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1.
The fragment of the VlhA1.2 gene was cloned from a Mycoplasma gallisepticum (MG) DNA library by serial PCRs after the same-sense mutagenesis of three TGA codons encoding tryptophan (Trp). Following transforming the generated plasmid of pKG-VlhA1.2, the recombinant VlhA1.2-GST fusion protein of 92 kDa was induced and recognized by anti-MG sera. After GST-affinity chromatographic purification, the VlhA-based colloidal gold immunochromatography assay (GICA) strips were generated. The GICA strips specifically detected anti-MG antibodies, but not antibodies against Mycoplasma synoviae and other positive sera against non-MG pathogens tested. The GICA strips were 128-fold more sensitive to detect anti-MG antibodies, as compared with traditional serological methods and were stored at 4 °C for 15 months without loss of their sensitivity and specificity. Analysis of sample revealed that, the GICA strips were highly sensitive, specific and stable for the on-site surveillance of MG infections by unskilled users.  相似文献   

2.
The present study describes the development of a specific Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) monoclonal antibody (MAb), 6E3, and its application in a sandwich ELISA (sELISA) format. Mab 6E3 reacted only to the 12 MmmSC within the 32 M. mycoides cluster strains and 12 representative strains of other bovine, ovine and caprine associated mycoplasmas examined. A capture/enrichment format of the sELISA that combined MAb 6E3 with a previously developed MAb 3H12 that cross reacted with Mmm Large Colony [Rodriguez, F., Ball, H.J., Finlay, D., Campbell, D., Mackie, D.P., 1996. Detection of Mycoplasma mycoides sub-species mycoides by monoclonal antibody-based sandwich ELISA. Veterinary Microbiology 51, 69–76], retained MmmSC specificity and improved the sensitivity from the 1.2 × 107 cfu/ml for a standard 2 h capture stage sELISA down to as low as 2 cfu/ml for a 72 h capture. A low level of false positives (1%) was observed when this assay was applied to 200 bovine respiratory and milk samples submitted for diagnostic investigation. This simple and specific sELISA provides a suitable assay for screening large numbers of samples for CBPP.  相似文献   

3.
The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund's modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund's complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (±S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 ± 97.5) were 77.25% (724 ± 98) in group 1, 88.02% (381 ± 97.5) in group 2, 38.92% (1943 ± 130.3) in group 3, 44.31% (1771.4 ± 102) in group 5, 59.28% (1295.2 ± 99.1) in group 7 and 55.69% (1409.5 ± 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate.  相似文献   

4.
Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (Ks) varied for different substrates. Substrate utilization patterns and Ks values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.  相似文献   

5.
Experiments were conducted to explore the use of a semiochemical bait to enhance exposure of Amblyomma variegatum Fabricius (Acari: Ixodidae) to different formulations of the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales). Initially, the relative efficacies of attraction-aggregation-attachment pheromone (AAAP), made up of o-nitrophenol, methyl salicylate and nonanoic acid in the ratio 2:1:8, 1-octen-3-ol and butyric acid, were evaluated in an olfactometer. Only AAAP and 1-octen-3-ol were found to elicit attractive responses to the tick. Simultaneous release of 1-octen-3-ol and AAAP together with CO2 from a trap in semifield plots attracted up to 94.0 ± 6% of adult ticks from a distance of 6 m, and up to 24.0 ± 5.1% from 8 m. Formulations of M. anisopliae (dry powder, oil, and emulsifiable) applied within the trap baited with AAAP, 1-octen-3-ol and CO2 resulted in high levels of contamination of the ticks attracted to the traps. However, 48 h after autoinoculation, 89.1 and 33.3% of conidia were lost in dry powder and oil formulations, respectively. Emulsifiable formulation showed least loss of propagules (17.1%). Samples of ticks attracted to the baited traps were transferred to plastic basins containing grass and maintained for 5 weeks. The experiment was conducted in rainy and dry seasons. Emulsifiable formulation gave the highest relative tick reduction in both seasons: 54.7 and 46.5% in rainy and dry seasons, respectively, followed by oil formulation (32.0 and 23.8%) and powder formulation (38.0 and 24.4%).  相似文献   

