Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

High Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Infection among the Dog Population in Thailand.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). In Thailand, three human cases of SFTS were reported in 2019 and 2020, but there was no report of SFTSV infection in animals. Our study revealed that at least 16.6% of dogs in Thailand were seropositive for SFTSV infection, and the SFTSV-positive dogs were found in several districts in Thailand. Additionally, more than 70% of the serum samples collected at one shelter possessed virus-neutralization antibodies against SFTSV and the near-complete genome sequences of the SFTSV were determined from one dog in the shelter. The dog SFTSV was genetically close to those from Thailand and Chinese patients and belonged to genotype J3. These results indicated that SFTSV has already spread among animals in Thailand.

A serological survey and characterization of Getah virus in domestic pigs in Thailand, 2017-2018.

Getah virus (GETV) is a mosquito-borne RNA virus belonging to the family Togaviridae, genus Alphavirus. GETV infection causes diarrhoea and death in piglets, and reproductive failure and abortion in sows. This study conducted a serological survey of GETV infection among domestic pig populations in Thailand. ELISA was used to analyse 1,188 pig serum samples collected from 11 provinces of Thailand during 2017-2018, with 23.1% of the samples being positive for anti-GETV antibodies. The positive ratio of anti-GETV antibodies was significantly higher in nursery (67.9%) and older stages (84.5%) of pigs than in finishing stage (14.2%). Furthermore, we successfully isolated GETV from one pig serum, designated as GETV strain GETV/SW/Thailand/2017, and determined the complete genome sequence (11,689 nt). Phylogenetic analysis demonstrated that our isolate was different from the recent GETV group spreading among pig populations in East Asia and formed a cluster with two GETV strains, namely YN12031 (China, 2015) and LEIV16275Mar (Far-East Russia, 2007). We concluded that two different GETV groups are currently spreading among pig populations in Asian countries.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

Getah virus epizootic among wild boars in Japan around 2012.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

Upregulation of Indian hedgehog gene in the uterine epithelium by leukemia inhibitory factor during mouse implantation.

Leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) are essential for embryo implantation in mice and are regulated by the actions of 17beta-estradiol (E2) and progesterone, respectively. The present study examined the effect of LIF on Ihh and Ihh-related factors in the uterine luminal epithelium during the implantation period using a DNA microarray. Expression of Ihh mRNA reached its peak on the forth day of pregnancy, and progesterone receptor (Pgr) mRNA decreased on the fifth day of pregnancy in wildtype mice. On the other hand, these changes in expression were not seen in LIF-/- mice. Ihh and Pgr mRNA were upregulated by LIF injection in delayed implantation mice. This up-regulation of Pgr was transient and preceded an increase of Ihh mRNA. Ihh mRNA also increased after E2 injection in delayed implantation mice of the LIF-/- genotype. E2 did not affect transcription of Pgr mRNA in the uterine luminal epithelium of delayed implantation LIF-/- mice. Using an antibody against the C-terminal epitope of Ihh, unprocessed Ihh proteins, but not C-terminal peptides, by autoproteolytic cleavage of Ihh were detected by western blot analysis. Unprocessed Ihh did not show quantitative changes between the wildtype and LIF-/- mice during the implantation period. Transcription of hedgehog acyltransferase was not influenced by LIF and E2 injection. In conclusion, LIF, which has a crucial role in E2 action for initiation of implantation, caused transient induction of Pgr mRNA and subsequent upregulation of Ihh mRNA, which mediates progesterone-Pgr actions for successful implantation.

Effects of bisphenol A (BPA) on placentation and survival of the neonates in mice.

The effects of bisphenol A (BPA) on placentation have not been fully determined. The aim of this study was to clarify the structural changes of the placenta, abortion rate, and survival of neonates after BPA administration in mice. BPA (10 mg/kg/day) was administered to pregnant mice (BPA mice) subcutaneously from the first day of pregnancy (Day 0) to Day 7 (8 days total). The number of embryos and weights of whole uteri were measured on Days 10 and 12. Morphological changes in the placentae were examined by light microscopy on the corresponding days of pregnancy. The number of neonates was also counted. Survival rates were periodically calculated for neonates from the first day after parturition (P-Day 0) to P-Day 56. The number of embryos and weight of the uterus on Days 10 and 12 were significantly decreased by BPA injection. No notable differences were recognized between the left and right uteri. The proportion of the labyrinthine zone per whole placenta in the BPA mice became lower than that in the controls, and that of the metrial gland was higher in the BPA mice. The intervillous spaces of the placenta were narrower in the BPA mice. Degenerative changes were found in the trophoblastic giant cells and spongiotrophoblast layers of the BPA mice. The number of BPA mouse neonates was drastically decreased within 3 days after birth, and no mice survived after P-Day 56. The results suggest that BPA not only disrupts placental functions and leads to abortion through chronic stimulation of gene expression by binding to DNA but that it also affects the mortality of neonates through indirect exposure of embryos.