Tick Rhipicephalus Microplus Research Articles

Vaccination of cattle with the Babesia bovis sexual-stage protein HAP2 abrogates parasite transmission by Rhipicephalus microplus ticks

The apicomplexan parasite Babesia bovis is responsible for bovine babesiosis, a poorly controlled tick-borne disease of global impact. The widely conserved gametocyte protein HAPLESS2/GCS1 (HAP2) is uniquely expressed on the surface of B. bovis sexual stage parasites and is a candidate for transmission-blocking vaccines (TBV). Here, we tested whether vaccination of calves with recombinant HAP2 (rHAP2) interferes with the transmission of B. bovis by competent ticks. Calves vaccinated with rHAP2 (n = 3), but not control animals (n = 3) developed antibodies specific to the vaccine antigen. Vaccinated and control animals were infested with Rhipicephalus microplus larvae and subsequently infected with virulent blood stage B. bovis parasites by needle inoculation, with all animals developing clinical signs of acute babesiosis. Engorged female ticks fed on the infected calves were collected for oviposition, hatching, and obtention of larvae. Transmission feeding was then conducted using pools of larvae derived from ticks fed on rHAP2-vaccinated or control calves. Recipient calves (n = 3) exposed to larvae derived from control animals, but none of the recipient calves (n = 3) challenged with larvae from ticks fed on rHAP2-vaccinated animals, developed signs of acute babesiosis within 11 days after tick infestation. Antibodies against B. bovis antigens and parasite DNA were found in all control recipient animals, but not in any of the calves exposed to larvae derived from HAP2-vaccinated animals, consistent with the absence of B. bovis infection via tick transmission. Overall, our results are consistent with the abrogation of parasite tick transmission in rHAP2-vaccinated calves, confirming this antigen as a prime TBV candidate against B. bovis.

Efficacy of hydroethanolic extract of Randia aculeata seed against the southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on naturally infested cattle under field conditions.

The cattle tick Rhipicephalus (Boophilus) microplus is a major problem of concern for cattle industry in tropical and subtropical areas. Control of cattle tick is based mainly on the use of chemical acaricides, which has contributed to the emerging problem of selection of resistant tick lineages. Plants have been used as an alternative to conventional acaricidal drugs. On the other hand, the acaricidal activity of hydroethanolic extract of Randia aculeata seed (EHRA) has been demonstrated against R. microplus under laboratory conditions. However, the utility of EHRA seed as a potential acaricidal needs to be determined under field conditions. For this reason, the aim of this study was to evaluate the efficacy of the EHRA against R. microplus sprayed on naturally infested calves, determine the effect of theEHRA seedon acetylcholinesterase activity in R. microplus larval and identify the chemical composition of EHRA. Forty-five male calves were divided in three groups and treated with: G1 water; G2 EHRA 20% w/v and G3 coumaphos 0.2% v/v. Acetylcholinesterase (AChE) activity in R. microplus larvae was determined by a colorimetric assay. The chemical composition of EHRA was accessed through HPLC/MS. Significantly fewer ticks were observed after 24h on the treated group compared to control group. EHRA significantly inhibitedin vitroAChE activity inR.microplus at all tested concentrations. Chlorogenic acid, vanillinic acid, p-coumaric acid, caffeic acid. rutin, quercetin, (-)-epicatechin, 4-hydroxybenzoic acid, quercetin, vanillin, 2,4-dimethoxy-6-methylbenzoic acid, scopoletin and ferulic acid were identified in the extract. The results provided new data for the elucidation of the mechanisms of EHRA acaricide action and to further evaluate the use as a new alternative control agent againstR. microplus under in vivo conditions.

Effect of a preventive strategic control program, with imidocarb dipropionate, against tick fever agents in dairy calves.

