Role of Hippoboscidae Flies as Potential Vectors of Bartonella spp. Infecting Wild and Domestic Ruminants

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.     

Bartonella species in bat flies (Diptera: Nycteribiidae) from western Africa

Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera: Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies removed from Eidolon helvum, the straw-coloured fruit bat, were screened for the presence of Bartonella by culture and PCR analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.     

Are hippoboscid flies a major mode of transmission of feather mites?

Feather mites (Astigmata) are distributed around the world, living on the feathers of birds, but their mechanisms for transmission among hosts are not fully understood. There is anecdotal evidence of feather mites attached to louseflies (Diptera: Hippoboscidae), suggesting that feather mites may use these flies as a mode of phoretic transmission among birds. Two bird-lousefly associations (alpine swift Apus melba-Crataerina melbae and feral pigeon Columba livia-Pseudolynchia canariensis) were inspected to test the hypothesis that feather mites use hippoboscid flies as major mode of transmission. Both bird species showed a high prevalence and abundance of feather mites and louseflies. However, no feather mites were found attached to the 405 louseflies inspected, although skin mites (Epidermoptidae and Cheyletiellidae) were found on louseflies collected from feral pigeons. This study suggests that feather mites do not use hippoboscid flies as a major mode of transmission among birds.     

The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers

Hippoboscoidea is a superfamily of Diptera that contains the Glossinidae or tsetse flies, the Hippoboscidae or louse flies, and two families of bat flies, the Streblidae and the Nycteribiidae. We reconstruct the phylogenetic relationships within Hippoboscoidea using maximum parsimony and Bayesian methods based on nucleotide sequences from fragments of four genes: nuclear 28S ribosomal DNA and the CPSase domain of CAD, and mitochondrial 16S rDNA and cytochrome oxidase I. We recover monophyly for most of the presently recognized groups within Hippoboscoidea including the superfamily as a whole, the Hippoboscidae, the Nycteribiidae, the bat flies, and the Pupipara (=Hippoboscidae+Nycteribiidae+Streblidae), as well as several subfamilies within the constituent families. Streblidae appear to be paraphyletic. Our phylogenetic hypothesis is well supported and decisive in that most competing topological hypotheses for the Hippoboscoidea require significantly longer trees. We confirm a single shift from a free-living fly to a blood-feeding ectoparasite of vertebrates and demonstrate that at least two host shifts from mammals to birds have occurred. Wings have been repeatedly lost, but never regained. The hippoboscoid ancestor also evolved adenotrophic viviparity and our cladogram is consistent with a gradual reduction in the motility of the deposited final instar larvae from active burrowing in the soil to true pupiparity where adult females glue the puparium within the confines of bat roosts.     

The role of some cyclorrhaphan flies as carriers of human helminths in Malaysia

The role of some adult flies (Diptera: Cyclorrhapha) as carriers of helminth parasites of man was studied at four sites in Malaysia: a refuse dump, where no helminth-positive flies were detected, and in three peri-domestic situations where four species of flies carried up to three types of nematodes. The dominant fly species Chrysomya megacephala (Fabricius) carried eggs of the roundworm Ascaris lumbricoides L., the pinworm Trichuris trichiura (L.) and hookworm on the adult external body surface and in the gut lumen, in association with Bukit Lanjan aborigines. Chrysomya rufifacies (Macquart) and Sarcophaga spp. also had Ascaris lumbricoides and Trichurus trichiura eggs in their gut contents. Human helminths were not recovered from Lispe leucospila (Wiedemann), Lucilia cuprina (Wiedemann) or the housefly Musca domestica L. In an urban slum area of Kuala Lumpur city, filariform larvae identified as the hookworm Necator americanus (Stiles) occurred in the intestines of the face-fly Musca sorbens Wiedemann (22 larvae per 100 flies) and of Chrysomya megacephala (4.5 larvae per 100 flies). This concentration of apparently infective N. americanus in M. sorbens, a fly which often breeds in faeces and browses on human skin, could have transmission potential.     

Bartonella spp. in deer keds, Lipoptena mazamae (Diptera: Hippoboscidae), from Georgia and South Carolina, USA

Deer keds, Lipoptena mazamae (Diptera: Hippoboscidae), were collected from white-tailed deer (Odocoileus virginianus) and humans in Georgia and South Carolina, USA (1 October 2001-6 January 2005) and screened for the presence of DNA from Bartonella spp. Forty deer keds were screened for Bartonella spp. by polymerase chain reaction using primers specific to the riboflavin synthase gene (ribC) of Bartonella. Bartonella species closely related to Bartonella schoenbuchensis and to the etiologic agent of cat-scratch disease (Bartonella henselae) were detected in 10 keds and one ked, respectively.     

