Salivary glands of the cat flea, Ctenocephalides felis: Dissection and microscopy guide.

Fleas are morphologically unique ectoparasites that are hardly mistaken for any other insect. Most flea species that feed on humans and their companion animals, including the cat flea (Ctenocephalides felis), have medical and veterinary importance. Besides facilitating blood acquisition, salivary biomolecules can modulate pathogen transmission. Thus, dissection of salivary glands is essential for comprehensive studies on disease vectors like the cat flea. Herein, we present the pictorial dissection protocol assisting future research targeting individual flea organs, for revealing their roles in vector competence and physiology. We provide a comprehensive guide, allowing researchers, even with limited practical experience, to successfully perform microdissection for collecting cat flea salivary glands. Furthermore, the protocol does not require expensive, sophisticated equipment and can be accomplished with routinely available tools. We illustrated expected results with morphological changes of salivary glands upon blood feeding as well as fluorescently stained these organs.

Cat Flea Coinfection with Rickettsia felis and Rickettsia typhi.

Purpose: Flea-borne rickettsioses, collectively referred to as a term for etiological agents Rickettsia felis, Rickettsia typhi, and RFLOs (R. felis-like organisms), has become a public health concern around the world, specifically in the United States. Due to a shared arthropod vector (the cat flea) and clinical signs, discriminating between Rickettsia species has proven difficult. While the effects of microbial coinfections in the vector can result in antagonistic or synergistic interrelationships, subsequently altering potential human exposure and disease, the impact of bacterial interactions within flea populations remains poorly defined. Methods: In this study, in vitro and in vivo systems were utilized to assess rickettsial interactions in arthropods. Results: Coinfection of both R. felis and R. typhi within a tick-derived cell line indicated that the two species could infect the same cell, but distinct growth kinetics led to reduced R. felis growth over time, regardless of infection order. Sequential flea coinfections revealed the vector could acquire both Rickettsia spp. and sustain coinfection for up to 2 weeks, but rickettsial loads in coinfected fleas and feces were altered during coinfection. Conclusion: Altered rickettsial loads during coinfection suggest R. felis and R. typhi interactions may enhance the transmission potential of either agent. Thus, this study provides a functional foundation to disentangle transmission events propelled by complex interspecies relationships during vector coinfections.

Critical roles of Rickettsia parkeri outer membrane protein B (OmpB) in the tick host.

Rickettsia parkeri is a pathogen of public health concern and transmitted by the Gulf Coast tick, Amblyomma maculatum. Rickettsiae are obligate intracellular bacteria that enter and replicate in diverse host cells. Rickettsial outer membrane protein B (OmpB) functions in bacterial adhesion, invasion, and avoidance of cell-autonomous immunity in mammalian cell infection, but the function of OmpB in arthropod infection is unknown. In this study, the function of R. parkeri OmpB was evaluated in the tick host. R. parkeri wild-type and R. parkeri ompBSTOP::tn (non-functional OmpB) were capillary fed to naïve A. maculatum ticks to investigate dissemination in the tick and transmission to vertebrates. Ticks exposed to R. parkeri wild-type had greater rickettsial loads in all organs than ticks exposed to R. parkeri ompBSTOP::tn at 12 h post-capillary feeding and after 1 day of feeding on host. In rats that were exposed to R. parkeri ompBSTOP::tn-infected ticks, dermal inflammation at the bite site was less compared to R. parkeri wild-type-infected ticks. In vitro, R. parkeri ompBSTOP::tn cell attachment to tick cells was reduced, and host cell invasion of the mutant was initially reduced but eventually returned to the level of R. parkeri wild-type by 90 min post-infection. R. parkeri ompBSTOP::tn and R. parkeri wild-type had similar growth kinetics in the tick cells, suggesting that OmpB is not essential for R. parkeri replication in tick cells. These results indicate that R. parkeri OmpB functions in rickettsial attachment and internalization to tick cells and pathogenicity during tick infection.

ATP-sensitive inward rectifier potassium channels regulate secretion of pro-feeding salivary proteins in the lone star tick (Amblyomma americanum).

