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Recent progress of the diagnosis for equine piroplasmosis
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9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
false positive results and the equipment requires a very controlled setting. In the food industry nucleic acid hybridization is used to rapidly and accurately identify contamination with Salmonella spp. This test targets the pathogen’s ribosomal RNA. The purpose of this study was to use this technology to detect Salmonella spp in fecal samples and environmental samples from an equine hospital and compare the results to bacterial culture. Routine fecal samples and environmental samples collected from April 2010- April 2011 at Rood and Riddle Equine Hospital were used for the study. The fecal samples were enriched in selenite broth and incubated for 24 hours, then plated on Hekton agar and incubated for 24 hours. The environmental samples were incubated in peptone broth for 24 hours, then tetrathionate broth for 24 hours. Afterwards the samples were plated and incubated on Hekton agar for 24 hours. After appropriate incubation any black colonies on the Hekton agar plates were gathered and transferred to the GeneQuenceÒ D2 automated system for nucleic acid hybridization and to BBL crystal identification system for the final phase of microbiological culture. The GeneQuenceÒ results were available within 2 hours whereas the bacterial culture results were available within 24 hours. A total of 1494 samples were analyzed. The results were as follows: 1274 samples were GeneQuenceÒ negative and culture negative, 210 samples GeneQuenceÒ positive and culture positive, four samples GeneQuenceÒ negative and culture positive, and six samples Gene QuenceÒ positive and culture negative. The overall sensitivity was 98.1% and the specificity was 99.5%. The positive predictive value was 97.2%, whereas the negative predictive value was 99.6%. GeneQuenceÒ results were available 1 day sooner and were almost as accurate as the bacterial culture. An advantage of GeneQuenceÒ over PCR testing is that there are fewer false positive results. A disadvantage is that bacterial culture is still required to identify an antimicrobial sensitivity. A rapid and reliable test for identification of Salmonella spp would be beneficial for equine veterinarians. Future projects include decreasing enrichment time thus making the results available sooner.
European Union Reference Laboratory for equine diseases: towards improved and harmonized diagnosis of West Nile disease C. Beck, S. Lowenski, S. Zientara, and S. Lecollinet ANSES, EU-RL for equine diseases, UMR1161 Virologie INRA, ANSES, ENVA, 23 avenue du Général de Gaulle, 94703 Maisons-Alfort, FRANCE
In July 2008, the European Commission officially designated the French Agency for food, environmental and occupational health safety (ANSES) as the European Union Reference Laboratory (EU-RL) for equine diseases (apart from African horse sickness). West Nile disease, exotic Equine Encephalitis, Vesicular stomatitis, glanders and melioidosis are studied at ANSES Laboratory for animal health located in Maisons-Alfort (near Paris), while the Laboratory for equine diseases located in Normandy is dedicated to Contagious Equine Metritis, Dourine, Equine
Viral Arteritis, Equine Herpes viruses and Equine Infectious Anaemia. The EU-RL on equine diseases aims at providing technical and scientific support to the European National Reference Laboratories and the European Commission, as well as improving, harmonizing and developing new diagnostic tools for the diagnosis of equine diseases (through workshops, trainings, proficiency tests). A proficiency test (PT) on West Nile (WN) serological and molecular diagnosis was organized in 2010, in order to assess the performances of different WN diagnosis tools and to improve the results homogeneity among European countries. 22 and 25 laboratories participated to the serology and RT-PCR proficiency test, respectively. This PT indicated that IgG or competition ELISA tests were well established, on the contrary to IgM ELISA and virus neutralization tests. Moreover, there is a need to develop more specific IgG ELISA. WN real-time RT-PCR diagnosis is functional and efficient in most EC countries, however some participants (8) failed to detect lineage 1b and 2 strains. The detection capacity of lineage 1b and 2 strains needs to be improved, in particular in Europe where a pathogenic lineage 2 virus has been identified since 2004. In conclusion, this European PT highlighted the need for improved, adapted and harmonized diagnosis on WN in Europe.
