Satisfactory breeding potential is transiently eliminated in beef bulls with clinical anaplasmosis.

BACKGROUND: Natural service breeding is common in U.S. cow-calf operations. Diseases impacting bull reproductive performance have significant economic consequences for producers. Anaplasmosis may be an underappreciated cause of poor reproductive performance in bulls. The primary systemic effects of bovine anaplasmosis including anemia, fever, and weight loss, can all result in unsatisfactory reproductive performance. The objective of this pilot study was to evaluate breeding soundness examination (BSE) outcomes and clinical changes in bulls during and upon resolution of clinical anaplasmosis. Anaplasma marginale-challenged bulls were observed for clinical disease and infection progression and changes in breeding soundness compared to uninfected control bulls for 16 weeks. RESULTS: All Anaplasma marginale-challenged bulls were PCR-positive, seropositive, and showed clinical signs by 3-, 17-, and 24-days post-challenge, respectively. Clinical signs of anaplasmosis included pallor, icterus, fever (≥ 40.2 °C), and weight loss. Acute anemia was observed in all challenged bulls with PCV nadirs ≤ 18% and peak percent parasitized erythrocyte ≥ 50%. Decreased scrotal circumference and poor semen quality (e.g., increased percentage of abnormal spermatozoa, decreased progressively motile sperm), were initially observed within days after onset of clinical anaplasmosis signs and continued weeks beyond disease resolution. Control bulls remained negative for A. marginale. CONCLUSION: This pilot study demonstrates that clinical anaplasmosis reduces breeding soundness in beef bulls. Anaplasmosis should be considered as a differential for bulls with decreased semen quality, especially within endemic areas. A 90 day or greater retest window is recommended for bulls of unsatisfactory breeding potential recently recovered from clinical anaplasmosis.

Analysis of Stage-Specific Protein Expression during Babesia Bovis Development within Female Rhipicephalus Microplus.

Arthropod-borne protozoan pathogens have a complex life cycle that includes asexual reproduction of haploid stages in mammalian hosts and the development of diploid stages in invertebrate hosts. The ability of pathogens to invade, survive, and replicate within distinct cell types is required to maintain their life cycle. In this study, we describe a comparative proteomic analysis of a cattle pathogen, Babesia bovis, during its development within the mammalian and tick hosts with the goal of identifying cell-surface proteins expressed by B. bovis kinetes as potential targets for the development of a transmission blocking vaccine. To determine parasite tick-stage-specific cell-surface proteins, CyDye labeling was performed with B. bovis blood stages from the bovine host and kinetes from the tick vector. Cell-surface kinete-stage-specific proteins were identified using 2D difference in gel electrophoresis and analyzed by mass spectrometry. Ten proteins were identified as kinete-stage-specific, with orthologs found in closely related Apicomplexan pathogens. Transcriptional analysis revealed two genes were highly expressed by kinetes as compared with blood stages. Immunofluorescence using antibodies against the two proteins confirmed kinete-stage-specific expression. The identified cell-surface kinete proteins are potential candidates for the development of a B. bovis transmission blocking vaccine.

Restriction of Francisella novicida genetic diversity during infection of the vector midgut.

The genetic diversity of pathogens, and interactions between genotypes, can strongly influence pathogen phenotypes such as transmissibility and virulence. For vector-borne pathogens, both mammalian hosts and arthropod vectors may limit pathogen genotypic diversity (number of unique genotypes circulating in an area) by preventing infection or transmission of particular genotypes. Mammalian hosts often act as "ecological filters" for pathogen diversity, where novel variants are frequently eliminated because of stochastic events or fitness costs. However, whether vectors can serve a similar role in limiting pathogen diversity is less clear. Here we show using Francisella novicida and a natural tick vector of Francisella spp. (Dermacentor andersoni), that the tick vector acted as a stronger ecological filter for pathogen diversity compared to the mammalian host. When both mice and ticks were exposed to mixtures of F. novicida genotypes, significantly fewer genotypes co-colonized ticks compared to mice. In both ticks and mice, increased genotypic diversity negatively affected the recovery of available genotypes. Competition among genotypes contributed to the reduction of diversity during infection of the tick midgut, as genotypes not recovered from tick midguts during mixed genotype infections were recovered from tick midguts during individual genotype infection. Mediated by stochastic and selective forces, pathogen genotype diversity was markedly reduced in the tick. We incorporated our experimental results into a model to demonstrate how vector population dynamics, especially vector-to-host ratio, strongly affected pathogen genotypic diversity in a population over time. Understanding pathogen genotypic population dynamics will aid in identification of the variables that most strongly affect pathogen transmission and disease ecology.