Understanding the physiological and molecular regulation of tick feeding is necessary for developing intervention strategies to curb disease transmission by ticks. Pharmacological activation of ATP-gated inward rectifier potassium (KATP) channels reduced fluid secretion from isolated salivary gland and blood feeding in the lone star tick, Amblyomma americanum, yet the temporal expression pattern of KATP channel proteins remained unknown. KATP channels were highly expressed in type II and III acini in off-host stage and early feeding phase ticks, yet expression was reduced in later stages of feeding. We next assessed KATP channel regulation of the secreted proteome of tick saliva. LC-MS/MS analysis identified 40 differentially secreted tick saliva proteins after exposure to KATP activators or inhibitors. Secretion of previously validated tick saliva proteins that promote tick feeding, AV422, AAS27, and AAS41 were significantly reduced by upwards of 8 log units in ticks exposed to KATP channel activators when compared to untreated ticks. Importantly, activation of KATP channels inhibited tick feeding and vice versa for KATP channel inhibitors. Data indicate KATP channels regulate tick feeding biology by controlling secretion of pro-feeding proteins that are essential during early feeding phases, which provides insights into physiological and molecular regulation of tick feeding behavior.

Revisiting the sialome of the cat flea Ctenocephalides felis.

The hematophagous behaviour emerged independently in several instances during arthropod evolution. Survey of salivary gland and saliva composition and its pharmacological activity led to the conclusion that blood-feeding arthropods evolved a distinct salivary mixture that can interfere with host defensive response, thus facilitating blood acquisition and pathogen transmission. The cat flea, Ctenocephalides felis, is the major vector of several pathogens, including Rickettsia typhi, Rickettsia felis and Bartonella spp. and therefore, represents an important insect species from the medical and veterinary perspectives. Previously, a Sanger-based sialome of adult C. felis female salivary glands was published and reported 1,840 expressing sequence tags (ESTs) which were assembled into 896 contigs. Here, we provide a deeper insight into C. felis salivary gland composition using an Illumina-based sequencing approach. In the current dataset, we report 8,892 coding sequences (CDS) classified into 27 functional classes, which were assembled from 42,754,615 reads. Moreover, we paired our RNAseq data with a mass spectrometry analysis using the translated transcripts as a reference, confirming the presence of several putative secreted protein families in the cat flea salivary gland homogenates. Both transcriptomic and proteomic approaches confirmed that FS-H-like proteins and acid phosphatases lacking their putative catalytic residues are the two most abundant salivary proteins families of C. felis and are potentially related to blood acquisition. We also report several novel sequences similar to apyrases, odorant binding proteins, antigen 5, cholinesterases, proteases, and proteases inhibitors, in addition to putative novel sequences that presented low or no sequence identity to previously deposited sequences. Together, the data represents an extended reference for the identification and characterization of the pharmacological activity present in C. felis salivary glands.

Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3' end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

The role of cofeeding arthropods in the transmission of Rickettsia felis.

Rickettsia felis is an emerging etiological agent of rickettsioses worldwide. The cosmopolitan cat flea (Ctenocephalides felis) is the primary vector of R. felis, but R. felis has also been reported in other species of hematophagous arthropods including ticks and mosquitoes. Canines can serve as a bacteremic host to infect fleas under laboratory conditions, yet isolation of R. felis from the blood of a vertebrate host in nature has not been realized. Cofeeding transmission is an efficient mechanism for transmitting rickettsiae between infected and uninfected fleas; however, the mechanism of transmission among different orders and classes of arthropods is not known. The potential for R. felis transmission between infected fleas and tick (Dermacentor variabilis) and mosquito (Anopheles quadrimaculatus) hosts was examined via cofeeding bioassays. Donor cat fleas infected with R. felis transmitted the agent to naïve D. variabilis nymphs via cofeeding on a rat host. Subsequent transstadial transmission of R. felis from the engorged nymphs to the adult ticks was observed with reduced prevalence in adult ticks. Using an artificial host system, An. quadrimaculatus exposed to a R. felis-infected blood meal acquired rickettsiae and maintained infection over 12 days post-exposure (dpe). Similar to ticks, mosquitoes were able to acquire R. felis while cofeeding with infected cat fleas on rats infection persisting in the mosquito for up to 3 dpe. The results indicate R. felis-infected cat fleas can transmit rickettsiae to both ticks and mosquitoes via cofeeding on a vertebrate host, thus providing a potential avenue for the diversity of R. felis-infected arthropods in nature.

Mycobacterium leprae Infection in Ticks and Tick-Derived Cells.

