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Comparison by experimental infections in cattle of a Dictyocaulus species occurring naturally in red deer and a dictyocaulus of bovine origin.

Dictyocaulus species larvae were obtained from young red deer which had become infected on pastures considered to be carrying the Dictyocaulus species indigenous to the red deer of Scotland. These larvae were cultured to third stage and transmitted to five bovine calves. Five other bovine calves were infected with third stage Dictyocaulus viviparus larvae of bovine origin. Microscopic appearances of both groups of larvae were indistinguishable and their lengths were similar. Results indicated that the Dictyocaulus species derived from deer induced milder though similar clinical and pathological responses in cattle than did the D viviparus derived from cattle. It was concluded that there are strains of different pathogenicity within the species D viviparus, that the deer derived Dictyocaulus species was a strain of D viviparus, and that the hazards to animal health associated with infection by D viviparus in farming systems where red deer and cattle may graze alternately are likely to be acceptable.

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Species-specific polymerase chain reaction for the differentiation of larvae from Dictyocaulus viviparus and Dictyocaulus eckerti.

Using substantial interspecific differences between the second internal transcribed spacer (ITS2) region within the rDNA gene of Dictyocaulus eckerti and Dictyocaulus viviparus a species-specific PCR was developed to distinguish between lungworm larvae of the two species from fallow deer and cattle. It was found that the method of DNA extraction was crucial for the sensitivity of the PCR. With serial dilutions of DNA extracted from 10,000 larvae the ITS2 fragment could be amplified from all dilutions down to a calculated amount of DNA equivalent to one larva. Using lower numbers of larvae, DNA from at least 100 larvae was necessary for a successful amplification. From this extraction a species-specific polymerase chain reaction (PCR) product was generated with a calculated amount of DNA equivalent to 33 larvae, whereas amplification of further diluted DNA was not successful. However, in a direct PCR single larvae could be detected after direct PCR amplification without preceding DNA extraction.

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[Comparative molecular biologic characterization of Dictyocaulus viviparus and Dictyocaulus eckerti].

For a comparative characterization of the lungworm species D. viviparus and D. eckerti which is not generally accepted as a separate species, the restriction fragment length polymorphism (RFLP) of the PCR amplified ribosomal second internal transcribed spacer (ITS2) and their sequences of both species have been examined. Ribosomal ITS2 DNA was amplified from genomic DNA of individual worms using primer that correspond to the conserved 3' and 5' ends of the ITS2 flanking 5.8S and 28S regions of Caenorhabditis elegans. PCR products were digested with restriction endonucleases AluI, NspI, SspI, BclI and MseI and separated electrophoretically on a 1% agarose gel. Each restriction enzyme produced a species specific fragment length pattern. PCR products were cloned into pCRII and sequenced. The length of the ITS2 varied between 403 (D. viviparus) and 481 bases (D. eckerti) with a GC content ranged from 25 to 33%. Intraspecific variations were low (0-1.5%). Interspecific differences occur at 112 bases. The sequence homology between D. viviparus and D. eckerti was found with 76.7%. ITS2 sequence differences between D. viviparus and D. eckerti by far exceeded intraspecific variations. Therefore both methods showed distinct differences between the lungworm species examined, thus proving that D. eckerti is correctly described as a separate species. Both species occur in deer and specific primers have been designed for both species that will be used in prevalence studies to investigate the actual role of deer in the transmission of D. viviparus to cattle.

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Molecular identification and prevalence of Dictyocaulus spp. (Trichostrongyloidea: Dictyocaulidae) in Swedish semi-domestic and free-living cervids.

