Proteome analysis of whole and water‐soluble proteins in masseter and semitendinosus muscles of Holstein cows

To assess both quantitative and qualitative differences between the slow‐ and fast‐type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two‐dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water‐soluble protein fraction. Two slow‐type myofibrillar proteins (myosin light chain‐1 slow‐b and myosin light chain‐2 slow), and aconitase‐2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast‐type myofibrillar proteins (myosin light chain‐1 fast, myosin light chain‐2 fast, myosin light chain‐3 fast and tropomyosin‐1), and three enzymes of glycolytic pathway (enolase‐3, aldolase‐A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow‐ and fast‐type muscles.     

Therapeutic effect of anti-TNF-α antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-α antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-α antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-α antibody and LVFX. Anti-TNF-α antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.     

Biosynthesis of estradiol-17β in the ovarian follicles of the red seabream Pagrus major during vitellogenesis

ABSTRACT: The red seabream Pagrus major is a useful experimental fish for studying the endocrine control of oogenesis in teleosts. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in the ovarian follicles of red seabream. Intact follicles were isolated during vitellogenesis and incubated in vitro with different radiolabeled steroid precursors. When 17-hydroxyprogesterone (17-P), dehydroepiandrosterone (DHEA), or androstenedione (AD) were used as precursors, both testosterone (T) and estrone (E1) were synthesized by follicles, leading to estradiol-17β (E2) production. Serum steroid levels measured by enzyme-linked immunosorbent assay showed that T, E1, and E2 were present in the circulation at levels ranging from 1 ng/mL to 2 ng/mL throughout the day during the spawning season. In vitro conversion of E1 into E2, however, was 15.8-fold greater than T conversion into E2, suggesting that E2 is synthesized mainly via E1 rather than T. The results showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, DHEA, AD, and E1. Thus, the study demonstrated the complete steroidogenic E2 synthesis pathway in the ovarian follicles of red seabream, and revealed that E1 is the major precursor of E2.     

Geographical distribution and seasonality of the prevalence of Leucocytozoon lovati in Japanese rock ptarmigans (Lagopus mutus japonicus) found in the alpine regions of Japan

In this study, we investigated the geographical distribution and seasonality of Leucocytozoon lovati infection in the Japanese rock ptarmigan (Lagopus mutus japonicus); this bird is one of the special natural monuments of Japan that inhabits the Japanese alpine regions. We examined blood samples from birds captured in the Kubiki, Hida, and Akaishi mountain ranges for three years from 2002 to 2005. Seventy-three blood samples from 42 males, 19 females, and 12 birds of unknown sex were used for this study. The rate of infection with L. lovati was 78.1% in the 73 birds examined. We demonstrated that the L. lovati infection was distributed across wide ranges of ptarmigan populations from the northern to the southern alpine zones. There was no sex bias in the prevalence ratio. The prevalence of L. lovati and the level of parasitization of the blood cells tended to increase from spring through summer; in contrast, a decrease was observed from summer through autumn. Although L. lovati infection was observed in a number of local populations inhabiting three mountainous regions, no infected birds were found in Mt. Johnen-dake and Mt. Maejohnen-dake. It is necessary to continue surveying the relationship between the population dynamics of the ptarmigan and the density of the arthropod vector from the perspective of in situ conservation of this endangered species.     

Phylogenetic comparison of Leucocytozoon spp. from wild birds of Japan

Eight species of Japanese birds were found to be infected with Leucocytozoon species using microscopic analysis. We used PCR and sequence analysis of the mitochondrial cytochrome b gene (cyt b) to compare the genetic background among these detected protozoa species. In 20 individuals of 22 samples, a single amplified band was detected from 6 of 8 bird species; 9 Japanese rock ptarmigans (Lagopus mutus japonicus), 4 large-billed crows (Corvus macrorhynchos), 2 carrion crows (C. corone), 2 scops owls (Otus scops), 1 Japanese grosbeak (Eophona personata), and 2 brown-eared bulbuls (Hypsipetes amaurotis), respectively. Phylogenetic analysis based on the partial cyt b sequences revealed that all Leucocytozoon isolates in Japan closely grouped with other Leucocytozoon species previously reported in the literature. Among the Japanese isolates, the phylogenetic tree suggested that L. lovati from the Japanese rock ptarmigan may be basal to the parasites found in other bird species. Our study is the first to identify the molecular relationships among Leucocytozoon parasites in the avifauna of Japan.     

