Modus operandi of oviposition in Dermacentor reticulatus (Acari: Ixodidae)

The process of oviposition in D. reticulatus was observed and found to be a sequence of exactly coordinated, interlocking events independent of the phase of oviposition. The average period of oviposition in the investigated ticks was 31.6 days at 20 °C and 95% relative humidity. The number of eggs deposited on each day increased until reaching a maximum on the fifth day of oviposition and then decreased continuously. As a result, most of the eggs were deposited during the initial phase of oviposition. The total number of eggs was proportional to the ticks' weight replenishment. Egg-laying commenced with the lowering of the capitulum and the simultaneous spread of the pedipalps which were lowered to the body wall embracing the genital aperture on both sides. Immediately afterwards the cuticular sac of Gene's organ was pushed out and retracted several times. At the cuticular sac's maximum extension, the vestibulum vaginae prolapsed, forming the ovipositor as an extended tube which handed over an egg to the two horns of the cuticular sac after a brief, but intensive, contact with the cuticular sac. Then the vestibulum vaginae invaginated, the pedipalps closed, and the cuticular sac was retracted. Finally, the egg was transported onto the dorsal area of the tick by means of a vigorous rising of the capitulum. During the course of oviposition most of the events, especially the period of egg embracement by the cuticular sac, were prolonged, as was the total time for laying one egg. Similarly, the intervals between successive egg-laying processes increased continuously.The number of eggs deposited was not dependent on the functional ability of Gene's organ, as shown by similar numbers of deposited eggs from ticks with and without mechanical blocking of the cuticular sac. But the participation of the organ in the process of oviposition proved to be a prerequisite for the viability of the eggs. Larvae developed and hatched only from those eggs which were deposited from ticks with an undisturbed Gene's organ. In comparison, eggs without contact to the cuticular sac of Gene's organ dried up and shrivelled immediately after being deposited and did not hatch. Consequently, it strongly suggests, together with the results from other studies, that Gene's organ covers the eggs with a secretion that prevents the loss of water.     

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 adultDermacentor marginatus and D.reticulatus ticks were investigated at a constant temperature of20 °C. It was also examined whether these ticks use liquidwaterto compensate water loss. Both Dermacentor spp. showed nosignificant differences in water content in relation to body mass. The meanwater 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 prolongedwith decreasing saturation deficits. On average, males survived longer thanfemales and D. marginatus ticks survived mostly longerthanD. reticulatus ticks. The 50% mortality period rangedbetween 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. inD. reticulatus. The critical equilibrium activity of unfedadults 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 slightlygained weight while most further lost weight. Liquid water was not attractivefor dehydrated or non-dehydrated ticks and drinking was not observed. Aftersubmerging in water for 2 d, most of the dehydrated ticks had gained weight.     

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. reticulatusticks 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°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°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.     

Isolation ofNosema slovaca (Microsporidiae) fromDermacentor reticulatus ticks (Acari: Ixodidae) collected in Hungary

Nosema slovaca (Microsporidiae) was isolated from a maleDermacentor reticulatus tick collected in the Mélykút locality, Bács-Kiskun province, Hungary. This parasitic protozoan caused an acute infection in partly engorgedD. reticulatus females when inoculated intracoelomically resulting in death of the ticks within 5–15 days post-infection. This strain ofNosema can be maintained in the laboratory by passaging between partly engorgedD. reticulatus females and is currently being studied for its potential as a biological control agent of ixodid ticks.     

Babesia canis vogeli: a novel PCR for its detection in dogs in Australia

Babesia canis vogeli is known to cause disease in dogs in Australia, and the rapid detection of various subspecies would enable effective treatment and management. A 21 bp oligonucleotide, "Bab-f" was proposed for the production of larger PCR products with high species specificity that would enable effective sequence analyses to yield subspecies identification. The new forward primer when paired with a previously reported "Babesia common" reverse primer generated a 394 bp product which was successfully amplified and provided subspecies differentiation by sequence analyses. Specificity and sensitivity were reported at 100% on a cohort of 55 dogs.     

Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia

Abstract. A total of 924 questing Dermacentor reticulatus (Fabricius), 504 Ixodes ricinus (L.), sixty Haemaphysalis concinna Koch and 718 mosquitoes (Aedes spp.) were examined in a floodplain forest ecosystem during the 1994-95 outbreak of tularaemia in South Moravia, Czech Republic. Francisella tularensis was not isolated from H.concinna ticks or Aedes spp. mosquitoes, whereas twenty-one isolates were recovered from the other haematophagous arthropods. Dermacentor reticulatus revealed a significantly higher infection rate (2.6%) than I.ricinus (0.2%). This tick species acts as principal vector for tularaemia in the enzootic focus. Monitoring of D.reticulatus for F.tularensis thus seems to be a very efficient approach in the surveillance of tularaemia in the flood-plain forest ecosystems of Europe.     

Cockroach allatostatin-like immunoreactivity in the synganglion of the American dog tick Dermacentor variabilis (Acari: Ixodidae)

Using immunocytochemistry based on a monoclonal antibodyagainst Diploptera punctata allatostatin I and horseradishperoxidase-diaminobenzidine reaction, the presence of allatostatin-likeimmunoreactivity is demonstrated in the synganglion of Dermacentorvariabilis females. The immunoreactive cells are located in theprotocerebral, cheliceral, palpal, stomodeal, postesophageal, and opisthosomalregions of the synganglion. Strongly immunoreactive granules accumulate in theboundary area of the subganglia in the preesophageal part of the synganglion.This suggests that the immunoreactive materials may be released directly fromthere. In addition, a putative neurohemal area is found in the anterior area ofthe opisthosomal ganglion, where abundant immunoreactive materials are stored.Weak immunoreactivity and fewer immunoreactive cells are seen in newly moltedfemales compared with one month old, unfed females. Thus, the immunoreactiveproducts may be depleted during molting and synthesized in females beforefeeding.     

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.     

Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): A review

Ticks play an important role in human and veterinary medicine, in particular due to their ability to transmit a wide spectrum of pathogenic micro-organisms of protozoal, rickettsial, bacterial and viral origin. Pathogens in ticks can be identified by conventional methods such as indirect immuno-fluorescence, isolation in cell culture or by using histological staining techniques. However, the advent of the polymerase chain reaction (PCR) has resulted in tremendous improvements in the specific and sensitive detection of pathogen DNA in ticks. In this paper, literature on DNA extraction methods, PCR protocols, primers and probes, which are in use for the successful detection and identification of pathogens in ticks, are critically reviewed. Some recommendations are also given towards the end of tht review.     

Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands

The effect of Anaplasma marginale antibodies ingested with the tick blood meal was tested on infected male ticks that were allowed to feed on cattle immunized with the erythrocytic stage of A. marginale. The experiments were done in two trials. Trial 1 was done using splenectomized calves (two calves per treated and control groups) while ticks in trial 2 were fed on intact yearling cattle (four cattle per treated and control groups). The cattle were immunized with purified outer membrane proteins of erythrocyte-derived A. marginale using saponin (trial 1) or monophosphoryl lipid-A-trehalose dicorynomycolate adjuvant (trial 2). The corresponding control cattle received adjuvant only. All cattle were challenged using Dermacentor andersoni males infected as adults that were allowed to feed for 7 days. In trial 1, the ticks were allowed to feed a second time on susceptible calves to test whether exposure of ticks to immunized cattle affected their ability to transmit anaplasmosis. Infections in fed ticks were monitored by determining the infection rates in salivary glands with an A. marginale-specific RNA probe and light microscopy. Vaccine-derived antibodies ingested with the tick blood meal did not appear to affect the development of A. marginale in previously infected ticks. The infection rates in the salivary glands were not significantly different among ticks fed on immunized versus adjuvant control cattle. When the vaccine-exposed ticks in trial 1 were allowed to feed a second time on susceptible calves, the resulting clinical symptoms of anaplasmosis were similar to those of the controls. There was no statistically significant effect of tick exposure to the anti-erythrocytic stage antibody on the development of salivary gland infection or transmission of A. marginale by ticks.     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

Fruit body formation ofBoletus reticulatus in pure culture

The ectomycorrhyzal fungusBoletus reticulatus formed young fruit bodies in pure culture on liquid and solid media. Primordia formation started 31–32 d after inoculation on liquid medium. The primordia developed into the young fruit bodies with convex pileus and clavate stipe 44 d after inoculation on liquid medium. The ability of this fungus to form fruit bodies declined at one and half years after isolation. Sufficient nutrient in medium is required for the fungus to form mature fruit bodies in pure culture.     

