João Luiz Horácio Faccini

Seasonal dynamics of ticks (Acari: Ixodidae) on horses in the state of São Paulo, Brazil

Natural tick infestations were assessed every 14 days on horses over a 2-year period. Amblyomma cajennense adult ticks were counted individually, without detachment from the horses. Larvae and nymphs of A. cajennense were collected using a rubber scraper that scratched engorged immature ticks from the host. Adult females of Anocentor nitens larger than 4mm length were counted on the horses. Blood samples were also obtained from the horses every 14 days and macroclimatic data were obtained for the study period. Infestations of A. cajennense demonstrated distinct peaks of activity for each of the three parasitic stages over each 12-month period, showing a 1-year generation pattern. Larvae predominated from April to July and nymphs from June to October. Adults predominated from October to March with a greater number of adult males than females. Although other studies on seasonal dynamics in the states of Rio de Janeiro and Minas Gerais were performed with the free-living stages of A. cajennense on pastures, the present study in the state of São Paulo, performed with the parasitic stages of A. cajennense on horses, showed similar results to those observed in other states. Infestations by A. nitens demonstrated distinct peaks of activity of adult females (>4 mm), suggesting different tick generations during the year. Infestation with A. nitens was much higher in the first year than the second year which may have been related to horse nutritional status and stocking rate. Although several climatic variables showed statistical significant correlation (r) with tick counts, the determination coefficients (R(2)) were always lower than 0.40, suggesting that any single significant variable (i.e. mean temperature) would not explain the tick distribution pattern over the year. The highest peaks of A. nitens females (>4 mm) were significantly associated with decrease in horse packed cell volumes (R(2)=0.603). The ears and the perineum, tail and groin region accounted for around 70% of all A. nitens females counted on the horses.

Risk factors to tick infestations and their occurrence on horses in the state of São Paulo, Brazil

From December 1998 to March 1999, 40 stud farms were studied in the state of São Paulo, Brazil. During visits to farms, horses reared under grazing conditions were examined for the presence of ticks. On each farm visit, horse pastures were closely inspected and a questionnaire was given to the farm supervisor with the purpose of gaining information about ecological and management variables (independent variables) that could be associated with the presence and infestation levels of ticks on the farm (dependent variables). Three tick species were found during the study. Anocentor nitens, Amblyomma cajennense and Boophilus microplus were present on horses from 38 (95%), 20 (50%) and four (10%) farms, respectively. All farms that had A. cajennense or B. microplus infestations also had A. nitens infestations. Only one of the four farms with B. microplus infestations on the horses also had A. cajennense infestations. Two farms had all horses free of ticks. There was a strong association between the presence of infestation by B. microplus on horses and the simultaneous use of a grazing area by cattle and horses (P = 0.000). There was no statistical association between any of the independent variables and the presence or infestation level of A. nitens on the horses (P > 0.20). The presence of A. cajennense was statistically associated with the presence of at least one mixed overgrowth pasture in the farm (P = 0.001). A mixed overgrowth pasture means the presence of undesired plants such as bushes and shrubs in the pasture. The presence of high levels of A. cajennense on horses was also associated with the presence of at least one mixed overgrowth pasture in the farm (P = 0.026). The regular use of acaricides was statistically associated with the presence of ticks on the horses (P < 0.05), making this procedure a result of the inefficacy of controlling ticks on the farms. The occurrence of human infestation by ticks was statistically associated with the presence of A. cajennense on the horses (P=0.000). The presence of at least one mixed overgrowth pasture on the farm was associated (P = 0.000) to either higher horse densities and to farms that did not mow all the pastures once a year, indicating that mowing all the pastures at least once a year can be considered a protective factor against the presence of mixed overgrowth pastures on the farm, and consequently, against the presence of A. cajennense on the horses.

