A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

The role of cattle in the transmission of cystic echinococcosis in a highly endemic area of the Peruvian Andes.

Cystic echinococcosis (CE) causes significant losses in Andean livestock production and affects Andean food security. However, more studies are needed to understand the epidemiology of the disease. In addition, the potential contribution of Andean cattle to the transmission of Echinococcus granulosus sensu lato needs to be known. This study aimed to determine the CE-prevalence and its association with risk factors, such as age and sex of the animals, the parasite load (number of cysts/organ) of condemned organs, and the viability and fertility of Echinococcus cysts from cattle in the Andes. The prevalence was examined in 348 cattle from an authorized slaughterhouse of Huancayo at 3300 m altitude. Cyst burden was determined by extracting all cysts from the total of the CE-infected organs. Cyst fertility and protoscolices viability were analysed from 90 randomly selected CE-infected organs. The CE prevalence was 35.6% (124/348; 95% CI: 30.6%-40.6%). There was no significant effect of age and sex on CE prevalence. CE was significantly more prevalent (p < 0.05) in lungs than livers, 34.8% (121/348; 95% CI: 29.8%-39.8%) vs 8.9% (31/348; 95% CI: 5.9%-11.9%). Most (75%) infected organs had one to five cysts. The mean cyst burden was significantly (p = 0.018) higher in the lungs than livers, 6.4 ± 4.9 vs 3.7 ± 2.9. Cyst fertility was 1.6% (10/608; 95% CI: 0.6%-2.6%). Despite the high CE prevalence, infected organs from Andean cattle play a minor role in CE transmission to dogs in the central Peruvian Andes.

Untargeted metabolomics to discriminate liver and lung hydatid cysts: Importance of metabolites involved in the immune response.

The Echinococcus granulosus sensu lato species complex is responsible for the neglected zoonotic disease known as cystic echinococcosis (CE). Humans and livestock are infected via fecal-oral transmission. CE remains prevalent in Western China, Central Asia, South America, Eastern Africa, and the Mediterranean. Approximately one million individuals worldwide are affected, influencing veterinary and public health, as well as social and economic matters. The infection causes slow-growing cysts, predominantly in the liver and lungs, but can also develop in other organs. The exact progression of these cysts is uncertain. This study aimed to understand the survival mechanisms of liver and lung CE cysts from cattle by determining their metabolite profiles through metabolomics and multivariate statistical analyses. Non-targeted metabolomic approaches were conducted using quadrupole-time-of-flight liquid chromatography/mass spectrometry (LC-QTOF-MS) to distinguish between liver and lung CE cysts. Data processing to extract the peaks on complex chromatograms was performed using XCMS. PCA and OPLS-DA plots obtained through multiple statistical analyses showed interactions of metabolites within and between groups. Metabolites such as glutathione, prostaglandin, folic acid, and cortisol that cause different immunological reactions have been identified both in liver and lung hydatid cysts, but in different ratios. Considering the differences in the metabolomic profiles of the liver and lung cysts determined in the present study will contribute research to enlighten the nature of the cyst and develop specific therapeutic strategies.

Self-emulsifying drug delivery systems (SEDDS) containing Cymbopogon citratus essential oil: Enhancing the stability and acaricide efficacy against Rhipicephalus (Boophilus) microplus.

The objectives of this study were to develop a self-emulsifying drug delivery system (SEDDS) to enhance the stability and efficacy of Cymbopogon citratus essential oil or lemongrass oil (LEO) against cattle tick larvae and engorged females. The system with the highest oil loading in SEDDS was composed of LEO (23.33%w/w), Tween 80: SGKH 4000 in a 2:1 ratio as surfactant (66.67%w/w), and propylene glycol as co-surfactant (10%w/w). The selected SEDDS-LEO has a particle size of 18.78 nm with a narrow size distribution (polydispersity index of 0.27). Notably, the stability of SEDDS was superior to that of the original oil, both during long-term storage and under accelerated conditions. SEDDS-LEO at oil concentrations ranging from 1.458% to 5.833% w/v showed a significantly higher percentage of egg-laying reduction against adult ticks compared with the original oil at the same concentrations (p < 0.05). Furthermore, SEDDS-LEO demonstrated greater larvicidal efficacy than the original oil, with lower LC50 and LC90 values of 0.91 mg/mL and 1.20 mg/mL, respectively, whereas the original oil's LC50 and LC90 values were 1.17 mg/mL and 1.74 mg/mL, respectively. Our findings indicate that SEDDS-LEO is a promising candidate for use as an acaricide in the control of tick populations in dairy cattle.

