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Equine neonatal salmonellosis 114 cases: clinical features, prognostic indicators, and racing performance of surviving Thoroughbreds (2004-2010)
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9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
for antemortem diagnosis of the pathogenic migrating larval stages of S. vulgaris. To identify potential diagnostic proteins, a cDNA library was constructed from migrating stages of S. vulgaris. The excretory-secretory antigen (ESA) fraction from S. vulgaris adult specimens was dialysed against PBS and used to immunise a rat. This hyperimmune serum was used to immunoscreen the cDNA library to identify immunogenic S. vulgaris proteins. Immunoreactive clones were rescreened, PCRs were performed and the PCR product was sequenced. An immunoreactive cDNA clone was subcloned into E.coli and the plasmid sequenced. The open-reading frame encoding the predicted mature part of the protein was amplified using high fidelity polymerase, and the PCR product was cloned into the pET22b expression vector. The resulting plasmid was transformed into BL21 expression cells, and expression of the recombinant protein was induced using isopropyl b-D-1-thiogalactopyranoside. The His-tagged recombinant protein was purified by affinity chromatography on immobilized cobalt. An indirect enzyme-linked immunosorbent assay (ELISA) was optimized by checkerboard titration using the recombinant protein as antigen. Antigen-specific total IgG and IgG(T) antibodies were evaluated. Intra- and interassay variability was evaluated as well as the diagnostic sensitivity and specificity of the ELISA. Antigen specific IgG(T) antibodies gave a better distinction between positive and negative horses than IgG. Using mean optic density (OD) values, the intraassay coefficient of variation (CV) values were 7 % and the interassay CV based on the mean of the OD values were 27 %. In comparison, when using the normalized percentage of positive (PP) control values, the interassay CV was 10 %. Using 100 serum samples from horses with infection status verified on necropsy, the diagnostic sensitivity and specificity were found to be 0.61 and 0.70, respectively, while using a cut-off value of 26 PP. The ELISA shows promise for diagnosing prepatent S. vulgaris infection with migrating larvae. The assay could be useful in clinical settings and as a research tool for epidemiological studies.
A case control study of foal diarrhoea: rotavirus detection by reverse transcription quantitative polymerase chain reaction in foals K.E. Bailey, S.J. Symes, C.A. Hartley, G.F. Browning, J.M. Devlin, and J.R. Gilkerson Centre for Equine Infectious Diseases, Faculty of Veterinary Science, The University of Melbourne, Victoria, 3010, Australia
Foal diarrhoea is both labour intensive and costly to manage, and although diarrhoea is a common disease in foals, the aetiological agent is often not determined. The potential infectious causes of foal diarrhoea are numerous, however few contemporary studies have investigated the clinical significance of those pathogens present in foals with and without diarrhoea. The aim of this study was to conduct a prospective age-matched case control investigation of a range of pathogens present in foals, while developing a sensitive molecular diagnostic screening panel. The results presented here describe a reverse transcription quantitative
polymerase chain reaction (RT-qPCR) to detect rotavirus in foal faeces. Faecal samples were collected on five Thoroughbred breeding farms in New South Wales, Australia, from foals with diarrhoea and from an age-matched control. A case was defined as any foal with diarrhoea on the day of sampling. A control was defined as a foal without diarrhoea on the day of sampling, born within 7 days of the case foal. Faecal samples were also collected from foals with gastroenteritis at an equine intensive care hospital, however, no control samples were collected for this population. To enable investigation of a range of bacterial and viral pathogens, faecal samples were aliquoted into RNAlater, glycerol and cooked meat broth. A Syto 9 based rotavirus RT-qPCR assay targeting an 87bp region of the rotavirus non-structural protein 3 1 was performed on nucleic acids extracted from faecal samples in RNAlater using the MoBio PowerSoil DNA Isolation Kit. Of the 1021 foals born on the farms during this study, 234 foals were sampled and included in the agematched case control series. Rotavirus RT-qPCR products with an appropriate dissociation curve were amplified from 25% of cases and 7% of controls. In this study, the odds of detecting rotavirus in a case were 4.49 times that of detecting rotavirus in a control (OR ¼ 4.49, 95%CI: 2.059.82). Of the 25 cases of foal diarrhoea from the equine intensive care hospital, 3 (12%) were rotavirus RT-qPCR positive. The results of this study indicate that rotavirus is an important pathogen in this foal population, despite use of an inactivated maternal vaccine on four of the five farms. The detection of rotavirus in control foals needs to be investigated further to establish the role of these foals in disease transmission. Importantly, in 75% of the cases in this study rotavirus was not detected and investigation of other infectious causes of foal diarrhoea in this sample set is ongoing.
