Borrelia theileri in Bovine in the northern and southeastern regions of Brazil.

The present study aimed to describe the occurrence of Borrelia spp. in cattle in the states of Minas Gerais and Pará in southeastern and northern Brazil, respectively. Bovine whole blood samples were examined by blood smear and polymerase chain reaction (PCR) to detect the flagellin B (flaB) gene of Borrelia spp. Frequencies of positive animals for Borrelia spp. were 1.52% (2/132) in the municipality of Unaí, Minas Gerais, and 14.2% (2/7) in the municipality of Marabá, Pará. Subsequent genetic sequencing confirmed that the detected spirochetes close to the species B. theileri. In both locations, the animals positive for B. theileri were also highly infested by Rhipicephalus microplus ticks. Despite the low frequency of Borrelia spp., the occurrence of this spirochete indicates that further studies are needed to determine the consequences in cattle herds.

Comparison of the efficacy of different methods to apply acaricides for control of Rhipicephalus (Boophilus) microplus.

The present study compared the efficacy of different methods to apply an acaricide formulation to control Rhipicephalus (Boophilus) microplus. To compare the methods, an acaricide blend containing three active ingredients (a pyrethroid and two organophosphates) was used. In experiment 1 (farm 1: Goiânia, GO, Brazil), three methods were tested: a backpack sprayer (BS), power sprayer (PS) and spray race (SR). In experiment 2 (farm 2: São José do Rio Pardo, SP, Brazil), two methods were tested: BS and PS. In both experiments, 10 cattle with similar tick burdens were used. On day 0 in both experiments, the animals were treated with the acaricide. On day +1 (only in experiment 1), +3, +7, +14, +21, +28 and +35 (only in experiment 2), tick counts were performed to determine the control efficacy. The time application, pressure (KPa), volume applied (L) and ergonomic aspects of each spraying system were also evaluated. The adult immersion test (AIT) using three different acaricide blends (combinations of pyrethroid + organophosphate) was performed to compare the susceptibility of strains of each farm. In experiment 1, all treatments significantly reduced (p < 0.05) the number of ticks on the animals, and PS resulted in the greatest acaricide efficacy since day +1. In experiment 2, both treatments (PS and BS) reduced (p < 0.05) the tick burden, and as observed in experiment 1, PS resulted in the best reduction. The application times were 4.5, 150 and 330 s, while pressures were 306.8, 4,826.3 and 220.6 KPa for SR, PS and BS, respectively. In the AIT, the efficacy values were between 99.8 and 100% for the tick strain form farm 1 (Goiânia), while for tick strain from farm 2 (São José do Rio Pardo), the efficacy was between 67.2 and 80.9%. We conclude that the sprayer methods chosen influences the efficacy of the acaricide. All sprayer methods were efficient for acaricide application; the best efficacy was obtained using the PS, while the SR resulted in good efficacy and lower application time. The strain from farm 2 was less susceptible to all acaricides tested.

Identification and Characterization of Jingmen Tick Virus in Ticks from Yunnan Imported Cattle.

Jingmen tick virus (JMTV) is a tick-borne segmented positive-sense ssRNA virus that can cause human disease. This virus has been confirmed to be widespread, having a wide host range. In human it can cause fever, headache, lymphadenopathy, and asthenia. Therefore, JMTV poses a threat to public health. In this study, we collected 478 ticks from imported cattle on three quarantine farms near the Yunnan border to detect medically significant tick-borne viruses. Our findings show that JMTV was the only detected virus, with an incidence rate of 56.67%. Phylogenetic analysis showed that our JMTV is more closely related to previously reported JMTV strains from Yunnan Province and neighboring Laos, implying that the tick-borne virus was most likely imported from Laos. In conclusion, we identified and characterized a novel JMTV strain in tick (Rhipicephalus microplus) from Yunnan imported cattle, emphasizing the importance of arbovirus quarantine of livestock imports.

Proteomic analysis of extracellular vesicles from tick hemolymph and uptake of extracellular vesicles by salivary glands and ovary cells.