Lungs of 102 roe deer (Capreolus capreolus), 136 moose (Alces alces), 68 fallow deer (Dama dama), and six red deer (Cervus elaphus) were examined during hunting seasons from 16 September 1997 to 1 March 2000. The aim was to determine the species composition and prevalence of Dictyocaulus lungworms in these hosts in Sweden. Worms were identified following polymerase chain reaction (PCR) amplification of the internal transcribed spacer of ribosomal DNA (ITS2), followed by hybridization with four species-specific oligonucleotides. In addition, 50 lungworms from five reindeer (Rangifer tarandus) from Norway were similarly analyzed. A total of 399 worms were recovered and analyzed representing a range of 29-128 worms per host species. All specimens from roe deer were identified as Dictyocaulus capreolus, whereas those from red deer and reindeer were identical with D. eckerti. From moose, 73 (81.1%) of the worms were identified as D. capreolus whereas 17 (18.9%) were D. eckerti. The ITS2 sequence of fallow deer lungworms differed significantly when compared with the ITS2 of D. viviparus, D. capreolus, and D. eckerti. This indicated that fallow deer in Sweden may be infected with a new genotype of Dictyocaulus spp. Consequently, a specific probe designed for the ITS2 from this Dictyocaulus sp. hybridized exclusively with samples from lungworms of fallow deer. Interestingly, no D. viviparus were found in any of these hosts. The prevalence of infection in each host was as follows: D. capreolus in roe deer (14.7%) and moose (10.6%); D. eckerti in moose (0.7%) and red deer (33.3%); and Dictyocaulus sp. in fallow deer (10.3%). Regardless of lungworm species, the overall prevalence of Dictyocaulus spp. in these hosts was 12.2%. Prevalence between male and female animals and among the different age groups did not differ significantly. Finally an enzyme linked immunosorbent assay (ELISA) specific for patent D. viviparus infection in cattle was utilized to analyze lung tissue fluids from infected animals. All samples from roe deer, red deer, and fallow deer were negative in the ELISA. However, three out of twelve (25%) samples from moose and 17 of 40 (43%) samples from cattle were positive. This indicated that moose anti-D. capreolus antibodies recognized the D. viviparus antigen and that anti-cattle immunoglobulin cross-reacted with moose antibodies.

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ITS2 sequences of Dictyocaulus species from cattle, roe deer and moose in Sweden: molecular evidence for a new species.

Total DNA was isolated from adult lungworms of the genus Dictyocaulus, collected from cattle, moose (Alces alces) and roe deer (Capreolus capreolus) in Sweden. The second ribosomal internal transcribed spacer was amplified with PCR, and DNA sequences were determined from nine individual worms that all came from different hosts in order to avoid analysis of siblings. The sequence data obtained were aligned and compared with similar data derived from German lungworm isolates from cattle and fallow deer (Cervus dama). These analyses clearly showed that specimens of the cattle lungworm, Dictyocaulus viviparus, were almost identical irrespective of their geographical origin. However, when the second internal transcribed spacer sequence of D. viviparus was compared with that of lungworms from moose and roe deer, major differences were noticed. Although lungworms collected from these cervids had identical second internal transcribed spacer sequences, they proved to be genetically different from Dictyocaulus eckerti of German fallow deer, displaying a 66.5% similarity. In an evolutionary tree, inferred by maximum likelihood analysis, the Dictyocaulus species from cattle and wild cervids clustered as compared with Dictyocaulus filaria from sheep. The study has thus demonstrated that A. alces and C. capreolus in Sweden are parasitised with a Dictyocaulus species that is different from D. viviparus and D. eckerti, indicating that we are dealing with a new species in moose and roe deer.

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Phylogeny of Dictyocaulus (lungworms) from eight species of ruminants based on analyses of ribosomal RNA data.

In this study, we conducted phylogenetic analyses of nematode parasites within the genus Dictyocaulus (superfamily Trichostrongyloidea). Lungworms from cattle (Bos taurus), domestic sheep (Ovis aries), European fallow deer (Dama dama), moose (Alces alces), musk ox (Ovibos moschatus), red deer (Cervus elaphus), reindeer (Rangifer tarandus) and roe deer (Capreolus capreolus) were obtained and their small subunit ribosomal RNA (SSU) and internal transcribed spacer 2 (ITS2) sequences analysed. In the hosts examined we identified D. capreolus, D. eckerti, D. filaria and D. viviparus. However, in fallow deer we detected a taxon with unique SSU and ITS2 sequences. The phylogenetic position of this taxon based on the SSU sequences shows that it is a separate evolutionary lineage from the other recognized species of Dictyocaulus. Furthermore, the analysis of the ITS2 sequence data indicates that it is as genetically distinct as are the named species of Dictyocaulus. Therefore, either this taxon needs to be recognized as a new species, or D. capreolus, D. eckerti and D. viviparus need to be combined into a single species. Traditionally, the genus Dictyocaulus has been placed as a separate family within the superfamily Trichostrongyloidea. The present molecular phylogenetic analyses support the placement as a separate family, but the current data do not support the placement of the Dictyocaulidae within the Trichostrongyloidea without a reassessment of the placement of the superfamily Strongyloidea. While D. eckerti has been regarded as the one and only lungworm species of cervids, this study showed that 4 host species including 3 members of Cervidae (moose, reindeer, red deer) and 1 Bovidae (musk ox) were infected with this parasite. Host ranges of D. viviparus (cattle), D. filaria (sheep) and D. capreolus (moose and roe deer) were more restricted. No clear pattern of co-evolution between the dictyocaulid taxa and their bovid and cervid hosts could be determined.

