Takashi Kurosawa

Theileria sergenti infection in the Bo-RBC-SCID mouse model

We have previously developed the Bo-RBC-SCID mouse model forTheileria sergenti infection. In the present study, this model was further examined to delineate the mode of parasite infection. The Bo-RBC-SCID mice were prepared by periodically transfusing uninfected bovine erythrocytes (Bo-RBCs) into splenectomized SCID mice via the intraperitoneal (i.p.) route. The mice, separated into three groups, were inoculated i.p., intravenously (i.v.), or subcutaneously (s.c.) withT. sergenti-infected Bo-RBCs. Examination of samples of peripheral blood demonstrated that the parasite infected mice inoculated via any one of the three routes. The mice inoculated i.v., however, developed parasitemia earlier than those inoculated i.p. or s.c. When Bo-RBC-SCID mice prepared without splenectomy were infected withT. sergenti, a high-level parasitemia appeared only once. After that, not only the level of parasitemia but also the number of Bo-RBCs in the peripheral blood rapidly decreased despite the continuation of Bo-RBC transfusions. The results suggest thatT. sergenti proliferates primarily in the circulating blood in Bo-RBC-SCID mice and that in response to the parasites growth, the spleen may play an important role in the removal of both parasitized and unparasitized Bo-RBCs from the blood circulation.

The Bo-RBC-SCID mouse model for evaluating the efficacy of anti-theilerial drugs

We have previously developed a mouse model which allowed the proliferation of Theileria sergenti in severe combined immunodeficiency (SCID) mice with circulating bovine erythrocytes (Bo-RBC). In the present study, this model was utilized to test the efficacy of anti-theilerial drugs. Bo-RBC-SCID mice were created by giving periodic transfusions of T. sergenti-free Bo-RBC, and subsequently infecting with T. sergenti. Three anti-protozoal compounds, Pamaquine (Yamanouchi Pharmaceutical Co. Ltd), Ganaseg (Japan CIBA-GEIGY Ltd) and Buparvaquone (Coopers Animal Health Ltd), were subcutaneously administered into the mice at doses recommended for cattle therapy. Blood examinations demonstrated that all three drugs significantly reduced the level of parasitemia although Ganaseg was effective only at a dose five times higher than that recommended for cattle therapy. Administration of the drugs neither caused any sign of acute toxicity nor changed the rate of Bo-RBC in the SCID mices circulating blood cells. The results indicate that the Bo-RBC-SCID mouse model may offer a useful in vivo system for evaluating the efficacy of anti-protozoal drugs against T. sergenti.

Ultrasonographic imaging of experimentally induced pancreatitis in cattle.

This study was conducted to determine the ultrasonographic patterns of pancreatitis evoked in cattle, with reference to laboratory and pathological findings. Using ultrasonographic guidance, acute necrotizing pancreatitis was induced in six cows by injecting chloroform into the pancreatic tissue. Ultrasonographic examination was then performed once daily for nine days. Pancreatic lesions were visible 24h after induction of pancreatitis, as represented by a uniform increase in echogenicity and by intralobular and subcapsular fluid accumulation. As the experiment progressed, patchy hypoechogenic foci appeared within the gland parenchyma. Amylase and lipase activities showed rapid increases. Post mortem examination revealed gross and microscopic necrotic and haemorrhagic lesions in the body and right lobe of the pancreas, accompanied by oedema and fibrosis. Ultrasonography was found to be extremely useful for the detection and characterization of experimentally induced pancreatitis and to monitor its progression in the cow. These findings are of potential value as a reference for the diagnostic workup of bovine pancreatitis, and ultrasonography is seen as a promising non-invasive technique for the diagnosis of suspected pancreatitis in cattle.

Sporogony of Theileria sergenti in the salivary glands of the tick vector Haemaphysalis longicornis.

Sporogony ofTheileria sergenti in the salivary glands of the tick vectorHaemaphysalis longicornis was observed by light and transmission electron microscopy. Fission bodies that developed from kinetes were observed as masses (approximately 32×15 μm) that occupied half of the acinous cell at 2 days after the infestation of ticks. Parasites possessed an irregular nucleus of low electron density, highly electron-dense granules, and mitochondrion-like bodies in the cytoplasm. Parasites developed into a multinucleate syncytium, increasing in size and complexity. Subsequently, the nuclei of parasites became round and highly electron-dense, and tubelike structures and rhoptries as precursors of the highly electron-dense granules appeared in the cytoplasm. Approximately 40,000 sporozoites arose via radial budding from the syncytium that had developed from kinetes. As inT. parva, no evidence of discrete secondary or tertiary sporoblasts was found inT. sergenti. The sporogony ofT. sergenti was extremely similar to that ofT. parva.

