World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of ectoparasiticides against myiasis causing parasites on ruminants

These guidelines have been prepared to assist in the planning, conduct and interpretation of studies for the assessment of efficacy of ectoparasiticides against the myiasis causing parasites of ruminants. These guidelines specifically focus on larvicidal efficacy against myiasis causing flies. Information is provided on the selection of animals, dose determination and dose confirmation studies, field studies, record keeping and result interpretation. These guidelines advocate the use of pen facilities for dose determination and dose confirmation studies for defining therapeutic and persistent efficacy. These guidelines are also intended to assist investigators on how to conduct specific experiments, to provide specific information for registration authorities involved in the decision-making process, to assist in the approval and registration of new ectoparasiticides, and to facilitate the world-wide adoption of standard procedures.     

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of ectoparasiticides against biting and nuisance flies on ruminants

These guidelines have been prepared to assist in the planning, conduct and interpretation of studies for the assessment of the efficacy of ectoparasiticides (excluding repellents) against the biting and nuisance dipteran flies of ruminants. Information is provided on the selection of animals, dose determination and dose confirmation studies, field studies, record keeping and result interpretation. These guidelines advocate the use of pen facilities for dose determination and dose confirmation studies. These guidelines also are intended to assist investigators on how to conduct specific studies, to provide specific information for registration authorities involved in the decision-making process, to assist in the approval and registration of new ectoparasiticides, and to facilitate the worldwide adoption of standard procedures.     

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of ectoparasiticides against biting lice, sucking lice and sheep keds on ruminants

These guidelines have been prepared to assist in the design, implementation and interpretation of studies for the assessment of the efficacy of ectoparasiticides against biting and sucking lice and sheep keds on ruminants. Information is provided on the selection of animals, dose determination, dose confirmation and field studies, record keeping and result interpretation. These guidelines advocate the use of pen facilities for dose determination and dose confirmation studies for defining therapeutic and persistent efficacy. These guidelines are also intended to assist investigators on how to conduct specific experiments, to provide specific information for registration authorities involved in the decision making process, to assist in the approval and registration of new ectoparasiticides, and to facilitate the world-wide adoption of standard procedures.     

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of acaricides against ticks (Ixodidae) on ruminants

These guidelines have been prepared to assist in the planning, conduct and interpretation of studies for the assessment of the efficacy of acaricides (excluding vaccines and other bio-control agents) against single and multi-host ticks (Ixodidae) on ruminants. Information is provided on the selection of animals, dose determination, dose confirmation and field studies, record keeping and result interpretation. The use of pen facilities is advocated for dose determination and confirmation studies for defining therapeutic and persistent efficacy. A minimum of two studies per tick species for which claims are sought is recommended for each dose determination and dose confirmation investigation. If dose confirmation studies demonstrate greater than 95% efficacy the sponsor may proceed to field studies, where a minimum of two studies per geographical location is preferred to confirm the therapeutic and persistent efficacy under field conditions. If dose confirmation studies demonstrate less than 95% efficacy then longer-term field studies can be conducted over two tick seasons with a minimum of two studies per geographical location. These studies can incorporate other control methods such as tick vaccines, to demonstrate stable long-term tick management. Specific advice is also given on conducting studies with paralysis ticks. These guidelines are also intended to assist investigators on how to conduct specific experiments, to provide specific information for registration authorities involved in the decision-making process, to assist in the approval and registration of new acaricides, and to facilitate the worldwide adoption of standard procedures.     

World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys

These guidelines have been written to aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against Eimeria species in chickens and turkeys. The information provided deals with many aspects of how to conduct controlled studies in battery cages (dose determination), floor pens (dose confirmation), and commercial facilities (field effectiveness studies), the selection of birds, housing, feeding, preparation of medicated rations, record keeping, diagnostic techniques, and methods for the preparation, maintenance and use of parasites. These guidelines are also intended to assist investigators in conducting specific studies, provide specific information for registration authorities involved in the decision-making process, assist in the approval and registration of new anticoccidial drugs, and facilitate the world-wide adoption of standard procedures.     

