Phylogeny for genera of Nematodirinae (Nematoda: Trichostrongylina).

Monophyly for the Nematodirinae, with 5 genera, Murielus, Rauschia, Nematodiroides, Nematodirus, and Nematodirella was confirmed based on comparative morphology and phylogenetic analysis of structural characters. This concept for the nematodirines excludes the monotypic Lamanema chavezi, but otherwise corroborates generic-level diversity as defined in prior studies. Exhaustive analysis resulted in 1 most parsimonious tree (36 steps; consistency index [CI] = 0.94; retention index [RI] = 0.93; excluding phylogenetically uninformative characters, CI = 0.92). As an inclusive or monophyletic group, Nematodirinae was diagnosed by 8 synapomorphies (7 are unequivocal): (1) large eggs, (2) long filiform spicules, (3) basal division of the dorsal ray, (4) symmetrical membrane enveloping the spicule tips, (5) fused structure of the spicule tips, (6) absence of the gubernaculum, (7) development of the third-stage larva within the egg, and (8) ornamentation in the form of discrete bosses on the bursa. Exclusion of Lamanema will require new assessments of historical biogeography and the evolution of host associations for the nematodirines.

Prevalence of Cryptosporidium spp. and Giardia spp. in five marine mammal species.

Cryptosporidium spp. and Giardia spp. are protozoan parasites that are often associated with severe diarrheal disease in a variety of mammals. Although these parasites have been extensively studied in terrestrial ecosystems, little is known about either parasite in the marine environment. Therefore, the objective of this study was to determine the prevalence of both Cryptosporidium spp. and Giardia spp. in 5 marine mammal species. Fecal samples were collected from 39 bowhead whales (Balaena mysticetus), 49 North Atlantic right whales (Eubalaena glacialis), 31 ringed seals (Phoca hispida), 22 bearded seals (Erignathus barbatus), and 18 beluga whales (Delphinapterus leucas) between 1998 and 2003. Using an immunofluorescent assay, parasites were detected in the feces of bowhead whales, right whales, and ringed seals, while neither parasite was detected in samples from bearded seals or beluga whales. Overall, prevalences were highest in ringed seals (Cryptosporidium spp., 22.6%; Giardia spp., 64.5%) and right whales (Cryptosporidium spp., 24.5%; Giardia spp., 71.4%) and lowest in bowhead whales (Cryptosporidium spp., 5.1%; Giardia spp., 33.3%). To our knowledge, this is the first report of Cryptosporidium spp. and Giardia spp. in either whale species and of Cryptosporidium spp. in the ringed seal.

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts, propagation in cell culture, and description of ultrastructural and genetic characteristics.

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.

Origin of stable flies (Diptera: Muscidae) on west Florida beaches: electrophoretic analysis of dispersal.

Stable fly adults were collected from 16 locations within the state of Florida and from locations in six other states in an attempt to determine the source of stable fly populations along Florida beaches on the Gulf of Mexico. Electrophoretic analyses were made of a minimum of 10 enzymes in each of 37 separate populations. Extremely low heterozygosity resulted in an inability to use standard genetic identity and distance procedures for determining the divergence of allopatric populations to establish the source of flies captured from beach areas where reproduction was unlikely. Comparisons of rare alleles in populations grouped geographically, computations of number of possible migrants, and analysis of conditional average frequency of alleles led to the conclusion that flies captured on Florida beaches come from a variety of sites, with locations northwest of the beaches contributing the majority of flies in outbreaks.

The prevalence of Cryptosporidium and Giardia spp. in fecal samples from free-ranging white-tailed deer (Odocoileus virginianus) in the southeastern United States.

The prevalence of Cryptosporidium sp. and Giardia sp. in fecal samples collected from free-ranging white-tailed deer (Odocoileus virginianus) was determined for animals in Virginia and Mississippi. The 34 Virginia samples came from young of the year (< or =6 months of age) presented to the Wildlife Center of Virginia, and the 360 Mississippi samples came from animals (> or =6 months of age) collected from 42 different sites across the state. Cryptosporidial oocysts were detected in 8.8% and 5.0% and Giardia sp. cysts were detected in 2.9% and 1.1% of the samples from Virginia and Mississippi, respectively. Analysis of data by collection site was not possible because of the small number of samples from some sites. Therefore, collection sites in Mississippi were grouped by the stream basin in which they occurred. Binary logistic regression analysis indicated the probability of infection with Cryptosporidium sp. decreased with increasing age of animal in all positive stream basins except 1, in which the pattern was reversed. This is the first report of either Cryptosporidium sp. or Giardia sp. in free-ranging white-tailed deer. The role of white-tailed deer in the epidemiology of these parasites is unknown. However, this study demonstrates that white-tailed deer shed cysts and oocysts of both parasites in the environment and must be considered potential sources of contamination.

