Diversity and genotypic analysis of tick-borne pathogens carried by ticks infesting horses in Korea.

Ticks account for an extensive range of health and welfare issues in horses. In addition, tick-borne pathogens (TBPs) limit global animal trading and equine sporting events. Here, we assess the prevalence, co-infectivity and risk factors of TBPs in horse ticks in Korea. A total of 245 hard ticks, including 103 male and 142 female adults, were obtained from horses on Jeju Island during the spring to autumn seasons of 2013-2019. All collected ticks were identified as adult Haemaphysalis longicornis. We screened and analyzed each tick for the presence of several TBPs by polymerase chain reaction (PCR) analysis. Among the 245 ticks, we detected genes for three TBPs, Candidatus Rickettsia longicornii (22.9%), Ehrlichia canis (0.4%) and Theileria luwenshuni (0.4%), while Anaplasma spp. was not detected. TBPs were most prevalent in ticks harvested during the autumn season, and more abundant in the female than male adults. This is the first report of the genera Ehrlichia, Rickettsia and Theileria in horse ticks in Korea. TBPs in horse ticks are likely a reservoir for zoonotic transmission to other animals, including humans. Our findings demonstrate the need for further understanding of the prevalence and epidemiology of TBPs in wild and domestic animals.

Borrelia Species Detected in Ticks Feeding on Wild Korean Water Deer (Hydropotes inermis) Using Molecular and Genotypic Analyses.

Lyme borreliosis is a vector-borne disease transmitted through the bite of ticks infected by Borrelia burgdorferi sensu lato group, including B. burgdorferi sensu stricto, B. afzelii, and B. garinii. The goal of the present study was to detect Borrelia species in ticks infesting wild Korean water deer (KWD; Hydropotes inermis Swinhoe), using molecular and genotypic analyses. In total, 48 ticks were collected from KWD, all of which were morphologically identified as Haemaphysalis longicornis Neumann that is dominant in Korea. Nested PCR was performed to detect the Borrelia-specific 5S (rrf)-23S (rrl) intergenic spacer region and the outer surface protein A (ospA) genes in ticks. Both rrf-rrl and ospA were amplified from one of the 48 ticks (2.1%) and were identified as B. afzelii. To our knowledge, this study constitutes the first molecular detection of B. afzelii in Haemaphysalis ticks in Korea. Because B. afzelii is a zoonotic tick-borne pathogen, understanding the molecular characteristics of this bacterium is important for preventing the transmission of Borrelia from ticks to other animals and humans.

Characteristics of DNase activities in excretory/secretory products of infective larvae of Haemonchus contortus.

While multiple DNase activities occur in the excretory/secretory products (ESPs) of the adult Haemonchus contortus, the DNase activities in ESPs of the infective larvae (L3) have not been studied. Thus, the DNase activities in ESPs of H. contortus L3 were investigated and compared to those of adults for developmental stage-specific analysis. The DNase activities had relative molecular masses (M rs) of 34 and 36 kDa upon zymographic analysis at pH 5.0 and 7.0 when the larvae were incubated for over 48 h. The 34 and 36 kDa DNases of L3 ESPs were also detected in adult ESPs with similar characteristics. However, the 37 and 38.5 kDa DNases of the adult ESPs were not detected in the L3 ESPs. Since the 37 and 38.5 kDa DNase activities were mainly detected in adult ESPs, these activities appear to be specific to the adult stage whereas the other ESP DNase activities appear to be expressed during multiple stages of the parasite's life cycle. While the difference in DNase activities of L3 and adults remains obscure, the role of DNase in larval development should be further clarified and the identification of stage-specific developmental markers will lead to the discovery of specific factors that stimulate larval development.

Hepatocellular carcinoma with metastasis to the spleen in a Holstein cow.

Hepatocellular carcinoma (HCC) with metastasis to the spleen in a Holstein cow was studied by histopathologic and immunohistochemical methods. The tumor was characterized by a pseudoglandular (acinar) pattern with an associated fibrous stroma. Individual cells often had a "hepatoid" appearance but were interspersed with scattered cells exhibiting a clear, periodic acid-Schiff (PAS)-positive cytoplasm and small eccentric nuclei. This pattern was present in nodules found in both liver and spleen. Moreover, hepatoid tumor cells were positive for alpha-fetoprotein. Immunohistochemical studies suggest that myofibroblasts were responsible for the production of fibrous septa surrounding the pseudoglandular structures of bovine HCC. In summary, our histologic and immunohistochemical findings support a diagnosis of primary HCC with splenic metastasis. Furthermore, the associated stromal response appears to be of a myofibroblast origin. The primary etiology of bovine HCC and the significance of the intralesional, PAS-positive clear cells remain undetermined.

