Novel Anaplasmataceae agents Candidatus Ehrlichia hydrochoerus and Anaplasma spp. Infecting Capybaras, Brazil.

We amplified Ehrlichia and Anaplasma DNA from Amblyomma dubitatum tick-infested capybaras (Hydrochoerus hydrochaeris) in southern Brazil. Sequencing of 16S rRNA, sodB, and groEL indicated a novel Ehrlichia species, and sequencing of 16S rRNA from 2 capybaras indicated a novel Anaplasma species. The tick vectors remain unknown.

'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.

Quantitative Proteomics Identifies Metabolic Pathways Affected by Babesia Infection and Blood Feeding in the Sialoproteome of the Vector Rhipicephalus bursa.

The negative impact of ticks and tick-borne diseases on animals and human health is driving research to discover novel targets affecting both vectors and pathogens. The salivary glands are involved in feeding and pathogen transmission, thus are considered as a compelling target to focus research. In this study, proteomics approach was used to characterize Rhipicephalus bursa sialoproteome in response to Babesia ovis infection and blood feeding. Two potential tick protective antigens were identified and its influence in tick biological parameters and pathogen infection was evaluated. Results demonstrate that the R. bursa sialoproteome is highly affected by feeding but infection is well tolerated by tick cells. The combination of both stimuli shifts the previous scenario and a more evident pathogen manipulation can be suggested. Knockdown of ub2n led to a significative increase of infection in tick salivary glands but a brusque decrease in the progeny, revealing its importance in the cellular response to pathogen infection, which is worth pursuing in future studies. Additionally, an impact in the recovery rate of adults (62%), the egg production efficiency (45.75%), and the hatching rate (88.57 %) was detected. Building knowledge on vector and/or pathogen interplay bridges the identification of protective antigens and the development of novel control strategies.

Molecular heterogeneity of Rhipicephalus sanguineus sensu lato and screening for Ehrlichia canis in mainland Portugal.

The present study aimed to expand knowledge regarding the molecular characterization of R. sanguineus s.l. in Portugal and to screen for ticks naturally infected with E. canis. A total of 113 R. sanguineus s.l. were collected questing or attached to domestic and wild animals from the 18 administrative regions of mainland Portugal. All the ticks were analyzed for the 16S rRNA and the partial sequences obtained showed high genetic similarities with specimens belonging to the temperate lineage. These sequences revealed eight haplotypes (H1‒H8), with a genetic distance ranging from 0.3% to 1.4%. A convenience sample representing approximately 75% of all the R. sanguineus s.l. ticks collected was tested for the presence of E. canis by qPCR for the dsb gene. No ticks were found to be infected with this pathogen. Accordingly, further studies are required to determine the role of the R. sanguineus s.l. temperate lineage in E. canis maintenance and transmission, as well as to elucidate if a different R. sanguineus s.l. lineage or other tick species act as E. canis vectors for dogs in Portugal.

Ferritin 1 silencing effect in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) during experimental infection with Ehrlichia canis.

Rhipicephalus sanguineus sensu lato (s.l.) is a very common ectoparasite of domestic dogs able to transmit several pathogens of human and veterinary importance. Tick infestations and tick-borne diseases (TBDs) remain a serious and persistent problem, due to the lack of efficient control measures. It is therefore vital that novel approaches to control are pursued. Whilst vaccination is recognised as a potential control method to reduce tick infestation, no anti-R. sanguineus vaccine is available. Ticks depend on their blood meals to obtain nutrients and to achieve sexual maturity, which exposes them to vast amounts of iron. Although an essential molecule for several biological processes, its excess can lead to oxidative stress. Iron homeostasis is achieved with the help of iron-binding proteins called ferritins, among others, present in several tick tissues and developmental stages. These evolutionarily conserved proteins regulate iron homeostasis by storing and releasing iron in a controlled manner. In this study the R. sanguineus ferritin 1 gene was silenced through RNA interference (RNAi) in adult females exposed to an experimental infection with Ehrlichia canis. The purpose of this study was to assess the role of this protein in tick feeding, ovary development, oogenesis, and pathogen acquisition. Our data has shown that silencing ferritin 1 alters tick competence to normally engorge and causes morphologic and histochemical changes in the ovaries (OV) and oocytes. Furthermore, our data revealed that no E. canis DNA was found in either experimental group. Determining the function of molecules that act in key biological processes, such as blood digestion or reproduction, and that could be considered potential tick antigens will contribute towards the improvement of current control measures against these ectoparasites and the pathogens they vector.

