Hirohisa Mekata

Development and application of a quantitative real-time PCR for the diagnosis of Surra in water buffaloes

Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a real-time PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA region in trypanosomes. The quantitative detection limit of the assay was 10(2) trypanosomes per mL of blood, and the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water buffaloes and horses were conducted. T. evansi was detected in 17/607 (2.8%) blood samples, with parasitemia levels ranging from >10(1) to 10(7) parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi.

Molecular detection of trypanosomes in cattle in South America and genetic diversity of Trypanosoma evansi based on expression-site-associated gene 6.

In South American countries, trypanosomiasis as a result of Trypanosoma evansi and Trypanosoma vivax infections causes significant economic losses in livestock. The objectives of this study were to characterize the epidemiology of bovine trypanosomiasis in South America and to draw a comparison between South American and Asian T. evansi isolates based on the polymorphisms in their transferrin receptor encoding gene 6. We assessed the prevalence rates of T. evansi and T. vivax infections in cattle in different regions of Peru and Bolivia using the polymerase chain reaction (PCR) and found that, in Lima and Pucallpa in the Republic of Peru, T. evansi infection rates were 5.8% (6/104) and 2.5% (5/195), respectively, while in Santa Cruz, Republic of Bolivia, the infection rate for T. evansi was 11.5% (59/510). The prevalence rates of T. vivax in Lima and Santa Cruz were 3.8% (4/104) and 0.9% (5/510), respectively. In T. evansi, uptake of host transferrin is mediated by a receptor derived from the two expression site-associated genes 6 and 7 (ESAG6 and ESAG7). We previously showed that the ESAG6 depicts genetic diversity among different isolates of T. evansi in Asia. In this study, we cloned and sequenced the ESAG6 genes from T. evansi isolates from South America, and found, in addition to some of the previously observed variants, 20 novel variants of ESAG6 genes which could be categorized into three new clades among the various isolates. To conclude, the results obtained in this study suggest that T. evansi isolates from South America are more diverse than the Asian isolates.

Evaluation of the natural perinatal transmission of bovine leukaemia virus

The perinatal transmission of bovine leukaemia virus (BLV) plays a critical role in the spread and persistence of BLV infection in cattle herds. The purpose of this study was to examine the frequency of perinatal infections in an area in Japan and investigate some risk factors associated with infection. Altogether, 129 calves born to BLV-infected cows in a herd in Japan were tested for infection immediately after birth and again at one month of age using nested PCR. Twenty-four calves (18.6 per cent) were infected with BLV, of which 14 (10.8 per cent) and 10 (7.7 per cent) calves were infected via the transplacental and the birth canal routes, respectively. Maternal viral loads, breed, the presence or absence of assistance during parturition and the number of births per dam were evaluated to investigate risk factors associated with infection. Maternal viral load was significantly correlated with the frequency of perinatal infection, and more than 40 per cent of newborn calves born to dams with high viral loads were infected with BLV. The results of this study could contribute towards developing effective eradication programmes by providing necessary data for replacement of breeding cow in the field.

Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies.

The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.

Horizontal transmission and phylogenetic analysis of bovine leukemia virus in two districts of Miyazaki, Japan

Horizontal transmission is recognized as a major infection route for bovine leukemia virus (BLV), and cattle with high viral loads are considered to be a major infectious source in a herd. However, a correlation between viral loads and the risk of infection has been insufficient to use as a foundation for BLV control strategies. In this report, we examined the epidemiology of BLV infection and the infectious source in a local area. In 2013–2014, BLV infection was investigated in 1,823 cattle from 117 farms in two adjacent districts, Miyazaki, Japan. Seropositive samples for BLV were detected with 88 cattle and in 14 farms. Phylogenetic analysis revealed that 94% of the isolates clustered into genotype I and the remaining isolate into genotype III. Among genotype I, genetically distinct strains were spread at each farm, and cattle infected with less than 3 copies/100 cells did not transmit BLV to other cattle for more than thirty months. This is the first report of concrete data of viral load in relation to viral horizontal transmission under the field condition. The data facilitate farmers and veterinarians understanding the status of BLV infected cattle. This research contributes to BLV infection control and the development of effective BLV eradication programs.

Identification of O serotypes, genotypes, and virulotypes of Shiga toxin-producing Escherichia coli isolates, including non-O157 from beef cattle in Japan.

Bovines are recognized as an important reservoir of Shiga toxin-producing Escherichia coli (STEC). Although STEC strains are significant foodborne pathogens, not all of the STEC held by cattle are pathogenic, and which type of STEC that will become epidemic in humans is unpredictable. Information about the prevalence of serotype and virulence gene distribution in beef cattle is insufficient to develop monitoring and controlling activities for a food safety and security program. Thus, this study investigated the prevalence of O157 and non-O157 STEC in Japanese beef cattle and characterized the isolates by the type of O antigen and several virulence markers to help predict the pathogenicity. In this study, 64.2% (176 of 274) of enrichment cultures of fecal samples collected from an abattoir and farms were stx1 and/or stx2 positive by PCR. STEC strains were isolated from 22.1% (39 of 176) of the positive fecal samples, and these isolates represented 17 types of O antigen (O1, O2 or O50, O5, O8, O55, O84, O91, O109, O113, O136, O150, O156, O157, O163, O168, O174, and O177). Two selective media targeting major STEC groups, cefixime-tellurite sorbitol MacConkey agar and CHROMagar O26/O157, allowed isolation of a variety of STEC strains. The most frequently isolated STEC was O113 (8 of 39), which has previously been reported as a cause of foodborne infections. Although most of the O113 STEC isolated from infected patients possessed the enterohemolysin (hlyA) gene, none of the O113 STEC cattle isolates possessed the hlyA gene. The second most common isolate was O157 (6 of 39), and all these isolates contained common virulence factors, including eae, tir, lpf1, lpf2, and hlyA. This study shows the prevalence of O157 and non-O157 STEC in Japanese beef cattle and the relationship of O antigen and virulotypes of the isolates. This information may improve identification of the source of infection, developing surveillance programs or the current understanding of virulence factors of STEC infections.

Campylobacter and Salmonella are prevalent in broiler farms in Kyushu, Japan: results of a 2‐year distribution and circulation dynamics audit

To elucidate the distribution and circulation dynamics of Campylobacter and Salmonella in Japanese chicken broiler flocks.

Six Novel O Genotypes from Shiga Toxin-Producing Escherichia coli.

Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny.

Nationwide Distribution of Bovine Influenza D Virus Infection in Japan

Cattle are major reservoirs of the provisionally named influenza D virus, which is potentially involved in the bovine respiratory disease complex. Here, we conducted a serological survey for the influenza D virus in Japan, using archived bovine serum samples collected during 2010–2016 from several herds of apparently healthy cattle in various regions of the country. We found sero-positive cattle across all years and in all the prefectural regions tested, with a total positivity rate of 30.5%, although the positivity rates varied among regions (13.5–50.0%). There was no significant difference in positivity rates for Holstein and Japanese Black cattle. Positivity rates tended to increase with cattle age. The herds were clearly divided into two groups: those with a high positive rate and those with a low (or no) positive rate, indicating that horizontal transmission of the virus occurs readily within a herd. These data demonstrate that bovine influenza D viruses have been in circulation for at least 5 years countrywide, emphasizing its ubiquitous distribution in the cattle population of Japan.

Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva.

Abstract Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo.