- Email: [email protected]
Development of a multilocus sequence typing method for analysis of Taylorella genus
S70
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
areas of the adrenal glands. Tissues were negative for equine herpesviruses 1 and 4 and equine arteritis virus. A gram-negative motile bacterium was isolated in pure culture from a range of tissues which was identified as Salmonella abortus equi (4,12:-:e, n, x). Isolates of the bacterium were sensitive to a broad range of antibiotics. Remaining in-foal mares were treated with trimethoprim and gentamicin. After initiation of antibiotic treatment only two further abortions occurred, on November 10th and February 10th. The source of S. abortus equi was not identified. However, since recipient mares came from a range of farms, one or more mares could have been a carrier that recommenced shedding Salmonella resulting from the intercurrent stress associated with relocation. There is a need for greater awareness of S. abortus equi as a potential cause of widespread abortion in the mare and of the importance of breeding farms having in place appropriate biosecurity and preventive measures including vaccination, to minimize the risk of future occurrences of abortion due to this pathogen.
Development of a multilocus sequence typing method for analysis of Taylorella genus F. Duquesne, L. Hébert, M.F. Breuil, C. Laugier, and S. Petry ANSES, Dozulé Laboratory for equine diseases, Unit Bacteriology and Parasitology, 14430 Goustranville, France
The Taylorella genus is composed of two species: Taylorella equigenitalis, the causative agent of the contagious equine metritis (a sexually-transmitted infection of horses, first reported in 1977 and notified to the World Organisation for Animal Health), and Taylorella asinigenitalis, considered as a nonpathogenic bacterium despite clinical signs of metritis and cervicitis on mares following an experimental intrauterine infection [1]. Using the recently published genome sequences [2], we developed a multilocus sequence typing (MLST) method for future epidemiological and phylogenetic studies of the Taylorella genus. We selected twenty housekeeping genes within T. equigenitalis MCE9 and T. asinigenitalis MCE3 genomes. Among them, seven were validated by the analysis of an internal sequence of z450bp, to define our MLST scheme. We performed PCR amplifications and sequencing on DNA extracted from 166 isolates (115 T. equigenitalis and 51 T. asinigenitalis) of diverse origins (Australia, France, United Kingdom, USA, Japan, United Arab Emirates) from 1977 to 2011. The sequences of each locus of each strain were aligned and compared (MEGA5 software). First, allele numbers were assigned to each unique sequence and then, a sequence type (ST) was attributed to each strain with a single combination of allele numbers. MLST data were analyzed by the eBURST algorithm. Our MLST scheme with seven loci shows 32 and 14 STs for T. equigenitalis and T. asinigenitalis, respectively. No ST was common between the two species despite the presence of common alleles for one of the six loci, reflecting potential allelic exchanges in the evolution of the Taylorella genus. We found one predominant ST for each species (ST1 and ST25), comprising 23% and 53% of
isolates per ST respectively. The 32 T. equigenitalis-STs were resolved into five clonal complexes and four single clones. The founding genotype (ST1) of the dominant clonal complex, grouping 18 STs, is composed of isolates from the first outbreaks late 70s in United Kingdom, France, Australia and USA. The 11 T. asinigenitalis-STs were resolved into tree clonal complexes and five single clones. The challenge of developing a single MLST scheme for T. equigenitalis and T. asinigenitalis was achieved despite z20% genetic differences between their genome [2]. Our first results show an organization into clonal complexes confirming the relevance of this method for future epidemiological and phylogenetic studies of the Taylorella genus. Its simplicity of implementation, its robustness and the use of a common MLST database (under development) will allow the comparison and the pooling of results between laboratories. References [1] Katz, et al. Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares. J Am Vet Med Assoc. 2000;216(12):1945-8. [2] Hébert L, et al. Genome sequence off Taylorella equigenitalis MCE9, the causative agent off contagious equine metritis. J. Bact 2011; 193(7):1785.
Evidence of T. equiperdum infection in the Italian Dourine outbreaks R. Lelli, P. Calistri, A. Giovannini, and V. Caporale Istituto “G. Caporale”, Teramo, Italy
Dourine is a sexually transmitted parasitic disease of equids caused by flagellate protozoa of the species Trypanosoma equiperdum. Dourine is endemic in many areas of Asia, Africa, Russia, part of Middle East, South America and south-eastern Europe. In Italy, it was first eradicated in the late-40s and again in the 70s of last century following an epidemic due to the import of infected animals from former USSR. A stallion undergoing routine serological testing for stud purposes was found positive for dourine in May 2011, in Sicilia and the following tracing back detected four further outbreaks. A surveillance plan based on the serological examination of all stallions and mares older than two years of age was implemented following to these events and two further outbreaks were detected in 2011. The Authors describe the epidemiological, clinical, pathological and laboratory observations in horses infected in the 2011 dourine epidemic in Italy. Two stallions and five mares showing clinical signs were transferred to the Istituto “G. Caporale”, Teramo (ICT), to both monitor the evolution of the disease and perform additional tests. Four of the five mares were euthanized and necropsied. Organs and tissues were sampled for histopathology. The cerebrospinal fluid (CSF) was tested for both anti-T. equiperdum antibodies and evidence of parasite presence. Blood, CSF, spleen, kidney, liver, udder, mammary secretion, lymph nodes, lung, skin wheals, genital organs, CNS, cranial nerves, joint fluid, urine were tested by a Real-Time PCR specific for the Trypanosoma subgenus. Serum samples were tested by two different
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
areas of the adrenal glands. Tissues were negative for equine herpesviruses 1 and 4 and equine arteritis virus. A gram-negative motile bacterium was isolated in pure culture from a range of tissues which was identified as Salmonella abortus equi (4,12:-:e, n, x). Isolates of the bacterium were sensitive to a broad range of antibiotics. Remaining in-foal mares were treated with trimethoprim and gentamicin. After initiation of antibiotic treatment only two further abortions occurred, on November 10th and February 10th. The source of S. abortus equi was not identified. However, since recipient mares came from a range of farms, one or more mares could have been a carrier that recommenced shedding Salmonella resulting from the intercurrent stress associated with relocation. There is a need for greater awareness of S. abortus equi as a potential cause of widespread abortion in the mare and of the importance of breeding farms having in place appropriate biosecurity and preventive measures including vaccination, to minimize the risk of future occurrences of abortion due to this pathogen.
