Seroprevalence for severe fever with thrombocytopenia syndrome virus among the residents of Miyazaki, Japan: An epidemiological study.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the SFTS virus (SFTSV). The Miyazaki Prefecture has the highest number of SFTS cases in Japan and requires countermeasures for prevention. In this study, we aimed to conduct an epidemiological survey in Miyazaki Prefecture to determine the exposure conditions of SFTSV by measuring the seroprevalence among residents of Miyazaki and to evaluate the factors that influence the endemicity of SFTS. METHODS: The survey was conducted between June 2014 and April 2019 in all 26 municipalities in Miyazaki Prefecture. SFTSV antibodies were detected using an enzyme-linked immunosorbent assay in the blood samples of 6013 residents (3184 men and 2829 women). A questionnaire-based survey of the living environment was also conducted. RESULTS: Multiple logistic regression analysis revealed that age and occupation were significant factors related to the proportion of participants with an optical density (OD) value > 0.2 and a seroprevalence of 0.9 % (54/6013). Seven seropositive individuals (0.1 %) with an OD value of >0.4 were identified (three men and four women, aged 54-69 years), and all were asymptomatic. One participant had a higher OD than the positive control. CONCLUSION: Although SFTS is endemic in Miyazaki Prefecture, Japan, its seroprevalence is relatively low. Since some risk areas in Miyazaki prefecture have been identified, it is important to enhance awareness of SFTS in residences and reduce contact with ticks, especially in high-risk areas.

The detection of long-lasting memory foot-and-mouth disease (FMD) virus serotype O-specific CD4+ T cells from FMD-vaccinated cattle by bovine major histocompatibility complex class II tetramer.

Foot-and-mouth disease (FMD) is a highly contagious, economically devastating disease of cloven-hooved animals. The development of long-lasting effective FMD vaccines would greatly benefit the global FMD control programme. Deep analysis of adaptive immunity in cattle vaccinated against FMD is technically challenging due to the lack of species-specific tools. In this study, we aimed to identify CD4+ T-cell epitopes in the FMD virus (FMDV) capsid and to phenotype the CD4+ T cells that recognize them using bovine major histocompatibility complex (BoLA) class II tetramer. A BoLA class II tetramer based on the DRA/DRB3*020:02 allele and FMDV antigen-stimulated PBMCs from bovine vaccinates were used to successfully identify four epitopes in the FMDV capsid, three of which have not been previously reported; two epitopes were identified in the structural protein VP1, one in VP3 and one in VP4. Specificity of the three novel epitopes was confirmed by proliferation assay. All epitope-expanded T-cell populations produced IFN-γ in vitro, indicating a long-lasting Th1 cell phenotype after FMD vaccination. VP3-specific CD4+ T cells exhibited the highest frequency amongst the identified epitopes, comprising >0·004% of the CD4+ T-cell population. CD45RO+ CCR7+ defined central memory CD4+ T-cell subpopulations were present in higher frequency in FMDV-specific CD4+ T-cell populations from FMD-vaccinated cattle ex vivo. This indicates an important role in maintaining cell adaptive immunity after FMD vaccination. Notably, FMDV epitope-loaded tetramers detected the presence of FMDV-specific CD4+ T cells in bovine PBMC more than four years after vaccination. This work contributes to our understanding of vaccine efficacy.

A descriptive survey of porcine epidemic diarrhea in pig populations in northern Vietnam.

Porcine epidemic diarrhea (PED) virus (PEDV) is a globally emerging and re-emerging epizootic swine virus that causes massive economic losses in the swine industry, with high mortality in piglets. In Vietnam, PED first emerged in 2009 and has now developed to an endemic stage. This is the first cross-sectional survey performed to evaluate the proportion of PEDV-positive swine farms in Vietnam from January 2018 to February 2019. Fecal samples from 327 pig farms in northern Vietnam were collected and tested for PEDV infection by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method. The proportion of PEDV-positive farms was 30.9% and PEDV-positive farms were distributed throughout the study area. The highest proportion of PEDV-positive farms was 70% (7/10) among nucleus production type farms (P < 0.05). Higher proportions of PEDV-positive farms were found in the Northeast and Red River Delta areas, which are the major areas of pig production (P < 0.05). The proportion of PEDV-positive farms was higher among larger farms (P < 0.05). Our findings illustrate the high proportion of PEDV-positive farms in the Vietnamese pig population and will help to better understand the epidemiological dynamics of PED infection, to estimate impact, and establish and improve prevention and control measures.

Coinfection of a lingual lesion with bovine papular stomatitis virus and bovine papillomavirus.

