Experimental infection of Korean native goats (Capra aegagrus hircus) with bovine viral diarrhea virus 1b.

BACKGROUND: Bovine viral diarrhea virus (BVDV) infects various ungulates and causes reproductive failure in infected goats. BVDV has been detected among goats in the Republic of Korea, but the route of transmission remains unclear. Here, we aimed to investigate whether BVDV-1b circulating among Korean cattle can be transmitted to Korean native goats (Capra aegagrus hircus) and characterize the outcomes of BVDV infection in these goats. RESULTS: Four goats were inoculated intranasally with the Korean noncytopathic (ncp) BVDV-1b strain. Two goats exhibited clinical signs of illness, including coughing and nasal discharge. Nasal swabs and blood were collected to screen for viral RNA and BVDV antibodies. Using the 5'-untranslated region (UTR), viral RNA was detected in the nasal swabs of two goats (Goat 1 and 3) on 12 day post-inoculation (dpi) and in the blood sample of one goat (Goat 1) on 7 and 19 dpi. Using the N-terminal protease (Npro) region, viral RNA was detected in the blood sample of Goat 1 on 7 and 12 dpi. Antibodies to BVDV were detected in Goats 1 and 3 on 16-21 dpi using enzyme-linked immunosorbent assay. Sequence analysis of the virus from nasal swabs and blood samples, which was detected via RT-PCR, using the 5'-UTR and Npro regions led to the identification of the strain as ncp BVDV-1b and revealed changes in the nucleotide sequence of these goats. CONCLUSIONS: Our results indicate that changes in the nucleotide sequence are associated with the establishment of BVDV infection in Korean native goats; these changes may be owing to a process required for the establishment of infection in a new host reservoir. Broadly, these findings highlight the importance of BVDV surveillance in ungulates other than cattle.

Experimental infection of mice with noncytopathic bovine viral diarrhea virus 2 increases the number of megakaryocytes in bone marrow.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes significant economic losses worldwide in the cattle industry through decrease in productive performance and immunosuppression of animals in herds. Recent studies conducted by our group showed that mice can be infected with BVDV-1 by the oral route. The purpose of this study was to assess the clinical signs, hematological changes, histopathological lesions in lymphoid tissues, and the distribution of the viral antigen after oral inoculation with a Korean noncytopathic (ncp) BVDV-2 field isolate in mice. METHODS: Mice were orally administered a low or high dose of BVDV-2; blood and tissue samples were collected on days 2, 5, and 9 postinfection (pi). We monitored clinical signs, hematological changes, histopathological lesions, and tissue distribution of a viral antigen by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) and then compared these parameters with those in ncp BVDV-1 infections. RESULTS: None of the infected mice developed any clinical signs of the illness. Significant thrombocytopenia was found in both low- and high-dose-inoculated mice on day 2 pi. Leukopenia was apparent only in low-dose-inoculated mice on day 2 pi, whereas lymphopenia was not observed in any ncp BVDV-2-infected animal. Viral RNA was found in the spleen in of low- and high-dose-inoculated mice by RT-PCR. According to the results of IHC, the viral antigen was consistently detected in lymphocytes of bone marrow and spleen and less frequently in bronchus-associated lymphoid tissue (BALT), mesenteric lymph nodes, and Peyer's patches. Despite the antigen detection in BALT and mesenteric lymph nodes, histopathological lesions were not observed in these tissues. Lympholysis, infiltration by inflammatory cells, and increased numbers of megakaryocytes were seen in Peyer's patches, spleens, and bone marrow, respectively. In contrast to ncp BVDV-1 infection, lympholysis was found in the spleen of ncp BVDV-2-infected mice. These histopathological lesions were more severe in high-dose-inoculated mice than in low-dose-inoculated mice. CONCLUSIONS: Our results provide insight into the pathogenesis of ncp BVDV-2 infection in mice. Collectively, these results highlight significant differences in pathogenesis between ncp BVDV-1 and ncp BVDV-2 infections in a murine model.