Reference [1] Pang XLL, Lee B, Boroumand N, et al. Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea. J. Med. Virol 2004;72:496-501.
Equine neonatal salmonellosis 114 cases: clinical features, prognostic indicators, and racing performance of surviving Thoroughbreds (2004-2010) B.S. Barr VMD, DACVIM 1, C.M. Childers DVM 1, and J. Cheetham VetMB, PhD, DACVS 2 1 Rood and Riddle Equine Hospital, Lexington, Kentucky, 2 Cornell University, Ithaca, New York
Salmonellosis is one of the most common causes of enteritis in the foal. The purpose of this study was to describe the clinical and clinicopathologic characteristics, treatments, and short-term outcome associated with equine neonatal salmonellosis, to assess risk factors for survival, and to evaluate the racing performance of Thoroughbred survivors. Medical records of Rood and Riddle Equine Hospital from 2004-2010 were searched to identify cases of equine neonatal salmonellosis, defined as a foal less than 14 days
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
of age with a positive fecal culture within 72 hours of hospitalization. Information was obtained from the medical record including clinical signs, clinicopathologic results, diagnostics, treatment and outcome. For surviving Thoroughbred foals, information regarding racing performance was obtained from each foal and all maternal siblings from an online database. Differences between the outcome groups (survivors or nonsurvivors) were determined using one-way ANOVA with Tukey’s post hoc tests where appropriate. For categorical variables Chi-squared tests were used. Statistical analysis was performed using and JMP (SAS Institute, Cary, North Carolina, USA). Significance was set at p < 0.05 throughout. There were a total of 114 foals that met the inclusion criteria. The two most common clinical signs at admission were diarrhea 56% (64/114) and fever 25% (29/114). Plasma was administered to 31% (35/114) of foals. The most common complications during hospitalization included the presence of at least one septic joint, 18% (20/114) and pneumonia, 6% (7/114). Fecal cultures were collected throughout hospitalization on 106/114 accompanying dams; 39% (44/106) cultured positive for Salmonella. Survivors had a significantly higher serum IgG at admission (mean 1222.6 mg/dl; SD 595.9) compared to nonsurvivors (mean 622.89 mg/dl; SD 537.52) (p¼0.017). The serum glucose was significantly different between the two groups. Foals that survived had a significantly higher glucose (mean 148.91 mg/dl; SD 38.24) compared to nonsurvivors (mean 110.31 mg/dl; SD 47.62) (p¼0.001). There were 79 out of 114 foals (69%) that survived to discharge from the hospital. Of the 35 foals that did not survive, 60% (21/35) were euthanized. Risk factors for nonsurvival included abnormal mucous membrane color, fever and a low WBC during hospitalization. The only prognostic indicator for survival identified was the presence of a capillary refill time of less than 2 seconds at admission. Of the 79 foals that were discharged from the hospital, 63 were Thoroughbreds. Of these registered horses, 74% (26/35) of hospitalized cases compared to 77% (135/175) of their maternal siblings started at least one race. There was no significant difference in regards to number of starts per year and total earnings per year between the two groups. In conclusion, foals with salmonellosis that are intensively managed have a fair prognosis for survival (69%). Foals that survive long enough to be registered with the Jockey Club raced at a level equivalent to their maternal siblings.
