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Counting of Anoplocephala eggs in equine faecal samples
S46
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
Polymorphism at the 167 and 200 Allele of the b-Tubulin Gene in Adults and Larvae of Cyathostomin sp R. Canever 1, U. Ioshitani 1, and M.B. Molento 1, 2 1 Federal University of Parana, UFPR, Curitiba, PR, Brazil, 2 National Institute of Science and Technology, INCT, Brazil
Cyathostomins are the most prevalent parasites in horses causing colic, diarrhea and death. The control is difficult due to the large number of different species and routine treatment has favor the selection of resistant population, especially in Brazil [1]. One of the problems is the late diagnostic to monitor resistance and molecular tools must be developed to detect specific alleles (SNPs). Point mutations on the b-tubulin isotype 1 gene may induce mutations on the 167, 198, and 200 codons, which are associated with benzimidazole (BZM) resistance. The TTC/TAC polymorphism at the 167 position has being incriminated as the mechanism of resistance to the BZMs [2]. The objective of this work was to determine the frequency of the polymorphism at the codons 167 and 200 of the b-tubulin isotype 1 gene in BZM-resistant adults and larvae of Cyathostomin sp. Twenty adult worms were collected from one horse from the Sao Jose da Serra Stud, a BZM-resistant (0% to fenbendazole) farm. The larvae were collected from 10 pool samples from BZM-resistant horse farms after faecal egg count reduction test (FECRT). Specific primers [3] were used to amplify the codons 167 and 200 for b-tubulin isotype 1 gene using, 167: forward primers 5’-GCTAACT CACTCACTTGGAGGA-3’; reverse: 5’-CTTTGGTGAGGGAACA ACG-3’ and 200: forward primers 5’- TACAATGCTACCCTAT CCGTTCAT-3’; 5’-GAAGTGAAGACGAGGGAATGGA-3’. Samples were sequenced with ABI3130 and analyzed with Mega 5. It was possible to identify the DNA amplified bands (167 and 200 ¼ TTC) to all samples (Figure 1) with double peaks 167 (6/20) and 200 (2/20) which corresponded to heterozygous worms (TTC/TAC) on chromatograms. All larvae had double peaks for the 167 and 7 for the 200. We found only the homozygous (TTC/TTC) susceptible genotype in adults and also the heterozygous (TTC/TAC) to benzimidazole in larvae. As suggested by other authors we consider that the mutation at the codon 167 may be involved with the mechanism of resistance in the Cyathostomin sp. and that molecular markers using SNPs could be used to detect BZMresistant worms, most certainly for the codon 167 in adults and larvae.
[2] Hodgkinson JE, Clark HJ, Kaplan RM, Lake SL, Matthews JB. The role of polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int J Parasitol 2008; 38(10):1149-60. [3] Blackhall WJ, Kuzmina T, von Samson-Himmelstjerna G. b-tubulin genotypes in six species of cyathostomins from anthelmintic-naïve Przewalski and benzimidazole-resistant brood horses in Ukraine. Parasitol Res 2011;109(4):1199-203.
Counting of Anoplocephala eggs in equine faecal samples J. Cima, and G.C. Coles School of Veterinary Sciences, University of Bristol, Langford House, Bristol BS40 5DU, UK
Figure 1. DNA bands of Cyathostomin sp b-tubulin isotype 1. Column 1: molecular marker (100 bp), columns 2 to 8: adults, 9 to 16: larvae.
A simple reliable method to count cestode eggs in equine faecal samples would be of value to possibly indicate a) numbers of mature worms present in the horse, b) whether horses required treatment and c) whether products were fully effective. Previous attempts at counting eggs of Anoplocephala perfoliata have not been very successful [1,2]. With the introduction of a novel egg counting technique sensitive to 1 e.p.g., FLOTAC [3], the possibility of using this technique to count cestode eggs in horse faecal samples was examined. Adult A.perfoliata and faeces from horses with negative worm burdens or known numbers of worms were collected from a licensed horse abattoir. Mature segments of worms were homogenised in tap water and washed through a 150mm sieve onto a 50 mm sieve. A known number of eggs were re-suspended and added to 10 g of faeces from uninfected horses. Counts were performed using the FLOTAC technique and different flotation solutions. When eggs were added directly to the FLOTAC cell the recovery was 100%. Percentage recovery of eggs added when undertaking the full FLOTAC process using three flotation solutions without faeces were: saturated sucrose 46%, saturated sodium chloride 21% and saturated zinc sulphate 13%. To eliminate the possibility of the eggs sticking during the process, Tween 20 was added to the flotation solutions at different concentrations. Recovery rates for the addition of 5% Tween 20 with no faeces added were: sucrose 81%, sodium chloride 79% and zinc sulphate 74%. When testing different solutions and Tween 20 concentrations, it was found that 10% Tween 20 and 50% sucrose produced the best results. With the FLOTAC technique using this combination and 10g faeces, a recovery rate of 72% was produced. When eggs were added to egg free faeces, eggs were always found in the sample, but the results were still variable. The repeatability of naturally infected faecal samples from heavily worm burdened horses was low, varying from horse to horse, suggesting eggs were not evenly distributed in the faeces. Further experiments with FLOTAC indicated that egg loss occurred at an early stage when the faecal suspension was first filtered suggesting that the coarse nature of the faecal fibres trapped the eggs. This was confirmed by the high recovery of eggs (88%) when added to cattle faeces. Novel methods of estimating worm burdens in horses are therefore required.
