Investigation of the parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Amblyomma sculptum (Acari: Ixodidae) ticks in the municipality of Salto, São Paulo, Brazil

The presence of capybaras (Hydrochoerus hydrochaeris) in urban and periurban areas has caused increased numbers of cases of Brazilian spotted fever. With the aim of investigating the presence of the parasitoid Ixodiphagus hookeri in Amblyomma sculptum ticks in the municipality of Salto, state of São Paulo, samples were collected monthly from 14 sites. Thirty samples were placed in containers for observation of the emergence of microhymenopterans and 88 samples were subjected to molecular testing to identify the presence of I. hookeri DNA. Neither dissections nor observation of emergence indicated any presence of I. hookeri larvae in ticks. Samples subjected to polymerase chain reaction (PCR) amplification of the mCOX I region of I. hookeri did not reveal its presence, although fragments corresponding to mRNA 16S of Amblyomma sculptum ticks were amplified in all samples tested.

'Candidatus Mycoplasma haematohydrochoerus', a novel hemoplasma species in capybaras (Hydrochoerus hydrochaeris) from Brazil.

Three different species of hemoplasmas have been described in rodents, Mycoplasma coccoides, 'Candidatus Mycoplasma haemomuris' and 'Candidatus Mycoplasma haemosphiggurus'. Additionally, potentially novel hemoplasma species have been detected in wild rodents from Brazil, including capybaras (Hydrochoerus hydrochaeris). Capybaras are the largest rodent in the world and are well adapted to live within close proximity to humans, which increases the risk to spread of zoonotic pathogens. Herein, we investigate the occurrence and genetic diversity of hemoplasmas infecting free-ranging capybaras from southern Brazil. Blood samples and ticks from 17 capybaras were collected. Packed cell volume and total plasma protein were measured, DNA was extracted, and further screened by species-specific and pan-hemoplasma PCR assays targeting the 16S rRNA gene of hemoplasmas. Sixteen out of 17 (94.12%; 95% CI: 73.02-98.95%) were anemic. Only one young female was hypoproteinemic. All capybaras were infested by adults and nymphs of Amblyomma dubitatum ticks. Using the PCR assay targeting the 16S rRNA gene of M. coccoides, 13/17 (76.47%; 95% CI: 52.74-90.44%) capybaras were positive for hemoplasmas. When DNA samples were tested by the pan-hemoplasma PCR, 16/17 (94.12%; 95% CI: 73.02-98.95%) animals were positive. One out of 11 (9.09%) adult ticks salivary glands tested positive for hemoplasma by the pan-hemoplasma PCR assay. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a novel hemotropic Mycoplasma sp. previously reported in capybaras from Brazil. Additionally, sequencing and phylogenetic analysis of the 23S rRNA gene from three hemoplasma-positive capybaras samples from a previous study performed in midwestern Brazil also confirm our findings. Based on phylogenetic and Neighbor-Net network analysis of the 16S rRNA and 23S rRNA genes, the name 'Candidatus Mycoplasma haematohydrochoerus' is proposed for this novel organism.

Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

Molecular Diagnosis and Prevalence of Trypanosoma vivax (Trypanosomatida: Trypanosomatidae) in Buffaloes and Ectoparasites in the Brazilian Amazon Region.

Trypanosoma vivax Ziemann is a parasite that affects both wild and domestic ungulates and is transmitted mechanically via tabanids and other blood-sucking insects in the Americas. A total of 621 blood samples from water buffaloes (Bubalus bubalis (Linnaeus) (Artiodactyla: Bovidae), and 184 ectoparasite samples (Amblyomma cajennense (Fabricius) sensu stricto and Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), and Haematopinus tuberculatus (Burmeister) (Phthiraptera: Haematopinidae)) were obtained from 60 farms in the State of Pará, Brazilian Amazon. Twelve buffalo blood samples (1.89%) and 11 ectoparasites (6%) were positive for T. vivax based on the cathepsin L-like gene. All sequences were 99% similar to T. vivax from northeastern Brazil (EU753788) in amplified PCR assays on each of the hosts tested.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Parâmetros biológicos do estágio larval de Amblyomma cajennense (Fabricius, 1787) (Acari: lxodidae) em coelhos / Biological parameters of the larval stage of Amblyomma cajennense (Fabricius, 1787) (Acari: lxodidae) in rabbits

Objetivando maiores informações sobre as fases parasitária e não parasitária do Amblyomma cajennense; foram coletadas 473 fêmeas ingurgitadas provenientes de eqüinos. As fêmeas ingurgitadas pesavam, em média, 601,96 ± 161 ,01 mg e realizaram posturas entre 100 e 444 mg, com média de 286,36 ± 91 ,85 mg. A fase não parasitária foi avaliada sob condições controladas de laboratório (temperatura de 27°C, umidade relativa do ar superior a 70% e 12 h de fotofase). Para cada 1g de fêmeas ingurgitadas correponderam 507,88 ± 88,61 mg de postura. O período de pré-oviposição oscilou entre 4 e 7 dias, com média de 5,30 ± 1,02 dias; a incubação dos ovos foi realizada em média em 33,04 ± 1,69 dias e o período de pré-eclosão foi de 37,94 ± 1,48 dias. A fase parasitária foi acompanhada a partir de infestações experimentais em coelhos, utilizando-se larvas de 3 a 18 dias de idade, provenientes das posturas das fêmeas coletadas. O ingurgitamento larval foi realizado entre 4 e 6 dias, com média de 5 dias. O período compreendido entre o desprendimento da metalarva e o término da ecdise larva/ninfa foi de 11 ,29 ± 1,19 dias e não foi afetado nem pelo sexo nem pela cor da pelagem do coelho utilizado para ingurgitamento larval. Do total de metalarvas obtidas, 95% completaram a ecdise.

In an attempt to obtain further information on both parasitic and non-parasitic phases of Amblyomma cajennense, 473 engorged te males from horses were collected. Females weighed 601.96 ± 161.01 mg on the average and made oviposition between 100 and 444 mg, with an average of 286.36 ± 91.85 mg. Non-parasitic phase was assessed under laboratory conditions (27°C temperatura, relativa air humidity above 70% and 12 hr photophase exposition). For 1g females there was a correspondence of 507.88 ± 88.61 mg oviposition. Preoviposition period ranged from 4 to 7 days, with an average of 5.30 ± 1.02 days; egg incubation was made in 33.04 ± 1.69 days on the average, and the pre-eclosion period was 37.94 ± 1.48 days. Parasitic phase was followed on the basis of experimental infestations in rabbits, using 3 to 18 day larvae from the oviposition of collected females. Larval ingurgitation was observed between 4 and 6 days with an average of 5 days. The period extending between loosening of metalarva and completion of larva/nymph ecdisis was 11.29 ± 1.19. This period was not affected by sex or hair colar of rabbit used for larvae ingurgitation. Out of the total of metalarvae obtained, 95% completed ecdisis.