Microstructure of poly(gamma-glutamic acid) produced by Bacillus subtilis consisting of clusters of D- and L-glutamic acid repeating units

Poly(gamma-glutamic acid) (PGA) produced by a strain of Bacillus subtilis was partially hydrolyzed into various oligopeptides so that the dipeptide fraction was isolated by the preparative thin-layer chromatography. HPLC analysis was applied to the detection of each of the four stereoisomers in this fraction using chemically synthesized authentic samples. The fraction consisted of N-gamma- d-glutamyl- d-glutamic acid, N-gamma- l-glutamyl- l-glutamic acid, N-gamma- d-glutamyl- l-glutamic acid, and N-gamma- l-glutamyl- d-glutamic acid at a ratio of 5.9:6.0:1.0:1.0. On the basis of this result, a model was proposed for the microstructure of the bacterial PGA, in which d- and l-glutamic acid repeating units are alternately linked in a single chain of the molecule.     

Gizzard nematodiasis in Japanese mountain hawk eagle (Spizaetus nipalensis)

In this paper, we report spontaneous gizzard nematodiasis in an adult Japanese mountain hawk eagle (Spizaetus nipalensis). Grossly, the gizzard had a black mucoid substance attached to the surface mucous membrane, and the heart was dilated. Histologically, immature larvae with yellow pigments invaded crypts of the mucous membrane. More developed larvae invaded the lamina propria and muscular layers and serosa of the gizzard, with pressure atrophy and cellular reaction (infiltration of heterophils and macrophages and proliferation of fibrous connective tissue). Moderate bronchopneumonia due to larvae invasion was also seen in the lung. The morphology suggests that the parasites may be nematodes, but the species of nematode could not be confirmed. The bird may have died from malabsorption and respiratory damage as a result of the gizzard and lung lesions.     

Detection of antibodies to avian pneumovirus by a micro-indirect immunofluorescent antibody test.

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.     

Low pathogenicity H5N2 avian influenza outbreak in Japan during the 2005-2006

At the end of May 2005, a low-pathogenicity avian influenza (LPAI) virus of subtype H5N2 was isolated for the first time from chickens in Japan. Through active and epidemiological surveillance, 5.78 million chickens on 41 farms were found to be affected and 16 H5N2 viruses were isolated. Antigenic analysis revealed antigenic similarity of these isolates. Phylogenetic analysis showed that they originated from a common ancestor and clustered with the H5N2 strains prevalent in Central America that have been circulating since 1994. Experimental infection of chickens with the index isolate (A/chicken/Ibaraki/1/05) demonstrated that this virus replicated efficiently in the respiratory tract without clinical signs, and dust-borne and/or droplet-borne transmission was considered as a possible mode of transmission. These results suggested that the H5N2 LPAI viruses isolated in Japan were highly adapted to chickens.     

Resistance phenotypes and genotypes among multiple-antimicrobial-resistant Salmonella enterica subspecies enterica serovar Choleraesuis strains isolated between 2008 and 2012 from slaughter pigs in Okinawa Prefecture,Japan

A total of 349 Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) strains, which were isolated between 2008 and 2012 from 349 pigs at two slaughterhouses in Okinawa Prefecture, Japan, were investigated for antimicrobial susceptibility and the presence of antimicrobial resistance genes. All isolates were resistant to at least four antimicrobial agents. The antimicrobial agents for which isolates showed a high incidence of resistance were as follows: ampicillin (100%) and streptomycin (100%), followed by gentamicin (99.7%), oxytetracycline (99.7%), sulfamethoxazole/trimethoprim (99.4%), nalidixic acid (40.1%) and oxolinic acid (40.1%). All isolates were sensitive to cefuroxime, ceftiofur, colistin, fosfomycin, enrofloxacin, orbifloxacin and danofloxacin. The predominant resistance phenotypes and genotypes were: resistance to ampicillin, streptomycin, gentamicin, oxytetracycline and sulfamethoxazole/trimethoprim (58.5%, 204/349) and blaTEM-strA-strB-aadA1-aadA2-aacC2-tet (B)-sul1-sul2-dhfrXII-dhfrXIII (36.1%, 126/349). The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of the quinolone-resistant isolates (n=12) showed amino acid substitutions of Ser-83→Phe or Asp-87→Tyr in GyrA and Ser-107→Ala in ParC. To our knowledge, this is the first report on the molecular characterization of antimicrobial resistance among S. Choleraesuis strains in Japan.     

