Evaluation of Anthelmintic Resistance and Exhaust Air Dust PCR as a Diagnostic Tool in Mice Enzootically Infected with Aspiculuris tetraptera.

The entry of infectious agents in rodent colonies occurs despite robust sentinel monitoring programs, strict quarantine measures, and stringent biosecurity practices. In light of several outbreaks with Aspiculuris tetraptera in our facilities, we investigated the presence of anthelmintic resistance and the use of exhaust air dust (EAD) PCR for early detection of A. tetraptera infection. To determine anthelmintic resistance, C57BL/6, DBA/2, and NCr nude mice were experimentally inoculated with embryonated A. tetraptera ova harvested from enzootically infected mice, followed by treatment with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 ppm piperazine in feed, or 2.1 mg/mL piperazine in water for 4 or 8 wk. Regardless of the mouse strain or treatment, no A. tetraptera were recovered at necropsy, indicating the lack of resistance in the worms to anthelmintic treatment. In addition, 10 of 12 DBA/2 positive-control mice cleared the A. tetraptera infection without treatment. To evaluate the feasibility of EAD PCR for A. tetraptera, 69 cages of breeder mice enzootically infected with A. tetraptera were housed on a Tecniplast IVC rack as a field study. On day 0, 56% to 58% of the cages on this rack tested positive for A. tetraptera by PCR and fecal centrifugation flotation (FCF). PCR from EAD swabs became positive for A. tetraptera DNA within 1 wk of placing the above cages on the rack. When these mice were treated with 150 ppm fenbendazole in feed, EAD PCR reverted to pinworm-negative after 1 mo of treatment and remained negative for an additional 8 wk. The ability of EAD PCR to detect few A. tetraptera positive mice was investigated by housing only 6 infected mice on another IVC rack as a field study. The EAD PCR from this rack was positive for A. tetraptera DNA within 1 wk of placing the positive mice on it. These findings demonstrate that fenbendazole is still an effective anthelmintic and that EAD PCR is a rapid, noninvasive assay that may be a useful diagnostic tool for antemortem detection of A. tetraptera infection, in conjunction with fecal PCR and FCF.

The use of epifluorescence microscopy and fluorescent dyes for visualization of Oxyuris equi eggs.

This study presents a new method for visualization of Oxyuris equi eggs collected by means of a clear adhesive tape applied to the perianal region of horses. The obtained results indicate that this protocol permits a quick, easy, clear and selective visualization of Oxyuris equi eggs even in the presence of feces and other material. Another advantage of this method is that it can be used with fluorescent dyes solubilized in water, which will stain biologic material without dissolving or altering the adhesive tape and is also environmentally safe. Other dyes currently used for staining biologic materials use organic solvents, which may be combined with acids or bases in their formulation, preventing their use with the tape method.

New species of Parapharyngodon (Nematoda: Pharyngodonidae) in Phymaturus spp. (Iguania: Liolaemidae) from Argentina.

Parapharyngodon sanjuanensis sp. nov. (Nematoda: Pharyngodonidae) from the large intestines of Phymaturus punae and Phymaturus williamsi (Squamata: Liolaemidae) from province of San Juan, Argentina, is described and illustrated. Parapharyngodon sanjuanensis sp. nov. is the 54th species assigned to the genus and the 8th from the Neotropical region. It differs from other species in the genus in that males possess 8 caudal papillae, 6 of which are large and pedunculate, 2 are small, almost inconspicuous; anterior lip echinate, posterior lip bilobate; females possess prominent vulva and short stiff tail spike.

A new species of Parapharyngodon (Nematoda: Pharyngodonidae) infecting Dermatonotus muelleri (Anura: Microhylidae) from Caatinga, Northeastern Brazil.

Parapharyngodon silvoi n. sp. (Nematoda: Pharyngodonidae) is described from the large and small intestine of the Muller's termite frog Dermatonotus muelleri (Boettger, 1885) from the biome Caatinga, Exu municipality, Pernambuco State, Brazil, Dermatonotus muelleri is a fossorial species with a specialized termite diet, and feeding and reproductive behavior occurring only during the wet season. The new species is distinguished from other species of the genus Parapharyngodon by showing ovary not coiled around the esophagus, morphology of anterior cloacal lip, spicule size and number of caudal papillae.

Exposure to chlorine dioxide gas for 4 hours renders Syphacia ova nonviable.