References
References
[1] Molento MB, Antunes J, Bentes RN, Coles GC. Anthelmintic resistant nematodes in Brazilian horses. Vet Rec 2008;162(12):384-5.
[1] Proudman CJ, Edwards GB. Validation of a centrifugation/flotation technique for the diagnosis of equine cestodiasis. Vet Rec 1992;131:71.
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95 [2] Skotarek SL, Colwell DD, Goater CP. Evaluation of diagnostic techniques for Anoplocephala perfoliata in horses from Alberta, Canada. Vet Parasitol 2010;172:249. [3] Cringoli G, Rinaldi L, Maurelli MP, Utzinger J. FLOTAC: new multivalent technique for quantitative copromicroscopic diagnostic of parasites in animals and humans. Nature Protocols 2010; 5:503.
The prevalence of helminths with tissue associated stages in horses in the south-west of England G.C. Coles, D. Fews, H.V.F. Jones, B. Hayes, and K. Shaddick School of Veterinary Sciences, University of Bristol, Langford House, Bristol BS40 5DU, UK
The transmission of a horse strain of Echinococcus granulosus occurs in the UK between hounds fed raw horse meat and horses with infection levels at 60% [1]. To reestimate the prevalence, livers of horses slaughtered at a licenced abattoir were inspected. The opportunity was also taken to examine for the presence of Strongylus vulgaris, Strongylus edentatus and Onchocerca cervicalis. Horses at the abattoir were primarily private horses brought in by owners with some animals from common grazing on moorland. A total 921 horses were visually inspected for the presence of E granulosus, S.edentatus, and Setaria equina. Of these 264 were also examined for S.vulgaris and O.cervicalis. Aneurisms caused by S.vulgaris were dissected out and numbers of larvae counted. To detect O.cervicalis fresh 8mm skin biopsies were collected and thin slices incubated over night at 37C. A questionnaire was sent out to 17 hunts on the feeding of raw meat and treatment of dogs with praziquantel. Hydatid cysts were present in 17.4% of livers. Most livers had only 1-2 cysts between 1 and 10 cms in diameter. As most horses were old when slaughtered this represents the probable rate of infection up to 20 years previously. One decade later E.granulosus cysts are still being found but at a reduced rate of 5.2% (in 1205 horses). 18% of kennels stated that they regularly fed raw horse meat and 59% said they did this occasionally. Only 53% stated that they were using a praziquantel based product so the infection rate is not really surprising. 5.7% of the 264 horses had S.edentatus in the peritoneal cavity and firm white foci in the liver. Of the 18 horses with aneurisms caused by S.vulgaris 3 had no worms and the remainder a mean of 16.7 worms (range 1-52). Although large strongyles have been greatly reduced in prevalence due to the use of macrocylic lactones they are clearly still present in the horse population possibly representing failure to use anthelmintics correctly. Small numbers of larvae of O.cervicalis were only found in October and not during the rest of the winter, S.equina was not found in any horses. The low numbers of O.cervicalis and lack of S.equina probably represents the widespread use of macrocyclic lactones and the disruption of their transmission. References [1] Thompson RCA, Smyth JD. Equine hydatidosis:a review of the current status in Great Britain and the results of an epidemiological survey. Vet Parasitol 1975;1:107.
