First detection of Borrelia and Rickettsia species from Ornithodoros ticks in the Republic of Korea.

Ticks are considered important vectors among arthropods and are linked to serious medical and veterinary health problems. In this study, we investigated tick-borne pathogens (TBPs) of Ornithodoros (Carios) sawaii and a newly identified Ornithodoros species from migratory bird nests in the uninhabited islands of the Republic of Korea (ROK). Ticks were collected from seabird nests with soil using a Tullgren funnel. Polymerase chain reaction (PCR) was performed using specific primer sets targeting genes of Borrelia spp., Rickettsia sp., Anaplasma phagocytophilum, Anaplasma bovis, and Bartonella spp. for molecular identification of TBPs, and two pathogens, Borrelia sp. and Rickettsia sp. were detected via PCR. Sequence data were analyzed and a phylogenetic analysis was conducted using the maximum-likelihood method in MEGA v.7. The detection rate of Borrelia sp. in O.(C.) sawaii was 6.8 % (5/74), and that of Rickettsia sp. in O. sawaii and the newly identified Ornithodoros species. was 36.5 % (27/74). Sequencing analysis revealed that the 16S ribosomal (r) RNA and flagellin genes of Borrelia sp., and the citrate synthase (gltA) and 17-kDa antigen gene of Rickettsia sp. were closely phylogenetically related to those of Borrelia turicatae and Rickettsia asembonensis. This is the first report identifying Borrelia sp. and Rickettsia sp. from O. sawaii, and Rickettsia sp. from the newly identified Ornithodoros species in the ROK, and these results imply that soft ticks (O. sawaii, and the newly identified Ornithodoros species) may function as pathogen carriers with important implications for public health throughout their distribution areas in Asia.

Efficient distribution of oral vaccines examined by infrared triggered camera for advancing the control of raccoon dog rabies in South Korea.

The field distribution of the oral rabies vaccine is effective in controlling the spread of rabies. The present study aimed to investigate efficient distribution locations based on the environment, contact rate, and consumption by target wildlife species in South Korea. The target species (Korean raccoon dogs, domestic dogs, and feral cats) accounted for 945 contacts (52.2%), in total 1,808 contacts. There were 863 (47.8%) contacts by non-target species. Raccoon dogs, a main reservoir of rabies in South Korea, had the highest contact rate (34.1%) among all species. The contact rate by target species was highest at riparian sites and bushy mountainous vegetation, where raccoon dogs are abundant. There was remarkable contact by raccoon dogs in mountainous areas below 150 m with bushy vegetation. Our results indicate that these locations are efficient areas for vaccine distribution, especially targeting the raccoon dog. Vaccines were continuously contacted with intervals ranging from one hour to one day. Vaccines at 94.4% of the distribution points were completely consumed within two weeks. The mean consumption rate was 95.2 ± 1.93% during the overall study period. These findings suggest that the oral rabies vaccine attracts wildlife including domestic dogs and feral cats. Our results suggest that low sections of mountainous areas with bushy vegetation and/or neighboring riparian areas are rich in target wildlife species (especially raccoon dogs) and are efficient locations for vaccine distribution to control rabies in South Korea.

First report of Newly Identified Ornithodoros Species in the Republic of Korea.

Ticks and tick-borne diseases are important issues worldwide because of their effects on animal and human health. The genus Ornithodoros, which is included in the family Argasidae, is typically associated with wild animals, including seabirds. In this study, samples from the nests of seabirds and surrounding soil were collected to investigate Ornithodoros spp. from 9 uninhabited islands in the western, eastern, and southern parts of Korea from April 2017 to October 2018. The islands are known as the breeding places of migratory and resident birds. Ticks were collected from soil and nest material of seabirds using a Tullgren funnel and identified using 16S rRNA and the cytochrome c oxidase 1 gene (COI), and host animals of soft ticks were identified using the mitochondrial DNA cytochrome b gene by a polymerase chain reaction. In the sequence identity of the 16S rRNA gene fragment of Ornithodoros sp., Ornithodoros sawaii was identified as the closest homologous sequence, and the new Ornithodoros sp. was newly identified. We found that the newly identified Ornithodoros sp. in the Republic of Korea was located in uninhabited islands used as breeding places by the black-tailed gull, Larus crassirostris.

