World association for the advancement of veterinary parasitology (WAAVP) guideline for testing the efficacy of ectoparasiticides for fish.

This guideline is intended to assist in the planning and execution of studies designed to assess the efficacy of ectoparasiticides for fish. It is the first ectoparasite-specific guideline to deal with studies set in the aquatic environment and therefore provides details for the maintenance of environmental standards for finfish. Information is included on a range of pre-clinical study designs as well as clinical studies in commercial/production sites, set within a regulatory framework. It provides information on the study animals, their welfare, husbandry and environmental requirements during the study. The most commonly pathogenic ectoparasites are presented with relevant points regarding life history, host challenge and numeric evaluation. Preparation and presentation of both topical and oral test treatments is provided, together with guidance on data collection and analysis. The guideline provides a quality standard or efficacy studies on finfish, which will assist researchers and regulatory authorities worldwide and contribute to the wider objective of harmonisation of procedures.

Epidemiological cut-off values for Flavobacterium psychrophilum MIC data generated by a standard test protocol.

Epidemiological cut-off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut-off values. For these agents, the cut-off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L(-1) for erythromycin, ≤2 mg L(-1) for florfenicol, ≤0.025 mg L(-1) for oxolinic acid (OXO), ≤0.125 mg L(-1) for oxytetracycline and ≤20 (1/19) mg L(-1) for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild-type observations were 'off scale', and therefore, statistically valid cut-off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut-off could not be determined. For flumequine, the putative wild-type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild-type and non-wild-type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents.

Florfenicol residues in rainbow trout after oral dosing in recirculating and flow-through culture systems.

Aquaflor is a feed premix for fish containing the broad spectrum antibacterial agent florfenicol (FFC) incorporated at a ratio of 50% (w/w). To enhance the effectiveness of FFC for salmonids infected with certain isolates of Flavobacterium psychrophilum causing cold water disease, the FFC dose must be increased from the standard 10 mg·kg⁻¹ body weight (BW)·d⁻¹ for 10 consecutive days. A residue depletion study was conducted to determine whether FFC residues remaining in the fillet tissue after treating fish at an increased dose would be safe for human consumption. Groups of Rainbow Trout Oncorhynchus mykiss (total n = 144; weight range, 126-617 g) were treated with FFC at 20 mg·kg⁻¹ BW·d⁻¹ for 10 d in a flow-through system (FTS) and a recirculating aquaculture system (RAS) each with a water temperature of ∼13°C. The two-tank RAS included a nontreated tank containing 77 fish. Fish were taken from each tank (treated tank, n = 16; nontreated tank, n = 8) at 6, 12, 24, 48, 72, 120, 240, 360, and 480 h posttreatment. Florfenicol amine (FFA) concentrations (the FFC marker residue) in skin-on fillets from treated fish were greatest at 12 h posttreatment (11.58 µg/g) in the RAS and were greatest at 6 h posttreatment (11.09 µg/g) in the FTS. The half-lives for FFA in skin-on fillets from the RAS and FTS were 20.3 and 19.7 h, respectively. Assimilation of FFC residues in the fillets of nontreated fish sharing the RAS with FFC-treated fish was minimal. Florfenicol water concentrations peaked in the RAS-treated tank and nontreated tanks at 10 h (453 µg/L) and 11 h (442 µg/L) posttreatment, respectively. Monitoring of nitrite concentrations throughout the study indicated the nitrogen oxidation efficiency of the RAS biofilter was minimally impacted by the FFC treatment.

Multidose pharmacokinetics of orally administered florfenicol in the channel catfish (Ictalurus punctatus).

Plasma disposition of florfenicol in channel catfish was investigated after an oral multidose (10 mg/kg for 10 days) administration in freshwater at water temperatures ranging from 24.7 to 25.9 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After the administration of florfenicol, the mean terminal half-life (t(1/2)), maximum concentration at steady-state (Css (max)), time of Css (max) (T(max)), minimal concentration at steady-state (Css (min)), and Vc /F were 9.0 h, 9.72 µg/mL, 8 h, 2.53 µg/mL, and 0.653 L/kg, respectively. These results suggest that florfenicol administered orally at 10 mg/kg body weight for 10 days could be expected to control catfish bacterial pathogens inhibited in vitro by a minimal inhibitory concentration value of <2.5 µg/mL.