Chemoprophylaxis with dipropionate imidocarb (IMD) is a method adopted to prevent cattle tick fever (TF). Sixty weaned dairy heifers (±60 days old), without previous exposure to Rhipicephalus microplus ticks, were housed in Tifton paddocks and were subsequently exposed to R. microplus ticks and monitored up to 315 days old. Thirty animals were kept as controls (T01) and 30 received five preventive strategic treatments with IMD at 21-day intervals (T02). The heifers were monitored weekly by means of packed cell volume (PCV) and blood smears to evaluate the presence of TF agents. Salvage treatments (ST) with diminazene and enrofloxacin were administered when animals showed PCV ≤ 24%. The A. marginale prevalence was 39.3% and 37.7%, B. bovis 6.0%, and 7.3%, and B. bigemina 16.3% and 13.7% for T01 and T02, respectively. Regarding PCV values, group T01 showed lower PCV than group T02, between 119 and 161 days of life, but when animals were 196, 210, 217, and between 252 to 301 days old, an inversion occurred. The IMD treatment protocol was effective in group T02 from day 91 to 175 while treatment was being administered, but from day 182 to 315 after the IMD treatment protocol was completed, the number of salvage treatments against TF agents performed in T02 group increased significantly. The sequential application of IMD treatments with intervals less than 21 days is not recommended.

Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

Therapeutic Management of Babesiosis in a Cow: A Case Report

Babesiosis is a haemoprotozoan disease intraerythrocytic protozoan parasites of the genus Babesia that infect a wide range of domestic and wild animals. It is one of the common tick borne haemoprotozoan diseases affecting the bovines in tropical and subtropical parts of Africa, Australia, America, and Asia including India (Kumar and Kala, 2018). In India, annual economic losses to livestock due to babesiosis are estimated to be about 57.2 million US dollars which is mainly caused by two most important species, i.e., Babesia bovis and Babesia bigemina (Bock et al., 2004). The cattle tick Rhipicephalus microplus is the main vector involved with the transmission and is the only known vector for bovine babesiosis (Souza et al., 2018). Due to universal distribution of the ixodid tick, babesiosis is considered as the second most widespread blood-borne disease of animals (Homer et al., 2000) and is prominently gaining increasing interest as an emerging zoonosis of humans also (Homer et al., 2000; Zintl et al., 2003). Clinical symptoms of Babesiosis are classical haemoglobinuria which is often present, with anaemia and jaundice that develops especially in more protracted cases (Bock et al., 2004). The disease has been recorded in all the cattle breeds but more commonly in exotic and crossbred cattle than in indigenous ones (Chakrabarti, 2003). The present communication describes the haemato-biochemical profile and therapeutic management of a non-descript native cow with babesiosis infection.

Biochemical characterization of a novel sphingomyelinase-like protein from the Rhipicephalus microplus tick

Sphingomyelinase D is a toxin present in venomous spiders and bacteria and is associated with infection symptoms in patients affected by spider bites. It was observed that in Ixodes scapularis ticks, sphingomyelinase-like protein secreted in saliva can modulate the host immune response, affecting the transmission of flavivirus to the host via exosomes. In this work, a sphingomyelinase D–like protein (RmSMase) from R. microplus, a tick responsible for economic losses and a vector of pathogens for cattle, was investigated. The amino acid sequence revealed the lack of important residues for enzymatic activity, but the recombinant protein showed sphingomyelinase D activity. RmSMase shows Ca2+ and Mg2+ dependence in acidic pH, differing from IsSMase, which has Mg2+ dependence in neutral pH. Due to the difference between RmSMase and other SMases described, the data suggest that RmSMase belongs to SMase D class IIc. RmSMase mRNA transcription levels are upregulated during tick feeding, and the recombinant protein was recognized by host antibodies elicited after heavy tick infestation, indicating that RmSMase is present in tick saliva and may play a role in the tick feeding process.

Comparative Study of Two Monoterpenes Effect on Rhipicephalus microplus Tick

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites for livestock in tropical and subtropical areas worldwide. This tick hurts the economy of the milk and meat production chain. In addition, it constitutes a vector for the transmission of Anaplasmosis and Babesiosis pathogens. The control of R. microplus populations is mainly based on the use of synthetic acaricides. However, using this control method presents a danger to humans and the environment and leads to the emergence of resistant tick populations. In this situation, searching for ecological and effective control alternatives is essential. Thus, plant extracts constitute a promising solution, particularly essential oils and their active compounds. Thus, the present study aims to assess the acaricidal activity of two monoterpenes (Thymol and 1.8 cineole) abundantly found in essential oils to find an alternative to synthetic acaricides. The acaricidal activity was determined according to the method of larval immersion test (LIT). Eight concentrations were tested and R software version 4.0.3 was used for data analysis. Results showed 100 % larval mortality rates for the two monoterpenes with LC50 and LC90 values of (0.28 and 0.64) and (0.64 and 2.66) respectively, for thymol and 1.8 cineole for the immersion time of 5 min. For 10 min of immersion, all LC values decreased for the two monoterpenes. These findings highlight the potential of the thymol and 1.8 cineole as an alternative for managing R. microplus tick.