6.
This laboratory study investigates the sub-adult developmental cycle of field collected Culicoides (Avaritia) imicola Kieffer (Diptera; Ceratopogonidae). The period required from blood-feeding field-collected females to the production of progeny adults occupied 34–56 days at 20 °C, 15–21 days at 25 °C and 11–16 days at 28 °C, demonstrating clear temperature dependence. When reared at 28 °C, C. imicola demonstrated higher variability in fecundity (between 2.4 and 20.6 eggs/female) and lower hatching rates (50.0–62.2%), although larval survival rates to pupation were low at all temperatures (20–30%). Similarly, the mean emergence rate from pupae was the highest at lower temperatures. These results highlight the difficulty in establishing and maintaining a laboratory colony of this species from field-collected material and results are discussed in reference to future research directions that may aid this process.  相似文献   

7.
A comparative study of seven isolates of Mycoplasma meleagridis indicated that they were indistinguishable morphologically. Two isolates, E2 and 8M92, induced hemagglutination of red blood cells of several different species while the others did not. Metabolic inhibition, growth inhibition and growth precipitation tests revealed minor differences among the seven isolates. According to these differences, isolates were divided into three groups: antiserum-sensitive isolate 1 466, less sensitive isolates N, 8M92, RY3, 529 and E2 and insensitive isolate 1940. One dimensional polyacrylamide gel electrophoresis of cell proteins revealed that all isolates of M. meleagridis had virtually identical patterns and that they were electrophoretically distinct from Mycoplasma gallisepticum and Mycoplasma synoviae. When nonhemagglutinating isolate N, and hemagglutinating isolate E2 were examined by simple immunoelectrophoresis, no differences were detected. However, minor antigenic differences were detected between the two strains by means of two dimensional immunoelectrophoresis.  相似文献   

8.
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9.

Background

The cell invasiveness of Mycoplasma gallisepticum, the causative agent of respiratory disease in chickens and infectious sinusitis in turkeys, may be a substantial factor in the well-known chronicity of these diseases and in the systemic spread of infection. To date, not much is known about the host factors and mechanisms involved in promotion or obstruction of M. gallisepticum adherence and/or cell invasion.In the current study, the influence of extracellular matrix (ECM) proteins such as fibronectin, collagen type IV and heparin, as well as plasminogen/plasmin, on the adhesion and cell invasion levels of M. gallisepticum to chicken erythrocytes and HeLa cells was investigated in vitro. Two strains, Rhigh and Rlow, which differ in their adhesion and invasion capacity, were analyzed by applying a modified gentamicin invasion assay. Binding of selected ECM molecules to M. gallisepticum was proven by Western blot analysis.

Results

Collagen type IV, fibronectin, and plasminogen exerted positive effects on adhesion and cell invasion of M. gallisepticum, with varying degrees, depending on the strain used. Especially strain Rhigh, with its highly reduced cell adhesion and invasion capabilities seemed to profit from the addition of plasminogen. Western and dot blot analyses showed that Rhigh as well as Rlow are able to adsorb horse fibronectin and plasminogen present in the growth medium. Depletion of HeLa cell membranes from cholesterol resulted in increased adhesion, but decreased cell invasion.

Conclusion

ECM molecules seem to play a supportive role in the adhesion/cell invasion process of M. gallisepticum. Cholesterol depletion known to affect lipid rafts on the host cell surface had contrary effects on cell adherence and cell invasion of M. gallisepticum.  相似文献   

10.
Two different species of hemoplasmas, Mycoplasma coccoides and M. haemomuris, are known to infect small rodents such as mice and rats. However, there are no previous reports of hemoplasma infection in capybara (Hydrochaeris hydrochaeris). The aim of our study was to determine whether these hemoplasmas might infect capybaras from Southern Brazil. Blood samples from 31 animals: 10 captive and 21 free-ranging capybaras were collected and packed cell volume and total plasma protein were measured. DNA was extracted and PCR assays for M. coccoides and M. haemomuris were performed. Using the M. coccoides-PCR assay 64% of the capybaras were positive, 80% free-ranging and 30% from captive animals. The prevalence of infection between the groups was significantly different (p = 0.001). Sequencing of the nearly entire 16S rRNA gene from the positive samples suggested a novel hemoplasma isolate with identity of 92% with M. coccoides and 86% with M. haemomuris. All capybara samples were negative for M. haemomuris infection. DNA of a housekeeping gene was successfully amplified from all samples. This is the first evidence of a hemoplasma infection in capybaras.  相似文献   