Papillomatous pastern dermatitis with spirochetes and Pelodera strongyloides in a Tennessee Walking Horse.

Papillomatous digital dermatitis is a common disease in cattle. The pastern dermatitis observed in a horse shared many of the gross characteristics of papillomatous digital dermatitis in cattle. Lesions included a mixture of proliferative and erosive changes, with a verrucose appearance in some areas. Microscopic similarities included pseudoepitheliomatous and papillomatous epidermal hyperplasia with hyperkeratosis, spongiosis of the epidermis, and intraepidermal spirochetes. The horse was also concurrently infected with Pelodera strongyloides. Papillomatous digital dermatitis in cattle is associated with poor husbandry practices. The environment of the affected horse was heavily contaminated with urine, manure, and other organic debris. Verrucous pododermatitis of horses may be the same as or similar to bovine papillomatous digital dermatitis, and these conditions have similar etiologies.

Parasitic gastritis in a llama (Lama glama) associated with inhibited larval Teladorsagia spp. (Nematoda: Trichostrongyloidea).

A 7-year-old female llama was experimentally infected with 500 Fasciola hepatica metacercariae then turned out onto pasture also grazed by domestic sheep. The llama was necropsied approximately 22 weeks post infection. Routine examination of the gastrointestinal tract revealed diffusely coalescing, umbilicated nodules covering the caudal one-fifth of the third stomach compartment. Microscopically, the mucosa was irregularly thickened. Numerous nematode larvae were present in glandular lumens, often extending to the base of the glands. Where nematodes were most numerous, decreased numbers of parietal cells, attenuation of glandular epithelium and increased collagen within the lamina propria were evident. Of a total of 6510 adult nematodes in the third stomach compartment, 51% were Teladorsagia spp., 47% were Trichostrongylus axei, and 2% were Camelostrongylus mentulatus. Except for C. mentulatus, three sheep that had grazed with the llama and were necropsied at approximately the same time, harbored the same nematode species. The total number of larvae in the llama was 56,710, with more than 97% being ostertagiine early fourth-stage larvae. Ostertagiine larvae were also present in the sheep. Based on the composition of the adult populations in the llama and sheep, the larvae were considered to be species of Teladorsagia. Additionally, the larger numbers of early fourth-stage larvae indicated that the development of Teladorsagia spp. in the llama is inhibited.

Development and application of a dot-ELISA test for the detection of serum antibodies to Fasciola hepatica antigens in llamas.

A microenzyme-linked immunosorbent assay (dot-ELISA) was developed to detect serum antibodies against Fasciola hepatica antigens in llamas. Sera from five F. hepatica-infected and 11 non-infected llamas were used in initial test development. Nitrocellulose filter disks containing F. hepatica excretory-secretory product were placed in 96-well microtiter plates, washed, blocked with Tween-20, then incubated with four-fold serial dilutions of llama sera. After incubation with rabbit anti-llama IgG followed by peroxidase-conjugated goat anti-rabbit IgG, addition of precipitable substrate resulted in purple dots on white background (positives) easily read by eye. The technique was further evaluated at titers of 1:512 using an additional six known positive and eight known negative llamas. Test results showed 6/6 known positive as positive and 8/8 known negative as negative. Sera were collected, at approximately weekly intervals, from three llamas experimentally infected with F. hepatica. The dot-ELISA detected antibodies to F. hepatica as early as the second week post-infection in all llamas. In a serologic survey of 256 llamas from an F. hepatica endemic area, the dot-ELISA detected antigen-specific serum antibodies to F. hepatica in 42 (16%) of the llamas. Although no difference was noted in antibody prevalence between sexes, prevalence increased in llamas over 6 months of age.

The epizootiology of gastrointestinal nematodes of cattle in selected areas of Oregon.