The purpose of our study was to evaluate the efficacy of chlorine dioxide gas for environmental decontamination of Syphacia spp. ova. We collected Syphacia ova by perianal cellophane tape impression of pinworm-infected mice. Tapes with attached ova were exposed to chlorine dioxide gas for 1, 2, 3, or 4 h. After gas exposure, ova were incubated in hatching medium for 6 h to promote hatching. For controls, tapes with attached ova were maintained at room temperature for 1, 2, 3, and 4 h without exposure to chlorine dioxide gas and similarly incubated in hatch medium for 6 h. Ova viability after incubation was assessed by microscopic examination. Exposure to chlorine dioxide gas for 4 h rendered 100% of Syphacia spp. ova nonviable. Conversely, only 17% of ova on the 4-h control slide were nonviable. Other times of exposure to chlorine dioxide gas resulted in variable effectiveness. These data suggest that exposure to chlorine dioxide gas for at least 4 h is effective for surface decontamination of Syphacia spp. ova.

Characterization of rat pinworm (Syphacia muris) epidemiology as a means to increase detection and elimination.

Rodent pinworms persist in many institutions, suggesting deficiencies in eradication and diagnostic processes. When pinworms are detected, treatment success is common, but false-negative test results during health surveillance or after treatment likely contribute to the continued presence of this parasite. PCR testing is not always practical, and increased information regarding the life cycle and general epidemiology of pinworm infestations could improve the sensitivity of traditional nonPCR detection methods and improve eradication efforts. We therefore investigated a pinworm (Syphacia muris) infestation in Sprague-Dawley rats (Rattus norvegicus) to develop a more accurate testing strategy. In addition, we sought to determine the duration of egg viability by using an in vitro hatching protocol to assess environmental persistence. Finally, we tested the ovicidal efficacy of a disinfectant used at our institution. Eggs were shed in higher numbers in the midafternoon as compared with other times of the day, and the sex of the host had no consistent effect on egg shedding. Egg shedding showed periodicity over time, with shedding decreasing to 0 at 2- to 3-wk intervals. Neither cecal examination nor tape tests alone reliably predicted pinworm infestation, and results of the 2 tests did not necessarily coincide. Eggs aged for as long as 7 mo remained viable, indicating a potential for recontamination from the environment. Finally, gaseous chlorine dioxide was an effective ovicidal agent, with a kill rate of 99.7%. These results suggest that strategies for S. muris eradication can be optimized to increase detection and elimination.

Distribution and reproductive strategies of Gyrinicola batrachiensis (Oxyuroidea: Pharyngodonidae) in larvae of eight species of amphibians from Nebraska.

In total, 462 tadpoles and salamander larvae of 8 species were examined for the presence of Gyrinicola batrachiensis from 5 locations in Nebraska. Infection by G. batrachiensis occurred in tadpoles of Rana blairi , Rana catesbeiana, Rana pipiens, and Bufo woodhousii. Tadpoles of Hyla chrysoscelis , Spea bombifrons, and Pseudacris maculata and larvae of Ambystoma mavortium were not infected with G. batrachiensis. Population structure, defined as prevalence, mean abundance, and mean intensity of G. batrachiensis, varied among tadpoles of different amphibian species and was determined by collection locality, developmental period of tadpole hosts, amphibian species co-occurrence, and different reproductive strategies of G. batrachiensis , or a combination. Gyrinicola batrachiensis observed in all ranid tadpoles and B. woodhousii tadpoles from where bufonids were the only anuran species present, confirmed to the didelphic haplodiploidy and monodelphic parthenogenetic reproductive strategies, respectively. However, tadpoles of B. woodhousii that co-occurred with tadpoles of R. pipiens at Cedar Creek were inconsistent with these predictions and contained both male and didelphic female nematodes, but at a low mean intensity (1.61 ± 0.70). Didelphic female nematodes from B. woodhousii tadpoles at Cedar Creek only produced thick-shelled eggs, whereas nematodes in R. pipiens tadpoles had a high mean intensity (14.88 ± 23.83) from this location and contained both thick-shelled and thin-shelled eggs in their respective uteri. More importantly, adult female nematodes from tadpoles of R. pipiens and B. woodhousii from Cedar Creek were morphologically more similar to each other than to female nematodes recovered from tadpoles of other anuran species, other locations, or both. These data suggest that when strains of G. batrachiensis are shared by tadpoles of different amphibian species that differ in developmental period, the nematodes have an intermediate reproductive strategy in amphibian species, with tadpoles having short development.