Superinfection Exclusion of the Ruminant Pathogen Anaplasma marginale in Its Tick Vector Is Dependent on the Time between Exposures to the Strains.

UNLABELLED: The remarkable genetic diversity of vector-borne pathogens allows for the establishment of superinfection in the mammalian host. To have a long-term impact on population strain structure, the introduced strains must also be transmitted by a vector population that has been exposed to the existing primary strain. The sequential exposure of the vector to multiple strains frequently prevents establishment of the second strain, a phenomenon termed superinfection exclusion. As a consequence, superinfection exclusion may greatly limit genetic diversity in the host population, which is difficult to reconcile with the high degree of genetic diversity maintained among vector-borne pathogens. Using Anaplasma marginale, a tick-borne bacterial pathogen of ruminants, we hypothesized that superinfection exclusion is temporally dependent and that longer intervals between strain exposures allow successful acquisition and transmission of a superinfecting strain. To test this hypothesis, we sequentially exposed Dermacentor andersoni ticks to two readily tick-transmissible strains of A. marginale The tick feedings were either immediately sequential or 28 days apart. Ticks were allowed to transmission feed and were individually assessed to determine if they were infected with one or both strains. The second strain was excluded from the tick when the exposure interval was brief but not when it was prolonged. Midguts and salivary glands of individual ticks were superinfected and transmission of both strains occurred only when the exposure interval was prolonged. These findings indicate that superinfection exclusion is temporally dependent, which helps to account for the differences in pathogen strain structure in tropical compared to temperate regions. IMPORTANCE: Many vector-borne pathogens have marked genetic diversity, which influences pathogen traits such as transmissibility and virulence. The most successful strains are those that are preferentially transmitted by the vector. However, the factors that determine successful transmission of a particular strain are unknown. In the case of intracellular, bacterial, tick-borne pathogens, one potential factor is superinfection exclusion, in which colonization of ticks by the first strain of a pathogen it encounters prevents the transmission of a second strain. Using A. marginale, the most prevalent tick-borne pathogen of cattle worldwide, and its natural tick vector, we determined that superinfection exclusion occurs when the time between exposures to two strains is brief but not when it is prolonged. These findings suggest that superinfection exclusion may influence strain transmission in temperate regions, where tick activity is limited by season, but not in tropical regions, where ticks are active for long periods.

Tick passage results in enhanced attenuation of Babesia bovis.

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.

Kansas beef bulls with chronic anaplasmosis demonstrate satisfactory breeding soundness outcomes at breeding soundness examination.

OBJECTIVE: The objectives of this study were to evaluate the prevalence of chronic Anaplasma marginale infection in beef bulls from eastern Kansas and compare breeding soundness parameters between A marginale-infected and uninfected bulls. We hypothesized that bulls with chronic anaplasmosis would have inferior breeding soundness exam (BSE) outcomes as a result of persistent A marginale infection or the consequence of initial clinical disease compared to uninfected bulls. ANIMALS: 535 client-owned beef bulls from eastern Kansas undergoing routine BSE. METHODS: Complete BSEs were conducted by participating veterinarians according to the second edition of the Society for Theriogenology Manual for Breeding Soundness Examination of Bulls. Blood samples were collected for PCV determination and analysis of A marginale infection status via quantitative PCR and cELISA. Logistic and linear regression methods were used to evaluate factors associated with A marginale infection status and BSE parameters. RESULTS: Prevalence of chronic A marginale infection was 46% (245/535) among bulls. Unsatisfactory BSE outcome was not statistically associated with chronic anaplasmosis in this study population, although more bulls with chronic anaplasmosis had unsatisfactory BSE outcomes (15.0 ± 2.4% vs 12.0 ± 2.2%). CLINICAL RELEVANCE: Chronic anaplasmosis is prevalent among eastern Kansas breeding bulls; however, no negative association between chronic anaplasmosis and breeding soundness at time of BSE was observed.

