeys' ophthalmic infections at a pH of 8.26, isotonic with the donkeys' tears pH, resulting in higher concentrations of tobramycin in tears and aqueous humor for a longer time.

Streptococcus equi subspecies equi from strangles suspected equines: molecular detection, antibiogram profiles and risk factors.

Strangles, caused by Streptococcus equi subspecies equi, is a highly infectious disease of equines causing major health issues and financial losses. The aim of the study was to detect the presence of the SeM gene in Streptococcus equi isolated from equine suspected of having strangles. A cross-sectional study design was conducted from July to December 2022 in five districts of the central Gondar zone, Ethiopia. One-hundred sixty swab samples were taken from animals that had been clinically suspected. The SeM gene was detected using polymerase chain reaction, and the antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. The binary logistic regression model was employed to test for statistical significance. In 31.87% (51/160) of the samples, Streptococcus equi species were isolated, and 31.37% (16/51) of these species carried the SeM gene. There was a significant amount of tetracycline (81.5%), erythromycin (81.5%), and vancomycin (75.5%) resistance among the 16 isolates. Strangles were more likely to be present in animals who shared feed containers (AOR = 7.59; 95% CI = 1.44-39.93), drank from the same water troughs (AOR = 7.74; 95% CI = 1.44-41.01), and spent the night together (AOR = 5.97; 95% CI 1.41-25.37). The findings of this study showed that the research areas harboured Streptococcus equi subspecies equi. Sharing feed containers and water troughs were potential sources of strangles infection; thus, these containers need to be cleaned regularly.

Do you see what I see? Testing horses' ability to recognise real-life objects from 2D computer projections.

The use of 2-dimensional representations (e.g. photographs or digital images) of real-life physical objects has been an important tool in studies of animal cognition. Horses are reported to recognise objects and individuals (conspecifics and humans) from printed photographs, but it is unclear whether image recognition is also true for digital images, e.g. computer projections. We expected that horses trained to discriminate between two real-life objects would show the same learnt response to digital images of these objects indicating that the images were perceived as objects, or representations of such. Riding-school horses (N = 27) learnt to touch one of two objects (target object counterbalanced between horses) to instantly receive a food reward. After discrimination learning (three consecutive sessions of 8/10 correct trials), horses were immediately tested with on-screen images of the objects over 10 image trials interspersed with five real object trials. At first image presentation, all but two horses spontaneously responded to the images with the learnt behaviour by contacting one of the two images, but the number of horses touching the correct image was not different from chance (14/27 horses, p > 0.05). Only one horse touched the correct image above chance level across 10 image trials (9/10 correct responses, p = 0.021). Our findings thus question whether horses recognise real-life objects from digital images. We discuss how methodological factors and individual differences (i.e. age, welfare state) might have influenced animals' response to the images, and the importance of validating the suitability of stimuli of this kind for cognitive studies in horses.

Effects of dietary zinc chloride hydroxide and zinc methionine on the immune system and blood profile of healthy adult horses and ponies.

The effects of dietary zinc on the immune function of equines have not been evaluated in detail so far. In the present study, eight healthy adult ponies and two healthy adult horses were fed a diet supplemented with either zinc chloride hydroxide or zinc methionine in six feeding periods of four weeks each (according to maintenance zinc requirement, 120 mg zinc/kg dry matter, and 240 mg zinc/kg dry matter, for both dietary zinc supplements, respectively). All animals received the six diets, with increasing amounts of zinc chloride hydroxide in the feeding periods 1-3, and with increasing amounts of zinc methionine in the feeding periods 4-6. At the end of each feeding period, blood samples were collected for a blood profile and the measurement of selected immune variables. Increasing dietary zinc chloride hydroxide doses increased the glutathione concentrations in the erythrocyte concentrate and the glutathione peroxidase activity in the erythrocyte lysate, decreased the numbers of total leukocytes and granulocytes in the blood, and also decreased the interleukin-2 concentrations in the plasma of the animals. The dietary supplementation of increasing doses of zinc methionine enhanced the mitogen-stimulated proliferative activity of peripheral blood mononuclear cells, and decreased the glutathione concentrations in the erythrocyte concentrate and the glutathione peroxidase activity in the plasma of the animals. The percentage of blood monocytes with oxidative burst after in vitro stimulation with E. coli decreased with increasing dietary zinc concentrations, independently of the zinc compound used. The blood profile demonstrated effects of the zinc supplements on the red blood cells and the bilirubin metabolism of the horses and ponies, which require further investigation. Overall, high doses of dietary zinc modulate the equine immune system, for the most part also depending on the zinc compound used.

