Acaricide resistance in livestock ticks infesting cattle in Africa: Current status and potential mitigation strategies.

In many African countries, tick control has recently been the responsibility of resource-poor farmers rather than central government veterinary departments. This has led to an increase in acaricide resistance, threatening the welfare of livestock farmers in sub-Saharan Africa. Resistance has evolved to the three classes of acaricides used most extensively in the continent, namely fourth-generation synthetic pyrethroids (SP), organophosphates (OP) and amidines (AM), in virtually all countries in which they have been deployed across the globe. Most current data are derived from research in Australia and Latin America, with the majority of studies on acaricide resistance in Africa performed in South Africa. There is also limited recent research from West Africa and Uganda. These studies confirm that acaricide resistance in cattle ticks is a major problem in Africa. Resistance is most frequently directly assayed in ticks using the larval packet test (LPT) that is endorsed by FAO, but such tests require a specialist tick-rearing laboratory and are relatively time consuming. To date they have only been used on a limited scale in Africa and resistance is often still inferred from tick numbers on animals. Rapid tests for resistance in ticks, would be better than the LPT and are theoretically possible to develop. However, these are not yet available. Resistance can be mitigated through integrated control strategies, comprising a combination of methods, including acaricide class rotation or co-formulations, ethnoveterinary practices, vaccination against ticks and modified land management use by cattle, with the goal of minimising the number of acaricide applications required per year. There are data suggesting that small-scale farmers in Africa are often unaware of the chemical differences between different acaricide brands and use these products at concentrations other than those recommended by the manufacturers, or in incorrect rotations or combinations of the different classes of chemicals on the market. There is an urgent need for a more evidence-based approach to acaricide usage in small-scale livestock systems in Africa, including direct measurements of resistance levels, combined with better education of farmers regarding acaricide products and how they should be deployed for control of livestock ticks.

Molecular survey of Babesia parasites in Kenya: first detailed report on occurrence of Babesia bovis in cattle.

BACKGROUND: Among protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers. METHODS: A species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis. RESULTS: A total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97-100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties. CONCLUSION: These findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established. GRAPHICAL ABTRACT.

Molecular survey of cattle ticks in Burundi: First report on the presence of the invasive Rhipicephalus microplus tick.

A recent research study on prevalence of tick-borne pathogens in Burundi reported high prevalence and endemicity of Theileria parva, Anaplasma marginale and Babesia bigemina infections in cattle. Detailed information about tick species infesting animals, their distribution and genetic diversity in Burundi is outdated and limited. This study therefore assessed the prevalence and genetic diversity of tick species infesting cattle across agroecological zones (AEZs) in Burundi. A cross-sectional study on the occurrence of tick species was conducted in 24 districts of Burundi between October and December 2017. Differential identification and characterization of ticks collected was conducted using tick morphological keys and molecular tools (cox1 and 12S rRNA gene). Chi-square test was used to test for association between agroecological zones and the prevalence of tick species. Phylogenetic relationships were inferred using bayesian and maximum likelihood algorithms. A total of 483 ticks were collected from the five AEZs sampled. Six tick species comprising of Rhipicephalus appendiculatus, R. sanguineus, R. evertsi evertsi, R. microplus, R. decoloratus and Amblyomma variegatum were observed. Rhipicephalus appendiculatus were the most prevalent ticks (~45%). A total of 138 specimens (28%) were found to be Rhipicephalus microplus, suggesting an emerging threat for cattle farmers. Twelve R. appendiculatus cox1 haplotypes were obtained from 106 specimens that were sequenced. Two cox1 haplotypes of R. microplus which clustered into previously reported Clade A were observed. Rhipicephalus sanguineus and R. evertsi evertsi ticks, the vectors of numerous zoonotic pathogens, were collected from cattle, which constitute a high risk for public health. These findings reveal an overlapping distribution of tick vectors in Burundi. The design of ticks and tick-borne diseases control strategies should consider the distribution of different vectors across the AEZs particularly the presence of the highly invasive R. microplus tick in Burundi and the potential risk of introducing the pathogenic Babesia bovis.

Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi.

BACKGROUND: Tick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi. METHODS: In a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively. RESULTS: The prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected . CONCLUSIONS: The findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

Molecular evidence confirms occurrence of Rhipicephalus microplus Clade A in Kenya and sub-Saharan Africa.

BACKGROUND: The tick vector Rhipicephalus microplus which transmits Babesia spp. and rickettsial pathogens has not been reported in Kenya since 1998. More recently, the pathogenic Babesia bovis has been detected in cattle blood DNA. The status of R. microplus in Kenya remains unknown. This study employed morphological and molecular tools to characterize R. microplus originating from Kenya and assess the genetic relationships between Kenyan and other African R. microplus genotypes. METHODS: Ticks were collected in south-eastern Kenya (Kwale County) from cattle and characterized to investigate the existence of R. microplus. Genetic and phylogenetic relationships between the Kenyan and other annotated R. microplus reference sequences was investigated by analysis of the cytochrome c oxidase subunit 1 (cox1) gene. To further characterize Kenyan ticks, we generated low coverage whole genome sequences of two R. microplus, one R. decoloratus and R. appendiculatus. A B. bovis specific TaqMan probe qPCR assay was used to detect B. bovis in gDNA from R. microplus ticks. RESULTS: Occurrence of R. microplus was confirmed in Kwale County, Kenya. The Kenyan R. microplus cox1 sequences showed very high pairwise identities (> 99%) and clustered very closely with reference African R. microplus sequences. We found a low genetic variation and lack of geographical sub-structuring among the African cox1 sequences of R. microplus. Four complete mitochondrial (mt) genomes for two R. microplus, one R. decoloratus and one R. appendiculatus were assembled from next generation sequence data. The mitochondrial genome sequences of the two Kenyan R. microplus ticks clustered closely with reference genome sequences from Brazil, USA, Cambodia and India forming R. microplus Clade A. No B. bovis was detected in the Kwale R. microplus DNA. CONCLUSIONS: These findings confirm the presence of R. microplus in Kenya and suggest that R. microplus Clade A is prevalent in cattle in sub-Saharan Africa. These and other recent findings of widespread occurrence of R. microplus in Africa provide a strong justification for urgent surveillance to determine and monitor the spread of R. microplus and vector competence of Boophilus ticks for B. bovis in Africa, with the ultimate goal of strategic control.

Mitochondrial and nuclear multilocus phylogeny of Rhipicephalus ticks from Kenya.

The morphological diversity of African ticks of the genus Rhipicephalus and subgenus Boophilus have been studied in detail. However, their taxonomy remains poorly resolved with limited molecular studies performed to improve inter-species discrimination. Herein, ribosomal cytochrome c oxidase I (COI), 12S ribosomal DNA (12S rDNA) and nuclear ribosomal DNA internal transcriber spacer 2 (ITS2) were analyzed in Rhipicephalus tick populations in Kenya. While the morphological and molecular criteria separated R. e. evertsi, R. pulchellus and R. appendiculatus from other members of the genus, except the morphologically similar sibling species R. zambeziensis, this was not the case for other tick populations. COI sequences of Rhipicephalus ticks from Ruma National Park (RNP) in Southwestern Kenya, that were morphologically similar to R. praetextatus/R. simus, a formed distinct clade and barcode gap group. 12S rDNA haplotypes of this population were 99% identical to a GenBank accession of R. muhsamae which is thought to be endemic in West and Central Africa. However, the ITS2 locus indicated that the RNP samples were genetically closest to ticks identified morphologically as R. praetextatus. The COI and 12S rDNA haplotype sequences of R. praetextatus clustered closely with R. simus reference sequences though the two species occurred in distinct barcode gap groups. Our results suggest that the R. simus/R. praetextatus/R. muhsamae comprise a closely related tick species complex found across sub-Saharan Africa and includes the yet to be described RNP population. More studies on the biology, ecology and genomics of all life stages of tick species in the complex may clarify their taxonomic status. A continent-wide study that combines morphology, DNA marker sequencing and emerging methods, such as mass spectrometry and whole-genome resequencing may reveal the diversity and distribution of taxa within the genus Rhipicephalus in sub-Saharan Africa.