BACKGROUND: Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that are important mediators of intercellular communication. Arthropods transport nutrients, signaling molecules, waste and immune factors to all areas of the body via the hemolymph. Little is known about tick hemolymph EVs. METHODS: Hemolymph was collected from partially fed Rhipicephalus haemaphysaloides and Hyalomma asiaticum ticks by making an incision with a sterile scalpel in the middle (between the femur and metatarsus) of the first pair of legs, which is known as leg amputation. EVs were isolated from hemolymph by differential centrifugation and characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteins extracted from the hemolymph EVs were analyzed by 4D label-free proteomics. The EVs were also examined by western blot and immuno-electron microscopy analysis. Intracellular incorporation of PHK26-labeled EVs was tested by adding labeled EVs to tick salivary glands and ovaries, followed by fluorescence microscopy. RESULTS: In this study, 149 and 273 proteins were identified by 4D label-free proteomics in R. haemaphysaloides and H. asiaticum hemolymph EVs, respectively. TEM and NTA revealed that the sizes of the hemolymph EVs from R. haemaphysaloides and H. asiaticum were 133 and 138 nm, respectively. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses of identified proteins revealed pathways related to binding, catalytic and transporter activity, translation, transport and catabolism, signal transduction and cellular community. The key EV marker proteins RhCD9, RhTSG101, Rh14-3-3 and RhGAPDH were identified using proteomics and western blot. The presence of RhFerritin-2 in tick hemolymph EVs was confirmed by western blot and immuno-electron microscopy. We demonstrated that PKH26-labeled hemolymph EVs are internalized by tick salivary glands and ovary cells in vitro. CONCLUSIONS: The results suggest that tick EVs are secreted into, and circulated by, the hemolymph. EVs may play roles in the regulation of tick development, metabolism and reproduction.

Molecular and serological evidence of Crimean-Congo hemorrhagic fever orthonairovirus prevalence in livestock and ticks in Cameroon.

Introduction: Despite a high fatality rate in humans, little is known about the occurrence of Crimean-Congo hemorrhagic fever virus (CCHFV) in Cameroon. Hence, this pioneer study was started with the aim of determining the prevalence of CCHFV in domestic ruminants and its potential vector ticks in Cameroon. Methods: A cross-sectional study was carried out in two livestock markets of Yaoundé to collect blood and ticks from cattle, sheep, and goats. CCHFV-specific antibodies were detected in the plasma using a commercial ELISA assay and confirmed using a modified seroneutralization test. Ticks were screened for the presence of orthonairoviruses by amplification of a fragment of the L segment using RT-PCR. Phylogeny was used to infer the genetic evolution of the virus. Results: Overall, 756 plasma samples were collected from 441 cattle, 168 goats, and 147 sheep. The seroprevalence of CCHFV was 61.77% for all animals, with the highest rate found in cattle (433/441, 98.18%) followed by sheep (23/147, 15.65%), and goats (11/168, 6.55%), (p-value < 0.0001). The highest seroprevalence rate was found in cattle from the Far North region (100%). Overall, 1500 ticks of the Rhipicephalus (773/1500, 51.53%), Amblyomma (341/1500, 22.73%), and Hyalomma (386/1500, 25.73%) genera were screened. CCHFV was identified in one Hyalomma truncatum pool collected from cattle. Phylogenetic analysis of the L segment classified this CCHFV strain within the African genotype III. Conclusion: These seroprevalence results call for additional epidemiological studies on CCHFV, especially among at-risk human and animal populations in high-risk areas of the country.

Density Functional Theory Calculations and Molecular Docking Analyses of Flavonoids for Their Possible Application against the Acetylcholinesterase and Triose-Phosphate Isomerase Proteins of Rhipicephalus microplus.

Ticks and tick-borne diseases constitute a substantial hazard to the livestock industry. The rising costs and lack of availability of synthetic chemical acaricides for farmers with limited resources, tick resistance to current acaricides, and residual issues in meat and milk consumed by humans further aggravate the situation. Developing innovative, eco-friendly tick management techniques, such as natural products and commodities, is vital. Similarly, searching for effective and feasible treatments for tick-borne diseases is essential. Flavonoids are a class of natural chemicals with multiple bioactivities, including the inhibition of enzymes. We selected eighty flavonoids having enzyme inhibitory, insecticide, and pesticide properties. Flavonoids' inhibitory effects on the acetylcholinesterase (AChE1) and triose-phosphate isomerase (TIM) proteins of Rhipicephalus microplus were examined utilizing a molecular docking approach. Our research demonstrated that flavonoids interact with the active areas of proteins. Seven flavonoids (methylenebisphloridzin, thearubigin, fortunellin, quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside), quercetagetin-7-O-(6-O-p-coumaroyl-ß-glucopyranoside), rutin, and kaempferol 3-neohesperidoside) were the most potent AChE1 inhibitors, while the other three flavonoids (quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside), isorhamnetin, and liquiritin) were the potent inhibitors of TIM. These computationally-driven discoveries are beneficial and can be utilized in assessing drug bioavailability in both in vitro and in vivo settings. This knowledge can create new strategies for managing ticks and tick-borne diseases.

Molecular detection and phylogenetic analysis of Anaplasma platys-like and Candidatus Anaplasma boleense strains from Argentina.

The present study aimed at the molecular detection of Anaplasma spp. in different samples obtained from cattle, goats and free-living Rhipicephalus microplus ticks from Argentina. DNA of members of the Anaplasmataceae family was detected by different PCR assays. The phylogenetic analyses of the obtained partial DNA sequences of the 16 S rDNA gene resulted in the identification of two different Anaplasma spp.: (I) Anaplasma platys-like bacteria (in blood sample from cattle and pools of R. microplus larvae and (II) Candidatus Anaplasma boleense (in blood samples from goats and one pool of R. microplus larvae of R. microplus). Candidatus A. boleense was found in two provinces that belong to different biogeographic regions, which leads to the conclusion that this bacterium may be widely distributed in Argentina. Interestingly, both Anaplasma spp. were found in the same R. microplus population in Chaco province, indicating that these two strains of Anaplasma are circulating in the same tick population. The results of this work represent the first report of the circulation of A. platys-like bacteria and Ca. A. boleense in domestic ruminants and free-living R. microplus ticks in Argentina. Further studies to determine the prevalence of infection, dispersion, clinical impact, transmission routes and cross-reactivity in serological tests of both Anaplasma species are needed.

Anaplasma species infecting questing ticks in the Iberá wetlands ecoregion, Argentina.

The present study evaluated the presence of Anaplasma species in questing ticks from six sites with opposing land usage (i.e., protected natural areas or livestock establishments) within the Iberá wetlands ecoregion in Argentina. The ticks were determined as Amblyomma dubitatum (n = 15,096), Rhipicephalus microplus (n = 399), Amblyomma triste (n = 134), Haemaphysalis juxtakochi (n = 5), and Amblyomma tigrinum (n = 1). Using a real-time PCR assay targeting the 16S rRNA gene, Anaplasma sp. was detected in A. dubitatum samples (one nymph, three nymph pools and one larvae pool) and one R. microplus larvae pool. The overall minimum infection rate (MIR) for Anaplasma sp. in questing A. dubitatum nymphs was 0.169% (0.175% in protected natural areas and 0% in livestock establishments). For R. microplus, overall Anaplasma sp. MIR was 0.25% (0.52% in protected natural areas and 0% in livestock establishments). Phylogenetic analysis positioned the Anaplasma sp. from A. dubitatum in the same clade as Anaplasma odocoilei, whereas the Anaplasma sp. from R. microplus was related to Anaplasma platys. In conclusion, these results support a possible role of A. dubitatum in the ecology of the Anaplasma agent reported to infect capybaras in the region.

Acaricidal action of ozone on larvae and engorged females of Rhipicephalus microplus: a dose-dependent relationship.

The tick Rhipicephalus microplus is a vector of infectious agents that causes great economic loss in the productivity of cattle herds. Several studies have sought natural compounds with acaricidal activity to control ticks, without allowing the development of resistance, without causing environmental damage, and without presenting toxicity to the hosts. The activity of ozone on the natural biomolecules of living beings has been studied as an alternative to control arthropods and acaricidal effects were shown on ticks. The aim of the present study was to assess the acaricidal effect on larvae and engorged females of R. microplus according to ozone dose. Larvae (n = 377) were distributed in 10 groups and engorged females (n = 284) were distributed in 14 groups. One group was used as control (not exposed to ozone) and the other groups were exposed to ozone gas for 5-105 min. Ozone had a dose-dependent acaricidal effect on both larvae and engorged females. Dosages between 355 and 2130 mg/L min had a delayed acaricidal effect (12-180 h), leading to the death of all engorged females before laying eggs, whereas doses between 3195 and 7455 mg/L min showed immediate acaricidal effect (5 min to 4 h). Doses between 1775 and 6390 mg/L min had an immediate (up to 5 min) acaricidal effect on the larvae of this species. Further studies should consider longer follow-up times during the assessment of the acaricidal activity against ticks.