Origin,acquisition and diversification of heritable bacterial endosymbionts in louse flies and bat flies

The γ‐proteobacterium Arsenophonus and its close relatives (Arsenophonus and like organisms, ALOs) are emerging as a novel clade of endosymbionts, which are exceptionally widespread in insects. The biology of ALOs is, however, in most cases entirely unknown, and it is unclear how these endosymbionts spread across insect populations. Here, we investigate this aspect through the examination of the presence, the diversity and the evolutionary history of ALOs in 25 related species of blood‐feeding flies: tsetse flies (Glossinidae), louse flies (Hippoboscidae) and bat flies (Nycteribiidae and Streblidae). While these endosymbionts were not found in tsetse flies, we identify louse flies and bat flies as harbouring the highest diversity of ALO strains reported to date, including a novel ALO clade, as well as Arsenophonus and the recently described Candidatus Aschnera chinzeii. We further show that the origin of ALO endosymbioses extends deep into the evolutionary past of louse flies and bat flies, and that it probably played a major role in the ecological specialization of their hosts. The evolutionary history of ALOs is notably complex and was shaped by both vertical transmission and horizontal transfers with frequent host turnover and apparent symbiont replacement in host lineages. In particular, ALOs have evolved repeatedly and independently close relationships with diverse groups of louse flies and bat flies, as well as phylogenetically more distant insect families, suggesting that ALO endosymbioses are exceptionally dynamic systems.     

Trypanosoma (Megatrypanum) melophagium in the sheep ked Melophagus ovinus from organic farms in Croatia: phylogenetic inferences support restriction to sheep and sheep keds and close relationship with trypanosomes from other ruminant species

Trypanosoma (Megatrypanum) melophagium is a parasite of sheep transmitted by sheep keds, the sheep-restricted ectoparasite Melophagus ovinus (Diptera: Hippoboscidae). Sheep keds were 100% prevalent in sheep from five organic farms in Croatia, Southeastern Europe, whereas trypanosomes morphologically compatible with T. melophagium were 86% prevalent in the guts of the sheep keds. Multilocus phylogenetic analyses using sequences of small subunit rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase, spliced leader, and internal transcribed spacer 1 of the rDNA distinguished T. melophagium from all allied trypanosomes from other ruminant species and placed the trypanosome in the subgenus Megatrypanum. Trypanosomes from sheep keds from Croatia and Scotland, the only available isolates for comparison, shared identical sequences. All biologic and phylogenetic inferences support the restriction of T. melophagium to sheep and, especially, to the sheep keds. The comparison of trypanosomes from sheep, cattle, and deer from the same country, which was never achieved before this work, strongly supported the host-restricted specificity of trypanosomes of the subgenus Megatrypanum. Our findings indicate that with the expansion of organic farms, both sheep keds and T. melophagium may re-emerge as parasitic infections of sheep.     

Bartonella quintana in head louse nits

The body louse is the principal vector of Bartonella quintana, the causative organism of trench fever, but B. quintana DNA has also been detected in adult head lice. Because there are no characteristics that distinguish the body louse from the head louse, we decided to test head louse nits collected from a homeless man for the presence of B. quintana DNA. All of the sampled nits tested positive by real-time PCR, and intergenic spacer region (ITS) gene sequences shared 100% similarity to the corresponding ITS fragment of the genome of B. quintana. The role of the head louse in the maintenance and transmission of B. quintana remains to be determined.     

Competence of the housefly,Musca domestica,as a vector of Microsporum canis under experimental conditions