Identification of a new bovine viral diarrhea virus subtype in the Republic of Korea.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is prevalent in Korean indigenous cattle, leading to substantial economic losses. This study was conducted to investigate the occurrence of BVDV. In 2016, a total of 143 blood samples were collected from asymptomatic Korean indigenous calves younger than 3-months of age from six different farms in the Republic of Korea (ROK). RESULTS: Eighty-seven calves (60.8%, 87/143) were tested positive for BVDV as evaluated by RT-PCR analysis. Phylogenetic analysis based on the 5'-untranslated region was used to classify these cases into three subtypes: BVDV-1b, BVDV-1o, and BVDV-2a. These results showed that BVDV-1b was the predominant subtype, while 2 samples clustered with BVDV-2a. Interestingly, one sample formed a separate group as a potentially new subtype, BVDV-1o. To our knowledge, this is the first report of BVDV-1o infection in Korean native calves. The BVDV-1o subtype identified in this study was closely related to cattle isolates obtained from Japan, indicating that this subtype is a new introduction to the ROK. CONCLUSIONS: This study provides useful information for carrying out epidemiological surveys of BVDV in the ROK and developing a vaccine for future use in the ROK, particularly for the first detection of BVDV-1o in Korean indigenous calves. Further studies are required to investigate the prevalence and pathogenicity of this BVDV-1o subtype.

Identification of zoonotic Giardia duodenalis in Korean native calves with normal feces.

Giardia duodenalis is an important zoonotic protozoan parasite that infects humans and other animals. This study was conducted to investigate the prevalence and molecular characterization of G. duodenalis infection in the feces of normal and diarrheic pre-weaned calves aged 1-60 days. Fecal samples were collected from 90 Korean native calves in 7 different regions in the central region of the Republic of Korea (ROK). Of the 90 fecal samples, 9 (10%, 9/90) were positive for G. duodenalis by PCR using the ß-giardin gene. Phylogenetic analysis identified G. duodenalis assemblages A and E in the farms examined and G. duodenalis with both assemblage A and E was detected in one farm. Assemblage A was detected in calves with normal feces, while assemblage E was frequently identified in calves with diarrhea. This is the first report of the identification of assemblage A in pre-weaned calves with normal feces in the ROK. These results suggest that G. duodenalis infection is closely associated with calf age and management systems including overcrowding, age-mixed grouping, and hygiene. Thus, giardiasis in calves is an important disease because of its zoonotic significance and should be screened in young animals. Further studies are necessary to elucidate the transmission routes of zoonotic G. duodenalis assemblages.

Genetic characterization of Theileria orientalis from cattle in the Republic of Korea.

Theileria orientalis is the causative agent of benign theileriosis, which is distributed mainly in Asian countries, and causes serious economic losses in the livestock industry. The present study was performed to investigate the epidemiology of T. orientalis infections in cattle in the Republic of Korea (ROK) and to characterize the genetic diversity of T. orientalis based on the major piroplasm surface protein (MPSP) genes. In 2015, between July and August, blood samples were collected from 138 asymptomatic cattle in four different geographical regions (Hoengseong, Jeongeup, Namwon, and Jeju island) in the ROK. In total, 57 blood samples (41.3 %, 57/138) were positive for T. orientalis based on PCR amplification of the MPSP gene. A high prevalence of T. orientalis infection was observed in Jeju island, whereas the infection rate was relatively low in Jeongeup. Phylogenetic analysis showed that isolates identified in this study belonged to four MPSP genotypes, specifically types 1, 2, 3, and 7. The distribution of the four genotypes varied considerably among the four regions; types 1, 2, and 3 were detected in Jeju island, whereas types 1 and 7 were found in Namwon, types 1 and 2 in Jeongeup, and type 2 in Hoengseong. To our knowledge, this is the first report on the identification of type 7 T. orientalis in cattle in the ROK. These results suggest that the MPSP genotypes detected in this study showed genetic diversity related to geographical location. Our findings revealed that the T. orientalis infection rate was relatively high, indicating that T. orientalis infection is closely associated with grazing. Of the four MPSP genotypes, the prevalence of the most pathogenic type 2 was relatively high in the ROK. Therefore, further studies should focus on the development of an effective monitoring and prevention program for T. orientalis.

Genome sequence of Coxiella burnetii KZQ2 isolated from a clinical strain in the Republic of Korea.

The genome of Coxiella burnetii KZQ2, isolated from clinical patients in Korea, is 2.04 MB long. Multispacer types were ST77, and phylogenetic tree analysis showed that KZQ2 is closely related to the CbuK_Q154 chronic strain isolated from human endocarditis patients in the USA.