Multiple congenital malformation in a Holstein calf.

A 10-day-old male Holstein dairy calf with orthopaedic abnormalities was unable to stand but was alert with a suckle reflex. At necropsy, the calf showed multiple defects, including partial agenesis of the left rib plate, deformed left scapula, shortened left humerus, agenesis of the left kidney, atresia ani and scoliosis. The cause of these anomalies could not be determined. This report is the first to describe partial agenesis of ribs in a calf.

Identical small subunit ribosomal RNA gene nucleotide sequence of bovine Theileria isolates (Korea and Japan) and Theileria buffeli (Marula, Kenya).

Small subunit ribosomal RNA (SSU rRNA) gene nucleotide sequences of bovine Theileria isolates from Korea (KLS and KCB) and Japan (JHS) were determined. The genes from each isolate were amplified by the polymerase chain reaction and the approximately 1.8 kb product cloned and sequenced by a modified dideoxynucleotide method. Overlapping gene segments produced with a series of primers were sequenced, resulting in a complete DNA sequence for both forward and reverse strands of the SSU rRNA genes of each isolate. SSU rRNA gene sequences (termed Type A) were identical among the bovine Theileria isolates from Korea and the isolate from Japan. A GenBank data library homology search showed the sequence to be the same as that listed as Theileria buffeli isolated from cattle in Marula, Kenya.

Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor.

IL-2 therapy is a potent inductive stimulus for nitric oxide (NO.) synthesis in mice and humans. It is not yet clear whether NO. can contribute to IL-2-induced therapeutic responses. The murine skin cancer Meth A is relatively resistant to lymphokine-activated killer (LAK) cell killing, allowing evaluation of the role of IL-2-induced NO. synthesis in vivo, without contribution by LAK cells. Subcutaneous IL-2 treatment of mice bearing i.p. Meth A tumor increased nitrite production by cells derived from ascites (63 +/- 14 microM vs 3.2 +/- 1.5 microM in untreated controls). N omega-monomethyl-L-arginine (MLA), NO. synthase inhibitor, prevented this increase. NO. production correlated in an inverse fashion with tumor cell proliferation in vitro. Evidence for IL-2-induced heme nitrosylation was demonstrated in tumor cells by electron paramagnetic resonance spectroscopy. By immunomagnetic depletion experiments, macrophages were implicated as a major source of NO. synthesis. Cytologic and flow-cytometric evaluation revealed that IL-2 treatment resulted in enhanced lymphocyte and macrophage recruitment into malignant ascites, and decreases in tumor cell recovery. MLA administration further increased host cell recovery. Subcutaneous IL-2 therapy increased urinary nitrate excretion up to eightfold in mice, and appeared to produce a significant survival advantage that was prevented by MLA administration.

Effectiveness and toxicity of protracted nitric oxide synthesis inhibition during IL-2 treatment of mice.

The current study was designed to characterize nitric oxide (NO.) synthesis during interleukin-2 (IL-2) treatment of mice, and to determine whether NO. mediated IL-2-induced "vascular leak." We developed a technique for chronic subcutaneous infusion of the NO. synthase inhibitor N omega monomethyl-L-arginine (MLA) via osmotic minipump to aid in further study of these processes. After IL-2 administration to C3H/HeN mice (180,000 IU i.p. b.i.d. for 5 days), NO. synthesis increased two-to-three fold, peaking on days 5-8. Administration of MLA reduced NO. synthesis in both IL-2-treated mice (from 2.7 to 1 microM/mouse/day), and normal mice (from 1 to 0.5 microM/mouse/day). This agent decreased IL-2-induced radiolabeled albumin accumulation in the liver after i.p. IL-2 administration (p < 0.02). MLA infusions resulted in minimal systemic toxicity in mice, as reflected by complete blood counts or serum chemistries. MLA also did not impair lymphokine-activated killer cell induction in vitro or in vivo, or alter IL-2-induced tumor responses in a 3-day pulmonary metastasis model. These experiments demonstrated that NO. is a mediator involved in the genesis of vascular permeability induced by IL-2 treatment. Studies designed to further evaluate the toxicity and usefulness of MLA infusions to modify this IL-2 induced toxicity appear to be warranted.