Concurrent treatment with a macrocyclic lactone and benzimidazole provides season long performance advantages in grazing cattle harboring macrocyclic lactone resistant nematodes.

In 2013, a 118-day study was initiated to investigate the efficacy of concurrent treatment at pasture turnout with an injectable macrocyclic lactone with activity up to 28 days and an oral benzimidazole, referred to as "conventional" anthelmintics, when compared to treatment with conventional macrocyclic lactone alone or an injectable macrocyclic lactone with extended activity of 100 days or longer. A group of 210 steers were obtained from a ranch in California and transported to Idaho, USA. A total of 176 steers with the highest fecal egg counts were blocked by pre-treatment body weights and pasture location. A total of 44 pasture paddocks were assigned with 4 steers per paddock with 12 paddocks per therapeutic treatment group and 8 paddocks per controls. The four treatments were injectable doramectin (Dectomax®, Zoetis Inc., 0.2 mg kg-1BW, SC), injectable doramectin concurrently with oral albendazole (Valbazen®, Zoetis Inc., 10 mg kg-1BW, PO), extended release injectable eprinomectin (LongRange™, Merial Limited, 1 mg kg-1BW, SC) or saline. Cattle were individually weighed and sampled for fecal egg count on Days 0, 31/32, 61, 88, and 117/118 with an additional fecal sample on Day 14. At conclusion, one steer per paddock was euthanized for nematode recovery. The results from the first 32 days found evidence of macrocyclic lactone resistance against injectable doramectin and extended release eprinomectin. During this period the concurrent therapy provided nearly 100% efficacy based on fecal egg count reduction and a 19.98% improvement in total weight gain compared to controls (P = 0.039). At the conclusion of the 118-day study and past the approved efficacy for the conventional anthelmintics, the concurrent therapy with conventional anthelmintics provided a 22.98% improvement in total weight gain compared to controls (P = 0.004). The 118-day improvement in weight gain for the extended release eprinomectin group (29.06% compared to control) was not statistically different from the concurrent therapy with conventional anthelmintics. The results indicate that concurrent treatment with a conventional macrocyclic lactone and benzimidazole may provide production benefits early in the grazing period that continue throughout the entire period for cattle harboring macrocyclic lactone resistant nematodes. By using two different anthelmintic classes together, macrocyclic lactone resistant parasites were effectively controlled early in the period. Furthermore, the use of an effective conventional anthelmintic treatment regimen without an extended period of drug release may help to promote refugia and decrease the further selection for anthelmintic resistant parasites.

American Association of Veterinary Parasitologists' review of veterinary fecal flotation methods and factors influencing their accuracy and use--is there really one best technique?

The principle of fecal flotation is based on the ability of a solution to allow less dense material (including parasite elements) to rise to the top. However, there are numerous factors that will influence the accuracy and use of such a theoretically simple technique. Whether or not centrifugation is used appears to have an impact on the ability to detect some parasites, but not others. Using a flotation solution with a relatively high specific gravity favors the simultaneous flotation of the diagnostic stages of many different parasites while, at the same time, making recognition of some more difficult because of distortion as well as the amount of debris in the preparation. Dilution methods tend to be less accurate because they require extrapolation; however, they are quicker to perform, in part, because of the cleaner preparation. Timing is a critical factor in the success of all flotation methods, as is technical ability of the personnel involved. Thus, simplicity, low costs and time savings have generally favored gravitational flotation techniques (including the McMaster technique and its modifications). How accurate the method needs to be is dependent upon the purpose of its use and choice of method requires an understanding of analytical sensitivity and expected levels of egg excretion. In some instances where the difference between, for example, 0 and 50 eggs per gram is insignificant with regards to management decisions, less accurate methods will suffice. In others, where the presence of a parasite means treatment of the animal regardless of the numbers of eggs present, methods with higher analytical sensitivities will be required, particularly for those parasites that pass few eggs. For other uses, such as the Fecal Egg Count Reduction Test, accuracy may become critical. Therefore, even though recommendations for standardized fecal flotation procedures have been promoted in the past, it is clear that the factors are too numerous to allow for the recommendation of one, or even a few, procedures for all purposes.

Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture-adjusted quantitative structure permeability relationship.

Topical application of ectoparasiticides for flea and tick control is a major focus for product development in animal health. The objective of this work was to develop a quantitative structure permeability relationship (QSPeR) model sensitive to formulation effects for predicting absorption and skin deposition of five topically applied drugs administered in six vehicle combinations to porcine and canine skin in vitro. Saturated solutions (20 µL) of (14) C-labeled demiditraz, fipronil, permethrin, imidacloprid, or sisapronil were administered in single or binary (50:50 v/v) combinations of water, ethanol, and transcutol (6 formulations, n = 4-5 replicates per treatment) nonoccluded to 0.64 cm(2) disks of dermatomed pig or dog skin mounted in flow-through diffusion cells. Perfusate flux over 24 h and skin deposition at termination were determined. Permeability (logKp), absorption, and penetration endpoints were modeled using a four-term Abrahams and Martin (hydrogen-bond donor acidity and basicity, dipolarity/polarizability, and excess molar refractivity) linear free energy QSPeR equation with a mixture factor added to compensate for formulation ingredient interactions. Goodness of fit was judged by r(2) , cross-validation coefficient, coefficients (q(2) s), and Williams Plot to visualize the applicability domain. Formulation composition was the primary determinant of permeation. Compounds generally penetrated dog skin better than porcine skin. The vast majority of permeated penetrant was deposited within the dosed skin relative to transdermal flux, an attribute for ectoparasiticides. The best QSPeR logKp model for pig skin permeation (r(2) = 0.86, q(2) s = 0.85) included log octanol/water partition coefficient as the mixture factor, while for dogs (r(2) = 0.91, q(2) s = 0.90), it was log water solubility. These studies clearly showed that the permeation of topical ectoparasiticides could be well predicted using QSPeR models that account for both the physical-chemical properties of the penetrant and formulation components.

Anthelmintic resistance in equine parasites--current evidence and knowledge gaps.

Anthelmintic resistance is becoming increasingly prevalent among equine nematode parasites. The first reports documenting resistance were published in the 1960s, just a short time after introduction of the first modern anthelmintics phenothiazine and thiabendazole. Several factors are known to influence development of resistance, but evidence specific to equine parasites is limited. Most current knowledge and applications have been extrapolated from research with trichostrongylid parasites of sheep. The number of cyathostomin species co-infecting horses adds to the complexity of investigating drug resistance but, given their apparent limited biological diversity, viewing these in a unispecific context remains a pragmatic approach. Factors affecting resistance development in cyathostomins include parasite seasonality, life span and fecundity, host immunity, and the existence of encysted stages. Further, parasite refugia have been shown to play a vital role in resistance development in other parasites, and likely is also important in equine parasites. Specific genetic factors for drug resistance and possible modes of inheritance have been identified for trichostrongylid nematodes, but it is widely accepted that several more remain undiscovered. Current evidence with equine and ruminant parasites suggests that fitness is not significantly compromised in drug resistant strains. Attempts to develop in vitro and molecular assays for diagnosing anthelmintic resistance in equine nematodes have had only limited success, standardized guidelines are sorely needed for performing the fecal egg count reduction test in horse populations. Taken together, this review illustrates the complexity of understanding anthelmintic resistance in equine nematodes, and emphasizes the need for further research.

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

These second edition guidelines, updated from the 2007 version (Marchiondo et al., 2007), 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. Major revisions to this second edition include guidelines on the assessment of systemic flea and tick products, an update of the geographical distribution of the common fleas and ticks species on dogs and cats, determination of flea and tick efficacy based on geometric versus arithmetic means with respect to geographic regulatory agencies, modification of tick categorization in the assessment of efficacy, expanded guidelines on repellency and anti-feeding effects, enhanced practical field study guidance, and considerations on the ranges of flea and ticks for infestations in laboratory studies. The term ectoparasiticide includes insecticidal and acaricidal compounds, as well as insect growth regulators. The range of biological activities 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 regulatory authorities involved in the approval and registration of new topical or systemic ectoparasiticides, 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 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.