Argasid and ixodid systematics: Implications for soft tick evolution and systematics, with a new argasid species list.

The systematics of the genera and subgenera within the soft tick family Argasidae is not adequately resolved. Different classification schemes, reflecting diverse schools of scientific thought that elevated or downgraded groups to genera or subgenera, have been proposed. In the most recent classification scheme, Argas and Ornithodoros are paraphyletic and the placement of various subgenera remains uncertain because molecular data are lacking. Thus, reclassification of the Argasidae is required. This will enable an understanding of soft tick systematics within an evolutionary context. This study addressed that knowledge gap using mitochondrial genome and nuclear (18S and 28S ribosomal RNA) sequence data for representatives of the subgenera Alectorobius, Argas, Chiropterargas, Ogadenus, Ornamentum, Ornithodoros, Navis (subgen. nov.), Pavlovskyella, Persicargas, Proknekalia, Reticulinasus and Secretargas, from the Afrotropical, Nearctic and Palearctic regions. Hard tick species (Ixodidae) and a new representative of Nuttalliella namaqua (Nuttalliellidae), were also sequenced with a total of 83 whole mitochondrial genomes, 18S rRNA and 28S rRNA genes generated. The study confirmed the utility of next-generation sequencing to retrieve systematic markers. Paraphyly of Argas and Ornithodoros was resolved by systematic analysis and a new species list is proposed. This corresponds broadly with the morphological cladistic analysis of Klompen and Oliver (1993). Estimation of divergence times using molecular dating allowed dissection of phylogeographic patterns for argasid evolution. The discovery of cryptic species in the subgenera Chiropterargas, Ogadenus and Ornithodoros, suggests that cryptic speciation is common within the Argasidae. Cryptic speciation has implications for past biological studies of soft ticks. These are discussed in particular for the Ornithodoros (Ornithodoros) moubata and Ornithodoros (Ornithodoros) savignyi groups.

Mitochondrial phylogeography and population structure of the cattle tick Rhipicephalus appendiculatus in the African Great Lakes region.

BACKGROUND: The ixodid tick Rhipicephalus appendiculatus is the main vector of Theileria parva, wich causes the highly fatal cattle disease East Coast fever (ECF) in sub-Saharan Africa. Rhipicephalus appendiculatus populations differ in their ecology, diapause behaviour and vector competence. Thus, their expansion in new areas may change the genetic structure and consequently affect the vector-pathogen system and disease outcomes. In this study we investigated the genetic distribution of R. appendiculatus across agro-ecological zones (AEZs) in the African Great Lakes region to better understand the epidemiology of ECF and elucidate R. appendiculatus evolutionary history and biogeographical colonization in Africa. METHODS: Sequencing was performed on two mitochondrial genes (cox1 and 12S rRNA) of 218 ticks collected from cattle across six AEZs along an altitudinal gradient in the Democratic Republic of Congo, Rwanda, Burundi and Tanzania. Phylogenetic relationships between tick populations were determined and evolutionary population dynamics models were assessed by mismach distribution. RESULTS: Population genetic analysis yielded 22 cox1 and 9 12S haplotypes in a total of 209 and 126 nucleotide sequences, respectively. Phylogenetic algorithms grouped these haplotypes for both genes into two major clades (lineages A and B). We observed significant genetic variation segregating the two lineages and low structure among populations with high degree of migration. The observed high gene flow indicates population admixture between AEZs. However, reduced number of migrants was observed between lowlands and highlands. Mismatch analysis detected a signature of rapid demographic and range expansion of lineage A. The star-like pattern of isolated and published haplotypes indicates that the two lineages evolve independently and have been subjected to expansion across Africa. CONCLUSIONS: Two sympatric R. appendiculatus lineages occur in the Great Lakes region. Lineage A, the most diverse and ubiquitous, has experienced rapid population growth and range expansion in all AEZs probably through cattle movement, whereas lineage B, the less abundant, has probably established a founder population from recent colonization events and its occurrence decreases with altitude. These two lineages are sympatric in central and eastern Africa and allopatric in southern Africa. The observed colonization pattern may strongly affect the transmission system and may explain ECF endemic instability in the tick distribution fringes.