First record of Rhipicephalus (Boophilus) microplus in Ghana, a potential risk to livestock production.

Ticks are a public health threat due to their tendency to spread pathogens that affect humans and animals. With reports of Rhipicephalus (Boophilus) microplus invasion in neighbouring countries, there is the risk of this species invading Ghana through livestock trade. Previous identification of tick species in Ghana has been based on morphological identification, which can be ineffective, especially with damaged tick specimens or engorged nymphs. This study focused on the Kassena-Nankana District, which serves as a trade route for cattle into Ghana, to determine the presence of R. microplus. Three genera of ticks were identified as Amblyomma (70.9%), Hyalomma (21.3%) and Rhipicephalus (7.8%). The engorged nymphs that could not be identified morphologically were analyzed using primers that target the mitochondrial 16S rRNA gene. This study reports the first record of R. (B.) microplus in Ghana. Furthermore, R. microplus constituted 54.8% of the Boophilus species collected in this study. This finding is an addition to the diverse tick species previously collected in Ghana, most of which are of veterinary and public health importance. With reports of acaricide resistance in R. microplus and its role in spreading infectious pathogens, the detection of this species in Ghana cannot be overlooked. Nationwide surveillance will be essential to ascertain its distribution, its effects on cattle production, and the control measures adopted.

Evaluation of the in vitro acaricidal effect of five organic compounds on the cattle fever tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important tick worldwide. Infestations with this tick can lead to direct damage and cattle mortality due to the transmission of potentially deadly pathogens. Management of this tick species has been focused on the use of synthetical acaricides; however, the emergence of acaricide resistance to single or multiple active ingredients has resulted in a need for novel acaricide compounds. Among potential avenues for the discovery of novel acaricides are plant-derived compounds. The efficacy of five organic compounds (nootkatone, Stop the Bites®, BioUD®, lavender oil, and cedarwood oil) was evaluated using larval immersion tests (LITs), repellency assays, and adult immersion tests (AITs). The results from the LITs indicate that three of the organic compounds (NootkaShield™, Stop the Bites, BioUD) led to significant mortalities at low concentrations (0.2, 0.02, and 0.08%, respectively). By comparison, lavender and cedar oil led to around 90% mortality at 10 and 1% concentrations, respectively. Similarly, NootkaShield, Stop the Bites, and BioUD had strong repellent properties with over 90% repellency at the two highest concentrations tested. Using the FAO 2004 guidelines, we evaluated the effectiveness of these organic compounds at reducing the fecundity of R. (B.) microplus and show that Nootkatone, Stop the Bites, and BioUD may significantly decrease tick populations (Drummond's index > 90% at concentrations of 5%), highlighting their potential as alternatives to synthetic acaricides for the control of cattle fever ticks.

Acaricides containing zein nanoparticles: A tool for a lower impact control of the cattle tick Rhipicephalus microplus.

Nanoformulations containing zein nanoparticles (ZN) can promote the stability and protection of molecules with acaricidal activity. The present study sought to develop nanoformulations with ZN associated with cypermethrin (CYPE) + chlorpyrifos (CHLO) + a plant compound (citral, menthol or limonene), characterize them, and verify their efficacy against Rhipicephalus microplus ticks. Additionally, we aimed to assess its safety in nontarget nematodes found in soil at a site subjected to contamination by acaricides. The nanoformulations were characterized by dynamic light scattering and nanoparticle tracking analysis. Nanoformulations 1 (ZN+CYPE+CHLO+citral), 2 (ZN+CYPE+CHLO+menthol), and 3 (ZN+CYPE+CHLO+limonene) were measured for diameter, polydispersion, zeta potential, concentration, and encapsulation efficiency. Nanoformulations 1, 2, and 3 were evaluated in a range from 0.004 to 0.466 mg/mL on R. microplus larvae and caused mortality > 80% at concentrations above 0.029 mg/mL. The commercial acaricide Colosso® (CYPE 15 g + CHLO 25 g + citronellal 1 g) was evaluated also from 0.004 to 0.512 mg/mL and resulted in 71.9% larval mortality at 0.064 mg/mL. Formulations 1, 2, and 3 at 0.466 mg/mL showed acaricidal efficacy of 50.2%, 40.5%, and 60.1% on engorged females, respectively, while Colosso® at 0.512 mg/mL obtained only 39.4%. The nanoformulations exhibited long residual period of activity and lower toxicity to nontarget nematodes. ZN was able to protect the active compounds against degradation during the storage period. Thus, ZN can be an alternative for the development of new acaricidal formulations using lower concentrations of active compounds.

