Motoshi Tajima

Reduced glutathione accelerates the oxidative damage produced by sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs.

The oxidative effects of sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs, were investigated in vitro using three types of canine erythrocytes, which are differentiated by the concentration of reduced glutathione and the composition of intracellular cations. After incubation with sodium n-propylthiosulfate, the methemoglobin concentration and Heinz body count in all three types of erythrocytes increased and a decrease in the erythrocyte reduced glutathione concentration was then observed. The erythrocytes containing high concentrations of potassium and reduced glutathione (approximately five times the normal values) were more susceptible to oxidative damage by sodium n-propylthiosulfate than were the normal canine erythrocytes. The susceptibility of the erythrocytes containing high potassium and normal reduced glutathione concentrations was intermediate between those of erythrocytes containing high concentrations of potassium and reduced glutathione and normal canine erythrocytes. In addition, the depletion of erythrocyte reduced glutathione by 1-chloro-2, 4-dinitrobenzene resulted in a marked decrease in the oxidative injury induced by sodium n-propylthiosulfate in erythrocytes containing high concentrations of potassium and reduced glutathione. The generation of superoxide in erythrocytes containing high concentrations of potassium and reduced glutathione was 4.1 times higher than that in normal canine erythrocytes when the cells were incubated with sodium n-propylthiosulfate. These observations indicate that erythrocyte reduced glutathione, which is known as an antioxidant, accelerates the oxidative damage produced by sodium n-propylthiosulfate.

GM1 gangliosidosis in shiba dogs

A six-month-old shiba dog with a one-month history of progressive motor dysfunction showed clinical signs of a cerebellar disorder, including ataxia, dysmetria and intention tremor of the head. Histopathological and ultrastructural studies revealed distended neurons packed with membranous cytoplasmic bodies throughout the central nervous system. The activities of lysosomal acid β-galactosidase in its leucocytes and liver were less than 2 per cent of the control levels, and the compound accumulated in the brain was identified as GM1 ganglioside. A sibling which died immediately after birth was shown to have a β-galactosidase deficiency in the brain and visceral organs. A family study revealed that the sire and dam of the probands were heterozygotes with approximately half of the normal level of β-galactosidase activity, suggesting an autosomal recessive pattern of inheritance.

Heinz Body Hemolytic Anemia With Eccentrocytosis From Ingestion of Chinese Chive (Allium tuberosum) and Garlic (Allium sativum) in a Dog

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.

A novel mutation in the gene for canine acid β-galactosidase that causes GM1-gangliosidosis in Shiba dogs

A homozygous recessive mutation, causing GM1-gangliosidosis in Shiba dogs, was identified as a deletion of C nucleotide 1668 in the gene for canine acid β-galactosidase, which was a novel mutation in canine GM1-gangliosidosis.

Molecular cloning and phylogenetic analysis of Babesia gibsoni heat shock protein 70

The Babesia gibsoni heat shock protein 70 gene (BGHsp70) was cloned by polymerase chain reaction (PCR) and sequenced. The length of the gene was 1938 bp and the predicted polypeptide was 646 amino acids long with a calculated molecular weight of 70,627. The amino acid sequences of BGHsp70 from 17 isolates were identical, though there were six types of polymorphisms among the corresponding nucleotide sequences. There was no intron in the BGHsp70 gene. Phylogenetic analysis of the amino acid sequence of Hsp70 showed that B. gibsoni was most closely related to B. bovis and lies within a phylogenetic cluster with Theileria. These results suggest that Hsp70 was well conserved among intraerythrocytic protozoa.

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks.

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.