The dynamics of macroparasite host-self-infection: a study of the patterns and processes of pinworm (Oxyuridae) aggregation.

OBJECTIVES: Among parasites, Taylor's power law identifies a tight relationship in aggregation of macroparasite infection intensity with few exceptions; notably, the nematode family Oxyuridae tends to have higher than expected aggregation. Oxyuridae infect a wide range of mammalian hosts and have a unique reproductive strategy that involves conventional horizontal transmission, as well as re-infection of an already infected host. We asked the question, do the unique aspects of pinworm life-history explain an exception to the widely observed patterns of aggregation of parasite populations? METHODS: We empirically examined the differences among Oxyuridae (genus: Syphacia) compared with other helminth (genus: Heligmosomoides) parasite aggregations in 2 rodent hosts with similar ecology: the yellow-necked mouse (Apodemus flavicollis) from Trento, Italy and the white-footed mouse (Peromyscus leucopus) from Pennsylvania, USA. To investigate the effects of pinworm life-history characteristics on generating aggregation, we present a stochastic model that explores aggregation under a range of host-self-infection, parasite death, and transmission scenarios. RESULTS: Oxyuridae parasites had consistently greater aggregation compared to other nematodes regardless of host or parasite species identity, and pinworm aggregation exceeded the range of macroparasite aggregation described previously. CONCLUSIONS: Our simulations demonstrate that host-self-infection, on its own, is sufficient to generate aggregation values greater than the predicted values.

A cost-effective and efficacious method of pinworm treatment for large colonies of mice.

Rodent pinworm infestations are common in modern animal facilities, and treatments to eradicate these nematodes are often costly and labor-intensive. The authors describe a method they developed to treat rodents with ivermectin using the automatic watering system available at their facility. This delivery method proved an efficacious and cost-effective means of eradicating Aspiculuris tetraptera from a large colony of mice. The system might also be used to provide other orally administered agents to mice and other species.

Evaluation of some plants used in Turkish folk medicine against parasitic infections for their in vivo anthelmintic activity.

Ethanolic and aqueous extracts obtained from nine plant species from seven families selected depending on their use in Turkish folk medicine, including Citrillus lanatus (Thunb.) Matsum. (seed), Jasminum fruticans L. (branches), Juniperus drupacea Labill. (fruits), Juniperus nana L. (fruit and leaves), Juniperus oxcycedrus L (fruit and leaves), Mentha longifolia L. (herba), Pinus nigra ssp. pallasiana (Lamb.) Richt. (fruits), Plantago lanceolata L. (leaves), and Zea mays L. (seed) were evaluated for their in vivo anthelmintic activity. Among the plant extracts studied, both ethanolic and aqueous extracts of Jasminum fruticans, Mentha longifolia and Pinus nigra ssp. pallasiana, the aqueous extracts of Zea mays, the ethanolic extracts of Citrillus lanatus, Juniperus drupacea (fruit), Juniperus oxcycedrus and Plantago lanceolata displayed significant anthelmintic activity against pinworms, Syphacia obvelata and Aspiculuris tetraptera, in mice. Rest of the extracts from plants did not show any remarkable anthelmintic activity. The results were considered significant at p<0.05.

A review of treatments for the eradication of pinworm infections from laboratory rodent colonies.

Oxyurids are common contaminants of both specific pathogen-free and conventional rodent colonies. This paper reviews the biology and life cycles of oxyurid parasites of laboratory rodents, mechanisms of action and potential adverse effects of commonly used treatments, and treatment modalities, both historical and recent.

Factors influencing the fecal egg and oocyst counts of parasites of wild European rabbits Oryctolagus cuniculus (L.) in Southern Western Australia.