Identification of multilocus genetic heterogeneity in Anaplasma marginale subsp. centrale and its restriction following tick-borne transmission.

Anaplasma marginale subsp. centrale was the first vaccine used to protect against a rickettsial disease and is still in widespread use a century later. As its use preceded development of either cryopreservation or cell culture, the vaccine strain was maintained for decades by sequential passage among donor animals, excluding the natural tick-borne transmission cycle that provides a selective pressure or population "bottleneck." We demonstrated that the vaccine strain is genetically heterogeneous at 46 chromosomal loci and that heterogeneity was maintained upon inoculation into recipient animals. The number of variants per site ranged from 2 to 11 with a mean of 2.8/locus and a mode and median of 2/locus; variants included single-nucleotide polymorphisms, insertions/deletions, polynucleotide tracts, and different numbers of perfect repeats. The genetic heterogeneity is highly unlikely to be a result of strain contamination based on analysis using a panel of eight gene markers with a high power for strain discrimination. In contrast, heterogeneity appears to be a result of genetic drift in the absence of the restriction of tick passage. Heterogeneity could be reduced following tick passage, and the reduced heterogeneity could be maintained in sequential intravenous and tick-borne passages. The reduction in vaccine strain heterogeneity following tick passage did not confer an enhanced transmission phenotype, indicating that a stochastically determined population bottleneck was likely responsible as opposed to a positive selective pressure. These findings demonstrate the plasticity of an otherwise highly constrained genome and highlight the role of natural transmission cycles in shaping and maintaining the bacterial genome.

Cytauxzoon felis: An Overview.

Cytauxzoon felis is a tick-transmitted, obligate, hemoprotozoal, piroplasmid pathogen of felids and the causative agent of cytauxzoonosis. It has a complex life cycle which includes a tick as its definitive host and a felid as its intermediate host. Since its first description in 1976, C. felis infections of felids have been reported in several southeastern and south-central U.S. states, overlapping with the ranges of its two known biological vectors, Amblyomma americanum (Lone star tick) and Dermacentor variabilis (American dog tick). Infected felids demonstrate disease as either an acute, often-fatal, infection, or a subclinical carrier infection. To develop effective C. felis transmission control strategies, the incidence of acute cytauxzoonosis, patient risk factors, the role of domestic cat carriers, and ecological variabilities need to be investigated further. Of equal importance is communicating these strategies for high-risk cat populations, including recommending year-round use of an acaricide product for all cats that spend any time outdoors. More studies are needed to further identify factors affecting C. felis and other Cytauxzoon spp. infection, transmission, disease progression, and treatment options and outcomes within the U.S. and globally. Here we provide an overview of C. felis highlighting its lifecycle within its definitive host, transmission to its intermediate host, symptoms and signs providing evidence of transmission, definitive diagnosis, current treatment and prevention strategies, and future considerations regarding this condition.

High levels of alpha-gal with large variation in the salivary glands of lone star ticks fed on human blood.