Efficacy of florfenicol for control of mortality associated with Francisella noatunensis subsp. orientalis in Nile tilapia, Oreochromis niloticus (L.).

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram-negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments or vaccines. The objective of this project was to determine the most efficacious concentration of florfenicol (FFC) [10, 15 or 20 mg FFC kg(-1) body weight (bw) per days for 10 days] administered in feed to control experimentally induced infections of Fno in Nile tilapia, Oreochromis niloticus (L.), reared in a recirculating aquaculture system. The cumulative mortality of fish that received 0, 10, 15 or 20 mg FFC kg(-1)  bw per day was 60, 37, 14 and 16%, respectively. Francisella noatunensis subsp. orientalis genome equivalents were detected in water from all challenged groups with slight reduction in the concentration in the florfenicol-treated groups 4 days after treatment. The mean LOG of CFU Fno mg(-1) spleen was 3-5 and was present in all challenged groups at necropsy 11 days after treatment (21 days after challenge). Results show that florfenicol administered at doses of 15 and 20 mg FFC kg(-1)  bw per days for 10 days significantly reduced mortality associated with francisellosis in Nile tilapia.

Single intravenous and oral dose pharmacokinetics of florfenicol in the channel catfish (Ictalurus punctatus).

Plasma distribution and elimination of florfenicol in channel catfish were investigated after a single dose (10 mg/kg) of intravenous (i.v.) or oral administration in freshwater at a mean water temperature of 25.4 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After i.v. florfenicol injection, the terminal half-life (t(1/2)), volume of distribution at steady state (V(ss)), and central volume of distribution (V(c)) were 8.25 h, 0.9 and 0.381 L/kg, respectively. After oral administration of florfenicol, the terminal t(1/2), C(max), T(max), and oral bioavailability (F) were 9.11 h, 7.6 µg/mL, 9.2 h, and 1.09, respectively. There was a lag absorption time of 1.67 h in oral dosing. Results from these studies support that 10 mg florfenicol/kg body weight in channel catfish is an efficacious dosage following oral administration.

Efficacy of emamectin benzoate against sea lice infestations of Atlantic salmon, Salmo salar L.: evaluation in the absence of an untreated contemporary control.

The efficacy of emamectin benzoate (SLICE) against sea lice infestations of Atlantic salmon, Salmo salar L., is typically assessed using untreated fish, or fish treated with alternative therapeutants, as controls. The State of Maine, USA, is currently under active management for the OIE-notifiable pathogen, infectious salmon anaemia virus (ISAV); consequently, neither control group is feasible in this region. Untreated salmon risk extensive damage from the ectoparasites, and threaten to increase vector-borne exposure or susceptibility of farms to ISAV; and the only treatment presently available in Maine is SLICE. However, because sea lice infestations are unlikely to resolve spontaneously, and response to treatment occurs within weeks, use of a pretreatment baseline is a reasonable alternative for confirmatory studies. We evaluated SLICE efficacy on Atlantic salmon farms in Cobscook Bay 2002-2005, in the absence of untreated controls, using pretreatment lice loads as a reference for calculation. Maximum efficacy ranged from 68% to 100% reduction from initial levels. Time-to-maximum efficacy ranged from 1 to 8 weeks after treatment initiation. Efficacy duration, measured between first reduction and first progressive rise in counts, ranged from 4 to 16 weeks.

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.

Efficacy of a topical spot-on containing 65% permethrin against the dog louse, Trichodectes canis (Mallophaga:Trichodectidae).

The efficacy of a 65% permethrin spot-on formulation (Defend EXspot, Schering-Plough Animal Health Corp., Union, NJ) against the dog louse, Trichodectes canis de Greer 1778, was studied. Fourteen dogs naturally infested with T. canis were evenly and randomly allocated to treatment with 65% permethrin administered at the label dose rate of 1 or 2 ml per dog or to an untreated control group. Louse counts were performed for each dog by gently back-combing the hair at six designated anatomic sites (head, tail, belly, each side, and an 8-cm strip the length of the body on the back), and lice were counted without removal on Days 0 (pretreatment), 7, 14, 21, and 28. Lice were eliminated from all dogs treated with the 65% permethrin spot-on within 7 days after treatment, and no subsequent reinfestations due to hatching of eggs were observed during the 28-day evaluation period. Untreated control dogs were subsequently treated with the 65% permethrin spot-on after the initial phase was completed and lice populations were evaluated as previously described. All lice were cleared from these dogs by Day 7, and there were no signs of reinfestation. No adverse reactions to treatment were noted during the study.

