Seasonal changes in serum total thyroxine, free thyroxine, and canine thyroid-stimulating hormone in clinically healthy beagles in Hokkaido.

The purpose of this study was to evaluate seasonal influences on thyroid hormone levels of healthy outdoor dogs in Hokkaido. We surveyed serum basal total thyroxine (tT4), free thyroxine (fT4), and canine thyroid-stimulating hormone (cTSH) levels, and tT4 levels after administration of TSH for a year. Basal tT4 levels decreased in January, and increased in August and September. fT4 levels increased in January and November. No significant seasonal variation was found in cTSH. tT4 levels after administration of TSH in August and November increased. These results suggested that the thyroid gland may have been activated in November. We should take seasonal variation into consideration when thyroid function is tested.     

Acquisition of quinolone resistance and point mutation of the gyrA gene in Campylobacter jejuni isolated from broilers and in vitro-induced resistant strains

A dramatic rise in the number of resistant Campylobacter to quinolones has been documented in human patients and domestic animals. In this study, the mechanism of acquisition of quinolone resistance was studied by detecting point mutations in the gyrA gene of Campylobacter strains obtained from broilers and strains with in vitro-induced resistance. The minimal inhibitory concentrations (MICs) of norfloxacin (NFLX) and ofloxacin (OFLX) for the strains that had no point mutation were slightly increased from the source strain (Campylobacter jejuni ATCC 33560). The MICs of nalidixic acid (NA), NFLX, and OFLX for the strains that had the point mutation at Thr-86 were 100 or 200 microg/ml, 50 microg/ml, and 25 microg/ml, respectively. The MIC of NA for the strain that had a point mutation at Asp-90 higher than those for the strains that had the point mutation at Thr-86, but the MICs of NFLX and OFLX were relatively lower than those for the strains that had point mutation at Thr-86. These findings suggest that the degree of antimicrobial resistance against NA, NFLX, and OFLX in the in vitro-induced C. jejuni strains was associated with the location of the point mutation in gyrA. On the other hand, a point mutation in all seven resistant strains isolated from broilers was located only at Thr-86, while the MICs of the three quinolones varied in each wild strain. This suggests that another mechanism might also be involved in the acquisition of quinolone resistance in C. jejuni wild strains.     

Effects of in vivo administration of anti-IL-10 or anti-IFN-gamma monoclonal antibody on the host defense mechanism against Plasmodium yoelii yoelii infection

Our previous reports indicated that C57BL/6 mice infected with a lethal variant of Plasmodium yoelii 17X (P. yoelii 17XL) produced high levels of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma) while mice infected with the nonlethal variant of the parasite did not produce detectable levels of IL-10. In the present study, the involvement of IL-10 and IFN-gamma in exacerbation or regulation of blood-stage malaria was investigated by using the lethal variant of P. yoelii 17XL and monoclonal antibodies (mAb) against the cytokines. C57BL/6 mice were injected intraperitoneally with a neutralizing anti-IL-10 mAb or anti-IFN-gamma mAb after inoculation with P. yoelii 17XL. Treatment of mice with anti-IL-10 mAb resulted in substantial prolongation of survival and 60% of treated mice survived while 100% of control mice died by day 11. On the contrary, treatment of mice with anti-IFN-gamma mAb exacerbated infection and all mice died after an earlier period than those treated with normal rat Ig. No differences in parasitemias were found between treated and untreated mice. To elucidate the involvement of nitric oxide in the host protection or exacerbation, mice were treated with aminoguanidine, an inhibitor of nitric oxide synthetase, after inoculation of P. yoelii 17XL. Neither mortality nor parasitemia was influenced by the treatment. These results indicate that an IFN-gamma response is associated with protective immunity in mice infected with P. yoelii 17XL, while an IL-10 response is associated with disease exacerbation during the infection.     

Epidemic of equine influenza among vaccinated racehorses in Japan in 2007

On August 2007, we encountered equine influenza epidemic by Florida sub-lineage strain (H3N8) in Japan Racing Association's facilities where 4142 racehorses in total were stabled. The number of new febrile cases sharply increased, but the occurrence was rapidly calmed down within 2 weeks. The morbidity rate in these facilities was 12.8% and the subclinical infection rate of healthy racehorses examined by rapid antigen detection tests was 19.4% at the early stage of epidemic. The serological studies along with the low morbidity rate and the existence of numbers of asymptomatically infected racehorses, suggested that the cross-reactivity of the antibodies provided by vaccination against the epidemic strain contributed to reducing the morbidity rate and duration of epidemic.     