Prolactin evokes lactational transmission of larvae in mice infected with Toxocara canis

We investigated the trans-lactational maternal–neonatal transmission of Toxocara canis larvae in mice, with particular interest in the role of prolactin in their migration to the mammary gland. Two female mice were infected with 300 T. canis eggs soon after delivery of 27 offspring. After 1 week of breast-feeding, seven larvae were recovered from 4 of 13 offspring. After 2 weeks of lactation, 101 larvae were recovered from all the remaining offspring. Daily prolactin administration (5 μg) was performed 2 weeks before T. canis infection and continued until 2 weeks after infection in six non-pregnant female mice, which resulted in larval accumulation in the mammary gland. Furthermore, prolactin administration in female mice that had been infected with T. canis 4 weeks prior to prolactin treatment induced migration of larvae into the mammary gland. These findings suggest that prolactin is a promoting factor contributing to lactational transmission of T. canis larvae in mice.     

Cold-hardiness of Dermacentor marginatus (Acari: Ixodidae)

The cold-hardiness of Dermacentor marginatus using laboratory-reared offspring of ticks collected in Germany was characterized. Investigations of unfed stages revealed that adult ticks suffered 50% mortality at –10°C after 4–5 months, but larvae and nymphs suffered mortality within few days, whereas –15°C was lethal for all stages within a very short period. Larval hatch and moulting of engorged larvae and nymphs did not occur at 10°C. Embryonic development of eggs with larval hatch was considerably reduced by exposure of eggs to 10°C. Engorged females did not lay eggs at 10°C, the oviposition capability, however, persisted over 6 months at 10°C, 5 months at 5°C, 3 months at 0°C and 2 months at –10°C without substantial decrease of the oviposition capacity or reduction of viable eggs. These results present evidence that unfed adult ticks are the ecoepidemiologically most effective stages, which are capable to tolerate long and extremely cold winters without substantial impairment of the population density. It is also considered that engorged females interrupt their oviposition at low and subzero temperatures delaying it for months and so contribute in bypassing winter conditions. None of the stages survived supercooling indicating that D. marginatus is freeze intolerant. Mean supercooling point (SCP) ranged between –26°C in eggs and –12, 6°C in engorged females. Compared with eggs, the SCP of the other stages was significantly higher. In conclusion, the SCP is considered to have no predictive value in the context with cold-hardiness.     

Identification of four genomic groups ofBorrelia burgdorferi sensu lato inIxodes ricinus ticks collected in a Lyme borreliosis endemic region of northern Croatia

We investigated the presence ofBorrelia burgdorferi sensu lato inIxodes ricinus ticks collected in a Lyme borreliosis (LB) endemic region of northern Croatia. Ticks (n=124) were collected at five locations and analysed by the polymerase chain reaction (PCR). A DNA fragment from the internal transcribed spacer (ITS2) ofI. ricinus was detected in all tick lysates, indicating that PCR inhibitors were not present.Borrelia burgdorferi sensu lato DNA was detected in 56 out of 124 ticks (45%). Four genomic groups were identified:Borrelia afzelii (n=26),Borrelia garinii (n=5), group VS116 (n=5) andB. burgdorferi sensu stricto (n=1). Mixed infections ofB. afzelii with group VS116 (n=10) andB. afzelii withB. burgdorferi sensu stricto (n=1) were also detected. Eight ticks containedB. burgdorferi sensu lato, which could not be typed. The detection ofB. afzelii andB. garinii in ticks was in agreement with manifestations of LB found locally. The occurrence of group VS116 in northern Croatia and in an earlier study in The Netherlands, infers that this genomic group may be well established in EuropeanI. ricinus.     

Kinetics of ingested host immunoglobulin G in hemolymph and whole body homogenates during nymphal development of Dermacentor variabilis and Ixodes scapularis ticks (Acari: Ixodidae)