Sympatric occurrence of Taenia saginata and Sarcocystis spp. in cattle from Narok County, Kenya: meat inspection findings with molecular validation.

The epidemiological picture of Taenia saginata infections in Kenya is fragmented with limited available data. Although Sarcocystis species are significant meat-borne parasites, few studies have explored their occurrence in Kenya. This study aimed to estimate the occurrence of bovine cysticercosis and screen for the presence of Sarcocystis spp. A meat inspection-based survey was conducted in ten abattoirs in Narok County, Kenya, and inspection for T. saginata cysticerci was limited to the Triceps brachii muscle. The apparent occurrence of the parasite was 5.4% (95% CI, 3.8, 7.6, n=573). Molecular confirmation of T. saginata was done via nested polymerase chain reaction targeting the mitochondrial 12S ribosomal RNA gene and restricted fragment length polymorphism. Sarcocystis species were identified using a multiplex polymerase chain reaction method targeting the 18S ribosomal RNA gene sequences and the mitochondrial cytochrome c oxidase subunit I gene. Of the 31 cystic lesions tested, 26/31 (83.9%) were confirmed to be T. saginata.Sarcocystis cruzi and S. hominis were detected in 8/31 (25.8%) and 1/31 (3.2%) of the cystic lesions, respectively. Co-infections of S. cruzi and T. saginata were found in 6/31 lesions (19.4%). The confirmation of bovine cysticercosis and S. hominis is suggestive of the presence of risky culinary and sanitation practices that facilitate transmission. This is the first report and molecular confirmation of Sarcocystis spp. in cattle in the country. The presence of both zoonotic S. hominis and pathogenic S. cruzi highlights an underexplored concern of veterinary and human health significance, warranting further epidemiological investigation.

Assessment of oxidative stress and tissue damage in Echinococcus granulosus naturally infected bovine liver.

Echinococcus granulosus is a zoonotic parasite infects many livestock species, especially cattle, sheep, goat and buffalo, causing cystic echinococcosis. The aim of this study was to demonstrate the presence of the parasite and parasitic tissue damage histopathologically and to determine the role of oxidative stress in the tissue damage through the immunohistochemical detection of the oxidative damage-marker malondialdehyde (MDA) and the antioxidant response-marker superoxide dismutase (SOD). The material of the study consisted of 20 liver samples with Echinococcus cysts and 10 E.granulosus- negative healthy liver samples obtained from different cattle at various times from slaughterhouses in Kirikkale province, Turkey. Histopathologically, Echinococcus cysts of various sizes were observed along with the surrounding fibrous connective tissue. Giant cells, mononuclear cells, and eosinophilic leukocytes were found between the fibrous connective tissue and the cyst. In the parenchymal tissue distant from the cyst, inflammatory changes were observed, including vacuolation and necrosis in hepatocytes, congestion and dilation sinusoidal capillaries. Immunohistochemically, MDA immunopositivity was observed in both hepatocytes surrounding the cyst and areas distant from the cyst, while SOD immunopositivity was mainly detected in fibrous connective tissue and hepatocytes surrounding the Echinococcus cysts. A significant increase in MDA immunoreactivity was observed in E.granulosus s.l.-infected livers. Although no statistically significant change was observed in SOD immunopositivity in the liver tissues with cystic echinococcosis, regional variations were noted. Germinal layer (GL) of Echinococcus cyst showed immunopositive staining for MDA, while laminated layer (LL) exhibited immunonegative staining. To the authors' best understanding, this study represents a pioneering effort in showcasing and evaluating the immunoreactivities of MDA and SOD within the liver tissue afflicted with Echinococcus cysts. Simultaneously, the examination extends to encompass tissue damage and the infiltration of inflammatory cells. This study highlights the role of oxidative stress in the pathogenesis of Cystic Echinococcosis (CE) and the need for further investigation of antioxidant defense mechanisms and their regional variations.