Leprosy is a zoonosis in the southern United States involving humans and wild armadillos. The majority of patients presenting with zoonotic strains of Mycobacterium leprae note extensive outdoor activity but only rarely report any history of direct contact with wild armadillos. Whether M. leprae is transmitted to new vertebrate hosts through the environment independently or with the aid of other organisms, e.g., arthropod vectors, is a fundamental question in leprosy transmission. The objectives of this study were to assess the potential for ticks to transmit M. leprae and to test if viable M. leprae can be maintained in tick-derived cells. To evaluate tick transmission, nymphal Amblyomma maculatum ticks were injected with isolated M. leprae. Infection and transmission were assessed by qPCR. Ticks infected as nymphs harbored M. leprae through vertical transmission events (nymph to adult and adult to progeny); and, horizontal transmission of M. leprae to a vertebrate host was observed. Mycobacterium leprae DNA was detected in multiple tick life cycle stages. Likewise, freshly isolated M. leprae (Thai-53) was used to infect a tick-derived cell line, and enumeration and bacterial viability were assessed at individual time points for up to 49 days. Evaluations of the viability of long-term cultured M. leprae (Thai-53 and Br4923) were also assessed in a mouse model. Tick-derived cells were able to maintain viable M. leprae over the 49-day course of infection and M. leprae remained infectious within tick cells for at least 300 days. The results of this study suggest that ticks themselves might serve as a vector for the transmission of M. leprae and that tick cells are suitable for maintenance of viable M. leprae for an extended period of time.

Unpacking the intricacies of Rickettsia-vector interactions.

Although Rickettsia species are molecularly detected among a wide range of arthropods, vector competence becomes an imperative aspect of understanding the ecoepidemiology of these vector-borne diseases. The synergy between vector homeostasis and rickettsial invasion, replication, and release initiated within hours (insects) and days (ticks) permits successful transmission of rickettsiae. Uncovering the molecular interplay between rickettsiae and their vectors necessitates examining the multifaceted nature of rickettsial virulence and vector infection tolerance. Here, we highlight the biological differences between tick- and insect-borne rickettsiae and the factors facilitating the incidence of rickettsioses. Untangling the complex relationship between rickettsial genetics, vector biology, and microbial interactions is crucial in understanding the intricate association between rickettsiae and their vectors.

Dynamic gene expression in salivary glands of the cat flea during Rickettsia felis infection.

The cat flea, Ctenocephalides felis, is an arthropod vector capable of transmitting several human pathogens including Rickettsia species. Earlier studies identified Rickettsia felis in the salivary glands of the cat flea and transmission of rickettsiae during arthropod feeding. The saliva of hematophagous insects contains multiple biomolecules with anticlotting, vasodilatory and immunomodulatory activities. Notably, the exact role of salivary factors in the molecular interaction between flea-borne rickettsiae and their insect host is still largely unknown. To determine if R. felis modulates gene expression in the cat flea salivary glands, cat fleas were infected with R. felis and transcription patterns of selected salivary gland-derived factors, including antimicrobial peptides and flea-specific antigens, were assessed. Salivary glands were microdissected from infected and control cat fleas at different time points after exposure and total RNA was extracted and subjected to reverse-transcriptase quantitative PCR for gene expression analysis. During the experimental 10-day feeding period, a dynamic change in gene expression of immunity-related transcripts and salivary antigens between the two experimental groups was detected. The data indicated that defensin-2 (Cf-726), glycine-rich antimicrobial peptide (Cf-83), salivary antigens (Cf-169 and Cf-65) and deorphanized peptide (Cf-75) are flea-derived factors responsive to rickettsial infection.

Evolution of Wolbachia mutualism and reproductive parasitism: insight from two novel strains that co-infect cat fleas.

Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae, wCfeT and wCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA, USA). wCfeT is basal to nearly all described Wolbachia supergroups, while wCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution of Wolbachia host associations. wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across the Wolbachia tree, indicating parallel evolution towards mutualism. Alternately, wCfeJ harbors a toxin-antidote operon analogous to the wPip cinAB operon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies. wCfeJ cinB and three adjacent genes are collectively similar to large modular toxins encoded in CI-like operons of certain Wolbachia strains and Rickettsia species, signifying that CI toxins streamline by fission of large modular toxins. Remarkably, the C. felis genome itself contains two CI-like antidote genes, divergent from wCfeJ cinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongst C. felis colonies, though fleas in wild populations mostly harbor wCfeT alone. Collectively, genomes of wCfeT, wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.

Comparative Analysis of Infection by Rickettsia rickettsii Sheila Smith and Taiaçu Strains in a Murine Model.