S47
Diarrhea in Thoroughbred foals is still a disease of concern in Argentina M. Ferrari 1, S. Miño 2, J. Raffo 1, J. Mondelli 1, M. Etchepareborda 1, A. Vissani 2, V. Parreño 2, and M. Barrandeguy 2 1 La Biznaga Stud Farm, Estación Islas, Buenos Aires, Argentina, 2 Instituto de Virología, CICVyA, INTA Castelar, Buenos Aires, Argentina
Diarrhea in foals is considered a major sanitary problem in thoroughbred breeding farms worldwide. The ethiology of diarrhea can be bacteria, viruses, parasites, or a range of non-infectious agents or conditions such as toxins, lactose intolerance, or “foal heat” diarrhea. During the 1990s, the morbidity rates of diarrhea in foals varied, reaching 100% in some Argentinean farms. Group A Rotavirus (RVA), G3 P[12] and G14 P [12], were associated to 39% of the outbreaks of foal diarrhea studied during a 17-year surveillance period. The introduction of a systematic Rotavirus vaccination program in pregnant mares significantly reduced the morbidity rates, reaching levels of only 15% in some thoroughbred farms. The aim of this work is to describe the occurrence of diarrhea in thoroughbred foals in a farm with high standard management in Argentina. The farm has a history of foal diarrhea outbreaks of variable intensity, which have occurred almost every year, and, as in other farms, the incidence and severity of diarrhea seem to have increased in the last years. The pregnant mares are vaccinated with a commercial inactivated rotavirus vaccine. The foals are born in a clean and disinfected stall, and under the care of a veterinarian, and get good-quality colostrums, in sufficient amounts, within the first 18 hours of life. Those mares with foal on foot are kept in a small individual paddock and under veterinarian supervision for 7 additional days. After this period the mares are grouped on pastured paddocks under daily veterinarian supervision. During the 2011 breeding season 40% (62/156) of the foals developed diarrhea. The first case took place in July 31st, and the last one in December 28th, while the maximum incidence occurred in October, when 24% (32/134) of the foals were affected. The age of affected foals ranged from 8 to 132 days, but in the majority of cases the age was between 10 and 20 days at the moment of the initiation of the disease. The duration of the disease was also variable, but the disease lasted, in average, 7 days. No deaths were registered. All of the affected foals received an immediate supportive symptomatic treatment. Twenty-one samples (diarrheic feces), obtained as soon as diarrhea was taken notice of (first day of disease), were analysed for RVA infection by commercial rapid test (FASTest ROTA Strip Megacor). RVA was detected in 38% (8/21) of the samples, and was characterized as G3 P[12]. In the present study, the number of diarrheic foals that did not shed the virus in feces on the first day of disease is remarkable, and emphasises the need for further research. Other infectious agents could be associated to diarrhea in young foals. Vaccine breakthrough should not be disregarded.
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
Polymorphism at the 167 and 200 Allele of the b-Tubulin Gene in Adults and Larvae of Cyathostomin sp R. Canever 1, U. Ioshitani 1, and M.B. Molento 1, 2 1 Federal University of Parana, UFPR, Curitiba, PR, Brazil, 2 National Institute of Science and Technology, INCT, Brazil
Cyathostomins are the most prevalent parasites in horses causing colic, diarrhea and death. The control is difficult due to the large number of different species and routine treatment has favor the selection of resistant population, especially in Brazil [1]. One of the problems is the late diagnostic to monitor resistance and molecular tools must be developed to detect specific alleles (SNPs). Point mutations on the b-tubulin isotype 1 gene may induce mutations on the 167, 198, and 200 codons, which are associated with benzimidazole (BZM) resistance. The TTC/TAC polymorphism at the 167 position has being incriminated as the mechanism of resistance to the BZMs [2]. The objective of this work was to determine the frequency of the polymorphism at the codons 167 and 200 of the b-tubulin isotype 1 gene in BZM-resistant adults and larvae of Cyathostomin sp. Twenty adult worms were collected from one horse from the Sao Jose da Serra Stud, a BZM-resistant (0% to fenbendazole) farm. The larvae were collected from 10 pool samples from BZM-resistant horse farms after faecal egg count reduction test (FECRT). Specific primers [3] were used to amplify the codons 167 and 200 for b-tubulin isotype 1 gene using, 167: forward primers 5’-GCTAACT CACTCACTTGGAGGA-3’; reverse: 5’-CTTTGGTGAGGGAACA ACG-3’ and 200: forward primers 5’- TACAATGCTACCCTAT CCGTTCAT-3’; 5’-GAAGTGAAGACGAGGGAATGGA-3’. Samples were sequenced with ABI3130 and analyzed with Mega 5. It was possible to identify the DNA amplified bands (167 and 200 ¼ TTC) to all samples (Figure 1) with double peaks 167 (6/20) and 200 (2/20) which corresponded to heterozygous worms (TTC/TAC) on chromatograms. All larvae had double peaks for the 167 and 7 for the 200. We found only the homozygous (TTC/TTC) susceptible genotype in adults and also the heterozygous (TTC/TAC) to benzimidazole in larvae. As suggested by other authors we consider that the mutation at the codon 167 may be involved with the mechanism of resistance in the Cyathostomin sp. and that molecular markers using SNPs could be used to detect BZMresistant worms, most certainly for the codon 167 in adults and larvae.