Feeding and survival of Culicoides sonorensis on cattle treated with permethrin or pirimiphos-methyl.

The persistence of permethrin (5% a.i.) and pirimiphos-methyl (27% a.i.), applied to the dorsum of calves in the field against Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), was estimated using a hair-blood-feeding bioassay in the laboratory. Hair clippings were taken before treatment and 3, 7, 14, 21, 28, 42 and 56 days after treatment from the dorsum, side and belly of treated and control calves. Laboratory-reared insects were allowed to feed through thin hair layers and a parafilm membrane on sheep blood warmed using a water-jacketed feeder. Some intoxication after exposure to hair was noted up to 28 days after treatment with permethrin and up to 14 days after treatment with pirimiphos-methyl. Hair from the dorsum caused more intoxication for a longer period than hair from other body regions. Permethrin and pirimiphos-methyl applied to the back did not significantly reduce overall engorgement (body regions pooled) after treatment. Permethrin residues on hair remained far higher on the back than other body regions and were related to insect intoxication and reduction in engorgement in the laboratory. Residues on belly hair never exceeded 12p.p.m. and did not result in significantly reduced feeding at any time. Engorged insects that exhibited sublethal intoxication from feeding through permethrin-treated hair did recover and matured numbers of eggs comparable to controls. Field trials using treated and control calves and enclosure nets showed that dorsal applications of 5% permethrin were not effective in reducing engorgement, despite some intoxication. Vacuum samples from a calf showed that C. sonorensis fed primarily on the belly. A 0.2% permethrin application on the belly (250 ml) did result in > 80% reduction of C. sonorensis in the enclosure nets at 3 and 7 days after treatment, but activity had subsided by 10 days after treatment. The utility of insecticidal treatments for suppression of this vector is discussed.

A field efficacy evaluation of emamectin benzoate for the control of sea lice on Atlantic salmon.

This study evaluated the efficacy of emamectin benzoate, 0.2% aquaculture premix, against sea lice on Atlantic salmon in eastern Canada. Salmon pens received either emamectin benzoate, orally, in feed at 50 micrograms/kg body weight/day for 7 consecutive days, or the same diet with no added medication. The site veterinarian had the option of administering a bath treatment with azamethiphos to any pen in the trial. The mean number of lice per fish was lower (P < 0.05) in the experimental group when measured 1, 3, 4, and 6 weeks after the start of medication. Treatment efficacy was 70%, 88%, 95%, and 61%, respectively. Three azamethiphos bath treatments were applied to each control pen during the trial, while the treatment pens received no bath treatment. No gravid female parasites were observed on any fish in the treatment group, while these life stages were observed on fish in the control group. Orally administered emamectin benzoate was palatable and highly effective for control of sea lice on salmon.

Efficacy of 65% permethrin applied as a topical spot-on against walking dandruff caused by the mite, Cheyletiella yasguri in dogs.

The efficacy of a 65% permethrin topically applied spot-on formulation (Defend EXspot Topical Remedy for Dogs, Schering-Plough Animal Health, Union, NJ) was determined against the dog mite, Cheyletiella yasguri (Smiley, 1965). Female dogs and their litters comprised the experimental unit, and all dogs in an experimental unit were treated on the same day 4 to 6 weeks after whelping. Mites and mite eggs were counted weekly on an untreated control group of six litters (15 pups) and on a group of six litters (14 pups) treated with 65% permethrin. Pups in the untreated control group maintained high numbers of Cheyletiella yasguri throughout the 14- to 21-day observation period. No mites or mite eggs were detected on dogs within 7 to 21 days after application of 65% permethrin. No adverse reactions were noted during the study. Clinical signs of infestation with C. yasguri--which included skin irritation, thickening of the stratum corneum, scratching with resultant scabs, pruritus, and flaky, scaly skin-were eliminated when mites were killed by the 65% permethrin formulation.