Tick bites, associated with the secretion of tick saliva containing the xenoglycan galactose-alpha-1, 3-galactose (alpha-gal or aGal), are recognized as the causal factors of alpha-Gal syndrome (AGS; or red meat allergy) in humans. AGS occurs after the increased production of IgE antibodies against aGal, which is found in most mammalian cells, except for the Old World monkey and humans. The aGal sensitization event has been linked to an initial tick bite, followed by consumption of red meat containing the aGal glycan, which triggers the onset of the allergic response resulting in urticaria, anaphylaxis, or even death. In North America, the lone star tick, Amblyomma americanum, has been identified as the main culprit for AGS. However, only a subset of the human population exposed to lone star tick bites develops AGS. This suggests the presence of unidentified variables associated with the sensitization event. To evaluate the quantitative variations of the aGal in ticks, we evaluated the differences in aGal levels in different strains of A. americanum ticks partially fed on different blood sources using an artificial feeding system and animal hosts. We found significantly higher aGal levels in the female ticks fed on human blood than those fed on the blood of other mammals with large variations among different tick populations and individuals. We propose that host-specific genetic components in the A. americanum ticks are involved in the production of high aGal epitope in the tick saliva, which provides a part of the explanation for the variables associated with the AGS sensitization event of the tick bite.

No evidence for a negative association between bovine leukemia virus status and fertility in Kansas beef herds: a cross-sectional study.

OBJECTIVE: Determine the association between bovine leukemia virus (BLV) status and fertility in beef cows. BLV-status was defined using 3 different testing strategies (ELISA-, quantitative polymerase chain reaction- [qPCR], and high proviral load [PVL]-status). Fertility was defined as the overall probability of pregnancy as well as the probability of becoming pregnant in the first 21 days of the breeding season. ANIMALS: Convenience sample of 2,820 cows from 43 beef herds. PROCEDURES: The association of BLV-status with the probability of becoming pregnant was evaluated with a multivariable logistic regression analysis that used pregnancy status as a binary outcome, herd nested within ranch as a random effect, and BLV-status (ELISA-, qPCR-, and PVL-status as separate models) and potential covariates (eg, age, Body Condition Score [BCS] category, and interactions) as fixed effects. RESULTS: Raw data revealed that 55% (1,552/2,820) of cows were classified as BLV-positive by ELISA, and 95.3% (41/43) of herds had a least 1 ELISA-positive cow. Classification as BLV ELISA-positive was positively associated with the probability of being pregnant; however, when qPCR or PVL were used to classify BLV-status, there was no association with the probability of being pregnant. None of the methods of classifying BLV-status were associated with the probability of becoming pregnant in the first 21 days of the breeding season. CLINICAL RELEVANCE: This study did not find evidence that testing beef cows for BLV-status using ELISA, qPCR, or a cut-off of 0.9 PVL and removing test-positive cows will improve cowherd fertility as described by the probability of becoming pregnant during the breeding season or becoming pregnant during the first 21 days of the breeding season.

Comparison of the initial and residual speed of Ixodes scapularis kill on dogs treated with a single dose of Bravecto® Chew (25 mg/kg fluralaner) or Simparica TRIO® (1.2 mg/kg sarolaner, 24 µg/kg moxidectin, 5 mg/kg pyrantel).

BACKGROUND: Compliant ectoparasiticide product use is a comprehensive way to control ticks and reduce the risk of tick-borne pathogen transmission to dogs. Because the systemically acting isoxazoline ectoparasiticides require tick attachment for drug delivery, fast speed of kill is essential to minimize tick-borne pathogen transmission risk. METHODS: Dogs of satisfactory tick-carrying capacity were randomly allocated to treatment groups and administered, per label instructions, Bravecto® Chews (minimum 25 mg/kg fluralaner), Simparica TRIO® (minimum 1.2 mg/kg sarolaner, 24 µg/kg moxidectin, 5 mg/kg pyrantel), or no treatment. Dogs were infested with approximately 50 unfed adult (35 female, 15 male) Ixodes scapularis on Day -2, 21 and 28. Live tick counts were performed at 4, 8, 12 and 24 h post-treatment (Day 0) and post-infestation on Day 21 and 28. Tick control efficacy was determined by comparing live tick means for each product-treated group to the untreated control group and each other at all time points using a linear mixed model. The percent of dogs free of live ticks was analyzed using the Fisher's exact test for treatment group comparison. RESULTS: The untreated control group maintained adequate tick infestations throughout the study. Using geometric means, an existing I. scapularis infestation was controlled by 99.7% and 93.0% 12 h post-treatment and by 100% and 99.5% 24 h post-treatment, for Bravecto® and Simparica TRIO®-treated dogs, respectively. Ixodes scapularis infestations were controlled more quickly for Bravecto®- compared to Simparica TRIO®-treated dogs on Day 21 at 8 h (efficacy 74.0% vs. 0.0%, p = 0.003) and 12 h (efficacy 99.2% vs. 39.4%, p < 0.001) post-infestation and Day 28 at 8 h (efficacy 92.2% vs. 0.0%, p < 0.001) and 12 h (efficacy 99.6% vs. 27.7%, p < 0.001) post-infestation. On Day 28 post-treatment, the efficacy of Bravecto® and Simparica TRIO® to control a new I. scapularis infestation was 100% and 96.6%, respectively, by 24 h post-infestation. Of product-treated dogs, 100% of Bravecto®-treated dogs were free of live ticks by 24 h post-treatment or post-infestation. No treatment-related adverse reactions occurred during the study. CONCLUSIONS: Ixodes scapularis infestations are controlled more quickly 21 and 28 days post-treatment for dogs administered a single dose of Bravecto® compared to dogs administered a single dose of Simparica TRIO®.

