Outline of the Completed Research

Microbiology


Application of loop-mediated isothermal amplification to detect pathogens causing lower respiratory bacterial infection in Thoroughbred racehorses and its antimicrobial susceptibility patterns (2014-2016)

 We developed five LAMP methods to detect bacteria causing lower respiratory tract infection in horses in this study, namely Streptococcus equi subsp. zooepidemicus, strains of the Bacteroides-Prevotella group, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Staphylococcus aureus. We then compared the clinical efficacies of three DNA extraction methods (Loopamp PURE DNA Extraction Kit, InstaGene Matrix, and boiling method); From the aspect of false-negative reaction, the Loopamp PURE DNA Extraction Kit was the most suitable DNA extraction method. The use of seven LAMP methods―the five new developed ones and two previously described ones targeting Escherichia coli and Pseudomonas aeruginosa―in combination with the Extraction Kit obtained high accordance rates with bacterial culture: 91.0% to 100% (using a threshold of 1×104 CFU/ml) and 85.1% to 100% (using 20 CFU/ml). In addition, we revealed the antimicrobial susceptibility patterns of those pathogenic bacteria.
 
These methods can be applied in the clinic with no special equipment and take no more than 90 min. Veterinary practitioners can select appropriate antimicrobials as the initial step in treatment through the quick identification of causative bacteria and antimicrobial susceptibility patterns. These swift and appropriate treatments will improve therapeutic efficacy in adult horses with lower respiratory bacterial infection.

Basic evaluation of the laser microdissection method for pathological diagnosis of equine infectious diseases (2012-2014)

 With a view to using the laser dissection method to diagnose equine infectious diseases, we examined various sampling conditions. The maximum fixation period that enabled extraction of tissue DNA fragments long enough to identify pathogens was 1 week in the case of formalin and 1 month in the case of Methacarn (methanol-Carnoy). A sample of at least 101 to 103 virus-infected cells or 101 to 103 bacterial cells was needed for analysis.

Determination of the whole-genome sequence of Salmonella Abortusequi (2012-2014)

 Equine paratyphoid is an infectious disease that is caused by Salmonella Abortusequi and is specific to the horse family. Abortion or multiple abscesses are major signs of infection. The disease is diagnosed by detecting the etiological agent or by serodiagnosis. However, these methods have some problems. In addition, conventional molecular epidemiological methods do not have enough resolution to discriminate the strains isolated in Japan. Here, we determined the whole-genome sequence of Salmonella Abortusequi in order to obtain basic data to solve these problems.
(1) Determination of the whole-genome sequence of L-2508 The whole-genome sequence of L-2508 (a representative strain of S. Abortusequi) was determined by using a next-generation sequencer and optical genome mapping. The size of the genome was 4,738,978 bp; the GC content was around 52%, and 4710 ORFs were detected. Compilation of a phylogenic tree based on the core genome sequence revealed that S. Abortusequi was close to S. Choleraesuis and S. Paratyphi C.
(2) Exploration of the S. Abortusequi gene We compared the whole-genome sequence of L-2508 with those of other serotypes of Salmonella spp. A region unique to S. Abortusequi was detected; the region seemed to be derived from a bacteriophage. None of the pathogenic genes was observed in S. Abortusequi but not in S. Typhimurium. However, several pathogenic genes were observed in S. Typhimurium but not in S. Abortusequi. These S. Typhimurium pathogenic genes might therefore be useful for developing a serological method to discriminate infections by the two organisms.
(3) Core-genome SNP-based phylogenic tree analysis of S. Abortusequi strains. We performed a phylogenic tree analysis based on single nucleotide polymorphisms (SNPs) of the core genome by using 25 S. Abortusequi strains isolated in various geographic areas and at various times. There were 1316 SNPs among the strains. Marked differences were observed between Japanese strains and foreign isolates. In addition, there were two lineages among the Japanese strains. Our results suggested that SNP-based molecular epidemiological analysis might be useful for discriminating Japanese endemic strains.