A multiepitope chimeric antigen from Rhipicephalus microplus-secreted salivary proteins elicits anti-tick protective antibodies in rabbit.

Rhipicephalus (Boophilus) microplus, the Cattle Fever Tick, causes significant economic losses in livestock in tropical and subtropical regions of the world. As the usual control strategy based on chemical acaricides presents different drawbacks, alternative control strategies have been considered for tick control. In recent decades, several tick proteins have been evaluated as targets for the development of anti-tick vaccines. Thus, in the present work, coding sequences from three different proteins present in tick saliva were employed together to construct a recombinant chimeric protein that was evaluated as an antigen in rabbit immunization. Then, the elicited antibodies were tested in a tick artificial feeding experiment to verify the protective effect against the parasites. In addition to Rhipicephalus microplus subtilisin inhibitor 7 (RmSI-7), a serine protease inhibitor member of the TIL (Trypsin Inhibitory Like) family, an interdomain region from the Kunitz inhibitor BmTI-A, and a new cysteine-rich AMP-like microplusin, called RmSEI (previously identified as an elastase inhibitor), were selected to compose the chimeric protein. Anti-chimeric IgG antibodies were able to affect R. microplus female egg production after artificial feeding. Moreover, antibodies elicited in infested tick-resistant and tick-susceptible cattle recognized the recombinant chimera. Additionally, the functional characterization of recombinant RmSEI was performed and revealed antimicrobial activity against gram-positive bacteria. Moreover, the antimicrobial protein was also recognized by antibodies elicited in sera from cattle previously exposed to R. microplus bites. Together, these data suggest that the chimeric protein composed of three salivary antigens is suitable for anti-tick vaccine development.

Encapsulation of entomopathogenic fungal conidia: evaluation of stability and control potential of Rhipicephalus microplus.

The use of chemical acaricides is the primary strategy to control tick infestations. Nonetheless, chemical resistance in ticks has been reported. Thus, complementary methods such as biological control using entomopathogenic fungi (EPF) have been investigated. EPF, although efficient, have their viability compromised when applied under natural conditions, which indicates that formulation development is essential. Some researchers have demonstrated the efficacy of ionic gelation in protecting EPF against deleterious abiotic factors. In the present study, we conducted the ionic gelation technique to encapsulate Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) conidia in 2% (EC 2%) and 3% (EC 3%) sodium alginate. Next, the quantity and viability of encapsulated conidia (EC) were determined. The morphology of particles was characterized by using Scanning Electron Microscopy (SEM). EC and non-encapsulated conidia (NEC) were stored at room temperature (26.8 °C) and in the freezer (-11.9 °C) to shelf-life testing. For UV-B irradiance tolerance and thermotolerance tests, EC and NEC were exposed to UV-B (6.0 or 8.0 kJ m - 2) and heat (42 ºC). In addition, biological parameters of Rhipicephalus microplus Canestrini (Acari: Ixodidae) engorged females exposed to EC were evaluated. The particles presented a spherical shape, more homogeneous (EC 2%) or heterogeneous (EC 3%). Encapsulation decreased (4.8×) the conidial concentration and did not affect their viability. On the other hand, encapsulation increased the shelf life of conidia at room temperature as well as their UV-B tolerance and thermotolerance (6 h). The fungal particles decreased the biological parameters of females more significantly than the NEC. As far as we know, we reported for the first time the use of the ionic gelation to encapsulate entomopathogenic fungi toward controlling R. microplus.

Prevalence of cattle ixodid ticks and acaricides efficacy evaluation in Amibara district of Afar Region, Ethiopia.

