Phenotypic and genotypic characterization of acaricide resistance in Rhipicephalus microplus field isolates from South Africa and Brazil.

Rhipicephalus (Boophilus) microplus is one of the most successful ticks infesting cattle around the world. This highly-invasive species transmits cattle parasites that cause cattle fever leading to a high socio-economic burden. Tick eradication programs have often failed, due to the development of acaricide resistance. Here we characterize acaricide resistance in a large number of tick isolates from regions in South Africa (KwaZulu Natal, Mpumalanga, Western & Eastern Cape provinces) and two Brazilian regions. By means of Larval Packet Tests (LPT's) acaricide resistance was evaluated against five commonly used acaricides (chlorfenvinphos, fipronil, deltamethrin, amitraz, and ivermectin). Furthermore, the coding region containing the knock down resistance (kdr) mutation, known to result in pyrethroid resistance, was sequenced. Resistance to at least one acaricide class was reported in each of the five regions, and a high proportion of tick isolates exhibited multi-resistance to at least two acaricide classes (range: 22.2-80.0%). Furthermore, resistance ratios (RR) showed high spatial variation (intercontinental, as well as regional) but low regional spatial autocorrelation. Previous and current acaricide use correlated with current RR, and several combinations of acaricide RR were positively correlated. Moreover, fipronil resistance tended to be higher in farms with more intense acaricide use. The kdr-mutations provided the ticks a fitness advantage under the selection pressure of synthetic pyrethroids based on population (kdr-allele frequency) and individual level data (genotypes). The data show the threat of acaricide (multi-)resistance is high in Brazil and South Africa, but acaricide specific levels need to be assessed locally. For this purpose, gathering complementary molecular information on mutations that underlie resistance can reduce costs and expedite necessary actions. In an era of human-caused habitat alterations, implementing molecular data-driven programs becomes essential in overcoming tick-induced socio-economic losses.

Meta-analysis of heritability estimates and genome-wide association for tick-borne haemoparasites in African cattle.

The control of tick-borne haemoparasites in cattle largely relies on the use of acaricide drugs against the tick vectors, with some vaccination also being used against selected pathogens. These interventions can be difficult in Africa, where accessibility and cost of vaccines can be issues, and the increasing resistance of tick vectors to the widely used acaricides is a complication to disease control. A potential complementary control strategy could be the exploitation of any natural host genetic resistance to the pathogens. However, there are currently very few estimates of the extent of host resistance to tick-borne haemoparasites, and a significant contributing factor to this knowledge gap is likely to be the difficulty of collecting appropriate samples and data in the smallholder systems that predominate livestock production in low- and middle-income countries, particularly at scale. In this study, we have estimated the heritability for the presence/absence of several important haemoparasite species (including Anaplasma marginale, Babesia bigemina, Babesia bovis, and Ehrlichia ruminantium), as well as for relevant traits such as body weight and body condition score (BCS), in 1,694 cattle from four African countries (Burkina Faso, Ghana, Nigeria, and Tanzania). Heritability estimates within countries were mostly not significant, ranging from 0.05 to 0.84 across traits and countries, with standard errors between 0.07 and 0.91. However, the weighted mean of heritability estimates was moderate and significant for body weight and BCS (0.40 and 0.49, respectively), with significant heritabilities also observed for the presence of A. marginale (0.16) and E. ruminantium (0.19). In a meta-analysis of genome-wide association studies (GWAS) for these traits, two peaks were identified as reaching the suggestive significance threshold (p < 1.91 × 10-7 and p < 1.89 × 10-7, respectively): one on chromosome 24 for BCS and one on chromosome 8 for the E. ruminantium infection status. These findings indicate that there is likely to be a genetic basis that contributes to pathogen presence/absence for tick-borne haemoparasite species, which could potentially be exploited to improve cattle resistance in Africa to the economically important diseases caused by these pathogens.

Assessment of genotyping array performance for genome-wide association studies and imputation in African cattle.

