Hemorrhagic disease caused by bovine viral diarrhea virus-2a in Korean Indigenous Cattle: case reports

Bovine viral diarrhea virus (BVDV), which is prevalent worldwide, is one of the most important viral pathogens and causes substantial economic loss to the livestock industry. Despite its importance, BVDV is largely unnoticed in the Republic of Korea (ROK). In this study, we report the case of a steer with BVDV that died suddenly due to severe enteritis. Two 1-year-old Korean indigenous cattle in the same herd presented severe hemorrhagic diarrhea. Case 1 had severe dehydration and died after 3 days, whereas case 2 had anorexia, depression, and severe diarrhea with mucus and blood. Only case 2 was necropsied, and BVDV2a was detected in the tissues of its alimentary tract. Gross lesions, including erosion, ulceration, and extensive hemorrhage, were observed in the digestive tract mucosa. Immunohistochemistry revealed marked positive staining for BVDV2a antigen in the large intestine. This report describes the first case of hemorrhagic disease caused by acute BVDV2a infection, which is characterized by high mortality in Korean indigenous cattle. This study will help establish vaccination and control strategies for BVDV in the ROK.

Serological evidence of Anaplasma spp., Borrelia burgdorferi and Ehrlichia canis in dogs from the Republic of Korea by rapid diagnostic test kits

Background@#Emergent and re-emergent canine tick-borne infections are attracting increasing attention worldwide. The rise in pet ownership and the close relationship between dogs and their owners are the most concerning factors because dogs may act as competent reservoirs for human tick-transmitted infectious agents. @*Objectives@#This study contributes to the epidemiological surveillance of canine ticktransmitted infections with zoonotic risk in the Republic of Korea (ROK) by investigating the seroprevalence of the pathogens, Anaplasma spp., Borrelia burgdorferi, and Ehrlichia canis. @*Methods@#Four hundred and thirty whole blood samples from domestic dogs were collected in seven metropolitan cities and nine provinces in the ROK and tested using SensPERT Ab test kits (VetAll Laboratories®) to detect seroreactive animals. @*Results@#The seroprevalence rates identified were 9.8% (42/430) for Anaplasma spp., 2.8% (12/430) for B. burgdorferi, and 1.4% (6/430) for E. canis. The risk factors evaluated in this study that could be associated with the development of a humoral immune response, such as sex, age, and history of tick exposure, were similar. There was only one exception for dogs seroreactive to Anaplasma spp., where the risk factor “tick exposure” was statistically significant (p = 0.047). @*Conclusions@#This serological survey exhibited the widespread presence of Anaplasma spp., B.burgdorferi, and E. canis throughout the ROK. Hence, dogs may play a key role as the sentinel animals of multiple zoonotic infectious agents in the country.

Euryhelmis squamula (Digenea: Heterophyidae) Recovered from Korean Raccoon Dog, Nyctereutes procyonoides koreensis, in Korea

In this study, we intended to describe an unrecorded species of heterophyid trematode recovered from the small intestine of a Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. A total of 13 small flukes were collected from a deceased Korean raccoon dog which was found in Chuncheon-si, Gangwon-do, Korea in May 2017. The trematode body were covered with many small spines, rectangular, broader than long, 807-1,103 µm long and 1,270-1,550 µm wide. Oral sucker in the anterior end slightly smaller than acetabulum. Pharynx muscular and well developed. Esophagus relatively long and sigmoid. Acetabulum small and located at median in anterior 2/5 portion. Ceca bifurcated at the anterior of genital pore and acetabulum and terminated at testis level. Testes larger, deeply lobed and located at the near of posterior end of body. Ovary small, triangular and located at the slight left of median and the anterior of left testis. Vitelline follicles dendritic and extend from the middle level of esophagus to the posterior portion of body. Eggs embryonated, operculated, small and 33-35×15-16 µm in size. Based on the morphological characteristics, the small heterophyid flukes recovered from the small intestines of Korean raccoon dog, N. procyonoides koreensis, were identified as Euryhelmis squamula (Digenea: Heterophyidae). Accordingly, this species of heterophyid flukes is to be a new trematode fauna in Korea by this study.