Recent progress of the diagnosis for equine piroplasmosis I. Igarashi, N. Yokoyama, and X. Xuenan National Research Center for Protozoan Diseases, OIE Reference Laboratories on equine piroplasmosis, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
Equine piroplasmosis, caused by intra-erythrocytic protozoa Theileria equi and Babesia caballi, is a tick-borne disease of equids. The disease is characterized by fever, anemia, icterus, and hemoglobinuria. Horses that recover from the initial infection often be carriers of parasites. In such case, it is very difficult to detect the parasites in microscopic examination, and the horses become potential disseminators of the parasites. Since the disease is distributed worldwide including Asia, Europe, Africa, South America, the disease has posed a threat to the horses in the piroplasmosis free area and severe restriction has imposed on international movement of equids to avoid the spread of the infection from an endemic area. Current diagnosis of equine piroplasmosis relies on microscopic examination, serological assays and other molecular tools. OIE is recommending the several diagnostic tests including microscopic detection, complement fixation test (CFT), the indirect fluorescent antibody test (IFAT) and competitive enzymelinked immunosorbent assay (cELISA). However, there is no gold standard reference test for equine piroplasmosis. Each country utilizes one or more tests from OIE recommending tests and each test has disadvantages. Microscopic detection from blood smears has been used for the most standard diagnosis of equine piroplasmosis; however, the technique is relatively laborious and it is difficult to detect the parasites in the case of low parasitemia. Several serological tests have
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
been used to detect specific antibodies against the infections, but these assays are also restricted due to their antibody-detected limitation and/or potential cross-reactivity to other pathogens. For example, reading of IFAT results is very difficult to read results and not objective at borderline between positive and negative titers. Results of IFAT may also change not only with different origin of antigens, but also with different lots of FITC-conjugated anti-horse IgG antibody. Recently, new diagnostics such as immunochromatographic test (ICT) and loop-mediated isothermal amplification (LAMP) assay have been developed for the detection of B. caballi and/or T. equi infections. ICT has similar sensitivity to ELISA and is a rapid and simple method to detect antibodies [1]. LAMP assay can rapidly amplify DNA samples within one hour under an isothermal condition (at 60 to 65 C). LAMP has higher specificity and sensitivity than those of PCR [2]. Therefore, both ICT and LAMP are practical pen-side diagnostic methods for equine piroplasmosis. The objectives of this presentation are to review diagnostic methods and problems of the current diagnostic methods, to introduce newly developed diagnostics, and to discuss how these methods can contribute the strengthening of quarantine for equine piroplasmosis. References [1] Huang X, et al. Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses. Clin Vaccine Immunol 2006;13:553-5. [2] Alhassan A, et al. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res. 2007;100:1165-8.
Development of one-step TaqManÒ real-time reverse transcription-PCR and conventional reverse transcription PCR assays for the detection of equine rhinitis A and B viruses Zhengchun Lu, Peter J. Timoney, Jena White, and Udeni B.R. Balasuriya Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546
Equine rhinitis virus A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. There is evidence that these two viral infections are prevalent in countries in which sero-surveillance studies have been undertaken. Currently there is a lack of rapid and reliable diagnostic methods for virus detection and antibody determination. The sensitivity of virus isolation varies between laboratories and is confounded by difficulties in isolating virus from the urine of carrier horses. The objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) capable of detecting and distinguishing ERAV from ERBV without the inherent problems associated with the current laboratory diagnosis of these infections. Three rRT-PCR assays targeting the 5’UTR of ERAV and ERBV were developed. One assay was specific for ERAV, whereas the remaining two assays were
S59
specific for ERBV. In addition, six cRT-PCR assays targeting the 5’-UTR and 3D polymerase regions of ERAV and ERBV were also developed. RNA extracted from prototype strains of ERAV and ERBV as well as 21 archived tissue culture fluid samples which were previously determined to be positive by virus isolation for ERAV or ERBV with mono-specific rabbit antisera were used to evaluate the sensitivity and specificity of these assays. Using serial decimal dilutions (101 to 1010) of ERAV and ERBV prototype strains, the detection limits of the rRT-PCR and cRT-PCR assays were evaluated and compared. The rRT-PCR and cRT-PCR assays targeting ERAV detected all ERAV isolates (n¼11) and did not cross-react with any ERBV isolates (n¼10). Similarly, the rRT-PCR and cRT-PCR assays targeting ERBV only detected ERBV isolates and did not cross-react with ERAV isolates. None of the rRT-PCR or cRT-PCR assays crossreacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit among all of these assays was 1 plaqueforming unit per ml (pfu/ml). The newly developed rRTPCR and cRT-PCR assays appear to provide improved diagnostic tools for the detection and differentiation of ERAV and ERBV. While the results of this study are very promising, they will need to be verified by testing a greater number of clinical specimens before each assay is fully validated for the detection of ERAV and/or ERBV.
