Natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens (Acari: Ixodidae)

The present paper reports the occurrence of natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens. Engorged females of ticks, collected from a naturally B. caballi-infected horse, were incubated at 27 degrees C and relative humidity over 83%. After a 6-day incubation period, Giemsa-stained smears prepared from hemolymph were examined microscopically under oil immersion. B. caballi infected ticks were dissected and samples of midgut tissue were examined by transmission electron microscopy, through which free sporokinetes were seen in the cytoplasm of gut epithelial cells. In addition, Encephalitozoon-like microsporidia were observed inside the parasitophorous vacuoles in the same cell in which sporokinetes of B. caballi were found and also in some neighbour cells. They presented different morphological stages, suggesting a sequential phases of development.     

Lethal Effect of Rickettsia rickettsii on Its Tick Vector (Dermacentor andersoni)

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88.3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4°C had a lower mortality (80.9%) than did those held at 21°C (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect of R. rickettsii may explain the low prevalence of infected ticks in nature and affect its enzootic maintenance.     

Tick (Dermacentor albipictus)-induced winter hair-loss in captive moose (Alces alces)

Five captive moose calves each infested with 42,000 larval Dermacentor albipictus, six calves each infested with 19,000-21,000, and five control moose were observed for changes in hair-loss, body condition and number, stages, and distribution of the tick. Winter hair-loss was observed only in moose infested with ticks and was correlated positively with the total number of adult ticks. Hair-loss associated with ticks was minimal from October to January, but rapidly increased from February to April when up to 44% of hair had been removed. The pattern of hair-loss was similar in all moose with the neck, shoulders, withers, and perianal areas losing the most hair. Moose with extensive premature hair-loss had less pericardial and abdominal visceral fat than moose with little or no hair-loss.     

Toxicity of solvents and surfactants to Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) and Dermacentor nitens (Neumann, 1897) (Acari: Ixodidae) larvae

The objective of this work was to verify the sensitivity of Amblyomma cajennense and Dermacentor nitens larvae to the solvents ethanol, methanol, acetone, xylol and dimethyl sulfoxide (DMSO) and the surfactant Tween 80. The first four solvents were tested at analytical purity while the DMSO and surfactant Tween 80 were tested at a concentration of 1%. The substances tested at analytical purity that caused high mortality were also tested at concentrations of 50%, 25% and 1%. The larval packet test was used, with 10 repetitions for each treatment. A control group was also formed with the same number of repetitions, in which the larvae were only exposed to distilled water. In the first experiment, only xylol was highly toxic at the concentrations tested, causing mortality above 90% for larvae of both species. In the second experiment, xylol at 1% and at 25% showed low toxicity to the A. cajennense and D. nitens larvae, respectively, since the percentage mortality was statistically similar to that of the control group (p>0.05).     

Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi

Monoclonal antibodies (mAbs) were produced against Babesia caballi (USDA strain) to define a species-specific antigen for use in diagnosis and vaccine development. Eight positive clones of B. caballi mAbs determined by indirect immunofluorescent antibody test were selected for purification and further characterisation. Confocal laser microscopy showed that the antigens recognised by the mAbs were located on the surface/cytoplasm, central part, and/or anterior end of B. caballi parasites, with five different reactive patterns. These mAbs seemed to be species-specific, since they did not cross-react with Babesia equi-infected erythrocytes or uninfected erythrocytes. In Western blotting analysis, 18, 20, 34, 36, 48, and 155 kDa proteins of B. caballi merozoites were recognised by six different mAbs. When added to in vitro cultures, four of the mAbs significantly inhibited the in vitro growth of B. caballi parasites. These results provide a rationale for evaluating antigens for the development of diagnostic methods or vaccines.     

Detection of Babesia caballi in Amblyomma variegatum ticks (Acari: Ixodidae) collected from cattle in the Republic of Guinea

A reverse line blot hybridisation (RLB) assay was applied to screen Amblyomma variegatum adult ticks (n = 504) collected from N'Dama cattle in the Republic of Guinea. In a PCR, the V1 hypervariable region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for species of the genera Anaplasma and Ehrlichia, and the V4 hypervariable region of the 18S rRNA gene was amplified with primers specific for members of the genera Theileria and Babesia. Amplified PCR products from A. variegatum ticks were hybridised onto a membrane, to which oligonucleotide probes species-specific for Ehrlichia/Anaplasma and Theileria/Babesia parasites were covalently linked. No pathogens belonging to Ehrlichia/Anaplasma species were found, while 10 DNA samples resulted positive for Babesia caballi and 5 samples for Theileria velifera. This is the first report of B. caballi in A. variegatum ticks. One of the B. caballi positive samples was sequenced. This new strain (BcabGuinea) showed a 97% similarity to the Z15104 B. caballi GenBank sequence.     

Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population

In Switzerland, the prevalence and incidence of equine piroplasma parasite (EPP) infections are unknown. In order to obtain a first insight into the prevalence, a representative sample of 689 sera of horses from Switzerland was serologically tested for the presence of antibodies directed against T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). A total of 50 (7.3%) horses were seropositive for EPP: overall, the seroprevalence of T. equi was significantly higher than that of B. caballi (p = 0.002). The seropositivities in indigenous horses (animals bred and raised in Switzerland) and in imported horses were 4.8% (11/230) and 8.5% (39/459), respectively. Unlike in indigenous horses, where no significant difference in seroprevalences could be observed between the two parasite species, the seroprevalence of T. equi was significantly higher (p < 0.001) than that of B. caballi in imported horses. Horses imported from France, Spain and Portugal exhibited a significantly higher seroprevalence, and horses imported from Germany a significantly lower seroprevalence of EPP compared to indigenous horses. There were no associations between sex, age, weight loss, surgery or blood transfusions with T. equi and B. caballi seroprevalences.The overall seroprevalence of 7.3% clearly shows that infection with EPP is a threat to the health of the horses in Switzerland. With the presumed expansion of permissive tick vectors, EPP infections will potentially increase in importance in the future. Therefore, continuous monitoring is mandatory.     

Estimation of the transmission dynamics of Theileria equi and Babesia caballi in horses

For the evaluation of the epidemiology of Theileria equi and Babesia caballi in a herd of 510 horses in SW Mongolia, several mathematical models of the transmission dynamics were constructed. Because the field data contain information on the presence of the parasite (determined by PCR) and the presence of antibodies (determined by IFAT), the models cater for maternal protection with antibodies, susceptible animals, infected animals and animals which have eliminated the parasite and also allow for age-dependent infection in susceptible animals. Maximum likelihood estimation procedures were used to estimate the model parameters and a Monte Carlo approach was applied to select the best fitting model. Overall, the results are in line with previous experimental work, and add evidence that the epidemiology of T. equi differs from that of Babesia spp. The presented modelling approach provides a useful tool for the investigation of some vector-borne diseases and the applied model selection procedure avoids asymptotical assumptions that may not be adequate for the analysis of epidemiological field data.     

Investigation of Ixodid ticks as vectors of Babesia caballi and Theileria equi (Protozoa: Apicomplexa) in central Italy

Babesia caballi and Theileria equi are widely recognized as causative agents of equine pirolasmosis (EP), an acute, sub‐acute, and chronic disease of equines, with relevant economic impact on horse trade worldwide. Although several studies on EP prevalence from central Italy have been published, data on ticks responsible for its transmission are still lacking. In this study, we identified a potential competent vector, investigating main features of its ecology together with EP infection rates. A two‐year sampling of questing ticks was carried out for the first time in Italy in an area known for high EP prevalence in horse sera, detecting the association between Rhipicephalus bursa and causative agents of EP. Most of the positive pools harbored a single infection (91.1%); mixed infections were also detected (8.9%). The infection rate for T. equi slightly decreased among years; B. caballi showed a lower, but increasing, infection rate. Tick phenology, climate variables, and peaks of EP prevalence indicated late May and second half of June as periods with the highest risk of new infections, especially during warm and dry days.     

First report of pathogenicity of entomopathogenic nematodes of the genus Heterorhabditis on partially engorged females of Dermacentor nitens (Acari: Ixodidae)

The aim of this study was to evaluate the effect of different concentrations of the entomopathogenic nematodes (EPNs) Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 on the reproductive biology of partially engorged females of Dermacentor nitens. Four groups were formed, with each group containing 10 females and exposed to concentrations of 0, 75, 300, and 1200 nematodes for each female. This procedure was performed separately for each nematode. The following biological parameters were evaluated: egg mass weight, egg production index, hatching percentage, and percentage of control. H. bacteriophora HP88 at the two highest concentrations (300 and 1200 EPNs/female) caused a reduction (p < 0.05) on the egg mass and egg production index. Was noted a significant reduction (p < 0.05) in the percentage of hatched in all the treated groups. For H. indica LPP1, all treatments resulted in decreased (p < 0.05) values for all the parameters. The percentages of controls obtained at concentrations of 75, 300, and 1200 EPNs/female were 56.3, 89.3, and 98.8 and 77.5, 77.1, and 95.9 for H. bacteriophora HP88 and H. indica LPP1, respectively. Therefore, it is concluded that these nematodes showed pathogenicity toward partially engorged females of D. nitens, thereby negatively affecting the reproductive biology of this tick.     