Establishment of methods of etiological diagnosis and serodiagnosis for equine proliferative enteropathy (2012-2014)

 Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis is an intestinal infectious disease often observed in foals. Because foals suffering from EPE generally show hypoproteinemia and developmental delay because of weight loss, this disease is of great concern in the horse-breeding areas of the US. Although the prevalence of the disease is unknown in the Japanese horse population, L. intracellularis has spread widely in the Japanese pig population as the etiological agent of porcine proliferative enteropathy (PPE). Diagnostic methods for EPE, such as PCR and indirect immunofluorescence antibody assay (IFA) have already established in the US but have not been validated in Japan. Here, we evaluated and compared the clinical efficacies of these methods with a view to introducing them into clinical diagnosis in Japan. We also attempted to modify those parts of the methods that were difficult to perform in this country.
 As methods for etiological diagnosis, we evaluated real time-PCR and nested PCR, which have been described in previous papers, by using a positive control made by adding the vaccine strain of L. intracellularis to the feces of a healthy horse. The two methods had the same sensitivity. We also evaluated two DNA extraction methods, Mo¨ller's method and a commercial kit (ZR Fecal DNA kit), as PCR-based methods. Mo¨ller's method was slightly superior to the commercial kit in terms of sensitivity. In addition, we examined the usefulness of tube-ELISA, which was under development for PPE diagnosis. The ELISA was able to differentiate L. intracellularis antigen from the positive control.
 For serodiagnosis, we developed a slide-SAB (streptavidin and biotin) method to detect L. intracellularis antibody. This method was a modification of IFA and had the advantage of availability of both the antigen and the required equipment in Japan. The results of slide-SAB were the same with those of IFA in sera obtained from horses and pigs. We also evaluated a commercial ELISA kit that had become licensed in Japan during this research period. The results obtained with the kit had a high level of concordance with those from IFA. We concluded that these methods, namely real time-PCR, nested PCR, tube-ELISA, slide-SAB, IFA, and the commercial ELISA kit, could all be used to diagnose EPE.

Improvement of high throughput and specific serological test for Salmonella Abortusequi. -Modification and evaluation of micro agglutination test and LPS -ELISA- (2011-2013)

 A tube agglutination test is official method for sero-diagnosis of equine paratyphoid (S. Abortusequi infection). Low throughput and the presence of non-specific reaction have been considered as difficult problems in the method. In this study, we modified and evaluate the sero-diagnosis methods, which are expected to contribute for the improvement of throughput and specificity.
 We modified a micro agglutination test (MAT) originally developed in 1986. Appropriate dilution buffer and its dilution rate of the antigen were phosphate buffered saline (PBS) and 15-fold, respectively. High correspondence rate was observed between modified MAT and TAT. This result suggests that the modified MAT is able to use for diagnosis of equine paratyphoid as same as TAT. On the other hand, ELISA previously reported in Canada was seemed to be impossible to put into practical use, since the method could not detect some of sero-positive samples confirmed by TAT.
 To reveal the mechanism of non-specific reaction observed in TAT, ELISA using anti-horse IgM, IgG, or IgA antibodies and using LPS antigen of S. Abortusequi or other gram negative equine pathogens were developed. By the analysis of these ELISAs, the presence of IgM, which might recognize multiple kind of LPSs, were strongly suggested as the cause of non-specific reaction in TAT. On the other hand, IgG and IgA specific to O4-LPS was detected in the serum obtained from S. Abortuseqi affected horses and the horse harboring 2-mercaptoethanol (2ME) resistant anti-O4 LPS antibody.
 It was confirmed that dithiothreitol (DTT) could be used as the substitute agent as 2ME. Modified MAT combined with DTT treatment (DTT-MAT) could detect O4 somatic antigen specific antibody from the S. Abortusequi challenged horse with negative result by TAT. This result suggests that DTT treatment might be more useful to detect the history of S. Abortusequi infection in horses.

Histopathological analysis of aborted fetuses and utero-placental lesions in EHV-1 infected pregnant mares (2011-2013)

 To elucidate the mechanism of abortion due to EHV-1 infection, we analyzed aborted fetuses and the lesions of uterus and placenta in experimental equine model of EHV-1 abortion.
 In aborted fetuses of experimental infection model, the histopathological lesions same as that of fetuses with natural EHV-1 infection were observed. Focal necrotic lesions with eosinophilic inclusion body were observed in liver, lung, thymus, systemic lymph node. EHV-1 positive cells were observed immunohistopathologically in these necrotic lesions and vascular endothelial cells of allantochorion in expelled placenta.
 In EHV-1 infected mares autopsied in the late stage of viremia, EHV-1 DNA was detected from uterus and attached placenta, but not from fetuses. Histopathological examination, Vasculitis was observed in small vessels that presented in lamina propria of uterus and EHV-1 positive cells were observed in endothelial cells of these vessels and infiltrating macrophages. Necrosis of villi and EHV-1 positive cells were observed in microcotyledons of uterus and placenta.
 In EHV-1 infected mares autopsied in the early stage of viremia, histopathological findings were similar to that observed in mares autopsied in the late stage of viremia, but the severity of lesions and distribution of EHV-1 positive cells were mild. In microcotyledon, magnitude of EHV-1 positive signals of infected cells was weak, and those cells were observed in the small focal area that connected trophoblasts with endometrial epithelium cells.
 These findings would contribute to clarify the mechanism how EHV-1 passes through the pracental barrirer.

