Two Clinical Cases of Feline Hemoplasmosis in Korea.

Feline hemotropic mycoplasmosis (hemoplasmosis) is an infection of the red blood cells caused by the Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm), and Candidatus Mycoplasma turicensis (CMt). The existence of Mhf, CMhm, and CMt has been demonstrated in feral cats in Korea using molecular methods, but no clinical cases have yet been reported. This study reports 2 clinical cases of hemotropic mycoplasmosis caused by CMhm and CMt in 2 anemic cats. The first case was a client-owned intact female domestic shorthair cat that presented with fever, pale mucous membranes, and normocytic normochromic non-regenerative anemia. Prior to referral, an immunosuppressive prednisolone dose was administered at the local veterinary clinic for 1 month. The cat was diagnosed with high-grade alimentary lymphoma. Organisms were found on the surface of the red blood cells on blood smear examination. The second case was of a rescued cat that presented with dehydration and fever. The cat had normocytic normochromic non-regenerative anemia. Necropsy revealed concurrent feline infectious peritonitis. Polymerase chain reaction assay targeting 16S rRNA revealed CMhm infection in case 1 and dual infection of CMhm and CMt in case 2. Normocytic normochromic non-regenerative anemia was observed in both cats before and during the management of the systemic inflammation. This is the first clinical case report in Korea to demonstrate CMhm and CMt infections in symptomatic cats.

Clinical efficacy of membrane-free stem cell extracts for the treatment of canine atopic dermatitis.

Despite various treatment options for canine atopic dermatitis (cAD), therapeutic limitations still exist, including adverse effects and low absorption ratios. This study evaluated the effects of a membrane-free stem cell extract (MFSCE) on the clinical signs of atopic dogs. Thirty client-owned dogs previously diagnosed with cAD were separated into placebo (n = 10) and MFSCE-treated groups (n = 20). The dogs were treated with a cream (MFSCE and placebo) via dermal administration twice daily for 14 days, and the clinical response was recorded on days 0, 7, and 14. The MFSCE-treated group showed significantly decreased severity of pruritus on day 14 compared to that on day 0. In addition, the erythema, pigmentation, skin dryness, and thickness were remarkably decreased in the MFSCE-treated group on day 14 compared to those on day 0 whereas no significant changes were observed in the placebo-treated group. No general clinical signs or adverse effects were observed in this study. These results suggest that MFSCE could be a surrogate treatment option for cAD.

Safety and immunological effects of recombinant canine IL-15 in dogs.

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 µg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3+CD8+ cytotoxic T lymphocytes, CD3+CD5dimCD21-, and non-B/non-T NK cell populations, without stimulating Treg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs.

Evaluation of treatment with a combination of mycophenolate mofetil and prednisolone in dogs with meningoencephalomyelitis of unknown etiology: a retrospective study of 86 cases (2009-2017).

BACKGROUND: Combination therapy with glucocorticoids and adjunctive immunomodulating drugs has been generally accepted as a standard treatment regimen for meningoencephalomyelitis of unknown etiology (MUE). We hypothesized that treatment with MMF as an adjunctive agent along with glucocorticoids would be effective and well-tolerated protocol in dogs with MUE. Eighty-six dogs with MUE between May 2009 and June 2017 were included (59 females and 27 males; mean age of 5.93 years; mean body weight of 3.83 kg). The medical records of dogs with MUE treated with prednisolone and MMF were retrospectively evaluated to determine the therapeutic response, survival time, and treatment-related adverse effects. RESULTS: A partial or complete response (CR) was recorded for 75 dogs. The overall median survival time from the initiation of treatment was 558 days. Dogs that showed CR with no relapse over the treatment period (from diagnosis to death) had significantly longer median survival times. A significantly higher mortality hazard ratio of 4.546 was recorded in dogs that failed to achieve CR. The interval between the onset of clinical signs and the clinical presentation was not significantly associated with CR, relapse rate, and survival time. Adverse effects included gastrointestinal upsets in 26 dogs (30.23%), sporadic infections in 17 dogs (19.77%), and pancreatitis in seven dogs (8.14%). CONCLUSIONS: The results suggest that adjunctive MMF treatment for MUE is safe and comparable to other immunosuppressive protocols. The treatment should focus on the achievement of CR and preventing relapse for successful management.

Clinical Case of a Transfusion-Associated Canine Mycoplasma haemocanis Infection in the Republic of Korea: A Case Report.

This report describes the first clinical case of a transfusion-associated Mycoplasma haemocanis infection in a dog in Korea. A 6-year-old male Maltese underwent a red blood cell transfusion for idiopathic immune-mediated hemolytic anemia. Eighteen days after the blood transfusion, the recipient's packed cell volume decreased and basophilic organisms were found on erythrocytes. A polymerase chain reaction and sequential analysis showed that both the donor dog and recipient dog had M. haemocanis. Six weeks after doxycycline administration, no organisms were detected and the recipient's anemia had improved.

Canine non-B, non-T NK lymphocytes have a potential antibody-dependent cellular cytotoxicity function against antibody-coated tumor cells.

