Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, ""Candidatus Rickettsia amblyommii,"" R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two ""Ca. Rickettsia amblyommii"" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of ""Ca. Rickettsia amblyommii,"" R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

Nymphs of the genus Amblyomma (Acari: Ixodidae) of Brazil: descriptions, redescriptions, and identification key

Together with the larval stage, the nymphal stage of ticks of the genus Amblyomma are the most aggressive ticks for humans entering areas inhabited by wildlife and some domestic animals in Brazil. However, due to the absence of morphological descriptions of the nymphal stage of most Brazilian Amblyomma species, plus the lack of an identification key, little or nothing is known about the life history of Amblyomma spp. nymphs in the country. In the present study, morphological description of the nymphal stage, illustrating important external characters through scanning electron microscopy, is provided for nymphs of 15 Amblyomma species that occur in Brazil, for which the nymphal stage had never been described: A. aureolatum, A. auricularium, A. calcaratum, A. coelebs, A. fuscum, A. humerale, A. incisum, A. latepunctatum, A. naponense, A. nodosum, A. ovate, A. pacae, A. pseudoconcolor, A. scalpturatum, A. varium. In addition, the nymphal stage of 12 Amblyomma species, which had been previously described, are redescribed: A. brasiliense, A. cajennense, A. dissimile, A. dubitatum, A. longirostre, A. oblongoguttatum, A. parked, A. parvum, A. romitii, A. rotundatum, A. tigrinum, A. triste. The descriptions and redescriptions totalized 27 species. Only 2 species (A. geayi, A. goeldii) out of the 29 Amblyomma species established in Brazil are not included in the present study. A dichotomous identification key is included to support taxonomic identification of the nymphal stage of 27 Amblyomma species established in Brazil. (C) 2010 Elsevier GmbH. All rights reserved.

Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations

The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. (C) 2009 Elsevier Ltd. All rights reserved.

Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n = 5), and Group B consisted of animals infected with R. vitalii (n = 7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n = 6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs. (C) 2011 Elsevier Inc. All rights reserved.

Carios fonsecai sp nov (Acari, Argasidae), a bat tick from the central-western region of Brazil

Males, females, and larvae of Carios fonsecai sp. nov. are described from free-living ticks collected in a cave at Bonito, state of Mato Grosso do Sul, Brazil. The presence of cheeks and legs with micromammillate cuticle makes adults of C. fonsecai morphologically related to a group of argasid species (mostly bat-associated) formerly classified into the subgenus Alectorobius, genus Ornithodoros. Examination of larvae indicates that C. fonsecai is clearly distinct from most of the previously described Carios species formerly classified into the subgenus Alectorobius, based primarily on its larger body size, dorsal setae number, dorsal plate shape, and hypostomal morphology. On the other hand, the larva of C. fonsecai is most similar to Carios peropteryx, and Carios peruvianus, from which differences in dorsal plate length and width, tarsal setae, and hypostome characteristics are useful for morphological differentiation. The mitochondrial 16S rDNA sequence of C. fonsecai showed to be closest (85-88% identity) to several corresponding sequences of different Carios species available in GenBank. Bats identified as Peropteryx macrotis and Desmodus rotundus were found infested by C. fonsecai larvae in the same cave where the type series was collected. C. fonsecai showed to be aggressive to humans in the laboratory.

Influence of Photoperiod and Temperature on the Larval Behavioral Diapause of Amblyomma cajennense (Acari: Ixodidae)

Larval behavioral diapause was shown to be the major factor controlling the 1-yr generation pattern of Amblyomma cajennense (F.) (Acari: Ixodidae) in Brazil. During fieldwork, this behavior was shown to coincide with long daylength (>12 h) and high mean ground temperature (approximate to 25 degrees C), which prevail during spring-summer in Brazil. The current study evaluated biological parameters of engorged females, their eggs, and the resultant larvae inside plastic pots planted with the grass Brachiaria decumbens Stapf. held in incubators set with different combinations of temperature and photoperiod. Both the long daylength (photoperiod 14:10 [L:D]h) and high temperature (25 degrees C) during larval hatching induced larval behavioral diapause, characterized by the confinement of hatched larvae on the ground below the vegetation for many weeks. When long daylength was present during hatching, but temperature was low (15 degrees C), larvae did not enter diapause. Similarly, when short daylength (10:14 or 12:12) was present during larval hatching, larvae did not enter diapause regardless whether temperature was high (25 degrees C). Termination of diapause was induced by shifting photoperiod from 14:10 to 12:12 or the temperature from 25 to 15 degrees C. When applied to field conditions, the present results indicate that both high ground mean temperature (approximate to 25 degrees C) and long daylength (>12 h) during spring-summer (October-March) are responsible for the induction and maintenance of A. cajennense larval behavioral diapause in the field. Furthermore, both the low ground mean temperature (-20 degrees C) and the short daylength (<12h) during autumn (April-May) are responsible for termination of larval behavioral diapause in the field.