Evaluation of a recombinant antigen ELISA for the diagnosis of cerebrospinal setariasis (2010-2013) (Joint research conducted with Kitasato University)

 Our goal was to develop an immunological technique for diagnosis of equine cerebrospinal setariasis; to achieve this, we developed and evaluated an ELISA-based method using a recombinant protein as the antigen.
 We generated a Setaria digitata cDNA library as well as antisera against diagnostic antigen-candidate proteins and cloned the gene for a candidate antigen protein based on immunoscreening. We then expressed and purified the recombinant protein in Escherichia coli for use as the ELISA antigen. We investigated the cerebrospinal fluid (CSF) from horses immunized with extract of S. digitata parasites and confirmed an increase in antibody titer.
 We confirmed the presence of the antibody in the CSF from several equine cases of cerebrospinal setariasis; however, we also noted instances in which the antibody was detected in the CSF from cases of other diseases. Thus, we demonstrated the feasibility of our ELISA-based method for diagnosis of cerebrospinal setariasis, either independently, using sequential CSF samples or in conjunction with other diagnostic methods.

Epizootiological and clinical microbiological studies on equine infectious diseases (2009-2013)

 Epizootiological surveys on infectious diseases that have a serious influence on race meetings were conducted during the years 2009-2013.
 According to the sero-epizootiological surveillance conducted in two training centers of JRA, the prevalence of equine rhinopneumonitis was rather small in each year, except for the year 2020 in the Miho training center when a relatively more number of febrile horses with sero-conversion was observed as compared to the other year.
 All of the nasal samples collected from febrile horses in JRA facilities during the investigation period showed negative results in RT-PCR for detecting H3N8 equine influenza virus.
 All the imported horses introduced into JRA facilities were examined serologically for foreign pathogens and obtained results were all negative.

Immunological analysis of flagella antigens of Salmonella Abortusequi for development of specific serodiagnosis method of equine paratyphoid (2007-2010)

 In this study, we evaluated usefulness of specific region of the flagella protein of S. Abortusequi and S. Typhimurium as an antigen for discriminately serodiagnosis method between equine paratyphoid and S. Typhimurium infection by immunological analysis. For the analysis, we constructed truncated flagella protein libraries and synthesized peptide libraries according to the amino acid sequence of the flagella protein of each flagellar phase in the serotypes. Significant increase of IgM antibody against the specific region of S. Abortusequi was observed in successive serum collection obtained from horses infected by experimental intrauterine administration of S. Abortusequi. In contrast, increase of specific IgM antibody was not significantly observed in successive serum collection obtained from horses infected by experimental oral administration of S. Abortusequi and S. Typhimurium. Increase of IgG antibody was not observed in these collections. High-level IgG antibody corresponding each specific region was detected in serum obtained from horses intramuscularly immunized by live or killed organisms. IgM or/and IgG antibody against S. Abortusequi were observed in serum obtained from horses kept in endemic region of equine paratyphoid and were not observed in serum from carrier horses of S. Abortusequi with low flagella expression. Conclusively, the specific region of the flagella protein of S. Abortusequi and S. Typhimurium seemed to be useful as the diagnosis antigen to discriminate between equine paratyphoid and S. Typhimurium infection. However, we also have to consider that specific antigens have a limitation of the application for the diagnosis because of some factors suppressing the production of the antibody against the specific region including administration antimicrobial agents at an initial phase of infection, degree of expression of flagella, and individual variation of host.

