House Flies Are Underappreciated Yet Important Reservoirs and Vectors of Microbial Threats to Animal and Human Health.

House flies are well recognized as filth-associated organisms and public nuisances. House flies create sanitation issues when they bridge the gap between microbe-rich breeding environments and animal/human habitations. Numerous scientific surveys have demonstrated that house flies harbor bacterial pathogens that pose a threat to humans and animals. More extensive and informative surveys incorporating next-generation sequencing technologies have shown that house fly carriage of pathogens and harmful genetic elements, such as antimicrobial resistance genes, is more widespread and dangerous than previously thought. Further, there is a strong body of research confirming that flies not only harbor but also transmit viable, and presumably infectious, bacterial pathogens. Some pathogens replicate and persist in the fly, permitting prolonged shedding and dissemination. Finally, although the drivers still have yet to be firmly determined, the potential range of dissemination of flies and their associated pathogens can be extensive. Despite this evidence, the house flies' role as reservoirs, disseminators, and true, yet facultative, vectors for pathogens have been greatly underestimated and underappreciated. In this review, we present key studies that bolster the house fly's role both an important player in microbial ecology and population biology and as transmitters of microbial threats to animal and human health.

Selection for, and characterization of, fluralaner resistance in the house fly, Musca domestica.

House flies, Musca domestica (L), are the mechanical vector of >100 human and animal pathogens, including those that are antibiotic-resistant. Given that house flies are associated closely with human and livestock activity, they present medical and veterinary health concerns. Although there are numerous strategies for control of house fly populations, chemical control has been favored in many facilities. Products with pyrethroid active ingredients have been used predominantly for >35 years in space sprays. As a result, strong selection for pyrethroid resistance has led to reduced control of many populations. Reliance on a limited number of insecticides for decades has created fly control problems necessitating the discovery and formulation of new control chemistries. Fluralaner is a relatively new insecticide and acaricide (first reported in 2010), belonging to the isoxazoline class. These insecticides target the glutamate- and gamma-aminobutyric acid-gated (GABA) chloride channels, which is a different mode of action from other insecticides used against house flies. Although is it not currently registered for house fly control in the United States, previous work has shown that fluralaner is highly toxic to house flies and that there was limited cross-resistance found in laboratory strains having high levels of resistance to other insecticides. Herein, we characterized the time and age dependency of fluralaner toxicity, detected cross-resistance in populations from across the United States, and selected a highly resistant (>11,000-fold) house fly strain. We found that the fluralaner LD50 of 18-24 h old flies was 2-fold higher than for 5-6 d old flies. This appears to be due to more rapid penetration of fluralaner into the 5-6 d old flies. Fluralaner resistance was inherited as an intermediate to incompletely dominant trait and was mapped to chromosomes 5 and 3. Resistance could be suppressed to 7-fold with piperonyl butoxide, suggesting that cytochrome P450 (CYP)-mediated detoxification was a major mechanism of resistance. Decreased penetration was also demonstrated as a mechanism of resistance. The utility of fluralaner for house fly control is discussed.

Vaccination of cattle with synthetic peptides corresponding to predicted extracellular domains of Rhipicephalus (Boophilus) microplus aquaporin 2 reduced the number of ticks feeding to repletion.