Life cycle of Ixodes luciae (Acari: Ixodidae) in the laboratory

The life cycle of Ixodes luciae was evaluated for five consecutive generations in the laboratory. Wild mice Calomys callosus and laboratory rats Rattus norvegicus were used as hosts for larvae and nymphs. For adult ticks, opossums Didelphis aurita were used as hosts. Off-host developmental periods were observed in an incubator at 27A degrees C and 95% RH. The life cycle of I. luciae lasted 95-97 days, excluding prefeeding periods. C. callosus, one of the natural host species for I. luciae immature stages, was shown to be much more suitable than the artificial host R. norvegicus. Significantly (P < 0.05), more larvae and nymphs successfully fed on C. callosus than on R. norvegicus. When tick-na < ve C. callosus were exposed to three consecutive larval infestations at 24-day intervals, recovery of engorged larvae were greater in the second and third infestations, indicating that previous infestations did not induce acquired resistance to ticks. Larval feeding period typically varied from 5 to 10 days on R. norvegicus, but was significantly (P < 0.05), longer on C. callosus (range, 7-34 days). The majority (71.7%) of I. luciae adult females successfully fed and oviposited after exposed to D. aurita. Mean engorged weight (581.9 mg; range, 237.1-796.0 mg) of these females were much higher than those previously reported for other New World Ixodes species. Our results are in accordance to the current literature that appoints opossums Didelphidae and small rodents (e.g., C. callosus) natural hosts for I. luciae immature and adult stages, respectively.

Rickettsia monteiroi sp nov., Infecting the Tick Amblyomma incisum in Brazil

Free-living adult Amblyomma incisum ticks were collected in an Atlantic rainforest area at Intervales State Park, State of Sao Paulo, Brazil. From an A. incisum specimen, rickettsiae were successfully isolated in Vero cell culture by the shell vial technique. Rickettsial isolation was confirmed by optical microscopy, transmission electron microscopy, and PCRs targeting portions of the rickettsial genes gltA, htrA, rrs, and sca1 on infected cells. Fragments of 1,089, 457, 1,362, and 443 nucleotides of the gltA, htrA, rrs, and sca1 genes, respectively, were sequenced. By BLAST analysis, the partial sequence of rrs of the A. incisum rickettsial isolate was closest to the corresponding sequence of Rickettsia bellii (99.1% similarity). The gltA partial sequence was closest to the corresponding sequences of ""Candidatus Rickettsia tarasevichiae"" (96.1% similarity) and Rickettsia canadensis (95.8% similarity). The htrA partial sequence was closest to the corresponding sequence of R. canadensis (89.8% similarity). The sca1 partial sequence was closest to the corresponding sequence of R. canadensis (95.2% similarity). Since our rickettsial isolate was genetically distinct from other Rickettsia species, we propose a new species designated Rickettsia monteiroi sp. nov. Phylogenetic analyses indicated that R. monteiroi belongs to the canadensis group within the genus Rickettsia, together with the species R. canadensis and ""Candidatus R. tarasevichiae"". Little or no antibody cross-reaction was observed between sera of R. monteiroi-inoculated guinea pigs and R. bellii-, Rickettsia rickettsii-, or R. canadensis-inoculated guinea pigs.

Prevalence of equine Piroplasmosis and its association with tick infestation in the State of Sao Paulo, Brazil

Serum samples were collected from 582 horses from 40 stud farms in the State of Sao Paulo and tick (Acari: Ixodidae) infestations were evaluated on them. Serum samples were subjected to the complement fixation test (CFT) and a competitive inhibition ELISA (cELISA) for Babesia caballi and Theileria equi. Logistic regression analyses were performed to construct multivariate models that could explain the dependent variable (horses positive for B. caballi or T equi) as a function of the independent variables (presence or abundance of each one of the rick species found on the farms). A higher overall prevalence of B. caballi (54.1%) than of T equi (21.6%) was found by the two tests. The ticks Dermacentor nitens Neumann, 1897, Amblyomma cajennense (Fabricius, 1787) and Rbipicephalus (Boopbilus) microplus (Canestrini, 1887) were present on horses on 38 (95%), 20 (50%), and 4 (10%) farms, respectively. Infestations by D. nitens were statistically associated with B. caballi-positive horses on the farms by either the CFT or cELISA. Infestations by A. cajennense were statistically associated with T equi-positive horses on the farms by either CFT or cELISA.