Activation of murine macrophages by membrane proteins from Tritrichomonas foetus grown on iron- and calcium-rich conditions.

Tritrichomonas foetus is a protozoan parasite that causes a venereal disease in cattle limiting reproduction by abortions and sterility. The immune response against this parasite is poorly understood. Since the iron and calcium ions are important regulators of the microenvironment of the urogenital tract in cattle, we decided to evaluate the role of these divalent cations on the antigenicity of membrane proteins of T. foetus on macrophage activation as one of the first inflammatory responses towards this pathogen. Colorimetric methods and ELISA were used to detect the nitric oxide and oxygen peroxide production and expression of cytokines in culture supernatant from macrophage incubated with membrane proteins from T. foetus cultured in iron- and calcium-rich conditions. qRT-PCR assays were used to evaluate the transcript expression of genes involved in the inflammatory response on the macrophages. The membrane proteins used for in vitro stimulation caused the up-regulation of the iNOS and NOX-2 genes as well as the generation of NO and H2 O2 in murine macrophages on a dependent way of the metal concentrations. Additionally, after stimulation, macrophages showed a considerable rise in pro-inflammatory cytokines and a downregulation of anti-inflammatory cytokines, as well as up-regulation in the transcription of the TLR4 and MyD88 genes. These data suggest that membrane proteins of T. foetus induced by iron and calcium can activate an inflammatory specific macrophage response via TLR4/MyD88 signalling pathway.

Bovine cysticercosis epidemiology and the economic impact of the triceps brachii incision in a South African export abattoir.

Taenia saginata is a zoonotic tapeworm of humans with bovines as its intermediate host (bovine cysticercosis). Traditional meat inspection is the main measure to identify the larval stage in carcasses and prevent human infection, but has a notoriously low sensitivity, especially in low prevalence settings. The legislation in multiple African countries mandates an incision in both triceps brachii muscles to detect the parasite as part of the normal post-mortem inspection, but this has an economic cost and is not universally mandated in other countries. The primary aim of this study was to investigate the epidemiology of bovine cysticercosis at an export abattoir in South Africa and determine the validity and cost of the triceps incision. Risk factors were investigated, and the effect of additional heart incisions on the current inspection. Four incisions were made into the heart in addition to the normal post-mortem inspection in 3353 carcasses. The proportion of positive animals with and without the cardiac and triceps incisions were compared using McNemar's chi-square tests, while risk factors were assessed using binary logistic regression. The economic impact of the triceps incision was estimated using a stochastic economic cost model. Thirty-three positive carcasses were identified for an apparent prevalence of 0.98 % (95 %CI, 0.69-1.36 %), while the true prevalence was estimated at 6.6 % (95 %CI, 4.3-8.8 %). All cysts were confirmed as T. saginata on histopathology with 70 % (95 %CI, 53-83 %) located in the heart. The additional cardiac incisions resulted in the detection of significantly more cases compared to the normal inspection method prevalence of 0.72 % (95 %CI, 0.47-1.05 %; P < 0.001). The apparent prevalence of T. saginata when omitting the triceps incision was not significantly lower compared to the prevalence when included in the inspection (P = 0.480). External feedlots (OR= 4.17, 95 %CI: 2.04-8.54, P < 0.001) and older animals (OR=3.90, 95 %CI: 1.17-13.03, P = 0.027) were associated with a positive detection. The current median annual financial cost to the food business operator from the triceps incision was estimated at $30387 (95 %CI: $0-$130696), with the proportion of deboned meat exported identified as the most important factor affecting cost (Spearman's rho=0.853). The identification of risk factors could aid in the development of a more effective risk-based inspection system. The current inspection should be modified to increase exposure of the heart and remove the triceps incisions. The latter should especially be considered given the minimal contribution to cysticercosis detection, the unsupported requirement for its inclusion specifically in Africa, and its economic impact, especially as beef exports increase.