Identification of Virulence Factors in Entomopathogenic Aspergillus flavus Isolated from Naturally Infected Rhipicephalus microplus.

Aspergillus flavus has been found to be an effective entomopathogenic fungus for various arthropods, including ticks. In particular, natural fungal infections in cattle ticks show promise for biocontrol of the Rhipicephalus (Boophilus) microplus tick, which is a major ectoparasite affecting cattle worldwide. Our study aimed to elucidate the specific entomopathogenic virulence factors encoded in the genome of an A. flavus strain isolated from naturally infected cattle ticks. We performed morphological and biochemical phenotyping alongside complete genome sequencing, which revealed that the isolated fungus was A. flavus related to the L morphotype, capable of producing a range of gene-coded entomopathogenic virulence factors, including ribotoxin, aflatoxin, kojic acid, chitinases, killer toxin, and satratoxin. To evaluate the efficacy of this A. flavus strain against ticks, we conducted experimental bioassays using healthy engorged female ticks. A morbidity rate of 90% was observed, starting at a concentration of 105 conidia/mL. At a concentration of 107 conidia/mL, we observed a 50% mortality rate and a 21.5% inhibition of oviposition. The highest levels of hatch inhibition (30.8%) and estimated reproduction inhibition (34.64%) were achieved at a concentration of 108 conidia/mL. Furthermore, the tick larval progeny that hatched from the infected tick egg masses showed evident symptoms of Aspergillus infection after incubation.

Dynamics of Infections in Cattle and Rhipicephalus microplus: A Preliminary Study.

Tick-borne pathogens (TBPs) pose a significant threat to livestock, including bovine species. This study aimed to investigate TBPs in cattle and ticks across four sampling points, utilizing real-time microfluidic PCR. The results revealed that Rhipicephalus microplus ticks were found infesting all animals. Among the detected TBPs in cattle, Anaplasma marginale was the most frequently identified, often as a single infection, although mixed infections involving Rickettsia felis, uncharacterized Rickettsia sp., and Anaplasma sp. were also observed. In ticks, A. marginale was predominant, along with R. felis, Rickettsia sp., and Ehrlichia sp. It is noteworthy that although A. marginale consistently infected all cattle during various sampling times, this pathogen was not detected in all ticks. This suggests a complex dynamic of pathogen acquisition by ticks. A phylogenetic analysis focused on the identification of Anaplasma species using amplified 16S rDNA gene fragments revealed the presence of A. marginale and Anaplasma platys strains in bovines. These findings underscore the presence of multiple TBPs in both cattle and ticks, with A. marginale being the most prevalent. Understanding the dynamics and phylogenetics of TBPs is crucial for developing effective control strategies to mitigate tick-borne diseases in livestock.

Worldwide comparison between the potential distribution of Rhipicephalus microplus (Acari: Ixodidae) under climate change scenarios.

The cattle tick Rhipicephalus microplus (Acari: Ixodidae) has demonstrated its ability to increase its distribution raising spatially its importance as a vector for zoonotic hemotropic pathogens. In this study, a global ecological niche model of R. microplus was built in different scenarios using Representative Concentration Pathway (RCP), Socio-Economic Pathway (SSP), and a climatic dataset to determine where the species could establish itself and thus affect the variability in the presentation of the hemotropic diseases they transmit. America, Africa and Oceania showed a higher probability for the presence of R. microplus in contrast to some countries in Europe and Asia in the ecological niche for the current period (1970-2000), but with the climate change, there was an increase in the ratio between the geographic range preserved between the RCP and SSP scenarios obtaining the greatest gain in the interplay of RCP4.5-SSP245. Our results allow to determine future changes in the distribution of the cattle tick according to the increase in environmental temperature and socio-economic development influenced by human development activities and trends; this work explores the possibility of designing integral maps between the vector and specific diseases.