BACKGROUND: There have been ongoing efforts to identify anti-tick vaccine targets to protect cattle from infestation with cattle fever ticks Rhipicephalus (Boophilus) microplus. Two commercial vaccines based on the tick gut protein Bm86 have had variable effectiveness, which has led to poor acceptance, and numerous studies have attempted to identify vaccine antigens that will provide more consistently effective protection. Transcriptomic analysis of R. microplus led to identification of three aquaporin genes annotated to code for transmembrane proteins involved in the transport of water across cell membranes. Previous work showed that vaccination with full-length recombinant aquaporin 1 (RmAQP1) reduced tick burdens on cattle. Targeted silencing of aquaporin 2 (RmAQP2) expression suggested it might also be a good anti-tick vaccination target. METHODS: Three synthetic peptides from the predicted extracellular domains of RmAQP2 were used to vaccinate cattle. Peptides were conjugated to keyhole limpet hemocyanin (KLH) as an antigenic carrier molecule. We monitored the antibody response with ELISA and challenged vaccinated cattle with R. microplus larvae. RESULTS: There was a 25% reduction overall in the numbers of ticks feeding to repletion on the vaccinated cattle. Immune sera from vaccinated cattle recognized native tick proteins on a western blot and reacted to the three individual synthetic peptides in an ELISA. The vaccinated calf with the highest total IgG titer was not the most effective at controlling ticks; ratios of IgG isotypes 1 and 2 differed greatly among the three vaccinated cattle; the calf with the highest IgG1/IgG2 ratio had the fewest ticks. Ticks on vaccinated cattle had significantly greater replete weights compared to ticks on controls, mirroring results seen with RNA silencing of RmAQP2. However, protein data could not confirm that vaccination had any impact on the ability of the tick to concentrate its blood meal by removing water. CONCLUSIONS: A reduced number of ticks feed successfully on cattle vaccinated to produce antibodies against the extracellular domains of RmAQP2. However, our predicted mechanism, that antibody binding blocks the ability of RmAQP2 to move water out of the blood meal, could not be confirmed. Further study will be required to define the mechanism of action and to determine whether these vaccine targets will be useful components of an anti-tick vaccine cocktail.

Comprehensive and Durable Modulation of Growth, Development, Lifespan and Fecundity in Anopheles stephensi Following Larval Treatment With the Stress Signaling Molecule and Novel Antimalarial Abscisic Acid.

The larval environment of holometabolous insects determines many adult life history traits including, but not limited to, rate and success of development and adult lifespan and fecundity. The ancient stress signaling hormone abscisic acid (ABA), released by plants inundated with water and by leaf and root fragments in water, is likely ubiquitous in the mosquito larval environment and is well known for its wide ranging effects on invertebrate biology. Accordingly, ABA is a relevant stimulus and signal for mosquito development. In our studies, the addition of ABA at biologically relevant levels to larval rearing containers accelerated the time to pupation and increased death of A. stephensi pupae. We could not attribute these effects, however, to ABA-dependent changes in JH biosynthesis-associated gene expression, 20E titers or transcript patterns of insulin-like peptide genes. Adult females derived from ABA-treated larvae had reduced total protein content and significantly reduced post blood meal transcript expression of vitellogenin, effects that were consistent with variably reduced egg clutch sizes and oviposition success from the first through the third gonotrophic cycles. Adult female A. stephensi derived from ABA-treated larvae also exhibited reduced lifespans relative to controls. Collectively, these effects of ABA on A. stephensi life history traits are robust, durable and predictive of multiple impacts of an important malaria vector spreading to new malaria endemic regions.

Rhipicephalus appendiculatus ticks transmit Theileria parva from persistently infected cattle in the absence of detectable parasitemia: implications for East Coast fever epidemiology.