In vitro insecticidal effects of fipronil and beta-cyfluthrin on larvae of the blowfly Lucilia sericata.

The insecticidal effects of the phenylpyrazole, fipronil, and a pyrethroid, beta-cyfluthrin, on larvae of the blowfly Lucilia sericata were determined in laboratory assays. When first stage larvae of L. sericata were reared on homogenized pig liver which had been treated with known amounts of test compounds, both fipronil and beta-cyfluthrin induced significant levels of mortality compared to acetone and water controls. However, fipronil was approximately 10 times more toxic than beta-cyfluthrin to L. sericata larvae following ingestion. Beta-cyfluthrin had little effect on mortality until concentrations of approximately 0.5 ppm were reached. In contrast, fipronil effected L. sericata mortality at a concentration of 0.05 ppm and 100% mortality was reached by 0.5 ppm. The lethal concentration (LC50) value for beta-cyfluthrin was 1.56 ppm as compared to 0.14 ppm for fipronil. Following contact of first and third stage larvae with cloth impregnated with known amounts of test compound, the mortality profiles of fipronil and beta-cyfluthrin were similar. At short contact times, the LC50 values for fipronil were lower than those for beta-cyfluthrin. However, at the highest contact time evaluated for the first stage larvae, 300 s, there was a reversal in this trend. The results suggest that the phenylpyrazole fipronil may represent a new potential insecticide for development against blowfly strike of sheep.

Chorion formation and ultrastructure of the egg of the cat flea (Siphonaptera: Pulicidae).

Oocyte development in adult female cat fleas, Ctenocephalides felis (Bouché), was studied by light and electron microscopy to determine the formation and ultrastructural morphology of the eggshell. As oocytes develop, somatic follicle cells from the lining of the ovariole migrate around the oocytes. The follicle cells produce electron-dense granules that form the vitelline membrane around the developing oocyte. Subsequently, electron-lucent granules containing an electron-dense core (precursors of the chorion) are produced from the rough endoplasmic reticulum that appear as dilated and clear linear clefts in the cytoplasm of the follicle cells. Exocytosis and coalescence of the granules around the oocyte as the follicle cells disintegrate give rise to the chorion. The chorion was found to consist of 4 distinct layers. The external surface of the egg shell consists of a particulate layer approximately 760 nm thick, composed of an electron-lucent layer of widely dispersed granules. Embedded in this layer are electron-dense spheres that project above the surface of this granular layer. Beneath this outermost layer is a band of electron-dense material, consisting of densely packed granules and is half as thick as the outer particulate layer. The 3rd layer consists of relatively thick, weakly laminated chorion, with a felt-like appearance due to a meshwork of microfibrils. Projections of this network of microfibrils form pillars that attach this layer to a thin relatively compact 4th or basal layer. The pillars and the air-filled cavities lying between the 3rd and 4th chorionic layers constitute the chorionic meshwork known as the palisades or trabecular layer that form the major respiratory organ of the eggshell. The trabecular layer is connected to the external environment by means of the lateral and anterior aeroplyes. The vitelline membrane lies between the chorion and oocyte and is a granular, uniform, moderately electron-dense layer measuring approximately 260 nm thick. The micropyle at the posterior of the flea egg consists of a rosette of 50-80 apertures and possesses an internal electron-dense plug between the chorion and the vitelline membrane. An aeropyle at the anterior end of the egg consists of a rosette of 40-50 apertures. An inconspicuous aeropyle appears as a cluster of hexagonal or polygonal-shaped plaques on the lateral surface of the chorion. Each plaque contains 3-8 pores.

Enhanced larval cyst growth of Echinococcus multilocularis in praziquantel-treated jirds (Meriones unguiculatus).