Abundance of intestinal parasites was monitored by fecal egg and oocyst counts for samples of wild rabbits Oryctolagus cuniculus with different levels of imposed female sterility from 12 populations in southwestern Australia. Differences in egg counts of Trichostrongylus retortaeformis between seasons and age groups were dependent on the sex of the host. Pregnancy may have been responsible for these differences because egg counts were consistently higher in intact females than in females surgically sterilized by tubal ligation. Egg counts for Passalurus ambiguus were influenced by season and host age but there were no differences between sexes or between intact and sterilized female rabbits. No differences were detected in the oocyst counts of the 8 species of Eimeria between male and female rabbits or between intact and sterilized females. Seasonal differences were detected in oocyst counts of Eimeria flavescens and Eimeria stiedai. The overwhelming determinant of coccidian oocyst counts was host age, with 6 species being much more abundant in rabbits up to 4 mo of age. There was a suggestion that egg counts of T. retortaeformis and oocyst counts of several species of Eimeria were reduced in populations where rabbit numbers had been depressed for at least 2 yr, but there was no evidence that short-term variations in rabbit numbers had a measurable effect on parasite abundance.

Evaluation of the association of parasitism with mortality of wild European rabbits Oryctolagus cuniculus (L.) in southwestern Australia.

Abundances of the parasitic nematodes Trichostrongylus retortaeformis and Passalurus ambiguus, and 8 Eimeria species were estimated by fecal egg and oocyst output in 12 discrete free-ranging populations of wild rabbits (Oryctolagus cuniculus) in southwestern Australia. Comparisons of parasite egg and oocyst counts were made between those rabbits known to have survived at least 2 mo after fecal samples were collected and those rabbits that did not survive. There were significant negative relationships between parasite egg and oocyst counts and survival when all age groups and collection periods were pooled for several species of coccidia and for T. retortaeformis. However, when the same comparisons were made within rabbit age groups and within collection periods, there were very few significant differences even where sample sizes were quite large. The differences indicated by the pooled analysis for coccidia were most likely due to an uneven host age distribution with respect to survival, combined with an uneven distribution of the oocyst counts with rabbit age. The result for T. retortaeformis was similarly affected but by a seasonal pattern. Parasitism by nematodes and coccidia did not appear to be an important mortality factor in these rabbit populations, at least at the range of host densities we examined. This suggests that other factors must have been responsible for the observed pattern of density-dependent regulation in these rabbits.

The effect of Toxoplasma gondii and other parasites on activity levels in wild and hybrid Rattus norvegicus.

Using both correlational and experimental evidence, the relationship between parasite load and host activity was assessed in brown rats, Rattus norvegicus. Two hypotheses were tested--(1) that parasites with indirect life-cycles, involving transmission between a prey and its predator, will alter the activity of the intermediate host so as to increase its susceptibility to predation by the definitive host and (2) that activity levels in parasitized rats would be increased rather than decreased. Four groups of rats (n = 140) were examined. One group (n = 50) were wild brown rats trapped from 3 UK farmsteads, with naturally occurring parasites. The others were purpose-bred wild/laboratory hybrid rats with experimentally induced parasitic infections of either (n = 15) adult-acquired or (n = 15) congenitally-acquired Toxoplasma gondii (an indirect life-cycle parasite), or (n = 15) Syphacia muris (a direct life-cycle parasite). Uninfected hybrid rats ( n = 45), matched for sex, age and weight, served as controls. Rats were housed individually in outdoor cages, and their activities were recorded on video-tapes for 6 non-consecutive 10 h nights. Exercise wheels were also available for the hybrid rats. Out of 6 parasite species detected in the wild rats, T. gondii was the only one which required predation by a definitive host to complete its life-cycle, and was also the only parasite to be associated with higher activity levels in infected than uninfected rats. Hybrid rats infected with T. gondii were also more active than those uninfected, whereas there were no differences in activity levels between S. muris infected and uninfected rats. This study shows that the indirect life-cycle parasite T. gondii can influence the activity of its intermediate host the rat. I suggest that this may facilitate its transmission to the cat definitive host.

Structure of the pinworm (Oxyurida: Nematoda) guild in the hindgut of the American cockroach, Periplaneta americana.