Susceptibility of inbred mice to Rickettsia parkeri.

Rickettsia parkeri, a member of the spotted fever group Rickettsia, is the causative agent of American boutonneuse fever in humans. Despite the increased recognition of human cases, limited information is available regarding the infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Toward the development of a viable transmission model and to further characterize the pathology associated with R. parkeri infection, inbred mouse strains (A/J, BALB/c, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with 10(5) low-passage-number R. parkeri (Portsmouth strain), and infection, gross pathology, and histopathology were scored. Additionally, a quantitative real-time PCR (qPCR) was performed to estimate rickettsial load in heart, lung, spleen, and liver tissues of infected mice at 19 days postinoculation. Of the A/J, BALB/c, and C3H/HeN mice, none displayed universal pathology consistent with sustained infection. Compared to age-matched control mice, the intravenously inoculated C3H/HeJ mice exhibited marked facial edema and marked splenomegaly upon gross examination, while the intradermally inoculated mice developed characteristic eschar-like lesions. The C3H/HeJ mice also exhibited the greatest concentrations of rickettsial DNA from heart, lung, liver, and spleen samples when examined by qPCR. The similarity of the pathology of human disease and sustained infection suggests that the C3H/HeJ strain of mice is a promising candidate for subsequent experiments to examine the tick transmission, dissemination, and pathology of R. parkeri rickettsiosis.

Cross-sectional study to describe bovine leukemia virus herd and within-herd ELISA prevalence and bovine leukemia virus proviral load of convenience-sampled Kansas beef cow-calf herds.

OBJECTIVE: Determine bovine leukemia virus (BLV) seroprevalence of adult female cattle in Eastern Kansas beef herds and the proviral load (PVL) of those cattle found to be ELISA positive. ANIMALS: Convenience sample of 2,845 cows from 44 beef herds. PROCEDURES: BLV serostatus was determined using an ELISA antibody test (gp-51; IDEXX). BLV quantitative PCR (qPCR) status and PVL were determined utilizing a qPCR test (SS1 qPCR test; CentralStar Laboratories). The association of age, herd size, and body condition score (BCS) category on the probability of being BLV positive was evaluated with a multiple variable logistic regression analysis that used BLV status as a binary outcome, herd nested within ranch as a random effect, and BCS, herd size, and age category as fixed effects. RESULTS: Forty-two of 44 herds had at least 1 BLV ELISA-positive cow (95.5% herd seroprevalence). Overall, 1,564 of the 2,845 cows were BLV ELISA positive (55.0% individual animal prevalence). No association between BLV ELISA status and herd size or BCS was identified. When evaluated by age, the model-adjusted probability of being BLV ELISA positive was lowest for heifers (1 year of age, first parity) and increased until 5 to 6 years of age. Of the 1,564 ELISA-positive animals, 838 were qPCR positive (53.6%). The model-adjusted probability of being qPCR positive was not associated with age, herd size, or BCS category. CLINICAL RELEVANCE: This study indicated that BLV-seropositive status both as a herd classification and individual animal classification was very common in this population. Because the percentage of BLV-seropositive cows varied between herds and by age, this study provides evidence that it is essential for investigators to control for herd and age in any analysis of the association of BLV serostatus and health and production outcomes of interest. Some BLV ELSIA-seropositive cows were classified as BLV negative by qPCR, and risk factors may differ between classification status by ELISA and qPCR.

Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts.

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle.

Dermacentor andersoni transmission of Francisella tularensis subsp. novicida reflects bacterial colonization, dissemination, and replication coordinated with tick feeding.

Ticks serve as biological vectors for a wide variety of bacterial pathogens which must be able to efficiently colonize specific tick tissues prior to transmission. The bacterial determinants of tick colonization are largely unknown, a knowledge gap attributed in large part to the paucity of tools to genetically manipulate these pathogens. In this study, we demonstrated that Francisella tularensis subsp. novicida, for which a complete two-allele transposon mutant library has been constructed, initially infects the midguts of 100% of acquisition-fed Dermacentor andersoni nymphs, with stable colonization and replication during a subsequent molt. Increased dissemination to and marked replication within the salivary gland was closely linked to a second (transmission) feed and culminated in secretion of bacteria into the saliva and successful transmission. Simultaneous testing of multiple mutants resulted in total bacterial levels similar to those observed for single mutants. However, there was evidence of a bottleneck during colonization, resulting in a founder effect in which the most successful mutant varied when comparing individual ticks. Thus, it is essential to assess mutant success at the level of the tick population rather than in individual ticks. The ability of F. tularensis subsp. novicida to recapitulate the key physiological events by which bacteria colonize and are transmitted by ixodid ticks provides a new genome-wide approach to identify the required pathogen molecules and pathways. The identification of specific genes and, more importantly, conserved pathways that function at the tick-pathogen interface will accelerate the development of new methods to block transmission.

AC-DC electropenetrography unmasks fine temporal details of feeding behaviors for two tick species on unsedated hosts.

Ticks are significant nuisance pests and vectors of pathogens for humans, companion animals, and livestock. Limited information on tick feeding behaviors hampers development and rigorous evaluation of tick and tick-borne pathogen control measures. To address this obstacle, the present study examined the utility of AC-DC electropenetrography (EPG) to monitor feeding behaviors of adult Dermacentor variabilis and Amblyomma americanum in real-time. EPG recording was performed during early stages of slow-phase tick feeding using an awake calf host. Both tick species exhibited discernable and stereotypical waveforms of low-, medium-, and high-frequencies. Similar waveform families and types were observed for both tick species; however, species-specific waveform structural differences were also observed. Tick waveforms were hierarchically categorized into three families containing seven types. Some waveform types were conserved by both species (e.g., Types 1b, 1c, 2b, 2c) while others were variably performed among species and individually recorded ticks (e.g., Types 1a, 2a, 2d). This study provides a proof-of-principle demonstration of the feasibility for using EPG to monitor, evaluate, and compare tick feeding behaviors, providing a foundation for future studies aimed at correlating specific feeding behaviors with waveforms, and ultimately the influence of control measures and pathogens on tick feeding behaviors.

AC-DC electropenetrography: fundamentals, controversies, and perspectives for arthropod pest management.

