IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.     

Discrepancy between minimal inhibitory concentration to enrofloxacin and mutations present in the quinolone-resistance determining regions of Mycoplasma gallisepticum field strains

This study evaluated the in vitro activity of diphenyl diselenide against 19 Pythium insidiosum isolates and the in vivo therapeutic response of rabbits with experimentally induced pythiosis. In vitro: susceptibility tests were performed using the broth macrodilution method in accordance with the CLSI document M38-A2. The criteria for interpretation were as follows: MIC-1 and MIC-2 (inhibition of 90% and 100% of mycelium growth, respectively) and the minimum fungicide concentration (MIC-3). In vivo: twenty rabbits were divided into four groups with five animals each and treated for 40 consecutive days: groups 1 and 2 (experimentally induced pythiosis) were treated with diphenyl diselenide (10mg/kg/day) and canola oil (1 mL/kg/day), respectively; groups 3 and 4 (controls) were treated with canola oil (1 mL/kg/day) and diphenyl diselenide (10mg/kg/day), respectively. Toxicity was evaluated using biochemical and haematological parameters. In vitro susceptibility tests showed that 89.4% of isolates had a MIC-1 ≤ 0.5 μg/mL, 84.2% of isolates had a MIC-2 ≤ 1.0 μg/mL and 94.7% of isolates had a MIC-3 ≤ 2.0 μg/mL. The in vivo assay suggested that this compound has a fungistatic activity, and the biochemical and haematological parameters indicated that there was no renal, hepatic or haematological toxicity. The comparison of the unsaturated iron binding capacity levels between animals with and without pythiosis suggested the involvement of iron metabolism in the pathogenesis of pythiosis. This study demonstrated the absence of detectable toxicity caused by diphenyl diselenide and the in vitro fungicidal and in vivo fungistatic activities of this drug, which makes it an option for future therapeutic approaches in the treatment of pythiosis.     

Effects of the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and liver biomarkers in the common carp (Cyprinus carpio)

The aim of the study was to evaluate the effect of subchronic exposure to the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and important liver biomarkers in the common carp (Cyprinus carpio). One year old fish were exposed for 28 days to LASSO MTX added to the tank water at concentrations of 240 and 2400 μg L⁻¹. The exposure did not affect fish biometric parameters. Glutathione-S-tranferase (GST) activity in liver (hepatopancreas) remained unchanged in exposed fish when compared to controls. However, significant induction of total cytochrome P 450 (CYP 450), ethoxyresorufin-O-deethylase (EROD) activity and elevated glutathione (GSH) in liver of exposed fish were detected.     

Immunization of calves with live and inactivated whole-cell vaccines against Salmonella typhimurium infection

Eight calves were immunized with live auxotrophic Salmonella typhimurium mutants (aro -SL 1479, gal E 3821) and twelve calves with phenol-killed whole-cell S. typhimurium vaccine, respectively. The clinical status of the animals was followed and serial reisolation of vaccine and challenge strains from faeces was attempted. The immunization of calves with the live aro- auxotrophic S. typhimurium SL 1479 mutant proved to be unsuitable due to the death of calves after revaccination. The calves immunized with live auxotrophic gal E S. typhimurium CCM 3821 mutant proved to be protected against challenge with virulent S. typhimurium 4/5 strain administered orally at a dose of 10(6) colony forming units (CFU). The postvaccination complications showed serious shortcomings. The immunization of calves with three doses of whole-cell inactivated vaccine containing 5 strains of S. typhimurium was effective against oral challenge with virulent S. typhimurium 4/5 at a dose of 10(6) CFU.     

Stabilization of Salmonella-specific dialyzable leukocyte extracts.

Activity of Salmonella-specific dialyzable leukocyte extracts (DLE) prepared from mesenteric lymphatic nodes of calves and stabilized with bovine albumin was studied in this work. The effect of ambient temperature and storage period on the activity of DLE was evaluated. Testing for DLE activity by means of capillary leukocyte migration inhibition (LMI) assay showed that DLE stabilized with albumin retained 60% of its activity for 12 months of storage at 4 degrees C. This level of activity was retained in the native DLE (without albumin) kept at -20 degrees C. DLE stabilized with albumin and stored for 12 months at 4 degrees C inhibited the penetration of salmonellae into the liver and spleen, and their colonization in the gastrointestinal tract was significantly reduced.     