Development of a rapid isothermal assay to detect Taylorella equigenitalis S.E. North, P. Das, P.R. Wakeley, and J. Sawyer Technology Transfer Unit, Specialist Scientific Support Department, Animal Health Veterinary Laboratories Agency – Weybridge, United Kingdom
Contagious Equine Metritis (CEM) is an infection of the equine genital tract, caused by the bacterium Taylorella equigenitalis. Identifying T. equigenitalis from equine genital swabs, and differentiating it from T. asinigenitalis, using culture is slow and requires the presence of live bacteria. We have developed a rapid isothermal assay for the detection of T. equigenitalis using Recombinase Polymerase Amplification (RPA). RPA reactions occur optimally at 37 C and do not require thermocycling equipment. The reactions can be monitored in real time, based on fluorescence readings, or the amplification products can be detected on a lateral flow device (LFD). Equine genital swabs were used to inoculate solid culture media before a simple boiling method was used to extract DNA from the swabs. This enabled comparisons between culture and the RPA assay. The assay was tested, in real time, against 60 equine genital swabs (30 positive and 30 negative, as determined by culture and real time PCR). The analytical specificity of the assay was determined by testing against a panel of 38 bacterial species, including equine commensals. The analytical sensitivity of the assay was calculated using a dilution series of target DNA. A second probe was introduced into the assay, specific for T. asinigenitalis, which enabled detection and differentiation of T. equigenitalis and T. asinigenitalis. The T. equigenitalis assay was adapted, by
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
false positive results and the equipment requires a very controlled setting. In the food industry nucleic acid hybridization is used to rapidly and accurately identify contamination with Salmonella spp. This test targets the pathogen’s ribosomal RNA. The purpose of this study was to use this technology to detect Salmonella spp in fecal samples and environmental samples from an equine hospital and compare the results to bacterial culture. Routine fecal samples and environmental samples collected from April 2010- April 2011 at Rood and Riddle Equine Hospital were used for the study. The fecal samples were enriched in selenite broth and incubated for 24 hours, then plated on Hekton agar and incubated for 24 hours. The environmental samples were incubated in peptone broth for 24 hours, then tetrathionate broth for 24 hours. Afterwards the samples were plated and incubated on Hekton agar for 24 hours. After appropriate incubation any black colonies on the Hekton agar plates were gathered and transferred to the GeneQuenceÒ D2 automated system for nucleic acid hybridization and to BBL crystal identification system for the final phase of microbiological culture. The GeneQuenceÒ results were available within 2 hours whereas the bacterial culture results were available within 24 hours. A total of 1494 samples were analyzed. The results were as follows: 1274 samples were GeneQuenceÒ negative and culture negative, 210 samples GeneQuenceÒ positive and culture positive, four samples GeneQuenceÒ negative and culture positive, and six samples Gene QuenceÒ positive and culture negative. The overall sensitivity was 98.1% and the specificity was 99.5%. The positive predictive value was 97.2%, whereas the negative predictive value was 99.6%. GeneQuenceÒ results were available 1 day sooner and were almost as accurate as the bacterial culture. An advantage of GeneQuenceÒ over PCR testing is that there are fewer false positive results. A disadvantage is that bacterial culture is still required to identify an antimicrobial sensitivity. A rapid and reliable test for identification of Salmonella spp would be beneficial for equine veterinarians. Future projects include decreasing enrichment time thus making the results available sooner.