Dynamics of protozoal excretion in the faeces of calves during the first 28 days after arrival at the fattening farm indicate infection before regrouping and show poor temporal correlation with diarrhoea.

BACKGROUND: Calves in dairy cattle production in Switzerland are transported to a fattening farm at the age of 3-5 weeks, and frequently suffer from diarrhoea within the first 14 days after arrival. To characterise the role of intestinal protozoa in this, we investigated the excretion dynamics of Eimeria, Cryptosporidium and Giardia during the first 28 days after the arrival and regrouping of calves at fattening farms. METHODS: A total of 610 faecal samples from 122 calves (mean age 37.3 days; mean body weight 79.8 kg) were collected on seven different fattening farms during the first 28 days after the arrival and regrouping of the animals. The farms were visited between January and April (cold season; n = 4) and between June and August (warm season; n = 3). The samples were collected rectally on days 1, 4, 7, 14 and 28, assessed for consistency, and analysed using the McMaster method for quantitative determination of the number of Eimeria oocysts per gram of faeces (OPG), flotation for morphological differentiation of the unsporulated Eimeria oocysts, a concentration method for the semi-quantitative determination of Giardia cysts, and modified Ziehl-Neelsen staining for semi-quantitative determination of Cryptosporidium oocysts. RESULTS: Overall, 50.8% (62/122) of the animals had diarrhoea during the study period. However, the faecal excretion of protozoal pathogens was neither associated with diarrhoea nor with body weight gain of the animals. Altogether, 90.2% (110/122) of the calves were Eimeria positive. Eimeria zuernii was excreted by 51 (41.8%) and Eimeria bovis by 68 (55.7%) animals. In the warm season more animals tested positive for Eimeria and OPGs were higher than in the cold season. There was no correlation between the age of the calves and the OPG values. Overall, 64.8% (79/122) of the calves excreted Eimeria oocysts within the first 7 days, indicating that they had been infected with the parasite on the dairy farm of origin. Eighty-nine calves (73.0%) excreted Giardia cysts, with more positive animals in the cold (80.3%) compared with the warm season (64.3%). Only Giardia duodenalis assemblage E was identified. Cryptosporidium oocysts were microscopically detected in 14 animals (11.5%) on five farms. Cryptosporidium spp. were present in a total of 12 animals, i.e. Cryptosporidium parvum in nine, Cryptosporidium ryanae in two, and Cryptosporidium bovis in one animal. CONCLUSIONS: A better understanding of the temporal dynamics of protozoal infections in calves is helpful for the implementation of appropriate measures to protect the health of these animals at a critical phase in their lives. Our results indicate that factors other than those examined in the present study contributed to the onset of diarrhoea in the calves.

Effectiveness of a fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) against Rhipicephalus microplus and sucking lice infesting cattle.