[2] Hodgkinson JE, Clark HJ, Kaplan RM, Lake SL, Matthews JB. The role of polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int J Parasitol 2008; 38(10):1149-60. [3] Blackhall WJ, Kuzmina T, von Samson-Himmelstjerna G. b-tubulin genotypes in six species of cyathostomins from anthelmintic-naïve Przewalski and benzimidazole-resistant brood horses in Ukraine. Parasitol Res 2011;109(4):1199-203.
Counting of Anoplocephala eggs in equine faecal samples J. Cima, and G.C. Coles School of Veterinary Sciences, University of Bristol, Langford House, Bristol BS40 5DU, UK
Figure 1. DNA bands of Cyathostomin sp b-tubulin isotype 1. Column 1: molecular marker (100 bp), columns 2 to 8: adults, 9 to 16: larvae.
A simple reliable method to count cestode eggs in equine faecal samples would be of value to possibly indicate a) numbers of mature worms present in the horse, b) whether horses required treatment and c) whether products were fully effective. Previous attempts at counting eggs of Anoplocephala perfoliata have not been very successful [1,2]. With the introduction of a novel egg counting technique sensitive to 1 e.p.g., FLOTAC [3], the possibility of using this technique to count cestode eggs in horse faecal samples was examined. Adult A.perfoliata and faeces from horses with negative worm burdens or known numbers of worms were collected from a licensed horse abattoir. Mature segments of worms were homogenised in tap water and washed through a 150mm sieve onto a 50 mm sieve. A known number of eggs were re-suspended and added to 10 g of faeces from uninfected horses. Counts were performed using the FLOTAC technique and different flotation solutions. When eggs were added directly to the FLOTAC cell the recovery was 100%. Percentage recovery of eggs added when undertaking the full FLOTAC process using three flotation solutions without faeces were: saturated sucrose 46%, saturated sodium chloride 21% and saturated zinc sulphate 13%. To eliminate the possibility of the eggs sticking during the process, Tween 20 was added to the flotation solutions at different concentrations. Recovery rates for the addition of 5% Tween 20 with no faeces added were: sucrose 81%, sodium chloride 79% and zinc sulphate 74%. When testing different solutions and Tween 20 concentrations, it was found that 10% Tween 20 and 50% sucrose produced the best results. With the FLOTAC technique using this combination and 10g faeces, a recovery rate of 72% was produced. When eggs were added to egg free faeces, eggs were always found in the sample, but the results were still variable. The repeatability of naturally infected faecal samples from heavily worm burdened horses was low, varying from horse to horse, suggesting eggs were not evenly distributed in the faeces. Further experiments with FLOTAC indicated that egg loss occurred at an early stage when the faecal suspension was first filtered suggesting that the coarse nature of the faecal fibres trapped the eggs. This was confirmed by the high recovery of eggs (88%) when added to cattle faeces. Novel methods of estimating worm burdens in horses are therefore required.
References
References
[1] Molento MB, Antunes J, Bentes RN, Coles GC. Anthelmintic resistant nematodes in Brazilian horses. Vet Rec 2008;162(12):384-5.
[1] Proudman CJ, Edwards GB. Validation of a centrifugation/flotation technique for the diagnosis of equine cestodiasis. Vet Rec 1992;131:71.
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95 [2] Skotarek SL, Colwell DD, Goater CP. Evaluation of diagnostic techniques for Anoplocephala perfoliata in horses from Alberta, Canada. Vet Parasitol 2010;172:249. [3] Cringoli G, Rinaldi L, Maurelli MP, Utzinger J. FLOTAC: new multivalent technique for quantitative copromicroscopic diagnostic of parasites in animals and humans. Nature Protocols 2010; 5:503.
