Experimental Infection of Ticks: An Essential Tool for the Analysis of Babesia Species Biology and Transmission

Babesiosis is one of the most important tick-borne diseases in veterinary health, impacting mainly cattle, equidae, and canidae, and limiting the development of livestock industries worldwide. In humans, babesiosis is considered to be an emerging disease mostly due to Babesia divergens in Europe and Babesia microti in America. Despite this importance, our knowledge of Babesia sp. transmission by ticks is incomplete. The complexity of vectorial systems involving the vector, vertebrate host, and pathogen, as well as the complex feeding biology of ticks, may be part of the reason for the existing gaps in our knowledge. Indeed, this complexity renders the implementation of experimental systems that are as close as possible to natural conditions and allowing the study of tick-host-parasite interactions, quite difficult. However, it is unlikely that the development of more effective and sustainable control measures against babesiosis will emerge unless significant progress can be made in understanding this tripartite relationship. The various methods used to date to achieve tick transmission of Babesia spp. of medical and veterinary importance under experimental conditions are reviewed and discussed here.

Transmission of the human relapsing fever spirochete Borrelia persica by the argasid tick Ornithodoros tholozani involves blood meals from wildlife animal reservoirs and mainly transstadial transfer

Borrelia persica transmitted by the argasid tick Ornithodoros tholozani causes human tick-borne relapsing fever in the Middle East and Central Asia. Infection is acquired often when visiting tick-infested caves and reported to be transmitted mainly transovarially between ticks occasionally infecting humans. To study the epidemiology of this infection, ticks were trapped in 24 caves in 12 geographic zones covering all of Israel and identified morphologically. DNA was extracted from larvae, nymphs and adult stages from each location and PCR followed by DNA sequencing was performed to identify Borrelia infection, tick species, and tick blood-meal sources. 51,472 argasid ticks were collected from 16 of 24 caves surveyed. 2,774 O. tholozani were analyzed and 72 (2.6%) from nine caves were PCR-positive for B. persica. Infection rates in male, female and nymphal ticks (4.4%, 3% and 3.2%, respectively) were higher than in larva (p<0.001) with only 3 (0.04%) positive larvae. Presence of blood-meal was associated with B. persica infection in ticks (p=0.003), and blood-meals of golden jackals, red foxes and Cairo spiny mouse were associated with infection (p≤0.043). PCR survey of 402 wild mammals revealed B. persica infection with the highest rate in social voles (22%), red foxes (16%), golden jackals (8%) and Cairo spiny mice (3%). In conclusion, although transovarial tick transmission of B. persica occurs at low levels, ticks apparently acquire infection mainly from wildlife canid and rodents and may eventually transmit relapsing fever borreliosis to humans who enter their habitat. Importance Borrelia persica is a spirochete that causes tick-borne relapsing fever in humans in an area that spans from India to the Mediterranean. Until now it was thought that the soft tick vector of this infection, Orhinthodoros tholozani, is also its main reservoir and it transmits B. persica mostly transovarially between tick generations. This study showed that tick infection with B. persica is associated with feeding blood from wild jackals, foxes and rodents, and that transovarial transmission is minimal. Since O. tholozani ticks are found in isolated caves and ruins, it is assumed that wild canids who migrate over long distances have a major role in the transmission of B. persica between remote tick populations, and its then maintained locally also by rodents and eventually transferred to humans during tick bites. Prevention of human infection could be achieved by restricting entrance of canines and humans to habitats with O. tholozani populations.

An Epidemiological Survey Regarding Ticks and Tick-Borne Diseases among Livestock Owners in Punjab, Pakistan: A One Health Context

Recent global changes have led to an increase in the spread of ticks and tick-borne diseases (TBDs) affecting domestic ruminants and humans, with an annual loss of US $13.9–$18.7 billion. The current study determined the perception and practices of livestock farmers regarding tick infestation. A total of 112 livestock farms were surveyed in Punjab, Pakistan, among which animals from 42 (37.5%) farms were infested with ticks. Only 28.6% (n = 32) of the dairy farmers were consulting veterinarians for ticks control, while 86.7% (n = 97) of the respondents did not consider biosecurity measures in the control of tick transmission. Most of the respondents, 71.4% (n = 80), did not consider manual tick removal from their animals (i.e., by hand, followed by physically crushing) as a risky practice for spreading zoonotic diseases. Improper disposal of bottles of acaricides in the farm drainage was also observed, putting the environment and aquatic life at risk. These wrong practices may contribute to high disease burdens and economic losses, increasing the possibility of transmission of zoonotic TBDs and pollution of the environment. Therefore, an integrated One Health approach is required for the control of TBDs through environmentally friendly approaches.