Repellency and efficacy of 65% permethrin and 9.7% fipronil against Ixodes ricinus.

Two topically applied spot-on products--65% permethrin (Defend Exspot Treatment for Dogs, Schering-Plough Animal Health, Union, NJ) and 9.7% fipronil (Frontline Spot On Dog, Merial Limited, Iselin, NJ)--used for canine flea and tick control were evaluated for repellency against Ixodes ricinus, the tick species that is the primary vector of Lyme disease in Europe. Eighteen dogs were randomly assigned to the following treatment groups (n = 6): (1) 65% permethrin, (2) 9.7% fipronil, or (3) untreated control. Dogs were exposed to ticks in individually assigned cages with a carpet that covered =70% of the cage bottom. Dogs in treatment groups 1 and 2 were treated in accordance with label directions for each re-spective product on study day 0. Fifty unfed, male and female adult ticks were placed in the cages 15 to 30 minutes before the dogs. The dogs were placed in the cages for a 2-hour exposure period at 2, 7, 14, 21, 28, 35, and 41 days after treatment. After a 2-hour exposure period, dogs were removed from the cages and live (attached and unattached) and dead ticks were counted on the dogs, on the carpets, and in the cages. Cages were thoroughly cleaned and new carpet was used for each tick exposure period. Treatment of dogs with 65% permethrin reduced tick numbers on dogs by 99.1% at 2 days, 99.0% at 1 week, 95.9% at 2 weeks, 88.5% at 3 weeks, 87.1% at 4 weeks, and 48.0% at 6 weeks after application. In contrast, treatment of dogs with 9.7% fipronil reduced tick numbers on dogs by 61.4% at 2 days, 51.6% at 1 week, 37.0% at 2 weeks, 33.7% at 3 weeks, 10.8% at 4 weeks, and 0% at 6 weeks after application. The efficacy of 65% permethrin was significant (P < or = .05) when compared to the control at all challenges, whereas the efficacy of 9.7% fipronil was not significant as compared to controls at 21, 28, 35, and 41 days after treatment. The 65% permethrin killed significantly more Ixodes ricinus ticks (P < or = .05) than 9.7% fipronil from 2 to 41 days after treatment. The 65% permethrin repelled 1.9-, 2.0-, 3.0-, 43.3-, 3.9-, 8.9-, and 17.3-fold more Ixodes ricinus than did 9.7% fipronil at 2, 7, 14, 21, 28, 35, and 41 days after treatment, respectively, and all differences in repellency were significant (P < or = .05).

Detection and quantification of Ornithodoros-specific anti-tick antibody by competitive inhibition ELISA.

The objective of this study was to develop a highly specific enzyme-linked immunosorbent assay (ELISA) for the serological detection of anti-Ornithodoros tick antibodies in animals. Affinity-purified rabbit anti-Ornithodoros IgG antibodies were employed in indirect competitive inhibition ELISA assays designed to measure the anti-Ornithodoros antibody titers in other animal species using the domestic goat (Capra hircus) as a large animal model. Repeated infestation of goats with Ornithodoros coriaceus was found to elicit the formation of antibodies capable of inhibiting the binding of the Ornithodoros-specific rabbit IgG. Western blot analysis of goat and rabbit anti-tick antisera demonstrated both animal species to respond immunologically to a set of 9 major protein bands in O. coriaceus salivary gland extracts. The results of these experiments demonstrate that a history of animal exposure to O. coriaceus may be detected serologically by competitive inhibition ELISA.

Effect of combing time on cat flea (Ctenocephalides felis) recovery from dogs.

Combing the haircoat to count fleas has been used to determine the efficacy of insecticides against fleas on dogs, but no standardization of method has been reported. In this study, the effect of combing time on flea recovery from dogs was examined. Six beagle dogs were infested with 100 unfed, adult Ctenocephalides felis (Bouché) on each of three consecutive days. A crossover design, balanced for carryover effects, was used to evaluate flea removal rates from each dog by comb-counting for three different time intervals; i.e. 5, 10 and 15 min. Each dog was combed once at each time interval on a different day, over three consecutive days. The results showed that the majority of fleas were recovered in the first 5 min of combing and there were no significant differences (P > or = 0.19) in the total number of fleas recovered between the 5, 10 or 15 min protocols. Moreover, the standard deviation and coefficient of variation increased with an increase in the amount of time spent combing, resulting in a decrease in precision for the longer protocols. Therefore, the comb time of 5 min provided a precise and accurate representation of the number of fleas present on an animal and could be useful as a standard measure of flea infestation levels in efficacy trials.