Analyses of mitochondrial genes reveal two sympatric but genetically divergent lineages of Rhipicephalus appendiculatus in Kenya.

BACKGROUND: The ixodid tick Rhipicephalus appendiculatus transmits the apicomplexan protozoan parasite Theileria parva, which causes East coast fever (ECF), the most economically important cattle disease in eastern and southern Africa. Recent analysis of micro- and minisatellite markers showed an absence of geographical and host-associated genetic sub-structuring amongst field populations of R. appendiculatus in Kenya. To assess further the phylogenetic relationships between field and laboratory R. appendiculatus tick isolates, this study examined sequence variations at two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (rRNA), and the nuclear encoded ribosomal internal transcribed spacer 2 (ITS2) of the rRNA gene, respectively. RESULTS: The analysis of 332 COI sequences revealed 30 polymorphic sites, which defined 28 haplotypes that were separated into two distinct haplogroups (A and B). Inclusion of previously published haplotypes in our analysis revealed a high degree of phylogenetic complexity never reported before in haplogroup A. Neither haplogroup however, showed any clustering pattern related to either the geographical sampling location, the type of tick sampled (laboratory stocks vs field populations) or the mammalian host species. This finding was supported by the results obtained from the analysis of 12S rDNA sequences. Analysis of molecular variance (AMOVA) indicated that 90.8 % of the total genetic variation was explained by the two haplogroups, providing further support for their genetic divergence. These results were, however, not replicated by the nuclear transcribed ITS2 sequences likely because of recombination between the nuclear genomes maintaining a high level of genetic sequence conservation. CONCLUSIONS: COI and 12S rDNA are better markers than ITS2 for studying intraspecific diversity. Based on these genes, two major genetic groups of R. appendiculatus that have gone through a demographic expansion exist in Kenya. The two groups show no phylogeographic structure or correlation with the type of host species from which the ticks were collected, nor to the evolutionary and breeding history of the species. The two lineages may have a wide geographic distribution range in eastern and southern Africa. The findings of this study may have implications for the spread and control of R. appendiculatus, and indirectly, on the transmission dynamics of ECF.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry for comprehensive indexing of East African ixodid tick species.

BACKGROUND: The tick population of Africa includes several important genera belonging to the family Ixodidae. Many of these ticks are vectors of protozoan and rickettsial pathogens including Theileria parva that causes East Coast fever, a debilitating cattle disease endemic to eastern, central and southern Africa. Effective surveillance of tick-borne pathogens depends on accurate identification and mapping of their tick vectors. A simple and reproducible technique for rapid and reliable differentiation of large numbers of closely related field-collected ticks, which are often difficult and tedious to discriminate purely by morphology, will be an essential component of this strategy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is increasingly becoming a useful tool in arthropod identification and has the potential to overcome the limitations of classical morphology-based species identification. In this study, we applied MALDI-TOF MS to a collection of laboratory and field ticks found in Eastern Africa. The objective was to determine the utility of this proteomic tool for reliable species identification of closely related afrotropical ticks. METHODS: A total of 398 ixodid ticks from laboratory maintained colonies, extracted from the hides of animals or systematically collected from vegetation in Kenya, Sudan and Zimbabwe were analyzed in the present investigation. The cytochrome c oxidase I (COI) genes from 33 specimens were sequenced to confirm the tentatively assigned specimen taxa identity on the basis of morphological analyses. Subsequently, the legs of ticks were homogenized and analyzed by MALDI-TOF MS. A collection of reference mass spectra, based on the mass profiles of four individual ticks per species, was developed and deposited in the spectral database SARAMIS™. The ability of these superspectra (SSp.) to identify and reliably validate a set of ticks was demonstrated using the remaining individual 333 ticks. RESULTS: Ultimately, ten different tick species within the genera Amblyomma, Hyalomma, Rhipicephalus and Rhipicephalus (Boophilus) based on molecular COI typing and morphology were included into the study analysis. The robustness of the 12 distinct SSp. developed here proved to be very high, with 319 out of 333 ticks used for validation identified correctly at species level. Moreover, these novel SSp. allowed for diagnostic specificity of 99.7 %. The failure of species identification for 14 ticks was directly linked to low quality mass spectra, most likely due to poor specimen quality that was received in the laboratory before sample preparation. CONCLUSIONS: Our results are consistent with earlier studies demonstrating the potential of MALDI-TOF MS as a reliable tool for differentiating ticks originating from the field, especially females that are difficult to identify after blood feeding. This work provides further evidence of the utility of MALDI-TOF MS to identify morphologically and genetically highly similar tick species and indicates the potential of this tool for large-scale monitoring of tick populations, species distributions and host preferences.