Patterns in the utilization of host immunoglobulin G (IgG) during nymphal development differed between Dermacentor varibilis (Say) and Ixodes scapularis Say ticks. In unfed nymphs of D. variabilis, host IgG was readily detectable in both hemolymph and whole body homogenates. In unfed nymphs of I. scapularis, host IgG was absent in hemolymph and at very low concentrations in whole body homogenates. Host IgG in unfed nymphs was undoubtedly the remnants of IgG acquired during the larval bloodmeal that persisted through metamorphosis to the nymphal stage. In both tick species, host IgG crossed the midgut into the hemocoel during the latter phases of engorgement. Concentrations of host IgG in I. scapularis declined considerably after replete nymphs molted to the adult stage. In contrast, concentrations of host IgG in D. variabilis remained elevated throughout metamorphosis to the adult stage. When larval D. variabilis were fed on a rat, then 2 months later as nymphs on a rabbit, the rat IgG (“old IgG”) present in unfed nymphs was totally replaced by rabbit IgG (“new IgG”) within 2 d of nymphs attaching to the rabbit. Presumably, the old IgG acquired from a previous bloodmeal was secreted via saliva into the new host. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunity of dogs against Babesia canis, its vector tick Dermacentor reticulatus, and Ixodes ricinus in endemic area

Previous epidemiological studies allowed us to accurately define endemic areas of canine babesiosis and tick distribution in southeastern France (Martinod, 1983). Using a micro-ELISA test 100 dogs sera were tested with 3 antigens: Babesia canis, Dermacentor reticulatus and Ixodes ricinus. Antibodies against B. canis and its vector D. reticulatus were detected in an endemic area, sometimes with high levels (optical density 1.38 and 0.80 respectively). A correlation factor and regression lines were found between ELISA activity of B. canis and vector tick antigens, even for dogs which never showed any babesiosis symptoms. These results were compared with those of an area without any babesiosis. Furthermore I. ricinus antigens detected ELISA activity in sera of dogs; some cross reactions were observed between I. ricinus and D. reticulatus antigen.     

Cell differentiation during early development of Nassarius reticulatus L. (Gastropoda,prosobranchia)

Summary In Nassarius reticulatus the nuclei and nucleoli undergo important morphological changes from the zygote to the 16-cell stage. In the zygote and in the trefoil (2-cell) and 4-cell stage, several agranular, fibrillar nucleolus-like bodies 1 m diameter are present in the interphase nucleus. Granular nucleoli first appear at the 8-cell stage. These nucleoli have fibrillar and granular regions. The granular regions are made up predominantly of ribosome-like osmiophilic granules. From the 16 and 32-cell stage onwards, the one or two spherical nucleoli of each nucleus measure 2.5 m in diameter and show a concentric organization with a very dense central region surrounded by a broad peripheral zone containing numerous granules, possibly of ribonucleoprotein. At the same time the number of ribosome-like particles increase in the karyoplasm and that of ribosomes in the cytoplasm. These findings are surprising because in eggs with radial cleavage, which have been subjected to more detailed analysis, the first granular nucleoli appear after the end of the cleavage, at the blastula or gastrula stage. The early appearance of granular nucleoli which seem to be characteristic of several eggs with spiral cleavage is discussed in connection with biochemical data on RNA-synthesis.This investigation was supported by the Deutsche Forschungsgemeinschaft and the Stiftung VolkswagenwerkWe wish to thank Mrs. C. Mehlis for valuable technical assistance and Prof. J. Bergerard for the excellent working conditions at the Station biologique at Roscoff (France)     

Immunocytochemical mapping of FMRFamide-like peptides in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis

FMRFamide-like immunoreactivity was studied in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis using immunocytochemistry based on the peroxidase-antigeroxidase method. FMRFamide-like immunoreactive cells are widely distributed in various regions of the tick synganglion including protocerebral, cheliceral, stomodeal, palpal, pedal I–IV, and opisthosomal regions in both species. However, there is one layer of immunoreactive cells located on the dorsal surface of the postoesophageal part of the synganglion that is found only in D. variabilis. Besides the immunoreactivity within the cell body and its axons, the neuropile and the neural lamella (the extracellular sheath of the synganglion) are rich in immunoreactive materials. Some coxal muscles are innervated by the FMRFamide-like immunoreactive processes of the nerve from the pedal ganglion.     

Extensive dermatophytoses caused by Trichophyton mentagrophytes and Microsporum canis in a patient with AIDS

Retrospective studies have shown the occurrence of episodes of deep or superficial fungal infections in 58 to 81% of HIV/AIDS patients as a result of impairment of cell immunity. We describe a case of disseminated cutaneous dermatophytoses caused by Trichophyton mentagrophytes and Microsporum canis in a patients with AIDS. Diagnostic and therapeutic problems in relation to this unsual presentation are emphasized as well as the importance of an early mycologic diagnosis to prescribe antifungal therapy.