Inter-stain interactions of a vector-borne parasite over its life cycle, "Borrelia afzelii", "Mus musculus", "Ixodes ricinus" model

Many pathogens consist of genetically distinct strains. When hosts are simultaneously infected with multiple strains the phenomenon is known as a mixed infection or a co-infection. In mixed infections, strains can interact with each other and these interactions between strains can have important consequences for their transmission and frequency in the pathogen population. Vector-borne pathogens have a complex life cycle that includes both a vertebrate host and an arthropod vector. As a result of this complexity, interactions between strains can occur in both the host and the vector. Interactions between strains in the vertebrate host are expected to influence transmission from the co-infected host to uninfected vectors. Conversely, interactions between strains in the arthropod vector are expected to influence transmission from the co-infected vector to the uninfected host. This thesis used the tick-borne bacterium, Borrelia afzelii, as a model system to investigate how co-infection and interactions between strains influence their transmission and lifetime fitness over the course of the tick-borne life cycle. B. afzelii is a common cause of Lyme disease in Europe, it is transmitted by the castor bean tick (Ixodes ricinus) and it uses small mammals (e.g. rodents) as a reservoir host. An experimental approach with two genetically distinct strains of B. afzelii (one Swiss stain, one Finnish strain) was used to investigate the effects of co-infection in both the host and the vector. In Chapter 1, lab mice were experimentally infected via tick bite with either 1 or 2 strains of B. afzelii. The infected mice were then fed upon by I. ricinus ticks from a laboratory colony to quantify host-to-tick transmission. qPCR was used to determine the presence and abundance of each strain in the ticks. Chapter 1 found that co-infection in the mice reduced the host-to-tick transmission success of the strains. This chapter also found that co-infection reduced the abundance of each strain in the tick. This is one of the first studies to show that co-infection is important for determining the abundance of the pathogen strains in the vector. In the lifecycle of B. afzelii, the bacterium is acquired by larval ticks that blood feed on an infected host. These larvae subsequently moult into nymphs that are responsible for transmitting the bacterium to the next generation of hosts. The bacterium has to persist inside the midgut of the nymph for a long time (8 – 12 months). Chapter 2 investigated whether nymphal ageing (1-month-old vs 4-month-old nymphs) under different environmental conditions (summer vs winter) influenced the interactions between strains in co-infected ticks. The spirochete abundance inside the nymph decreased with nymphal age, but there was no effect of the environmental conditions investigated. In Chapter 3, the presence and abundance of the two strains of B. afzelii were quantified in the tissues of 6 different organs (bladder, left ear, right ear, heart, ankle joint, and dorsal skin) that were harvested from the co-infected and singly infected mice. This study showed that co-infection in the mouse host reduced the prevalence of the Finnish strain in the host tissues (but the Swiss strain was not affected by co-infection). Chapter 3 found a positive relationship between the prevalence (or abundance) of each strain in the mouse tissues and the host-to-tick transmission of each strain. External tissues (e.g. ears) were more important for host-to-tick transmission than internal organs (e.g. bladder). Chapter 3 enhances our understanding of the biology of mixed infections by showing the causal links between co-infection in the host, the distribution and abundance of the strains in host tissues and the subsequent host-to-tick transmission success of the strains. Chapter 4 investigated how co-infection in the arthropod vector influences vector-to-host transmission success. A second infection experiment was performed, where naïve mice were exposed to nymphs that were either co-infected or infected with one of the two strains (i.e., using the nymphs generated in Chapters 1 and 2). The infection status of the mice was then tested using the same qPCR-based methods. Importantly, Chapter 4 confirmed that the negative effect of co-infection in the mouse on host-to-tick transmission (observed in Chapters 1, 2, and 3) had real fitness consequences for subsequent tick-to-host transmission. Ticks that had fed on co-infected mice were much less likely to transmit their strains to the host because these strains were less common inside these co-infected ticks. Chapter 4 did not find evidence that co-infection in the nymph influenced the nymph-to-host transmission success of each strain. This Chapter did find that there was a two-fold difference in nymph-to-host transmission success between the two strains. This work provides evidence for the idea that vector-borne pathogen strains can exhibit trade-offs across the different steps of their complex life cycles. In the co-infected mice, the Swiss strain had higher host-to-tick transmission success than the Finnish strain. Conversely, the Finnish strains had higher spirochete loads in the tick vector and had tick-to-host transmission success. Thus, the Swiss and Finnish strains are specialized on the host versus the vector, respectively.