Unmanaged tick and sucking lice infestations negatively impact the health and production potential of cattle. Described herein are two non-interference dose confirmation studies evaluating the efficacy of a single administration of a new fixed-dose combination injectable (FDCI) endectocide consisting of 0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride, against either laboratory-induced Rhipicephalus microplus infestations in Australia or naturally acquired sucking lice (Linognathus vituli) infestations in the US. This FDCI is available as Dectomax V® in Australia and New Zealand and as Valcor® in the United States. To evaluate therapeutic efficacy against R. microplus, 12 calves were each exposed to 10 infestations of ∼5000 larvae per infestation between Days -24 and -2. Calves were either treated on Day 0 with the FDCI or left untreated (control). Additional R. microplus infestations of ∼5000 larvae were conducted on Day 2 and then three times weekly to also evaluate persistent efficacy of the FDCI. Tick collections were conducted daily from Day -3. Group mean live tick counts, egg production, and egg viability were analyzed for significant differences between the two groups. To determine efficacy of the FDCI against lice, 24 cattle with active sucking lice infestations based on Day -7 counts were allocated to two groups and treated on Day 0 with either saline (control) or the FDCI. Lice counts were conducted weekly from Day 14 through 42 and again on Day 56. Mean group lice counts on each count day were compared between treatment groups. In the R. microplus study presented here, cattle in Queensland, Australia treated with the FDCI (Dectomax V®) showed > 90 % reduction in tick counts based on arithmetic means within 48 h of treatment when compared to untreated cattle, and counts were > 95 % reduced from post-treatment Day 5 through Day 30. In the sucking lice study conducted in the US, the FDCI (Valcor®) displayed 100 % efficacy against sucking lice infestations (L. vituli) from first count day (Day 14 post-treatment) through Day 35 and then 99.9 % efficacy through Day 56 post-treatment. No treatment-related adverse events were reported for cattle in either study. Using R. microplus and sucking lice as representative ectoparasites, these studies demonstrate the ectoparasite activity of doramectin is retained in the new FDCI.

Toxic effect of Callistemon viminalis essential oil on the ovary of engorged Rhipicephalus microplus female ticks (Acari: Ixodidae).

Rhipicephalus microplus is the main ectoparasite of cattle and its parasitism can reduce weight gain of hosts, in addition to causing anemia, increasing the risk of myiasis, and resulting in the transmission of Babesia bovis and Anaplasma marginale, among other pathogens. The use of synthetic chemicals plays an important role in controlling these ticks. However, its frequent and indiscriminate use has contributed to the selection of resistant strains, resulting in greater interest in the search for natural-origin products. The shrub Callistemon viminalis (Myrtaceae), also known as weeping bottlebrush, is known for its antibacterial, acaricidal, repellent, and antifungal activities, but there are no reports in the literature about its effects on the internal morphology of ticks. This study aimed to extract and characterize the essential oil obtained from the leaves of C. viminalis. Additionally, its effects on the ovary morphology of engorged R. microplus were assessed through histological, histochemical, and morphometric techniques. Exposure to C. viminalis caused dose-dependent morphological changes, such as cellular alterations in the epithelial layer lining the ovary lumen and the pedicel, irregularity of the chorion and oocyte shape, changes in protein and carbohydrate content, decrease in oocyte size, reduction in the size of the nucleus as well as cytoplasmic and nucleolar vacuolation. Thus, C. viminalis essential oil exhibited a toxic effect on the reproductive system of R. microplus, which may result in the reproductive impairment of this tick species.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages.

Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.

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.

Biochemical characterisation of Cytochrome P450 oxidoreductase from the cattle tick, Rhipicephalus microplus, highlights potential new acaricide target.

Management of the cattle tick, Rhipicephalus microplus, presents a challenge because some populations of this cosmopolitan and economically important ectoparasite are resistant to multiple classes of acaricides. Cytochrome P450 oxidoreductase (CPR) is part of the cytochrome P450 (CYP450) monooxygenases that are involved in metabolic resistance by their ability to detoxify acaricides. Inhibiting CPR, the sole redox partner that transfers electrons to CYP450s, could overcome this type of metabolic resistance. This report represents the biochemical characterisation of a CPR from ticks. Recombinant CPR of R. microplus (RmCPR), minus its N-terminal transmembrane domain, was produced in a bacterial expression system and subjected to biochemical analyses. RmCPR displayed a characteristic dual flavin oxidoreductase spectrum. Incubation with nicotinamide adenine dinucleotide phosphate (NADPH) lead to an increase in absorbance between 500 and 600 nm with a corresponding appearance of a peak absorbance at 340-350 nm indicating functional transfer of electrons between NADPH and the bound flavin cofactors. Using the pseudoredox partner, kinetic parameters for both cytochrome c and NADPH binding were calculated as 26.6 ± 11.4 µM and 7.03 ± 1.8 µM, respectively. The turnover, Kcat, for RmCPR for cytochrome c was calculated as 0.08 s-1 which is significantly lower than the CPR homologues of other species. IC50 (Half maximal Inhibitory Concentration) values obtained for the adenosine analogues 2', 5' ADP, 2'- AMP, NADP+and the reductase inhibitor diphenyliodonium were: 140, 82.2, 24.5, and 75.3 µM, respectively. Biochemically, RmCPR resembles CPRs of hematophagous arthropods more so than mammalian CPRs. These findings highlight the potential of RmCPR as a target for the rational design of safer and potent acaricides against R. microplus.

