Genetic evidence against a morphologically suggestive conspecificity of Dermacentor reticulatus and D. marginatus (Acari:Ixodidae).

International Journal for Parasitology 25: 1413-1419. Dermacentor reticulatus and D. marginatus exhibit overlapping phenotypes. The possibility of conspecificity was investigated on the nucleotide level by comparing DNA sequences of the second internal transcribed spacer (ITS 2) of the rDNA gene. The inter-specific polymorphism was more than 20-times greater than the intra-specific polymorphism of 3 D. reticulatus strains of different geographic origins. Furthermore, the degree of polymorphisms between D. reticulatus and D. marginatus was found to be of the same order of magnitude as that between D. andersoni and D. variabilis, for which separate species status is accepted. These genomic findings do not support a possible conspecificity of D. reticulatus and D. marginatus.

Genetic evidence suggests that Psoroptes isolates of different phenotypes, hosts and geographic origins are conspecific.

The second internal transcribed spacer of the rRNA gene was characterised in 15 Psoroptes isolates collected from the ears or bodies of rabbits, goats, sheep and cattle originating from four continents. Morphologically, the isolates were differentiated as Psoroptes cuniculi, Psoroptes ovis and Psoroptes cervinus. Genotypically, the isolates were highly homogeneous, except for the existence of different rDNA classes. In view of previous phenotypic data, a possible conspecificity of these species is proposed.

Molecular analyses suggest monospecificity of the genus Sarcoptes (Acari: Sarcoptidae).

To clarify the taxonomic status of mites of the genus Sarcoptes, the second internal transcribed spacer (ITS-2) of the rRNA gene, as well as phenotypic characters, were investigated in 23 isolates from nine host species in four continents. Phenotypic differences among isolates were observed, but the range of variation within each isolate precluded the differentiation of individual mites. Genotypically, there was no delimitation between distinct genotypic groups and no correlation with host species or geographic origin was evident. These results support the conspecificity of the mites investigated and confirm the view that the genus Sarcoptes consists of a single, heterogenous species.

Detection of a new pathogenic Babesia microti-like species in dogs.

Small babesiae in dogs are generally considered to belong to Babesia gibsoni. Here we describe the genotypic characterisation of small piroplasms found in the blood of a dog which suffered from clinical babesiosis. Pairwise identities as well as distance, parsimony and maximum likelihood analyses of the 18S rDNA clearly demonstrated that this isolate was only distantly related to the other canine piroplasms characterised genetically so far, including B. gibsoni. It was more closely related to B. microti, B. rodhaini, and Theileria equi. It is concluded that the small canine piroplasms described in this study represent a hitherto unknown species and that the fauna of piroplasms occurring in dogs is more diverse than assumed so far.

Relationships between species of the Rhipicephalus sanguineus group: a molecular approach.

The biosystematic status of species belonging to the Rhipicephalus sanguineus group is difficult to determine by phenotypic methods and has been subject to frequent revisions and ongoing debate. Yet, the differentiation of species within this complex is of clinical importance especially in veterinary medicine due to different host specificities of the transmitted pathogens. In order to generate quantifiable and comparable data independent of environmental influences, a section of the second internal transcribed spacer (ITS2) of the rRNA gene was sequenced for 6 species belonging to the Rhipicephalus sanguineus complex: Rhipicephalus sanguineus sensu stricto, R. turanicus, R. rossicus, R. pumilio, R. pusillus, and R. camicasi. Rhipicephalus sanguineus s. str. and R. turanicus as well as R. pumilio and R. rossicus were found to have a close genetic relationship compatible with possible conspecificities. Conversely, R. pusillus was found to have the most distant relationship to the other species.

Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle.

The reservoirs and the routes of transmission of Enterocytozoon bieneusi are still unknown. In humans, it is the most commonly found microsporidial species. It has also been found repeatedly in pigs, too. The first detection of E. bieneusi in cattle is reported herein. Two distinct genotypes were characterized and compared with 4 other genotypes from humans, 6 from pigs, and 1 from a cat. From these 13 E. bieneusi genotypes known to date, 25 polymorphic sites could be identified in the internal transcribed spacer of the rRNA gene. The spectrum of polymorphisms within and between each of the 4 host species indicates a close relationship between E. bieneusi strains from humans and pigs, whereas those from cattle are more distantly related. The data suggest the absence of a transmission barrier between pigs and humans for this pathogen.