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classes of drugs [1, 2]. New alternative control strategies are needed and biological control with the fungi Duddingtonia flagrans is an appropriate option. The fungi pass through the intestinal tract of the horse acting on the parasite free-living stages. The chlamydospores need to grow in particular environmental conditions for the hyphae to be able to predate the eggs and larvae [3]. The objective of this study was to evaluate the activity of D. flagrans against nematode parasites in naturally infected horses. The study was performed at the Sao Jose da Serra Stud in Pinhais, Brazil. Twenty Thoroughbred animals were divided based on their faecal egg count (EPG) values in 3 treatment groups and one control (n¼5). Chlamydospores of a Brazilian isolate of the D. flagrans were orally administered together with grindedcorn at G1: 250.000, G2: 500.000, and G3: 1.000.000 per kilogram of live weight. The animals were treated daily during 5 days. Faecal samples were collected during the 5 days of treatment and 5 days after treatment for EPG counts, which was performed with a 1:25 concentration, individually, as well as the coproculture. Recovered larvae from coproculture were counted on triplicates from each sample, calculating the average and standard deviation. Statistical analysis was performed using Tukey test with GraphPad Prims 5 at 5% significance (P < 0.05). The average EPG counts from all groups had a large (53%) daily variation during the experiment and did not show any statistical difference between groups (data not shown). Cyathostomum sp. was the predominant (above 94%) genus present at the faecal cultures in all groups. The fungi started to have an effect on the L3 stages 4 days after initiating the treatment reducing more than 97% (P < 0.01) compared to the Control group. The predatory effect of the hyphae persisted for 4 days after the treatment was interrupted significantly (P < 0.01) reducing larvae number (Figure 1). Duddingtonia flagrans showed a significant activity against infecting free-living larvae of Cyathostomins of horses.
Biological control using the fungi Duddingtonia flagrans against cyathostomins of horses A. Buzatti 1, C.P. Santos 2, U.Y. Yoshitani 1, L.K. Sprenger 1, F. Kloster 1, J.D. Antunes 3, and M.B. Molento 1, 4 1 Federal University of Parana, UFPR, Curitiba, PR, Brazil, 2 North Fluminense State University, 3 DMV, Haras Sao Jose da Serra, Brazil, 4 National Institute of Science and Technology, INCT, Belo Horizonte, MG, Brazil
Gastrointestinal parasitism in horses can affect all categories with evident clinical signs. Control is made with the regular use of chemical products, which has cause resistance to all
Figure 1. Average and standard error count of infecting larvae per day after Duddingtonia flagrans treatment in Cyathostomins of horses.
References [1] Molento MB, Antunes J, Bentes RN, Coles GC. Anthelmintic resistant nematodes in Brazilian horses. Vet Rec 2008;162(12):384-5. [2] Kaplan RM. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol 2004;20:477-81. [3] Grønvold J, et al. J. Biological control of nematode parasites in cattle with nematode-trapping fungi: a survey of Danish studies. Vet Parasitol 1993;48:311-25.
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
for antemortem diagnosis of the pathogenic migrating larval stages of S. vulgaris. To identify potential diagnostic proteins, a cDNA library was constructed from migrating stages of S. vulgaris. The excretory-secretory antigen (ESA) fraction from S. vulgaris adult specimens was dialysed against PBS and used to immunise a rat. This hyperimmune serum was used to immunoscreen the cDNA library to identify immunogenic S. vulgaris proteins. Immunoreactive clones were rescreened, PCRs were performed and the PCR product was sequenced. An immunoreactive cDNA clone was subcloned into E.coli and the plasmid sequenced. The open-reading frame encoding the predicted mature part of the protein was amplified using high fidelity polymerase, and the PCR product was cloned into the pET22b expression vector. The resulting plasmid was transformed into BL21 expression cells, and expression of the recombinant protein was induced using isopropyl b-D-1-thiogalactopyranoside. The His-tagged recombinant protein was purified by affinity chromatography on immobilized cobalt. An indirect enzyme-linked immunosorbent assay (ELISA) was optimized by checkerboard titration using the recombinant protein as antigen. Antigen-specific total IgG and IgG(T) antibodies were evaluated. Intra- and interassay variability was evaluated as well as the diagnostic sensitivity and specificity of the ELISA. Antigen specific IgG(T) antibodies gave a better distinction between positive and negative horses than IgG. Using mean optic density (OD) values, the intraassay coefficient of variation (CV) values were 7 % and the interassay CV based on the mean of the OD values were 27 %. In comparison, when using the normalized percentage of positive (PP) control values, the interassay CV was 10 %. Using 100 serum samples from horses with infection status verified on necropsy, the diagnostic sensitivity and specificity were found to be 0.61 and 0.70, respectively, while using a cut-off value of 26 PP. The ELISA shows promise for diagnosing prepatent S. vulgaris infection with migrating larvae. The assay could be useful in clinical settings and as a research tool for epidemiological studies.