Abstract The role of Musca domestica Linnaeus as a vector of the dermatophyte Microsporum canis was investigated under experimental laboratory conditions. About 400 4‐day‐old M. domestica flies were divided into two groups. Group A consisted of about 200 infected flies and group B comprised about 200 uninfected flies that were used as controls. Each trial was run three times. Flies from group A were fed for 24 h with a solution of ultra‐high temperature‐treated (UHT) milk containing about 10⁶ colony‐forming units (CFU) per mL of M. canis (infected milk inoculum [IMI]). The control group (group B) was fed with only UHT milk spiked with a teaspoon of honey. Microsporum canis was detected from faeces, vomitus, external surfaces and internal organs of 20 adult flies, eggs, first‐, second‐ and third‐stage (L1, L2, L3) larvae and pupae of each group, as well from 20 adult newly emerged flies (NEFs; from infected generations only). Samples were collected at 2, 4, 6 and 24 h post‐infection (p.i.) (i.e. the times at which IMI was available) and on 2, 5, 7 and 8 days p.i. from adult flies, faeces and vomitus. Eggs, L1, L2, L3 and pupae were processed as soon as they appeared. Equivalent samples were taken from group B. All the samples were individually cultured. Microsporum canis was not isolated from the control group, from eggs, larvae, pupae or NEFs, or from faeces and vomitus, although it was detected on the body surface (26.2%) and internal organs (26.9%) of adult flies. The highest positivity for M. canis was detected on flies within the first 6 h p.i. (i.e. 57.2% on the body surface and 71.6% in the internal organs). No M. canis was isolated at 24 h p.i., but it was isolated from the body surface only at 2 and 5 days p.i. The results presented provide evidence that M. domestica transmits M. canis mechanically with its outer body surface for up to 5 days p.i., but does not do so through its vomitus and faeces or transovarially. The role played by M. domestica in the epidemiology of human and animal dermatophytoses is discussed.     

Domestic cattle as a non-conventional amplifying host of vesicular stomatitis New Jersey virus

The role of vertebrates as amplifying and maintenance hosts for vesicular stomatitis New Jersey virus (VSNJV) remains unclear. Livestock have been considered dead-end hosts because detectable viraemia is absent in VSNJV-infected animals. This study demonstrated two situations in which cattle can represent a source of VSNJV to Simulium vittatum Zetterstedt (Diptera: Simuliidae) by serving: (a) as a substrate for horizontal transmission among co-feeding black flies, and (b) as a source of infection to uninfected black flies feeding on sites where VSNJV-infected black flies have previously fed. Observed co-feeding transmission rates ranged from 0% to 67%. Uninfected flies physically separated from infected flies by a distance of up to 11 cm were able to acquire virus during feeding although the rate of transmission decreased as the distance between infected and uninfected flies increased. Acquisition of VSNJV by uninfected flies feeding on initial inoculation sites at 24 h, 48 h and 72 h post-infection, in both the presence and absence of vesicular lesions, was detected.     

Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), occurs via oral secretions and excreta

The MdSGHV is a double-stranded DNA virus that replicates in the salivary glands of infected adult house flies. Transmission of this non-occluded, enveloped virus is believed to be mediated orally via deposition and consumption of oral secretions composed of salivary gland secretions and crop contents. In this study, transmission electron micrographs of crops from infected flies showed numerous enveloped virions in the crop lumen adjacent to the cuticular intima, as well as on the hemocoel side in close vicinity to muscle cells. Oral treatments of newly emerged flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. Virus released via oral secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection. Using quantitative real-time PCR, MdSGHV DNA was quantified in oral secretions and excreta obtained from viremic flies. Between 2 and 4 days post-infection (dpi), viral copy numbers in oral secretions increased exponentially and from 5 to 21 dpi each infected fly released an average 10⁶ MdSGHV copies per feeding event. Excreta samples collected overnight from individual infected flies at 5 dpi contained an average 6.5 × 10⁵ viral copies. Low but detectable infection rates were produced when newly emerged flies were challenged with excreta samples. In summary, evaluation of the quantity and infectivity of MdSGHV released by individual infected house flies clearly showed that deposition of oral secretions and excreta onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies.     

Pathogenicity of Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo) Vuillemin,to three adult fruit fly species: Ceratitis capitata (Weidemann), C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Diptera :Tephritidae)

The pathogenicity of two isolates of Beauveria bassiana and 12 of Metarhizium anisopliae towards adult fruit flies, Ceratitis capitata and Ceratitis rosa var. fasciventris was tested in the laboratory. Fruit flies were exposed to dry conidia evenly spread on velvet material covering the inner side of a cylindrical plastic tube. All isolates tested were pathogenic to both species of fruit flies. Mortality ranged from 7 to 100% in C. capitata and from 11.4 to 100% in C. rosa var. fasciventris at 4 days post-inoculation. Six isolates, M. anisopliae ICIPE 18, 20, 32, 40, 41 and 62, were highly pathogenic to both C. capitata and C. rosa var. fasciventris. The LT90 values of the most pathogenic isolates ranged between 3-4 days in both insects. Because of the difficulties in rearing C. cosyra, only the isolates that were highly pathogenic to both C. rosa var. fasciventris and C. capitata were tested against adult C. cosyra. They caused mortality of between 72-78% at 4 days post-inoculation. The LT90 values in all the isolates did not exceed 4 days. One of the most pathogenic isolates, M. anisopliae ICIPE 20, was evaluated against C. capitata and C. rosa var. fasciventris in cage experiments using three autoinoculators (maize cob, cheesecloth and Petri dish) in an autoinoculative device consisting of plastic mineral bottle. Mortality of between 70-93% was observed in flies of both species that were captured from the cages and held under laboratory conditions. These results indicate the possibility of fruit fly suppression with entomopathogenic fungi using an autoinoculative device.     