Ticks (Acari: Ixodidae) Infesting Birds in an Atlantic Rain Forest Region of Brazil

Brazil has the third richest bird diversity of the world; however, there are few data concerning ticks (Acari: Ixodidae) parazitizing birds. The aim of the study was to report tick infestations on wild birds from an Atlantic rain forest region of Brazil. During 2 yr, ticks were collected from birds and from the environment in 12 forest sites. A total of 1,725 birds were captured representing 80 species from 24 families. In total, 223 (13%) birds were found infested by immature stages of Amblyomma ticks: 1,800 larvae and 539 nymphs. The prevalence of ticks was higher among-birds from the families Thamnophilidae, Conopophagidae, and Momotidae. The most common tick parasitizing birds was Amblyomma nodosum Koch. Other tick species, Amblyomma coelebs Neumann, Amblyomma cajennense (F.), Amblyomma ovale Koch, Amblyomma longirostre (Koch), Amblyomma calcaratum Neumann, and Amblyomma naponense (Packard), were found sporadically. Among free-living ticks collected in the environment, A. cajennense was the most common, followed by A. coelebs, A. naponense, Amblyomma brasilense Aragao, and Hemaphysalis juxtakochi Cooley.

HOST RECORDS FOR THE IMMATURE STAGES OF THE SOUTH AMERICAN TICK, AMBLYOMMA FUSCUM (ACARI: IXODIDAE)

This work reports free-living opossums (Didelphis aurita and Didelphis albiventris) and a rodent species (Thrichomys laurentius) naturally infested by the immature stages of Amblyomma fuscum Neumann, 1907 in Brazil. Previously the only host record for the A. fuscum immature stages was for a single nymph collected on an opossum D. aurita in the state of Sao Paulo. Herein are presented two new host records (D. albiventris and T. laurentius) for A. fuscum. Our results indicate that opossums (Didelphis spp.), and one small rodent species (T. laurentius) are major hosts for immature stages of A. fuscum in Brazil. Based on the known feeding habits of immature stages of A. fuscum. coupled with previous reports of the adult stage parasitizing humans, A. fuscum is a potential vector of spotted fever group rickettsiae.

Hepatozoon canis infecting dogs in the State of Espirito Santo, southeastern Brazil

From May 2007 to March 2008, blood samples were collected from 92 healthy dogs living in 21 households (17 farms in rural area, and 4 homes in urban area) in 6 counties of the State of Espirito Santo, southeastern Brazil. In addition, ticks were collected from these dogs. A mean of 4.4 +/- 3.0 dogs (range: 1-12) were sampled per household; 78 and 14 dogs were from rural and urban areas, respectively. Polymerase chain reaction (PCR) designed to amplify fragments of the 18S rDNA gene of Babesia spp or Hepatozoon spp revealed amplicons of the expected size in 20 (21.7%) dogs for Babesia, and 54 (58.7%) dogs for Hepatozoon. All Babesia-positive dogs were also Hepatozoon-positive. Among the 21 households, 15 (71.4%) from 3 counties had at least one PCR-positive dog, including 13 farms (rural area) and 2 homes (urban area). A total of 40 PCR products from the Hepatozoon-PCR, and 19 products from the Babesia-PCR were submitted to DNA sequencing. All generated sequences from Hepatozoon-PCR were identical to each other, and to corresponding 18S rDNA sequences of H. canis in GenBank. Surprisingly, all generated sequences from the Babesia PCR were also identical to corresponding 18S rDNA sequences of H. canis in GenBank. Dogs from 10 rural and 2 urban households were found infested by Rhipicephalus sanguineus ticks. Immature of Amblyomma cajennense ticks were found in dogs from only 4 rural households (also infested by R. sanguineus). All but one household with R. sanguineus-infested dogs had at least one Hepatozoon-infected dog. Statistical analysis showed that the presence of ticks (i.e. R. sanguineus) infesting dogs in the households was significantly (P < 0.05) associated with at least one PCR-positive dog. There was no significant association (P > 0.05) between PCR-positive dogs and urban or rural households. Canine hepatozoonosis caused by H. canis is a high frequent infection in Espirito Santo, Brazil, where it is possibly vectored by R. sanguineus. Since all infected dogs were found apparently healthy, the pathogenicity of H. canis for dogs in Espirito Santo is yet to be elucidated. (C) 2009 Elsevier B.V. All rights reserved.