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestation on dogs and cats

These guidelines are intended to assist the planning and conduct of laboratory and clinical studies to assess the efficacy of ectoparasiticides applied to dogs or cats for the purpose of treating, preventing and controlling flea and tick infestations. The term ectoparasiticide includes insecticidal and acaricidal compounds, as well as insect growth regulators. The range of biological activities accruing from animal treatment that are considered include: repellency and anti-feeding effects, knockdown, speed of kill, immediate and persistent lethal effects, and interference with egg fertility and subsequent development of off-host life cycle stages. Information is provided on the selection of animals, dose determination, dose confirmation and field studies, record keeping, interpretation of results and animal welfare. These guidelines are also intended to assist registration authorities involved in the approval and registration of new parasiticides, and to facilitate the worldwide adoption of harmonized procedures.     

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of equine anthelmintics

These guidelines have been designed to assist in the planning, operation and interpretation of studies which would serve to assess the efficacy of drugs against internal parasites of horses. Although the term anthelmintic is used in the title and text, these guidelines include studies on drug efficacy against larvae of horse bot flies, Gasterophilus spp, which are non-helminth parasites commonly occurring in the stomach of horses. The advantages, disadvantages and application of critical and controlled tests are presented. Information is also provided on selection of animals, housing, feed, dose titration, confirmatory and clinical trials, record keeping and necropsy procedures. These guidelines should assist both investigators and registration authorities in the evaluation of compounds using comparable and standard procedures with the minimum number of animals.     

World association for the advancement of veterinary parasitology (WAAVP): second edition of guidelines for evaluating the efficacy of equine anthelmintics.

These guidelines have been designed to assist in the planning, operation and interpretation of studies which would serve to assess the efficacy of drugs against internal parasites of horses. Although the term anthelmintic is used in the title and text, these guidelines include studies on drug efficacy against larvae of horse bot flies, Gasterophilus spp., which are non-helminth parasites commonly occurring in the stomach of horses. The advantages, disadvantages and application of critical and controlled tests are presented. Information is also provided on selection of animals, housing, feed, dose titration, confirmatory and clinical trials, record keeping and necropsy procedures. These guidelines should assist both investigators and registration authorities in the evaluation of compounds using comparable and standard procedures with the minimum number of animals.     

World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the effectiveness of anthelmintics in chickens and turkeys

These guidelines have been prepared to assist in the planning, operation and interpretation of studies designed to assess the effectiveness of drugs against helminth parasites of chickens and turkeys. They are the first to be compiled under the auspices of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for these parasites. The advantages and disadvantages of the widely used critical and controlled tests are discussed. Information is provided on the selection of animals for experiments, animal housing, feed, dose determination studies, confirmatory and field trials, record keeping and necropsy procedures. This document should help investigators and those involved in product approval and registration in conducting and evaluating studies concerned with determining the effectiveness and safety of anthelmintic drugs.     

Confirmation of the efficacy of a novel formulation of metaflumizone plus amitraz for the treatment and control of fleas and ticks on dogs

A novel spot-on formulation containing metaflumizone plus amitraz (ProMeris/ProMeris Duo for Dogs, Fort Dodge Animal Health, Overland Park, KS) was evaluated in four laboratory studies to confirm efficacy against fleas and ticks on dogs for 1 month. Three different strains of cat flea (Ctenocephalides felis felis) and four tick species were used. Rhipicephalus sanguineus and Dermacentor variabilis were evaluated concurrently in two studies and Ixodes scapularis and Amblyomma americanum in one study each. In all studies, dogs were randomly allocated to treatment groups and compared with nontreated dogs. One study also included a placebo treatment and a commercial product containing fipronil plus S-methoprene. All treatments were applied to the skin at a single spot between the scapulae on Day 0. Dogs were infested with fleas and/or ticks prior to treatment and then reinfested at weekly intervals for 6 weeks after treatment and evaluated for efficacy at 1 or 2 days after treatment and each reinfestation. These studies confirmed that treatment with ProMeris for Dogs at the proposed commercial dose rate rapidly controlled existing infestations of fleas and ticks on dogs. Treatment provided control of reinfesting fleas for up to 6 weeks and at least 4 weeks control of ticks. Efficacy was confirmed in a variety of dog breeds against three different flea strains and four common species of ticks found on dogs in the United States.     