European Union Reference Laboratory for equine diseases: towards improved and harmonized diagnosis of West Nile disease C. Beck, S. Lowenski, S. Zientara, and S. Lecollinet ANSES, EU-RL for equine diseases, UMR1161 Virologie INRA, ANSES, ENVA, 23 avenue du Général de Gaulle, 94703 Maisons-Alfort, FRANCE
In July 2008, the European Commission officially designated the French Agency for food, environmental and occupational health safety (ANSES) as the European Union Reference Laboratory (EU-RL) for equine diseases (apart from African horse sickness). West Nile disease, exotic Equine Encephalitis, Vesicular stomatitis, glanders and melioidosis are studied at ANSES Laboratory for animal health located in Maisons-Alfort (near Paris), while the Laboratory for equine diseases located in Normandy is dedicated to Contagious Equine Metritis, Dourine, Equine
Viral Arteritis, Equine Herpes viruses and Equine Infectious Anaemia. The EU-RL on equine diseases aims at providing technical and scientific support to the European National Reference Laboratories and the European Commission, as well as improving, harmonizing and developing new diagnostic tools for the diagnosis of equine diseases (through workshops, trainings, proficiency tests). A proficiency test (PT) on West Nile (WN) serological and molecular diagnosis was organized in 2010, in order to assess the performances of different WN diagnosis tools and to improve the results homogeneity among European countries. 22 and 25 laboratories participated to the serology and RT-PCR proficiency test, respectively. This PT indicated that IgG or competition ELISA tests were well established, on the contrary to IgM ELISA and virus neutralization tests. Moreover, there is a need to develop more specific IgG ELISA. WN real-time RT-PCR diagnosis is functional and efficient in most EC countries, however some participants (8) failed to detect lineage 1b and 2 strains. The detection capacity of lineage 1b and 2 strains needs to be improved, in particular in Europe where a pathogenic lineage 2 virus has been identified since 2004. In conclusion, this European PT highlighted the need for improved, adapted and harmonized diagnosis on WN in Europe.
Recent progress of the diagnosis for equine piroplasmosis I. Igarashi, N. Yokoyama, and X. Xuenan National Research Center for Protozoan Diseases, OIE Reference Laboratories on equine piroplasmosis, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
Equine piroplasmosis, caused by intra-erythrocytic protozoa Theileria equi and Babesia caballi, is a tick-borne disease of equids. The disease is characterized by fever, anemia, icterus, and hemoglobinuria. Horses that recover from the initial infection often be carriers of parasites. In such case, it is very difficult to detect the parasites in microscopic examination, and the horses become potential disseminators of the parasites. Since the disease is distributed worldwide including Asia, Europe, Africa, South America, the disease has posed a threat to the horses in the piroplasmosis free area and severe restriction has imposed on international movement of equids to avoid the spread of the infection from an endemic area. Current diagnosis of equine piroplasmosis relies on microscopic examination, serological assays and other molecular tools. OIE is recommending the several diagnostic tests including microscopic detection, complement fixation test (CFT), the indirect fluorescent antibody test (IFAT) and competitive enzymelinked immunosorbent assay (cELISA). However, there is no gold standard reference test for equine piroplasmosis. Each country utilizes one or more tests from OIE recommending tests and each test has disadvantages. Microscopic detection from blood smears has been used for the most standard diagnosis of equine piroplasmosis; however, the technique is relatively laborious and it is difficult to detect the parasites in the case of low parasitemia. Several serological tests have
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
been used to detect specific antibodies against the infections, but these assays are also restricted due to their antibody-detected limitation and/or potential cross-reactivity to other pathogens. For example, reading of IFAT results is very difficult to read results and not objective at borderline between positive and negative titers. Results of IFAT may also change not only with different origin of antigens, but also with different lots of FITC-conjugated anti-horse IgG antibody. Recently, new diagnostics such as immunochromatographic test (ICT) and loop-mediated isothermal amplification (LAMP) assay have been developed for the detection of B. caballi and/or T. equi infections. ICT has similar sensitivity to ELISA and is a rapid and simple method to detect antibodies [1]. LAMP assay can rapidly amplify DNA samples within one hour under an isothermal condition (at 60 to 65 C). LAMP has higher specificity and sensitivity than those of PCR [2]. Therefore, both ICT and LAMP are practical pen-side diagnostic methods for equine piroplasmosis. The objectives of this presentation are to review diagnostic methods and problems of the current diagnostic methods, to introduce newly developed diagnostics, and to discuss how these methods can contribute the strengthening of quarantine for equine piroplasmosis. References [1] Huang X, et al. Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses. Clin Vaccine Immunol 2006;13:553-5. [2] Alhassan A, et al. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res. 2007;100:1165-8.
