Blocking transmission of vector-borne diseases

Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission.


Introduction
Blood-feeding ectoparasites are responsible for severe aggravation through their constant attempts to get blood from their hosts. Besides causing discomfort, allergic reactions, skin damage and pain, many ectoparasites are also vectors of life-threatening or debilitating diseases caused by the transmission of a wide variety of pathogens, i.e. viruses, bacteria, protozoans, and worms, adding to their economic and emotional impact on human and animal health (Mehlhorn, 2008). Therefore, requirements for new ectoparasitic drugs should include not only the control of ectoparasites for a certain period of time, but also address their ability to block the transmission of the various vector-borne pathogens by a rapid onset of action. In this scope, "speed of kill" has become an important commercial differentiator for recent marketed products Wengenmayer et al., 2014;Beugnet et al., 2016;Blair et al., 2016;) and many studies have been designed for testing the ability of those products to block transmission of some important pathogens of cats like Bartonella henselae (Bradbury and Lappin, 2010), and of dogs like Dipilidium caninum (Fourie et al., 2013a), Leishmania infantum (Brianti et al., 2014), Ehrlichia canis (Jongejan et al., 2015), Borrelia burgdorferi, Anaplasma phagocytophilum (Honsberger et al., 2016), and Babesia canis Taenzler et al., 2016). These studies all report a complete prevention of pathogen transmission by fast elimination of the vector. These promising results confirm that a rapid onset of action should be an essential component of a novel drug profile. However, due to the diversity and specificity of vector parasite interactions, the blocking characteristics of those ectoparasiticides may not be sufficient to control other major pathogen transmitted by vectors to human, companion and farm animals. The arthropod can be either a mechanical vector, that is a simple carrier for dispersion, or a biological vector, within which the pathogen undergoes asexual and/or sexual multiplication before being transferred to a mammalian host. In the latter situation, pathogens need time to undergo development inside the vector and reach their infective stage. This depends to a major part on environmental conditions like temperature and humidity, and on the ability of the vector to survive long enough to harbor the matured infectious stage to be transmitted at next bite. Blocking pathogen transmission during that period has been tried with success as seen with Ixodes scapularis and Borrelia burgdorferi (Eisen and Dolan, 2016). Treating only the mammalian host with an efficient drug is a simpler option, but ensuring that a new drug is able to reliably block pathogen transfer remains very challenging. Nevertheless, there is a window of time for an ectoparasitic drug to prevent disease transmission (Fig. 1). This time period differs in length for each pathogen and vector, and can last from mere seconds to weeks.
Here we catalog a fair number of ectoparasite vectors and the respective transmitted pathogens of medical and veterinary importance. In addition, we complement that list with published information on the pathogen transmission time. Based on these results we propose several characteristics for an effective ectoparasitic drug profile.

Vector-borne transmitted pathogens
Major pathogens of medical and veterinary importance are listed in Tables 1e3. A short description of their development in the vector and timing of transmission is given when available. For each category of mammalian hosts, vectors are listed according to their importance in disease transmission. Many pathogens are zoonotic, with companion or farm animals becoming reservoirs in close contact with human populations, thus highlighting the practicality of employing common strategies for both human and animals to control pathogen transmission. The tables demonstrate the diversity of the vector e pathogen interactions. In most cases, the pathogen will undergo a multiplication, a change in morphology, and a maturation in the vector. Very often, the infectious pathogen is waiting in the vector's salivary glands and will be passed on to the host together with the saliva immediately at bite. On the other hand, there are a few organisms, like Rickettsia sp (Hayes and Burgdorfer, 1982), Anaplasma sp (Hodzic et al., 1998;Katavolos et al., 1998;des Vignes et al., 2001), Borrelia sp (Kahl et al., 1998;des Vignes et al., 2001), or Babesia sp. (Homer et al., 2000;Zintl et al., 2003), that need an activation step for migration into the salivary glands, multiplication within the salivary glands, or a maturation phase all triggered by the onset of the blood meal. Interestingly, these pathogens all mature in ticks, which are slow blood-feeding arthropods and typically need days of host attachment to fully engorge.