A study was initiated to (1) determine species of gastrointestinal nematodes present in cattle in selected areas of Oregon, (2) examine seasonal abundance of these nematodes as an indicator of periods of transmission, and (3) determine when developmental inhibition occurs in the major genera of nematodes encountered. Four study sites were chosen: Corvallis, Langlois, near Fort Rock, and southeast of Klamath Falls on the Oregon-California border. Eight sets of three to four tracer calves each were introduced onto pasture at each site over a 2 year period with time of turn out onto pasture corresponding to late spring, mid-summer, late fall and late winter. At Corvallis, a distinct seasonality in parasite transmission was evident, peaking during the fall and winter. At Langlois, transmission was fairly constant throughout the year. No discernible patterns were evident at either Klamath Falls or Fort Rock. Eight nematode genera were found during the study. Ostertagia, Cooperia, Nematodirus and Trichostrongylus were present at all study sites and were the most common genera at each. Trichuris was found at all sites except Klamath Falls. Oesophagostomum was present in tracers only from Langlois and Corvallis while Haemonchus was found only at Klamath Falls and Fort Rock. Capillaria was only present at Klamath Falls. Where possible, specific transmission patterns for Nematodirus, Cooperia, and Ostertagia were determined for each site. Nematodirus was transmitted fairly steadily at both Langlois and Corvallis but was quite variable at Fort Rock. Developmental arrest was detected in this genus at all study sites during the fall and/or winter. Cooperia exhibited the most seasonally defined pattern of transmission, peaking during the fall and winter at Langlois, Corvallis and Klamath Falls. Hypobiotic larvae of Cooperia were present during the fall and/or winter only at Langlois and Corvallis. Peak transmission of Ostertagia at Langlois and Corvallis occurred during the fall and winter. At Fort Rock, transmission was lowest in the fall and increased in the winter. Hypobiotic larvae were evident in the fall and winter at Corvallis, Fort Rock and Klamath Falls. These data suggest Type II ostertagiasis may occur in late winter through spring in these areas. Hypobiotic larvae of Ostertagia were not detected at Langlois. The lack of appropriate environmental stimuli is one possible explanation for the apparent lack of hypobiosis at that site.

Ostertagia ostertagi challenge of calves vaccinated with Haemonchus placei intestinal homogenate.

The protective capacity of vaccination with Haemonchus placei whole gut homogenate against challenge with the non-blood-feeding nematode Ostertagia ostertagi was evaluated in calves. Ten helminth-free calves were randomly assigned to two groups. Group 1 received 100microg H. placei intestinal homogenate in the adjuvant 5% dextran sulfate/PBS, while Group 2 received the adjuvant alone. Injections were administered subcutaneously on Days 0 and 28. All calves were challenged with approximately 26,100 O. ostertagi larvae on Day 42. Serum antibody response and counts of nematode eggs per gram of feces (EPG) were determined throughout the study. Calves were necropsied at 5.5 weeks post-challenge for recovery of nematodes. Although significant increases were detected in both serum IgG(1) and IgG(2) of Group 1 calves (p<0.05), there was no significant difference in the total number of O. ostertagi recovered from the two groups (p>0.05). Lengths of adult nematodes were not significantly different between groups nor were the numbers of eggs present in adult females recovered from each group significantly different (p>0.05). There were also no significant differences between groups regarding fecal egg counts (p>0.05). Results suggest either: (1) the antigens targeted by the induced antibodies were not present in O. ostertagi; (2) the antigens targeted by the induced antibodies were present, but not essential to O. ostertagi survival; or (3) the antigen was present and essential, but amount of antibody ingested was insufficient to cause damage to the nematode.

Vaccination of calves with Haemonchus placei intestinal homogenate.

The ability of adult Haemonchus placei intestinal homogenate to confer protection against homologous challenge infection was evaluated. Calves were immunized twice with 100 microg H. placei intestinal protein in 5% dextran-sulfate/PBS (vaccinates) or PBS alone (controls) and were challenged with approximately 3300 infective H. placei larvae. There was no significant difference between groups in the total number of nematodes recovered but significantly fewer (p < 0.001) adult females were recovered from vaccinates. The proportion of fourth-stage larvae in vaccinates was significantly greater (p < or = 0.05) than in controls. Lengths of adult male and female nematodes were significantly shorter (p < 0.001) in vaccinated calves, and the numbers of eggs present in the uteri of female nematodes from vaccinates were significantly decreased (p < 0.001). Counts of nematode eggs per gram of feces (EPG) of vaccinates were significantly less than that for controls on Days 29-49 post-challenge (p < or = 0.05). Vaccinates had significant increases in serum IgG1 and IgG2 log(10) titers (p < or = 0.05) but not in serum IgM. EPG, numbers of females, and size of males and females were negatively correlated with increased mean post-challenge IgG1 and IgG2 titers. Reduction in binding of periodate-treated gut homogenate by immune serum indicated a carbohydrate specific component in the immune response.