Multi-locus genotyping reveals absence of genetic structure in field populations of the brown ear tick (Rhipicephalus appendiculatus) in Kenya.

Rhipicephalus appendiculatus is an important tick vector of several pathogens and parasitizes domestic and wild animals across eastern and southern Africa. However, its inherent genetic variation and population structure is poorly understood. To investigate whether mammalian host species, geographic separation and resulting reproductive isolation, or a combination of these, define the genetic structure of R. appendiculatus, we analyzed multi-locus genotype data from 392 individuals from 10 geographic locations in Kenya generated in an earlier study. These ticks were associated with three types of mammalian host situations; (1) cattle grazing systems, (2) cattle and wildlife co-grazing systems (3) wildlife grazing systems without livestock. We also analyzed data from 460 individuals from 10 populations maintained as closed laboratory stocks and 117 individuals from five other species in the genus Rhipicephalus. The pattern of genotypes observed indicated low levels of genetic differentiation between the ten field populations (FST=0.014±0.002) and a lack of genetic divergence corresponding to the degree of separation of the geographic sampling locations. There was also no clear association of particular tick genotypes with specific host species. This is consistent with tick dispersal over large geographic ranges and lack of host specificity. In contrast, the 10 laboratory populations (FST=0.248±0.015) and the five other species of Rhipicephalus (FST=0.368±0.032) were strongly differentiated into distinct genetic groups. Some laboratory bred populations diverged markedly from their field counterparts in spite of originally being sampled from the same geographic locations. Our results demonstrate a lack of defined population genetic differentiation in field populations of the generalist R. appendiculatus in Kenya, which may be a result of the frequent anthropogenic movement of livestock and mobility of its several wildlife hosts between different locations.

Micro- and minisatellite-expressed sequence tag (EST) markers discriminate between populations of Rhipicephalus appendiculatus.

Biological differences, including vector competence for the protozoan parasite Theileria parva have been reported among populations of Rhipicephalus appendiculatus (Acari: Ixodidae) from different geographic regions. However, the genetic diversity and population structure of this important tick vector remain unknown due to the absence of appropriate genetic markers. Here, we describe the development and evaluation of a panel of EST micro- and minisatellite markers to characterize the genetic diversity within and between populations of R. appendiculatus and other rhipicephaline species. Sixty-six micro- and minisatellite markers were identified through analysis of the R. appendiculatus Gene Index (RaGI) EST database and selected bacterial artificial chromosome (BAC) sequences. These were used to genotype 979 individual ticks from 10 field populations, 10 laboratory-bred stocks, and 5 additional Rhipicephalus species. Twenty-nine markers were polymorphic and therefore informative for genetic studies while 6 were monomorphic. Primers designed from the remaining 31 loci did not reliably generate amplicons. The 29 polymorphic markers discriminated populations of R. appendiculatus and also 4 other Rhipicephalus species, but not R. zambeziensis. The percentage Principal Component Analysis (PCA) implemented using Multiple Co-inertia Analysis (MCoA) clustered populations of R. appendiculatus into 2 groups. Individual markers however differed in their ability to generate the reference typology using the MCoA approach. This indicates that different panels of markers may be required for different applications. The 29 informative polymorphic micro- and minisatellite markers are the first available tools for the analysis of the phylogeography and population genetics of R. appendiculatus.

