Validity of species status of the parasitic mite Otodectes cynotis.

A combined molecular and phenotypic approach was used to determine whether ear mites of the genus Otodectes (Acari: Psoroptidae) belong to a single species. The second internal transcribed spacer (ITS 2) of the rDNA of 16 isolates from 11 cats, two dogs, one arctic fox and two ferrets originating from four different continents was characterized. In addition, mites from dog, cat and arctic fox were investigated morphologically. Sequence comparisons revealed five different, but closely related genotypes which did not segregate according to host species or geographical origin. Morphologically, mites of the three host species did not differ significantly in their body or leg sizes. These investigations support the view that ear mites of the genus Otodectes from different hosts and geographical origins belong to a single species, Otodectes cynotis (Hering).

Zoonotic potential of Enterocytozoon bieneusi.

The reservoirs and the modes of transmission of the most frequent microsporidial species in humans, Enterocytozoon bieneusi, are still unknown. We have examined fecal samples of 26 humans and 350 animals from 37 species to find 18 samples containing this parasite from humans, cats, pigs, cattle, and a llama. Genotypic characterization of the internal transcribed spacer of the rRNA gene resulted in 14 different genotypes, 6 of them previously undescribed. Phylogenetic analysis revealed the lack of a transmission barrier between E. bieneusi from humans and animals (cats, pigs, and cattle). Thus, E. bieneusi appears to be a zoonotic pathogen.

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.

Taxonomic reconsideration of the genus Chorioptes Gervais and van Beneden, 1859 (Acari: Psoroptidae).

The present review of the literature on mites of the genus Chorioptes Gervais and Van Beneden, 1859 argues for a support of the validity of C. bovis (Hering, 1845) and C. texanus Hirst, 1924 based on biological, morphological and molecular genetic studies. However, the validity of three further species. C. crewei Lavoipierre, 1958, C. mydaus Fain, 1975 and C. panda Fain and Leclerc, 1975, is regarded as questionable because discriminations of mites, which were described as isolated cases only, were based on morphological features while transfer or cross-breeding studies were not done.

Studies on survival and water balance of unfed adult Dermacentor marginatus and D. reticulatus ticks (Acari: Ixodidae).

The water content, the survival time at various relative humidities (r.h.) and the critical equilibrium activity of unfed adult Dermacentor marginatus and D. reticulatus ticks were investigated at a constant temperature of 20 degrees C. It was also examined whether these ticks use liquid water to compensate water loss. Both Dermacentor spp. showed no significant differences in water content in relation to body mass. The mean water content of D. marginatus and D. reticulatus was 54.6% and 54.7%, respectively, in females and 56.3% and 57.0%, respectively, in males. The survival time of unfed adults prolonged with decreasing saturation deficits. On average, males survived longer than females and D. marginatus ticks survived mostly longer than D. reticulatus ticks. The 50% mortality period ranged between 40 d at 33% r.h. and 420 d at 95% r.h. in D. marginatus, and between 43 d at 33 r.h. and 366 d at 95% r.h. in D. reticulatus. The critical equilibrium activity of unfed adults was estimated to be 0.84 for both species and was independent of sex. When dehydrated adult D. marginatus and D. reticulatus ticks were offered liquid water, only a few slightly gained weight while most further lost weight. Liquid water was not attractive for dehydrated or non-dehydrated ticks and drinking was not observed. After submerging in water for 2 d, most of the dehydrated ticks had gained weight.

Species of the genus Psoroptes (Acari: Psoroptidae): a taxonomic consideration.

The biosystematic status of mite species belonging to the genus Psoroptes Gervais, 1841 is difficult to determine by phenotypic methods and has been subject to taxonomic revisions and ongoing debate. At present, the existence of five species, P cuniculi (Delafond, 1859), P. ovis (Hering, 1838). P. equi (Hering, 1838), P. cervinus Ward, 1915 and P. natalensis Hirst, 1919, is generally accepted. This classification is based mainly on the host species, the localization of the mites on their hosts and morphological characters of male mites. However, a critical review of the literature indicates that the features used to discriminate between the five species are not unequivocal: (a) the localization of mite populations on host animals is not completely strict, (b) the lengths of the outer opisthosomal setae of male mites, which are the main morphological features used for species discrimination, overlap between the five postulated species, and (c) host specificity cannot be deduced from results of transfer experiments. Rather, conspecificity of the members of the genus Psoroptes has to be presumed which is supported by molecular genetic analyses. On these grounds and on rules of priority P. cervinus Ward, 1915, P. cuniculi (Delafond, 1859), P. natalensis Hirst, 1919 and P. ovis (Hering, 1838) are seen as synonyms of P. equi (Hering, 1838).

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.

The application of a lentiviral vector for gene transfer in fetal human hepatocytes.

BACKGROUND: The applications of traditional retroviral vectors are limited because proviral integrations into the host genome require DNA synthesis. Lentiviruses are considered to be advantageous because of their ability to infect non-dividing cells. METHODS: To demonstrate the potential of lentiviral vectors, we used a human immunodeficiency virus (HIV)-1 virus encoding the green fluorescence protein (GFP) to infect fetal human hepatocytes. GFP-expressing cells were transplanted into the liver of Balb/C SCID mice via intrasplenic injection. RESULTS: Primary fetal hepatocytes incorporated the GFP reporter with high (30-40%) efficiency. A cell line derived from human fetal liver (HFL) exhibited similar transduction efficiency to the lentiviral vector. To demonstrate the relationship between lentiviral gene transfer and cell proliferation, cells were subjected to gamma-irradiation, which attenuated the replication of primary fetal hepatocytes. However, lentiviral gene transfer was unaffected by this decrease in cell proliferation. GFP expression in transduced cells was preserved during multiple passages in cell culture. When GFP-expressing cells were transplanted into the liver of Balb/C SCID mice via intrasplenic injection, GFP expression was observed throughout the 3 week duration of the study. CONCLUSION: These studies establish that human hepatocytes are amenable to lentiviral gene transfer with sustained transgene expression. Incorporation of lentiviral vectors will be helpful in testing strategies for hepatic gene therapy.

'Babesia gibsoni' of dogs from North America and Asia belong to different species.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0.5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related 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.

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.

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.

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.

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.

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.

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.

[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.

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.

[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.

[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.