Serum from Theileria sergenti-infected cattle accelerates the clearance of bovine erythrocytes in SCID mice

Anemia is a major clinical sign of Japanese bovine theileriosis caused byTheileria sergenti. To investigate the possible factors causing anemia in cattle, we developed a clearance test for bovine erythrocytes (Bo-RBC) in severe combined immunodeficient (SCID) mice. Clearance of Bo-RBC in the SCID mice was significantly accelerated when the mice were inoculated with a serum sample obtained from an infected calf during a highly parasitized phase but not when they were injected with a serum sample obtained during the convalescence phase. Acceleration of the clearance of Bo-RBC was also observed in mice treated with merozoite extract. Furthermore, the clearance of Bo-RBC that had been treated with merozoite extract was accelerated. A significant hemolytic activity in infected serum (highly parasitezed phase) was observed. Activities sufficient to accelerate the clearance of Bo-RBC in SCID mice and to induce in vitro hemolysis of Bo-RBC were also observed with a merozoite extract fromT. sergenti. The results suggest a possible linkage between the in vitro hemolysis of Bo-RBC and the acceleration of Bo-RBC clearance in SCID mice.

Ultrasonographic diagnosis of omasal leiomyoma in a cow

GASTROINTESTINAL (GI) tumours in cattle are rare. In 1·3 million animals examined at slaughter in the UK, only three tumours occurred specifically in the GI tract (Anderson and Snadison 1969). Among 1400 cattle examined postmortem in New York, neoplasms were found in the forestomachs of eight animals (0·6 per cent), the only forms being fibroma and lymphosarcoma (Bertone and others 1985). Other reports of GI tumours in cattle include papilloma and squamous cell carcinoma in Kenya (Plowright 1955, Plowright and others 1971) and northern England (Jarrett 1973). Smooth muscle tumours of the GI tract have been found to develop mostly in the small intestine in dogs and cats (Engle and Brodey 1969, ter Haar and others 1998) and cows (Bertone 1990); none has been reported in the forestomachs of cows. Ultrasonography has proved to be the most effective and least invasive diagnostic method of detecting GI tumours in dogs and cats (Paoloni and others 2002), and it has also been used increasingly as part of the diagnostic evaluation of cattle with a variety of GI disorders, such as traumatic reticuloperitonitis (Braun and others 1993). However, the ultrasonographic features of GI tumours in cattle have not yet been delineated. This short communication describes the ultrasonographic, laboratory and clinicopathological findings of an omasal leiomyoma in a cow. A six-year-old Holstein cow, weighing 534 kg, presented with intermittent fever and a one-month history of inappetence. Clinical signs included anorexia, depression and ataxia. The cow remained thin and frequently ran a rectal temperature of 38·8 to 39·5°C; but heart and respiratory rates were within normal ranges (55 bpm and 16 breaths per minute, respectively). Rumen movements were inaudible. The back grip test, pain percussion test on the reticulum and pole test on the sternum elicited only a mild reaction. The left and right sides of the thorax and the right side of the abdomen were clipped and shaved. Ultrasonic coupling gel was liberally applied to the wet skin to ensure good contact and a 3·5 MHz linear transducer was firmly held against the body wall with the cow in a standing position. With the transducer placed over the left ventral thorax, the reticular contractions were monitored for three minutes, as described by Braun and Götz (1994). The entire ventral thorax right and left of the sternum and the right and left thoracic walls were then scanned up to the elbow joints. The outer contours of the reticulum were visualised and the echogenic changes recorded. The omasum was scanned medially and to the right of the ventral midline. The reticular biphasic contractions were not detectable, the reticular contour was uneven and a loss of normal layering was observed. Deposits of echogenic material appeared on the reticular wall, cavitated by hypoechogenic fluid, and a space-occupying mass was identified within the lumen of the omasum. The echotexture of the lesion was inconsistent and echogenic, and the margins were asymmetric (Fig 1). Blood gas analysis showed no abnormalities. Echocardiographic tracings demonstrated atrial fibrillation, a deep QRS complex and irregular T waves. Haematology showed leucocytosis (21,300 leucocytes/μl, reference range 4000 to 12,000/μl) and neutrophilia (18,851 neutrophils/μl, reference range 600 to 4000/μl). Serum chemistry revealed hypoalbuminaemia (8·6 g/litre, reference range 30·3 to 35·3 g/litre) and hyperproteinaemia (100 g/litre, reference range 21 to 36 g/litre), attributed to hypergammaglobulinaemia (73·4 g/litre, reference range 16·9 to 22·5 g/litre), which suggested a chronic inflammatory disease or neoplasia. These findings supported differential diagnoses of traumatic reticuloperitonitis, reticular abscess, reticular adhesions, pleuritis, and pulmonary and hepatic diseases. Ultrasonography of the forestomachs and liver narrowed the diagnosis to either traumatic reticuloperitonitis or a space-occupying mass in the omasum. The results also revealed a high activity of gamma glutamyl transferase (62 U/litre, reference value 6·1 to 17·4 U/litre), indicating a degree of liver impairment, which may have also accounted for the hypoalbuminaemia. All laboratory values were referenced from Kaneko and others (1997). The cow had a poor prognosis and was euthanased. Postmortem examination revealed fibrinous, thick-walled inflammatory deposits interspersed with jelly-like serous fluid adhering to the reticulum, omasum and abomasum, as well as to the rumen. A pedunculated, grape-like mass (18 x 14 cm) was attached to the ventral omasal wall (Fig 2). No ulceration or abrasions were observed on the surface of the mass and the cut surface had a lobulated appearance. Neither a diaphragmatic reticular adhesion nor any metallic foreign body or trace was observed. A specimen from the omasal mass was fixed in buffered 10 per cent formalin, embedded in paraffin, stained with haematoxylin and eosin and examined histologically. There was a proliferation of smooth muscle cells in the omasal muscularis in intertwisting fascicles of well differentiated cells; the fascicles arranged in an exceedingly irregular pattern, transversing at intersecting and obtuse angles to one another. The smooth muscle cells had abundant cytoplasm and irregular fusiform nuclei with vesiculated chromatin. There was marked submucosal oedema with diffuse infiltration of lymphocytes, macrophages and a few neutrophils. Mitotic figures were scarce. The histological picture was compatible with a diagnosis of leiomyoma (Fig 3). Leiomyomas, benign tumours of smooth muscle, are the most common gynaecological tumours in domestic animals FIG 1: Longitudinal sonogram of an omasal leiomyoma imaged at the seventh intercostal space. Note the asymmetric, inhomogenous and the echogenic patterns of the tumour. The mass appeared to project into the omasal lumen. Lt Lateral, Md Medial, 1 Right ventral thoracic wall, 2 Sternal part of the diaphragm and peritoneum, 3 Liver, 4 Omasal wall, 5 Omasal lumen