Epidemiological Investigation of Tick Species from Near Domestic Animal Farms and Cattle, Goat, and Wild Boar in Korea.

This study aimed to investigate the tick species and give background for tick-borne investigations in Korea. Ticks were collected from the area within 2 km radius of the 4 domestic animal farms, where they were located in mountainous areas and raising animals on pasture, and from animal bodies in 2014 and 2015. In total, 7,973 nymphal and adult ticks were collected from the farms - 7,758 Haemaphysalis longicornis, 198 Haemaphysalis flava, and 17 Ixodes nipponensis, and 1,763 were collected from animals - 729 H. longicornis from cattle; 569 H. longicornis from goats; and 297 H. longicornis, 118 H. flava, 1 I. nipponensis, and 49 Amblyomma testudinarium from wild boars. As more species of ticks were collected from wild boars than domesticated animals and their habitats, various animal hosts should be considered while investigating tick species.

Molecular Detection of Anaplasma, Bartonella, and Borrelia theileri in Raccoon Dogs (Nyctereutes procyonoides) in Korea.

Anaplasmosis, cat-scratch disease, and Lyme disease are emerging vector-borne infectious diseases in Korea. Although the prevalence of vector-borne pathogens (VBPs) in domestic animals and vector arthropods has been documented, there is limited information on the presence of VBPs in wild animals. The raccoon dog (Nyctereutes procyonoides), a wild canid found in East Asia and Europe, represents a potential wildlife reservoir for zoonotic diseases. To investigate the prevalence of VBPs in raccoon dogs, 142 carcasses and 51 blood samples from captured raccoon dogs were collected from 2003 to 2010 and from 2008 to 2009, respectively, in Korea. In addition, 105 Haemaphysalis flava (14 larvae, 43 nymphs, 32 males, and 16 females) and nine Haemaphysalis longicornis (all female) were collected from three raccoon dogs. Samples of the spleen and blood were tested for the presence of VBPs by using nested polymerase chain reaction. Among the samples collected from 193 raccoon dogs and 114 ticks, two samples were positive for Anaplasma phagocytophilum, four for Anaplasma bovis, two for Borrelia theileri, and two for Bartonella henselae. To the best of our knowledge, this study is the largest survey of raccoon dogs aimed at the analysis of VBPs in this species. Moreover, the present study represents the first identification of A. phagocytophilum, B. henselae, and B. theileri in raccoon dogs in their native habitat (East Asia).

Relationship between sociability toward humans and physiological stress in dogs.

Sociability is an essential trait for dogs to successfully interact with humans. In this study, the relationship between sociability and physiological stress was examined. Additionally, whether differences exist between companion dogs (C group) and shelter dogs (S group) was examined. Overall, healthy 37 dogs (C group=21 and S group=16) were examined. After 5 min of walking, the dog and the owner (or the chief manager) rested freely in the experimental location for 5 min. The behavioral test with 6 categories was conducted to evaluate sociability over 4 min. The establishment of two groups (H group=dogs with high sociability; L group=dogs with low sociability) was supported by the statistical results of the behavioral tests. Saliva was collected before (P1) and after the test period (P2), and salivary cortisol levels were determined and statistically analyzed. The cortisol concentrations at P2 and the differences in concentrations between P1 and P2 (P2-P1) in the groups with high sociability were significantly lower than those in the groups with low sociability. These results may demonstrate that sociable dogs adapt more comfortably to strangers and unfamiliar situations. Meanwhile, there were significant differences in hormonal results between the C and S groups. For this reason, their sociability should be evaluated using behavioral and physiological assessments before re-adoption to ensure their successful adaptation.

Identification of Tick Species Collected from Wild Boars and Habitats of Wild Boars and Domestic Pigs in the Republic of Korea.