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin 'M' (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.

Hematology and biochemical values in equines naturally infected with Theileria equi in Nigeria.

Equine piroplasmosis (EP) is a tick-borne disease of equines with resultant economic consequences. To date, little information is available regarding the effects of EP on the health of equids in Nigeria. Therefore, this study was carried out to ascertain the effects of natural sub-clinical infection with T. equi on the hematology and biochemical profiles of horses and donkeys. Blood and serum samples were subjected to hematology and serum biochemistry analyses, respectively. Data corresponding to PCR-positive (infected) and PCR-negative (uninfected) was computed to ascertain changes in any of the parameters. Hematological significant findings (P<0.001) in both horses and donkeys include decreased packed cell volume (PCV), red blood cell (RBC) count, hemoglobin (Hb) concentration, and white blood cell (WBC) counts in the infected group compared to the uninfected group. In horses, the PCV (%) and Hb (g/dL) values were 40.6±1.0 and 13.4±1.7 in the uninfected group compared with 30.8±0.6 and 10.1±0.3 in the infected group respectively and differs significantly (P<0.001). Similarly, in the donkeys, the PCV (%) and Hb (g/dL) values were 32.2 ± 0.5 and 10.5 ± 0.2 in the uninfected group compared with 28.5±0.7 and 9.5±0.2 in the infected group respectively with significant difference (P<0.001). Additionally, we observed a slight decrease in WBC counts (×103/µL) in the infected group in horses (9.9±0.4) and donkeys (10.2±4.3) compared with 10.5±2.0 and 13.0±0.8 from the uninfected respectively. Serum biochemical profiles revealed slight increase in the levels of total proteins principally the globulin fractions in the infected group in both equids. The findings from this study will be helpful in the diagnosis, prognosis, and treatment evaluation of equines in Nigeria.

Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.

BACKGROUND: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for "naked eye" end-point detection when testing 'real-world' clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. RESULTS: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91-100%. CONCLUSIONS: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing 'real-world' samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

Screening and detection of chromosomal copy number alterations in the domestic horse using SNP-array genotyping data.

Chromosomal abnormalities are a common cause of infertility in horses. However, they are difficult to detect using automated methods. Here, we propose a simple methodology based on single nucleotide polymorphism (SNP)-array data that allows us to detect the main chromosomal abnormalities in horses in a single procedure. As proof of concept, we were able to detect chromosomal abnormalities in 33 out of 268 individuals, including monosomies, chimerisms, and male and female sex-reversions, by analyzing the raw signal intensity produced by an SNP array-based genotyping platform. We also demonstrated that the procedure is not affected by the SNP density of the array employed or by the inbreeding level of the individuals. Finally, the methodology proposed in this study could be performed in an open bioinformatic environment, thus permitting its integration as a flexible screening tool in diagnostic laboratories and genomic breeding programs.

How Can Nutrition Help with Gastrointestinal Tract-Based Issues?

Many horses are fed differently than their wild ancestors. They often have limited access to pasture and are fed conserved forage and concentrates rich in starch and sugars, in only 2 meals per day. Feeding practices in contrast to natural feeding behavior can lead to gastrointestinal issues. Standard nutritional evaluation is warranted because of its important role in prevention and in treatment and management of diseases. When medical and nutritional treatments are combined, success rates are higher. New techniques to characterize equine microbiota have been used, allowing for microbiota manipulation to prevent and treat intestinal diseases.

Molecular evidence of Babesia caballi and Theileria equi in equines and ticks in Nigeria: prevalence and risk factors analysis.