Transmission time is considerably different between insects and ticks
When considering ectoparasites in relation to the pathogens transmitted and the time needed to transfer the pathogens after biting the host, a clear difference between insects and ticks is noticeable (Tables 1e3). Many, if not all holometabolic insects like mosquitoes (Vlachou et al., 2006), tsetse flies (Van Den Abbeele et al., 1999), fleas (Gage and Kosoy, 2005), or sand flies (Bates, 2007), which undergo complete metamorphosis, almost always transfer the respective pathogens immediately at bite. By contrast, some ticks can require host attachment time periods of several hours, extending up to days in some instances before transmission of pathogens occurs. As hard ticks (Ixodidae), sometimes also referred to as hardbacked ticks, feed only once before molting to the next stage, ingested pathogens will have to survive the molting process and be transferred transstadially (i.e. Babesia sp, Homer et al., 2000;Ehrlichia sp, Paddock andChilds, 2003, Stich et al., 2008). It may be difficult for the pathogen to develop, migrate, or mature while the physical and metabolic changes take place during the vector's molting process. The pathogen will also have to survive for an extended period in the tick vector that might not find the next host immediately and could stay unfed for weeks or months. Those micro-organisms may then need a reactivation from some kind of dormant condition to resume their development. Temperature change due to the tick attachment to a warm-blooded animal (Hayes and Burgdorfer, 1982), or fresh blood entering the tick may be the signal for the pathogen to multiply (Hodzic et al., 1998), migrate to salivary glands (Kahl et al., 1998), or finish its maturation and be ready for transmission (Homer et al., 2000). This last step might take hours or days, and gives opportunities to block the Fig. 1. Generic sketch for transmission of diseases by ectoparasites (vectors). Blocking of transmission can in principle occur at every stage, but most drugs aim to interfere during "Attachment" phase and/or "Feeding & Transmission" phase.  Immediate transmission at next bite once proventricule blockage is achieved.

Gage and Kosoy, 2005
Rickettsia felis Cat flea typhus Transmitted by C. felis. Ingestion by feeding on an infected host. Multiplication in midgut cells and dissemination in the flea tissues, including ovaries and salivary glands. Migration to salivary glands takes 7e14 days but transmission has been reported to occur as soon as 12 h after infection feeding (surely within 24 h) via co-feeding with infected fleas. This early phase transmission seems to be mechanical. Transovarial transmission also occurs in the flea vector. Mosquitoes (Anopheles gambiense) now also suspected to be vector.  transfer. In this context, fast-acting ectoparasiticides could be effective at preventing disease transmission in ticks (Reichard et al., 2013;Fourie et al., 2013a;Brianti et al., 2014;Jongejan et al., 2015;Beugnet et al., 2014;Honsberger et al., 2016;Taenzler et al., 2016). In soft ticks (Argasidae), also referred to as softbacked ticks, pathogens face similar conditions as in hard ticks (i.e. survival through molting, long periods of fasting, transstadial transmission) but also have to adapt to additional constraints. Soft ticks like Ornithodoros are fast blood-feeders that need only minutes to fully engorge. Adults feed many times, and females lay eggs in small batches after each blood meal. They develop through more than one nymphal stage, increasing the number of opportunities for transmitting pathogens during their life-span (Schwan and Piesman, 2002). Fast-feeding implies that pathogens cannot go through an activation step during the blood meal like that previously discussed for hard ticks, but rather have to be ready in the salivary glands to be transferred as soon as feeding starts. As an example, Borrelia duttoni infecting soft ticks is transmitted from within 30 s to a few minutes after feeding starts (Dworkin et al., 2008), whereas B. burgdorferi is only transmitted by hard ticks after 24 he48 h on average (des Vignes et al., 2001;Schwan and Piesman, 2002). Thus a drug with an onset of action within a few hours might be sufficient for blocking transmission by hard ticks, but not for preventing transmission by soft ticks. In the latter case, preventing the vector from accessing the host with a repellent could be a more effective solution.
Some major pathogens of hemimetabolic insects like true bugs (Reduviidae) or lice (Phthiraptera) can develop in either immature stages or adults. Trypanosoma cruzi (Krinsky, 2008), Rickettsia prowazekii (Houhamdi et al., 2002), or Bartonella quitana (Byam and Lloyd, 1920) are transmitted to their host via infected feces rubbed on wounded skin. Killing the vector before it gets time to produce infected feces could be possible using a drug with very fast onset of action. In the case of lice, such a drug could also have a massive impact on lice populations that do not move easily from one host to another, and therefore reduce the inflammation and scratching that are the real cause of infection. In Reduviidae, blocking transmission via killing the insect before releasing infected feces may also work. However, as Reduviidae are fast feeders, release of the feces could occur within the first minutes of a blood meal. It remains to be demonstrated if preventing access to a host and subsequent biting with a repellent drug can effectively block T. cruzi transmission.
Pathogens of most holometabolic insects develop and multiply in an adult individual that has a life expectancy on the order of days or weeks. Their development can start immediately after ingestion and needs to reach the infective stage within the life-span of the insect vector. In these cases the pathogen strategy appears to be different and infectious stages are transmitted often within seconds to the mammalian host. Blocking transmission is therefore more challenging, and avoiding insect bite via a repellent drug could be the best option.

