微小牛蜱Bm86基因的真核表达

采用RT-PCR技术从微小牛蜱饥饿幼蜱破解物中扩增到Bm86基因，将其与巴斯德毕赤酵母分泌型表达载体pPlC9K重组构建了重组表达载体pPIC9K-Bm86，测序正确后将其用SacⅠ内切酶线性化后采用电穿孔法转化巴斯德毕赤酵母菌Gs115，经G418抗性筛选高拷贝重组菌株后用甲醇诱导表达，SDSPAGE和Western-blotting分析结果表明，诱导表达的培养上清液中表达出具有反应活性的68ku重组Bm86蛋白，目的蛋白约占培养上清液中蛋白总量的32％以上，诱导96h目的蛋白的表达量为0．36mg／mL。     

Control of Boophilus microplus ticks in cattle calves by immunization with a recombinant Bm86 glucoprotein antigen preparation.

Forty Egyptian native cattle calves of 4-6 months old randomly allocated into two groups of twenty animals each were used to assess the effect of immunization of animals with a recombinant Bm86 antigen derived from Boophilus microplus ticks on induction of immunity that could protect calves during tick season. The immunization protocol involved two injections administered intramuscularly, the first was applied with complete Freund's adjuvant and the second was given with incomplete Freund's adjuvant two months later. Control calves were given saline plus adjuvant. Immunization reduced the number of adult ticks developing from a subsequent challenge infestation by 78% in immunized calves. Vaccination also, significantly reduced the weight of adult ticks in immunized calves (30.51%). The results of skin delayed hypersensitivity reaction revealed that the diameter of sites injected with the recombinant Bm86 antigen was significantly larger in immunized calves than those in controls. Analysis of the immune response indicated that there was a significant increase in the level of IgG and IgA antibodies in serum of immunized calves and protection from reinfestation was correlated with the levels of circulating antibodies.     

Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep

Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production.     

Resistance of Holstein-Friesian cows to infestation by the cattle tick (Boophilus microplus)

In two experiments, the milk production of 59 Holstein-Friesian cows in mid lactation was measured over 1 week before artificial infestation with 2,500 or 5,000 Boophilus microplus larvae. Host resistance, the proportion of female ticks applied but not engorging was estimated from weekly counts of engorging female ticks of 4.5-8. 0mm long. Mean host resistance was 79 and 67% in two experiments. Host resistance was not significantly related to milk yield before infestation, to early pregnancy, or to parity. Culling the 10% of cattle with the least resistance to ticks would result in removal of 19-21% of ticks in a herd. The results suggest that selection to improve the existing low levels of resistance to B. microplus might be undertaken without compromising milk production. However, low levels of resistance among the cattle studied and difficulties in assessment of resistance are likely to limit the usefulness of selection within the Holstein-Friesian breed.     

Synthetic pyrethroid resistance in field samples in the cattle tick (Boophilus microplus)

First evidence of resistance to synthetic pyrethroids in the cattle tick in Australia, other than the pre-existing cross-resistance in DDT-resistant ticks, has emerged. Three new strains have been investigated and characterised. The first, detected in central Queensland, showed increased ability to detoxify pyrethroids resulting in a marginal loss of efficacy of all pyrethroid tickicides tested, except flumethrin. Subsequently two more strains have been detected, with serious implications for chemical tick control. The first, from southern Queensland, has a high level of specific resistance to flumethrin. The second, from central Queensland, has a broad spectrum resistance encompassing all pyrethroids currently in use. The toxicology and biochemistry of these new resistant strains have been investigated and alternative tickicides evaluated. The significance of these latest developments is discussed.     

Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin)

Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 degrees C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks.     

Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene

Plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tick Boophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50microg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of immunoglobulin isotypes, significant levels of IgG2a and IgG2b were observed in mice immunized with 50microg of pBMC2. Measurement of interleukine (IL) levels (IL-4, IL-5, IL-12(p40)) and interferon-gamma (IFN-gamma) in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-gamma. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-gamma secretion. In all trials, mice injected intramuscularly with 50microg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus.     

Boophilus microplus: passage of bovine immunoglobulins and albumin across the gut of cattle ticks feeding on normal or vaccinated cattle

Cattle were vaccinated with antigens from adult female Boophilus microplus and haemolymph was collected from female ticks which had engorged on these animals and on matched control cattle. Radio-immunoassay for bovine plasma proteins in haemolymph from ticks fed on control cattle showed low concentrations of IgG1 and albumin. There was a significant increase in bovine plasma proteins passing across the gut in ticks fed on vaccinated cattle, with an average of 150 times more albumin and four to five times more IgG1 in the haemolymph. Ticks with obviously damaged gut had the highest concentrations of bovine plasma proteins but apparently undamaged ticks from vaccinated cattle also had elevated protein concentrations.     

Efficacy of topically applied ivermectin against Boophilus microplus (Canestrini, 1887) in cattle

Systemic efficacy of ivermectin applied topically was evaluated against the cattle tick, Boophilus microplus. Twenty cattle with induced infestations were randomly allocated to five groups of equal size based on the numbers of engorged female ticks which fell through the slatted floor of individual pens during the 3 days prior to treatments. Control cattle were non-medicated. Cattle in three groups were given ivermectin at 200, 500 or 1000 mcg kg-1 in a topical formulation applied along the backline from the withers to the rump; cattle in the fifth group were given ivermectin in an injectable formulation subcutaneously at 200 mcg kg-1. Individual 24-h tick collections were made three times weekly for 5 weeks after treatment. Engorged female B. microplus were incubated to determine effects on reproduction. Based on the numbers of engorged female B. microplus collected following treatments, overall efficacy of ivermectin applied topically at 200, 500 and 1000 mcg kg-1 was 50, 85 and 91%, respectively, whereas ivermectin given at 200 mcg kg-1 subcutaneously was 80% effective. The index of reproduction for ivermectin given topically was reduced by 84, 94 and 95%, respectively, and that for ivermectin subcutaneously was 94%. No significant differences (P greater than 0.05) were found for these variables between ivermectin given topically at 500 or 1000 mcg kg-1 versus 200 mcg kg-1 given subcutaneously.     

