Delivery of engineered interleukin-2 (IL-2) variants (muteins) is thought to be a promising cancer therapy in humans and mice. Our previous study indicated that bovine IL-2 (boIL-2) has a great potential to elicit NK cell activity for which distribution of IL-2 receptors on the target cell surface influences signal transduction. We developed nine boIL-2 muteins and examined the influence of the muteins on bovine peripheral blood mononuclear cells in vitro. On bovine peripheral mononuclear cells, NK cells strongly expressed CD122, followed by CD8+ T cells, while CD4+ T cells and γδ T cells did not show significant CD122 expression. All boIL-2 muteins showed decreasing in binding to boIL-2 receptor α, CD25, while maintaining their ability to bind to boIL-2 receptor βγ, CD122/CD132, heterodimer. The mutein F44A and E63A suppressed CD4+ T cell expansion but maintained the NK cell expansion. These results indicate that boIL-2 muteins can alter immunological outcomes and may be used for clinical intervention for a disease progression.

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.

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.

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.

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.

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.

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.

AbstractBackgroundMiyazaki 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.ObjectivesA 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.MethodsThe 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).ResultsSeroprevalences 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.ConclusionsThe 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.

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.

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.

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.

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.

A cross-sectional study was used to identify and assess prevalence and phenotypic antimicrobial resistance (AMR) profiles of Escherichia coli and other enterobacteria isolated from healthy wildlife and livestock cohabiting at a 10,000 acres game ranch near Lusaka, Zambia. Purposive sampling was used to select wildlife and livestock based on similarities in behavior, grazing habits and close interactions with humans. Isolates (n = 66) from fecal samples collected between April and August 2018 (n = 84) were examined following modified protocols for bacteria isolation, biochemical identification, molecular detection, phylogenetic analysis, and antimicrobial susceptibility testing by disc diffusion method. Data were analyzed using R software, Genetyx ver.12 and Mega 6. Using Applied Profile Index 20E kit for biochemical identification, polymerase chain reaction assay and sequencing, sixty-six isolates were identified to species level, of which Escherichia coli (72.7%, 48/66), E. fergusonii (1.5%, 1/66), Shigella sonnei (22.7%, 14/66), Sh. flexinerri (1.5%, 1/66) and Enterobacteriaceae bacterium (1.5%, 1/66), and their relationships were illustrated in a phylogenetic tree. Phenotypic antimicrobial resistance or intermediate sensitivity expression to at least one antimicrobial agent was detected in 89.6% of the E. coli, and 73.3% of the Shigella isolates. The E. coli isolates exhibited the highest resistance rates to ampicillin (27%), ceftazidime (14.3%), cefotaxime (9.5%), and kanamycin (9.5%). Multidrug resistance (MDR) was detected in 18.8% of E. coli isolates while only 13.3% Shigella isolates showed MDR. The MDR was detected among isolates from impala and ostrich (wild animals in which no antimicrobial treatment was used), and in isolates from cattle, pigs, and goats (domesticated animals). This study indicates the possible transmission of drug-resistant microorganisms between animals cohabiting at the wildlife–livestock interface. It emphasizes the need for further investigation of the role of wildlife in the development and transmission of AMR, which is an issue of global concern.

Enzootic bovine leucosis (EBL) is a neoplastic disease of cattle caused by Bovine leukaemia virus (BLV). EBL causes great economic losses, so a fast and reliable diagnostic method is critical for understanding the status of BLV. This will allow us to control BLV infections efficiently and mitigate economic losses. In this study, we established a direct diagnostic test for BLV using dried blood-spotted filter papers without sample pre-treatment. The study was based on 159 clinical blood specimens collected in EDTA from one farm in Kyushu, Japan. The blood-spotted filter papers were used as the template for direct filter PCR. When an ELISA was used as the diagnostic gold standard, the sensitivity and specificity of the direct filter PCR were 90.1% and 97.5%, respectively. The kappa value for the direct filter PCR and real-time PCR methods was 0.97. The dried blood samples spotted onto filter papers were stable for at least 10 days at room temperature, even when the samples were from cattle with a low BLV proviral load. Direct filter PCR is a rapid, easy, reliable and cost-effective diagnostic test that directly detects the BLV proviral genome in clinical blood specimens without DNA extraction. Moreover, it simplifies the collection, transportation and storage procedures for clinical blood specimens.

