Molecular phylogenetic analysis of Theileria species detected from Japanese serows (Capricornis crispus) in Iwate Prefecture, Japan

In the present study, we tried to detect DNA for ribosomal RNA genes of piroplasma parasites from the liver or blood of 43 Japanese serows (Capricornis crispus) in Iwate Prefecture of Japan by polymerase chain reaction. Approximately 500-bp amplicons were obtained in 35 (81.4%) of the 43 samples by amplification for V4 hyper-variable regions of the 18S rRNA gene, and the amplicons were considered to be DNA of Theileria species. The complete nucleotide sequences (1,700 or 1709 bp) of the 18S rRNA gene were determined in 20 samples and were divided into 5 genotypes that were phylogenetically separated into two different lineages showing a polyphyletic relation. The Theileria DNAs of the two different lineages were considered to be those of distinct species.     

Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan

A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 μg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum β-lactamase (ESBL). The two bovine isolates produced bla_CTX-M-2, while the nine poultry isolates produced bla_CTX-M-25 (4), bla_SHV-2 (3), bla_CTX-M-15 (1) and bla_CTX-M-2 (1). Thus, our results showed that several types of ESBL were identified and three types of β-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased animals in Japan.     

Characterization of monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (PSGL-1)

Thirteen different monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (cPSGL-1) were obtained by immunization of rats with cells of a canine lymphoma cell line (Ema). O-sialoglycoprotein endopeptidase treatment of Ema cells showed that all of these antibodies recognized O-glycosylated peptides of canine PSGL-1. Experiments using deletion or point mutants of cPSGL-1 indicated that these antibodies could be categorized into several groups based on their cPSGL-1 recognition characteristics. These anti-cPSGL-1 monoclonal antibodies will be useful for analysis of the canine P-selectin and PSGL-1 system.     

Detection of chromogranin A in the adrenal gland extracts of different animal species by an enzyme-linked immunosorbent assay using Thomsen-Friedenreich antigen-specific Amaranthus caudatus lectin

The reactivity of different lectins with crude chromogranin A (CgA) obtained from different animals, namely, cow, horse, dog, pig, and dolphin, was examined to identify lectin(s) that would be useful as coating reagent(s) in a sandwich enzyme-linked immunosorbent assay (ELISA). Of the different lectins studied, the Amaranthus caudatus lectin (ACA), which is specific for the Thomsen-Friedenreich (T)-antigen (Galβ1-3GalNAc), was found to react with the CgA from different animals by western blotting. Purified rabbit anti-bovine CgA antibody was also found to cross-react with the crude CgA preparations. On the basis of these findings, a sandwich ELISA was developed with ACA as the coating reagent and anti-bovine CgA antibody as the probing antibody. Using this method, concentration-dependent curves ranging from 0.003 μg/mL to 25 μg/mL and from 0.02 μg/mL to 25 μg/mL were obtained for bovine CgA and canine CgA, respectively. Similarly, concentration-dependent curves were obtained for the equine, swine, and dolphin crude CgA extracts. Thus, ACA is concluded to be a valuable reagent for CgA detection in crude extracts from different animal species, and for CgA isolation/purification.     

A cytotoxic antibody produced by allo-skin transplantation in cats

A high titer of cytotoxic antibody against donor lymphocytes, tentatively designated as anti-F1, was found in one cat who received 3 times of skin allograft from the same donor. The anti-F1 antibody titer was 1:16, and the antibody activity was mostly detected in the IgG fraction. A cross-absorption test using erythrocytes from F1-positive animals revealed that the F1(+) antigen recognized by anti-F1 antibody was expressed on erythrocytes as well as lymphocytes. The frequencies of F1(+) and F1(-) types in 81 healthy cats, involving all the cats used for allo-immunization in the present study, were 30.9 and 69.1%, respectively. Moreover, F1 antigen was considered to be a hereditary antigen from family studies.     

Randomized clinical trial to evaluate the effectiveness of enrofloxacin as a second‐line antibiotic for treatment of acute Escherichia coli mastitis

The objective of the present study was to evaluate the effectiveness of enrofloxacin (ERFX) as a second‐line antibiotic for treatment of acute Escherichia coli (E. coli) mastitis. Forty‐two cows with naturally occurring acute E. coli mastitis were enrolled. On the first day of treatment (day 0), empirically selected antibiotics (oxytetracycline: n = 32, kanamycin: n = 10) were administered. Although systemic signs improved in 10 cows (first‐line group), the signs remained unchanged or worsened in 32 cows on day 1, including two cows that were found dead. The 30 surviving cows were randomly assigned to second‐line groups constituting an ERFX group (n = 19) or a control group (n = 11) that was treated with other antibiotics. Response to each treatment was evaluated by measuring clinical signs from day 0 to day 3, subsequent quarter milk recovery, and the 60‐day survival rate. Appetite on day 3 was significantly better in the ERFX group compared to the control group. No significant differences were observed in the 60‐day survival rate or the subsequent milk recovery between the ERFX group and the control group. Thus, the use of ERFX as a second‐line antibiotic for the treatment of acute E. coli mastitis could induce a rapid appetite recovery.