Non-cytopathic bovine viral diarrhoea virus 2 induces autophagy to enhance its replication.

BACKGROUND: Bovine viral diarrhoea virus (BVDV) is an important viral pathogen that has an economic impact on the livestock industry worldwide. Autophagy is one of the earliest cell-autonomous defence mechanisms against microbial invasion, and many types of viruses can induce autophagy by infecting host cells. OBJECTIVES: The aim of this study was to identify the role of autophagy in the pathogenesis of non-cytopathic (ncp) BVDV2 infection. METHODS: Madin-Darby bovine kidney (MDBK) cells were treated with ncp BVDV2, rapamycin, or 3-methyladenine (MA) and ncp BVDV2 and then incubated at 37°C for 24 h. Cells were harvested, and the effects of autophagy were determined by transmission electron microscopy (TEM), confocal laser microscopy, western blotting and qRT-PCR. Apoptotic analysis was also performed using western blotting and flow cytometry. RESULTS: In ncp BVDV2-infected MDBK cells, more autophagosomes were observed by TEM, and the number of microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein puncta was also increased. The ncp BVDV2-infected cells showed significantly enhanced conversion of LC3-I to LC3-II, as well as upregulation of autophagy-related proteins, including ATG5 and Beclin 1, and substantial degradation of p62/SQSTM1. These results are similar to those induced by rapamycin, an autophagy inducer. E2 protein expression, which is associated with viral replication, increased over time in ncp BVDV2-infected cells. Inhibition of autophagy by 3-MA in ncp BVDV2-infected MDBK cells downregulated the expressions of LC3-II, ATG5 and Beclin 1 and prevented the degradation of p62/SQSTM1. Moreover, the expressions of phosphorylated Akt and procaspase-3 were significantly increased in ncp BVDV2-infected cells. In addition, the mRNA level of protein kinase R (PKR) was significantly reduced in ncp BVDV2-infected cells. CONCLUSIONS: Our results demonstrate that ncp BVDV2 infection induced autophagy in MDBK cells via anti-apoptosis and PKR suppression. Therefore, autophagy may play a role in establishing persistent infection caused by ncp BVDV.

First report of Anaplasma phagocytophilum infection in Holstein cattle in the Republic of Korea.

Global warming has increased the incidence and risk of tick-borne diseases in domestic animals and humans in the Republic of Korea (ROK). In this study, we investigated the prevalence of Anaplasma phagocytophilum in Holstein cattle (n = 214) in the ROK using specific PCR assays. A. phagocytophilum infection was detected in only two animals (0.93%, 2/214). Our findings showed that PCR assay using the 16S rRNA gene, but not groEL, was suitable for detection of A. phagocytophilum in cattle. Phylogenetic analysis based on the16S rRNA gene showed that A. phagocytophilum was divided into two clades. Clade 1 included Korean isolates, such as those from dogs, cats, Korean water deer, and ticks, while A. phagocytophilum identified in Holstein cattle formed clade 2. Our results suggest that there is genetic variability among isolates of A. phagocytophilum circulating in the ROK. This is the first study to report A. phagocytophilum infection in Holstein cattle in the ROK. As A. phagocytophilum has zoonotic potential, additional epidemiological studies are needed to investigate the prevalence and genetic characterization of A. phagocytophilum from different regions and hosts.

Molecular detection of Anaplasma bovis in Holstein cattle in the Republic of Korea.

Anaplasmosis is a tick-borne infectious disease that affects both human and animal health. This study was performed to characterize and investigate the prevalence of infection with Anaplasma bovis in Holstein cattle originating from two regions in the Republic of Korea (ROK). Blood samples (n = 151; 80 from Namwon and 71 from Jeju Island) were analyzed by polymerase chain reaction, and the prevalence of A. bovis infection was compared before and after grazing. In Namwon, A. bovis infection was not detected, while in the Jeju Island, A. bovis infection was detected in three of 13 animals after grazing. Phylogenetic analysis revealed that the A. bovis isolates had homology (97.1-99.7%) with a Korean spotted deer (Cervus nippon) isolate and Haemaphysalis longicornis tick isolates identified in the ROK. A. bovis infection has not previously been diagnosed in cattle in the ROK. This study shows that A. bovis infection in the Jeju Island is closely related to grazing.