BACKGROUND: In cattle, genome-wide association studies (GWAS) have largely focused on European or Asian breeds, using genotyping arrays that were primarily designed for European cattle. Because there is growing interest in performing GWAS in African breeds, we have assessed the performance of 23 commercial bovine genotyping arrays for capturing the diversity across African breeds and performing imputation. We used 409 whole-genome sequences (WGS) spanning global cattle breeds, and a real cohort of 2481 individuals (including African breeds) that were genotyped with the Illumina high-density (HD) array and the GeneSeek bovine 50 k array. RESULTS: We found that commercially available arrays were not effective in capturing variants that segregate among African indicine animals. Only 6% of these variants in high linkage disequilibrium (LD) (r2 > 0.8) were on the best performing arrays, which contrasts with the 17% and 25% in African and European taurine cattle, respectively. However, imputation from available HD arrays can successfully capture most variants (accuracies up to 0.93), mainly when using a global, not continent-specific, reference panel, which partially reflects the unusually high levels of admixture on the continent. When considering functional variants, the GGPF250 array performed best for tagging WGS variants and imputation. Finally, we show that imputation from low-density arrays can perform almost as well as HD arrays, if a two-stage imputation approach is adopted, i.e. first imputing to HD and then to WGS, which can potentially reduce the costs of GWAS. CONCLUSIONS: Our results show that the choice of an array should be based on a balance between the objective of the study and the breed/population considered, with the HD and BOS1 arrays being the best choice for both taurine and indicine breeds when performing GWAS, and the GGPF250 being preferable for fine-mapping studies. Moreover, our results suggest that there is no advantage to using the indicus-specific arrays for indicus breeds, regardless of the objective. Finally, we show that using a reference panel that better represents global bovine diversity improves imputation accuracy, particularly for non-European taurine populations.

Transmission of Anaplasma phagocytophilum (Foggie, 1949) by Ixodes ricinus (Linnaeus, 1758) ticks feeding on dogs and artificial membranes.

BACKGROUND: The interplay of speed of activity of acaricidal products and tick-borne pathogen transmission time is the major driver for disease prevention. This study aimed to investigate the time required for transmission of Anaplasma phagocytophilum by adult Ixodes ricinus ticks in vivo on dogs, and to confirm the time required for transmission observed in vivo, in vitro. METHODS: Nymphs of I. ricinus were experimentally infected with an A. phagocytophilum strain of canine origin. Dogs were allocated to 6 groups of 3 dogs each. Groups 1-5 were infested with 50 A. phagocytophilum-infected female adult ticks on Day 0. Ticks were removed post-infestation at 3, 6, 12, 24 and 48 h. Dogs in Group 6 were infested with 60 A. phagocytophilum-infected female adult ticks (left on dogs until engorged). Dogs were observed daily for general health and clinically examined on Day 0, and weekly from Day 14. Blood was collected for qPCR and serological analysis on Day 0 (pre-challenge) and weekly thereafter. In the in vitro study each artificial feeding chamber was seeded with 10 adult ticks (5 male/5 female), attachment assessed, and blood pools sampled for qPCR at 6 h intervals up to 72 h after first tick attachment. RESULTS: Anaplasma phagocytophilum specific antibodies and DNA were detected in all 3 dogs in Group 6. No A. phagocytophilum-specific antibodies or DNA were detected in any dogs in Groups 1-5. All dogs remained healthy. Female tick attachment in 60 artificial feeding chambers over 72 h ranged between 20-60%. Anaplasma phagocytophilum DNA was detected in the blood collected from 5% of chambers sampled at 6 h, with the highest number of positive samples (16.3%) observed at 36 h. CONCLUSIONS: Transmission of A. phagocytophilum by I. ricinus ticks starts within a few hours after attachment but establishment of infections in dogs is apparently dependent on a minimum inoculation dose that was only observed when ticks attached for greater than 48 h. These findings highlight the need for acaricidal products to exert a repellent and/or rapid killing effect on ticks to forestall transmission and subsequent disease.

Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection.

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis.

Heterologous coexpression of the benzoate-para-hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeasts.

Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large-scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self-sufficient P450s for their potential as whole-cell biocatalysts. Most P450s are, however, not self-sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450-reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450-CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad-range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole-cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450-based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole-cell biocatalyst was emphasized, supporting earlier findings.

Analysis of Dipylidium caninum tapeworms from dogs and cats, or their respective fleas - Part 1. Molecular characterization of Dipylidium caninum: genetic analysis supporting two distinct species adapted to dogs and cats.

A 28S rDNA PCR detection assay was previously developed to identify Dipylidium caninum DNA inside single fleas collected from both cats and dogs. Sequence analysis of the 28S rDNA fragment indicated two genetically distinct variations of the target region. The two genotypes, so-called "D. caninum canine genotype" and "D. caninum feline genotype", based on host origin, are further investigated and described in this paper. Restriction fragment length polymorphism (RFLP) analysis and hydrolysis probe-based genotyping assays were developed and validated for genotyping D. caninum DNA. The complete mitochondrial (mt) genome of the "feline genotype" was sequenced and compared to the D. caninum mt genome available in GenBank. The molecular characterization of D. caninum isolates collected from infected fleas, and also proglottids collected from dogs and cats, confirmed the existence of two distinct genotypes. These genotypes are related to host origin (dogs or cats), irrespective of their geographical origin, and they present a biological adaptation to their respective host, as confirmed by the comparison of biological development and host preference in another study. The genetic differences (Part 1, present paper) and biological observations (Part 2, in this journal) enabled us to suggest the existence of two distinct species within D. caninum, which will have to be clarified.

Analysis of Dipylidium caninum tapeworms from dogs and cats, or their respective fleas - Part 2. Distinct canine and feline host association with two different Dipylidium caninum genotypes.

Initial investigations suggested the existence of two distinct genotypes of Dipylidium caninum from infected cat fleas (Ctenocephalides felis). One genotype was found almost always (> 95%) in fleas collected from, and proglottids shed by, domestic dogs. The other was found almost always (> 95%) in fleas collected from, and proglottids shed by, domestic cats. Molecular investigations (Part 1, in this journal) confirmed the presence of two distinct genotypes. Due to the apparent host association observed, these were referred to as the "D. caninum canine genotype" and the "D. caninum feline genotype". The current article reports on an in vivo experimental infection study assessing the host-parasite interaction for each genotype. Mixed infections with the two genotypes in both dogs and cats were conducted. The specific genotyping of proglottids allowed us to assess the specific prepatent periods, prolificity, and longevity of each genotype in dogs versus cats. The possible hybridisation was also studied through molecular evaluation of the proglottids expelled by infected dogs and cats. Results demonstrate a clear distinct host interaction. The canine D. caninum genotype occurred at a higher frequency in dogs, with a shorter prepatent period and a longer lifespan; and the feline genotype occurred at a higher frequency in cats, with a shorter prepatent period and a longer lifespan. The absence of any hybrids in the mixed infections of both dogs and cats confirm the hypothesis of two distinct genotypes, suggesting the possibility of two distinct species within Dipylidium caninum.

Occurrence of Dipylidium caninum in fleas from client-owned cats and dogs in Europe using a new PCR detection assay.

Ctenocephalides fleas are not only the most prevalent ectoparasites of dogs and cats but also the intermediate host of the cestode Dipylidium caninum. Due to the poor sensitivity of coproscopy to diagnose cat and dog infestation by Dipylidium, few epidemiological data are available on its prevalence among pet populations. A new PCR method was developed to specifically identify D. caninum rDNA inside single fleas. The PCR test was then applied to 5529 fleas of Ctenocephalides genus, 2701 Ctenocephalides felis fleas (1969 collected on 435 cats and 732 on 178 dogs) and 2828 Ctenocephalides canis fleas collected from 396 dogs. Precisely, 4.37% of cats were infested by a flea population infected with D. caninum. Out of the 1969 C. felis from cats, 2.23% were found to be infected with Dipylidium. From the 396 dogs infested with C. canis, 9.1%% were infested with the Dipylidium infected fleas, which is significantly higher than the observation made in cats (p=0.03). Moreover, 3.1% of the C. canis fleas were found to be infected with Dipylidium, which is not significantly different than in C. felis. Looking at the number of infected fleas in the positive samples (at least one PCR positive flea in a sample), the infestation rate in samples was varied from 3 to 100% with an average of 19.7% which is in favour of easy and regular Dipylidium reinfestations of both cats and dogs in households. For the first time, the spread of D. caninum between fleas and dogs and cats is confirmed throughout Europe.