Phenotypic evaluation of genetic resistance to the tick Rhipicephalus (Boophilus) microplus in Argentine Creole cattle

The objective of this work was to characterize the Argentine Creole cattle breed through the identification of individual phenotypic variations in the levels of infestation with Rhipicephalus (Boophilus) microplus. We evaluated 179 heifers exposed to successive artificial infestations from 2015 to 2018, achieving a total of 663 observations. Tick counts were assessed with the linear mixed model, considering year of evaluation, time of infestation, dam's age and nutritional status during the evaluated period as fixed effects. The average tick count value obtained allowed to classify the breed as highly resistant to the tick charge (99.3%). Although the previous nutritional condition of the animals did not affect the individual charge response, weight gain during the trial showed a significantly negative correlation. We conclude that the Argentine Creole breed is an attractive genetic alternative for cattle breeding in endemic regions, either as a pure breed or a cross-breed.

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.

In Vitro and In Silico Protocols for the Assessment of Anti-Tick Compounds from Pinus roxburghii against Rhipicephalus (Boophilus) microplus Ticks.

Pinus roxburghii, also known by the name "Himalayan chir pine," belongs to the Pinaceae family. Rhipicephalus (Boophilus) microplus tick is one of the most significant bovine ectoparasites, making it a major vector of economically important tick-borne diseases. The researchers conducted adult immersion tests (AIT) and larval packet tests (LPT) to investigate the acaricidal effect of P. roxburghii plant extract on R. (B.) microplus and its potential modulatory function when used with cypermethrin. Eggs were also assessed for their weight, egg-laying index (IE), hatchability rate, and control rate. After exposure to essential extract concentrations ranging from 2.5 to 40 mg/mL for 48 h, adult female ticks' oviposition inhibition and unfed R. (B.) microplus larvae's mortality rates were analyzed. Engorged females exposed to P. roxburghii at 40 mg/mL had reduced biological activity (oviposition, IE) compared to positive and negative controls. A concentration of 40 mg/mL of P. roxburghii caused 90% mortality in R. (B.) microplus larvae, whereas cypermethrin (the positive control) caused 98.3% mortality in LPT. In AIT, cypermethrin inhibited 81% of oviposition, compared to the 40 mg/mL concentration of P. roxburghii, which inhibited 40% of the ticks' oviposition. Moreover, this study assessed the binding capacity of selected phytocompounds with the targeted protein. Three servers (SWISS-MODEL, RoseTTAFold, and TrRosetta) recreated the target protein RmGABACl's 3D structure. The modeled 3D structure was validated using the online servers PROCHECK, ERRAT, and Prosa. Molecular docking using Auto Dock VINA predicted the binding mechanisms of 20 drug-like compounds against the target protein. Catechin and myricetin showed significant interactions with active site residues of the target protein, with docking scores of -7.7 kcal/mol and -7.6 kcal/mol, respectively. In conclusion, this study demonstrated the acaricidal activity of P. roxburghii extract, suggesting its potential as an alternative natural acaricide for controlling R. (B.) microplus.

Epidemiological analysis of cattle ticks and tick-borne pathogens in Gadag district, Karnataka state in India.

In the present study, epidemiological analysis on 839 ticks collected from 50 cattle in Gadag district, Karnataka state, tick identification and detection of tick-borne pathogens was conducted by PCR, sequencing, and phylogeny. The morphological identification revealed that Haemaphysalis spp. [48.6%], Rhipicephalus spp. [48.4%], and Hyalomma spp. [3.0%] tick genera in Gadag district. Further, a higher infestation of Haemaphysalis spp. [69.0%] and Rhipicephalus spp. [62.3%] in Shirahatti and Gadag taluk, respectively was observed. Based on the taluk-wise and tick genus-wise analysis, a higher number of ticks was present in the dewlap region of cattle body sites, except for Hyalomma spp., the majority of which was present in the neck. Tick genus prevalence was 45.1, 42.7%, and 12.2 for Haemaphysalis spp., Rhipicephalus spp., and Hyalomma spp., respectively. The mean tick per cattle was 11.6, 11.0, and 2.5 for Rhipicephalus spp., Haemaphysalis spp., and Hyalomma spp., respectively. The prevalence of Anaplasma marginale, Babesia spp., and Rickettsia rickettsii was 8.0, 6.4, and 6.4%, respectively in the tick DNA samples and was negative for Ehrlichia and Theileria spp. The sequencing of the cytochrome oxidase subunit 1 gene revealed the presence of Haemaphysalis bispinosa, Rhipicephalus decoloratus, and Rhipicephalus microplus tick species in the Gadag district. The phylogenetic analysis revealed the tick species have similarities and identity with the isolates from India and neighboring countries. Thus, the study provides knowledge on tick genus distribution and tick-borne pathogens in Gadag district, Karnataka which will help in developing the control and prevention strategies by the policymakers and for profitable dairy farming by farmers.