11.
Addition of anti-immunoglobulin M (anti-IgM), G (anti-IgG) and A (anti-IgA) sera to the haemagglutination-inhibition (HI) test (anti-Ig HI test) forMycoplasma gallisepticum resulted in 2- to 8-fold increases in the HI titres. On investigating the anti-Ig HI reaction using IgM and IgG antibodies separated by affinity chromatography, it was confirmed that, in the enhanced HI titres, specificity existed between the chicken Ig classes having antibody activity and the antisera used in the test. Four days after inoculation ofM. gallisepticum, the anti-Ig HI reaction was markedly enhanced by anti-IgM antiserum in the intravenously inoculated chickens and by anti-IgA serum in the nasally inoculated chickens. Ten days after inoculation ofM. gallisepticum marked enhancement of the reaction was produced by anti-IgG serum in both intravenously and nasally inoculated chickens, but the enhancement of the anti-Ig HI reaction diminished from the second week after inoculation.  相似文献   

12.
Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.  相似文献   

13.
An experiment was conducted with eight ruminally-cannulated cows using a crossover design with 2 periods to determine the effects of yeast supplementation on rumen fermentation. Holstein dairy cows in late lactation were either supplemented with 0.5 g/hd/d of Saccharomyces cerevisiae, an active dry yeast (CNCM-1077, Levucell SC20 (r) SC, Lallemand Animal Nutrition) or not supplemented (control). A basal diet consisting of 60% forage and 40% concentrate (DM basis) was fed once daily to both groups of cows throughout the entire experiment. Ruminal pH was measured continuously every 22 min using a pH probe that was placed in the ventral rumen sac for 6 consecutive days. Volatile fatty acid and ammonia N concentrations in the rumen were measured on days 5 or 6 of the 12-d period for each cow and DM intake was monitored throughout the experiment. Data were analyzed using a mixed-effects model with repeated measures. There were no differences in dry matter intake between treatments. Mean ruminal pH was greater (P < 0.05) when yeast was supplemented (6.53 ± 0.07) compared with the control (6.32 ± 0.07). Average maximum and minimum ruminal pH were also greater (P < 0.05) when yeast was supplemented (7.01 ± 0.09 and 5.97 ± 0.08, respectively) compared with the control (6.80 ± 0.09 and 5.69 ± 0.09, respectively). Time spent under the subacute acidosis threshold, pH less than 5.6, was lower (P < 0.05) with yeast supplementation compared with control cows. No difference was observed for ruminal ammonia N concentrations (mean = 14.0 ± 1.2 mg/dL) between treatments. Total VFA concentration (mM) in the rumen tended to be lower (P = 0.10) in the yeast-supplemented cows (107.3 ± 6.35) than in the control cows (122.4 ± 6.35), which could be related to the greater pH observed with yeast supplementation. Supplementing dairy cows with active dry yeast in the current experiment increased the mean, minimum and maximum ruminal pH; decreased time spent in subacute rumen acidosis, and tended to decrease total VFA concentration in the rumen compared with control cows.  相似文献   

14.
Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents.The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog.  相似文献   

15.
Two recombinant Mycobacterium bovis BCG (rBCG) strains carrying the Eimeria tenella rhomboid gene (Rho) delivered by extrachromosomal vector pMV261 and integrative vector pMV361 were evaluated for their ability to protect chickens against E. tenella challenge. The chickens were immunized intranasal with BCG, rBCG pMV261-Rho, or rBCG pMV361-Rho twice at a 2-week interval. All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+ and CD8+ cells compared to the control (P < 0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge. But vaccination with rBCG pMV261-Rho induced higher specific antibody titers and produced greater protection rate (56.04%) than rBCG pMV361-Rho group (P < 0.05). These results indicated that M. bovis BCG is a novel vaccine vector to express and present antigens of E. tenella, and rBCG has a potential as vaccine in chickens.  相似文献   