Hemagglutinin-neuraminidase gene of genotype VII Newcastle disease virus strains isolated in Japan

Recently, genotype VII of Newcastle disease virus (NDV) has become the most prevalent NDV genotype in Asia. Here the hemagglutinin-neuraminidase (HN) gene of genotype VII NDV strains isolated in Japan was analyzed. Notably, point amino acid substitutions in the HN protein at position 347, which is located on the major linear epitope of the HN protein, were found in two strains. However, by a hemagglutination inhibition assay, major antigenic differences did not exist between the studied strains. Additionally, chickens vaccinated with the B1 strain did not exhibit clinical effects after challenge with variants possessing the substitution at position 347 (E to K), whereas all unvaccinated chickens subjected to this challenge died within 5 days.     

Experimental assessment of the pathogenicity of H5N1 influenza A viruses isolated in Japan

We examined the pathogenicity for chickens of two H5N1 avian influenza viruses isolated in Japan, A/chicken/ Yamaguchi/7/2004 (Ck/Yamaguchi/7/04) isolated from outbreaks in commercial layer chickens, and A/duck/Yokohama/aq10/ 2003 (Dk/Yokohama/aq10/03) isolated from duck meat imported from China. All chickens inoculated intranasally with either strain died, and the viruses were reisolated from all organs examined. However, both the mean time of onset of clinical signs and the mean death time of Ck/Yamaguchi/7/04 were shorter than those of Dk/Yokohama/aq10/03.     

Conserving the Eastern Hellbender Salamander

Through a mail survey of 541 residents and riparian landowners in the area surrounding the Blue River watershed of southern Indiana, we examined perceptions and intended behaviors toward the eastern hellbender (Cryptobranchus alleganiensis alleganiensis), an aquatic salamander experiencing drastic population declines due to anthropogenic causes. While anecdotal reports attribute hellbender mortality and removal to anglers and pet collectors, only 5% of respondents reported these negative behaviors. Misconceptions among anglers and the general public about the eastern hellbender were less prevalent than anticipated. Anglers, recreational boaters, riparian landowners, and respondents who were previously familiar with the hellbender displayed more positive attitudes toward the animal than other groupings of individuals. Data collected in this article shaped a detailed education and outreach campaign based on the principles of community-based social marketing.     

Pathology of fatal highly pathogenic H5N1 avian influenza virus infection in large-billed crows (Corvus macrorhynchos) during the 2004 outbreak in Japan

Highly pathogenic H5N1 avian influenza viruses were isolated in 9 large-billed crows that died in Kyoto and Osaka prefectures in Japan from March to April in 2004. We studied 3 of the 9 crows using standard histologic methods, immunohistochemistry, and virus isolation. The most prominent lesions were gross patchy areas of reddish discoloration in the pancreas. The consistent histologic lesions included severe multifocal necrotizing pancreatitis, focal degeneration and necrosis of neuron and glial cells in the central nervous system, and focal degeneration of cardiac myocytes. All of these tissues contained immunohistochemically positive influenza viral antigens. The virus was isolated from the brain, lung, heart, liver, spleen, and kidney of the crows examined. Thus we concluded that highly pathogenic avian influenza virus was associated with clinical disease, severe pathologic changes, and death in the 3 crows.     

Psychiatric investigation of 18 bereaved pet owners

This survey explores the grief associated with the loss of a pet, and was carried out using a self-administered questionnaire. The questionnaires were handed out to 50 bereaved pet owners attending a public animal cremation service, and we received 18 responses. Participants responded within 0 to 44 (median 4) days of the death of their pet. Although most mental health problems immediately following mourning are presumed to be normal grief reactions, on the basis of several psychiatric scales, 8 of the 16 valid responses indicated depression and/or neurosis. Statistical analyses showed that the following factors were significantly associated with grief reactions: age of owner, other stressful life events, family size, age of deceased animal, rearing place, and preliminary veterinary consultation.