BACKGROUND: The antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector immune cells actively lyse antibody-coated target cells. The ADCC of tumor cells is employed in the treatment of various cancers overexpressing unique antigens, and only natural killer (NK) cells are known to be major effectors of antibody mediated ADCC activity. Canine NK cells are still defined as non-B, non-T large granular lymphocytes because of the lack of information regarding the NK cell-restricted specific marker in dogs, and it has never been demonstrated that canine NK cells have ADCC ability against tumor cells. In the present study, we investigated whether canine non-B, non-T NK cells have ADCC ability against target antibody-coated tumor cells, using cetuximab and trastuzumab, the only human antibodies reported binding to canine cancer cells. RESULTS: Activated canine non-B, non-T NK cells (CD3-CD21-CD5-TCRαß-TCRγδ-) for 13~17 days ex vivo showed ADCC ability against trastuzumab- or cetuximab-coated target tumor cells expressing various levels of human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR). Trastuzumab and cetuximab induced significant ADCC responses of canine NK cells even in CMT-U334 and CF41.Mg cells expressing low levels of HER-2 and/or EGFR, as well as in SKBR3 and DU145 cells overexpressing HER-2 and/or EGFR. The trastuzumab-mediated ADCC activity of NK cells was significantly enhanced by treatment with rcIL-21. CONCLUSIONS: The results of this study suggest that canine non-B, non-T NK lymphocytes have a potential ADCC function and that combinational strategies of monoclonal antibodies with either cytokines, which activate NK cells in vivo, or adoptive transfer of NK cells may be a feasible method for amplifying the efficacy of immunotherapy against malignant cancers even with very low expression of target molecules in dogs.

Evaluation of the efficacy of ivermectin against Theileria orientalis infection in grazing cattle.

BACKGROUND: Raising cattle on pastures is known to be beneficial for animal welfare and cost reduction. However, grazing is associated with the risk of contracting tick-borne diseases, such as theileriosis. Here, the efficacy of ivermectin against these diseases and associated clinical symptoms were evaluated. RESULTS: A total of 68 cattle from a grazing cattle farm were selected and divided into two groups: the control group (17 cattle) with no preventive treatment and the ivermectin-treated group (51 cattle) in which cattle were treated with pour-on ivermectin prior to grazing. The infection rates of Theileria orientalis and the red blood cell (RBC) profile (e.g., RBC count, hematocrit value, and hemoglobin concentration) were compared in the spring (before grazing) and summer (during grazing) between the two groups. Based on PCR amplification of the major piroplasm surface protein (MPSP) gene, 12 cattle were positive for T. orientalis infection. Phylogenetic analysis revealed that the isolates identified in this study consisted of three MPSP types (1, 2, and 7). The T. orientalis infection rate in the control group during grazing was 3-fold higher than that in the ivermectin-treated group. Moreover, differences in RBC parameters during grazing were greater in the control group than in the ivermectin-treated group. In particular, the hematocrit value was significantly reduced in the control group. CONCLUSIONS: The results of this study demonstrated that ivermectin had protective effects against T. orientalis infection and RBC hemolysis in grazing cattle.

Use of computed tomography and minimum intensity projection in the detection of lobar pneumonia mimicking lung lobe torsion in a dog.

A 10-year-old female spayed Dachshund was referred with progressive coughing for 1 month. The dog was tentatively diagnosed with right middle lung torsion based on pleural effusion, vesicular emphysema, abruptly ending bronchus in consolidated right middle lung, and no contrast enhancement of the affected lobe on radiography and computed tomography (CT). There was no evidence of torsion upon thoracotomy, and histological examination confirmed lobar pneumonia. The CT images were reevaluated using minimum intensity projection and revealed normal bronchial courses. The minimum intensity projection technique can be to assist in evaluation of the bronchial tree for dogs with suspected lung lobe torsion and other pulmonary diseases.

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.

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 investi- gated the prevalence of Eimeria spp. in pre-weaned native Korean calves and determined the correlation between diar- rhea 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.

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.

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.

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.

Identification of bovine viral diarrhea virus infection in Saanen goats in the Republic of Korea.

Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of livestock and causes substantial economic losses to the livestock industry worldwide. BVDV is not necessarily species specific and is known to infect domesticated and wild ruminants. In the present study, BVDV infection was identified in two Saanen goats from one farm, and two different viral subtypes were found, BVDV-1a and BVDV-2a. Each isolate was closely related to cattle isolates identified in the Republic of Korea. The two sequences obtained in this study were not consistent with border disease virus (BDV). The incidence of BVDV in this farm apparently occurred in the absence of contact with cattle and may be associated with grazing. This study demonstrates that BVDV infection may be possible to transmit among goats without exposure to cattle. Therefore, this result indicates that Saanen goats may act as natural reservoirs for BVDV. This is the first report of BVDV-1a infection in a Saanen goat.

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.

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.

Molecular Prevalence of Acarapis Mite Infestations in Honey Bees in Korea.

Acarapis mites, including Acarapis woodi, Acarapis externus, and Acarapis dorsalis, are parasites of bees which can cause severe damage to the bee industry by destroying colonies and decreasing honey production. All 3 species are prevalent throughout many countries including UK, USA, Iran, Turkey, China, and Japan. Based on previous reports of Acarapis mites occurring in northeast Asia, including China and Japan, we investigated a survey of Acarapis mite infestations in honey bees in Korean apiaries. A total of 99 colonies of Apis mellifera were sampled from 5 provinces. The head and thorax of 20 bees from each colony were removed for DNA extraction. PCR assays were performed with 3 primer sets, including T, A, and K primers. Results indicated that 42.4% (42/99) of samples were Acarapis-positive by PCR assay which were sequenced to identify species. Each sequence showed 92.6-99.3% homology with reference sequences. Based on the homology, the number of colonies infected with A. dorsalis was 32 which showed the highest infection rate among the 3 species, while the number of colonies infected with A. externus and A. woodi was 9 and 1, respectively. However, none of the Acarapis mites were morphologically detected. This result could be explained that all apiaries in the survey used acaricides against bee mites such as Varroa destructor and Tropilaelaps clareae which also affect against Acarapis mites. Based on this study, it is highly probable that Acarapis mites as well as Varroa and Tropilaelaps could be prevalent in Korean apiaries.

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.