A case control study of foal diarrhoea: rotavirus detection by reverse transcription quantitative polymerase chain reaction in foals K.E. Bailey, S.J. Symes, C.A. Hartley, G.F. Browning, J.M. Devlin, and J.R. Gilkerson Centre for Equine Infectious Diseases, Faculty of Veterinary Science, The University of Melbourne, Victoria, 3010, Australia
Foal diarrhoea is both labour intensive and costly to manage, and although diarrhoea is a common disease in foals, the aetiological agent is often not determined. The potential infectious causes of foal diarrhoea are numerous, however few contemporary studies have investigated the clinical significance of those pathogens present in foals with and without diarrhoea. The aim of this study was to conduct a prospective age-matched case control investigation of a range of pathogens present in foals, while developing a sensitive molecular diagnostic screening panel. The results presented here describe a reverse transcription quantitative
polymerase chain reaction (RT-qPCR) to detect rotavirus in foal faeces. Faecal samples were collected on five Thoroughbred breeding farms in New South Wales, Australia, from foals with diarrhoea and from an age-matched control. A case was defined as any foal with diarrhoea on the day of sampling. A control was defined as a foal without diarrhoea on the day of sampling, born within 7 days of the case foal. Faecal samples were also collected from foals with gastroenteritis at an equine intensive care hospital, however, no control samples were collected for this population. To enable investigation of a range of bacterial and viral pathogens, faecal samples were aliquoted into RNAlater, glycerol and cooked meat broth. A Syto 9 based rotavirus RT-qPCR assay targeting an 87bp region of the rotavirus non-structural protein 3 1 was performed on nucleic acids extracted from faecal samples in RNAlater using the MoBio PowerSoil DNA Isolation Kit. Of the 1021 foals born on the farms during this study, 234 foals were sampled and included in the agematched case control series. Rotavirus RT-qPCR products with an appropriate dissociation curve were amplified from 25% of cases and 7% of controls. In this study, the odds of detecting rotavirus in a case were 4.49 times that of detecting rotavirus in a control (OR ¼ 4.49, 95%CI: 2.059.82). Of the 25 cases of foal diarrhoea from the equine intensive care hospital, 3 (12%) were rotavirus RT-qPCR positive. The results of this study indicate that rotavirus is an important pathogen in this foal population, despite use of an inactivated maternal vaccine on four of the five farms. The detection of rotavirus in control foals needs to be investigated further to establish the role of these foals in disease transmission. Importantly, in 75% of the cases in this study rotavirus was not detected and investigation of other infectious causes of foal diarrhoea in this sample set is ongoing.
Reference [1] Pang XLL, Lee B, Boroumand N, et al. Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea. J. Med. Virol 2004;72:496-501.
Equine neonatal salmonellosis 114 cases: clinical features, prognostic indicators, and racing performance of surviving Thoroughbreds (2004-2010) B.S. Barr VMD, DACVIM 1, C.M. Childers DVM 1, and J. Cheetham VetMB, PhD, DACVS 2 1 Rood and Riddle Equine Hospital, Lexington, Kentucky, 2 Cornell University, Ithaca, New York
Salmonellosis is one of the most common causes of enteritis in the foal. The purpose of this study was to describe the clinical and clinicopathologic characteristics, treatments, and short-term outcome associated with equine neonatal salmonellosis, to assess risk factors for survival, and to evaluate the racing performance of Thoroughbred survivors. Medical records of Rood and Riddle Equine Hospital from 2004-2010 were searched to identify cases of equine neonatal salmonellosis, defined as a foal less than 14 days
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
of age with a positive fecal culture within 72 hours of hospitalization. Information was obtained from the medical record including clinical signs, clinicopathologic results, diagnostics, treatment and outcome. For surviving Thoroughbred foals, information regarding racing performance was obtained from each foal and all maternal siblings from an online database. Differences between the outcome groups (survivors or nonsurvivors) were determined using one-way ANOVA with Tukey’s post hoc tests where appropriate. For categorical variables Chi-squared tests were used. Statistical analysis was performed using and JMP (SAS Institute, Cary, North Carolina, USA). Significance was set at p < 0.05 throughout. There were a total of 114 foals that met the inclusion criteria. The two most common clinical signs at admission were diarrhea 56% (64/114) and fever 25% (29/114). Plasma was administered to 31% (35/114) of