Isolation and measurement of carbonic anhydrase isoenzymes in erythrocytes of dogs

OBJECTIVE: To purify canine carbonic anhydrase (CA) isoenzymes CA-I and CA-II and to determine concentrations of CA-I and CA-II in erythrocytes of Beagles and dogs native to Japan. SAMPLE POPULATION: Blood samples from 116 Beagles, including 24 pregnant Beagles, and blood samples from 29 dogs native to Japan. PROCEDURE: Canine CA-I and CA-II were purified by use of column chromatography. Concentrations of CA-I and CA-II in erythrocytes of dogs were determined, using an ELISA. RESULTS: Mean (+/- SD) concentrations of CA-I and CA-II in erythrocytes of Beagles were 3.21+/-0.86 and 1.63+/-0.39 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male Beagles than female Beagles. In contrast, mean concentration of CA-II was greater in female Beagles than male Beagles. Furthermore, concentration of CA-II was greater in pregnant female Beagles than male or nonpregnant female Beagles. Mean concentrations of CA-I and CA-II in erythrocytes of dogs native to Japan were 11.03+/-4.39 and 3.29+/-0.91 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male dogs from Japan than female dogs from Japan. CONCLUSIONS AND CLINICAL RELEVANCE: The ELISA used in this study proved to be precise and sensitive for determining CA-I and CA-II concentrations in dogs. The ELISA may enable study of changes in isoenzymes associated with hereditary or metabolic disorders of blood or other body fluids, using only a small sample. Measurement of the concentrations of CA isoenzymes in dogs may be of diagnostic value.     

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre‐ and postnatal development

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre‐ and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.     

Efficacy of thiabendazole, mebendazole, levamisole and ivermectin against gullet worm, Gongylonema pulchrum: in vitro and in vivo studies

In vitro and in vivo studies were conducted to evaluate the effects of thiabendazole, mebendazole, levamisole and ivermectin against Gongylonema pulchrum. For in vitro assays, third-stage larvae (L3) incubated with the drugs were administered orally to mice and the ability of larvae to invade the gastric mucosa of the animals was examined. After incubation, only those larvae treated with high concentrations of levamisole (1 and 10 microg/ml) were tightly coiled with intestines exhibiting morphological abnormalities. Good dose-response data for the drugs tested was observed at the time of worm recovery from mice, with no worms recovered at the two highest concentrations of levamisole. In vivo efficacy of the drugs against adult worms was evaluated in six groups of three rabbits, each of which was infected with 30 L3 of G. pulchrum and treated with thiabendazole at 100 mg/kg for 3 days, mebendazole at 70 mg/kg for 3 days, levamisole as a single dose of 8 mg/kg, and subcutaneously injected ivermectin as a single dose of 0.2 mg/kg or vehicles of the drugs (control) at 4 months post-infection. Necropsy 14 days after treatment revealed that levamisole, mebendazole and ivermectin reduced worm burdens by 63.2%, 22.8% and 25.8%, respectively, with no reductions in worms observed with thiabendazole. The surviving worms were principally found in the esophagus with the remainder distributed among the buccal mucosa, the tongue, and/or pharyngeal mucosa in all groups. A number of morphologically abnormal eggs were observed within the uterus and ovijector in female worms recovered from the thiabendazole-treated group. These findings suggest that levamisole exhibits in vivo efficacy against G. pulchrum infection and that the larval invasion tests using mice could be used to screen for anthelmintic susceptibility of nematodes.     

Cathepsin gene expression in mouse placenta during the latter half of pregnancy

Gene expressions and their interaction are complex and have not been definitely clarified in the placenta. To identify interactions of gene products previously not studied, we applied cDNA subtraction analyses to the placenta between days 12 and 16, days 12 and 14, days 14 and 16 of pregnancy. Among subtracted cDNAs cathepsin M, Q and R in PECs were specifically identified on days 14 and 16 pregnancy. All of these gene expressions exhibited a similar pattern to the mPL-II gene expression determined by northern blot and RT-PCR analyses. By means of in situ hybridization, these mRNAs were localized in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Double staining studies of cathepsin Q or cathepsin R mRNA by in situ hybridization followed by immunohistochemical staining of mPL-II in the same section revealed that signals for cathepsin Q and cathepsin R mRNAs were colocalized in mPL-II immunopositive trophoblast cells in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Possible association of cathepsins with mPL-II may play important roles in placental functions during the latter half of pregnancy in mice.     

The effect of estrogen on phosphorylation of prolactin in the mouse pituitary gland

Several studies have indicated that prolactin (PRL) assumes oligomeric, proteolytically cleaved, phosphorylated and glycosylated forms. Phosphorylated PRL (PPRL) is considered to be the most important posttranslationally modified form in the rat. In the present study, we examined whether or not PRL is present in the mouse pituitary gland in the phosphorylated form. Mouse pituitary PRL was digested with acid phosphatase, resolved by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue, and then immunoblotted against the anti-PRL, anti-phosphoserine and anti-phosphothreonine antibodies. We also examined whether PRL is phosphorylated by protein kinases and semi-quantified the ratios of PPRL to PRL in the pituitary gland. The results indicated that three types of PRL are present in the pituitary glands of both male and female mice. One was non-phosphorylated (isoform 1), and the other two were immunoreactive to anti-phosphoserine (isoform 2) and/or anti-phosphothreonine (isoform 3) antibodies. The ratio between isoforms 2 and 1 of the 30-day-old female mice was higher than that of the 20-day-old female mice. However, the ratios among the three isoforms in the male pituitary glands did not differ with age. The ratio of PPRL to isoform 1 was obviously reduced after ovariectomy (OVX), and it recovered with estrogen replacement. These results suggest that estrogen influences PRL phosphorylation in female mice.