Experimental Infection of Rhipicephalus sanguineus Ticks with the Bacterium Rickettsia rickettsii, Using Experimentally Infected Dogs

We evaluated if Rickettsia rickettsii-experimentally infected dogs could serve as amplifier hosts for hipicephalus sanguineus ticks. In addition, we checked if Rh. sanguineus ticks that acquired Ri. rickettsii from dogs could transmit the bacterium to susceptible hosts (vector competence), and if these ticks could maintain the bacterium by transstadial and transovarial transmissions. Uninfected larvae, nymphs, and adults of Rh. sanguineus were allowed to feed upon three groups of dogs: groups 1 (G1) and 2 (G2) composed of Ri. rickettsii-infected dogs, infected intraperitoneally and via tick bites, respectively, and group 3 composed of uninfected dogs. After larval and nymphal feeding on rickettsemic dogs, 7.1-15.2% and 35.8-37.9% of the molted nymphs and adults, respectively, were shown by polymerase chain reaction (PCR) to be infected by Ri. rickettsii, confirming that both G1 and G2 dogs were efficient sources of rickettsial infection (amplifier host), resulting in transstadial transmission of the agent. These infected nymphs and adults successfully transmitted Ri. rickettsii to guinea pigs, confirming vector competence after acquisition of the infection from rickettsemic dogs. Transovarial transmission of Ri. rickettsii was observed in engorged females that had been infected as nymphs by feeding on both G1 and G2 dogs, but not in engorged females that acquired the infection during adult feeding on these same dogs. In the first case, filial infection rates were generally <50%. No tick exposed to G3 dogs was infected by rickettsiae in this study. No substantial mortality difference was observed between Ri. rickettsii-infected tick groups (G1 and G2) and uninfected tick group (G3). Our results indicate that dogs can be amplifier hosts of Ri. rickettsii for Rh. sanguineus, although only a minority of immature ticks (<45%) should become infected. It appears that Rh. sanguineus, in the absence of horizontal transmission, would not maintain Ri. rickettsii through successive generations, possibly because of low filial infection rates.

Detection of Anaplasma phagocytophilum in Brazilian dogs by real-time polymerase chain reaction.

Anaplasma phagocytophilum was detected in dogs from Brazil in the municipalities of Seropédica and Itaguaí, Rio de Janeiro state, by real-time polymerase chain reaction (PCR) using SYBR Green to detect the amplification. Of 253 samples, 18 (7.11%) were positive, with a threshold cycle (Ct) ranging from 31 to 35 cycles. The PCR product from a positive sample was cloned and sequenced. The sequence obtained demonstrated 100% identity with other A. phagocytophilum sequences published in the GenBank database. The analytical sensitivity of RT-PCR using SYBR Green system was able to detect 3 plasmid copies when defined numbers of plasmid copies containing 122 base pairs from the msp2 gene were used. The assay was considered specific when DNA from bacteria (Anaplasma platys, Anaplasma marginale, Ehrlichia canis, Neorickettsia risticii, Rickettsia rickettsii) closely related to A. phagocytophilum was placed in the reaction. These results demonstrate that the canine granulocytic anaplasmosis agent is present in regions in which dogs could be a source of infection for tick vectors. The current study reports the detection of A. phagocytophilum, a zoonotic agent responsible for Human granulocytic anaplasmosis, in Brazilian dogs.

Candidatus Rickettsia andeanae, a spotted fever group agent infecting Amblyomma parvum ticks in two Brazilian biomes.

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.

Life cycle of the tick Haemaphysalis leporis-palustris (Acari: Ixodidae) under laboratory conditions

The life cycles of two separate populations (colonies A and B) of the rabbit tick, Haemaphysalis leporis-palustris, were studied under laboratory conditions. Domestic New Zealand rabbits, Oryctolagus cuniculus, and wild rabbits, Sylvilagus brasiliensis, were used as hosts for ticks from colony B and only O. cuniculus rabbits were used as hosts for ticks from colony A. Developmental periods were observed in an incubator at 27 ± 1°C and RH 90 ± 5%. Larvae from colonies A and B fed for 8.0 ± 3.7 days and 8.5 ± 1.3 days, respectively, on O. cuniculus. On S. brasiliensis larvae from colony B fed for 7.2 ± 1.3 days. Nymphs from colony A fed for 8.1 ± 1.4 days on O. cuniculus and nymphs from colony B fed for 8.1 ± 1.0 days on S. brasiliensis. Only one engorged nymph from colony B was recovered from O. cuniculus. Females from colony A fed for 20.9 ± 5.9 days on O. cuniculus and females from colony B fed for 18.6 ± 2.4 days on O. cuniculus and 18.7 ± 3.7 days on S. brasiliensis. Engorged larvae from colony A required 13.7 ± 3.7 days to molt while engorged larvae from colony B required 11.8 ± 3.0 and 11.5 ± 1.8 days to molt, after having fed on O. cuniculus and S. brasiliensis, respectively. Engorged nymphs from colonies A and B required 16.3 ± 1.9 days and 14.7 ± 1.4 days to molt, respectively. Engorged females from colonies A and B required 4–7 and 3–5 days, respectively, to start oviposition. Mean egg incubation periods lasted for 33–34 days. For ticks from colony B, host species accounted for significant differences (p < 0.05) in larval and nymphal feeding periods, oviposition weights and CEIs. Significant differences (p < 0.05) between the two colonies when ticks fed on O. cuniculus were observed for larval and nymphal feeding and premolt periods, engorged female and oviposition weights and conversion efficiency indexes (CEI). S. brasiliensis were always a more suitable host for H. leporis-palustris than O. cuniculus. Significantly more larvae and nymphs engorged and molted when fed on S. brasiliensis (p < 0.001). Females fed S. brasiliensis were more successful to lay fertile eggs and showed the highest engorged and egg mass weights, and the highest CEIs. Data of H. leporis-palustris fed on wild rabbits (one of its natural host species) are reported for the first time.