Euryhelmis squamula (Digenea: Heterophyidae) Recovered from Korean Raccoon Dog, Nyctereutes procyonoides koreensis, in Korea

In this study, we intended to describe an unrecorded species of heterophyid trematode recovered from the small intestine of a Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. A total of 13 small flukes were collected from a deceased Korean raccoon dog which was found in Chuncheon-si, Gangwon-do, Korea in May 2017. The trematode body were covered with many small spines, rectangular, broader than long, 807-1,103 µm long and 1,270-1,550 µm wide. Oral sucker in the anterior end slightly smaller than acetabulum. Pharynx muscular and well developed. Esophagus relatively long and sigmoid. Acetabulum small and located at median in anterior 2/5 portion. Ceca bifurcated at the anterior of genital pore and acetabulum and terminated at testis level. Testes larger, deeply lobed and located at the near of posterior end of body. Ovary small, triangular and located at the slight left of median and the anterior of left testis. Vitelline follicles dendritic and extend from the middle level of esophagus to the posterior portion of body. Eggs embryonated, operculated, small and 33-35×15-16 µm in size. Based on the morphological characteristics, the small heterophyid flukes recovered from the small intestines of Korean raccoon dog, N. procyonoides koreensis, were identified as Euryhelmis squamula (Digenea: Heterophyidae). Accordingly, this species of heterophyid flukes is to be a new trematode fauna in Korea by this study.

Morphological and Molecular Characterization of Toxocara apodemi (Nematoda: Ascarididae) from Striped Field Mice, Apodemus agrarius, in Korea

Adult ascarid worms from the field mice, Apodemus agrarius, were observed with a light and scanning electron microscope, and molecularly analized with 18S rRNA gene. In the scanning electron microscope, 3 prominent labia were present in the anterior end of male and female worms, but the interlabia and gubernaculum were absent. Scanning electron micrographs showed cervical alae as vestigial organs that looked like a slightly uplifted superficial sewing stitch. Total 6 pairs of post-cloacal papillae were observed on the tail of the male worms. The tail of female worms was blunt and conical shape with a spine-like structure, mucron. The eggs were sub-globular, coated with the albuminous layer and 73 by 82 μm in average size. The superficial pits of T. apodemi egg (mean 8.6×6.7 μm) are obviously bigger than those of Toxocara spp. The partial sequence of 18S rRNA showed the sequence homology of Toxocara canis (99.6%), Toxocara cati (99.4%), Toxascaris leonina (99.4%), and Toxocara vitulorum (99.2%). Conclusively, it was confirmed that ascarid nematodes, Toxocara apodemi, recovered from striped field mice in Korea are taxonomically conspecific relationship with genus Toxocara and genetic divergence from other Toxocara species.

Electrolyte and acid-base imbalance in native calves with enteropathogenic diarrhea

Abstract: Diarrhea is the most common cause of death in calves, and remains a major health challenge. Although there are many studies on the related pathogens, the understanding of the clinicopathological changes is limited. This study aimed to identify the pathogens and observe the clinicopathological changes in electrolytes and acute phase proteins (APPs) associated with diarrhea.Blood samples and fecal samples were collected from 141 calves for the determination of APPs, electrolyte and acid-base status and identification of enteropathogens, respectively. Single or co-infections with enteropathogens, including virus (bovine viral diarrhea virus, coronavirus, and rotavirus), Eimeria, Cryptosporidium, and Escherichia coliK99 were detected in both non-diarrheic and diarrheic calves. Levels of APPs such as serum amyloid A, haptoglobin and fibrinogen were comparable between diarrheic and nondiarrheic calves. Hypoglycemia, high blood urea, electrolytes and acid-base imbalance (hyponatremia, hypochloremia, and decreased bicarbonate), and strong ion difference (SID) acidosis showed a significant association in diarrheic calves (p < 0.01). Particularly, significant hyponatremia, bicarbonate loss, SID acidosis, hypoglycemia, and elevated blood urea nitrogen were found in rotavirusinfected calves. Monitoring the clinicopathological parameters of APPs and electrolyte levels could be vital in the clinical management of diarrheic calves.