An inhibitor from the argasid tick Ornithodoros moubata of cell adhesion to collagen.

An inhibitor of the adhesion of human platelets to collagen was identified in soluble extracts of the soft tick Ornithodoros Moubata and purified by four chromatographic steps. The isolated inhibitor, TAI (Tick Adhesion Inhibitor), is a approximately 15-kDa protein that completely blocks the adhesion of platelets to collagen-coated microtiter plates with an IC50 of 8 nM. In the same concentration range it does not inhibit collagen-induced platelet aggregation or platelet adhesion to fibrinogen and has a partial inhibitory effect on platelet adhesion to fibronectin. TAI also blocks the adhesion of human endothelial cells to collagen, thus its inhibitory effect is not limited to platelets. TAI competes for the binding to platelets of a radiolabeled monoclonal antibody against the platelet glycoprotein Ia-IIa integrin complex. Based on its selective activity and small size, TAI is a promising new molecule for exploring cell-collagen interactions.

A further comparison of the thumb-counting and comb-counting techniques used to determine Ctenocephalides felis infestation levels on dogs.

A comparison was made to determine whether thumb-counting or comb-counting was more accurate for determining flea infestation levels on dogs when performed for equal periods of time. To accomplish this, ten beagle dogs were each infested with 100 adult fleas, Ctenocephalides felis. After the fleas were allowed to disperse for 1 h the dogs were examined using the thumb-counting method. The time required to cover each dog and the number of fleas counted were recorded. Thumb-counting times ranged from 3.0 to 4.8 min. Each of the dogs was then examined by the comb-counting method for the same amount of time it had been thumb-counted. The thumb-counting method detected a geometric mean of five (range, 0-13) fleas per dog, while comb-counting recovered a mean of 73.5 (range, 57-87) fleas per dog. These results were significantly different (P < 0.01), indicating that the differences in accuracy previously recorded for the two methods are independent of time. The standard deviations for both methods were also statistically significantly different, suggesting that comb-counting is also more precise than the thumb-counting method.

Attempted transovarial and venereal transmission of African swine fever virus by the Iberian soft tick Ornithodoros (Pavlovskyella) marocanus (Acari: Ixodoidea: Argasidae).

Transovarial transmission experiments were conducted with three groups of Ornithodoros (Pavlovskyella) marocanus Velu; one group consisted of 27 pairs of adults that had been fed as larvae on a pig with a viremia of 10(7.4) HAd50/ml of African swine fever virus (ASFV). The second and third groups each consisted of 100 pairs of adults fed on a viremic pig (10(4.5) HAd50/ml) as adults. The first group underwent five gonotrophic cycles over a 554-d period. The second and third groups underwent three and two gonotrophic cycles, respectively. All larvae were fed in individual cohorts on naive pigs and the resulting nymphs were assayed by cohort for ASFV. None of the larvae transmitted ASFV to naive pigs by bite and ASFV was not isolated in swine buffy coat cultures from any cohort of nymphs. Therefore, O. marocanus does not exhibit transovarial transmission of ASFV. Venereal transmission experiments were conducted with pairs of O. marocanus in which either the female (100 pairs) or the male (100 pairs), respectively, had fed on a viremic pig (10(4.5) HAd50/ml). Both groups underwent at least two gonotrophic cycles over a 470-d period, were sampled periodically for the presence of ASFV, and the progeny were tested for the presence of ASFV. Venereal transmission from male to female occurred in 10% (1/10) of O. marocanus after the first gonotrophic cycle, but not after the second or third gonotrophic cycle, and transovarian transmission in these groups was not observed. Venereal transmission from infected females to uninfected males did not occur. ASFV persisted through five gonotrophic cycles over a 554-d period in 30% of adults fed on a viremic pig as larvae. ASFV was cleared during three gonotrophic cycles within a year from nearly all ticks fed on a viremic pig as adults. Virus-induced mortality rats of 12-80% occurred among ticks fed on viremic animals, whereas, no mortality was seen in ticks fed on uninfected animals. ASFV infection in ticks did not effect feeding frequency, egg-hatch rate, or the oviposition rate among females fed on a viremic pig as adults. The oviposition rate for females fed on a viremic pig as larvae was reduced by 63.4%. Parthenogenesis was not observed among O. marocanus. The mean gonotrophic cycle duration for pig-fed O. marocanus at 27 degree C was 19.8 d and the mean fecundity was 88.3 +/- 17.9 eggs/female/gonotrophic cycle.