Temporal and Spatial Patterns of <Emphasis Type="Italic">Bartonella</Emphasis> Infection in Black-tailed Prairie Dogs (<Emphasis Type="Italic">Cynomys ludovicianus</Emphasis>)

We describe the temporal dynamics and spatial distribution of Bartonella in black-tailed prairie dogs (Cynomys ludovicianus) based on a longitudinal study conducted in 20 black-tailed prairie dog (BTPD) colonies in Boulder County, CO from 2003 to 2005. Bartonella infection was widely distributed in all colonies with an overall prevalence of 23.1%, but varied by colony from 4.8% to 42.5% and by year from 9.1 to 39.0%, with a marked increase in Bartonella activity in 2005. Levels of bacteremia varied from 40 to 12,000 colony forming units (CFU) per milliliter of BTPD blood, but were highly skewed with a median of 240 CFU. Bartonella infection rates were unimodal with respect to BTPD body mass, first increasing among growing juveniles, then declining among adults. Infection rates exhibited a sigmoidal response to body mass, such that 700g may prove to be a useful threshold value to evaluate the likelihood of Bartonella infection in BTPDs. Bartonella prevalence increased throughout the testing season for each year, as newly emerged juveniles developed bacteremia. Data from recaptured animals suggest that Bartonella infections did not persist in individual BTPDs, which may explain the relatively low prevalence of Bartonella in BTPDs compared to other rodent species. No association was found between Bartonella prevalence and host population density. Prevalence did not differ between males and females. The spatio-temporal pattern of Bartonella infection among colonies suggests epizootic spread from northern to central and southern portions of the study area. The potential significance of the BTPD-associated Bartonella for public health needs to be further investigated.     

Louse flies of Eleonora’s falcons that also feed on their prey are evolutionary dead‐end hosts for blood parasites

Host shifts are widespread among avian haemosporidians, although the success of transmission depends upon parasite‐host and parasite‐vector compatibility. Insular avifaunas are typically characterized by a low prevalence and diversity of haemosporidians, although the underlying ecological and evolutionary processes remain unclear. We investigated the parasite transmission network in an insular system formed by Eleonora's falcons (the avian host), louse flies that parasitize the falcons (the potential vector), and haemosporidians (the parasites). We found a great diversity of parasites in louse flies (16 Haemoproteus and 6 Plasmodium lineages) that did not match with lineages previously found infecting adult falcons (only one shared lineage). Because Eleonora's falcon feeds on migratory passerines hunted over the ocean, we sampled falcon kills in search of the origin of parasites found in louse flies. Surprisingly, louse flies shared 10 of the 18 different parasite lineages infecting falcon kills. Phylogenetic analyses revealed that all lineages found in louse flies (including five new lineages) corresponded to Haemoproteus and Plasmodium parasites infecting Passeriformes. We found molecular evidence of louse flies feeding on passerines hunted by falcons. The lack of infection in nestlings and the mismatch between the lineages isolated in adult falcons and louse flies suggest that despite louse flies’ contact with a diverse array of parasites, no successful transmission to Eleonora's falcon occurs. This could be due to the falcons’ resistance to infection, the inability of parasites to develop in these phylogenetically distant species, or the inability of haemosporidian lineages to complete their development in louse flies.     