Babesia caballi and Theileria equi are biological agents responsible for equine piroplasmosis (EP). We conducted a robust and extensive epidemiological study in Nigeria on the prevalence and risk factors of EP. Blood (468, both horses and donkeys) and ticks (201 pools) were screened using polymerase chain reaction (PCR). DNA of equine piroplasms was observed in tick pools with B. caballi amplified in Rhipicephalus evertsi evertsi only [minimum infection rate (MIR) of 7.6%] while T. equi was observed in R. e. evertsi (MIR, 61.6%), Hyalomma dromedarii (MIR, 23.7%) and H. truncatum (MIR, 50.0%). Overall results showed that 196/468 (41.9%) animals were positive for equine piroplasms (both B. caballi and T. equi). The prevalence for T. equi was 189/468 (40.4%) compared to 7/468 (1.5%) for B. caballi. In the horses and donkeys, respectively, the prevalence for T. equi was (39.9%; 112/281) and (41.2%; 77/187) compared with (1.4%; 4/281) and (1.6%; 3/187) due to B. caballi. Our analysis showed that location (Jigawa state), Talon breed, horses used for work and reproduction, unsatisfactory husbandry practices, contact with other mammals are risk factors that associated positivity to T. equi infection in horses, whilst horses kept on intensive management appeared to be less prone to infection. On the other hand, Jangora breed of donkeys and location (Jigawa state) are risk factors to infection with T. equi in donkeys. Findings suggest the persistence of EP in equids and ticks in Nigeria.

Equines as reservoirs of human fascioliasis: transmission capacity, epidemiology and pathogenicity in Fasciola hepatica-infected mules.

Fascioliasis is a zoonotic disease caused by liver flukes transmitted by freshwater lymnaeid snails. Donkey and horse reservoir roles have been highlighted in human endemic areas. Liver fluke infection in mules has received very limited research. Their role in disease transmission, epidemiological importance and Fasciola hepatica pathogenicity are studied for the first time. Prevalence was 39.5% in 81 mules from Aconcagua, and 24.4% in 127 from Uspallata, in high-altitude areas of Mendoza province, Argentina. A mean amount of 101,242 eggs/mule/day is estimated. Lymnaeids from Uspallata proved to belong to ribosomal DNA internal transcribed spacer (ITS) markers ITS-1 and ITS-2 combined haplotype 3C of Galba truncatula. These lymnaeids were experimentally susceptible to infection by egg miracidia from mules. Infectivity, number of cercariae/snail and shedding period fit the enhanced F. hepatica/G. truncatula transmission pattern at very high altitude. This indicates that the mule is able to maintain the F. hepatica cycle independently. Individual burdens of 20 and 97 flukes were found. Mule infection susceptibility is intermediate between donkey and horse, although closer to the latter. Anatomo-pathology and histopathology indicate that massive infection may cause mule death. Haematological value decreases of red blood cells, haemoglobin, leucocytes and lymphocytes indicate anaemia and strong immunosuppression. Strongly increased biochemical marker values indicate liver function alterations. The mule probably played a role in the past exchanges with Chile and Bolivia through Mendoza province. Evidence suggests that mules could contribute to the spread of both F. hepatica and G. truncatula to human fascioliasis-endemic areas in these countries.

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins.

Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI-TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex-specific profiles within both morphospecies could not be reliably discriminated using MALDI-TOF MS. In conclusion, MALDI-TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.

Anthelmintic resistance and novel control options in equine gastrointestinal nematodes.

Control of equine nematodes has relied on benzimidazoles (BZs), tetrahydropyrimidines and macrocyclic lactones. The intensive use of anthelmintics has led to the development of anthelmintic resistance (AR) in equine cyathostomins and Parascaris equorum. Field studies indicate that BZ and pyrantel resistance is widespread in cyathostomins and there are also increasing reports of resistance to macrocyclic lactones in cyathostomins and P. equorum. The unavailability of reliable laboratory-based techniques for detecting resistance further augments the problem of nematode control in horses. The only reliable test used in horses is the fecal egg count reduction test; therefore, more focus should be given to develop and validate improved methodologies for diagnosing AR at an early stage, as well as determining the mechanisms involved in resistance development. Therefore, equine industry and researchers should devise and implement new strategies for equine worm control, such as the use of bioactive pastures or novel feed additives, and control should increasingly incorporate alternative and evidence-based parasite control strategies to limit the development of AR. This review describes the history and prevalence of AR in equine nematodes, along with recent advances in developing resistance diagnostic tests and worm control strategies in horses, as well as giving some perspective on recent research into novel control strategies.

Sero-epidemioloical survey on African horse sickness virus among horses in Khartoum State, Central Sudan.