[Endemic risk of Babesia canis by Dermacentor reticulatus in Germany. An epidemiologic study]. / Endemisierungsrisiko von Babesia canis durch Dermacentor reticulatus in Deutschland. Eine epidemiologische Studie.

During the period of January 1995 to January 1997, Babesia canis infections were diagnosed in 127 dogs, 65 of them had stayed in foreign countries where Dermacentor reticulatus is endemic, namely France, Austria and Hungary, or in the area Offenburg/Kehl/Freiburg i. Breisgau (Germany). Two dogs being positive for Babesia never had left Germany before and had not been in the region Offenburg/Kehl/Freiburg i. Breisgau. Concurrent tick infestations were reported for all of the 67 dogs. For 10 dogs which had travelled to Hungary and France the ticks were diagnosed as D. reticulatus. In Germany, ecological conditions are suitable for D. reticulatus and Babesia canis. Therefore, as a consequence of the spreading of these parasites the emergence of new endemic foci has to be expected.

Evidence for the reproductive isolation of Dermacentor marginatus and Dermacentor reticulatus (Acari: Ixodidae) ticks based on cross-breeding, morphology and molecular studies.

The species status of Dermacentor marginatus and Dermacentor reticulatus was evaluated by scanning electron microscope (SEM) examination of adult ticks, cross-breeding experiments and molecular biological analysis of eggs derived from transspecific pairings. The SEM investigations including the morphometric quantification of phenotypic features resulted in an unequivocal differentiation of adult D. marginatus and D. reticulatus ticks. The cross-breeding experiments demonstrated that irrespective of whether female ticks of both species were applied with con- or transspecific male ticks or without males to sheep, they engorged and laid eggs. The larvae, however, developed only in eggs which originated from conspecific matings. A nested polymerase chain reaction (PCR) of the second internal transcribed spacer (ITS2) using the DNA of eggs from transspecific pairings and sequencing of the PCR products revealed two different genotypes. The genotypes of eggs originating from D. marginatus and D. reticulatus females of these pairings differed. However, the eggs deposited by D. marginatus always possessed the same two genotypes as did the eggs produced by D. reticulatus. These results argue for a strict reproductive isolation of D. marginatus and D. reticulatus and, therefore, for a separate species status.

Effect of temperature and humidity on longevity of unfed adults and on oviposition of engorged females of Dermacentor reticulatus (Ixodidae).

The effect of ambient temperature and relative humidity on longevity of unfed adult ticks and on oviposition of engorged female ticks of Dermacentor reticulatus was examined. The 50% mortality time of unfed adult ticks, incubated at 5 degrees C, 10 degrees C, 20 degrees C and 27 degrees C and humidities of 15%, 50% and 100%, increased irrespective of their sex with rising relative humidity and decreasing temperature. It was longest at 5 degrees C and 100% r.h. being 617.8 days and shortest at 27 degrees C and 15% r.h. amounting only to 33.6 days. All unfed ticks survived an incubation of 150 days at -10 degrees C and 15%, 50% and 100% r.h. as well as an exposure of 180 days at 0 degrees C and 100% r.h. At 0 degrees C the percent survival was 75% for the males and 40% for the females at 50% r.h. but 100% for males and 95% for the females at 15% r.h. after exposure of 180 days. Oviposition of engorged females occurred at a temperature range of 10-27 degrees C, at 10 degrees C merely at 50% and 100% r.h. The percentage of ovipositioning females amounted to at least 80% apart from 10 degrees C and 50% r.h. being only 10%. The egg index and the egg conversion index as well as the larvae index and the larvae conversion index generally increased with rising temperature and relative humidity. The indices were lowest at 10 degrees C and 50% r.h. and were highest at 20 degrees C and 27 degrees C at 100% r.h. The reproduction capability of engorged female ticks, held at -10 degrees C, 0 degrees C and 5 degrees C and 15%, 50% and 100% r.h. and transferred to 20 degrees C and 95% r.h., persisted for 10 weeks at -10 degrees C and for 2 weeks at 0 degrees C at the most, but for at least 20 weeks at 5 degrees C. There were no significant differences of the preoviposition periods, egg indices and larvae indices as well as of the minimum embryonic developmental times and hatching rates with regard to the preceding incubation conditions and the length of the ticks' exposure.