Redescription of larva, nymph and adults of Ixodes (I.) loricatus Neumann, 1899 (Acari: Ixodidae) based on light and scanning electron microscopy

Ticks of the genus Ixodes are poorly known in the Neotropical zoogeographical region, from where only 45 species have been recorded. In Brazil, the genus is currently represented by eight species, four of which are known only from this country. This paper presents a redescription of all active stages in the life-cycle of I. loricatus, based on optical and scanning electron microscopy. In addition, the relationship of I. loricatus to other Neotropical Ixodes is presented and discussed.

Immature argasid ticks: diagnosis and keys for Neotropical region

Many argasid tick species are known only through their larval descriptions, in which the chaetotaxy, together with other external morphological characteristics, has been used to separate genera and species. However, the illustrations of these features are based on optical microscopy alone and many of these features are not clearly defined. Because of the difficulties in determining the larval and nymph stages of some genera, we have prepared illustrated keys for the immature stages of argasids, including an up-to-date list of the known species of the Neotropical region. We have also included an illustrated key for larvae of the Ornithodoros species from Brazil, based on scanning electron microscopy.

Diagnoses of and illustrated key to the species of Ixodes Latreille, 1795 (Acari: Ixodidae) from Brazil

The current Brazilian Ixodes fauna is composed of the following eight species: I. amarali Fonseca, 1935; I. aragaoi Fonseca, 1935; I. auritulus Neumann, 1904; I. fuscipes Koch, 1844; I. loricatus Neumann, 1899; I. luciae Sénevet, 1940; I. paranaensis Barros-Battesti, Arzua, Pichorim & Keirans, 2003; and I. schulzei Aragão & Fonseca, 1951. Further studies are needed to establish the taxonomic status of I. serrafreirei Amorim, Gazeta, Bossi & Linhares, 2003, a recently proposed species based solely on the nymphal stage. We present an up-to-date key to adults of the currently valid Brazilian species of Ixodes based on scanning electron microscopy. The relationships between Brazilian and other Neotropical Ixodes are also discussed.

Efeito dos fungos entomopatogenicos Metarhizium anisopliae e Beauveria bassiana em ovos de Rhipicephalus sanguineus (acarlixodidae)

This experiment was conducted to evaluate the effects of two species of fungi Beauveria bassiana and Metarhizium anisopliae on the eggs of the tick Rhipicephalus sanguineus at constant (27 ± 1° C and > 80% RH) and variable (25 ± 5° C and > 60% RH), temperatures and humidities. Eggs of R. sanguineus were immersed in four concentrations (108, 107, 106, 105 conidia/ml) of two isolated of B. bassiana (Bb 986 and Bb 747) and three isolated of M. anisopliae ( Ma 959, Ma E9 and Ma 319). The incubation and hatch periods of all treated eggs and controls were statistically not significant (P> 0.05). Otherwi- se, the percentage of hatch of larvae in all treated eggs was inversely proportional to the concentration of conidia/ml. The percentage of hatch ranged from 0 to 26.66% for the 108 concentration and from 6.66 to 100% for the 105 concentration. The percentage of eclosion of eggs maintened at variable temperatures and humiditis were lower as compared with eggs maintened at 27 ± l° C and > 80% RH (P< 0.05). The DL 50 and 90 for inhibicion of eclosion of larvae were also lower at variable temperatures and humidities (P<0.05).