Jirds (Meriones unguiculatus) inoculated intraperitoneally with cystic material of Echinococcus multilocularis were given daily oral treatments of praziquantel at 300 mg/kg of body weight (bw) or dimethyl sulfoxide vehicle for five-day treatment regimens starting at 29 days postinoculation (PI) up to 69 days PI. At 39 or 49 days PI, the growth of the larval cystic mass (LCM) in jirds following a single or two five-day treatment regimens was significantly enhanced (P < 0.05) by 129.0% (2.3-fold) or 102.9% (2.0-fold), respectively. At 59 or 69 days PI following three or four five-day treatments with praziquantel, LCM growth was enhanced by 47.8% (1.5-fold) and 44.1% (1.4-fold), respectively, but was no longer significantly different than that in control jirds. A single five-day treatment on 29-33 days PI (with necropsy at 69 days PI) significantly enhanced the growth of the LCM by 87.6% (1.9-fold). Parasites from praziquantel treatment regimens examined ultrastructurally showed consistent damage to the germinal membrane evidenced by vacuolization and rupture of syncytial cytoplasm, rupture and coalescence of the electron-lucent vesicles just below the microvilli of the tegumental surface, and swelling and rounding of mitochondria. At 39 days PI, increased blebbing of the germinal membrane into the lumen of the LCM in praziquantel-treated animals was observed by scanning electron microscopy. The treatment-induced blebs were identified as nucleated germinal cells by transmission electron microscopy and appeared to be responsible for metastasis and enhanced growth of the LCM. Although praziquantel damaged the ultrastructural integrity of the LCM, treatment failed to inhibit larval cyst growth or protoscolex development.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovicidal and larvicidal modes of action of fenoxycarb against the cat flea (Siphonaptera: Pulicidae).

The mode of action of the insect growth regulator, fenoxycarb, on embryogenesis of cat flea eggs, Ctenocephalides felis (Bouché), was evaluated biologically and morphologically. Newly oviposited flea eggs were aged for 0, 3, 6, 12, 24, 48, or 72 h and exposed to fenoxycarb (1.1 micrograms/cm2) on filter paper disks for 0 (60 s), 2, 4, 8, 12, or 24 h at 23 +/- 1 degree C and 70 +/- 3% RH. Following exposure, samples of flea eggs were processed for microscopic examination and seeded onto carpet swatches containing flea-rearing medium in order to assess egg hatch, and larval, pupal, and adult development. Fenoxycarb exhibited embryocidal activity against eggs in early blastoderm formation, blastokinesis, and advanced larval development up to hatching. The ovicidal effect of fenoxycarb was not restricted to any specific developmental stage of embryogenesis, and no significant relationship was found between duration of exposure and lethal or inhibitory effects. Indeed, exposure for as little as 60 s produced a lethal inhibition of embryonic development. Conventional microscopy and ultrastructural observations of flea eggs exposed to fenoxycarb showed extensive cellular and tissue damage of the developing embryos, including membrane lysis, burst cells, pycnotic nuclei with coalesced and clumped heterochromatin, swollen and ruptured mitochondria, cellular autolysis, and collapse and discoloration of the eggshell. Fenoxycarb also exhibited larvicidal activity against newly emerged and 24-h-old flea larvae. The midguts of larvae exposed to fenoxybarb appeared disrupted and distorted with large gaps. These data document the ovicidal and larvicidal modes of action of fenoxycarb against the cat flea.

Significance of the distribution of 57Co-vitamin B12 in Spirometra mansonoides (Cestoidea) during growth and differentiation in mammalian intermediate and definitive hosts.

