Charles H. Courtney

Unusual population genetics of a parasitic nematode : mtDNA variation within and among populations

Very little is known about the distribution of genetic variance within and among populations of parasitic helminths. In this study we used mitochondrial DNA (mtDNA) restriction fragment analysis to describe the population genetic structure of Ostertagia ostertagi, a nematode parasite of cattle, in the United States. Estimates of within‐population mtDNA diversity are 5 to 10 times greater than typical estimates reported for species in other taxa. Although populations are genetically differentiated for a key life–history trait, greater than 98% of the total genetic diversity is partitioned within populations, and the geographic distribution of individual mtDNA haplotypes suggests high gene flow among populations.

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

Guidelines have been designed to assist in the planning, operation and interpretation of studies for the assessment of the efficacy of drugs against helminth parasites of dogs and cats. The advantages, disadvantages and application of critical and controlled tests are presented. Information is also provided on the selection of animals, housing, feeding, dose-titration, confirmatory and clinical trials, record keeping and necropsy procedures. These guidelines should assist both investigators and registration authorities involved in the evaluation of anthelmintics to employ comparable and standard procedures and will have the added benefit of minimising the numbers of animals needed for such tests.

Comparison of heartworm antigen test kit performance in dogs having low heartworm burdens.

Sensitivity and specificity of four in-clinic heartworm antigen test kits, AbboScreen (Abbott Laboratories), Snap PF (IDEXX Laboratories), Solo Step (HESKA Corporation), Witness (Synbiotics Corporation) and two heartworm antigen microwell plate assays, DiroCHEK (Synbiotics) and PetChek PF (IDEXX) were compared in a blinded study using serum or plasma drawn from 237 random source dogs, including 140 with necropsy-confirmed, low worm burden infections (minimum 1 worm, maximum 10, mean 2.3, median 3) and 97 confirmed heartworm-free at necropsy. In general, microwell format tests were more sensitive than membrane format tests and tests using ELISA technology were more sensitive than tests using lateral flow immunochromatographic technology. Percent sensitivity and specificity, respectively, were PetChek PF 76 and 97, DiroCHEK 71 and 94, SNAP PF 67 and 98, Solo Step 60 and 98, and AbboScreen 52 and 96. The Witness test protocol was changed by the manufacturer midway through the study, and the newer version of this test kit arrived containing a package insert alerting the user to a change in procedure, which purportedly resulted in improved sensitivity. PetChek was significantly more sensitive than all other tests except DiroCHEK and the new version of Witness. DiroCHEK was significantly more sensitive than all tests except PetCheck, SNAP and the new version of Witness. Snap was more sensitive than AbboScreen and the old version of Witness. Differences in specificity were not significant (P>0.05).

Haemonchus placei and Haemonchus contortus are distinct species based on mtDNA evidence

Debates continue over the extent to which the parasitic trichostrongylids Haemonchus placei and Haemonchus contortus hybridise in nature, and whether they deserve species status. Mitochondrial ND4 gene sequences from individuals of each putative species collected from populations around the United States indicate that the two species are highly differentiated at the mtDNA level. Furthermore, there was no evidence of introgressive hybridisation occurring in wild populations.

A repetitive DNA probe for the sensitive detection of Fasciola hepatica infected snails

Epizootiologic studies on F. hepatica frequently use microscopic techniques for the detection of infected snails, however, the poor efficiency, sensitivity, and specificity associated with these techniques limit their usefulness. A DNA-based test for the identification of snails infected with larval stages of F. hepatica would solve these problems and enable a level of detection accuracy previously unavailable. We have cloned and sequenced a 124 bp fragment of repetitive DNA from F. hepatica which hybridizes specifically with DNA of F. hepatica but not with DNA of its snail intermediate hosts Fossaria cubensis and Pseudosuccinea columella, or with DNA of Fascioloides magna and Paramphistomum liorchis, ruminant trematodes which share the same intermediate host and same enzootic range as F. hepatica. Using this 124 bp fragment as a probe, infection in snails was detected immediately following miracidial penetration, thus a sensitivity equivalent to the minimum biologic unit of the parasite was achieved. This 124 bp repeated sequence belongs to a large family of 124 bp repeats that share a high level of sequence identity and constitute approximately 15% of the F. hepatica genome. We also report here the development of a quick and inexpensive DNA extraction protocol for use in field-collected snails. Thus, we have developed both a highly sensitive and specific DNA probe and a means to use the probe in a large epizootiologic study of F. hepatica where thousands of field-collected snails need to be assayed for infection.

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of > 99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.

Resistance to experimental infection with Fasciola hepatica in exotic and domestic breeds of sheep

Abstract Significant breed differences were detected in fecal egg counts and fluke counts following experimental infection of five breeds of sheep with metacercariae of F. hepatica . Two experiments compared the responses of Barbados Blackbelly, St. Croix, Florida Native, Finn X Rambouillet and Targhee sheep to primary and challenge infections (Expt. 1) and to multiple infections (Expt. 2). Barbados Blackbelly sheep were more susceptible to infection than the other breeds in both experiments. St. Croix and Florida Native sheep were the most resistant breeds in Expt. 1, while Targhee and Florida Native were the most resistant in Expt. 2. There was no evidence of acquired immunity in any breed in either experiment.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin.

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonal transmission of equine cyathostomes in warm climates

Few studies investigating the seasonal transmission of equine cyathostomes have been done in warm climates. Two Australian studies used experimentally-infected plots to determine hatching, development and survival of free living stages of equine cyathostomes. Four studies in the southern United States used pasture larval counts, and in some instances tracer animals, to determine seasonal availability of infective cyathostome larvae on naturally-infected pastures. With the exception of the dry Australian tropics, a general pattern of peak transmission of cyathostomes during the cooler seasons of the year and minimal transmission during the warmest seasons was observed. Infective larvae and developing stages survived poorly in hot weather, although the rate of development was most rapid during that time. In contrast, infective larvae and developing stages survived well in cool weather, although the rate of development was slower. Adequate moisture was crucial to cyathostome transmission in warm climates, thus hot, dry weather effectively sterilized a pasture, whereas cool, moist weather was optimum for transmission. These data suggest that suppression of cyathostome egg output in feces of horses beginning shortly before the onset of cooler and/or more moist weather, and continued through the favorable period for development and survival of larvae on pasture - usually the autumn and winter should provide adequate control of these parasites. However, the efficacy of such seasonal control programs has yet to be adequately tested against that of traditional year round treatments.

Coccidia of sandhill cranes, Grus canadensis.

Eimeria gruis Yakinoff and Matschoulsky 1935, Eimeria reichenowi Yakimoff and Matschoulsky 1935, and an Adelina species are described from sandhill cranes in the United States. E. gruis was found in the feces of 11 of 14 Florida sandhill cranes (Grus canadensis pratensis) and 62 of 72 greater sandhill cranes (G. c. tabida) from Florida, 5 of 14 greater sandhill cranes from Arizona, and 4 of 16 lesser sandhill cranes (G. c. canadensis) from Texas. E. reichenowi was found in the feces of 12 of 14 Florida sandhill cranes and 66 of 72 greater sandhill cranes from Florida, 4 of 14 greater sandhill cranes from Arizona, and 5 of 16 lesser sandhill cranes from Texas. Adelina sp. was found in the feces of 3 of 14 Florida sandhill cranes and 2 of 72 greater sandhill cranes from Florida. The Adelina species is considered to be a spurious parasite of the cranes.