Tick is one of the most important arthropods in the transmission of vector-borne diseases. In this study, we investigated the abundance and species of ticks associated with swine and their habitats to assess the risk of spread of tick-borne diseases in host species, such as wild boars. Ticks were collected from 24 grazing or traditionally reared domestic pig farms and 8 habitats of wild boars in 8 provinces and 1 city in the Republic of Korea, by using the dragging and flagging methods. Ticks were also collected directly from 49 wild boars by using fine forceps. A total of 9,846 hard ticks were collected, including 4,977 Haemaphysalis longicornis, 4,313 Haemaphysalis flava, 508 Ixodes nipponensis, 1 Ixodes turdus, and 47 Amblyomma testudinarium. A total of 240 hard ticks were collected from 49 wild boars, including 109 H. flava, 84 H. longicornis, and 47 A. testudinarium. A total of 578 hard ticks were collected from areas around domestic pig farms. Only 2 hard tick species, 546 H. longicornis and 32 H. flava, were collected from these areas. A total of 9,028 hard ticks were collected from wild boars of 8 habitats, including 4,347 H. longicornis, 4,172 H. flava, 508 I. nipponensis, and 1 I. turdus. A. testudinarium was collected only from wild boars, and I. nipponensis and I. turdus were collected only from the habitats of wild boars.

Elevated cortisol content in dog hair with atopic dermatitis.

Canine atopic dermatitis (CAD) is a chronic relapsing inflammatory skin disease occurring in 10% of the canine population. Although most studies have focused on the pathophysiological mechanism involved in CAD, the detrimental impact of CAD on quality of life has received only little attention. Hair cortisol analysis is becoming a valuable tool in monitoring chronic stress. To further validate this approach in CAD, we compared the hair cortisol concentration of atopic dogs with that of healthy conditioned dogs. The extent and severity of cutaneous lesions of atopic dermatitis were assessed according to modified CADESI-03 scores. In addition, skin barrier function was evaluated by measuring transepidermal water loss (TEWL) and stratum corneum conductance. The correlation between CAD severity and hair cortisol concentration was evaluated. The level of hair cortisol evaluated by ELISA assay showed that the atopic dermatitis group had significantly increased cortisol levels compared to that of the healthy control group. A significant positive correlation was identified between hair cortisol level and the CADESI score in CAD patients. The TEWL value of the cubital flexor of the forelimb in the atopic group was significantly higher compared to the healthy controls. These findings imply that the hair cortisol analysis can be an effective and objective biomarker in assessment of long-term stress of CAD patients.

Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Wild Animals and Ixodidae Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic to central-northeastern China, southern Japan, and the Republic of Korea (ROK). To investigate SFTSV infections, we collected serum samples and ticks from wild animals. Using serum samples and ticks, SFTSV-specific genes were amplified by one-step RT-PCR and nested PCR and sequenced. Indirect immunofluorescence assay (IFA) was performed to analyze virus-specific antibody levels in wild animals. Serum samples were collected from a total of 91 animals: 21 Korean water deer (KWD), 3 Siberian roe deer, 5 gorals, 7 raccoon dogs, 54 wild boars (WBs), and 1 carrion crow. The SFTSV infection rate in wild animals was 3.30% (3 of 91 animals: 1 KWD and 2 WBs). The seropositive rate was 6.59% (6 of 91 animals: 5 KWD and 1 WB). A total of 891 ticks (3 species) were collected from 65 wild animals (9 species). Of the attached tick species, Haemaphysalis longicornis (74.86%) was the most abundant, followed by Haemaphysalis flava (20.20%) and Ixodes nipponensis (4.94%). The average minimum infection rate (MIR) of SFTSV in ticks was 4.98%. The MIRs of H. longicornis, H. flava, and I. nipponensis were 4.51%, 2.22%, and 22.73%, respectively. The MIRs of larvae, nymphs, and adult ticks were 0.68%, 6.88%, and 5.53%, respectively. In addition, the MIRs of fed and unfed ticks were 4.67% and 4.96%, respectively. We detected a low SFTSV infection rate in wild animals, no differences in SFTSV infection rate with respect to bloodsucking in ticks, and SFTSV infection for all developmental stages of ticks. This is the first report describing the detection of SFTSV in wild animals in the ROK.