Eprinomectin pour-on for control of Boophilus microplus (Canestrini) ticks (Acari: Ixodidae) on cattle

The efficacy of a commercial pour-on formulation of eprinomectin, a macrocyclic lactone, against experimental infestations of Boophilus microplus (Canestrini) ticks was evaluated in two trials involving 27 Bos taurus calves. The first trial was designed to evaluate the effects of a single treatment at a dose of 0.5 mg/kg of body weight against standard size B. microplus females (4.5-8.0 mm long). A significant reduction in tick numbers (P<0.05, Wilcoxon test) was observed between treated calves as compared to untreated ones from Day 3 (44% efficacy) after treatment to the end of the trial on Day 28 (96.9%), with a peak efficacy of 97.1% on Day 21. In the second trial the effect of eprinomectin on standard size tick numbers, engorgement weight and fertility of female ticks from calves with a single treatment dose of 1 mg/kg on Day 0 and calves treated twice at a dose of 0.5 mg/kg on Days 0 and 4 was evaluated. An efficacy >93% was obtained from Day 2 to Day 28 after treatment in calves treated twice at 0.5 mg/kg, and to the end of the trial (Day 35) in calves treated once with 1 mg/kg. The 1mg/kg treatment provided >98% residual efficacy for at least 7 days. During the first part of the second trial the efficacy of eprinomectin resulted from a dramatic adverse effect on engorgement weight and fertility of female ticks, with 100% control on Day 5 (dosage of 1 mg/kg) and on Days 6 and 7 (two doses of 0.5 mg/kg). Following Day 7, most of the effect was due to reduction in the number of standard size female ticks.     

Moxidectin pour-on for control of natural populations of the cattle tick Boophilus microplus (Acarina: Ixodidae)

Fifty Bos taurus x Bos indicus heifers naturally infested with Boophilus microplus ticks were divided into two groups of 25 heifers each. Individuals of one group were treated with moxidectin 0.5% pour-on at a dosage of 500 microg of moxidectin/kg body weight and heifers from the other group remained as untreated controls. An efficacy higher than 95% was found on days 7-21 after treatment by using female ticks 4.5-8.0 mm long as the main infestation parameter. A lower, but significant efficacy (p < 0.05) was also found on days 1 (32.3% efficacy) and 27 (73.4% efficacy) post-treatment. Significantly (p < 0.05) lower numbers of immature ticks were also observed on heifers of the treated group from days 7 through 27 after treatment. A lower engorgement weight of female ticks from treated heifers was found on days 1 and 21 after treatment. Treatment also affected reproductive performance (oviposition, egg hatch and number of eggs laid) of female ticks collected on Day 1.     

The productivity effects of cattle tick (Boophilus microplus) infestation on cattle, with particular reference to Bos indicus cattle and their crosses

In response to uncertainty among cattle producers in Australia regarding the need to treat Bos indicus and B. indicus crossbreeds, the scientific literature relating to the productivity effects of Boophilus microplus on cattle of all breeds was reviewed. Estimates of the mean effect of each engorging tick (damage coefficient, d) were made from a simple analysis of the reported data. On average, each engorging female tick is responsible for the loss of 1.37 +/- 0.25 g bodyweight in B. taurus cattle. The comparable value for B. taurusxB. indicus cattle is 1.18 +/- 0.21 g/engorging tick. These values were not statistically significantly different, indicating that if a threshold approach to tick control were taken, then the threshold number of standard ticks would be the same regardless of cattle genotype. No studies provided useable estimates of the effect of tick infestation on pure B. indicus cattle. An economic threshold for treatment, below which acaricide application is not beneficial, can be predicted, using known values for the cost of acaricide application and the price of beef. However, the application of a threshold approach to control has not been embraced by government advisers and runs contrary to the accepted principals of strategic control programs.     

An estimate of the economic effects of cattle tick (Boophilus microplus) infestation on Queensland dairy farms

OBJECTIVE: To establish the cost to the Queensland dairy industry of cattle tick infestation and its control, excluding the costs incurred from control measures directed specifically at tick fever and morbidity and mortality arising from tick fever. STUDY DESIGN: Economic models are described that have been based on empirical data relating to liveweight and milk yield loss, and on a survey of control practices and tick infestation. The first two models were designed to estimate costs of control and losses resulting from tick infestation on a single dairy farm. The third model developed estimates of the cost of tick infestation for each of four regions within the tick-infested area of Queensland. RESULTS: The overall cost to the Queensland dairy industry of the cattle tick (excluding the costs associated specifically with tick fever) and based on 1998 management practices, was $4,096,000 per annum. About 49% of this cost was related to the costs of control and 51% to losses in production. CONCLUSION: Cattle tick infestation represents a significant impost on dairy producers in Queensland, and although the actual cost will change as deregulation results in economic changes in the industry, infestations of ticks will continue to be expensive to control.