Development of a multilocus sequence typing method for analysis of Taylorella genus F. Duquesne, L. Hébert, M.F. Breuil, C. Laugier, and S. Petry ANSES, Dozulé Laboratory for equine diseases, Unit Bacteriology and Parasitology, 14430 Goustranville, France
The Taylorella genus is composed of two species: Taylorella equigenitalis, the causative agent of the contagious equine metritis (a sexually-transmitted infection of horses, first reported in 1977 and notified to the World Organisation for Animal Health), and Taylorella asinigenitalis, considered as a nonpathogenic bacterium despite clinical signs of metritis and cervicitis on mares following an experimental intrauterine infection [1]. Using the recently published genome sequences [2], we developed a multilocus sequence typing (MLST) method for future epidemiological and phylogenetic studies of the Taylorella genus. We selected twenty housekeeping genes within T. equigenitalis MCE9 and T. asinigenitalis MCE3 genomes. Among them, seven were validated by the analysis of an internal sequence of z450bp, to define our MLST scheme. We performed PCR amplifications and sequencing on DNA extracted from 166 isolates (115 T. equigenitalis and 51 T. asinigenitalis) of diverse origins (Australia, France, United Kingdom, USA, Japan, United Arab Emirates) from 1977 to 2011. The sequences of each locus of each strain were aligned and compared (MEGA5 software). First, allele numbers were assigned to each unique sequence and then, a sequence type (ST) was attributed to each strain with a single combination of allele numbers. MLST data were analyzed by the eBURST algorithm. Our MLST scheme with seven loci shows 32 and 14 STs for T. equigenitalis and T. asinigenitalis, respectively. No ST was common between the two species despite the presence of common alleles for one of the six loci, reflecting potential allelic exchanges in the evolution of the Taylorella genus. We found one predominant ST for each species (ST1 and ST25), comprising 23% and 53% of
isolates per ST respectively. The 32 T. equigenitalis-STs were resolved into five clonal complexes and four single clones. The founding genotype (ST1) of the dominant clonal complex, grouping 18 STs, is composed of isolates from the first outbreaks late 70s in United Kingdom, France, Australia and USA. The 11 T. asinigenitalis-STs were resolved into tree clonal complexes and five single clones. The challenge of developing a single MLST scheme for T. equigenitalis and T. asinigenitalis was achieved despite z20% genetic differences between their genome [2]. Our first results show an organization into clonal complexes confirming the relevance of this method for future epidemiological and phylogenetic studies of the Taylorella genus. Its simplicity of implementation, its robustness and the use of a common MLST database (under development) will allow the comparison and the pooling of results between laboratories. References [1] Katz, et al. Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares. J Am Vet Med Assoc. 2000;216(12):1945-8. [2] Hébert L, et al. Genome sequence off Taylorella equigenitalis MCE9, the causative agent off contagious equine metritis. J. Bact 2011; 193(7):1785.
Evidence of T. equiperdum infection in the Italian Dourine outbreaks R. Lelli, P. Calistri, A. Giovannini, and V. Caporale Istituto “G. Caporale”, Teramo, Italy
Dourine is a sexually transmitted parasitic disease of equids caused by flagellate protozoa of the species Trypanosoma equiperdum. Dourine is endemic in many areas of Asia, Africa, Russia, part of Middle East, South America and south-eastern Europe. In Italy, it was first eradicated in the late-40s and again in the 70s of last century following an epidemic due to the import of infected animals from former USSR. A stallion undergoing routine serological testing for stud purposes was found positive for dourine in May 2011, in Sicilia and the following tracing back detected four further outbreaks. A surveillance plan based on the serological examination of all stallions and mares older than two years of age was implemented following to these events and two further outbreaks were detected in 2011. The Authors describe the epidemiological, clinical, pathological and laboratory observations in horses infected in the 2011 dourine epidemic in Italy. Two stallions and five mares showing clinical signs were transferred to the Istituto “G. Caporale”, Teramo (ICT), to both monitor the evolution of the disease and perform additional tests. Four of the five mares were euthanized and necropsied. Organs and tissues were sampled for histopathology. The cerebrospinal fluid (CSF) was tested for both anti-T. equiperdum antibodies and evidence of parasite presence. Blood, CSF, spleen, kidney, liver, udder, mammary secretion, lymph nodes, lung, skin wheals, genital organs, CNS, cranial nerves, joint fluid, urine were tested by a Real-Time PCR specific for the Trypanosoma subgenus. Serum samples were tested by two different