Epidemiology of Ostertagia in the Northwestern USA.

The two most significant environmental factors that influence the epidemiology of Ostertagia are temperature and moisture. These factors vary more in the west and northwest than in any other part of the USA because of extreme regional differences in climate, topography, and land use. Consequently, patterns of Ostertagia transmission and inhibition also vary widely from region to region and from year to year. Recent data require revisions of previously accepted concepts of northern/southern transmission and inhibition. In particular, the flaw of liberal extrapolation of information from one region to another has now been recognized. Inhibition may not occur along the coast of the Pacific Northwest. Although not completely delineated, the transition zone between northern and southern patterns of inhibition appears to be significantly wider and more variable than previously recognized; Oregon data suggest a possible indeterminate and variable band extending north of the 45th parallel and south of the 43rd parallel. Summer inhibition has now been documented in areas of Oregon and Montana. Other than the recent Oregon and Montana studies, data on this transition zone in the Rocky Mountain region and high plains are sparse to non-existent. In contrast to previously accepted doctrine, the fall rise in egg count generally exceeds the traditionally accepted spring rise in many areas. From the Pacific Northwest to the Midwest, year-round transmission patterns have been observed. The presence of geothermal ground water sources produces microclimates that favor larval survival in many areas.(ABSTRACT TRUNCATED AT 250 WORDS)

First report of Lamanema chavezi (Nematoda: Trichostrongyloidea) in llamas (Lama glama) from Argentina.

Lamanema chavezi is a characteristic helminth of llamas and alpacas in Peru and Chile. However, its overall geographic range has not been well defined. This report documents the presence of L. chavezi in llamas from Argentina.

Effect of ivermectin delivered from a sustained-release bolus on the productivity of beef cattle in Oregon.

The effect of ivermectin delivered from a sustained-release bolus (I-SRB) on the weight gain of beef cattle through a grazing season was evaluated using 20 yearling beef steer calves randomly divided into two groups of ten animals each. Calves in the control group each received a placebo bolus, while those in the treatment group each received an I-SRB designed to release 12 mg ivermectin day-1 for approximately 90 days. All animals were weighted and samples of feces were collected from the rectum at monthly intervals, beginning on Day 0, until trial termination (Day 148). Pasture larval counts were also conducted on herbage collected on each sampling date. On Day 119, two control and two treated calves were removed from pasture, housed in isolation for 3 weeks, then necropsied for recovery of gastrointestinal nematodes. Three sets of parasite-naive tracer calves were utilized to evaluate the initial, interim and final levels of pasture contamination by nematode larvae. A fourth set was used to evaluate the level of pasture contamination the following spring. The use of the I-SRB resulted in a greater than 99% reduction in fecal egg counts of trichostrongyles and numbers of gastrointestinal nematodes in the treated principals, as well as an average daily gain advantage of 0.114 kg over the 148 day period. A 67-98% reduction in pasture larval nematode contamination occurred on pastures grazed by the treated animals, as indicated by the parasite burden in tracer calves and pasture larval counts. The treatment effect was eventually lost by the following spring since tracer calves on the treated pasture had only 33% fewer nematodes than those on the control pasture.

Influence of ivermectin and clorsulon treatment on productivity of a cow-calf herd on the southern Oregon coast.

The reproductive performance of beef cows and the weight gain of their calves was evaluated after oral administration of clorsulon or clorsulon in combination with the subcutaneous administration of ivermectin. One hundred and fifty pregnant cows harboring infections of Fasciola hepatica were assigned to one of three treatment groups: Group 1 comprising 50 unmedicated controls; Group 2, 50 cows treated with clorsulon orally at 7 mg kg-1; Group 3, 50 cows treated with clorsulon orally at 7 mg kg-1 and ivermectin subcutaneously at 200 micrograms kg-1. Weights and body condition scores of the cows were measured and fecal and blood samples were taken at trial initiation and days 158 and 270. Pregnancy status was also determined at day 270. Weights and body condition scores were measured for the calves at days 158 and 270. Adjusted 205 day weaning weight of the calves was calculated and analyzed for differences between treatment groups. Four sets of tracer calves were used periodically throughout the trial to monitor the helminth challenge to the herd. Both gastro-intestinal nematodes and liver flukes were transmitted to the tracer calves during the entire trial. Even in the face of continual helminth challenge, beef cows treated with combined clorsulon/ivermectin conceived approximately 2 weeks earlier than their untreated counterparts and their calves had better body condition scores and weights than untreated calves.