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Baermannization of Dictyocaulus spp. from faeces of cattle, sheep and donkeys.

Faeces were collected per rectum from calves infected with Dictyocaulus viviparus (D.v.), from lambs infected with Dictyocaulus filaria (D.f.) and donkeys infected with Dictyocaulus arnfieldi (D.a.). In one experiment, the influence of storage temperature before Baermannization was investigated. Recovery rate for D.v. was approximately 80% after 24 h at 4 degrees C or 16 degrees C but only 40% at 20 degrees C. After two days at 20 degrees C the rate had fallen to 20%. Recovery rates for D.f. decreased so markedly during the first 12 h at 4, 16 and 20 degrees C that storage can not be recommended. Losses in the recovery rates of D.a. appeared insignificant after 48 h at 4 degrees C but not at 16 degrees C and 20 degrees C. In experiment II the time taken for larvae to emerge from a 10-g sample as well as the sedimentation time in Baermann tubes was investigated. The bulk of the D.v. larvae remained in the faeces for about 10 h whereas D.f. larvae emerged during the first few hours. D.a. larvae were intermediate in this respect. Sedimentation of the bulk of larvae from all three species took place within a few hours with the modified Baermann technique used.

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Identification of Dictyocaulus spp. in ruminants by morphological and molecular analyses.

Lungworms of the genus Dictyocaulus from cattle, roe deer, and moose in Sweden were subjected to morphological and molecular analyses. The objectives of the study were to investigate whether mixed or monospecific Dictyocaulus infections occur in Swedish cattle and whether wild cervids may act as reservoirs. The morphological characters examined were thickness and shape of the buccal capsule wall (BCW) and total spicular length (TSL). Morphometry was also done on the total body length, and BCW thickness and length. In the molecular identification, we used a PCR-linked hybridization assay to probe worm DNA with species-specific oligonucleotide probes to the second internal transcribed spacer (1TS2). The results showed that the BCW shape was the most reliable morphological character for identification. Significant differences were observed in this character, but an overlap occurred between lungworms from each of the host species. With the hybridization assay, all lungworms from cattle were identified as D. viviparus, whereas those from roe deer represented a novel Dictyocaulus species demonstrating that each host had a monospecific lungworm infection. In moose, 61 (78.2%) worms belonged to the new species and 17 (21.8%) were D. eckerti. This study shows the usefulness of hybridization assay as an epidemiological tool for the specific identification of lungworms of cattle and wild cervids.

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Dictyocaulus capreolus n. sp. (Nematoda: Trichostrongyloidea) from roe deer, Capreolus capreolus and moose, Alces alces in Sweden.

Dictyocaulus capreolus n. sp. recovered from roe deer, Capreolus capreolus and moose, Alces alces in Sweden is described and figured. Morphological studies revealed the new species to be closest to D. eckerti and D. africanus on the basis of mouth shape, all three species having an elongate mouth opening. The other species of the genus, including D. viviparus, all have a circular to oval mouth opening. Dictyocaulus capreolus n. sp. can be distinguished from D. eckerti and D. africanus on the basis of the morphology of the buccal capsule and the bursa. These morphological studies support earlier evidence of the presence of a new species of Dictyocaulus in roe deer and moose that could be distinguished from D. eckerti and D. viviparus using either a PCR-linked hybridization assay or image analysis software to study the dimensions of the buccal capsule.

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Dictyocaulus species: cross infection between cattle and red deer.

AIM: To discover whether cross infection between red deer (Cervus elaphus) and cattle is possible with either a bovine isolate of the cattle lungworm, Dictyocaulus viviparus, or with a cervine isolate of the lungworm, Dictyocaulus eckerti which is thought to be maintained primarily in deer. METHOD: Twelve cattle and 12 red deer were reared parasite-free from birth. At 3-4 months of age, half of each species (n=6) were experimentally infected with D. viviparus and the other half with D. eckerti. The course of infection was monitored for 34 days, after which the animals were slaughtered and the lungs removed to assess levels of infection. RESULTS: Faecal larval counts demonstrated that patent Dictyocaulus infections occurred in all groups. At necropsy, adult worms were found in the lungs in all groups except the cattle that were infected with D. eckerti. The largest numbers of adult worms were found in the red deer infected with D. eckerti. CONCLUSION: It was demonstrated that both cattle and red deer could be infected with either D. viviparus or D. eckerti. However, D. eckerti larvae that originated from deer established more successfully in deer and D. viviparus larvae that originated from cattle established more successfully in cattle.