To date, there have been no reports of coinfection with bovine papular stomatitis virus (BPSV) and bovine papillomavirus (BPV) in the same lesion. In the present study, one lingual papilloma-like sample was collected at an abattoir from the tongue of a 31-month-old Japanese black cow. Coinfection with BPSV and BPV was confirmed by histopathology, immunohistochemistry, PCR and RT-PCR. The evidence for coinfection with BPSV and BPV in the same lesion and an association of BPV with lingual papillomatosis will contribute to future epidemiological studies of these two viruses.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks.

BACKGROUND: Since late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013-2016, were sequenced and analyzed. RESULTS: Phylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics. CONCLUSIONS: These results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.

Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: Porcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd. RESULTS: In this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed. CONCLUSIONS: The developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.

Comparative analysis of LTR and structural genes in an equine infectious anemia virus strain isolated from a feral horse in Japan.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

Identifying Pathogen and Allele Type Simultaneously in a Single Well Using Droplet Digital PCR.

In the transmission control of chronic and untreatable livestock diseases such as bovine leukemia virus (BLV) infection, the removal of viral superspreaders is a fundamental approach. On the other hand, selective breeding of cattle with BLV-resistant capacity is also critical for reducing the viral damage to productivity by keeping infected cattle. To provide a way of measuring BLV proviral load (PVL) and identifying susceptible/resistant cattle simply and rapidly, we developed a fourplex droplet digital PCR method targeting the BLV pol gene, BLV-susceptible bovine major histocompatibility complex (BoLA)-DRB3*016:01 allele, resistant DRB3*009:02 allele, and housekeeping RPP30 gene (IPATS-BLV). IPATS-BLV successfully measured the percentage of BLV-infected cells and determined allele types precisely. Furthermore, it discriminated homozygous from heterozygous carriers. Using this method to determine the impact of carrying these alleles on the BLV PVL, we found DRB3*009:02-carrying cattle could suppress the PVL to a low or undetectable level, even with the presence of a susceptible heterozygous allele. Although the population of DRB3*016:01-carrying cattle showed significantly higher PVLs compared with cattle carrying other alleles, their individual PVLs were highly variable. Because of the simplicity and speed of this single-well assay, our method has the potential of being a suitable platform for the combined diagnosis of pathogen level and host biomarkers in other infectious diseases satisfying the two following characteristics of disease outcomes: (i) pathogen level acts as a critical maker of disease progression; and (ii) impactful disease-related host genetic biomarkers are already identified. IMPORTANCE While pathogen-level quantification is an important diagnostic of disease severity and transmissibility, disease-related host biomarkers are also useful in predicting outcomes in infectious diseases. In this study, we demonstrate that combined proviral load (PVL) and host biomarker diagnostics can be used to detect bovine leukemia virus (BLV) infection, which has a negative economic impact on the cattle industry. We developed a fourplex droplet digital PCR assay for PVL of BLV and susceptible and resistant host genes named IPATS-BLV. IPATS-BLV has inherent merits in measuring PVL and identifying susceptible and resistant cattle with superior simplicity and speed because of a single-well assay. Our new laboratory technique contributes to strengthening risk-based herd management used to control within-herd BLV transmission. Furthermore, this assay design potentially improves the diagnostics of other infectious diseases by combining the pathogen level and disease-related host genetic biomarker to predict disease outcomes.

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA.

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

Identification of a DRB3*011:01-restricted CD4+ T cell response against bovine respiratory syncytial virus fusion protein.

Although Human Respiratory Syncytial Virus (HRSV) is a significant cause of severe respiratory disease with high morbidity and mortality in pediatric and elderly populations worldwide there is no licensed vaccine. Bovine Respiratory Syncytial Virus (BRSV) is a closely related orthopneumovirus with similar genome structure and high homology between structural and nonstructural proteins. Like HRSV in children, BRSV is highly prevalent in dairy and beef calves and known to be involved in the etiology of bovine respiratory disease, in addition to being considered an excellent model for HRSV. Commercial vaccines are currently available for BRSV, though improvements in efficacy are needed. The aims of this study were to identify CD4+ T cell epitopes present in the fusion glycoprotein of BRSV, an immunogenic surface glycoprotein that mediates membrane fusion and a major target of neutralizing antibodies. Overlapping peptides representing three regions of the BRSV F protein were used to stimulate autologous CD4+ T cells in ELISpot assays. T cell activation was observed only in cells from cattle with the DRB3*011:01 allele by peptides from AA249-296 of the BRSV F protein. Antigen presentation studies with C-terminal truncated peptides further defined the minimum peptide recognized by the DRB3*011:01 allele. Computationally predicted peptides presented by artificial antigen presenting cells further confirmed the amino acid sequence of a DRB3*011:01 restricted class II epitope on the BRSV F protein. These studies are the first to identify the minimum peptide length of a BoLA-DRB3 class II-restricted epitope in BRSV F protein.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100.