Seroprevalence of Toxoplasma gondii in llamas (Lama glama) in the northwest USA.

Serum samples from 283 llamas (Lama glama) from Oregon, Washington State and Idaho were tested for antibodies to Toxoplasma gondii using the modified agglutination test. Antibodies were found in 95 (33.5%) llamas. Percent seropositivity in serum dilutions of 1:25, 1:50, 1:500, and 1:5000 was 9.5%, 18.3%, 4.9%, and 0.7%, respectively.

Risk factors associated with the presence of Sarcocystis neurona sporocysts in opossums (Didelphis virginiana).

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM) in horse in the Americas. The only known definitive host for this parasite in the United States is the opossum (Didelphis virginiana); however, despite the importance of the disease, the epidemiology of the parasite in the definitive host is poorly understood. To begin addressing these data gaps, potential risk factors were evaluated for their association with the presence of sporocysts of S. neurona in opossums live-trapped in March 1999 and November 1999 to May 2000. Sporocysts of S. neurona were found in 19 of the 72 animals examined. Potential risk factors evaluated were locality, trap date, age, gender, the presence of young in the pouch of females, and body condition score. Variables that were associated with the presence of S. neurona sporocysts were used in logistic regression analysis. Of the factors examined, season and body condition score were associated with increased odds of an animal harboring sporocysts.

Efficacy of the morantel sustained release trilaminate matrix against gastrointestinal nematodes in beef calves.

The effectiveness of the morantel sustained release trilaminate (MSRT) in controlling gastrointestinal nematodes through a grazing season was evaluated using 60 yearling beef stocker calves randomly divided into 2 groups of 30 animals each. In April 1985, the calves comprising the treatment group each received an MSRT designed to release morantel tartrate continuously for 90 days while those of the control group remained unmedicated. All animals were weighed and samples of rectal feces were taken at 14-day intervals, beginning on Day 0, until trial termination (Day 168). At trial termination, 10 control and 10 treated calves were necropsied for recovery of gastrointestinal nematodes. Three sets of parasite-naïve tracer calves were utilized to evaluate the initial, interim and final levels of pasture contamination by nematode larvae. Overall, the use of the MSRT resulted in a 75.5% reduction (P less than 0.001) in output of nematode eggs from the principals, an 81.8% reduction (P less than 0.001) in numbers of gastrointestinal nematodes in principals (at trial termination), and a 96.9% reduction (P less than 0.05) of pasture larval nematode contamination (as indirectly indicated by parasite burdens in tracer calves). The mean weight advantage of treated calves was 16.6 kg per head (P less than 0.001).

Prevalence of Sarcocystis neurona sporocysts in opossums (Didelphis virginiana) from rural Mississippi.

Sarcocystis species sporocysts were found in intestinal scrapings from 24 of 72 opossums (Didelphis virginiana) from rural Mississippi. The number of sporocysts in each opossum varied from a few ( < 100000) to 187 million. Sporocysts from 24 opossums were bioassayed for Sarcocystis neurona infections by feeding to gamma-interferon knockout (KO) mice. S. neurona was detected in the brains of KO mice fed sporocysts from 19 opossums by immunohistochemical staining with anti-S. neurona specific polyclonal rabbit serum, and by in vitro culture from the brains of KO mice fed sporocysts. The isolates of S. neurona from opossums were designated SN16-OP to SN34-OP. Merozoites from 17 of 19 isolates tested at the 25/396 locus were identical to previously described S. neurona isolates from horses. The high prevalence of S. neurona sparocysts in D. virginiana suggests that this opossum constitutes an ample reservoir of infection in the southern United States.

Subclinical cyathostomiasis and unidentified helminthiasis in a juvenile emu (Dromaius novaehollandiae).

The nematode Cyathostoma variegatum (a lungworm) was present in the distal trachea and proximal bronchi of an 8-mo-old emu that was euthanatized after fracturing the left tibiotarsus. Clinical signs of infection were not present prior to euthanasia and abnormalities were not noted on gross or histologic examination of the respiratory tract. The absence of respiratory signs in this and other emus underscores the need for routine fecal examinations to interrupt the inadvertent spread of this potentially life-threatening parasite. In addition to the lungworms, large helminth eggs were present in renal and reproductive tissue. Egg morphology was suggestive of species of Deletrocephalus or Paradeletrocephalus. The mechanism by which these eggs became lodged in this ectopic site is unknown.