Comparison of thumb-counting and comb-counting methods to determine Ctenocephalides felis infestation levels on dogs.

Comb-counting and thumb-counting were compared in a cross-over study to determine which was more accurate for quantifying flea infestation levels on dogs. Twenty beagle dogs were used in the study and infested with either 50 or 100 adult fleas (Ctenocephalides felis). Two groups of five dogs each were infested with either 50 or 100 fleas per dog, and then comb-counted with a fine-toothed flea comb for 8 min periods. An additional two groups of five dogs each were also given 50 or 100 fleas, and then thumb-counted. The counting time for this technique is both lower and more variable because the fleas are only observed and not captured; thus, the speed at which the dog is covered must be increased in order to prevent counting the same fleas more than once. The mean time of thumb-counting per dog was 3.2 min. Fleas removed during comb-counting were placed back on the dog they were taken from after the count was concluded. At the cross-over point, the ten dogs that had been comb-counted were then thumb-counted and the ten dogs that had been thumb-counted were comb-counted. The results showed that comb-counting recovered significantly (P < or = 0.05) more fleas than did thumb-counting. On dogs given 50 and 100 fleas, comb-counting gave mean percentage recoveries of 67.6% and 75.4%, respectively, whereas thumb-counting found means of 8.8% and 7.7%, respectively. The order in which the counting methods were employed produced no significant effect (P > 0.05) on the number of fleas counted.

Disagregin is a fibrinogen receptor antagonist lacking the Arg-Gly-Asp sequence from the tick, Ornithodoros moubata.

A platelet aggregation inhibitor was identified in the salivary gland of the tick Ornithodoros moubata and isolated by gel filtration and reverse phase high pressure liquid chromatography. The purified inhibitor is a approximately 6-kDa protein, which we have named disagregin. It inhibits ADP-stimulated platelet aggregation in plasma with an IC50 = 104 +/- 17 nM. Disagregin also inhibits platelet aggregation induced by other agonists, including collagen, epinephrine, platelet-activating factor, thrombin, and the thrombin receptor peptide SFLLRNPNDKYEPF. It does not, however, affect platelet shape change induced by these agonists or thrombin-induced dense granule release. Disagregin inhibits platelet aggregation by binding to the platelet fibrinogen receptor. 125I-Disagregin forms a specific complex with both subunits of the fibrinogen receptor, glycoproteins IIb and IIIa, in the presence of a chemical cross-linker. It binds to unstimulated platelets with a Kd = 42.5 +/- 7.5 nM (23,800 +/- 1600 sites/platelet) and to ADP-stimulated platelets with Kd = 39.4 +/- 6.6 nM (24,050 +/- 1500 sites/platelet). Unlabeled disagregin and the snake venom disintegrin echistatin both compete for this binding. Disagregin also completely blocks platelet adhesion to fibrinogen while partially inhibiting platelet adhesion to fibronectin and having little effect on platelet adhesion to collagen. Disagregin had no effect on the adhesion of human umbilical cord vein endothelial cells to fibrinogen or vitronectin. These cells lack the glycoprotein IIb-IIIa complex; therefore, this result is consistent with the ability of disagregin to bind selectively to platelet glycoproteins IIb and IIIa. Sequence analysis of disagregin revealed 60 residues composing a unique protein. Unlike other fibrinogen antagonists it does not contain the Arg-Gly-Asp cell recognition sequence or a conservative substitution, and it has no structural homology with the Arg-Gly-Asp-containing snake venom disintegrins. Thus, disagregin is unique both in structure and function and may serve as a useful tool for the design of therapeutically useful antithrombotic agents.