A cross sectional and randomized controlled trial study was conducted starting from July 2021 to July 2022 to determine the prevalence of ixodid ticks, and evaluate the efficacy of commonly used acaricides (amitraz 12.5% and diazinon 60%) in vitro and in vivo on cattle ticks in Amibara district. A total of 372 cattle were visited randomly from six kebeles of the district which were selected based on their livestock population. A total of 4330 adult ticks were collected and identified to the species level with stereomicroscope. For the invitro acaricide efficacy test, 90 engorged female ticks were collected from infested herds and grouped into three, each group having ten engorged ticks; two groups for the two test acaricides and the third groups were control to evaluate these acaricides in the laboratory. All the groups were replicated three times and incubated for seven days with similar temperature and humidity. Thirty-six infested cattle were selected and again grouped into three randomly, 12 each for the invivo test. The first groups of animals were sprayed with amitraz, the second with diazinon and the third groups were left untreated. Ticks were counted and recorded before, day 3, day 7, day 14 and day 21 of spraying. A total of 360 (96.7%) of the visited cattle were infested with ticks. Amblyomma. Rhipicephallus, Hyalomma and Bophillus were the genera of ticks identified with a prevalence of 44.9%, 41.4%, 7.5% and 6.2% respectively. A total of 15 species of ixodid ticks were identified from which Rhipicephallus pulchellus (39.86%) was dominant followed by Amblyomma lepidum (26.65%) and Amblyomma gemma (14.36%). Diazinon 60% EC and amitraz 12.5% were significantly inhibited the egg laying ability of engorged ticks' in vitro with percent inhibition of 100% and 99.1% respectively. On live animal, both acaricides showed statistically significant difference from the control group (p- value <0.05) with maximum 97.37% (amitraz) and 93.33% (diazinon) at day 7 and minimum 73.3% (amitraz) and 73.7% (diazinon) efficacy. However, there was no statistically significant difference on egg laying inhibition and tick count reduction between the two acaricides (p-value >0.05). Therefore, awareness should be created about the high prevalence as well as the application, dosage and dilution of the acaricides to pastoralists, community animal health workers and para veterinarians in the study area. Cattle owners, veterinarians and concerned government officials should address infested herds with manufacturer recommended dosage of these acaricides to control the tick infestation.

Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite.

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.

MALDI-TOF MS as a tick identification tool in a tertiary hospital in Spain.

In Spain, as in other countries, the spectrum of tick-borne diseases and their number have increased in recent years. The tick identification, at species level, can be challenging outside research centers although this information is very usufull for decisions making. The performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in tick identification of specimens collected from patients have been seldomly reported. The aim of the present study was to desing a protein-extraction protocol and build a tick-legs reference spectra. This protocol was then validated using specimens from both patients and non-patient sources. Nine species of ticks that usually bites humans in Spain were included: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hyalomma marginatum, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus pusillus and Rhipicephalus sanguineus sensu lato. Other less-frequent biting species were also included: Haemaphysalis inermis, Haemaphysalis concinna, Hyalomma scupense, Ixodes frontalis, Ixodes hexagonus, and Argas sp. specimens were identified by PCR and sequencing of a fragment of the 16S rRNA gene of ticks. In the tests performed with non-patient collected specimens, a 100% correlation was observed between molecular methods and MS, while in the tests performed with ticks collected from patients a 92.59% correlation was observed. Misidentification was observed only in two of I. ricinus nymphs (identified as Ctenocephalides felis). Therefore, mass- spectrometry can be confidently used as a tick identification tool in a hospital setting for the rapid identification of tick vectors.

The ovaries of ivermectin-resistant Rhipicephalus microplus strains display proteomic adaptations involving the induction of xenobiotic detoxification and structural remodeling mechanisms.

Controlling Rhipicephalus microplus is among the most significant challenges for livestock production worldwide. The indiscriminate use of acaricides stimulates the selection of resistant tick populations and is therefore ineffective. Understanding the molecular foundations of resistance could help inform the search for new alternatives for tick control. Although the ovary has been suggested as a relevant target organ for tick control, there are few existing studies that focus on tick ovarian tissue. Therefore, we conducted a comparative proteomic analysis on ovaries of R. microplus strains with differential resistance to ivermectin. In resistant ticks, we observed the over-accumulation of proteins involved in several biological processes, including translation, proteolysis, transport, cellular organization, differentiation, and xenobiotic detoxification. We also observed the accumulation of many structural and extracellular proteins such as papilin-like protein, which glycosylation increase its stability-based molecular modeling. Therefore, we propose that ovaries of ivermectin-resistant ticks overcome the negative impact of ivermectin through the activation of detoxification mechanisms and structural proteins associated with the remodeling of the ovary's extracellular matrix. SIGNIFICANCE: Understanding the molecular foundation of ivermectin resistance in Rhipicephalus microplus represents an essential step in cattle farming, which could provide clues and alternatives for tick control. Excessive use of chemicals like ivermectin allows the generation of resistant tick strains in different countries. However, limited molecular information is available concerning the tick's resistance to ivermectin. Detailed proteomics scrutiny in various tick organs will provide more comprehensive molecular information. Thus, we conducted an ovary comparative proteomic-based TMT-SPS-MS3 approach. We highlight in ivermectin-resistant ticks the over-accumulation of structural proteins and enzymes connected to detoxification mechanisms.