African swine fever (ASF) is emerging in Vietnam and poses a continuing severe threat to the swine industry. A histopathological study of clinical samples collected during the May to July 2019 outbreak of ASF was performed to determine the characteristic lesions. We analysed samples from eight ASFV-infected farms. Histopathological results revealed the characteristic lesions of the acute to the subacute clinical form of ASF. Immunohistochemical results showed ASFV viral antigen distribution in mononuclear cells/macrophage in various organs, hepatocytes and renal tubular epithelium. Molecular analysis of partial capsid protein 72 gene revealed that ASFV strain from the eight separate outbreaks belonged to genotype II.

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.

The cattle industry is suffering economic losses caused by bovine leukemia virus (BLV)and enzootic bovine leukosis (EBL), the clinical condition associated with BLV infection. Thispathogen spreads easily without detection by farmers and veterinarians due to the lack of obviousclinical signs. Cattle movement strongly contributes to the inter-farm transmission of BLV. Thisstudy quantified the farm-level risk of BLV introduction using a cattle movement analysis. Ageneralized linear mixed model predicting the proportion of BLV-infected cattle was constructedbased on weighted in-degree centrality. Our results suggest a positive association between weightedin-degree centrality and the estimated number of introduced BLV-infected cattle. Remarkably, theintroduction of approximately six cattle allowed at least one BLV-infected animal to be added to thefarm in the worst-case scenario. These data suggest a high risk of BLV infection on farms with ahigh number of cattle being introduced. Our findings indicate the need to strengthen BLV controlstrategies, especially along the chain of cattle movement.

Porcine epidemic diarrhea virus(PEDV) causes enteritis, vomiting, watery diarrhea, and high mortality in suckling pigs, threatening the swine industry. Porcine epidemic diarrhea (PED) re-emerged globally in 2013 in many important swine-producing countries in Asia and the Americas. Several studies have identified the risk factors for the spread of PEDV in acute outbreaks. However, limited information is available on the risk factors for the transmission of PEDV in endemic regions. We hypothesized that poor biosecurity, location, and some social or cultural practices are the main risk factors for PEDV transmission in the Vietnamese pig population. The aim of this study was to evaluate the potential risk factors for the transmission of PEDV in an endemic area in Vietnam. In this case-control study, questionnaires containing 51 questions were completed for 92 PEDV-positive and 95 PEDV-negative farms. A logistic regression analysis was performed to assess the risk factors associated with PEDV infection. Province and the total number of pigs were included as random effects to determine their influence on the risk of PEDV infection. Twenty-nine variables of interest that have been associated with PEDV status were analyzed in a univariate analysis (P<0.20), with backward stepwise selection. Only three of these 29 variables in four models remained significant PEDV risk factors in the final model: farrow-to-wean production type, distance from the farm to the slaughterhouse (<1,000 m), and the presence of chickens on site (P<0.05). This is the first study to identify the main risk factors for PEDV infection in an endemic area. Our findings suggest that hygiene measures should be strictly implemented on farms for the effective control and prevention of PEDV infection.

Teat papillomatosis is one important infectious disease affecting cattle health and results in significant economic losses especially in the dairy industry. Although there is a large number of commercial cattle herds in China, limited information is available for molecular epidemiological investigation of bovine papillomaviruses (BPVs). In October 2017, an outbreak of teat papillomatosis occurred in the Shandong Province of China. Samples were collected and diagnosed with PCR, and 3 full-length viral genomes were amplified from tissue samples collected from 3 outbreak farms. Analysis results revealed that the outbreak was associated with BPV type 10. This is the first report of BPV-10 infection in China and will contribute to the molecular epidemiological study of the disease.