Myxobolus cerebralis in Tubifex tubifex from a whirling disease epizootic in Montana.

Aquatic oligochaetes from a whirling disease enzootic area in southwest Montana were examined for infection with Myxobolus cerebralis. Anterior portions of oligochaetes were preserved for specific identification, whereas DNA was purified from posterior portions. The purified DNA was used in a nested polymerase chain reaction (PCR) assay specific for M. cerebralis small-subunit ribosomal DNA. Naturally infected oligochaetes were identified by a resulting 415-bp fragment of amplified parasite DNA. A total of 704 oligochaetes was tested from April through September 1997. Eighteen (2.6%) oligochaetes were infected with M. cerebralis as defined by the nested PCR assay. Two of these were identified as mature Tubifex tubifex, and the others were likely T. tubifex but were immature and lacking the diagnostic reproductive structures. This is the first report of T. tubifex naturally infected with M. cerebralis.

Fascioloidiasis in game-ranched elk from Montana.

The distribution of Fascioloides magna in game-ranched elk and the potential for spread of the parasite through movement of infected animals was examined in Montana (USA). Fecal samples (n = 448) collected from captive elk on 29 game ranches were examined for eggs of F. magna by fecal sedimentation. Eggs were detected in elk on 5 ranches. This suggests that F. magna has been translocated by infected game-ranched elk. The wide distribution of snail intermediate hosts for F. magna in Montana indicates a potential to spread the parasite to other captive cervids domestic livestock or free-ranging wildlife.

Seasonal transmission of Fasciola hepatica in Montana, USA, with detection of infected intermediate hosts using a DNA-based assay.

A cashmere goat ranch in the Beaverhead Valley, southwest Montana, USA reported acute fasciolosis in March 1992. The ranch was used as a study site to gather seasonal transmission data for the liver fluke, Fasciola hepatica. Testing of snails for infection with a nucleic acid-based assay, and use of tracer sheep at the site has shown the seasonal transmission of the parasite is only in late autumn. The snail responsible for transmission was identified as Lymnaea modicella although another known intermediate host species, Lymnaea bulimoides, was found in one collection at the study site. Tracer sheep used over a 12-month period became infected with F. hepatica while on pasture during a period between September 10 and November 12, 1993. During the 28 months of study, 3072 individual lymnaeid snails were tested for infection. One sample of L. modicella containing 25 snails, collected in August 1994 contained liver fluke ribosomal RNA.

Distribution of potential intermediate hosts for Fasciola hepatica and Fascioloides magna in Montana, USA.

A collection of lymnaeid snails in Montana was made over a 3 year period, in conjunction with a state-wide survey of the distribution of liver flukes in Montana. Collection areas were selected based on reports of infected cattle, sheep or wildlife, and with the intent of covering all geographic regions of the state. Snails were found at all 97 of the locations chosen for collections, with lymnaeids collected at 71 of the locations. The 97 sites were located in 28 of Montana's 56 counties. Nine lymnaeid species were collected, five of which have been reported either as natural or experimental intermediate hosts for Fasciola hepatica or Fascioloides magna. The two snail species most widely distributed over the areas enzootic for the flukes were Lymnaea modicella and Lymanaea caperata.

Efficacy of ivermectin pour-on against Ostertagia ostertagi infection and residues in the American bison, Bison bison.

Sixteen American bison, Bison bison, were artificially infected with 10(5) infective stage larvae of Ostertagia ostertagi on 21 April 1993. At 42 days post-infection eight bison were treated with 0.5% ivermectin pour-on (500 micrograms/kg bodyweight) and eight treated with the carrier only. Bison were necropsied 17 and 18 days post-treatment (21 and 22 June 1993, respectively). Mean (+/- SE) of 5,413 (+/- 1,716) adults and 565 (+/- 305) immature O. ostertagi were recovered at necropsy from bison treated with the carrier. No O. ostertagi were detected in bison treated with ivermectin pour-on. Based on the levels of the ivermectin marker metabolite in liver and adipose tissue 18 days post-treatment, the established bovine withdrawal time of 48 days appears adequate to insure that violative residues do not occur.

Detection of Fasciola hepatica in infected intermediate hosts using RT-PCR.

Fasciola hepatica, the common bile duct fluke, is an economically important parasite of domestic livestock. Current research interest is directed toward an understanding of the parasite's biology at the intermediate host level. To permit study of seasonal transmission patterns and parasite/intermediate host interactions, a fasciolid-specific assay has been developed to detect infected snail vectors. This assay uses the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify specifically a region of F. hepatica small-subunit rRNA, followed by hybridization to an F. hepatica-specific probe. The assay does not cross-react with 2 trematodes outside of the Fasciolidae but does detect Fascioloides magna rRNA. Sequence alignment with additional small-subunit rRNAs shows Fasciolopsis buski would also cross-react with the assay. The detection limit of the assay is 10 fg of fluke total RNA with 5 micrograms of snail RNA added as background. Additionally, the assay detects individual infected snails immediately after miracidial exposure and throughout the parasite's development period.

Enteric coccidia of Cashmere goats in southwestern Montana, USA.

A survey of enteric coccidia was made in a Cashmere goat herd in Montana, USA. Eimerian oocysts were found in 97.2% of 616 fecal samples. Newly weaned wethers and does had higher oocyst counts than yearling wethers. Nine Eimeria species were identified, with Eimeria arloingi, Eimeria ninakohlyakimovae and Eimeria alijevi jointly comprising 88.3% of all oocysts recovered. These three species and Eimeria hirci were present in all specimens examined. Prevalence of the other species was as follows: Eimeria caprina, 88.2%; Eimeria jolchijevi, 70.6%; Eimeria christenseni, 32.4%; Eimeria caprovina, 29.4%; Eimeria apsheronica 26.5%.

Range of heartworm (Dirofilaria immitis) infection in Montana dogs.

The serological presence of heartworm in dogs of Montana was studied in a 3-yr survey. Serum samples were provided by veterinary practitioners throughout Montana or were submitted to the state diagnostic laboratory. Sera from 3,490 dogs were tested using an enzyme-linked immunosorbent assay (ELISA) for circulating adult heartworm antigen. Twenty-four serum samples were positive for heartworm antigen. Two were from dogs that had never been outside the state. Nineteen additional positive dogs, 7 of which presumably had never been out of the state, were reported through correspondence with veterinary practitioners. Because suitable vectors for Dirofilaria immitis exist in Montana, there is potential for propagation of heartworm.

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.