In vitro effects of four tropical plants on three life-cycle stages of the parasitic nematode, Haemonchus contortus

Alcoholic extracts of four tropical plants (Zanthoxylum zanthoxyloides, Newbouldia laevis, Morinda lucida and Carica papaya) were screened in vitro for potential anti-parasitic effects against eggs, infective larvae and adult Haemonchus contortus. Significant effects were obtained with all four plants but differences were observed depending on the parasitic stage. The effects of the four plant extracts were similar on egg hatching and were dose dependent. In contrast, no dose-response relationship was found for infective larvae and adult worms, although more potent effects were usually observed with the highest concentrations. Using a larval inhibition migration test, extracts of fagara (Z. zanthoxyloides) were found to be less active against Haemonchus infective larvae than were the other plants. N. laevis was found to be highly and rapidly effective against adult worms. Overall, these in vitro results suggest that these four plants, traditionally used by small farmers in Western Africa, do possess anti-parasitic properties. These effects remain to be confirmed through in vivo studies.     

Dose determination of a novel formulation of metaflumizone plus amitraz for control of cat fleas (Ctenocephalides felis felis) and brown dog ticks (Rhipicephalus sanguineus) on dogs

A novel spot-on formulation containing metaflumizone and amitraz (ProMeris/ProMeris Duo for Dogs, Fort Dodge Animal Health, Overland Park, KS) was evaluated in a laboratory study to determine the appropriate dose for efficacy against fleas and ticks on dogs for 1 month. Thirty-six Beagles were randomly allocated to six equal groups and individually housed. One group remained nontreated. Another was treated with a placebo formulation (solvents with no active ingredients). Three groups of dogs were treated topically with the metaflumizone plus amitraz formulation (150mg of each of metaflumizone and amitraz/ml), at volumes providing doses of 10, 20 and 40mgeachactive/kg. The final group was treated with a commercial spot-on providing 6.7mgfipronil/kg. All treatments were applied to the skin at a single spot between the scapulae on Day 0. Dogs were infested with 50 adult brown dog ticks (Rhipicephalus sanguineus) on each of Days -2, 5, 12, 19, 26, 33 and 40, and with 100 cat fleas (Ctenocephalides felis felis) on Days -1, 6, 13, 20, 27, 34 and 41. Dogs were examined and parasites "finger counted" on Day 1 to estimate knock down efficacy, and all animals were comb counted to determine the numbers of viable fleas and ticks on Days 7, 14, 21, 28, 35 and 42. There were no significant differences in parasite counts between the nontreated control and the placebo-treated control groups for either fleas or ticks (P>0.05) except for very slight reductions on Day 7 for fleas and Day 14 for ticks, demonstrating that the formulation excipients had no activity. The qualitative finger counts on Day 1 indicated that all of the insecticidal treatments resulted in a noticeable reduction in flea and tick numbers within 1 day of treatment. All of the metaflumizone and amitraz treatments and fipronil resulted in significantly lower flea and tick numbers relative to nontreated controls on all posttreatment count days (P<0.05). For the metaflumizone plus amitraz treatments, mean flea and tick counts for the 10mg/kg dose were significantly higher than those for the 20mg/kg dose (P<0.05) from Day 21 on. There was no significant advantage provided by the 40mg/kg dose over the 20mg dose throughout the entire study (P>0.05). The two higher metaflumizone plus amitraz doses provided >95% control of fleas and >90% control of ticks for at least 35 days after treatment, and this level of control was similar to that of the commercial fipronil product. The 20mg/kg dose was selected as the minimum commercial dose rate to provide effective flea and tick control for at least 1 month following a single treatment.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.     