Biology and life cycle of Amblyomma incisum (Acari: Ixodidae)

Amblyomma incisum Neumann is a major tick species in the Atlantic Forest of Brazil. Tapir is the main host for adult ticks and a high aggressiveness of nymphs to humans has been reported. In this work data on the biology and life cycle of this tick species is presented for the first time. It was shown that horse is a suitable host for A. incisum adults and rabbit for larvae and nymphs. It was also shown that A. incisum is a big tick species (mean engorged female weight of 1.96 g) with a long life cycle which lasts 262.3 days when maintained at 27A degrees C and 85% RH. These laboratory conditions were, however, inappropriate and egg hatching rate (1.2%) was very low. Nevertheless egg hatching of ticks in a forest patch increased considerably (72.2%) indicating that this A. incisum population is highly dependent on a forest-like environment.

Ehrlichia canis in dogs attended in a veterinary hospital from Botucatu, Sao Paulo State, Brazil

This study investigated the etiology of canine ehrlichiosis and possible clinical and epidemiological data associated with the infection in 70 dogs suspect of ehrlichiosis attended at the Veterinary Hospital of the Sao Paulo State University in Botucatu city during 2001 and 2002. Dogs were evaluated by clinical-epidemiological and hematological data and molecular analysis by partial amplification and DNA sequencing of the ehrlichial dsb gene. E. canes DNA was amplified and sequenced in 28 (40.0%) dogs. Dogs younger than 12 months old showed significantly higher infection rates (65.0%; P < 0.05). Diarrhea, apathy, and anorexia were the major clinical signs observed in 55.2% (P = 0.05), 47.0% (P > 0.05), and 42.4% (P > 0.05) of the PCR-positive dogs, respectively. Twenty-five anemic (<5.5 x 10(6) RBC.mu L(-1)), and 8 leukopenic (<5.5 x 10(3) WBC.mu L(-1)) dogs were PCR-positive (P > 0.05). All 28 PCR-positive dogs showed thrombocytopenia (<175 x 10(3) platelets.mu L(-1)) and revealed statistical significance (P < 0.05). E. canis was the only Ehrlichia species found in dogs in the studied region, with higher infection rates in younger dogs, and statistically associated with thrombocytopenia.

Elevated progesterone concentrations enhance prostaglandin F(2 alpha) synthesis in dairy cows

The objective was to evaluate the influence of varying plasma progesterone (P(4)) concentrations throughout the luteal phase in dairy cows on PGF(2 alpha) production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF(2 alpha); PGFM) following treatment with estradiol-17 beta (E(2)) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n = 9), received 20 mg LH (n = 7), or had ovarian follicles larger than 6 mm aspirated (n = 8). In Experiment 2, cows on Day 6 were untreated (n = 9) or received 5000 IU hCG (n = 10). In Experiments 1 and 2, all cows received 3 mg E(2) on Day 17, and blood samples were collected every 30 min from 2h before to 10h after E(2). Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n = 4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n = 7) received the alternate sequence. Blood was collected daily for measurement Of P(4) 4 h after concentrate feeding. On Day 17, blood was collected from 1 h before to 1 h after a 100 IU OT injection. In Experiment 1, both plasma P(4) and release Of PGF(2 alpha) were similar between LH-treated and control cows (P > 0.10). In Experiment 2, plasma P4 was elevated to a greater extent on Day 17 in cows treated with hCG (P < 0.05) and plasma PGFM was also greater in hCG-treated animals (treatment x time interaction; P < 0.05). In Experiment 3, there was a group x period interaction (P < 0.01) for plasma P(4), indicating that less concentrate feeding was associated with greater plasma P(4). Release of PGF(2 alpha) in response to OT was greater for cows receiving less concentrate (group x period interaction; P < 0.05). In conclusion, dairy cows with more elevated blood P(4) concentrations released more PGF(2 alpha) in response to E(2) or OT. (c) 2008 Elsevier B.V. All rights reserved.