Closing the Gaps to Understand the Tick Transmission of Anaplasma marginale among Giant Anteaters (Myrmecophaga tridactyla) in Argentina

Anaplasma marginale, a well-known cattle pathogen of tropical and subtropical world regions, has been previously molecularly characterized in a giant anteater (Myrmecophaga tridactyla) from Corrientes, Argentina. Ticks or other hematophagous arthropod involved in the wild transmission cycle remained unknown. The aim of the present study was to analyze the simultaneous occurrence of A. marginale in blood samples and ticks from giant anteaters from Corrientes in order to investigate if ticks could be relevant in the transmission among these mammals. Blood samples from 50 giant anteaters collected in different years and 26 ticks Amblyomma dubitatum and A. sculptum were studied through the molecular amplification of two unequivocal species-specific genes from A. marginale: msp5 and msp1β. Twenty five giant anteaters and tick organs (salivary glands, gut and oviduct) from 11 ticks tested positive to the A. marginale DNA amplification. The further molecular characterization through MSP1a tandem repeats analysis revealed the presence of genotypes circulating among giant anteaters that had been previously identified in cattle blood samples from the same geographical region. These results confirm the presence of A. marginale in giant anteaters in Corrientes and suggests that A. dubitatum and A. sculptum ticks could be involved in the transmission among giant anteaters. Future studies will determine the role of these tick species in the wild transmission cycle in the study area and the eventual connection with the domestic cycle.

Prevalence of Cytauxzoon felis Infection-Carriers in Eastern Kansas Domestic Cats

Cytauxzoon felis is a hemoprotozoal tick-transmitted pathogen of felids. Felids that survive acute disease often remain infected and serve as reservoirs for subsequent tick transmission to other susceptible felines. States adjacent to Kansas have identified C. felis-domestic cat carriers while statewide awareness and concern of cytauxzoonosis have increased. The objective of this study was to determine the prevalence of C. felis-carriers in the eastern Kansas domestic cat population using a sensitive quantitative PCR assay targeting the C. felis Cox3 mitochondrial gene. An overall C. felis infection prevalence of 25.8% was determined for asymptomatic domestic cats in eastern Kansas. Significantly more C. felis-carrier cats were identified in spring and fall, suggesting a seasonal fluctuation of survivors. Additionally, a greater percentage of feral and owned cats were positive for C. felis compared to rescue/rescinded cats. This study demonstrates that C. felis-domestic cat carriers are common among cats that spend at least a portion of time outdoors in eastern Kansas, and that more cats likely survive cytauxzoonosis than expected. Understanding the role of domestic cat carriers of C. felis is essential in developing cytauxzoonosis mitigation strategies, including recommending year-round use of acaricide products for all cats that spend any time outdoors.

Borrelia afzelii does not suppress the development of anti-tick immunity in bank voles

Vector-borne pathogens manipulate their vertebrate hosts to enhance their transmission to arthropod vectors. The ability of vertebrate hosts to develop acquired immunity against arthropod vectors represents an existential threat for both the vector and the pathogen. The purpose of the study was to test whether the tick-borne spirochete bacterium Borrelia afzelii could suppress the development of acquired immunity to its tick vector Ixodes ricinus in the bank vole Myodes glareolus, which is an important host for both the tick and the pathogen. We created a group of B. afzelii-infected bank voles and an uninfected control group by exposing lab-reared animals to infected or uninfected ticks. At 1, 2, and 3 months post-infection, all bank voles were infested with larval I. ricinus ticks. The bank voles developed a strong antibody response against tick salivary gland extract proteins. This anti-tick immunity had negative effects on tick fitness traits including engorged larval weight, unfed nymphal weight, larva-to-nymph molting time and larva-to-nymph molting success. Infection with B. afzelii did not suppress the development of acquired immunity against I. ricinus ticks. The development of anti-tick immunity was strongly correlated with a dramatic temporal decline in both the bacterial abundance in the host ear tissues and the host-tick transmission success of B. afzelii. Our study suggests that the development of anti-tick immunity in bank voles has important consequences for the density of infected ticks and the risk of Lyme borreliosis.ImportanceMany pathogens enhance their persistence and transmission by suppressing the immune system of their host. We used an experimental infection approach to test whether the Lyme disease pathogen, Borrelia afzelii, could suppress the development of acquired immunity against its tick vector (Ixodes ricinus) in the bank vole (Myodes glareolus), but found no evidence for this phenomenon. Uninfected and B. afzelii-infected bank voles both developed a strong IgG antibody response against tick salivary gland extract following repeated infestations with I. ricinus ticks. The development of anti-tick immunity was negatively correlated with the abundance of B. afzelii in ear tissue biopsies and with host-to-tick transmission to I. ricinus ticks. Our study suggests that anti-tick immunity in the bank vole reduces the prevalence of this important tick-borne pathogen.

EhrlichiaIsolate from a Minnesota Tick: Characterization and Genetic Transformation

ABSTRACTEhrlichia murissubsp.eauclairensisis recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species ofEhrlichia. The isolate could be grown in a variety of cultured cell lines and was effectively transmitted betweenIxodes scapularisticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely,Amblyomma americanumticks were not susceptible to infection by theEhrlichia. Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. AHimar1transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks.IMPORTANCEEhrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as theEhrlichia murissubsp.eauclairensis, a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate’s cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.