Molecular, biological, and morphometric comparisons between different geographical populations of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

In this study, different geographical populations of Rhipicephalus sanguineus sensu lato were compared by molecular, biological, and morphometric methods. Phylogenetic trees were constructed using 12S and 16S rDNA sequences and showed two distinct clades: one composed of ticks from Brazil (Jaboticabal, SP), Cuba (Havana) Thailand (Bangkok) and the so-called "tropical strain" ticks. The second clade was composed of ticks from Spain (Zaragoza), Argentina (Rafaela, Santa Fe) and the so-called "temperate strain" ticks. Morphometric analysis showed good separation between females of the two clades and within the temperate clade. Males also exhibited separation between the two clades, but with some overlap. Multiple biological parameters revealed differences between the two clades, especially the weight of the engorged female. These results confirm the existence of at least two species under the name "R. sanguineus".

Oocyte maturation in the sloth's giant tick Amblyomma varium (Acari: Ixodidae) in an ecological context.

The sloth's giant tick Amblyomma varium Koch, which is a neotropical species that inhabits tropical rainforests, is the largest tick reported to date. The adult stage of this tick parasitizes mammals from the families Bradypodidae and Magalonychidae (Xenarthra) nearly exclusively. This study aimed to describe morphological and histological features of the reproductive system and the oocyte maturation process of this tick species. The ovary of A. varium is a long single tubular organ that is horseshoe-shaped, winding and arranged in the posterior part of the body. Two oviducts are connected to the ovary on each side; these thicken at certain region forming the uterus (common oviduct), followed by a muscular connecting tube, vagina and genital aperture. A large number of oocytes at different stages of development are attached to the ovary wall by the pedicel, as they reach maturity they are released into the ovary lumen and from there to the genital aperture. These oocytes develop simultaneously and asynchronically along the ovary. Amblyomma varium oocytes were classified into five development stages (i.e., I-V), and specific characteristics were observed; the processes of yolk and chorion deposition begin early in oocytes stage II, and oocytes V exhibit a very thick chorion and eggs of a large size. These characteristics are likely adaptations that enhance the survival and the reproductive success of this extremely host-specific tick, which is limited to a particular environment.

Copulation is necessary for the completion of a gonotrophic cycle in the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

Ovarian development and egg maturation are essential stages in animal reproduction. For bisexual ixodid ticks, copulation is an important prerequisite for the completion of the gonotrophic cycle. In this study, we aimed to characterize the morpho-histological changes in the ovary and oocytes of the tick Rhipicephalus sanguineus, together with the identification of feeding and reproductive parameters associated with mating. Virgin and cross-mated females (with R. turanicus males) weighed 60% less at full engorgement than females mated conspecifically. In addition, the oocytes of these females did not develop to the same advanced stages as those of the conspecifically mated females. Sequencing of a 250-bp ITS-2 fragment in eggs that originated from a cross between an R. sanguineus female and an R. turanicus male showed a genotype similar (except by a deletion of 1 thymine) to that observed in the mother, arguing against fertilization by a trans-specific male. These findings suggest that male sex peptides are species-specific molecules that influence both full engorgement and oocyte maturation. Mechanical stimulation of the gonopore alone was insufficient for the completion of the entire process of vitellogenesis.

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae).

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I-III and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption.

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 27 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.

Biological aspects of Amblyomma brasiliense (Acari: Ixodidae) under laboratory conditions.

Although Amblyomma brasiliense Aragão 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20 degrees C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.