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

Association and evidence for linked recognition of type IV secretion system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale.

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+ T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale.

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements.

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.

A pooled testing system to rapidly identify cattle carrying the elite controller BoLA-DRB3*009:02 haplotype against bovine leukemia virus infection.

As genetically resistant individuals, the "elite controllers" (ECs) of human immunodeficiency virus infection have been focused on as the keys to developing further functional treatments in medicine. In the livestock production field, identifying the ECs of bovine leukemia virus (BLV) infection in cattle is desired to stop BLV transmission chains on farms. Cattle carrying the bovine leukocyte antigen (BoLA)-DRB3*009:02 allele (DRB3*009:02) have a strong possibility of being BLV ECs. Most of cattle carrying this allele maintain undetectable BLV proviral loads and do not shed virus even when infected. BLV ECs can act as transmission barriers when placed between uninfected and infected cattle in a barn. To identify cattle carrying DRB3*009:02 in large populations more easily, we developed a pooled testing system. It employs a highly sensitive, specific real-time PCR assay and TaqMan MGB probes (DRB3*009:02-TaqMan assay). Using this system, we determined the percentage of DRB3*009:02-carrying cattle on Kyushu Island, Japan. Our pooled testing system detected cattle carrying the DRB3*009:02 allele from a DNA pool containing one DRB3*009:02-positive animal and 29 cattle with other alleles. Its capacity is sufficient for herd-level screening for DRB3*009:02-carrying cattle. The DRB3*009:02-TaqMan assay showed high-discriminative sensitivity and specificity toward DRB3*009:02, making it suitable for identifying DRB3*009:02-carrying cattle in post-screening tests on individuals. We determined that the percentage of DRB3*009:02-carrying cattle in Kyushu Island was 10.56%. With its ease of use and reliable detection, this new method strengthens the laboratory typing for DRB3*009:02-carrying cattle. Thus, our findings support the use of BLV ECs in the field.

A survey of bovine leukemia virus resistant bovine leukocyte antigen (BoLA)-DRB3*009:02 allele-carrying Japanese Black cattle in two prefectures in Japan.

The bovine leukocyte antigen (BoLA) DRB3*009:02 allele is strongly associated with a low/undetectable bovine leukemia virus (BLV) proviral load. Understanding the status of cattle possessing DRB3*009:02 allele is key for BLV control by breeding. We performed a survey of DRB3*009:02-carrying cattle in two prefectures in Japan using a TaqMan assay developed previously. The allele was found in 3.8% (confidence interval (CI): 3.3-4.3) of 6020 Japanese Black female cattle. A prefecture-level difference was found: the allele was observed in 8.6% CI: 7.5-9.9) of 2242 cattle of the birth prefecture B in Kyushu/Okinawa region, and this percentage was significantly higher than those of prefecture C in Kyushu/Okinawa region (1.3% (CI: 0.4-3.4) of 319) and prefecture A in Chugoku region (0.9% (CI: 0.6-1.4) of 2741), respectively. Consideration on the difference in possession of DRB3*009:02 allele is needed to establish the more efficient control strategy of BLV infection in Japanese Black cattle.

Serological and molecular survey of tick-borne zoonotic pathogens including severe fever with thrombocytopenia syndrome virus in wild boars in Miyazaki Prefecture, Japan.

BACKGROUND: Miyazaki Prefecture is one of the hotspots of severe fever with thrombocytopenia syndrome (SFTS) cases and related deaths in Japan since 2013 and other pathogens of tick-borne diseases (TBDs). Japanese spotted fever and scrub typhus are also endemic in this region. OBJECTIVES: A total of 105 wild boars, hunted in 2009, were serologically examined as sentinels for TBDs to indirectly demonstrate the potential hazard of ticks transmitting pathogens to humans in the studied area. METHODS: The collected blood and spleens of the wild boars underwent serological and molecular tests for SFTSV, Rickettsia japonica (Rj) [antibody to spotted fever group rickettsiae (SFGR) were tested by using species-common antigen], and Orientia tsutsugamushi (Ot). RESULTS: Seroprevalences of SFTSV, SFGR, and Ot were 41.9%, 29.5%, and 33.3%, respectively. SFTS viral RNA was identified in 7.6% of the sera, whereas DNA of Rj or Ot was not detected in any sample. In total, 43.8% of the boars possessed an infection history with SFTSV (viral gene and/or antibody). Of these, 23.8% had multiple-infection history with SFGR and/or Ot. CONCLUSIONS: The high prevalence of SFTSV in wild boars might reflect the high risk of exposure to the virus in the studied areas. In addition, SFTSV infection was significantly correlated with Ot infection, and so were SFGR infection and Ot infection, indicating that these pathogens have common factors for infection or transmission. These data caution of the higher risk of SFTSV infection in areas with reported cases of other TBDs.

