Effects of a novel recombinant Gonadotropin-Releasing Hormone-1 vaccine on the reproductive function of mixed-breed dogs (Canis familiaris) in Taiwan.

Immunocastration is an effective alternative to surgical castration for controlling the population of animals. As gonadotropin-releasing hormone (GnRH) regulates the reproductive endocrine system in mammals, it is a target antigen for vaccine formulation. Through this study, we evaluated the effectiveness of a recombinant subunit GnRH-1 vaccine for the immunocastration of the reproductive function of 16 mixed-breed dogs (Canis familiaris) provided voluntarily by different households. All the dogs were deemed clinically healthy prior to and during the experiment. A specific anti-GnRH immune response was detected at Week 4, which was maintained for at least 24 weeks after vaccination. Moreover, decreased levels of sexual hormones (testosterone as well as progesterone and estrogen, respectively) were observed in both male and female dogs. Estrous suppression was apparent in female dogs, and testicular atrophy and poor semen quality (concentration, abnormality, and viability) were observed in male dogs. In conclusion, the recombinant subunit GnRH-1 vaccine could successfully suppress fertility and delay the estrous cycle in canines. These results support the efficacy of the recombinant subunit GnRH-1 vaccine; thus, it is a suitable candidate for fertility control in dogs.

Effects of a Recombinant Gonadotropin-Releasing Hormone Vaccine on Reproductive Function in Adult Male ICR Mice.

Gonadotropin-releasing hormone (GnRH) regulates the reproductive endocrine system in mammals. The GnRH immunocontraception vaccine can aid animal population control and management. We evaluated a recombinant GnRH fusion protein with the adjuvant MONTANIDE ISA 206 VG as a GnRH vaccine in adult male ICR mice by evaluating anti-GnRH antibodies; concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone; testis size and histomorphology; and semen quality. Response was assessed after intramuscular administration of the vaccine to mice in weeks 0, 4, and 8. The vaccine induced specific antibody response by week 5, with peak of antibody levels observed by week 13 and a declining level thereafter until the end of the study at week 24. Furthermore, it reduced serum FSH, LH, and testosterone concentrations. The vaccinated mice exhibited testicular atrophy and reduced sperm quality, concentration, morphology, and viability compared to control males. The outcomes of pairings of treated males with untreated females revealed reduced mating, pregnancy rates and number of litters compared to control pairings. Assessment of this GnRH vaccine in different species could assist its development for future applications.

Molecular Characteristics of Carnivore protoparvovirus 1 with High Sequence Similarity between Wild and Domestic Carnivores in Taiwan.

Carnivore protoparvovirus 1 (CPPV-1) is a DNA virus causing gastrointestinal disease and immunosuppression in various terrestrial carnivores. Domestic dogs and cats are considered the primary CPPV-1 reservoirs. The habitat overlap of wild carnivores and free-roaming dogs increases the threat of CPPV-1 transmission between them. This study explored the CPPV-1 distribution among wild carnivores in Taiwan through PCR screening and compared the partial capsid protein (VP2) gene sequences from wild and domestic carnivores. In total, 181 samples were collected from 32 masked palm civets (Paguma larvata), 63 Chinese ferret badgers (Melogale moschata), and 86 crab-eating mongooses (Herpestes urva), from 2015 to 2019 were screened for CPPV-1. The average prevalence of CPPV-1 was 17.7% (32/181), with the highest prevalence in masked palm civets (37.5%). In addition, a masked palm civet was coinfected with two CPPV-1 strains. Among the 33 partial VP2 gene sequences, 23 were identical to the sequences amplified from domestic dogs and cats in Asia, and the remaining 10 were identified for the first time. This study supported the circulation of CPPV-1 strains with the same genomic features as domestic carnivores that are also in wild carnivores from the same environment in Taiwan by molecular data. Therefore, further population control and health management of free-roaming domestic carnivores are recommended.

Association between stress and bilateral symmetrical alopecia in free-ranging Formosan macaques in Mt. Longevity, Taiwan.