Pathogenicity of three formulations of entomopathogenic fungi for control of adult Haematobia irritans (Diptera: Muscidae)

Powder formulations of three species of entomopathogenic fungi were evaluated for their pathogenic effect upon adult horn flies, Hematobia irritans (L.) (Diptera: Muscidae). Flies were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. (strain GHA), Metarhizium anisopliae (Metschnikoff) Sorokin (strain ESCI), and Paecilomyces fimosoroseus (Wize) Brown & Smith (strain ARSEF 3581) in the laboratory. At 4 d postexposure, flies treated with B. bassiana had an average of 98.4% mortality versus 43.5% from treatment with M. anisopliae and 13.0% from treatment with P. fiimosoroseus. At 7 d postexposure, flies treated with B. bassiana had an average of 100.0% mortality compared with 73.0% from treatment with M. anisopliae and 33.3% from treatment with P.fumosoroseus. Mean lethal time (LT50) was 2.70, 4.98, and 7.97 d for B. bassiana, M. anisopliae, and P. fiumosoroseus, respectively. Entomopathogenic fungi such as B. bassiana and M. anisopliae may have the potential for controlling populations of horn flies. These studies indicate that B. bassiana and M. anisopliae were not only pathogenic to adult horn flies, but they caused mortality in a short time.     

Bartonellosis,an increasingly recognized zoonosis

Cat scratch disease is the most common zoonotic infection caused by Bartonella bacteria. Among the many mammals infected with Bartonella spp., cats represent a large reservoir for human infection, as they are the main reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Bartonella spp. are vector‐borne bacteria, and transmission of B. henselae by cat fleas occurs mainly through infected flea faeces, although new potential vectors (ticks and biting flies) have been identified. Dogs are also infected with various Bartonella species and share with humans many of the clinical signs induced by these infections. Although the role of dogs as source of human infection is not yet clearly established, they represent epidemiological sentinels for human exposure. Present knowledge on the aetiology, clinical features and epidemiological characteristics of bartonellosis is presented.     

Molecular phylogenetic analysis of nycteribiid and streblid bat flies (Diptera: Brachycera, Calyptratae): implications for host associations and phylogeographic origins

Bat flies are a small but diverse group of highly specialized ectoparasitic, obligatory bloodsucking Diptera. For the first time, the phylogenetic relationships of 26 species and five subfamilies were investigated using four genes (18S rDNA, 16S rDNA, COII, and cytB) under three optimality criteria (maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference). Tree topology tests of previous hypotheses were conducted under likelihood (Shimodaira-Hasegawa test). Major findings include the non-monophyly of the Streblidae and the recovery of an Old World- and a New World-Clade of bat flies. These data ambiguously resolve basal relationships between Hippoboscidae, Glossinidae, and bat flies. Recovered phylogenies resulted in either monophyly (Bayesian approach) or paraphyly (MP/ML topologies) of the bat flies, thus obscuring the potential number of possible associations with bats throughout the history of this group. Dispersal-vicariance analysis suggested the Neotropical region as the possible ancestral distribution area of the New World Streblidae and the Oriental region for the Old World bat flies. The genes examined show conflicting support across the nodes of the tree, particularly in the basal positions. Additionally, there is poor character support among all genes for the nodes associated with early hippoboscoid diversification. This results in extremely short basal branches, adding support to the idea of a rapid radiation among the four major groups of Hippoboscoidea.     

Geographic and host range of the nematode Soboliphyme baturini across Beringia

The nematode Soboliphyme baturini Petrov, 1930, was found to represent a single species with a relatively broad geographic range across Beringia and northwestern North America on the basis of the assessment of molecular sequence data for adult and juvenile parasites. Refuted are hypotheses suggesting that several cryptic species could be partitioned either among an array of mustelid definitive hosts or across the vast region that links North America and Eurasia. Host specificity for this species is examined on the basis of a comprehensive list for definitive hosts, derived from new field surveys and existing literature for S. baturini. Only 5 mustelids (Gulo gulo, Martes americana, M. caurina, M. zibellina, and Neovison vison) appear to have significant roles in the life history, persistence, and transmission of this nematode. Soboliphyme baturini readily switches among M. americana, M. caurina, Mustela erminea, or N. vison at any particular locality throughout its geographic range in North America, although Martes spp. could represent the source for nematodes in a broader array of mustelids. Molecular analyses (243 base pairs of mitochondrial gene nicotinamide dehydrogenase [ND4]) suggest that hypotheses for host specificity across an array of mustelid definitive hosts are not supported. The life cycle of S. baturini is explored through a review of diet literature for 2 marten species, M. americana and M. caurina, and other mustelids across the Holarctic. Shrews (Soricomorpha: Soricidae) comprise >8% of prey for these species of Martes, suggesting their putative role as paratenic hosts. Juvenile nematodes found in the diaphragms of soricids are genetically identical to adult S. baturini found in the stomachs of mustelids at the same locations in both Asia and North America, corroborating a role in transmission for species of Sorex.