16.
《Veterinary microbiology》2015,175(2-4):265-274
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17.
Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC50) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC50 (concentration required to inhibit 50% of viral cytopathic effect). CC50s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC50 values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC50 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.  相似文献   

18.
Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.  相似文献   

19.
Vibrio tasmaniensis, Vibrio splendidus and Vibrio neptunius species were distributed worldwide and associated with aquaculture and have been reported as the cause of diseases in aquatic organisms. Polyphasic analyses for bacterial identification are not feasible for routine diagnostic because of the time involved. The aim of this study is to design three PCR primer sets that can assist with fast detection of these species. They were designed from the 16S ribosomal RNA gene, and PCR conditions were found. Each PCR test successfully identified all the tested strains of each target species. The combined specificity of V. tasmaniensis and V. splendidus primer sets offered the best coverage (86%) in terms of separating target organisms from other related species. The primer set of V. tasmaniensis showed a lower sensitivity limit (500 fg of DNA) than the V. splendidus set (1 pg) and both sets gave positive amplification using homogenized tissues from inoculated clams, with 102 and 104 cfu/g of clam, respectively. The primer set of V. neptunius was highly specific, showing only cross-reaction with V. parahaemolyticus species from 44 tested species. Its sensitivity limit was 100 pg of DNA. A small number of biochemical tests were proposed concurrently with the PCR to differentiate the cross-reacting bacteria. The time of detection of the three tested species was reduced and the further affected animals can be diagnosed in a rapid fraction of time. The detection of virulent strains of V. tasmaniensis pointed to the risk of mollusc culture outbreaks.  相似文献   

20.
The concentration of condensed tannins (CT) in sulla (Hedysarum coronarium L.) is moderate and overall regarded as beneficial. However, the intake of this forage can reduce diet digestibility, particularly during flowering phase. An experiment was run to assess the effect of CT on feeding behaviour, intake, diet digestibility and performance of dairy sheep rotationally grazing sulla at flowering phase. Twenty-four late-lactating sheep were blocked in two homogeneous groups and submitted to the following treatments: i) twice daily drenching with 200 g/day of a 50% w/v water solution of an anti-tannic substance, polyethylene glycol, group PEG; ii) twice daily drenching with 200 g/day of water, group CON (Control group). All the sheep rotationally grazed as a flock two sulla plots from April to June (8 weeks in total). Sward height, herbage mass, botanical and chemical composition of the herbage were measured at the beginning and the end of each grazing period. The feeding behaviour (3 sheep per group) was continuously monitored for 24 h in 6 weeks using the IGER behaviour recorders. Herbage DM intake (DMI), dietary DM digestibility (DMD) and apparent CP digestibility (CPD) were estimated on 8 sheep per group by the n-alkane method. On average, PEG group had longer total grazing (503 ± 12 vs 460 ± 12 min, P < 0.05) and eating time (425 ± 13 vs 391 ± 13 min, P < 0.07) than CON group. Moreover PEG group showed shorter inter-meal intervals (41 ± 3 vs 52 ± 3, min, P < 0.05) and higher number of daily meals than CT-exposed group (24 ± 1 vs 19 ± 1 min, P < 0.01). The herbage DMI was not affected by the treatment whereas DMD (74.60 ± 3.48 vs 58.30 ± 3.01%), and CPD (60.14 ± 4.83 vs 38.21 ± 4.83%) were both increased by PEG administration (P < 0.05) confirming the negative effect of sulla CT on these variables. Milk yield tended to be higher in PEG than CON (1331 ± 45 vs 1205 ± 59 ml, P < 0.11). Milk protein content was similar between groups while milk fat content was higher in CON than PEG ewes (6.61 ± 0.15 vs 6.11 ± 0.15%, P < 0.05), being the reverse true for milk urea (46.04 ± 1.27 vs 53.04 ± 0.76%, P < 0.001). To conclude, this experiment shows that when sulla is grazed at flowering as monoculture, dietary CT can exert negative effects on DM and CP apparent digestibility, in this study partially counterbalanced by a better metabolic utilisation of the nutrients up-taken.  相似文献   

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