Studying the intimate association of arthropods with their physical substrate is both important and challenging. It is important because substrate is a key determinant for organism fitness; challenging because the intricacies of this association are dynamic, and difficult to record and resolve. The advent of electropenetrography (EPG) and subsequent developments allowed researchers to overcome this challenge. Nonetheless, EPG research has been historically restricted to piercing-sucking hemipteran plant pests. Recently, its potential use has been greatly broadened for additional pests with instrument advances. Thus, blood-feeding arthropods and chewing feeders, as well as non-feeding behaviors like oviposition by both pests and parasitoids, are novel new targets for EPG research, with critical consequences for integrated pest management. EPG can explain mechanisms of crop damage, plant or animal pathogen transmission, and the effects of insecticides, antifeedants, repellents, or transgenic plants and animals, on specific behaviors of damage or transmission. This review broadly covers the principles and development of EPG technology, emphasizing controversies and challenges remaining with suggested research to overcome them. In addition, it summarizes 60+ years of basic and applied EPG research, and previews future directions for pest management. The goal is to stimulate new applications for this unique enabling technology. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Effect of Protracted Free-Choice Chlortetracycline-Medicated Mineral for Anaplasmosis Control on Escherichia coli Chlortetracycline Resistance Profile from Pastured Beef Cattle.

Anaplasmosis is an economically-significant, hemolytic, tick-borne disease of cattle caused by Anaplasma marginale which can cause clinical anemia and death. Current control options are limited, and FDA-approved antimicrobial control options do not have a defined duration of use. A practical and routinely used anaplasmosis control method involves feeding free-choice chlortetracycline (CTC)-medicated mineral to pastured cattle for several months. Constant antimicrobial use poses the risk of expediting the development and dissemination of antimicrobial resistance in off-target commensal bacteria in the bovine gastrointestinal tract. The objective of this study was to determine the CTC-susceptibility of Escherichia coli isolated from anaplasmosis endemic beef cattle herds provided different FDA-approved free-choice CTC-medicated mineral formulations, all intended to provide cattle a dosage of 0.5 to 2.0 mg CTC/lb bodyweight per day. A closed-herd, comprised of Hereford-Angus cows, naturally endemic for anaplasmosis, were grazed in five different pastures with one herd serving as an untreated control group. The other cattle herds were randomly assigned one of four FDA-approved CTC-medicated mineral formulations (700, 5000, 6000, and 8000 g CTC/ton) labeled for "the control of active anaplasmosis" and provided their respective CTC-medicated mineral formulation for five consecutive months. Fecal samples were collected monthly from a subset of cows (n = 6 or 10) per pasture. Fecal samples were cultured for E. coli isolates and the minimal inhibitory concentration of CTC was determined. Baseline CTC-susceptibility of E. coli was variable among all treatment and control groups. The susceptibility of E. coli isolates was significantly different between study herds over the treatment period (p = 0.0037 across time and 0.009 at the final sampling time). The interaction between study herds and treatment period was not significant (p = 0.075).

Failure to Eliminate Persistent Anaplasma marginale Infection from Cattle Using Labeled Doses of Chlortetracycline and Oxytetracycline Antimicrobials.

Bovine anaplasmosis, caused by the intracellular rickettsial pathogen Anaplasma marginale, is the most prevalent tick-transmitted disease of cattle worldwide. In the U.S., tetracycline antimicrobials are commonly used to treat and control anaplasmosis. Oxytetracycline, administered by injection, is indicated for treatment of clinical anaplasmosis in beef and dairy cattle and calves. Chlortetracycline, administered orally, is indicated for control of active anaplasmosis infection in beef and nonlactating dairy cattle. Tetracyclines have been demonstrated to be effective for treating active anaplasmosis, but their ability to eliminate A. marginale at currently approved therapeutic doses or dosing regimens remains unclear. In the absence of approved dosing regimens for A. marginale clearance, a study was conducted to determine the effect of approved oxytetracycline and chlortetracycline indications on A. marginale bacteremia. Fifteen animals with persistent anaplasmosis were enrolled and divided into three treatment groups. Group 1 (n = 6) received oral chlortetracycline (1.1 mg/kg bodyweight) administered via hand-fed medicated feed for 60 consecutive days. Group 2 (n = 6) received injectable oxytetracycline administered subcutaneously at 19.8 mg/kg bodyweight three times in 3-week intervals. Group 3 (n = 3) served as an untreated control. After 60 days, bacteremia failed to permanently decrease in response to treatment. This result indicates that clearance of A. marginale is unlikely to be reliably achieved using currently approved tetracycline-based regimens to manage anaplasmosis.

Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation.

Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis, a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis.