Masateru Koiwa

Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites.

At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.

Hemophagocytic histiocytic sarcoma in a Japanese black cow.

At necropsy, an 11-year-old Japanese Black cow with anemia, leukocytopenia, and progressive hind limb ataxia had marked diffuse splenomegaly and multiple masses in the thoracic vertebrae. Histologically, neoplastic erythrophagocytic histiocytes were in the splenic red pulp, vertebral masses, and blood vessels of the liver and lungs. The spinal cord was compressed by the vertebral masses. Clinicopathological, macroscopic, and histologic findings were consistent with hemophagocytic histiocytic sarcoma. Vertebral involvement with spinal cord compression and resultant hind limb ataxia is an unusual presentation for this tumor, which has been described mainly in dogs and cats.

Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis

Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death‐1 (PD‐1)/programmed death‐ligand 1 (PD‐L1) pathway in immunosuppression in bovine mycoplasmosis.

Intussusception of the spiral colon associated with fibroserous granulation in a heifer

MECHANICAL obstruction to the passage of intestinal contents can be caused by a luminal mass, an intrinsic lesion of the bowel wall or extrinsic compression (Kohn 1992). Such obstructions may be induced by factors including intussusception, volvulus, adhesions or hernias (Pearson 1973, Robertson 1979, Van der Velden 1984). Intussusception involves the invagination of the gastrointestinal tract into the lumen of the adjacent segment of the intestine (Pearson 1971). In adult cattle and human beings, the cause of intussusception seems to be intraluminal or intramural lesions such as papillomas, abscesses, parasitic nodules or tumours, since intramural or intraluminal growths or masses have often been observed within intussusceptions (Bosshart 1930, Maldonado and others 2004). However, Pearson (1971) reported that only two of 15 cases of intussusception in cattle were found to have predisposing lesions. However, almost all such cases described in the literature were adult cattle affected in the small intestine. Since this finding has been attributed to the length and mobility of the jejunal mesenteric attachments, Constable and others (1997) suggested the possibility that a remnant of the yolk sac and yolk stalk located near the intussusception might act in a similar way to an intramural mass and facilitate the development of the intussusception. Intussusception of the colon has only rarely been reported in cattle (Hamilton and Tulleners 1980, Strand and others 1993). Hamilton and Tulleners (1980) described a 10-monthold heifer with intussusception of the spiral colon accompanied by signs of colic for 18 hours before presentation. The heifer recovered after the lesion was reduced by gentle traction. Strand and others (1993) reported the apparent lack of either a detectable predisposing lesion or an abnormality of the mesenteric sheet in a three-year-old bull at postmortem examination. The bull had no history of colic, but the intussusception in its colon was highly necrotic and covered with fibrin deposits; these authors considered that the owner had not noticed that his bull had colic. To the authors’ knowledge, there are no reports confirming a predisposing lesion in cases of intussusception of the colon. This short communication describes a secondary intussusception due to a thickened spiral colon, complicated by diarrhoea, in a heifer. On April 21, 2004, a four-month-old Holstein heifer weighing 89 kg presented with signs of hypodynamia, reduced appetite, distention of the hypogastrium and green, mucosanguineous diarrhoea. Its heart rate was 96 bpm, its respiratory rate was 20 breaths/minute, and its rectal temperature was 38·4°C. Haematological and serum biochemical analyses, made on the fourth day after presentation, indicated severe hypoproteinaemia and anaemia (Table 1). The animal’s condition progressed to dysentery with mucus being passed on the fifth day after presentation. Coccidial oocysts and whipworm eggs were detected in the faeces by coprological examination on days 2 and 13. No rotavirus, coronavirus or Salmonella species was detected in the faeces, and bovine viral diarrhoea virus could not be detected in the peripheral blood mononuclear cells on day 14. Three to 6 kg/100 g bodyweight/day of anthelmintic (Daimeton; Daiichi Pharmaceutical) were administered orally for 18 days to treat the parasitic infection and indirect transfusion, using whole blood from three donors, was carried out on days 6, 13 and 15 by intravenous administration, because of the heifer’s severe anaemia. To avoid any possibility of producing an agglutination-haemolysis reaction before the indirect transfusion, matching tests were carried out by mixing a sample of the serum of the recipient with the blood cells of the three donors. The heifer gradually became unable to get to its feet readily on day 18. It produced progressively smaller amounts of faeces, and showed progressive abdominal distension on day 21, and finally dysstasia on day 22. As the heifer failed to improve and because its prognosis was poor, it was euthanased and examined postmortem on day 23. Postmortem examination revealed distension of the proximal colon to the central flexure of the colon, and an adhesion between the dorsal sac of the rumen and the spiral loop of the colon. Examination of the distended region revealed an intussusception impacted into the spiral loop of the colon, beginning 50 cm distal from the proximal loop of the colon. The intussuscipiens occupied approximately 40 cm adjacent to the centripetal gyrus of the spiral loop. Transverse sections of the intussusception revealed that the walls of the intussuscipiens were fully dilated and the serosa of the intussusceptum was severely thickened (Fig 1). The lumen between each mucomembranous aspect was severely stenosed, and the mural lesion of the intussusceptum aspect was severely compromised and covered with a granulating, gangrenous layer. The mural gangrene was more severe at the distal invagination than at the proximal invagination of the intussusception. The intestine cranial to the intussusception was markedly distended with gas, a little fluid and ingesta. After the postmortem examination, tissue specimens from the intestine were fixed in 10 per cent neutral-buffered formalin and embedded in paraffin, and thin sections (4 μm) were stained with haematoxylin and eosin for microscopic examination. Histological examination of the intussusceptum revealed severe fibroserous hyperplasia with granulation tissue in the lamina subserosa (Fig 2a), and effusions of fibrin were sometimes found under the adjacently apposed serous Veterinary Record (2007) 160, 376-378