Urogonimus turdi (Digenea: Leucochloridiidae) from the White’s Thrush, Zoothera aurea, in the Republic of Korea

Avian trematodes, Urogonimus turdi (Digenea: Leucochloridiidae), were collected from the intestine of wild birds, Zoothera aurea, 2013–2017 in the Daejeon Metropolitan City, Korea. The body was ellipsoidal, attenuated and/or round ends, 1,987–2,120 long and 819–831 μm wide. The oral sucker was subterminal, rounded anteriorly, and 308–425×351–432 μm in size; the prepharynx and esophagus were almost lacking; pharynx was well-developed, 142–179×78–170 μm in size; intestine narrow, bifurcating just after pharynx, ascending to the oral sucker before looping posteriorly and terminating near the posterior end; ventral sucker larger, in almost median, 536–673×447–605 μm and approximately 1.5 times larger than oral sucker. A phylogenetic tree constructed with 18S ribosomal RNA showed inter- and intraspecific relationships. Based on these morphological and molecular findings, we report here a U. turdi from White’s thrushes in Korea.

Prevalence of coronavirus from diarrheic calves in the Republic of Korea

Objective: To investigate the prevalence of bovine coronavirus (BCoV), bovine rotavirus, and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea. Methods: Samples were obtained from 50 normal and 93 diarrheic (56 semi-formed, 28 loose, and 9 watery feces) calves in six different regions of northern and southern Korea. These fecal samples were tested for BCoV, bovine rotavirus, and bovine viral diarrhea virus by RT-PCR. Results: Among the three pathogens examined, infection with BCoV was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio (OR)=9.3, 95% confidence interval (CI): 1.1-78.9; P=0.02). Infection with BCoV alone (OR=2.9; 95% CI: 1.1-7.6; P=0.03) or co-infection of BCoV with bovine viral diarrhea virus (OR=3.6; 95% CI: 1.0-12.4; P=0.04) was significantly associated with the development of loose feces. Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves (OR=0.2; 95% CI: 0.1-0.4; P=0.00) and calves that had not been fed colostrum (OR=0.2; 95% CI: 0.1-0.7; P=0.02), respectively. Conclusions: The present study suggests that BCoV is involved in calf diarrhea in the Republic of Korea. Therefore, grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.

Molecular Identification of Sarcocystis grueneri in Wild Korean Water Deer (Hydropotes inermis argyropus)

The cysts of Sarcocystis grueneri were detected and characterized from the cardiac muscles of the Korean water deer (Hydropotes inermis argyropus). Of the 38 heart muscle samples examined by light microscopy, 10 were found infected with the cysts of Sarcocystis sp. The cysts appeared oval to spherical shape and measured 110–380 μm in length and 90–170 μm in width. A phylogenetic tree of the 18S rRNA sequences (1.5 kb) revealed a close relationship of the infected cysts to genus Sarcocystis. The 18S rRNA sequence of the infected cysts showed 100% identity to S. grueneri and 97% to S. capracanis. Here, we first report the S. grueneri infections in the Korean water deer.

Detection and Identification of Sarcocystis cruzi (Protozoa: Apicomplexa) by Molecular and Ultrastructural Studies in Naturally Infected Korean Cattle (Bos taurus coreanae) from Daejeon, Korea

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately 0.21–1.25 μm in length and 0.05–0.07 μm in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.

Epidemiological Survey on Eimeria spp. Associated with Diarrhea in Pre-weaned Native Korean Calves