BACKGROUND: African horse sickness virus (AHSV) is an infectious non contagious insect-transmitted double-stranded (ds) RNA orbivirus of the family Reoviridae. AHSV causes an often fatal hemorrhagic infection with high mortality among selected breeds of Arabian horses. This study was conducted to avail some information with regard to the prevalence and associated risk factors of AHSV among ecotype breeds of horses in central Sudan. METHODS: Sera were collected from 320 horses, which were selected randomly from four localities and employed in the study. A competitive enzyme-linked immunosorbent assay (cELISA) was used to screen sampled sera for AHSV-specific immunoglobulin G (Ig G) antibodies. RESULTS: Seropositivity to AHSV Ig G was detected in 275 out of the 320 horse sera, thus accounting for a prevalence rate of 85.9%. Potential risk factors to AHSV infection were reported to be associated with horse breed (OR = 5.0, CI = 0.07-2.104, p-value = 0.039) and activity of the horse (OR = 3.21, CI = 0.72-1.48, p- value = 0.008). CONCLUSIONS: The high prevalence of AHSV in Khartoum State of Central Sudan necessitates the need for continuous surveillance for AHSV infection to prevent a possible disease outbreak in this region of the African continent.

Sex chromosomal abnormalities associated with equine infertility: validation of a simple molecular screening tool in the Purebred Spanish Horse.

Chromosomal abnormalities in the sex chromosome pair (ECAX and ECAY) are widely associated with reproductive problems in horses. However, a large proportion of these abnormalities remains undiagnosed due to the lack of an affordable diagnostic tool that allows for avoiding karyotyping tests. Hereby, we developed an STR (single-tandem-repeat)-based molecular method to determine the presence of the main sex chromosomal abnormalities in horses in a fast, cheap and reliable way. The frequency of five ECAX-linked (LEX026, LEX003, TKY38, TKY270 and UCDEQ502) and two ECAY-linked (EcaYH12 and SRY) markers was characterized in 261 Purebred Spanish Horses to determine the efficiency of the methodology developed to be used as a chromosomal diagnostic tool. All the microsatellites analyzed were highly polymorphic, with a sizeable number of alleles (polymorphic information content > 0.5). Based on this variability, the methodology showed 100% sensitivity and 99.82% specificity to detect the most important sex chromosomal abnormalities reported in horses (chimerism, Turner's syndrome and sex reversal syndromes). The method was also validated with 100% efficiency in 10 individuals previously diagnosed as chromosomally aberrant. This STR screening panel is an efficient and reliable molecular-cytogenetic tool for the early detection of sex chromosomal abnormalities in equines that could be included in breeding programs to save money, effort and time of veterinary practitioners and breeders.

Cross-sectional surveillance of Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels and other mammals in Egypt, August 2015 to January 2016.

A cross-sectional study was conducted in Egypt to determine the prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) in imported and resident camels and bats, as well as to assess possible transmission of the virus to domestic ruminants and equines. A total of 1,031 sera, 1,078 nasal swabs, 13 rectal swabs, and 38 milk samples were collected from 1,078 camels in different types of sites. In addition, 145 domestic animals and 109 bats were sampled. Overall, of 1,031 serologically-tested camels, 871 (84.5%) had MERS-CoV neutralising antibodies. Seroprevalence was significantly higher in imported (614/692; 88.7%) than resident camels (257/339; 5.8%) (p < 0.05). Camels from Sudan (543/594; 91.4%) had a higher seroprevalence than those from East Africa (71/98; 72.4%) (p < 0.05). Sampling site and age were also associated with MERS-CoV seroprevalence (p < 0.05). All tested samples from domestic animals and bats were negative for MERS-CoV antibodies except one sheep sample which showed a 1:640 titre. Of 1,078 camels, 41 (3.8%) were positive for MERS-CoV genetic material. Sequences obtained were not found to cluster with clade A or B MERS-CoV sequences and were genetically diverse. The presence of neutralising antibodies in one sheep apparently in contact with seropositive camels calls for further studies on domestic animals in contact with camels.

VectorTest™ West Nile Virus Antigen Assay in an Inhibition Platform as Field Screening Tool for Flavivirus Group-Specific Antibodies in Brazilian Equines.