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

Polymeric immunoglobulin receptor expression and local immunoglobulin A production in bovine sublingual, submandibular and parotid salivary glands.

The submandibular and parotid glands are the main sources of immunoglobulins A (IgAs) in human and rat saliva. These glands express the polymeric immunoglobulin receptor (pIgR), which transports IgAs into saliva. The main source of IgAs in saliva and pIgR expression in salivary glands has not been well documented in cattle. Expressions of pIgR were determined in the major bovine salivary glands (sublingual, submandibular, and parotid) by RT-PCR for mRNA and by Western blot analysis and immunohistochemistry (IHC) using an anti-human pIgR antibody for protein. The protein detected with the antibody was identified by nano-liquid chromatography-quadrupole time of flight mass spectrometry. Additionally, the distribution of Ig-producing plasma cells was analyzed by IHC. RT-PCR showed that pIgR was expressed in the sublingual and submandibular glands, but not in the parotid gland. Higher protein levels were observed in sublingual glands than in submandibular glands by Western blot. By IHC, pIgR was mainly located on the apical side of the cytoplasmic membrane in the sublingual gland, whereas it was observed only on the basal side in the submandibular gland. The highest density of plasma cells expressing IgAs was observed in the sublingual gland. These results suggest that the sublingual gland plays an important role in first-line defence of the oral cavity in cattle in contrast to humans and rats.

Analysis of Trace and Major Elements in Bronchoalveolar Lavage Fluid of Mycoplasma Bronchopneumonia in Calves

The aim of this study was to evaluate the reliability and effectiveness of direct determination of trace and major element concentrations in bronchoalveolar lavage fluid samples from Holstein calves with Mycoplasma bronchopneumonia (n = 21) and healthy controls (n = 20). The samples were obtained during bronchoscopy using a standard examination method. A total of 18 elements (aluminum, bromine, calcium, chlorine, chromium, copper, iron, potassium, magnesium, manganese, molybdenum, nickel, phosphorous, sulfur, silicon, strontium, titanium, and zinc) were detected by particle-induced X-ray emission. The average bromine, iron, potassium, magnesium, and phosphorous concentrations were higher in calves with bronchopneumonia than in controls (p < 0.05). They were found to have higher amounts of calcium and zinc, and a higher zinc–copper ratio than that in healthy calves (p < 0.001). Based on the receiver operating characteristics curves, we propose a diagnostic cutoff point for zinc–copper ratio for identification of Mycoplasma pneumonia of 8.676. Our results indicate that assessment of the elemental composition of broncholaveolar lavage fluid is a promising diagnostic tool for Mycoplasma bronchopneumonia.

