BackgroundAnaplasma phagocytophilum is an emerging pathogen of humans, dogs and other animals, and it is transmitted by ixodid ticks. The objective of the current study was a) detect A. phagocytophilum in dogs and ixodid ticks using real-time Polymerase Chain Reaction (qPCR); and b) Determine important variables associated to host, environment and potential tick vectors that are related to the presence of A. phagocytophilum in dogs domiciled in Rio de Janeiro, Brazil.MethodsWe tested blood samples from 398 dogs and samples from 235 ticks, including 194 Rhipicephalus sanguineus sensu lato, 15 Amblyomma cajennense, 8 Amblyomma ovale and 18 pools of Amblyomma sp. nymphs. A semi-structured questionnaire was applied by interviewing each dog owner. Deoxyribonucleic acid obtained from ticks and dog buffy coat samples were amplified by qPCR (msp2 gene). The sequencing of 16S rRNA and groESL heat shock operon genes and a phylogenetic analysis was performed. The multiple logistic regression model was created as a function of testing positive dogs for A. phagocytophilum.ResultsAmong the 398 blood samples from dogs, 6.03% were positive for A. phagocytophilum. Anaplasma phagocytophilum was detected in one A. cajennense female tick and in five R. sanguineus sensu lato ticks (four males and one female). The partial sequences of the 16S rRNA, and groESL genes obtained were highly similar to strains of A. phagocytophilum isolated from wild birds from Brazil and human pathogenic strains. The tick species collected in positive dogs were R. sanguineus sensu lato and A. cajennense, with A.cajennense being predominant. Tick infestation history (OR = 2.86, CI = 1.98-14.87), dog size (OR = 2.41, IC: 1.51-12.67), the access to forest areas (OR = 3:51, CI: 1.52-16.32), hygiene conditions of the environment in which the dogs lived (OR = 4.35, CI: 1.86-18.63) and Amblyomma sp. infestation (OR = 6.12; CI: 2.11-28.15) were associated with A. phagocytophilum infection in dogs.ConclusionsThis is the first report of A. phagocytophilum in ixodid ticks from Brazil. The detection of A. phagocitophylum in A. cajennense, an aggressive feeder on a wide variety of hosts, including humans, is considered a public health concern.

This study aimed to evaluate the in vitro effect of the entomopathogenic fungi Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana sensu lato (s.l.) on two distinct populations of Rhipicephalus microplus, from two different experimental farms. Bioassays were performed with engorged females, eggs and larvae. Fungal infection was evaluated based on biological parameters of treated engorged females, percentage of hatch from treated eggs, and percentage of mortality and mean lethal time (LT90) of treated larvae. When the treatments were compared between the two populations, there were significant differences in the following parameters: pre-oviposition period, hatching period and egg production index. Moreover, the results showed that B. bassiana s.l., isolate Bb 986, was more virulent than M. anisopliae s.l., isolate 959, for engorged females, showing a control percentage of 49%. In the bioassay with eggs, the hatching percentage ranged from 3.1% to 49.5% in one population and from 3.4% to 42.7% in the other, with no significant difference between the two populations. In the bioassay with unfed larvae, the mortality percentage ranged from 91.8% to 98.7% in one population and from 71.0% to 94.0% in the other. The LT(90) varied from 19.52 to 27.51 days in one of the populations and 22.89 to 37.31 days in the other. These results suggest that populations of R. microplus show distinct variation in their susceptibility to B. bassiana s.l. and M. anisopliae s.l.

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 10 8 conidia mL −1 ). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control.

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