Identification of Anaplasma marginale outer membrane protein antigens conserved between A. marginale sensu stricto strains and the live A. marginale subsp. centrale vaccine.

Live vaccination with Anaplasma marginale subsp. centrale (synonym for Anaplasma centrale) induces protection against severe disease upon challenge with A. marginale sensu stricto strains. Despite over a century of field use, the targets of protective immunity remained unknown. Using a broad proteomic approach, we identified the proteins in a challenge sensu stricto strain that were bound by the relevant antibody isotype induced by live vaccination with Anaplasma marginale subsp. centrale. A core of 15 proteins was identified in vaccinated animals across multiple major histocompatibility complex (MHC) haplotypes. This core separated into two structural/functional classes: "housekeeping" proteins involved in replication and metabolism and outer membrane proteins (OMPs). Orthologous proteins of both classes were identified within the vaccine strain and among sensu stricto strains. In contrast to the broad conservation among strains in the sequences of the housekeeping proteins, there was significantly greater divergence in the OMPs and greater divergence in both OMP sequences and the encoding locus structure between the vaccine strain and the sensu stricto strains than among the sensu stricto strains. The OMPs bound by live vaccine-induced antibody overlapped with OMPs that were immunogenic in animals vaccinated with inactivated vaccines and subsequently protected against bacteremia and disease. The identification of this core set of OMPs is consistent with the hypothesis that "subdominant" immunogens are required for vaccine-induced protection against A. marginale and provides clear direction for development of a safer, more effective vaccine.

A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by Babesia bovis merozoites.

OBJECTIVE: The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA. METHODS: Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay. RESULTS: RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival. CONCLUSION: B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.

Characterization of bovine interleukin-2 stably expressed in HEK-293 cells.

Interleukin 2 (IL-2) is a pleotropic cytokine and well-known as a T cell growth factor in immunology. It is now known to exert both immunostimulatory and immunosuppressive effects, optimizing immunological microenvironments for effector and regulatory T cell responses. The immunomodulatory role of IL-2 is critical for deciding whether or not T cell responses against specific antigens result in protection. We have established a mammalian cell line (HEK-293) stably expressing bovine IL-2 (boIL-2) (designated as HEK-293/boIL-2), using the piggyBac transposon system. The concentration of recombinant bovine IL-2 (rboIL-2) in the culture supernatant of HEK-293/boIL-2 reached 100 ng/ml on day 7 and showed similar proliferative activity to recombinant human IL-2 (rhuIL-2) for bovine peripheral mononuclear blood cells. Although rhuIL-2 has been often used to activate bovine T cells, our results indicate that characteristics of the T cell activation through rboIL-2 and huIL-2 appear slightly but significantly different. Interestingly, the rboIL-2/anti-boIL-2 monoclonal antibody (C5) (rboIL-2/C5) complex strongly induced proliferation of bovine NKp46+cells, natural killer (NK) cells, in vitro. This indicates that the rboIL-2/C5 complex could function as an IL-2 agonist specifically to increase the NK cell population, which in turn could enhance the activity of NK cells leading to protective immunity.

Relationship between Allelic Heterozygosity in BoLA-DRB3 and Proviral Loads in Bovine Leukemia Virus-Infected Cattle.

Enzootic bovine leukosis is a lethal neoplastic disease caused by bovine leukemia virus (BLV), belongs to family Retroviridae. The BLV proviral load (PVL) represents the quantity of BLV genome that has integrated into the host's genome in BLV-infected cells. Bovine leukocyte antigen (BoLA) class II allelic polymorphisms are associated with PVLs in BLV-infected cattle. We sought to identify relationships between BoLA-DRB3 allelic heterozygosity and BLV PVLs among different cattle breeds. Blood samples from 598 BLV-infected cattle were quantified to determine their PVLs by real-time polymerase chain reaction. The results were confirmed by a BLV-enzyme-linked immunosorbent assay. Restriction fragment length polymorphism-polymerase chain reaction identified 22 BoLA-DRB3 alleles. Multivariate negative binomial regression modeling was used to test for associations between BLV PVLs and BoLA-DRB3 alleles. BoLA-DRB3.2*3, *7, *8, *11, *22, *24, and *28 alleles were significantly associated with low PVLs. BoLA-DRB3.2*10 was significantly associated with high PVLs. Some heterozygous allele combinations were associated with low PVLs (*3/*28, *7/*8, *8/*11, *10/*11, and *11/*16); others were associated with high PVLs (*1/*41, *10/*16, *10/*41, *16/*27, and *22/*27). Interestingly, the BoLA-DRB3.2*11 heterozygous allele was always strongly and independently associated with low PVLs. This is the first reported evidence of an association between heterozygous allelic combinations and BLV PVLs.