The ability of an oral formulation of afoxolaner to block the transmission of Babesia canis by Dermacentor reticulatus ticks to dogs.

BACKGROUND: Canine babesiosis due to Babesia canis is an endemic disease in many European countries. A vaccine is available in some countries, but it does not prevent the infection and just helps in reducing the gravity of clinical signs. Therefore, the major way to help preventing the disease is by controlling tick infestations on dogs.To assess the preventive efficacy of afoxolaner (NexGard®), a new oral anti- flea and tick product, against Babesia canis infected adult Dermacentor reticulatus in an experimentally controlled study. METHODS: Sixteen healthy mixed breed adult dogs, negative for Babesia canis antibodies were included in a single centre, randomized, blinded and controlled study to evaluate the impact of treatment with afoxolaner on the transmission of Babesia canis to dogs exposed to Dermacentor reticulatus. The dogs were randomly allocated into two groups of 8 dogs each. One group remained untreated. In the other group, dogs were treated orally with a novel formulation of afoxolaner (NexGard®) on day 0. All dogs were infested each by 50 adult Dermacentor reticulatus ticks (equal sex ratio) at days 7, 14, 21 and 28. The Dermacentor reticulatus ticks were confirmed to harbour Babesia canis by Polymerase Chain Reaction (PCR). RESULTS: The treatment was well tolerated by all dogs without any adverse effects. Babesia canis was transmitted by D. reticulatus to all untreated control dogs, confirmed following demonstration of hyperthermia, detection of B. canis parasites in blood smears and PCR assay from blood and serology. These confirmed infected dogs were subsequently treated with imidocarb and diminazene. The treated dogs remained negative based on all criteria until the last study, Day 56, confirming that the oral treatment of dogs with NexGard® prevented transmission of Babesia canis and development of clinical babesiosis for up to 28 days. CONCLUSION: This is the first demonstration that an oral acaricidal treatment may prevent the transmission of a pathogen despite the need for the tick to attach and start feeding before being killed by the acaricide.

Development and evaluation of an ITS1 "Touchdown" PCR for assessment of drug efficacy against animal African trypanosomosis.

Animal African trypanosomoses (AAT) are caused by flagellated protozoa of the Trypanosoma genus and contribute to considerable losses in animal production in Africa, Latin America and South East Asia. Trypanosoma congolense is considered the economically most important species. Drug resistant T. congolense strains present a threat to the control of AAT and have triggered research into discovery of novel trypanocides. In vivo assessment of trypanocidal efficacy relies on monitoring of treated animals with microscopic parasite detection methods. Since these methods have poor sensitivity, follow-up for up to 100 days after treatment is recommended to increase the chance of detecting recurrent parasitaemia waves. Molecular techniques are more amendable to high throughput processing and are generally more sensitive than microscopic detection, thus bearing the potential of shortening the 100-day follow up period. The study presents a "Touchdown" PCR targeting the internal transcribed spacer 1 of the ribosomal DNA (ITS1 TD PCR) that enables detection and discrimination of different Trypanosoma taxa in a single run due to variations in PCR product sizes. The assay achieves analytical sensitivity of 10 parasites per ml of blood for detection of T. congolense savannah type and T. brucei, and 100 parasites per ml of blood for detection of T. vivax in infected mouse blood. The ITS1 TD PCR was evaluated on cattle experimentally infected with T. congolense during an investigational new veterinary trypanocide drug efficacy study. ITS1 TD PCR demonstrated comparable performance to microscopy in verifying trypanocide treatment success, in which parasite DNA became undetectable in cured animals within two days post-treatment. ITS1 TD PCR detected parasite recrudescence three days earlier than microscopy and had a higher positivity rate than microscopy (84.85% versus 57.58%) in 66 specimens of relapsing animals collected after treatments. Therefore, ITS1 TD PCR provides a useful tool in assessment of drug efficacy against T. congolense infection in cattle. As the assay bears the potential for detection of mixed infections, it may be applicable for drug efficacy studies and diagnostic discrimination of T. vivax and T. congolense against other pathogenic trypanosomes, including T. brucei, T. evansi and T. equiperdum.