The pinworm (Oxyurida: Nematoda) guild in a laboratory colony of Periplaneta americana was investigated to answer the following questions. (1) Is guild structure stable with time? (2) Is there evidence of interaction among the species? (3) Is there evidence for niche diversification with respect to spatial distribution or developmental stage of host? Four species were found: Thelastoma periplaneticola, Thelastoma bulhoesi, Hammerschmidtiella diesingi and Leidynema appendiculatum; females of the first two could not be distinguished and they were lumped and referred to as Thelastoma sp.; 328 mid-instar hosts were dissected between September 1987 and February 1990, and the number of adult females of each pinworm species was recorded to investigate changes in guild structure through time. Seventy-five percent of hosts had Thelastoma sp., 62% had H. diesingi and 40% had L. appendiculatum; mean intensities were 6.5 (Thelastoma), 2.9 (H. diesingi) and 1.9 (L. appendiculatum). Relative numbers were stable with time and intensities of the species were similar whether they occurred alone in the host or in conjunction with other species. Thelastoma sp. and L. appendiculatum occurred together less often than expected and their numbers in individual hosts were negatively correlated. Both species occurred alone more often than expected. Surprisingly, H. diesingi and L. appendiculatum occurred together more often than expected. Intestines of 44 roaches were frozen in liquid nitrogen and divided into 5 equal sections to study longitudinal distributions of the species in the gut. Most L. appendiculatum and H. diesingi were found in the first, whereas most Thelastoma sp. occurred in the second segment. However, Thelastoma sp. dominated all segments of the gut. Worms were counted in 192 hosts belonging to 4 categories (early instar, late instar, adult male and adult female). Single species infections were more common in early instar hosts but there was no evidence that different species preferred different host stages. Modal distributions in male L. appendiculatum and T. bulhoesi suggest that these species may exhibit intraspecific interference competition similar to that observed in other pinworms. Such effects may be more important than interspecific effects in maintaining guild stability.

Haplodiploidy in the Oxyurida: decoupling the evolutionary processes of adaptation and speciation.

Host distribution and life history variation in the Oxyurida are discussed in the context of haplodiploidy. The Oxyurida have the broadest host distribution of any zooparasitic nematode group but have retained a virtually unchanged life cycle. Like other haplodiploids, oxyuridians have life histories that maintain viscous population structures. It is postulated that this has facilitated speciation but may have discouraged major evolutionary innovation in the group.

Long-term population dynamics of pinworms (Syphacia obvelata and Aspiculuris tetraptera) in mice.

The population dynamics of concurrent infections of Syphacia obvelata and Aspiculuris tetraptera (Nematoda) in laboratory mice were investigated under conditions of constant re-exposure over periods of 56 and 115 days. The results indicate that A. tetraptera burdens equilibrate at a higher level than S. obvelata burdens and that both parasites become aggregated in the mouse population. Parasite burdens were higher following short-term (7 day) exposure of uninfected tracer mice to both parasites when compared with parasite burdens attained over long-term exposure, indicating probable development of immunity. A significant positive correlation was detected between numbers of immature S. obvelata and immature A. tetraptera for both experimental and tracer mice.

The life-cycle of Syphacia muris Yamaguti (Nematoda: Oxyuroidea) in the laboratory rat.

The life-cycle of Syphacia muris is described in primary infections of Wistar rats. The life-cycle of S. muris is completed within seven days and no moults were discovered inside the egg. Instead each of two moults were observed outside the egg up to 24 and 40 hours after infection, and TEM studies suggest a third moult occurs up to 64 hours. The development and maturation of the larval stages are described for the first time using both light and electron microscopy. Differences in the size and growth of female S. muris compared with previously published figures could be due to differences in the strains of rats used.

[Haplodiploidy of Oxyurida. Effects of this phenomenon in the life cycle]. / L'haplodiploidie des oxyurida. Incidence de ce phénomène dans le cycle évolutif.

Chromosome behavior during gametogenesis and early cleavage is studied in two species of Thelastomatidae (Oxyurida), Hammerschmidtiella sp. and Thelastoma sp., parasites of diplopods. Both species are shown to be haplodiploid: male with 4 Thelastoma) or 5 (Hammerschmidtiella) chromosomes and double this number in the corresponding female. Haplodiploidy is now known in the 4 families of the Oxyurida and the author concludes that this form of reproduction is characteristic of the entire order. The Oxyuridan life cycle seems simple: 2 moults in the egg, no intermediate host, no extra-intestinal migration. However, several findings (some quite recent) indicate that this life cycle is often complicated by phenomena very closely adapted to host biology. The following particularities are analysed: 1) male progenesis; 2) laying of eggs in agglomerated packets; 3) peri-anal egg deposition; 4) poecilogony; 5) spanandry; 6) traumatic extra-genital insemination; 7) thelytoky. In a final chapter the author relates these adaptations to host biology and attempts to explain how they may have come to be favoured by natural selection during the course of evolution.