The prevalence of helminths with tissue associated stages in horses in the south-west of England G.C. Coles, D. Fews, H.V.F. Jones, B. Hayes, and K. Shaddick School of Veterinary Sciences, University of Bristol, Langford House, Bristol BS40 5DU, UK
The transmission of a horse strain of Echinococcus granulosus occurs in the UK between hounds fed raw horse meat and horses with infection levels at 60% [1]. To reestimate the prevalence, livers of horses slaughtered at a licenced abattoir were inspected. The opportunity was also taken to examine for the presence of Strongylus vulgaris, Strongylus edentatus and Onchocerca cervicalis. Horses at the abattoir were primarily private horses brought in by owners with some animals from common grazing on moorland. A total 921 horses were visually inspected for the presence of E granulosus, S.edentatus, and Setaria equina. Of these 264 were also examined for S.vulgaris and O.cervicalis. Aneurisms caused by S.vulgaris were dissected out and numbers of larvae counted. To detect O.cervicalis fresh 8mm skin biopsies were collected and thin slices incubated over night at 37C. A questionnaire was sent out to 17 hunts on the feeding of raw meat and treatment of dogs with praziquantel. Hydatid cysts were present in 17.4% of livers. Most livers had only 1-2 cysts between 1 and 10 cms in diameter. As most horses were old when slaughtered this represents the probable rate of infection up to 20 years previously. One decade later E.granulosus cysts are still being found but at a reduced rate of 5.2% (in 1205 horses). 18% of kennels stated that they regularly fed raw horse meat and 59% said they did this occasionally. Only 53% stated that they were using a praziquantel based product so the infection rate is not really surprising. 5.7% of the 264 horses had S.edentatus in the peritoneal cavity and firm white foci in the liver. Of the 18 horses with aneurisms caused by S.vulgaris 3 had no worms and the remainder a mean of 16.7 worms (range 1-52). Although large strongyles have been greatly reduced in prevalence due to the use of macrocylic lactones they are clearly still present in the horse population possibly representing failure to use anthelmintics correctly. Small numbers of larvae of O.cervicalis were only found in October and not during the rest of the winter, S.equina was not found in any horses. The low numbers of O.cervicalis and lack of S.equina probably represents the widespread use of macrocyclic lactones and the disruption of their transmission. References [1] Thompson RCA, Smyth JD. Equine hydatidosis:a review of the current status in Great Britain and the results of an epidemiological survey. Vet Parasitol 1975;1:107.
S47
Diarrhea in Thoroughbred foals is still a disease of concern in Argentina M. Ferrari 1, S. Miño 2, J. Raffo 1, J. Mondelli 1, M. Etchepareborda 1, A. Vissani 2, V. Parreño 2, and M. Barrandeguy 2 1 La Biznaga Stud Farm, Estación Islas, Buenos Aires, Argentina, 2 Instituto de Virología, CICVyA, INTA Castelar, Buenos Aires, Argentina
Diarrhea in foals is considered a major sanitary problem in thoroughbred breeding farms worldwide. The ethiology of diarrhea can be bacteria, viruses, parasites, or a range of non-infectious agents or conditions such as toxins, lactose intolerance, or “foal heat” diarrhea. During the 1990s, the morbidity rates of diarrhea in foals varied, reaching 100% in some Argentinean farms. Group A Rotavirus (RVA), G3 P[12] and G14 P [12], were associated to 39% of the outbreaks of foal diarrhea studied during a 17-year surveillance period. The introduction of a systematic Rotavirus vaccination program in pregnant mares significantly reduced the morbidity rates, reaching levels of only 15% in some thoroughbred farms. The aim of this work is to describe the occurrence of diarrhea in thoroughbred foals in a farm with high standard management in Argentina. The farm has a history of foal diarrhea outbreaks of variable intensity, which have occurred almost every year, and, as in other farms, the incidence and severity of diarrhea seem to have increased in the last years. The pregnant mares are vaccinated with a commercial inactivated rotavirus vaccine. The foals are born in a clean and disinfected stall, and under the care of a veterinarian, and get good-quality colostrums, in sufficient amounts, within the first 18 hours of life. Those mares with foal on foot are kept in a small individual paddock and under veterinarian supervision for 7 additional days. After this period the mares are grouped on pastured paddocks under daily veterinarian supervision. During the 2011 breeding season 40% (62/156) of the foals developed diarrhea. The first case took place in July 31st, and the last one in December 28th, while the maximum incidence occurred in October, when 24% (32/134) of the foals were affected. The age of affected foals ranged from 8 to 132 days, but in the majority of cases the age was between 10 and 20 days at the moment of the initiation of the disease. The duration of the disease was also variable, but the disease lasted, in average, 7 days. No deaths were registered. All of the affected foals received an immediate supportive symptomatic treatment. Twenty-one samples (diarrheic feces), obtained as soon as diarrhea was taken notice of (first day of disease), were analysed for RVA infection by commercial rapid test (FASTest ROTA Strip Megacor). RVA was detected in 38% (8/21) of the samples, and was characterized as G3 P[12]. In the present study, the number of diarrheic foals that did not shed the virus in feces on the first day of disease is remarkable, and emphasises the need for further research. Other infectious agents could be associated to diarrhea in young foals. Vaccine breakthrough should not be disregarded.