Seroepidemiological survey for antibody to Borrelia burgdorferi in cows

Antibody to Borrelia burgdorferi was examined in 380 healthy and 38 clinical cases of cows from Hokkaido and Shizuoka in Japan. In healthy animals, IgG and IgM antibody to B. burgdorferi HO14 strain were found in 44 cows (14.6%) and 24 cows (8.0%) from Hokkaido. In contrast, antibody‐positive case was not observed except for only 1 case which was IgM positive (1/79: 1.3%) in cows from Shizuoka. Mean antibody levels of healthy animals in Hokkaido and Shizuoka were 0.651 and 0.263 (IgG antibody to HO14 strain), 0.642 and 0.169 (IgG to HP3 strain), 0.613 and 0.367 (IgM to HO14 strain) and 0.582 and 0.286 (IgM to HP3 strain). The differences of the antibody levels between cows from Hokkaido and Shizuoka were significant. Seasonal difference was found in seropositive cows from Hokkaido. The rate of seropositive cows was high in summer (23.4% in June and 11.8% in July) but low in winter (0% in January and February). The pattern was discussed to be associated with activation of ticks. One of 4 cows with arthritis showed significantly higher IgG antibody level than that of healthy cows and cows with some disease, although the serum was collected from Shizuoka where antibody‐positive animals for B. burgdorferi were rare among healthy cows. This high IgG antibody may suggest that the arthritis of such cows was caused by infection with B. burgdorferi. Two of 7 cows with unclassified abortion showed positive antibody reaction in Hokkaido. These cases, however, may not be related to the B. burgdorferi infection because the positive rate was similar to those of healthy cows in the same season.

Cleavage of the NS2-3 protein in the cells of cattle persistently infected with non-cytopathogenic bovine viral diarrhea virus

The NS2‐3 of BVDV is cleaved in cultured cells infected with cp BVDV but not in those infected with ncp BVDV when tested more than 10 hours post infection. However, it is not known whether cleavage of NS2‐3 occurs in vivo. In the present study, cleavage of NS2‐3 in cattle persistently infected with BVDV was investigated. All BVDV isolated from PI animals were of the ncp biotype, and NS2‐3 proteins were detected in bovine fetal muscular cells infected with these viruses. On the other hand, in the leukocytes of those PI animals, NS3 proteins, products of the cleavage of NS2‐3 proteins, were detected. In addition, the NS3 proteins were also detected in leukocytes artificially infected with ncp BVDV. These results reveal that the NS2‐3 protein of BVDV is cleaved in leukocytes. Furthermore, NS3 proteins were detected in many tissues of PI cattle, such as lymphoid tissue, brain, thyroid, lung, and kidney. These results suggest that the NS2‐3 protein of ncp BVDV cleaves in vivo.

Classification of new BVDV isolates from Philippine water buffalo using the viral E2 region.

Characterization of bovine viral diarrhea virus (BVDV) isolates has been focused of several studies this last decade. Until now lots of new strains are being unfolded maybe due to the viral fast mutation ability. As we focused our research on water buffalo immunology, we were able to identify a probable new BVDV isolates. RNA was extracted from water buffalo blood in the Philippines. The extracted RNA was reverse‐transcribed and synthesized cDNA. Oligonucleotide primers from the viral E2 region were used to amplify the target viral gene and later purified, cloned and sequenced. The E2 region with 420 bp nucleotides long was compared with existing published sequences in the GenBank. Based on the constructed phylogenetic tree, the isolated strain showed to be a BVDV type 1b along with Osloss and CP7 strains. Further classification of the new isolates was done within the BVDV type 1b1 group, which was compared with other strains in the sub‐group. The analysis revealed that Lamspringe/738, KE9 and 2543/87 were the closest with 92% homology. Additional study is being done to further qualify and quantify the extent of the existence of this new BVDV isolates in water buffalo in the Philippines. This is the first report of BVDV in the Philippines and first concerning BVDV in Philippine water buffalo. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

HEAT SHOCK RESPONSE OF BABESIA GIBSONI HEAT SHOCK PROTEIN 70

mRNA and protein expression profiles for heat shock protein 70 (Hsp70) of Babesia gibsoni (BgHsp70) exposed to either high or low temperatures, were examined by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. In the present study, a commercially available anti-human Hsp70 antibody that could recognize recombinant BgHsp70 was used. BgHsp70 was detected in the parasites cultured under standard conditions at 37 C by Western blotting and immunostaining of thin smears of infected erythrocytes. These results suggested that BgHsp70 was expressed constitutively at the erythrocyte stage. BgHsp70 levels were elevated when the parasites were incubated at 42 C for 1 hr. In contrast, its mRNA amount was decreased and its protein amount was unchanged when the parasites were incubated at 32 C for 1 hr. Moreover, the level of parasitemia of B. gibsoni incubated at either 42 C or 32 C was almost the same as that at 37 C. These results indicated that the exposure of B. gibsoni to elevated temperatures might result in increased expression of BgHsp70 and that the exposure of the intraerythrocytic parasites to decreased temperatures might not induce the overexpression of BgHsp70.