Development of one-step TaqManÒ real-time reverse transcription-PCR and conventional reverse transcription PCR assays for the detection of equine rhinitis A and B viruses Zhengchun Lu, Peter J. Timoney, Jena White, and Udeni B.R. Balasuriya Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546
Equine rhinitis virus A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. There is evidence that these two viral infections are prevalent in countries in which sero-surveillance studies have been undertaken. Currently there is a lack of rapid and reliable diagnostic methods for virus detection and antibody determination. The sensitivity of virus isolation varies between laboratories and is confounded by difficulties in isolating virus from the urine of carrier horses. The objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) capable of detecting and distinguishing ERAV from ERBV without the inherent problems associated with the current laboratory diagnosis of these infections. Three rRT-PCR assays targeting the 5’UTR of ERAV and ERBV were developed. One assay was specific for ERAV, whereas the remaining two assays were
S59
specific for ERBV. In addition, six cRT-PCR assays targeting the 5’-UTR and 3D polymerase regions of ERAV and ERBV were also developed. RNA extracted from prototype strains of ERAV and ERBV as well as 21 archived tissue culture fluid samples which were previously determined to be positive by virus isolation for ERAV or ERBV with mono-specific rabbit antisera were used to evaluate the sensitivity and specificity of these assays. Using serial decimal dilutions (101 to 1010) of ERAV and ERBV prototype strains, the detection limits of the rRT-PCR and cRT-PCR assays were evaluated and compared. The rRT-PCR and cRT-PCR assays targeting ERAV detected all ERAV isolates (n¼11) and did not cross-react with any ERBV isolates (n¼10). Similarly, the rRT-PCR and cRT-PCR assays targeting ERBV only detected ERBV isolates and did not cross-react with ERAV isolates. None of the rRT-PCR or cRT-PCR assays crossreacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit among all of these assays was 1 plaqueforming unit per ml (pfu/ml). The newly developed rRTPCR and cRT-PCR assays appear to provide improved diagnostic tools for the detection and differentiation of ERAV and ERBV. While the results of this study are very promising, they will need to be verified by testing a greater number of clinical specimens before each assay is fully validated for the detection of ERAV and/or ERBV.
Development of a rapid isothermal assay to detect Taylorella equigenitalis S.E. North, P. Das, P.R. Wakeley, and J. Sawyer Technology Transfer Unit, Specialist Scientific Support Department, Animal Health Veterinary Laboratories Agency – Weybridge, United Kingdom
Contagious Equine Metritis (CEM) is an infection of the equine genital tract, caused by the bacterium Taylorella equigenitalis. Identifying T. equigenitalis from equine genital swabs, and differentiating it from T. asinigenitalis, using culture is slow and requires the presence of live bacteria. We have developed a rapid isothermal assay for the detection of T. equigenitalis using Recombinase Polymerase Amplification (RPA). RPA reactions occur optimally at 37 C and do not require thermocycling equipment. The reactions can be monitored in real time, based on fluorescence readings, or the amplification products can be detected on a lateral flow device (LFD). Equine genital swabs were used to inoculate solid culture media before a simple boiling method was used to extract DNA from the swabs. This enabled comparisons between culture and the RPA assay. The assay was tested, in real time, against 60 equine genital swabs (30 positive and 30 negative, as determined by culture and real time PCR). The analytical specificity of the assay was determined by testing against a panel of 38 bacterial species, including equine commensals. The analytical sensitivity of the assay was calculated using a dilution series of target DNA. A second probe was introduced into the assay, specific for T. asinigenitalis, which enabled detection and differentiation of T. equigenitalis and T. asinigenitalis. The T. equigenitalis assay was adapted, by