Effect of temperature and humidity on egg hatch, moulting and longevity of larvae and nymphs of Dermacentor reticulatus (Ixodidae).

Larvae of Dermacentor reticulatus hatched at a temperature range of 20-34 degrees C, but a high egg hatch success of > 90% was only achieved at 20 degrees C and 27 degrees C and 100% r.h. The developmental capability of the eggs at low or freezing temperatures persisted up to 3 weeks at -10 degrees C and up to 8 weeks at 5 degrees C. The 50% mortality period of unfed larvae and nymphs increased with rising humidity and was longest at 5 degrees C and 100% r.h., amounting to 83.5 days for larvae and 108 days for nymphs. Engorged larvae and nymphs only moulted in the temperature range of 10-27 degrees C. The percent moulting success was low at 10 degrees C and increased with rising ambient humidity at 20 degrees C and 27 degrees C reaching more than 90% at 100% r.h. After incubation at low or freezing temperatures and subsequent transfer to 20 degrees C and 100% r.h. engorged larvae did not moult after exposure to -10 degrees C, but they tolerated 0 degrees C and 5 degrees C up to 3 and 10 weeks, respectively, and developed into nymphs. Engorged nymphs moulted into adults even after preceding incubation at -10 degrees C, their metamorphosis capability persisted for a maximum of 4 and 17 weeks at 0 degrees C and 5 degrees C, respectively.

[Imported parasites in dogs: Dirofilaria repens and Dipetalonema reconditum]. / Eingeschleppte Parasiten bei Hunden: Dirofilaria repens und Dipetalonema reconditum.

Between June 1993 and May 1996 infections with filariae were diagnosed in 80 dogs. Five animals were infected with Dirofilaria repens and three other dogs with Dipetalonema reconditum. One of the D. reconditum positive dogs was also infected with Dirofilaria immitis. An epidemiological analysis of the cases was possible for three D. repens and two D. reconditum infected dogs. Two of the dogs with D. repens infections had been imported from Italy or Greece, the third dog had been taken by its owner to Hungary and former Yugoslavia. The dogs infected with D. reconditum were imported from Corse or Spain. Regarding the other 72 dogs with filariae, 45 animals were infected with D. immitis. For 27 dogs being negative in the D. immitis ELISA, microfilariae have been demonstrated. However, a histochemical species differentiation of the microfilariae was not possible because fresh blood smears were not available.

[Dermacentor ticks in France and Germany. Molecular biological differences in species, ecology and epidemiological implications]. / Dermacentor-Zecken in Frankreich und Deutschland. Zur molekularbiologischen Artdifferenzierung, Okologie und epidemiologischen Bedeutung.

Dermacentor reticulatus and D. marginatus, two ixodid tick species occurring in France and Germany, exhibit morphologically overlapping phenotypes. The results of sequence analyses of the hypervariable rDNA gene region ITS 2 do not support conspecifidity. As in France, D. reticulatus has to be characterized as an epidemiologically effective vector of Babesia canis in Germany as well, with the endemic area being located at Kehl/Offenburg/Lahr/Emmendingen/Freiburg in Breisgau. Ecological investigations indicate that a spreading of D. reticulatus and, as a possible consequence, that of B. canis in Germany has to be expected.

[The brown dog tick, Rhipicephalus sanguineus (Ixodidae), in Germany: an epidemiologic study and control measures]. / Die Braune Hundezecke, Rhipicephalus sanguineus (Ixodidae), in Deutschland: eine epidemiologische Studie und Bekämpfungsmassnahmen.

Between January and December 1995 infestations with Rhipicephalus sanguineus were established in 22 dogs in Germany. Out of these 22 animals 2 originated from the endemic area of this tick species, Spain and the Dominican Republic and 11 dogs accompanied their owner to the Mediterranean countries, 5 to Italy, 3 to France, 2 to Spain and 1 animal to Greece. Six dogs had never left Germany and with 3 dogs the way of infestation could not be reconstructed. In 16 cases an abundant occurrence of R. sanguineus in homes was reported, whereby in 5 homes also men were infected. An infection with Ehrlichia canis was diagnosed in the dog which originated from Spain and in one animal which had never left Germany.