Dialyzable leukocyte extract used in the prevention of Salmonella infection in calves.

The protective effect of dialyzable leukocyte extract (DLE) was investigated in the experimental model of Salmonella infection in calves. DLE was obtained from the lymphatic nodes and spleens of fattening bulls immunized with whole-cell Salmonella vaccine (designated DLEs-im), from the same organs of calves immunized and subsequently infected with Salmonella typhimurium (DLEs-inf), and from non-immunized fattening bulls (DLEn). Three doses of DLEs-inf and DLEs-im applied intravenously at 3-day intervals induced protection in all calves against infection. There were statistically significant differences in the immunological, clinical and microbiological parameters. Three doses of DLEs-inf injected intramuscularly at 3-day intervals provided a protective effect; however, one calf died. The intravenous application of DLEn induced low protection against experimental Salmonella infection and two calves died. The results indicate that the preparation of antigen-specific DLE may be possible via immunization of fattening bulls.     

Selective method for cyanobacterial bloom removal: hydraulic jet cavitation experience

The aim of this study was to investigate the suitability of hydraulic jet cavitation as a method for cyanobacterial water-bloom management. Effects of cavitation were studied on laboratory culture of the cyanobacterium Microcystis aeruginosa, on a culture of a green alga Chlorella kessleri (as a non-target species) as well as on a real cyanobacterial biomass with Microcystis sp. as a dominant species. Our results suggested that the cavitation treatment of cyanobacteria is capable of causing the disintegration of their gas vesicles. Using this treatment, up to 99 % removal efficiency of cyanobacteria was achieved. Moreover, no effect on cyanobacterial membrane integrity or metabolic activity was detected by flow cytometry; thus, hydraulic cavitation seems to be harmless from the viewpoint of possible release of cyanotoxins into the water column. The green algae (here C. kessleri) were not affected negatively by the cavitation, and thus, they may still act as the natural nutrient competitors of cyanobacteria in lakes, ponds or reservoirs treated by cavitation.     

Attitudes of small-scale forest owners to their properties in an ageing society: Findings of survey in Yamaguchi prefecture, Japan

Populations of most developed countries have been ageing, and the populations of Japanese mountain villages are estimated to have reached into a super-ageing society. In particular, because forestry is unprofitable and due to the economic recession in Japan, many small-scale forest owners face the problems of ageing. For policy-makers, it is important to assess the socioeconomic impacts of forest owners’ ageing in order to ensure the sustainable management of forests. A survey was conducted of forest owners in Yamaguchi Prefecture, which is famous for overall depopulation and ageing of the rural population. It was found that 83% of 687 plantation forest owners who responded in the survey were 60 years or older and 76% did not have forestry income over the past three years, but 81% had kept up ownership of their forest as the traditional family property. In terms of forest management intentions, the respondents were found to consist of four types, namely ‘family management’, ‘commissioned management’, ‘de-accession’ and ‘possession without proper management’. Differences in evaluations of hypothetical policies were found among the four types.     

Determination and Health-Risk Evaluation of Nitroxynil Residues in the Edible Tissue of Cattle

A specific Polarographic method with a sensitivity of ≥ 2 jig/kg (ppb) has been used to determine the plasma and tissue concentrations of nitroxynil (NTX), which is used against Parafilaria bovicola in cattle. After treatment with the therapeutic dose on NTX (2 × 20 mg/kg b.w., s,c), there was an initial rapid decrease in the plasma concentration followed by a slower elimination phase. The plasma levels of NTX were 8 mg/kg (ppm) and 3 mg/kg after 6 weeks and 2 months, respectively. The muscle and other edible tissue from treated cattle contained 0.1–0.3 mg/kg NTX after 2 months, and jxg/kg amounts were still detectable 3 months after the injection. Based on available pharmacological and toxicological data, a 3-months withdrawal time for NTX in cattle is proposed.