A retrospective study and gene analysis of canine sterile panniculitis

In this study, a retrospective analysis was conducted to assess the current aspects and predisposing factors of canine sterile panniculitis. Miniature dachshund, neutered, and younger dogs appeared to be predisposed. In addition, histories of previous surgery and injection were associated in 46.5% of the cases, with several types of surgical suture materials used. About 88% of the dogs had multifocal lesions, frequently with signs of systemic illnesses. Whereas systemic immunosuppressive therapy was effective in most dogs, surgical excision of lesions was rarely curative. In order to prevent recurrences, over 65% of the cases required prolonged immunosuppressive therapy. Polymorphism of canine alpha(1)AT gene was investigated as a candidate gene for sterile panniculitis. Eight polymorphisms were discovered in seven Miniature dachshunds by direct nucleotide sequencing, which included a 12-bp deletion, three non-synonymous single nucleotide polymorphisms, and four silent substitutions. Genotyping of the two polymorphisms, c.109_120del12 and c.483A>C, which identified at high incidence in the dachshunds, was conducted in 83 dogs of 6 popular breeds. The frequencies of neither polymorphism differed between Miniature dachshunds and other breeds, suggesting that neither is responsible for developing panniculitis.     

Characterization of Escherichia coli strains obtained from layer chickens affected with colibacillosis in a commercial egg-producing farm

The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.     

Isolation of a Megatrypanum trypanosome from sika deer (Cervus nippon yesoensis) in Japan

A trypanosome was isolated from a sika deer (Cervus nippon yesoensis) in Hokkaido, Japan, during the primary culture of sika deer renal cells. This is the first report of isolation of a Megatrypanum trypanosome from Japanese Cervidae. The trypanosome, designated TSD1, was propagated and maintained in Eagle's modified essential medium containing 20% fetal bovine serum with sika deer renal cells as feeder. The TSD1 trypanosome was morphometrically similar to Trypanosoma cervi, which is commonly isolated from American and European deer. PCR analysis with primers for 18S ribosomal DNA and nucleotide sequencing showed that TSD1 is a member of genus Trypanosoma, subgenus Megatrypanum. Phylogenetically TSD1 is closely related to T. theileri, a common trypanosome of cattle, but is distinguishable from T. theileri by some morphometrical and biological features.     

Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene

OBJECTIVE: To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1). SAMPLE POPULATION: Madin-Darby canine kidney (MDCK) epithelial cell line. PROCEDURES: The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus-based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays. RESULTS: P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1. CONCLUSIONS AND CLINICAL RELEVANCE: Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.     

Common variable immune deficiency in a Pomeranian with Pneumocystis carinii pneumonia

A Pomeranian dog, 1 year- and 8 month-old neutered female, was presented with persistent respiratory distress and recurrent generalized demodicosis. Physical examination revealed cyanosis, rough respiratory sounds, multifocal alopecia and dermal erosions on the dorsal side of the forelimbs, perineal area and skin around the eyes. A severe diffuse interstitial lung pattern was observed on thoracic radiographs. The blood examination revealed neutrophilia and hypoglobulinemia. Serum immunoglobulin concentrations of IgG and IgA were low. Histopathological examination revealed severe diffuse interstitial pneumonia with Pneumocystis carinii infection. Severe lymphoid depletion was observed in the spleen and other organs with lymphoid follicles consisted mainly of CD3-positive T cells and few cells of B-cell lineage. B-cell hypoplasia with subsequent antibody deficiency was suspected.