Transcutaneous ultrasound-guided pancreatic biopsy in cattle and its safety: a preliminary report

This study describes a free-hand technique for percutaneous pancreatic biopsy in cattle with ultrasound-guidance using a 14G spinal biopsy needle. Its safety was evaluated based on 36 consecutive procedures. To assess the immediate effects of pancreatic biopsy, 31 cows were necropsied shortly after the procedure and examined. The remaining five cows were examined daily for eight days and then necropsied and examined. No life-threatening complications nor clinically detectable abnormalities were observed. Changes indicative of inflammation were not apparent in total and differential WBC counts or in total protein and fibrinogen concentrations. A small increase of amylase activity was detected in only one cow kept for the eight-day observation period. Serum lipase activity increased significantly on day four after biopsy. Urea nitrogen, creatinine, glucose and the activities of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltransferase remained within reference ranges. Changes in the peritoneum and pancreas observed at necropsy were negligible. We conclude that percutaneous ultrasound-guided pancreatic biopsy did not appear to influence the cows condition adversely and the procedure provided an excellent method of obtaining a pancreatic specimen for histological examination. The procedure was considered safe, fast, cost-effective, and practical when performed properly. We believe that the technique can be used in cows with suspected pancreatic disease for making an ante mortem diagnosis.

Detection of Theileria sergenti schizonts in bovine lymph node

The growth of Theileria sergenti schizonts in bovine lymph nodes was examined by experimental infection. Six naive Holstein calves were inoculated with a sporozoite stabilate into the superficial cervical and subiliac lymph nodes. Biopsy samples from the inoculated calves were collected every 24 h for 10 days. Histological examination demonstrated the schizont of T. sergenti on day 4 post-inoculation. The growth of schizonts in the lymph node was observed on days 4-8 post-inoculation. Electron microscopic observation disclosed that the core of the schizont has a high density central core surrounded by numerous electron-dense spherules.

温度感作により誘発された Haemaphysalis longicornis若ダニ唾液腺内における Theileria sergentiの発育

The parthenogenetic Haemaphysalis longicornis larvae engorged on cattle naturally infected with Theileria sergenti were reared at 24 degrees C. The resultant nymphal ticks were incubated at 37 degrees C to clear the effect of incubation on the development and maturation of sporozoites. The sporozoites in the salivary glands of the nymphal ticks exposed to 37 degrees C for 16 days were observed by the methyl green pyronin staining method. The ticks exposed to 37 degrees C were ground up in a mortar and the supernatant of the tick suspension in PBS was inoculated into cattle. The cattle showed parasitemia and specific antibody response 18 days after inoculation. Consequently, the parasites in the tick salivary glands became infective to cattle by incubating infected. H. longicornis nymphs at 37 degrees C.

ホルスタイン牛のWolff-Parkinson-White症候群の一例

A case of Wolff-Parkinson-White (WPW) syndrome in a Holstein-Freisian cow aged 10-year-old was examined in detail. In electrocardiogram (ECG), the P-wave was the same configuration in both the normal and abnormal ECG. The PR-interval shortened from 0.2 to 0.1 second and the duration of the QRS-complex prolonged from 0.1 to 0.12 second compared with normal ECG. The delta wave, characterized in WPW syndrome, could not be recognized. In echocardiogram, notches were recognized at the early stage of ventricular contraction in the interventricular septum. This cow was, therefore, diagnosed as type B WPW syndrome. The abnormal ECG disappeared by the administration of procainamide. It was strongly indicated that the ventricular contraction showing abnormal ECG was generated only by the stimulation through an accessory pathway in this cow.