Efficacy of a novel formulation of metaflumizone for the control of fleas (Ctenocephalides felis) on cats

A novel spot-on formulation containing metaflumizone (ProMeris for Cats, Fort Dodge Animal Health, Overland Park, KS) was evaluated in five laboratory studies to determine the duration of residual efficacy in cats against fleas after a single spot treatment. In each study, eight domestic shorthair cats were randomly allocated to each treatment group and individually housed. One group in each study remained non-treated. In one study, an additional group of eight cats was treated with a placebo formulation. Cats were treated topically with metaflumizone formulation to provide a dose of at least 40mg metaflumizone/kg. Cats were infested with 100 cat fleas (Ctenocephalides felis felis) once per week for approximately 8 weeks. Cats were comb counted 48h after treatment and each infestation to determine the number of viable fleas present. There were no significant differences in flea counts between the non-treated control and the placebo-treated control (P>0.05) other than a 26% reduction at week 1, demonstrating that the formulation excipients had no activity. Metaflumizone treatment resulted in significantly lower flea numbers relative to non-treated controls on all post-treatment count days (P<0.05). Metaflumizone provided >90% control of flea infestations up to 7 weeks following a single treatment.     

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.     

环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立简

为寻求一种快速灵敏的环形泰勒虫病PCR检测方法,基于环形泰勒虫裂殖体表面蛋白（Theirelia annulata surface protein,TaSP）基因序列保守区设计特异性引物,通过PCR技术扩增出该基因长为393 bp的高免疫原性区片段。用该引物对环形泰勒虫、中华泰勒虫、瑟式泰勒虫、尤氏泰勒虫、吕氏泰勒虫、绵羊泰勒虫、马泰勒虫、驽巴贝斯虫、牛巴贝斯虫基因组模板进行特异性试验,对环形泰勒虫基因组模板进行梯度稀释后扩增,以确定试验的敏感性,同时用本试验建立的方法与常规显微镜镜检方法对150份血清样品进行检测。特异性试验结果显示,在被检测的9个样本中,只有环形泰勒虫基因组模板中扩增出了符合大小的特异核苷酸片段;敏感性试验结果表明,PCR对环形泰勒虫的扩增效率可达到10^-10;通过对150份血清样品的检测,并与血涂片方法进行比较,结果显示PCR方法具有特异性强、敏感度高等特点,适用于牛环形泰勒虫病的检测。     

Anthelmintic activity of Cocos nucifera L. on intestinal nematodes of mice

In this study, we evaluated the anthelmintic activity of the liquid extracted from the bark of the green coconut (LBGC), as well as butanol extract obtained from LBGC, on mouse intestinal nematodes. Thirty-six naturally infected mice were distributed into six groups receiving the following treatments: Group I: 1000 mg/kg of LBGC; Group II: 2000 mg/kg of LBGC; Group III: 500 mg/kg of butanol extract; Group IV: 1000 mg/kg of butanol extract; Group V: 0.56 mg/kg febendazole; and Group VI: 3% dimethylsulfoxide. The chemical composition of the LBGC and its butanol extract was determined by phytochemical tests. A dose of 1000 mg/kg of butanol extract had 90.70% efficacy in reducing the mouse worm burden (p < 0.05). Phytochemical tests revealed the presence of triterpens, saponnins and condensed tannins in the LBGC and butanol extracts. These results suggest that Cocos nucifera extracts may be useful in the control of intestinal nematodes.     

Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

Oocysts of Cryptosporidium parvum are resistant to environmental conditions and many disinfectants. A combination of cell culture and quantitative real time PCR (cc–qPCR) is established for evaluation of anticoccidial disinfectants against C. parvum. C. parvum oocysts were treated with disinfectants, washed and oocysts were incubated with HCT-8 cell monolayers in the presence of excystation medium for 3 h. Subsequently, unbound parasites were removed by washing with growing medium and the infected monolayers were further maintained in fresh growing medium for 48 h. Genomic DNA was extracted from each sample and qPCR performed targeting a specific sequence of the 70 kDa heat shock protein gene in order to quantify development. Treatment of oocysts with cresolic disinfectants demonstrated dose dependent reduction of viability of oocysts. More than 98% inactivations were recorded with at least 2% concentration of cresolic disinfectants after 2 h of treatment. Bleach (sodium hypochlorite) at 6% solution induced 92.7% inactivation of C. parvum oocysts after 2 h. Thermally treated oocysts (56 and 70 °C for 20 min) demonstrated complete inactivation, whereas at 38 °C no inactivation was observed. Application of Neopredisan^® 135-1 and Aldecoc^® TGE (4% for 2 h) as recommended according to the current guidelines stipulated by DVG (German Veterinary Society) consistently inactivated more than 99.5% of oocysts. The suggested cc–qPCR method appeared to be suited for standardized testing of inactivation measures, particularly for evaluation of chemical disinfectants and thus cc–qPCR is proposed as an alternative to the established chicken infectivity model for Eimeria tenella for testing anticoccidial disinfectants. A minimum inactivation of 99.5% in cc–qPCR model is claimed as a suitable threshold for certification of chemical products for disinfection of coccidia oocysts.     

Growth pattern and partial proteome of Mycobacterium avium subsp. paratuberculosis during the stress response to hypoxia and nutrient starvation

Mycobacterium avium subsp. paratuberculosis is an important pathogen that causes Johne's disease in animals and has been implicated in Crohn's disease in man yet few data exist on its physiological adaptation in either the host or the environment. In this study, the proteomic responses of the two distinct strains of M. a. paratuberculosis, cattle (C) and sheep (S), to hypoxia and starvation were studied in vitro. Nutrient starvation inhibited growth of both strains and was lethal for S strain after 12 weeks. Hypoxia induced a state of very low metabolic activity but rapid resuscitation occurred upon restoration of an aerobic atmosphere, consistent with the dormancy response of other mycobacteria. A total of 55 protein spots differentially expressed in response to starvation and/or hypoxic stress in one or both strains were identified from 2D gels and classified based on biological function. Antioxidant enzymes, oxidoreducatse enzymes and proteins involved in amino acid metabolism, fatty acid metabolism, ATP and purine biosynthesis, proteolysis, cell wall synthesis, protein synthesis, signal recognition and hypothetical proteins with putative functions including dormancy response regulators and universal stress proteins were identified. These proteins are potential screening targets for future diagnosis, prevention and control of M. a. paratuberculosis infection and their identification will assist understanding the pathogenesis of diseases caused by this organism.     

麻叶荨麻醇提液对大鼠离体子宫运动的影响

为探究荨麻对子宫运动的影响及其可能机制,本试验用生物信号处理系统测定了浓度递增(0.5%、1.0%、2.0%、4.0%、8.0%和16.0%)的麻叶荨麻(Urtica cannabina,UC)醇提液对未孕大鼠离体子宫收缩频率、收缩张力和收缩幅度的影响,并用特异性M-受体阻断剂硫酸阿托品(atropine,A,2.5 μg/mL)和2.0% UC联合用药,分为3个处理: UC单独处理、UC+A和A+UC,观察UC对子宫收缩的影响是否与M-受体有关.结果显示,在UC浓度递增到4.0%时收缩频率较用药前显著增加(P <0.05);浓度递增到4.0%、8.0%时,收缩幅度较用药前极显著降低(P <0.01);但浓度递增UC对子宫收缩张力影响不显著(P >0.05).一次性加入2.0% UC后子宫收缩张力迅速增加并接近僵直,之后逐渐降低.无论UC+A或A+UC处理都使子宫收缩频率较用药前明显加快,收缩张力极显著增加(P <0.01),但2个联合用药处理组之间差异不显著(P >0.05);UC+A和A+UC处理组收缩张力较UC单独用药组均极显著降低(P <0.01).结果表明,UC可兴奋未孕大鼠离体子宫,A可缓解UC引起的子宫僵直或张力增加,但不能阻断UC引起的子宫收缩频率增加.提示UC对未孕大鼠离体子宫的兴奋作用和激动与M-受体有一定关系,但可能还存在其他机制.