Journal Article↗

Efficacy of Michel's 'dose and move' system against Dictyocaulus viviparus infections in cattle using moxidectin as anthelmintic.

Two grazing experiments were performed to study the effect of moxidectin in a 'dose and move' system on Dictyocaulus viviparus infections in calves. In the first experiment, three groups of four calves were experimentally infected with 20 larvae of D. viviparus 7 weeks before moxidectin treatment of two of these groups. A sufficient suppression of Dictyocaulus infections was observed in a 'dose and move' group, but also in a group which stayed on contaminated pasture after treatment. In contrast, high faecal larval counts and lungworm disease were observed in July-August in a non-treated pasture control group. Development of immunity against lungworm was sufficient in all three groups, on pasture. In the second experiment, four out of 26 calves, including two groups of six calves and four tracer calves, were experimentally infected with 20 larvae of D. viviparus 7 weeks before moxidectin treatment combined with a move of one of the groups. No lungworm disease was observed in this 'dose and move' group. In a pasture control group high faecal larval counts and severe clinical disease were observed in August-September and one calf had to be euthanized. Although a mean burden of 129 immature lungworms was present at the time of treatment, development of immunity appeared to be low in the 'dose and move group'. In contrast, high levels of immunity had developed in the pasture control group.

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Parasitic bronchitis in goats and the possible use of Dictyocaulus filaria vaccine for its control.

Parasitic bronchitis is widely prevalent in migratory flocks of small ruminants in the northwest Himalayan regions of India. The prevalence data collected from 5554 goats, maintained in 31 villages in different agroclimatic regions of the Himalayas, showed that the prevalence of the disease in goats varied from 18.7 to 47.6% with an overall prevalence of 21.8%. Interestingly, 27.6% of goats maintained at an altitude of 2700-3900 m above mean sea level in Kargil (Jammu and Kashmir), where the climate is cold and dry for the major part of the year, were positive for the lungworm infections. The common lungworms observed were Dictyocaulus filaria, Protostrongylus rufescens, Varestrongylus pneumonicus and occasionally Muellerius spp. The kids were more susceptible to lungworm infections than adult goats. In experimental studies, it was seen that goats were more susceptible to Dictyocaulus filaria infection than sheep and two vaccine doses comprising 1000 and 2000 gamma-attenuated D. filaria (ovine strain) infective larvae conferred 97% protection in male Beetal kids against a homologous challenge dose of 4200 normal D. filaria larvae. The importance of simultaneous control of the disease in goats and sheep is discussed.

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Vaccine development and diagnostics of Dictyocaulus viviparus.

Parasitic bronchitis is a serious disease of cattle and is caused by the nematode, Dictyocaulus viviparus. For over 30 years, a radiation-attenuated larval vaccine has been used for prevention of this disease. This vaccine has been used with considerable success in the UK and parts of Western Europe, however, it has several disadvantages. It has a short shelf-life and the vaccine has to be produced annually necessitating the use of donor calves. Following vaccination, calves must receive further boosting from natural challenge to maintain protective immunity. Sales of the irradiated larval vaccine have decreased dramatically since the 1970s. This is thought to be due to increased reliance of farmers on anthelmintic programmes to control lungworm infection. It is possible that, under certain circumstances, these programmes do not allow sufficient parasite exposure to stimulate protective immunity to further Dictyocaulus challenge. This is borne out by the recent documented increase in the number of outbreaks of parasitic bronchitis in the UK. A stable vaccine against D. viviparus that is capable of stimulating a more prolonged immunity would be beneficial. Recent research has been directed at identification and isolation of components thought to be involved in parasite survival in the host and examination of their potential as vaccine candidates. One of these components is acetylcholinesterase (AChE), an enzyme secreted by adult worms. This review describes the development of the secreted AChE as a vaccine candidate, as well as documenting recent developments in the immunodiagnosis of D. viviparus.

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Dictyocaulus viviparus in calves: quantitation of antibody activities in sera and respiratory secretions by immuno-enzymatic analysis.

An enzyme-linked immunosorbent assay (Elisa) which is in detail described here has been adapted to trace antibody production in different fluids from infected calves. Seven calves were infected both with Dictyocaulus viviparus, and with several intestinal nematodes. Antibodies against an antigen prepared from Dictyocaulus viviparus adult worms were first detected during the 5th week post-infection, both in serum and nasal secretions, and were also observed in bronchial washings of the same calves slaughtered 53 days post-infection. IgA antibodies were never evident in the serum but were found in limited quantities in nasal secretions. In bronchial washings at slaughtering, a significant part of antibody activity is accounted for by the local IgA production.

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