Establishment and characterization of a diploid cell line from the tick, Dermacentor parumapertus Neumann (Acarina: Ixodidae)

Establishment of a continuous cell line (RML-14) from embryonic tissues of the tick Dermacentor parumapertus Neumann is reported. The culture medium employed consisted of a combination (2:1) of Eagle's and L-15 (Leibovitz) media supplemented with 20% fetal bovine serum, 10% tryptose phosphate broth, and 0.1% bovine plasma albumin. At the 8th passage, 99% of dividing cells had the female chromosome complement, among which more than 70% had a diploid chromosome number of 22. At the 13th passage, cell population showed approximately a 3-fold increase during the first 8 days of culture. As of December 1976, had been subcultured 40 times.     

Seasonal analysis of the number of aeropiles in Anocentor nitens (Neumann, 1897) (Acari: Ixodidae) from the State of Rio de Janeiro, Brazil

Variation between aeropile numbers of the right and left peritrematic plate in male and female Anocentor nitens (Neumann, 1897) is reported from a site in Brazil. From January to December 1998, 146 males and 247 females of A. nitens were recovered from Equus caballus L. in Silva Jardim District, State of Rio de Janeiro. Asymmetry of numbers of aeropiles between right and left plates occurred in 83.6% of the males and 82.2% of the females. Differences in the number of aeropiles between the sexes were not significant. Quantitative variation of aeropiles was correlated to the period of recovery, with significant asymmetry detected in August-September and November-December, mainly in males. Results suggest an adaptation, especially in the male ticks, that expresses itself as greater variation in the number of aeropiles in some periods of the year.     

Differences in the Life Cycles Between a Vaccine Strain and an Unmodified Strain of Babesia bovis (Babes, 1889) in the Tick Boophilus microplus (Canestrini)*

SYNOPSIS. Developmental forms of 2 strains of Babesia bovis (Babes) were studied in the tick vector Boophilus microplus (Canestrini). One strain (designated T) was shown to be infective for the tick, and the other (NT) to have lost infectivity for the tick, because of repeated blood passaging in cattle. Parasites of the 2 strains in gut contents of adult female ticks were similar during the first 16 h post-repletion (PR), but thereafter their structure differed. From 16–64 h PR, the majority of T strain parasites were spherical and without processes. During the next 32 h elongate forms and vermicules developed. Fission bodies were seen within epithelial cells of the gut by 96 h PR. T-strain parasites in gut contents decreased in number from ～ 96 h and were difficult to find at 144 h, the time of the final observation. In contrast, NT strain parasites were plentiful throughout the period of observation. They were predominantly spherical, ranging in diameter from 1.5 to 15 μm. Forms with obvious processes measuring up to 81 μm in length were seen in large numbers at seemingly regular intervals from 16–144 h PR, suggesting that a process of development and divisions was being repeated. No vermicules or fission bodies were seen. T-strain, but not NT strain parasites, were seen in hemolymph and ova of the ticks and in their larval progeny. It is suggested that continuous blood passaging of the NT strain had resulted in selection of parasites incapable of penetrating gut epithelial cells of the tick.     

Development and characterization of 12 polymorphic microsatellite loci in the American dog tick (Dermacentor variabilis)

Twelve polymorphic microsatellite loci were developed for the American dog tick (Dermacentor variabilis), an important vector of infectious diseases in humans and animals. Four multiplexed panels comprising the loci were developed and 45 ticks collected from two raccoons (Procyon lotor) were genotyped. The number of alleles per locus ranged from nine to 30, and single locus heterozygosities ranged from 0.18 to 0.93. Data generated using these markers will further our understanding of factors affecting gene flow in D. variabilis, thus helping to elucidate the transmission dynamics of diseases associated with this vector.     

Internal and External Mycoflora of the American Dog Tick, Dermacentor variabilis (Acari: Ixodidae), and Its Ecological Implications

Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.