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.

Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus.

BACKGROUND: Babesia bovis, an intra-erythrocytic apicomplexan parasite, is one of the causative agents of bovine babesiosis, the most important tick-borne disease of cattle in tropical and subtropical regions. Babesia bovis has a complex life-cycle that includes sexual development within the tick vector. The development of a transmission blocking vaccine to control bovine babesiosis requires the identification of antigens displayed on the surface of the parasite during its development within tick vectors. Four B. bovis cysteine-rich GCC2/GCC3 domain protein (BboGDP) family members were previously identified and are differentially expressed as discrete pairs by either blood stages or kinetes. In this study we focused on two family members, BboGDP1 and -3, that are expressed by Babesia parasites during tick infection. METHODS AND RESULTS: Transcription analysis using quantitative PCR demonstrated that BboGDP1 and -3 were upregulated in in vitro-induced sexual stage parasites and during parasite development in the tick midgut. Moreover, protein expression analysis of BboGDP1 and -3 during the development of sexual stages in in vitro culture was consistent with their transcription profile. Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of BboGDP1 and -3 on in vitro-induced sexual stage parasites. In addition, fixed immunofluorescence analysis showed reactivity of anti-BboGDP1 and -3 polyclonal antibodies to kinetes. CONCLUSIONS: The collective data indicate that BboGDP1 and -3 are expressed by kinetes and on the surface of sexual stages of the parasites. The identified parasite surface membrane proteins BboGDP1 and -3 are potential candidates for the development of a B. bovis transmission blocking vaccine.

A preliminary study on placental damage associated to experimental neosporosis in BALB/c mice.

Neospora caninum is a protozoan parasite which can infect a range of animals, including dogs, cattle, and sheep. Bovine neosporosis, which mainly causes abortion in cattle, results in substantial economic losses worldwide. To study the effects of N. caninum infection on the placenta, a pregnant mouse model for N. caninum infection was established. The litter size (8.6 ± 1.5) and the number of live pups (6.4 ± 1.8) of infected dams were significantly lower compared with those of non-infected dams. Trophoblast cell shrinkage and a large number of apoptosomes were detected in the placentas of the infected group. The parasite load in the placental tissue was significantly higher with time after infection. Likewise, apoptosis of placental trophoblast cells significantly increased with time after infection. Among the 66 apoptotic genes detected in this study, eight genes, including Bcl-2, were significantly differentially expressed by about > tenfold in infected and uninfected mice. The expression of BAX and tumor necrosis factor-alpha (TNF-α) was upregulated in the placental cells of the infected mice, whereas the expression of BCL-2 was downregulated. Enzyme-linked immunosorbent assays (ELISAs) showed that apoptotic protease caspase-3 level was significantly increased in placental cell suspension, and insulin-like growth factor (IGF)-2 level was significantly reduced. Acetylcholine (ACH) and placental prolactin (PL) levels were initially decreased but eventually increased. In summary, infection of mice with N. caninum caused apoptotic damage to the placental tissues, cells, and genes and affected the normal physiological functions of placenta, which may largely explain the adverse pregnancy outcomes caused by N. caninum infection in mice.

Absence of anti-Besnoitia spp. specific antibodies in European wild lagomorphs from the Iberian Peninsula.