Epidemiological and clinical microbiological studies on equine infectious diseases (2004-2008)

 Here, study was mainly conducted on epidemiological surveys about infectious diseases that have a serious influence on race meetings and diagnostic methods of equine infectious diseases. The principal research results were as follows.
1. A febrile disease that occurred as a mass outbreak at a draft-horse
racecourse in Hokkaido in 2005 was surveyed. It was suggested that the disease might have been caused by equine coronavirus infection.
2. It was demonstrated that a collective occurrence of Salmonella Typhimurium infection amongst Thoroughbred foals in the Hokkaido breeding area in 2005 was caused by the multiple drug resistant DT104 type.
3. The circumstances of an equine influenza epidemic in JRA facilities in 2007 were clarified.
4. The neutralization test that is a kind of serological diagnosis method for
equine viral arteritis was standardized in joint research with the Epizootic Research Center, JRA, the Animal Quarantine Service and the National Institute of Animal Health.
5.Two methods of detecting Clostridium difficile genes (the PCR method and the LAMP method) were applied in the field.

Molecular genetic analysis of the presentation mechanism of Streptococcus equi subsp. equi PEPK antigens (2006-2008)
(Joint research conducted with National Institute of Animal Health)

 While Streptococcus equi subsp. equi (S. equi) has bacterial surface proteins called SzPSe antigens, those of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus), which is in a substrain relationship with the former, are called SzP antigens. Both SzPSe antigens and SzP antigens have PEPK repeated sequences. However, while PEPK antigens react to the serum of horses infected with strangles, they do not react to that of S. zooepidemicus infected horses. This study was conducted to clarify the reason for this. In the first year, it was confirmed that knockout mutant strains of S. equi do not produce SzPSe antigens. In the second year, it was demonstrated that the SzPSe antigens of S. equi are expressed only by SzPSe genes. In the final year, it was confirmed that SzPSe antigens are only secreted in culture supernatant of S. equi. From these results, it was suggested that the difference in the secretion of SzPSe or SzP antigens between S. equi and S. zooepidemicus might be related to the production of specific anti-PEPK antibodies in horses with strangles.

Development of an ELISA test for diagnosis of strangles using peptide antigens (2004-2006)

Purpose
 Strangles is a contagious disease peculiar to animals in the genus equus, and is caused by S.equi. It tends to more readily affect young horses and can be fatal. While a serum diagnostic method that can specifically diagnose strangles is thought to be effective in preventing the introduction of this disease from overseas and its spread inside Japan, no specific method yet exists anywhere in the world. Therefore, we established a new serum diagnostic method using specific PEPK repeated peptide antigens for strangles, resulting from previous research, and studied serum diagnostic method for strangles.

Results
 1. As a control for the study, we obtained the necessary serum of S. zooepidemicus infected horses through an infection experiment.
 2. We proved that PEPK 5-times repeated peptide antigens are ideal as ELISA antigens, and established an ELISA measurement method for strangles antibodies using these antigens.
 3. Using the serum of horses infected with S.equi, the serum obtained from the infected horses with S. zooepidemicus and those of healthy horses, we investigated and compared antibody titers against PEPK repeated antigens. As a result, we could clearly differentiate the serum of horses infected with the strangles bacillus from the other sera.
 4. After searching for the expression and localization of PEPK antigens in S.equi, it was confirmed that S. equi expresses antigens on the surface of the bacteria and in cultured supernatant.
 5. We inoculated horses with two types of strangles vaccine (component vaccine: Nos. 1, 2 and 3; live vaccine: Nos. 4, 5 and 6), and studied their side effects and changes in antibody titers against the PEPK 5-times-repeated-domain of SzPSe. As a result, no side effects were seen to be caused by vaccine inoculation. In the antibody titers against the PEPK 5-times-repeated-domain of SzPSe, on the other hand, a rise in antibody titers was recognized in all three horses in the component vaccine inoculated group, although the degree was relatively mild. In the live vaccine inoculated group, a mild rise was observed in one of the three horses, but there was no change in the other two.
 6. When we carried out a challenge test for strangles bacillus on six yearlings variously inoculated with two types of strangles vaccine, symptoms of strangles were observed in all six horses. Meanwhile, antibody titers against PEPK repeated peptide antigens rose moderately in horses inoculated with component vaccine and mildly in horses inoculated with live vaccine. We also carried out experimental infections with S equi using six yearlings, and investigated changes in their antibody titers over a period of 8 months. The result was that, although a conspicuous rise in antibody titers was recognized in three of the six horses, the titers diminished in a relatively short time after inoculation with S equi.