Current methods for detecting Flavivirus antibodies are enzyme-linked immunosorbent assays (ELISAs) and neutralization tests, both of which require laboratories and trained staff. We evaluated the VectorTest™ West Nile Virus Antigen Assay in an inhibition platform (VecTest-inhibition assay [VIA]) as a simpler screening method for detecting antibodies for a variety of flaviviruses among a population of equines from Brazil. We found that the VIA is a field-deployable rapid method with 100% sensitivity and 64% specificity compared with blocking ELISA for the detection of group-specific Flavivirus antibodies in equine serum samples. The VIA is a potentially useful field test for rapid field-based Flavivirus antibody detection in equine serum samples.

Oral creatine supplementation on performance of Quarter Horses used in barrel racing.

The aim of this study was to evaluate the effects of oral creatine supplementation on the athletic performance of equines used for barrel racing. Ten healthy Quarter Horses, or Quarter Horse crossbred, weighing 429.7 ± 25.3 kg and with mean age of 3.8 ± 1.2 years, were used. Animals were evaluated in four different moments (M1, M2, M3, M4), and between M3 and M4, they were supplemented with 28 g of creatine/100 kg of body weight, orally, for 45 days. Although significant alterations for LDH activity, plasma glucose and packed cell volume were observed, it was possible to conclude that there was no improvement in the athletic performance for the animals used on the experiment, as there were no changes in time scores, heart rate and plasma lactate, variables considered as performance indicators, before and after supplementation.

[Development of PCR methods for detection of EAV infection]. / Entwicklung einer PCR zum Nachweis der EAV-Infektion.

The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

Le but de ce travail était de développer, comme alternative à l'isolation, une méthode de RT-PCR (en temps réel) pour le diagnostic rapide de l'EAV et pour la caractérisation des souches virales. Pour cela, on a adapté deux méthodes de RT-PCR conventionnelles et une de RT-PCR en temps réel, de manière à ce qu'un spectre aussi large que possible d'isolats soit démontrable. Les lignées de dilution avec la souche Bucyrus ont montré une sensibilité cent fois plus élevée avec la RT-PCR en temps réel et avec la RT-PCR heminested qu'avec la RT-PCR simple. L'efficacité des méthodes a pu être démontrée avec 11 surnageants de cultures cellulaires de divers isolats d'EAV et 7 échantillons de sperme positifs à l'EAV. L'analyse phylogénétique des séquences des échantillons par rapport à des séquences connues laisse penser qu'il existe plus de sous-groupes d'EAV que décrit jusqu'à ce jour.

First report of ivermectin resistance in cyathostomins (small strongyles) of horses in Argentina.

In Argentina, as in the rest of the world, cyathostomins are the most common nematodes parasitizing horses. Control is based almost exclusively on the administration of benzimidazoles, pyrimidines, and macrocyclic lactones. However, intensive use of these drugs is resulting in the development of anthelmintic resistance (AR). For example, AR to benzimidazoles is currently distributed throughout Argentina, while incipient AR to pyrimidines (pyrantel embonate) is appearing in areas where this drug is used. Macrocyclic lactones and especially ivermectin, are by far the most used drugs by the vast majority of equine premises in the country. Although ivermectin has been used since 1982, its efficacy against equine strongylid parasites has remained very high until the present. In this study we report for the first time, the presence of a cyathostomin population with resistance to ivermectin in adult horses belonging to an equine premise located in central Argentina. Fecal egg count reduction tests (FECRT) were performed following the most recent guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for the diagnosis of anthelmintic resistance (research protocol) and resistance was considered when the Upper 90% Credible Interval fell below the expected efficacy threshold of 99.9%. Calculations were carried out using two different online calculation interfaces suggested by WAAVP. For the 14-day post-treatment interval, ivermectin efficacy was 79.5% (90% Credible Interval: 68.1-88.8) and 79.3% (74.2-83.6.3%) with the two methods, respectively. At 19 days post treatment, fecal egg count reductions were 68.6% (50.5-83.1) and 68.4% (61.9-74.1), respectively. At both intervals, this cyathostomin population fullfilled the criteria for AR. These findings suggest dispersion of ivermectin resistant cyathostomins in Argentina. Given the widespread use of macrocyclic lactones, it is important that veterinarians and the equine industry promote a more selective and evidence-based use of these drugs and establish routine monitoring to determine anthelmintic field efficacy to detect treatment failures as early as possible and avoid potential health problems as well as further spread of resistant genes.