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 para‑sodium 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 para‑sodium 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.

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 5-105min. Ozone had a dose-dependent acaricidal effect on both larvae and engorged females. Dosages between 355 and 2130mg/L min had a delayed acaricidal effect (12-180h), leading to the death of all engorged females before laying eggs, whereas doses between 3195 and 7455mg/L min showed immediate acaricidal effect (5min to 4h). Doses between 1775 and 6390mg/L min had an immediate (up to 5min) 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.

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.

Evaluation of Acaricidal Activity of Cinnamomum camphora (F. Lauraceae) Essential Oil Nanoemulsion Against Cattle Tick Rhipicephalus microplus

The present study aims at preparing nanoemulsions of plant essential oil Cinnamomum camphora as acaricidal agent against cattle tick Rhipicephalus microplus larvae. In the initial step, the phytochemical screening of oil was done through gas chromatography and mass spectrometry depicting major compounds as α-pinene (53.99%) and camphor (20.89%) in C. camphora oil. Different ratios of Cinnamomum camphora oil and surfactant Tween 80 (oil:surfactant- 1:0.25, 1:0.5, 1:1, 1:1.5, 1:2 and 1:2.5) were used for preparing stable nanoemulsion through ultrasonication approach. Thermodynamic stability analysis exhibited that CA3 ratio (1:1) to be the most stable which was further characterized using Dynamic light scattering study through Nano zeta sizer indicating droplet size of 101.60 nm, polydispersity index as 0.50 and zeta potential value measured as -8.59 mV. Spectral analysis through Fourier Transform Infrared spectrometry for functional group interaction and Transmission electron microscopy for morphology of nanoemulsion droplets was also done for CA3 nanoemulsion. Acaricidal activity of stable nanoemulsion was evaluated against R. microplus larvae using larval packet assay and repellency rod test with concentration ranging from 500 ppm to 20000 ppm and 500 ppm to 25000 ppm respectively. Highest concentration (20000 ppm) showed 77% mean mortality with LC₅₀ (3364.46 ppm) after 24 hour. CA3 showed promising repellent activity that ranged from 27% to 87% at various concentrations at different time intervals. These findings proved that Cinnamomum camphor essential oil nanoemulsion could be developed and used against R. microplus larvae as a safer, greener and nanoacaricide product.

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.

Field efficacy of Metarhizium anisopliae oil formulations against Rhipicephalus microplus ticks using a cattle spray race

Rhipicephalus microplus tick is the main ectoparasite of cattle in Brazil. The exhaustive use of chemical acaricides to control this tick has favored the selection of resistant tick populations. Entomopathogenic fungi, as Metarhizium anisopliae, has been described as a potential biocontroller of ticks. Therefore, the aim of this study was to evaluate the in vivo efficacy of two oil based formulations of M. anisopliae for the control of the cattle tick R. microplus under field conditions using a cattle spray race as a method of treatment. Initially, in vitro assays were carried out with an aqueous suspension of M. anisopliae, using mineral oil and/or silicon oil. A potential synergism between oils and fungus conidia for tick control was demonstrated. Additionally, the usefulness of silicon oil in order to reduce mineral oil concentration, while improving formulation efficacy was illustrated. Based on the in vitro results, two formulations were selected for use in the field trial: MaO1 (107 conidia/mL plus 5% mineral oil) and MaO2 (107 conidia/mL plus 2.5% mineral oil and 0.01% silicon oil). The adjuvants concentrations (mineral and silicon oils) were chosen since preliminary data indicate that higher concentrations caused significant mortality in adult ticks. For this, 30 naturally infested heifers were divided into three groups based on previous tick counts. The control group did not receive treatment. The selected formulations were applied on animals using a cattle spray race. Subsequently, tick load was evaluated weekly by counting. The MaO1 treatment significantly reduced the tick count only on day +21, reaching approximately 55% efficacy. On the other hand, MaO2 showed significantly lower tick counts on days +7, +14, and +21 after treatment, with weekly efficacy achieving 66%. The results showed a substantial reduction of tick infestation, up to day +28, using a novel formulation of M. anisopliae based in the mixture of two oils. Moreover, we have shown, for the first time, the feasibility of employing formulations of M. anisopliae for large-scale treatment methods, such as a cattle spray race, which in turn, may increase the use and adhesion to biological control tools among farmers.