The distribution of labeled cyanocobalamin (CN-[57Co]Cbl = [57Co]-vitamin B12) in pleurocercoids and adult tapeworms of Spirometra mansonoides was studied during development in mice 22 days days PI, respectively. Plerocercoid scolices, obtained by cutting away their bodies or by in vitro enzymatic dissolution of the bodies, were pulsed with CN- magnitude of 57Co Cbl for 1h at 37 degrees C and reimplanted subcutaneously into mice or given per os to cats. In regenerated plerocercoids, the highest concentration of magnitude of 57Co Cbl occurred in the scolex and then decreased posteriorly in the newly-formed tissues of the body. Approximately 60% of the total magnitude of 57Co Cbl present remained concentrated in the scolex following body regeneration plerocercoids and adult tapeworms of Spirometra mansonoides was studied during development in mice 22 days post-infection (PI) and in cats 16 days PI, respectively. Plerocercoid scolices, obtained by cutting away their bodies or by in vitro enzymatic dissolution of the bodies, were pulsed with CN-[57Co]Cbl for 1 h at 37 degrees C and reimplanted subcutaneously into mice or given per os to cats. In regenerated plerocercoids, the highest concentration of [57Co]Cbl occurred in the scolex and then decreased posteriorly in the newly-formed tissues of the body. Approximately 60% of the total [57Co]Cbl present remained concentrated in the scolex following body regeneration for up to 109 days PI. This high [57Co]Cbl concentration in the plerocercoid scolex was bound to protein and appears to be maintained by a complex homeostatic mechanism in association with directional transport of [57Co]Cbl to the scolex with ultimate depletion along the length of the body.(ABSTRACT TRUNCATED AT 250 WORDS)

On the status of Eimeria nieschulzi oocysts embedded in resin eleven years ago: a permanent method for preserving coccidian oocysts.

Sporulated oocysts of Eimeria nieschulzi that were fixed and mounted on glass slides in polymerized resin in 1976 are examined. Size, shape, and integrity of oocysts and sporocysts are compared to similar observations we made in 1977 and reported in 1978 (Journal of Parasitology 64: 163-164). Our conclusion is that the methods we reported on in 1978 provide one opportunity to produce permanent specimens of sporulated oocysts that could be made available for deposit in nationally accredited museums.

Efficacy of dichlorvos, fenbendazole, and ivermectin in swine with induced intestinal nematode infections.

Anthelmintic efficacies of dichlorvos, fenbendazole, and ivermectin were compared in specific-pathogen-free crossbred weanling pigs inoculated with Ascaris suum, Trichuris suis, and Oesophagostomum dentatum. On postinoculation day (PID) 50, 24 pigs in each treatment group were treated orally with 43 mg of dichlorvos/kg of body weight, 3 X 3 mg of fenbendazole/kg, or 300 micrograms of ivermectin/kg, SC. Twenty-four pigs were not treated. On posttreatment day 7 (PID 57), 12 pigs from each treatment group (phase I) were slaughtered, and the anthelmintic efficacy of each treatment was determined. Efficacies against A suum, T suis, and O dentatum, respectively, were: dichlorvos, 100%, 99.9%, and 100%; fenbendazole, 100%, 99.8%, and 100%; and ivermectin, 98.7%, 53.9%, and 87.6%. Weight gains and feed conversions of the remaining pigs were monitored until they reached market weight (phase II). The average weight gains (kg) and feed conversions (kg of feed/kg of gain) at posttreatment day 81 (PID 131), respectively, were: 73.6 and 3.44 for nontreated controls, 78.9 and 3.31 for dichlorvos-treated pigs, 72.1 and 3.36 for fenbendazole-treated pigs, and 74 and 3.48 for ivermectin-treated pigs. Differences in average weight gains and feed conversions were not significant (P greater than 0.05).

In vivo efficacy and ultrastructural effects of mitomycin C against experimental alveolar hydatid disease.

The in vivo efficacy and ultrastructural effects of mitomycin C were determined against alveolar hydatid disease in experimentally infected animals and compared to mebendazole treatment. Mitomycin C inhibited the mean cyst mass of treated versus control animals by 84.1% which was statistically significant at the alpha = 0.01 level. Mebendazole given daily inhibited the mean cyst mass by 80.1%, while mebendazole administration on the same treatment schedule as that used for mitomycin C inhibited the mean cyst mass by 70.4%. Ultrastructurally, mitomycin C was not observed to affect the tegumental microtriches (microvilli) or the microtubular system. However, an increase in the number and accumulation of round to oval electrondense vesicles was observed within the subtegument. These inclusion bodies became vacuolated, subsequently degenerated, and formed myelin-like figures. Mitomycin C, like mebendazole, was only cystistatic in its effects on the cyst stage of Echinococcus multilocularis as evidenced by the growth of treated cyst material following inoculation into helminth-free animals.