Phylogeography and demographic history of Amblyomma variegatum (Fabricius) (Acari: Ixodidae), the tropical bont tick.

The genetic diversity of Amblyomma variegatum (Fabricius) from four Caribbean islands and five African countries was compared by analyzing the sequences of three gene fragments, two mitochondrial (12SrDNA and D-Loop-DL), and one nuclear (intergenic transcribed spacer 2 [ITS2]). Genetic variability of the ITS2 DNA fragment consisted of only uninformative single nucleotide mutations, and therefore this gene was excluded from further analyses. Mitochondrial gene divergences among African populations and between Caribbean and African populations were very low. Nevertheless, the data suggest that A. variegatum is divided into distinct East and West African groups, the western group including all Caribbean samples. Phylogenetic analyses of the 12SrDNA and DL gene sequences showed that the West African A. variegatum clustered in a well-supported monophyletic clade, distinct from eastern paraphyletic lineages. Sequences of A. variegatum from the Caribbean were embedded in the West African clade, which supports the known West African historical origin for these ticks.

Early bactericidal activity of a moxifloxacin and isoniazid combination in smear-positive pulmonary tuberculosis.

BACKGROUND: In vitro and animal studies have shown that moxifloxacin-containing combinations may improve the bactericidal efficacy of antituberculosis regimens. PATIENTS AND METHODS: We measured the decline in the sputum viable count of 13 patients who were given a combination of moxifloxacin 400 mg daily and isoniazid 300 mg daily. RESULTS: The time required to reduce the viable count by 50% (vt50) was 0.38 days (95% CI -0.03-0.78 days, SEM 0.13) and the mean early bactericidal activity (EBA) was 0.60 log10 cfu/day (95% CI 0.23-0.97, SEM 0.14). This compares with the vt50 calculated for isoniazid and moxifloxacin alone in the same population of 0.48 and 0.88 days, respectively. The EBA values for isoniazid and moxifloxacin alone were 0.77 and 0.53 log10 cfu/day, respectively. CONCLUSIONS: The combination of moxifloxacin and isoniazid is not antagonistic, but the combination does not significantly enhance bactericidal activity above that of isoniazid alone.

The bactericidal activity of moxifloxacin in patients with pulmonary tuberculosis.

Patients in whom acid-fast bacilli smear-positive pulmonary tuberculosis was newly diagnosed were randomized to receive 400 mg moxifloxacin, 300 mg isonaizid, or 600 mg rifampin daily for 5 days. Sixteen-hour overnight sputa collections were made for the 2 days before and for 5 days of monotherapy. Bactericidal activity was estimated by the time taken to kill 50% of viable bacilli (vt50) and the fall in sputum viable count during the first 2 days designated as the early bactericidal activity (EBA). The mean vt50 of moxifloxacin was 0.88 days (95% confidence interval [CI], 0.43-1.33 days) and the mean EBA was 0.53 (95% CI 0.28-0.79). For the isoniazid group, the mean vt50 was 0.46 days (95% CI, 0.31-0.61 days) and the mean EBA was 0.77 (95% CI, 0.54-1.00). For rifampin, the mean vt50 was 0.71 days (95% CI, 0.48-0.95 days) and the mean EBA was 0.28 (95% CI, 0.15-0.41). Using the EBA method, isoniazid was significantly more active than rifampin (p < 0.01) but not moxifloxacin. Using the vt50 method, isoniazid was more active than both rifampin and moxifloxacin (p = 0.03). Moxifloxacin has an activity similar to rifampin in human subjects with pulmonary tuberculosis, suggesting that it should undergo further assessment as part of a short course regimen for the treatment of drug-susceptible tuberculosis.