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease caused by Rickettsia rickettsii, which is widely distributed throughout the Americas. Over 4000 cases of RMSF are recorded annually in the United States, while only around 100 cases are reported in Brazil. Conversely, while case fatality rates in the United States oscillate around 5%, in Brazil they can surpass 70%, suggesting that differences in tick vectoring capacity, population sensitivity, and/or variability in virulence of the rickettsial strains may exist. In this study, we compared the susceptibility of C3H/HeN mice to two highly virulent strains of R. rickettsii, one from the United States (Sheila Smith) and the other from Brazil (Taiaçu). Animals inoculated with the Taiaçu strain succumbed to infection earlier and exhibited severe histological lesions in both liver and spleen sooner than mice infected with the Sheila Smith strain. These differences in survival and signs of the disease are not related to a greater proliferation of the Taiaçu strain, as there were no significant differences in the rickettsial load in mice tissues inoculated with either strain. The present study is the first step to experimentally assess differences in fatality rates of RMSF in two different regions of the American continent.

A chromosome-level assembly of the cat flea genome uncovers rampant gene duplication and genome size plasticity.

BACKGROUND: Fleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e., the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. RESULTS: By combining Illumina and PacBio sequencing of DNA derived from multiple inbred female fleas with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ~ 38% of protein-coding genes with two or more copies and over 4000 tRNA genes. A broad range of genome size determinations (433-551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly, broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable "genome-in-flux" phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads, we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. CONCLUSION: Rampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts-potential tools for blocking pathogen transmission-these oddities highlight a unique and underappreciated disease vector.

The complete mitochondrial genome of the cat flea, Ctenocephalides felis.

The cat flea, Ctenocephalides fells, is widely recognized as a global veterinary pest and a vector of pathogenic bacteria. We recently reported on the C. felis nuclear genome, which is characterized by over 38% protein coding gene duplication, extensive tRNA gene family expansion, and remarkable gene copy number variation (CNV) between individual fleas. Herein, we describe the assembly of the C. felis mitochondrial genome, a novel resource for comparative genomics of fleas and other insects. The order and content of mitochondrial genes is highly consistent with four previously sequenced flea mitochondrial genomes, limiting CNV to siphonapteran nuclear genomes.

Inward rectifier potassium (Kir) channels mediate salivary gland function and blood feeding in the lone star tick, Amblyomma americanum.

BACKGROUND: Tick feeding causes extreme morbidity and mortality to humans through transmission of pathogens and causes severe economic losses to the agricultural industry by reducing livestock yield. Salivary gland secretions are essential for tick feeding and thus, reducing or preventing saliva secretions into the vertebrate host is likely to reduce feeding and hinder pathogen life cycles. Unfortunately, the membrane physiology of tick salivary glands is underexplored and this gap in knowledge limits the development of novel therapeutics for inducing cessation of tick feeding. METHODOLOGY: We studied the influence of inward rectifier potassium (Kir) channel subtypes to the functional capacity of the isolated tick salivary gland through the use of a modified Ramsay assay. The secreted saliva was subsequently used for quantification of the elemental composition of the secreted saliva after the glands were exposed to K+ channel modulators as a measure of osmoregulatory capacity. Lastly, changes to blood feeding behavior and mortality were measured with the use of a membrane feeding system. PRINCIPAL FINDINGS: In this study, we characterized the fundamental role of Kir channel subtypes in tick salivary gland function and provide evidence that pharmacological inhibition of these ion channels reduces the secretory activity of the Amblyomma americanum salivary gland. The reduced secretory capacity of the salivary gland was directly correlated with a dramatic reduction of blood ingestion during feeding. Further, exposure to small-molecule modulators of Kir channel subtypes induced mortality to ticks that is likely resultant from an altered osmoregulatory capacity. CONCLUSIONS: Our data contribute to understanding of tick salivary gland function and could guide future campaigns aiming to develop chemical or reverse vaccinology technologies to reduce the worldwide burden of tick feeding and tick-vectored pathogens.

Spotted Fever Group Rickettsia Infection and Transmission Dynamics in Amblyomma maculatum.

Tick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novel Rickettsia species or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG) Rickettsia species is transmitted at higher levels during tick feeding. Using Amblyomma maculatum cohorts infected with Rickettsia parkeri or "Candidatus Rickettsia andeanae," a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers of R. parkeri than of "Ca Rickettsia andeanae" rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localized R. parkeri, but not "Ca Rickettsia andeanae," in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed to R. parkeri than in those exposed to "Ca Rickettsia andeanae." The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

A Tangled Web: Origins of Reproductive Parasitism.