Identification of the G184C, C190A and T2134A mutations in the para-sodium channel gene of the southern cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to cypermethrin in northern Veracruz, Mexico.

The southern cattle tick (Rhipicephalus microplus) represents one of the ectoparasites with the greatest distribution worldwide. Infestations by this arthropod can cause a decrease in the production of meat and milk, as well as anaemia and the transmission of bacterial and parasitic agents. For this reason, several active molecules have been developed to control these arthropods. A widely used group of ixodicides are pyrethroids, especially cypermethrin, which have knockdown effects on ticks. Resistance to cypermethrin has been reported in ticks since the 2000s; it was registered for the first time in Mexico in 2009. Even though multiple studies have evaluated resistance with conventional tests, there are few studies in Mexico that have identified the presence of single nucleotide polymorphisms (SNPs) associated with resistance. Hence, the aim of this work was to monitor three mutations associated with resistance in the sodium/chlorine channel in eight populations of ticks from northern Veracruz. Engorged adult females were collected from which genomic DNA was extracted. Subsequently, three mutations in domains II and III of para­sodium channel gene were detected by conventional PCR and sequencing. Global alignments were done with the reference sequences deposited in GenBank. A total of 116 engorged females were analysed, of which 10 tested positive for G184C and C190A of domain II of the para­sodium channel gene. T2134A was present in domain III in a single production unit. This is the first work where molecular monitoring of cypermethrin resistance has been carried out in the northern zone of the state of Veracruz.

Analysis of the tick communities associated to domestic mammals in rural areas of the Yungas montane forest from Argentina.

The aim of this work was to describe the tick community associated to domestic mammals in rural areas from the Yungas lower montane forest of Argentina. The circulation of tick-borne pathogens was also analyzed. Samples of ticks parasitizing cattle, horses, sheep and dogs were carried out in different seasons, and questing ticks were collected from vegetation and analyzed to detect the presence of Rickettsia, Ehrlichia, Borrelia and Babesia by a battery of different PCRs. The structure of the tick communities was analyzed through the Chao1 species richness estimator, the Shannon-Wiener index and the Horn index of community similarity. Eight tick species were collected in the study area: Amblyomma sculptum, Rhipicephalus microplus, Amblyomma hadanii, Dermacentor nitens, Amblyomma ovale, Haemaphysalis juxtakochi, Ixodes pararicinus and Rhipicephalus sanguineus sensu stricto. However, A. sculptum was by far the dominant species in the tick assemblages analyzed, and this was reflected in the low diversity values obtained. Dermacentor nitens, A. sculptum and R. microplus were the three species associated to horses. The predominance of A. sculptum was also observed in the tick samples obtained from dogs, even on two tick species, namely A. ovale and R. sanguineus s.s., which have dogs as the principal domestic host. Rhipicephalus microplus and A. sculptum were the most abundant ticks on cattle, while few specimens of I. pararicinus, A. hadanii and D. nitens were found on bovines. Dermacentor nitens ticks were found to be infected with B. caballi, which indicate the circulation of this pathogen of horses in the Yungas area. The detection of a strain of Borrelia sp. belonging to the B. burgdorferi s.l. complex in I. pararicinus is consistent with previous findings made in Argentina, but the public health relevance of this vector-microorganism association is far from being similar to that occurs in the northern hemisphere because there are practically no records of these tick species parasitizing humans in South America. The tick community of rural areas of the Yungas lower montane forest is composed by species which are potential vectors of pathogenic microorganism with veterinary and public health importance, circulating in a human-wildlife-livestock interface.