Since 2013, a high incidence of bilateral symmetrical alopecia has been observed in free-ranging Formosan macaques (Macaca cyclopis) in Mt. Longevity, Taiwan. We hypothesized that stress induces alopecia in this population. To verify our hypothesis, we evaluated the histopathological characteristics of skin biopsy and used a validated enzyme immunoassay (EIA) for fecal glucocorticoid metabolite (FGM) analysis, which act as an indicator of stress experienced by the individual. Follicular densities were lower (2.1-3.0 mm2) in individuals with symmetrical alopecia than in those with normal hair conditions (4.7 mm2). Furthermore, anagen to catagen/telogen ratios were lower in individuals with alopecia (0-1.4) than in those with normal hair (4.0). The histopathological characteristics of alopecia were similar to those of telogen effluvium, which indicates that stress is one of the possible etiologies. On the basis of the analytical and biological validation of EIAs for FGM analysis, 11ß-hydroxyetiocholanolone was considered suitable for monitoring adrenocortical activity in both sexes of Formosan macaques. The mean concentrations (standard error; sample size) of 11ß-hydroxyetiocholanolone were 2.02 (0.17; n = 10) and 1.41 (0.10; n = 31) µg/g for individuals with and without alopecia, respectively. Furthermore, the results of logistic regression analysis show that 11ß-hydroxyetiocholanolone (p = 0.012) concentration was positively associated with alopecia. Thus, stress was the most likely to trigger symmetrical alopecia in Formosan macaques in Mt. Longevity. Although stress can decrease the fitness of an individual, considering the population status of Formosan macaques in Taiwan is stable and alopecia was only observed in our study area, which is isolated from other populations, the impact on the total population of Formosan macaque in Taiwan is limited. Nonetheless, stress-induced immunosuppression and alopecia might affect the local abundance and increase zoonosis risk due to frequent human-macaque contact in Mt. Longevity. Future studies are suggested to focus on the causative factor of stress and the effects of stress and alopecia on the health and welfare in the Formosan macaques.

Molecular survey of selected viral pathogens in wild leopard cats (Prionailurus bengalensis) in Taiwan with an emphasis on the spatial and temporal dynamics of carnivore protoparvovirus 1.

The leopard cat (Prionailurus bengalensis) was listed as an endangered species under the Wildlife Conservation Act in Taiwan in 2009. However, no study has evaluated the possible direct or indirect effects of pathogens on the Taiwanese leopard cat population. Here, we targeted viral pathogens, including carnivore protoparvovirus 1 (genus Protoparvovirus), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), coronaviruses (CoVs), and canine distemper virus (CDV), through molecular screening. The spatial and temporal dynamics of the target pathogens were evaluated. Through sequencing and phylogenetic analysis, we clarified the phylogenetic relationship of viral pathogens isolated from leopard cats and domestic carnivores. Samples from 23 live-trapped leopard cats and 29 that were found dead were collected from 2015 to 2019 in Miaoli County in northwestern Taiwan. Protoparvoviruses and CoVs were detected in leopard cats, and their prevalence (95% confidence interval) was 63.5% (50.4%-76.6%) and 8.8% (0%-18.4%), respectively. Most of the protoparvovirus sequences amplified from Taiwanese leopard cats and domestic carnivores were identical. All of the CoV sequences amplified from leopard cats were identified as feline CoV. No spatial or temporal aggregation of protoparvovirus infection in leopard cats was found in the sampling area, indicating a wide distribution of protoparvoviruses in the leopard cat habitat. We consider sympatric domestic carnivores to be the probable primary reservoir for the identified pathogens. We strongly recommend management of protoparvoviruses and feline CoV in the leopard cat habitat, particularly vaccination programs and population control measures for free-roaming dogs and cats.

Co-circulation of both low and highly pathogenic avian influenza H5 viruses in current poultry epidemics in Taiwan.

Highly pathogenic avian influenza (HPAI) A(H5) viruses belonging to clade 2.3.4.4c of the A/goose/Guangdong/1/96-like (Gs/GD) lineage caused severe global outbreaks in domestic birds from 2014 to 2015, that also represented the first incursions of Gs/GD viruses into Taiwan and the USA. However, few studies have investigated the circulation of clade 2.3.4.4c viruses after 2015. Here, we describe Gs/GD clade 2.3.4.4c and Mexican-like H5N2 viruses that were isolated in Taiwan during active surveillance conducted in chicken farms from February to March 2019. Phylogenetic analysis demonstrated two distinct genome constellations of the clade 2.3.4.4c H5 viruses, with the internal genes of one of the new genotypes closely related to a virus isolated from a pintail (Anas acuta) in Taiwan, providing the first direct evidence that migratory birds play a role in importing viruses into Taiwan. Our study also confirmed the co-circulation of Gs/GD clade 2.3.4.4c and Mexican-like H5 lineage viruses in Taiwan, presenting a rare case where Gs/GD viruses developed sustained transmission alongside another enzootic H5 lineage, raising the possibility that homosubtypic immunity may mask virus transmission, potentially frustrating detection, and the implementation of appropriate control measures. To eradicate H5 viruses from poultry in Taiwan, further studies on the effect of co-circulation in poultry of low pathogenic avian influenza and HPAI viruses are needed. Furthermore, only with continued surveillance efforts globally can we fully discern dispersal patterns and risk factors of virus transmission both to and within Taiwan.