BACKGROUND: East Coast fever (ECF) is a devastating disease of cattle and a significant constraint to improvement of livestock production in sub-Saharan Africa. The protozoan parasite causing ECF, Theileria parva, undergoes obligate sexual stage development in its tick vector Rhipicephalus appendiculatus. Tick-borne acquisition and transmission occurs transstadially; larval and nymphal ticks acquire infection while feeding and transmit to cattle when they feed after molting to the next stage. Much of the current knowledge relating to tick-borne acquisition and transmission of T. parva has been derived from studies performed during acute infections where parasitemia is high. In contrast, tick-borne transmission during the low-level persistent infections characteristic of endemic transmission cycles is rarely studied. METHODS: Cattle were infected with one of two stocks of T. parva (Muguga or Marikebuni). Four months post-infection when parasites were no longer detectable in peripheral blood by PCR, 500 R. appendiculatus nymphs were fed to repletion on each of the cattle. After they molted to the adult stage, 20 or 200 ticks, respectively, were fed on two naïve cattle for each of the parasite stocks. After adult ticks fed to repletion, cattle were tested for T. parva infection by nested PCR and dot blot hybridization. RESULTS: Once they had molted to adults the ticks that had fed as nymphs on Muguga and Marikebuni infected cattle successfully transmitted Theileria parva to all naïve cattle, even though T. parva infection was not detectable by nested PCR on salivary gland genomic DNA of a sample of individual ticks. However, a salivary gland homogenate from a single Marikebuni infected tick was able to infect primary bovine lymphocytes. Infection was detected by nested p104 PCR in 3 of 4 calves and detected in all 4 calves by T. parva 18S nested PCR/dot blot hybridization. CONCLUSION: We show that R. appendiculatus ticks are able to acquire T. parva parasites from infected cattle even in the absence of detectable parasitemia. Although infection was undetectable in a sample of individual ticks, cumulatively as few as 20 ticks were able to transmit T. parva to naïve cattle. These results have important implications for our understanding of T. parva transmission by R. appendiculatus in ECF endemic regions.

Abscisic acid: new perspectives on an ancient universal stress signaling molecule.

Few biological molecules have as far reaching and dynamic effects as abscisic acid (ABA). In this review, we draw together the often segregated fields of plant, animal, and human biology to highlight ABA biosynthesis, signaling and physiological effects with examples of host-pathogen interactions to emphasize the cross-kingdom biology of this ancient signaling molecule.

Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae.

Napier grass Stunt Disease (NSD) is a severe disease of Napier grass (Pennisetum purpureum) in Eastern Africa, caused by the leafhopper-transmitted bacterium Candidatus Phytoplasma oryzae. The pathogen severely impairs the growth of Napier grass, the major fodder for dairy cattle in Eastern Africa. NSD is associated with biomass losses of up to 70% of infected plants. Diagnosis of NSD is done by nested PCR targeting the phytoplasma DNA, which is difficult to perform in developing countries with little infrastructure. We report the development of an easy to use, rapid, sensitive and specific molecular assay for field diagnosis of NSD. The procedure is based on recombinase polymerase amplification and targets the imp gene encoding a pathogen-specific immunodominant membrane protein. Therefore we followed a two-step process. First we developed an isothermal DNA amplification method for real time fluorescence application and then transferred this assay to a lateral flow format. The limit of detection for both procedures was estimated to be 10 organisms. We simplified the template preparation procedure by using freshly squeezed phloem sap from Napier grass. Additionally, we developed a laboratory serological assay with the potential to be converted to a lateral flow assay. Two murine monoclonal antibodies with high affinity and specificity to the immunodominant membrane protein IMP of Candidatus Phytoplasma oryzae were generated. Both antibodies specifically reacted with the denatured or native 17 kDa IMP protein. In dot blot experiments of extracts from infected plant, phytoplasmas were detected in as little as 12,5 µg of fresh plant material.

Detection of Theileria parva in tissues of cattle undergoing severe East Coast fever disease show significant parasite DNA accumulation in the spleen.

Infiltration and proliferation of Theileria parva infected lymphocytes in bovine host lymphoid organs is one of the hallmarks of T. parva infection. The relative abundance of parasites within infected host tissues, both lymphoid and non-lymphoid is however unknown. Using quantitative PCR, we have shown that significantly higher concentrations of T. parva DNA are detected in the spleens of cattle undergoing severe disease compared to other organs.

Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission.