Ultrastructural characterization of serum-induced changes in the tegument of Taenia taeniaeformis.

The objective of this study was to characterize complement-dependent damage to the tegument of isolated metacestodes of Taenia taeniaeformis caused by exposure to immune or normal rat serum (IRS and NRS, respectively). Metacestodes of T. taeniaeformis (34- and 69-day-old) from rats were incubated for 1 hr in 0.85% physiological saline solution (PSS), IRS, NRS, heat-inactivated at 56 C for 1 hr (delta) IRS, or delta NRS and then fixed for 2 hr in 3% glutaraldehyde. The larvae were then prepared for freeze-etching, thin sectioning, and SEM by standard techniques. Freeze-etch replicas of PSS-, delta IRS-, and delta NRS-treated larvae showed no damage, whereas those of IRS- and NRS-treated metacestodes exhibited vesiculation in the extracellular matrices, segmentation or "beading" of the microthrix tip, significant reductions in the number of intramembranous particles (IMP) in the P face of the membrane of the microthrix base, and changes in the pattern of IMP distribution in the P face of the base. Similar results were obtained from larvae prepared for thin sectioning and SEM. Additionally, thin-sectioned preparations demonstrated that in some cases the entire tegument was stripped away in IRS- and NRS-treated metacestodes. Our results have provided supportive evidence that complement-mediated lysis of larvae of T. taeniaeformis is not enhanced by the presence of antibody in serum, and we also characterized ultrastructurally the types of tegumental damage that may contribute to lysis. In addition, a possible defense mechanism used by the parasite to counter immunological attack by host phagocytic cells is proposed.

Fine structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (cestoda).

Freeze-etch replicas of the protoscolex tegument of Echinococcus multilocularis were examined and compared with conventional thin sections by TEM. The microtopography of the protoscolex tegument was also examined by SEM. The protoscolex consisted of morphologically-distinct, apical and basal tegumentary regions, the latter of which lacked microtriches. The hook area of the apical region contained long, slender, filamentous microtriches that obscured the hook arrangement. These microtriches were structurally different from those found on the suckers and rostellum of the protoscolex. Freeze-etch replicas of the tegumental membrane of the sucker and rostellar microtriches showed that the protoplasmic (P) and exoplasmic (E) faces of the microthrix base and tip contained numerous intramembranous particles (IMP). The densities of the IMP on both the P and E faces of the microthrix tip were approximately twice the number of the larger diameter IMP found on the P and E faces of the microthrix base. No freeze-etch replicas of the microtriches from the hook area were obtained. The basal tegumentary region of the protoscolex consisted of irregularly-distributed, knoblike processes that were variable in size and shape, and contained an electron-dense cap. The IMP on the P face of the knoblike processes measured approximately the same diameter as those on the P face of the microthrix base. However, their density was about half that of the latter. The density of IMP on the E face of the knoblike processes could not be determined from the freeze-etch replicas.

Freeze-etch characterization of the teguments of three metacestodes: Echinococcus granulosus, Taenia crassiceps, and Taenia taeniaeformis.

The objective of this study was to characterize the teguments of metacestodes of Echinococcus granulosus, Taenia crassiceps, and Taenia taeniaeformis using the freeze-etch technique. Metacestodes of E. granulosus (19 mo old), T. crassiceps (28 days old), and T. taeniaeformis (34 days old) from gerbils, mice and rats, respectively, were fixed for 2 hr in 3% glutaraldehyde and then prepared for freeze-etching and thin sectioning by standard techniques. Freeze-etch replicas of the teguments of all three species displayed morphologic characteristics that were generally in agreement with previous ultrastructural work, although some new features and interpretations arose from use of this technique. For each species there was a concentric ring structure within the microthrix base, and cytoplasmic extensions of the perikarya into the distal tegument were membrane-bound rather than confluent bridges; these extensions frequently branched within the tegument. In addition, channels running from the proximal tegumental membrane to, and opening at the distal surface of, the tegument were seen in thin sections.