While typically a flea parasite and opportunistic human pathogen, the presence of Rickettsia felis (strain LSU-Lb) in the non-blood-feeding, parthenogenetically reproducing booklouse, Liposcelis bostrychophila, provides a system to ascertain factors governing not only host transitions but also obligate reproductive parasitism (RP). Analysis of plasmid pLbAR, unique to R. felis str. LSU-Lb, revealed a toxin-antitoxin module with similar features to prophage-encoded toxin-antitoxin modules utilized by parasitic Wolbachia strains to induce another form of RP, cytoplasmic incompatibility, in their arthropod hosts. Curiously, multiple deubiquitinase and nuclease domains of the large (3,841 aa) pLbAR toxin, as well the entire antitoxin, facilitated the detection of an assortment of related proteins from diverse intracellular bacteria, including other reproductive parasites. Our description of these remarkable components of the intracellular mobilome, including their presence in certain arthropod genomes, lends insight on the evolution of RP, while invigorating research on parasite-mediated biocontrol of arthropod-borne viral and bacterial pathogens.

Role of Sca2 and RickA in the Dissemination of Rickettsia parkeri in Amblyomma maculatum.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro, similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia-free Amblyomma maculatum, the natural vector of R. parkeri, was exposed to wild-type, R. parkeri rickA::tn, or R. parkeri sca2::tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeririckA::tn or R. parkerisca2::tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

Correlation between fecal egg count, presence of Strongylus vulgaris, and body score of feral horses on Fort Polk, Louisiana.

Approximately 700 feral horses, dubbed "trespass horses" by the United States Army, occupy Fort Polk, Louisiana and the surrounding Kisatchie National Forest. These horses are considered a nuisance and hazard, and the military is seeking to remove the horses via adoption. The aim of this research was to evaluate the fecal egg count (FEC), body condition score (BCS), and the presence of Strongylus vulgaris within this previously unstudied horse population prior to removal. The feral horse data was compared to domestic horses living on a single farm in the same area. A modified McMaster FEC, Henneke body scoring via photography, and PCR were used to evaluate 10 domestic horses and 28 feral horses. A significantly higher FEC was identified for feral horses when compared to domestic horses (p = 0.004), and 69.2% of feral horses were positive for S. vulgaris while all domestic horses tested negative. Additionally, no correlation was found between FEC and BCS for domestic (p = 0.213) or feral (p = 0.099) horses, and no association was found between FEC and S. vulgaris presence (p = 0.21) or BCS and S. vulgaris presence (p = 0.52). This study provides insight into S. vulgaris and strongyle prevalence in a previously unstudied group of horses and indicates a need for anthelmintic treatment and monitoring of the feral horses once they are adopted.

Microbial Invasion vs. Tick Immune Regulation.

Ticks transmit a greater variety of pathogenic agents that cause disease in humans and animals than any other haematophagous arthropod, including Lyme disease, Rocky Mountain spotted fever, human granulocytic anaplasmosis, babesiosis, tick-borne encephalitis, Crimean Congo haemorhagic fever, and many others (Gulia-Nuss et al., 2016). Although diverse explanations have been proposed to explain their remarkable vectorial capacity, among the most important are their blood feeding habit, their long term off-host survival, the diverse array of bioactive molecules that disrupt the host's natural hemostatic mechanisms, facilitate blood flow, pain inhibitors, and minimize inflammation to prevent immune rejection (Hajdusek et al., 2013). Moreover, the tick's unique intracellular digestive processes allow the midgut to provide a relatively permissive microenvironment for survival of invading microbes. Although tick-host-pathogen interactions have evolved over more than 300 million years (Barker and Murrell, 2008), few microbes have been able to overcome the tick's innate immune system, comprising both humoral and cellular processes that reject them. Similar to most eukaryotes, the signaling pathways that regulate the innate immune response, i.e., the Toll, IMD (Immunodeficiency) and JAK-STAT (Janus Kinase/ Signal Transducers and Activators of Transcription) also occur in ticks (Gulia-Nuss et al., 2016). Recognition of pathogen-associated molecular patterns (PAMPs) on the microbial surface triggers one or the other of these pathways. Consequently, ticks are able to mount an impressive array of humoral and cellular responses to microbial challenge, including anti-microbial peptides (AMPs), e.g., defensins, lysozymes, microplusins, etc., that directly kill, entrap or inhibit the invaders. Equally important are cellular processes, primarily phagocytosis, that capture, ingest, or encapsulate invading microbes, regulated by a primordial system of thioester-containing proteins, fibrinogen-related lectins and convertase factors (Hajdusek et al., 2013). Ticks also express reactive oxygen species (ROS) as well as glutathione-S-transferase, superoxide dismutase, heat shock proteins and even protease inhibitors that kill or inhibit microbes. Nevertheless, many tick-borne microorganisms are able to evade the tick's innate immune system and survive within the tick's body. The examples that follow describe some of the many different strategies that have evolved to enable ticks to transmit the agents of human and/or animal disease.