foals. The most common complications during hospitalization included the presence of at least one septic joint, 18% (20/114) and pneumonia, 6% (7/114). Fecal cultures were collected throughout hospitalization on 106/114 accompanying dams; 39% (44/106) cultured positive for Salmonella. Survivors had a significantly higher serum IgG at admission (mean 1222.6 mg/dl; SD 595.9) compared to nonsurvivors (mean 622.89 mg/dl; SD 537.52) (p¼0.017). The serum glucose was significantly different between the two groups. Foals that survived had a significantly higher glucose (mean 148.91 mg/dl; SD 38.24) compared to nonsurvivors (mean 110.31 mg/dl; SD 47.62) (p¼0.001). There were 79 out of 114 foals (69%) that survived to discharge from the hospital. Of the 35 foals that did not survive, 60% (21/35) were euthanized. Risk factors for nonsurvival included abnormal mucous membrane color, fever and a low WBC during hospitalization. The only prognostic indicator for survival identified was the presence of a capillary refill time of less than 2 seconds at admission. Of the 79 foals that were discharged from the hospital, 63 were Thoroughbreds. Of these registered horses, 74% (26/35) of hospitalized cases compared to 77% (135/175) of their maternal siblings started at least one race. There was no significant difference in regards to number of starts per year and total earnings per year between the two groups. In conclusion, foals with salmonellosis that are intensively managed have a fair prognosis for survival (69%). Foals that survive long enough to be registered with the Jockey Club raced at a level equivalent to their maternal siblings.
S31
classes of drugs [1, 2]. New alternative control strategies are needed and biological control with the fungi Duddingtonia flagrans is an appropriate option. The fungi pass through the intestinal tract of the horse acting on the parasite free-living stages. The chlamydospores need to grow in particular environmental conditions for the hyphae to be able to predate the eggs and larvae [3]. The objective of this study was to evaluate the activity of D. flagrans against nematode parasites in naturally infected horses. The study was performed at the Sao Jose da Serra Stud in Pinhais, Brazil. Twenty Thoroughbred animals were divided based on their faecal egg count (EPG) values in 3 treatment groups and one control (n¼5). Chlamydospores of a Brazilian isolate of the D. flagrans were orally administered together with grindedcorn at G1: 250.000, G2: 500.000, and G3: 1.000.000 per kilogram of live weight. The animals were treated daily during 5 days. Faecal samples were collected during the 5 days of treatment and 5 days after treatment for EPG counts, which was performed with a 1:25 concentration, individually, as well as the coproculture. Recovered larvae from coproculture were counted on triplicates from each sample, calculating the average and standard deviation. Statistical analysis was performed using Tukey test with GraphPad Prims 5 at 5% significance (P < 0.05). The average EPG counts from all groups had a large (53%) daily variation during the experiment and did not show any statistical difference between groups (data not shown). Cyathostomum sp. was the predominant (above 94%) genus present at the faecal cultures in all groups. The fungi started to have an effect on the L3 stages 4 days after initiating the treatment reducing more than 97% (P < 0.01) compared to the Control group. The predatory effect of the hyphae persisted for 4 days after the treatment was interrupted significantly (P < 0.01) reducing larvae number (Figure 1). Duddingtonia flagrans showed a significant activity against infecting free-living larvae of Cyathostomins of horses.
Biological control using the fungi Duddingtonia flagrans against cyathostomins of horses A. Buzatti 1, C.P. Santos 2, U.Y. Yoshitani 1, L.K. Sprenger 1, F. Kloster 1, J.D. Antunes 3, and M.B. Molento 1, 4 1 Federal University of Parana, UFPR, Curitiba, PR, Brazil, 2 North Fluminense State University, 3 DMV, Haras Sao Jose da Serra, Brazil, 4 National Institute of Science and Technology, INCT, Belo Horizonte, MG, Brazil
Gastrointestinal parasitism in horses can affect all categories with evident clinical signs. Control is made with the regular use of chemical products, which has cause resistance to all
Figure 1. Average and standard error count of infecting larvae per day after Duddingtonia flagrans treatment in Cyathostomins of horses.
References [1] Molento MB, Antunes J, Bentes RN, Coles GC. Anthelmintic resistant nematodes in Brazilian horses. Vet Rec 2008;162(12):384-5. [2] Kaplan RM. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol 2004;20:477-81. [3] Grønvold J, et al. J. Biological control of nematode parasites in cattle with nematode-trapping fungi: a survey of Danish studies. Vet Parasitol 1993;48:311-25.