A broad-range yeast expression system reveals Arxula adeninivorans expressing a fungal self-sufficient cytochrome P450 monooxygenase as an excellent whole-cell biocatalyst.

The feasibility of using a single vector to clone a cytochrome P450 monooxygenase (P450) in different yeasts and then compare whole-cell hydroxylase activity was investigated. A broad-range yeast expression vector using the ylTEFp to drive expression of the cloned gene and the scTEFp to drive the hygromycin resistance marker gene was used to clone the genes encoding two self-sufficient P450s, CYP102A1 and CYP505A1. Both genes were cloned into Saccharomyces cerevisiae, Kluyveromyces marxianus, Yarrowia lipolytica (two strains) and Arxula adeninivorans. 4-Hexylbenzoic acid (HBA), which is subterminally hydroxylated by both CYP102A1 and CYP505A1, was used to compare whole-cell hydroxylase activity of transformants. Kluyveromyces marxianus and A. adeninivorans exhibited activity with both CYP102A1 and CYP505A1, while S. cerevisiae only displayed CYP102A1 activity and Y. lipolytica only CYP505A1 activity. The highest CYP102A1 activity (0.8 mM HBA converted in 24 h) was observed with concentrated resting-cell suspensions of S. cerevisiae. The CYP505A1 activity observed with growing cultures of A. adeninivorans was however at least 12 times higher than the CYP102A1 activity of S. cerevisiae with up to 2 mM HBA converted within 6 h. The use of K. marxianus and A. adeninivorans for P450 expression has not previously been reported.

Cloning of an epoxide hydrolase-encoding gene from Rhodotorula mucilaginosa and functional expression in Yarrowia lipolytica.

Epoxide hydrolases (EHs), especially those of fungal origin, have the ability to catalyse the enantioselective hydrolysis of epoxides to their corresponding diols. Recombinant DNA technology has been used extensively to overproduce these catalysts for the efficient hydrolytic kinetic resolution of epoxides, which serve as high-value intermediates in the fine chemicals and pharmaceutical industries. Degenerate primers, based on data from available EH-encoding gene sequences, in conjunction with inverse PCR, were used to amplify the genomic EH-encoding gene from Rhodotorula mucilaginosa. The 2347 bp genomic sequence revealed a 1979 bp ORF containing nine introns. The cDNA sequence revealed an 1185 bp EH-encoding gene that translates into a 394 amino acid protein exhibiting low sequence homology towards the known EH proteins. The EH gene from R. mucilaginosa was functionally expressed in Yarrowia lipolytica using a constitutive integrative expression cassette. Whole-cell biotransformation of (2,3-epoxypropyl)benzene, using the recombinant EH, revealed activity and selectivity far superior to any other activity and selectivity reported in literature using wild-type organisms. The GenBank Accession No. for the R. mucilaginosa EH gene is AY627310.

Cloning and sequencing of an epoxide hydrolase gene from Rhodosporidium paludigenum.

Epoxide hydrolase (EH) activity was recently described in yeasts and highly selective hydrolysis of epoxides was observed during whole cell biotransformations. To expand the available molecular data regarding yeast EHs, the EH encoding gene from Rhodosporidium paludigenum (CBS 6565) was isolated, cloned and sequenced. The genomic EH sequence revealed a 1600 bp sequence interrupted by six introns. cDNA sequence analysis revealed an open reading frame of 1236 bp with a deduced polypeptide length of 411 amino acids. The deduced amino acid sequence revealed a relative high degree of sequence homology compared to the amino acid sequence of the EH from Rhodotorula glutinis.