Besnoitia besnoiti is an apicomplexan parasite whose life cycle is not completely understood. It is assumed that this parasite might have an indirect life cycle with a carnivore as a definitive host able to shed oocysts after the ingestion of mature cysts in tissues of an infected intermediate host. Cattle and wild cervids on the Iberian Peninsula can act as intermediate hosts of B. besnoiti, and exposure to the parasite has been demonstrated in equids. In this study, we aimed to assess the presence of members of the genera Besnoitia in wild lagomorphs from the Iberian Peninsula and the potential role of these host species in the life cycle of B. besnoiti, as all the animals were sampled from 19 regions of the Iberian Peninsula where cases of bovine besnoitiosis have been previously detected. Serum samples (Oryctolagus cuniculus: n = 552; Lepus europaeus: n = 122) were first analysed by ELISA and subsequently confirmed by Western blot (WB). Specific antibodies against B. besnoiti were not found in any sampled animal by WB. In addition, lung samples from a subset of wild rabbits (n = 16) were tested by PCR and Besnoitia spp. DNA was not detected. These results suggest that Besnoitia spp. are unlikely to circulate in wild lagomorphs in the Iberian Peninsula. Thus, lagomorphs are not expected to play a key role in the biological cycle of B. besnoiti. Further studies are necessary to assess whether different micromammal species, such as rodents, can serve as natural reservoirs of Besnoitia spp. in other European regions.

Meta-analysis of the prevalence of bovine cystic echinococcosis in China during decade.

BACKGROUND: Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by Echinococcus granulosus sensu lato (E. granulosus s.l.) larvae. It is mainly prevalent in western agricultural and pastoral areas in China, seriously affecting the development of public health and animal husbandry. METHODS: This study conducted a systematic evaluation and meta-analysis of bovine CE in China in the past 10 years (2011-2020). Five databases (PubMed, ScienceDirect, CNKI, Wanfang Data, VIP Chinese Journal Database) were used to collect the relevant references, and finally 77 published articles were obtained. RESULTS: The total prevalence of bovine CE was estimated to be 9.62% during decade, and decreased year by year after reaching its peak in 2014 (18.75%). The highest E. granulosus s.l. infection rate was found in Qinghai province (14.79%). The infection rate of cattle >4 years old (15.84%) was significantly higher than that of other age groups, and cattle raised by grazing was significantly higher than that of other methods (17.00%). In the groups of geographical and climatic factors, the prevalence of CE was higher in regions with high-altitude (20.76%), cold (20.76%) and rainy areas (11.33%). CONCLUSION: This study found that old age and grazing were two important factors for the high incidence of bovine CE. The prevalence of CE is higher in low temperature, rainy and high altitude areas, where may be more suitable for the survival of E. granulosus s.l. eggs.

An epidemiological study of hydatid cyst of Echinococcus granulosus isolated from sheep, goats and cattle in Sulaimani province, Kurdistan Regional-Iraq.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of Echinococcus granulosus sensu lato with medical importance and economic effect. This study was carried out in Sulaimani province, Kurdistan Region, Iraq from October 2020 to November 2021. The study focused on the prevalence of cystic echinococcosis in slaughtered animals. The total prevalence was 8% (1324/16524), with a high rate in sheep (5.3%) and low rates in goats and cattle (1.9% and 0.76%, respectively). With respect to abattoirs involved in the study, the highest prevalence was detected in Kalar abattoir (16.8%) and the lowest in Sulaimani abattoir (4.4%). Among the infected animals, sheep in Kalar abattoir recorded the highest prevalence, while cattle in Sulaimani abattoir recorded the lowest prevalence (11.7% and 0.4%, respectively). Males of sheep, cattle, and female goats showed higher prevalence (66.7%, 76.2%, and 58.6%, respectively). Regarding organ involvements, males of cattle and sheep showed high liver involvement (81.25% and 72.2%, respectively), while females of goat showed the highest liver involvement (76%). Females of cattle and sheep showed high lung involvement (46.7% and 28.5%, respectively), but the males of goats showed the highest lung involvement (53.8%).