Drug profile for blocking pathogen transmission
The principal feature of an ectoparasitic drug aiming to block transmission should certainly be a very fast onset of action. This requirement is generally understood by the animal health industry, and most products marketed recently have been tested and compared for the speed of their onset of action Wengenmayer et al., 2014;Beugnet et al., 2016;Blair et al., 2016;. Recent compounds deriving from the fairly new chemical class of isoxazolines (Weber and Selzer, 2016) exhibit their ectoparasitic action against both, insects and acari of veterinary importance, within hours, and certainly reduce the risk of  which further multiply and produce a promastigote secretory gel. Some attach and transform into haptomonad promastigotes. Some differentiate into the infective metacyclic promastigotes. The gel containing the infective metacyclic forms obstructs the anterior midgut, forcing regurgitation at next bite prior to feeding, releasing the pathogen into the host. One-2 weeks are needed between ingestion of amastigotes and regurgitation of the infective metacyclic promastigotes. Vector gets infected at larval stage through cestode egg ingestion. Development in fleas is temperature dependent. With temperature lower than 30 C, the infective metacestode is not ready when the adult fleas emerge. The flea will need to survive and stay on a host a few days to allow completion of the development of the metacestode, triggered by the higher temperature of the host. Blood meal has no effect on development. Dog infection through ingestion of the parasitized flea.
Immediate transmission once the infective larvae is mature.

Granulocytic anaplasmosis
Transmitted by Ixodes sp. Persistence in the vector through transstadial transmission, but not transovarial. Acquisition by the vector within 24 h blood feeding. Multiplication in vector during and after acquisition feeding, and triggered again by next blood meal.
Transmission does not take place before 36 h-48 h tick feeding, but was shown in the lab to occasionally occur occasionally within 24 h of attachment already. Hodzic et al., 1998;Katavolos et al., 1998;des Vignes et al., 2001 Ehrlichia Heartwater Transmitted by Amblyomma sp. Persistence in the vector through transstadial transmission. Transstadial transmission can happen over one or more stages depending on tick species. No interstadial transmission reported and transovarial transmission not sure. Bacteria ingested with the blood meal and enter the gut cells into which they multiply by binary fission in inclusion bodies. Migration to other organs like hemocytes, Malpighian tubules and salivary glands. Bacteria colonies detected in salivary glands only after transmission feeding start.
Transmission reported to occur from the 2nd day of feeding in nymphs, and from the 4th day in adult ticks. Kocan and Bezuidenhout, 1987;Bezuidenhout, 1987;Allsopp, 2010 Borrelia Ingested gametocytes fuse to give rise to immobile zygotes that transform into mobile kinetes, They enter the hemolymph, disseminate into various tissues including muscles, epidermis, Malpighian tubules and ovaries in adults. They undergo an additional asexual multiplication step and further dissemination as secondary ookinetes.
In salivary glands, kinetes continue to multiply asexually. Maturation into infective haploid sporozoites happens only after transmission feeding starts.
In nymph ticks, sporozoites were detected in salivary glands from the 3rd day of feeding.
Like other Babesia, transmission is delayed to the second half of the tick blood meal. Transmission reported from day 3 of feeding. Donnelly and Pierce, 1975;Homer et al., 2000;Zintl et al., 2003 Babesia  Ticks: transmission does not take place before 36 h-48 h tick feeding, but was shown in the lab to occasionally occur after 24 h. Kocan et al., 2004;Kocan et al. 2010 Hard and soft ticks (Ixodidae and Argasidae) *Coxiella burnetti Q fever Transmitted by many tick genera. Persistence in the vector through transovarial and transstadial transmission. Multiplication in midgut cells. The bacteria are released in tick feces when the tick begins to feed again. Transmission via an arthropod vector is very rare, occurs mostly through aerosol or from parturient fluids released by infected vertebrate hosts. The pathogen persists in the environment for weeks, and can be spread by the wind.
Timing not known in feeding ticks. Maurin and Raoult, 1999 Tsetse flies disease transmission of hard tick pathogens that are not immediately passed on to the host such as Babesia sp Taenzler et al., 2016) or Borrelia sp. (Honsberger et al., 2016). Such a beneficial effect was shown especially for canine borreliosis (Honsberger et al., 2016;Weber and Selzer, 2016). Based on those results, one could hypothesize that isoxazolines may also be able to prevent human borreliosis (Lyme disease). However, to date many unknowns remain, including the pharmacokinetic behavior and safety of the drug in humans. Although effective at eliminating some tick infestations and consequently blocking pathogen transmission, systemic ectoparasiticides may be more limited in controlling those pathogens that are transmitted within a few hours or immediately after the vector's bite. For example, in a comparative study on the ability to block Ehrlichia canis transmission from Rhipicephalus sanguineus ticks by orally administered isoxazolines compared against topically applied products containing synthetic pyrethroids, the tested systemic isoxazoline ectoparasiticides gave insufficient protection of dogs from pathogen transfer (Jongejan et al., 2016). Despite being considered "old drugs", synthetic pyrethroids (i.e. permethrin, deltamethrin, flumethrin) exhibit features that would in principle be close to an ideal drug profile. In addition to having a fast onset of action on many insects and tick species, some pyrethroids are also irritant or repulsive for a variety of ectoparasites (Mencke, 2006). It appears that a combination of repellency and parasiticidal activity could be the best way to prevent pathogen transmission, independently from the transfer time at bite. Synthetic pyrethroids have been shown to efficiently block transmission of Leishmania sp. in dogs by repelling and killing sandflies (Ferroglio et al., 2008;Brianti et al., 2014). They are also widely used for impregnating bed nets and clothing to prevent insect bites and disease transmission to humans (Curtis et al., 2003;Banks et al., 2014). They have been added to some recently marketed products for companion animals, to act as repellents and/or speed up the onset of action (Beugnet et al., 2016;Blair et al., 2016). However, wide-spread resistance in many vectors (including mosquitoes, lice, true bugs, and ticks) and safety issues (Anad on et al., 2009;Peterson et al., 2011) disqualify them for longer-term use and motivate the search for novel drugs displaying an equivalent profile with improved safety. Designing and developing new and safe ectoparasiticide drugs able to effectively block fast transmitted vector-borne pathogens is still on a wish list and remains extremely challenging. In our opinion, such novel ectoparasitic drug for animal health, should combine features of fast killing, long persistency and repellency to both acari and insects. Additional constraints may be encountered if any new ectoparasiticide should be considered for human use. Beyond identifying a relevant application, it is not clear if humans would accept a persistent drug exposure to achieve a long lasting protection period. In principle, repellency combined with long-term persistence is very difficult to achieve in a single compound, constituting a challenge as big as achieving very rapid onset of action. In addition, a drug with only repellent activity would have the disadvantage of having no impact on vector populations. The fast killing and long lasting persistence already achieved with the isoxazolines would allow prevention of important tick-borne diseases. Additional repellency or deterrent activity would be efficacious at preventing insect-borne pathogens that are transmitted rapidly upon biting. Combining all of these activities would be the ideal profile for an ectoparasiticide. Achieving that goal might not be possible with a single chemical entity but may be possible with a combination of molecules, bearing in mind the challenges of maintaining a good safety profile for the host and for the environment. Hurdles remain extremely high however, and other complementary measures targeting the pathogen itself via specific drugs or vaccines should definitely be investigated in parallel.