Evaluation of effects of olfactory and auditory stimulation on separation anxiety by salivary cortisol measurement in dogs.

Separation anxiety (SA) is a serious behavioral problem in dogs. In this study, salivary cortisol was studied to determine if the owner's odor or voice could reduce SA in dogs. Twenty-eight dogs with SA were divided into three groups: group 1 (control), group 2 (with owner's clothes during the separation period; SP) and group 3 (a recording of the owner's voice was played during SP). The dog's saliva was collected after the owner and their dog were in the experimental room for 5 min (PRE). The dog was then separated from the owner for 20 min and saliva collected four times at intervals of 5 min (SP1-4). Finally, the owner was allowed back into the room to calm the dog for 5 min, after which saliva was collected (POST). Evaluation of salivary cortisol concentrations by ELISA revealed that the ratios of SP1 concentration to PRE or POST concentrations were significantly higher in group 1 than in group 2 or 3. Additionally, the concentrations of SP1-PRE and SP1-POST among groups differed significantly. These findings indicate that the owner's odor or voice may be helpful to managing stress in dogs with SA.

Home-care treatment of swimmer syndrome in a miniature schnauzer dog.

A 50-day-old, female miniature schnauzer dog was presented for astasia, dorsoventral flattening of the thorax, hypoplasia of hind-limb muscles, stiffness of hind-limb joints, paddling leg motion, and panting. The dog was diagnosed with swimmers syndrome. The dog recovered completely following 40 days of home-care treatment that involved environmental and nutritional management along with intensive physiotherapy.

Traitement à domicile du syndrome du chiot nageur chez un Schnauzer miniature. Une chienne Schnauzer miniature âgée de 50 jours est présentée pour de l'astasie, un aplatissement dorsiventral du thorax, l'hypoplasie des muscles des pattes arrières, une rigidité des articulations des pattes arrières, un mouvement de pédalage des pattes et des halètements. La chienne a été diagnostiquée avec le syndrome du chiot nageur. La chienne s'est complètement rétablie après 40 jours de traitements à domicile qui ont inclus une gestion de l'environnement et de la nutrition avec une physiothérapie intensive.(Traduit par Isabelle Vallières).

GC/MS analysis of high-performance liquid chromatography fractions from Sophora flavescens and Torilis japonica extracts and their in vitro anti-neosporal effects on Neospora caninum.

We analyzed alcoholic extracts of herbs possessing anti-neosporal activity against Neospora (N.) caninum. To identify the chemical components of Sophora (S.) flavescens and Torilis (T.) japonica associated with anti-neosporal activity, specific fractions were isolated by high-performance liquid chromatography (HPLC). In vitro activity of the fractions against N. caninum was then assessed. Gas chromatography/ mass spectrometry (GC/MS) was used to identify and quantify specific anti-neosporal molecules in the herbal extracts. Almost all HPLC fractions of S. flavescens and T. japonica had higher levels of anti-neosporal activity compared to the not treated control. Active constituents of the extracts were sophoridane, furosardonin A, and tetraisopropylidene-cyclobutane in S. flavescens; 5,17-ß-dihydroxy-de-A-estra-5,7,9,14-tetraene, furanodiene, and 9,12-octadecadienoic acid (Z,Z)-(CAS,1) in T. japonica.

Molecular detection of Bartonella grahamii and B. schoenbuchensis-related species in Korean water deer (Hydropotes inermis argyropus).