Genotypic status of Babesia microti within the piroplasms.

For resolution of the controversial taxonomic status of Babesia microti in relation to other Babesia and Theileria spp. a phylogenetic analysis of an American and a German B. microti strain was performed on the basis of sequences of the small-subunit rRNA gene (rDNA) using distance-matrix, maximum-parsimony, and maximum-likelihood algorithms. Both B. microti isolates clearly separated from a group containing other Babesia spp. as well as from a second group consisting of Theileria spp. Interestingly, the B. microti isolates clustered in a monophyletic group together with other piroplasm species of unclear taxonomic status, B. rodhaini, and a recently described small canine piroplasm species. These results support the existence of a third taxonomic entity of equal rank besides the Babesiidae and Theileriidae.

Diagnostic DNA amplification from individual tick eggs, larvae and nymphs.

The species of single tick eggs, larvae and nymphs was determined by PCR amplification and characterization of the hypervariable, second transcribed spacer (ITS2) of the multicopy ribosomal RNA gene (rDNA). Engorgement of larvae and nymphs did not preclude species identification. The method is generally applicable for ixodid and argasid ticks and can be used for epidemiological studies requiring the identification of individuals from pre-adult stages.

Genetic differentiation of mites of the genus Chorioptes (Acari: Psoroptidae).

In an effort to clarify the species status of mites of the genus Chorioptes the second internal transcribed spacer of the rRNA gene was characterized in 14 isolates from cattle, horse, sheep and llama of different geographic origins. The genotypes segregated into two clearly separated groups of DNA sequences. In addition, two phenotypes could be distinguished by the lengths of the outer opisthosomal setae of male adults which had previously been designated as Chorioptes bovis and Chorioptes texanus. The bipartite division of genotypes and phenotypes correlated completely in all isolates. Nine out of ten cattle isolates from three continents were determined to be C. texanus including the first description in Europe and Northern America. Chorioptes texanus appears to have a wider geographic distribution than previously known. Chorioptes bovis was found in four different host species. The apparent lack of host specificity of both species implicates a potential that mites are dispersed freely in a wide range of hosts and this might have contributed to the wide geographic distribution of these species.

Studies on the critical water mass and the rehydration potential of unfed adult Dermacentor marginatus and D. reticulatus ticks (Acari: Ixodidae).

In unfed adult Dermacentor marginatus and D. reticulatus ticks survival and capability to restore water balance after loss of high percentages of exchangeable body water were investigated. Furthermore, it was examined how frequently dehydrated ticks of these species were able to rehydrate by uptake of atmospheric water vapour. The critical water mass, defined as the water mass remaining in a tick at the nonambulatory state, differed between light and heavy weight groups and averaged 62.4 and 55.8%, respectively, of the total body water of fully hydrated ticks in females, and 54.4 and 51.1%, respectively, in males of D. marginatus. In D. reticulatus, the corresponding figures were 55.9 and 54.7% in females and 52.1 and 52.7% in males. All ticks survived dehydration to 50, 75 or 100% of the critical water mass, and 96.7% of the D. marginatus ticks and 95.8% of the D. reticulatus ticks compensated water losses during subsequent incubation at 95% relative humidity (r.h.) and 20 degrees C. Unfed females and males of both Dermacentor spp. were capable to balance water loss very frequently over a period of several months. When ticks were repeatedly dehydrated at 0% r.h. for 7 days and rehydrated at 95% r.h. and 20 degrees C, females and males of D. marginatus reached the 50% mortality after 22 and 29 cycles of de- and rehydration, respectively, during 211 and 285 days, respectively. In D. reticulatus, 50% of females and males survived 23 and 17 cycles, respectively, during 248 and 186 days, respectively. Rehydration weights were as high or even higher as those of ticks kept at permanent 95% r.h.

Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs.

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene were characterized in eight Babesia canis isolates of differing geographic origin, vector specificity, and pathogenicity to dogs. The genotypes determined by sequencing segregated into three clearly separated groups close to or near the species level and correspond to the previously proposed subspecies B. canis canis, B. canis vogeli, and B. canis rossi. The three genotypes can be distinguished by Sau96I digestion of the polymerase chain reaction (PCR)-amplified rDNA target.