Moraxella bovis
Bovine keratoconjunctivitis Transmission via direct contact, though feces or regurgitation of the bacteria by the vector. Regurgitation seems to play a major role. Bacteria accumulate in the fly crop. Immediate transmission, with success depending on fly numbers feeding at same time. Glass and Gerhardt, 1984 Tabanids, mosquitoes, fleas, hard ticks Bacteria *Francisella tularensis

Tularemia
Main ways of transmission via tick bites and direct contact with a contaminated animal, mainly rabbits and hares, but occurs also via insect bites, ingestion of contaminated food or aerosol.
Ticks: Dermacentor variabilis is the main vector. Persistence in the vector through transstadial transmission although infected nymph ticks suffer high mortality due to the pathogen. Transovarial transmission also reported. Ticks: can occur within 1 day after an adult tick infected as nymph begins to feed. Reese et al., 2011

Conclusion
Meeting the requirements for new ectoparasiticides, including prevention of transmission of pathogens, is challenging if possible at all. Transmission in companion animals of some major tick-borne pathogens can be now controlled with compounds of the isoxazoline class because of their fast onset of action. Extending the use of this class of molecules to humans and farm animals may help to control some tick-borne zoonotic diseases. For other pathogens, mainly those transferred to the host by insects immediately at bite and by soft ticks, the speed of kill by isoxazolines is insufficient to effectively prevent pathogen transmission. Most insect vectors have little time for feeding before being chased away or being killed by the host, and therefore, in most cases, blood feeding and associated pathogen transmission begins immediately upon landing. In this situation, drugs having repellent or deterrent activity that hinders the vector from biting or landing on the host would be more successful at preventing disease transmission. Solutions could, therefore, be different depending on the vector, the associated pathogens and the speed of transmission. In an ideal situation, a drug or a combination of chemical entities should prevent the vector from access, or at least from biting the host. If the vector eventually succeeds in reaching the host, killing by the drug should happen very rapidly. Repellent efficacy combined with parasiticidal activity seems to be the ideal drug profile for successfully preventing vector-borne diseases in humans, pets and livestock. This easy statement unfortunately hides major difficulties especially if the repellent effect has to be long-lasting for weeks or months. Due to those substantial difficulties, the search for new vaccines or drugs targeting the pathogen should not be left aside. Novel alternative approaches, for example ones based on regulators of the immune system like the Toll pathway of the vector (Garver et al., 2009) should also continue to be explored.