BACKGROUND: Rhipicephalus appendiculatus is the primary vector of Theileria parva, the etiological agent of East Coast fever (ECF), a devastating disease of cattle in sub-Saharan Africa. We hypothesized that a vaccine targeting tick proteins that are involved in attachment and feeding might affect feeding success and possibly reduce tick-borne transmission of T. parva. Here we report the evaluation of a multivalent vaccine cocktail of tick antigens for their ability to reduce R. appendiculatus feeding success and possibly reduce tick-transmission of T. parva in a natural host-tick-parasite challenge model. METHODS: Cattle were inoculated with a multivalent antigen cocktail containing recombinant tick protective antigen subolesin as well as two additional R. appendiculatus saliva antigens: the cement protein TRP64, and three different histamine binding proteins. The cocktail also contained the T. parva sporozoite antigen p67C. The effect of vaccination on the feeding success of nymphal and adult R. appendiculatus ticks was evaluated together with the effect on transmission of T. parva using a tick challenge model. RESULTS: To our knowledge, this is the first evaluation of the anti-tick effects of these antigens in the natural host-tick-parasite combination. In spite of evidence of strong immune responses to all of the antigens in the cocktail, vaccination with this combination of tick and parasite antigens did not appear to effect tick feeding success or reduce transmission of T. parva. CONCLUSION: The results of this study highlight the importance of early evaluation of anti-tick vaccine candidates in biologically relevant challenge systems using the natural tick-host-parasite combination.

Draft Genome Sequence of "Candidatus Phytoplasma oryzae" Strain Mbita1, the Causative Agent of Napier Grass Stunt Disease in Kenya.

Phytoplasmas are bacterial plant pathogens with devastating impact on agricultural production worldwide. In eastern Africa, Napier grass stunt disease causes serious economic losses in the smallholder dairy industry. This draft genome sequence of " ITALIC! CandidatusPhytoplasma oryzae" strain Mbita1 provides insight into its genomic organization and the molecular basis of pathogenicity.

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.

Sequence diversity between class I MHC loci of African native and introduced Bos taurus cattle in Theileria parva endemic regions: in silico peptide binding prediction identifies distinct functional clusters.

There is strong evidence that the immunity induced by live vaccination for control of the protozoan parasite Theileria parva is mediated by class I MHC-restricted CD8(+) T cells directed against the schizont stage of the parasite that infects bovine lymphocytes. The functional competency of class I MHC genes is dependent on the presence of codons specifying certain critical amino acid residues that line the peptide binding groove. Compared with European Bos taurus in which class I MHC allelic polymorphisms have been examined extensively, published data on class I MHC transcripts in African taurines in T. parva endemic areas is very limited. We utilized the multiplexing capabilities of 454 pyrosequencing to make an initial assessment of class I MHC allelic diversity in a population of Ankole cattle. We also typed a population of exotic Holstein cattle from an African ranch for class I MHC and investigated the extent, if any, that their peptide-binding motifs overlapped with those of Ankole cattle. We report the identification of 18 novel allelic sequences in Ankole cattle and provide evidence of positive selection for sequence diversity, including in residues that predominantly interact with peptides. In silico functional analysis resulted in peptide binding specificities that were largely distinct between the two breeds. We also demonstrate that CD8(+) T cells derived from Ankole cattle that are seropositive for T. parva do not recognize vaccine candidate antigens originally identified in Holstein and Boran (Bos indicus) cattle breeds.

Immunization of cattle with Ra86 impedes Rhipicephalus appendiculatus nymphal-to-adult molting.

Commercial vaccines based on the tick gut protein Bm86 have been successful in controlling the one-host tick Rhipicephalus (Boophilus) microplus and provide heterologous protection against certain other non-target ixodid tick species. This cross protection, however, does not extend to the three-host tick R. appendiculatus, the vector of the protozoan parasite Theileria parva. When transmitted to cattle, T. parva causes the often fatal disease East Coast fever. Here, we used insect cell-expressed recombinant versions of the R. appendiculatus homologs of Bm86, named Ra86, to vaccinate cattle. We measured multiple fitness characteristics for ticks that were fed on cattle Ra86-vaccinated or unvaccinated. The Ra86 vaccination of cattle significantly decreased the molting success of nymphal ticks to the adult stage. Modeling simulations based on our empirical data suggest that repeated vaccinations using Ra86 could reduce tick populations over successive generations. Vaccination with Ra86 could thus form a component of integrated control strategies for R. appendiculatus leading to a reduction in use of environmentally damaging acaricides.