Distribution of Carnivore protoparvovirus 1 in free-living leopard cats (Prionailurus bengalensis chinensis) and its association with domestic carnivores in Taiwan.

Carnivore protoparvovirus 1 (CPPV-1) is widespread among free-living carnivores, and CPPV-1 infection may directly or indirectly impact on the population of endangered carnivore species. In this study, we used molecular screening of viral capsid protein 2 (VP2) from 2015 to 2017, to assess the prevalence of CPPV-1 infection in 9 live-trapped (LT) and 17 vehicle collision (VC)-affected free-living leopard cats (Prionailurus bengalensis chinensis). In addition, we conducted the phylogenetic analysis to evaluate the possible transmission of CPPV-1 between domestic carnivores and leopard cats. We identified the circulation of feline parvovirus and variants of canine parvovirus (CPV), including CPV-2a, CPV-2b, and CPV-2c, in the free-living leopard cat population. The partial sequences of different variants of VP2 obtained from the leopard cats were identical with those obtained from the domestic dogs and cats in Taiwan. Our result suggested that CPPV-1 was currently transmitted between domestic carnivores and leopard cats in Taiwan. A plan of conservation measures based on vaccination program for domestic carnivores, strict controls on populations of free-living dogs and cats and limiting road development only to low-risk areas for leopard cats should be encouraged.

ENTAMOEBA SPP. IN WILD FORMOSAN ROCK MACAQUES (MACACA CYCLOPIS) IN AN AREA WITH FREQUENT HUMAN-MACAQUE CONTACT.

Entamoeba is a genus of gastrointestinal protozoon that is transmitted through contaminated food and water. This protozoon is commonly found in human and nonhuman primates. Contact between humans and Formosan rock macaques (Macaca cyclopis) has become more frequent due to food provisioning; accordingly, concerns regarding zoonotic pathogen transmission through the fecal-oral route have increased. For example, surveillance of intestinal parasites in wild Formosan rock macaques indicated that Entamoeba infection was the most prevalent type of intestinal parasite infection. The morphologies of pathogenic and nonpathogenic species are difficult to distinguish. In this study, we collected fecal samples from wild Formosan rock macaques in the Shoushan National Nature Park (Kaohsiung, Taiwan) and adopted both morphologic and molecular methods for Entamoeba species identification. In total, we collected 208 fecal samples with a 57.7% (120/208, 95% confidence interval: 50.9-60.4%) prevalence of Entamoeba infection. Four Entamoeba species were identified: three nonpathogenic species, Entamoeba coli (19%), Entamoeba chattoni (50%), and Entamoeba hartmanni (11%), and one potentially pathogenic species, Entamoeba nuttalli (20%). Our study revealed the risk of zoonotic transmission of these Entamoeba species to humans. To address relevant public health and wildlife conservation concerns, further research is required to fully understand the virulence of E. nuttalli isolated from Formosan rock macaques.

Encephalitis induced by a newly discovered ruminant rhadinovirus in a free-living Formosan sambar deer (Rusa unicolor swinhoei).

We documented a case of a free-living Formosan sambar deer (Rusa unicolor swinhoei) infected with a newly discovered ruminant Rhadinovirus (RuRv). Non-purulent encephalitis was the primary histological lesion of the sambar deer. We conducted nested PCR to screen for herpesvirus using generic primers targeting the DNA polymerase gene. In addition, we found that DNA polymerase gene of the sambar deer RuRv was present in the macrophage distributed in the Virchow Robin space with histopathologic lesions by chromogenic in-situ hybridization (CISH). The phylogenetic analysis indicated a high similarity between the viral sequence isolated from fallow deer and our case. This result suggests the possibility of cross-species transmission from other exotic Cervidae reservoir to the Formosan sambar deer.