Measurement of plasma chromogranin A concentrations for assessment of stress responses in dogs with insulin-induced hypoglycemia

OBJECTIVE: To determine whether cross-reactivity exists between canine chromogranin A (CgA) and anti-human CgA antibody and investigate the usefulness of plasma CgA concentration measurements as an index of acute stress responses in dogs. ANIMALS: 12 healthy Beagles. PROCEDURE: Canine CgA was extracted and purified from canine adrenal glands of cadaver dogs for studying cross-reactivity with anti-human CgA antibody. Western blotting with anti-human CgA antibody was performed. Blood samples were collected from dogs at 0, 10, 20, 30, 40, 60, 120, and 180 minutes after IV administration of saline (0.9% NaCl) solution or insulin. Canine plasma CgA concentrations were determined by use of a CgA ELISA kit with rabbit antiserum against the carboxy-terminal fragment of human CgA. Plasma cortisol and catecholamine (ie, norepinephrine and epinephrine) concentrations were measured by use of an ELISA and a high-performance liquid chromatography method, respectively. RESULTS: Purified canine CgA was specifically detected by use of western blot analysis and an ELISA with anti-human CgA antibody. An increase in plasma CgA concentrations was observed in insulin-induced hypoglycemic dogs. Changes in plasma CgA concentration were correlated with changes in plasma cortisol or catecholamine concentrations of hypoglycemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the CgA ELISA kit for determination of human plasma CgA concentrations is applicable to the measurement of canine plasma CgA concentrations. Canine plasma CgA concentrations, along with measurements of plasma cortisol and catecholamine concentrations, correctly reflect insulin-induced hypoglycemic stressed conditions in dogs. Measurement of canine plasma CgA concentrations may provide a useful index for evaluation of an acute stress response.     

Genotyping of Giardia intestinalis from domestic and wild animals in Japan using glutamete dehydrogenase gene sequencing

To determine the genotypes of Giardia intestinalis from domestic and wild animals in Japan, Giardia isolates obtained from feces of 24 dogs kept in households and breeding kennels, three companion cats, five dairy calves and three wild monkeys, Macaca fuscata, were genotyped using the 177 bp sequence of the glutamete dehydrogenase gene (gdh). The genotypes were assemblages A, C, D or A/D for dog isolates, Assemblage F for cat isolates, assemblages A or E for calf isolates and assemblage B for monkey isolates. This is the first report on the genotypes of Giardia isolates from cats, calves and wild monkeys in Japan.     

In vitro susceptibility and a new point mutation associated with tylosin-resistance in Japanese canine intestinal spirochetes

The in vitro susceptibilities of six commonly used antimicrobial agents against 29 isolates of intestinal spirochetes isolated from dogs in Japan were examined by the agar dilution technique. In addition, the genetic basis of tylosin resistance in in vitro selected resistant mutants of two reference strains and three tylosin-susceptible field isolates obtained by three successive subcultures on blood agar containing 1 microg/ml of tylosin was investigated. Carbadox was the most active (MIC: < 0.00625) of all the antimicrobial agents. Although all the isolates were susceptible to tylosin, some were resistant to erythromycin. Tiamulin, lincomycin and dimetridazole were also very active against the isolates. All the resistant isolates did not harbor any plasmids. In vitro selected tylosin-resistant mutants of previously tylosin-susceptible isolates showed a new mutation in which their adenine at the base position equivalent to 2062 of 23S rDNA of Escherichia coli has been replaced by cytosine. These findings may both provide guidance towards the proper choice of antimicrobial agents for the treatment of canine intestinal spirochetosis, and add to the understanding of the genetic basis of tylosin resistance.     

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.     

Gastroduodenal adenocarcinomas and rectal adenoma in a cougar (Felis concolor) infected with Helicobacter-like organisms and spirochetes

A 14-year-old female cougar died from gastroduodenal adenocarcinomas and rectal adenoma. At necropsy, polypoid tumor masses of various sizes were scattered on the mucosal surfaces of the stomach, duodenum, and rectum. Histologically, the gastric tumor was diagnosed as an intestinal type adenocarcinoma and the tumor cells metastasized to the mesenteric lymph nodes, spleen, and lung. Helicobacter-like organisms were detected in the lumina lined by foveolar epithelium. In the duodenum, the carcinoma cells were localized in the limina propria and many of them were intensely positive for proliferating cell nuclear antigen (PCNA). In contrast, the rectal adenoma had a lower number of PCNA-positive cells. In the rectum, chronic inflammation with numerous spirochetes was also noted. These results indicated that the occurrence of the gastrointestinal tumors might be associated with the bacterial infection described above.     

Serological survey of leptospirosis in sows with premature birth and stillbirth in Chiba and Gunma prefectures of Japan

In 1996 and 1997, the seroprevalence against Leptospira in parturient sows with premature birth or stillbirth from two herds was investigated. In three out of four sow serum samples obtained in Gunma Prefecture, the antibody titers to Leptospira interrogans serovar copenhageni (M20) were higher than 10,000 (the reciprocal of the serum dilution). Furthermore, the antibody titers to L. interrogans serovar canicola (Hond Utrecht IV) were significantly high in the three sows and the titers ranged from 1,000 to 3,000. In sows obtained in Chiba Prefecture, significantly high antibody titers to serovar copenhageni (M20) were confirmed in eight out of 40 sows, and antibody titers greater than 10,000 in six of them. Significantly high antibody titers to L. interrogans serovar icterohaemorrhagiae (RGA) and L. canicola (Hond Utrecht IV) were confirmed in four and 18 out of the 40 sows, respectively, compared with the titers to the other serovars. These findings may indicate the prevalence of leptospirosis in pig herds in both Gunma and Chiba Prefectures.     

Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.     

Comparison of the hemagglutination of formalinized American channel catfish (Ictalurus punctatus) erythrocytes between formalinized and live Aeromonas hydrophila isolated from the catfish rearing in Kasumigaura lake in Japan

Formalinized Aeromonas (A.) hydrophila agglutinated loosely with the formalinized American channel catfish erythrocytes (FACCE), while live A. hydrophila agglutinated tightly with the FACCE. There was a significant difference on the number of attaching bacterial cells to the FACCE (p<0.01) (n=40 erythrocytes) between formalinized and live A. hydrophila. The other bacteria such as Salmonella (S.) Typhimurium ST-5, Escherichia (E.) coli V-517 and Staphylococcus (S.) hyicus ATCC1249 used in this experiment did not attach the FACCE.     

Analysis of DNA relationships among Aeromonas species by RAPD (randomly amplified polymorphic DNA) typing

Genetic differences between a collection of aeromonads were studied in two laboratories by analysis of randomly amplified polymorphic DNA (RAPD). A single randomly designed primer, generated reproducible profiles of genomic DNA in both laboratories for Aeromonas salmonicida subspecies salmonicida, although the profiles differed between laboratories. Analysis of atypical strains of A. salmonicida and isolates of the A. hydrophila group produced scattered profiles in both laboratories. The uniform fingerprints produced for A. salmonicida subspecies salmonicida indicate genomic homogeneity. The scattered RAPD profiles of the motile aeromonads demonstrate the genomic diversity of this group. A group of unspeciated motile aeromonads gave uniform fingerprints, suggesting the possibility of a genomically homogeneous species. Although the RAPD technique is susceptible to the effects of minor technical variations, this study has demonstrated that where there is DNA similarity, it can be recognized, and where there is diversity, differentiation can be made. RAPD promises to be useful in epidemiological studies for rapid identification of bacteria where a source of reference DNA is available and may be useful in preliminary investigations of relatedness within groups.