We determined the prevalence of Bartonella spp. and investigated which species of Bartonella naturally infects Korean water deer (KWD, Hydropotes inermis argyropus) in the Republic of Korea (ROK). A total of 70 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife (CGRB) in the ROK between 2008 and 2009. Nested PCRs were performed using the rpoB gene and internal transcribed spacer (ITS) region primers to amplify the DNA fragment of Bartonella. Using ITS-based nested PCR, Bartonella grahamii and Bartonella schoenbuchensis-related species were detected in 11 (15.8%) and 9 (12.9%) of 70 KWD spleens, respectively. The 11 B. grahamii amplicons were classified into 2 genotypes by sequence analysis. Using rpoB-based nested PCR, B. grahamii was detected in 5 (7.1%) of 70 KWD spleen samples. This is the first report of B. grahamii and B. schoenbuchensis in KWD, suggesting that KWD may act as reservoirs for the spreading of Bartonella spp. in the ROK.

Immunolocalization of cytoskeletal proteins in the testes of two Asian cervids: water deer (Hydropotes inermis) and Reeves' muntjac (Muntiacus reevesi).

In order to gather basic reproductive information of the water deer and Reeves' muntjac, the immunolocalization of the cytoskeleton proteins in the testes and epididymides of these two species was investigated. The distribution pattern of cytoskeletal proteins in these two species was similar. The desmin was detected in the peritubular myoid cells of the testes and the sub-epithelial cells of the epididymal ducts. Vimentin was observed in the myoid cells, Leydig cells and perinuclear region of the Sertoli cells. Intense immunoreactions for α-smooth muscle actin were restricted to the smooth vascular muscle cells and the peritubular myoid cells in the testes. From the present results, it appears that these distribution patterns of cytoskeletal proteins may be common in the cervids.

Detection of antibodies against the rabies virus in Korean raccoon dogs (Nyctereutes procyonoides koreensis).

Rabies has been reported in dogs, cattle, and raccoon dogs in Korea, especially in the provinces of Gyeonggi and Gangwon. The aim of this study was to investigate the seroprevalence of the rabies in wild Korean raccoon dogs by using 50 blood samples collected in Gyeonggi and Gangwon provinces. Antibodies against rabies were measured by using a fluorescent antibody virus neutralization test. Of the 50 blood samples tested, 20 raccoon dogs (40%) tested positive. Seropositive animals were distributed in Paju, Yangpyeong, Goyang, Yangju, Yanggu, and Hoengseong cities.

Effects of alprazolam on capture stress-related serum cortisol responses in Korean raccoon dogs (Nyctereutes procyonoides koreensis).

The purpose of this study was to evaluate the effect of alprazolam on the stress that Korean raccoon dogs (Nyctereutes procyonoides koreensis) may experience while caught in a live trap by measuring their serum cortisol response. The animals were placed in a live trap with or without being pretreated with oral doses of alprazolam. In both groups, pre-trap blood samples were initially collected without anesthesia before the animals were positioned in the live trap; then post-trap blood samples were collected after the animals had remained in the live trap for 2 h. Changes in cortisol levels were observed using a chemiluminescent immunoassay. The level of cortisol increased in the control group and decreased in the alprazolam-pretreatment group (p < 0.05). In this study, we demonstrated that alprazolam pretreatment reduced stress during live trap capture.

New genetic variants of Anaplasma phagocytophilum and Anaplasma bovis from Korean water deer (Hydropotes inermis argyropus).

Wild deer are one of the important natural reservoir hosts of Anaplasma species, which cause granulocytic anaplasmosis in equines, canines, and humans. The objective of the present study was to determine whether and what species of Anaplasma naturally infect Korean water deer (KWD) in the Republic of Korea. A total of 66 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife in Korea between March 2008 and May 2009. Polymerase chain reaction (PCR) was performed using 16S ribosomal (r)RNA, with ankA, groEL, and msp2 gene primers to amplify the genes of Anaplasma and Ehrlichia. Using 16S rRNA-based nested PCR, Anaplasma phagocytophilum and Anaplasma bovis were detected in 42 (63.6%) and 23 (34.8%) of 66 KWD spleens, respectively. The 42 A. phagocytophilum were classified into five genotypes and the 23 A. bovis were classified into two genotypes by sequence analysis. By ankA-, groEL-, and msp2-based nested PCR, A. phagocytophilum was detected in 1 (1.5%), 7 (10.6%), and 3 (4.6%) of 66 samples, respectively. These gene sequences had only one genotype. Five of seven obtained 16S rRNA gene sequences have never been identified. The ankA, groEL, and msp2 obtained gene sequences represented new genotypes. This is the first report of A. phagocytophilum and A. bovis in KWD, suggesting that they may act as reservoirs for anaplasmosis zoonotic pathogens.