Bovine coccidiosis is one of the most important parasitic diseases affecting calf productivity. Here, we investigated the prevalence of Eimeria spp. in pre-weaned native Korean calves and determined the correlation between diarrhea and Eimeria spp. Fecal samples were collected from individual calves (288 normal and 191 diarrheic) in 6 different farms. Of the 479 samples, Eimeria oocysts were detected in 124 calves (25.9%). Five Eimeria spp. were identified; E. zuernii (18.8%) was the most prevalent, followed by E. auburnensis (12.5%), E. bovis (7.5%), E. subspherica (5.8%), and E. bukidnonensis (1.0%). A significant correlation was observed between diarrhea and mixed infection with more than 2 Eimeria spp. (odds ratio [OR]=2.21; 95% confidence interval [CI]: 1.09–4.49; P=0.03) compared to single infection (OR=1.29; 95% CI: 0.77–2.15; P=0.33). Of the 5 Eimeria spp. identified, E. subspherica (95% CI: 1.24–5.61; P=0.01) and E. bukidnonensis (95% CI: 825.08–1,134.25; P=0.00) strongly increased the risk of diarrhea by 2.64-fold and 967.39–fold, respectively, compared to other species. Moreover, mixed infection with E. auburnensis and E. bukidnonensis was significantly associated with diarrhea (OR=2,388.48; 95% CI: 1,009.71–5,650.00; P < 0.00) in pre-weaned native Korean calves. This is the first report to demonstrate the importance of E. bukidnonensis associated with diarrhea in pre-weaned native Korean calves. Further epidemiological studies should investigate the prevalence of E. bukidnonensis and the association between E. bukidnonensis and diarrhea.

Prevalence of coronavirus from diarrheic calves in the Republic of Korea

Objective:To investigate the prevalence of bovine coronavirus (BCoV),bovine rotavirus,and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea.Methods:Samples were obtained from 50 normal and 93 diarrheic (56 semi-formed,28 loose,and 9 watery feces)calves in six different regions of northern and southern Korea.These fecal samples were tested for BCoV,bovine rotavirus,and bovine viral diarrhea virus by RT-PCR.Results:Among the three pathogens examined,infection with BCoV was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio (OR)=9.3,95％ confidence interval (CI):1.1-78.9;P=0.02).Infection with BCoV alone (OR=2.9;95％ CI:1.1-7.6;P=0.03) or coinfection of BCoV with bovine viral diarrhea virus (OR=3.6;95％ CI:1.0-12.4;P=0.04) was significantly associated with the development of loose feces.Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves (OR=0.2;95％ CI:0.1-0.4;P=0.00) and calves that had not been fed colostrum (OR=0.2;95％ CI:0.1-0.7;P=0.02),respectively.Conclusions:The present study suggests that BCoV is involved in calf diarrhea in the Republic of Korea.Therefore,grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.

Prevalence of severe fever with thrombocytopenia syndrome virus among ticks surveyed at Mt. Gwanak, Korea

This study was performed to investigate the distribution of ticks and the rate of infection with severe fever with thrombocytopenia syndrome (SFTS) virus in ticks collected at Mt. Gwanak and the Seoul National University campus, Korea. Ticks (n = 273) were collected from May to October and included 76 Haemaphysalis longicornis (4 adult females, 72 nymphs), 49 Haemaphysalis flava (9 adult females, 3 adult males, 37 nymphs), and 148 Haemaphysalis spp. larvae. SFTS virus detection was performed by using one-step RT PCR and nested PCR. The SFTS virus was detected in 7 samples (1 Haemaphysalis longicornis nymph, 3 Haemaphysalis flava nymphs, and 3 Haemaphysalis spp. larva). The overall minimum field infection rate was 2.6%, whereas the minimum field infection rates of adult, nymphal, and larval ticks were 0%, 3.2%, and 2.0%, respectively. For a more accurate indication of the prevalence of SFTS virus in Korea, further in-depth investigations of tick species and SFTS virus occurrence over a larger area and longer period are needed.

Hematological Changes Associated with Theileria orientalis Infection in Korean Indigenous Cattle