Inflammation-related microRNA expression level in the bovine milk is affected by mastitis

MicroRNA (miRNA) in tissue and liquid samples have been shown to be associated with many diseases including inflammation. We aimed to identify inflammation-related miRNA expression level in the bovine mastitis milk. Expression level of inflammation-related miRNA in milk from mastitis-affected and normal cows was analyzed using qPCR. We found that expression level of miR-21, miR-146a, miR-155, miR-222, and miR-383 was significantly upregulated in California mastitis test positive (CMT+) milk. We further analyzed these miRNA using a chip-based QuantStudio Digital PCR System. The digital PCR results correlated with those of qPCR, demonstrating upregulation of miR-21, miR-146a, miR-155, miR-222, and miR-383 in CMT+ milk. In conclusion, we identified miRNA that are upregulated in CMT+ milk. These miRNA exhibited sensitivity and specificity greater than 80% for differentiating between CMT+ milk and normal milk. Our findings suggest that inflammation-related miRNA expression level in the bovine milk was affected by mastitis, and miRNA in milk have potential for use as biomarkers of bovine mastitis.

Epithelioid Cells in Mediastinal Lymph Nodes of Cattle without Cancer

Epithelioid cells are found in lymph nodes in cases of metastatic carcinoma; however, epithelioid cells with benign features have also been discovered incidentally in lymph nodes. Epithelioid cells were observed in mediastinal lymph nodes of cattle without cancer during routine diagnostic necropsy. To explain this finding, the authors evaluated the prevalence and histopathologic, immunohistochemical, and ultrastructural features retrospectively using mediastinal lymph nodes from 110 cattle and found that 66 of the lymph nodes contained epithelioid cells. In all 66 nodes, most of the epithelioid cells were individual or aggregated in nodal sinuses; in 21 nodes, some epithelioid cells formed tubular structures. The individual and aggregated epithelioid cells were mostly considered to be mesothelial in origin by immunohistochemical and ultrastructural examination; presumably, they entered the lymph nodes via lymphatic vessels, because they were found in afferent lymphatic vessels and nodal sinuses. Although the presence of epithelioid cells in lymph nodes prompts suspicion of metastatic carcinoma, the epithelioid cells in these bovine lymph nodes did not disrupt nodal architecture, lacked atypia or mitotic figures, and did not invade nodal parenchyma.

Testicular yolk sac tumor of myxomatous, reticular, and polyvesicular vitelline type in a newborn calf

Yolk sac tumors (YSTs) are rare neoplasms of germ cell origin. In humans, the tumors primarily occur in the testes or ovaries, but occasionally develop at other sites. The neoplastic cells of YSTs form many histological patterns resembling embryonal structures, and the World Health Organization classification lists 11 such patterns: reticular, macrocystic, endodermal sinus, papillary, solid, glandular-alveolar, myxomatous, sarcomatoid, polyvesicular vitelline, hepatoid, and parietal. Among domestic animals, only 2 cases of YST, which were of testicular and abdominal cavity origin, have been reported in calves. In both cases, neoplastic cells had epithelial properties and disseminated metastases in the abdomen. In the present study, the enlarged testis of a newborn calf, which was subsequently diagnosed as YST and exhibited myxomatous, reticular, and polyvesicular vitelline histological patterns, is described. There was no metastasis in this case, and histological and immunohistochemical features varied from previous cases of YST.