Assessment of experimental saccular aneurysm using selective angiography in common carotid artery of rabbits.

In order to study the treatment of aneurysms, the technique of making experimental aneurysms in laboratory animals must be established. In our study, to examine the feasibility of making experimental aneurysm and selective angiography on the common carotid artery in rabbits and to determine the size of experimental aneurysm after surgery, saccular aneurysms were fashioned on the right common carotid artery in 17 rabbits using a vein pouch technique. Selective angiography of the common carotid artery was performed immediately after surgery, and at 1 week, 4 weeks, and 8 weeks after surgery. Also, histological changes in the aneurysms were observed. In 16 rabbits with established successful experimental aneurysm, no differences were found in diet intake and behavior before and after surgery. The patency of the carotid artery was confirmed by selective angiography. The average size of the aneurysm immediately after surgery was similar to that of 1 week postoperatively in selective angiography, however it increased with time at 4weeks and 8 weeks. Histologically, infiltration of inflammatory cells and hemorrhage were found at the junction of the carotid artery and the vein pouch at 1 week, which disappeared at 4 weeks and 8 weeks. This study suggests experimental saccular aneurysm using the vein pouch technique might form aneurysms similar to that of the human in its properties such as increment of size, and selective angiography might be suitable for assessment of experimental aneurysm. Therefore, this animal model may be suitable for investigating new treatment methodologies for human aneurysms.

Canine behavioral problems and their effect on relinquishment of the Jindo dog.

The purpose of this study was to evaluate behavior problems of the Jindo dog, the native dog of Korea, based on an owner's survey and their effect on pet relinquishment. To live a better life with their own pet and prevent relinquishment, it is important to understand the innate behavior characteristics of dog breed and the potential causes of relinquishment. Information concerning various factors and demonstration of the five most common behavior problems was collected via 189 completed questionnaires. No factors significantly affected the demonstration of behavior problem. A total 151 of 189 dogs had behavior problems (79.9%) and 38 dogs did not have behavior problems (20.1%). Among 151 dogs, 139 dogs showed single behavior problem (92.1%). They were 'excessive excitability' (46.8%), 'excessive vocalization' (30.2%), 'inappropriate elimination' (17.3%), 'destructive behavior' (4.3%), and 'aggressive behavior' (1.4%), respectively. In addition, 12 dogs showed two concurrent behavior problems (7.9%) According to the results, the relinquishment of Jindo dogs was not significantly associated with canine behavior problems, which is the single greatest risk factor of relinquishment in general. The possible reasons for potential behavior problems include improper raising, lack of socialization, and insufficient dog training classes, therefore canine behavior would be improved by owner education.

Prevalence of Bartonella henselae and Bartonella clarridgeiae in cats and dogs in Korea.

Blood, saliva, and nail samples were collected from 54 dogs and 151 cats and analyzed for the presence of Bartonella henselae with a novel nested polymerase chain reaction (PCR) method. Bartonella (B.) henselae was detected in feral cat blood (41.8%), saliva (44.1%), and nail (42.7%) samples. B. henselae was also detected in pet cat blood (33.3%), saliva (43.5%), and nail (29.5%) samples and in pet dog blood (16.6%), saliva (18.5%), and nail (29.6%) samples. Nine samples were infected with B. clarridgeiae and 2 were co-infected with B. henselae and B. clarridgeiae of blood samples of dogs. This report is the first to investigate the prevalence of B. henselae and B. clarridgeiae in dogs and cats in Korea, and suggests that dogs and cats may serve as potential Bartonella reservoirs.