Tick-borne pathogens can cause serious problems in grazing cattle. However, little information is available on tick-mediated diseases in cattle grazing on mountains. Thus, this study aimed to understand the potential problems related to tick-borne diseases in grazing cattle through the investigation of prevalent tick-transmitted infections, and their associated hematological changes, in terms of season and grazing type in Korean indigenous cattle (=Hanwoo). Hanwoo cattle from 3 regions of the Republic of Korea (=Korea) were either maintained indoors or placed on grassy mountains from spring to fall of 2014 and 2015. Cattle that grazed in mountainous areas showed a greater prevalence of tick-borne infections with an increased Theileria orientalis infection rate (54.7%) compared to that in non-grazing cattle (16.3%) (P < 0.001). Accordingly, the red blood cell (RBC) count and hematocrit (HCT) values of grazing cattle were significantly lower than those of non-grazing cattle throughout the season (P < 0.05). Moreover, RBC, hemoglobin (Hb), and HCT of T. orientalis-positive group were significantly lower than those of T. orientalis-negative group (P < 0.05). T. orientalis is a widespread tick-borne pathogen in Korea. Grazing of cattle in mountainous areas is closely associated with an increase in T. orientalis infection (RR=3.4, P < 0.001), and with consequent decreases in RBC count and HCT. Thus, these findings suggest that the Hanwoo cattle in mountainous areas of Korea are at a high risk of infection by T. orientalis, which can lead to hematological alterations. This study highlights the necessity of preventive strategies that target T. orientalis infection.

Prevalence of Anaplasma and Bartonella spp. in Ticks Collected from Korean Water Deer (Hydropotes inermis argyropus)

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.

Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province, Korea

The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.

Detection of Anaplasma sp. in Korean Native Goats (Capra aegagrus hircus) on Jeju Island, Korea

Anaplasma species are obligate intracellular pathogens that can cause tick-borne diseases in mammalian hosts. To date, very few studies of their occurrence in Korean native goats (Capra aegagrus hircus) have been reported. In the present study, we investigated Anaplasma infection of Korean native goats on Jeju Island, Republic of Korea, and performed phylogenetic analysis based on the 16S rRNA gene sequences. Our results showed that Anaplasma infection was found mostly in adult female goats. The phylogenetic tree revealed that the 7 sequences identified in Korean native goats could belong to Anaplasma sp. and were distinct from A. marginale, A. centrale, and A. ovis. The results indicated that the sequences identified to belong to Anaplasma were closely related to sequences isolated from goats in China and were clustered within the same group. To our knowledge, this is the first study to detect Anaplasma sp. infection in Korean native goats.

A Horsehair Worm, Gordius sp. (Nematomorpha: Gordiida), Passed in a Canine Feces

Nematomorpha, horsehair or Gordian worms, include about 300 freshwater species in 22 genera (Gordiida) and 5 marine species in 1 marine genus (Nectonema). They are parasitic in arthropods during their juvenile stage. In the present study, the used gordian worm was found in the feces of a dog (5-month old, male) in July 2014. Following the worm analysis using light and scanning electron microscopes, the morphological classification was re-evaluated with molecular analysis. The worm was determined to be a male worm having a bi-lobed tail and had male gonads in cross sections. It was identified as Gordius sp. (Nematomorpha: Gordiidae) based on the characteristic morphologies of cross sections and areole on the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out, and the gordiid worm was assumed to be close to the genus Gordius based on a phylogenic tree analysis.

Differential diagnosis of Salmonella gallinarum and S. pullorum using PCR-RELP

Salmonellosis in poultry of Korea is a significant health problem, which causes substantial economic losses. The most common causative agents of chicken salmonellosis ar S. gallinarum and S. pullorum. Traditional methods used to detect Salmoenella spp. In chicken are tedious, time consuming and confer little guarantee of sensitivity and species specificity. Therefore, a rapid and sensitive method for the differentiation of Salmonella serogroup D was assessed. We first amplified the rfbS genes by PCR and characterized the amplified product by nucleotide sequence analysis. The homology of nucleotide sequence was 99.7% between S. gallinarum and S. pullorum rfbS genes. Further comparisons of the sequences of S. gallinarum, S. gallinarum fied strain, S. pullorum and S. typhi(GenBank Accession No.M29682) showed a homology of 98.3%. The predicted amino acid sequence homology was 97.1%, 97.1% and 97.5%, respectively. Based on this comparison of these nucleotide sequences, we found unique restriction enzyme sites within the rfbS genes of S. gallinarum and S. pullorum. Thus, the PCR products could be further digested with restriction enzymes TfiI and PleI for use in a restriction fragment length polymorphism (RELP) technique. This method can be applied in the differential diagnosis between S. gallinarum and S. pullorum.