28. The protection of European dogs against infection with Lyme disease spirochaetes

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1 28. The protection of European dogs against infection with Lyme disease spirochaetes K. Emil Hovius Amphipoda BV Ecovet, Heike 9, 5508 PA Veldhoven, the Netherlands; Abstract In this chapter the natural, innate immunity of dogs, the added protection offered by tick repellents and acaricidal products, and the activated immunity provided by vaccines against Lyme disease spirochaetes are discussed. The dog s innate immune defence to spirochaetes is incomplete. Only one Borrelia species is killed by serum complement. This means that feral dogs may have a role to play as a reservoir in the ecology of Lyme disease spirochaetes. However, only a minority (5%) of dogs develop Lyme disease symptoms, the reason for which is probably breed related. Thus added protection seems appropriate. Recently acaricidal products have improved in efficacy and in ease of application. Client compliance with the application regime may greatly influence the outcome and care must be taken, at time of reapplication, to prevent ticks from having an opportunity to reinfest. Vaccination with the recently improved newest vaccines which target the European Borrelia species and its related strains can ensure a yearlong active immunity. It is anticipated that the new vaccines will exert nearly 100% protection if they are applied in the way the manufacturer s instructions recommend and even more surely with an extra booster. Experience with the new vaccines has not yet resulted in published field trials. More knowledge of the effects of vaccination is needed in Europe; knowledge which could also benefit the development of human vaccine production. Keywords: acaricides, anti-osp A, anti-osp C, canine borreliosis, One Health, serum resistance, vaccines Introduction The dog is the one household animal that is most often let free to traverse its favoured playing/ hunting grounds, these are usually parks or wooded areas that are not far from the neighbourhood where the dog lives in proximity with its owners. Companion animals in general are daily in close contact with humans and, as such, may put their owners at risk of tick bites from tick infested areas. For these reasons, the protection of dogs against ticks is a rewardable precondition against the risk, not only for the dog itself, but for its owners too. The dog is possibly a competent reservoir host for Lyme spirochaetes and it is not unlikely that feral dogs still roaming in some parts of Europe are playing a local role in Lyme disease ecology. Therefore, it is relevant to explore the dogs own defence against ticks, Borrelia infections and disease. Perhaps, in the near future, vaccination against ticks could be a realistic option (Klouwens et al. 2016). Anti-Borrelia vaccines are already on the market and millions of dogs have been vaccinated across North America and Europe. For dog breeds that are sensitive to the development of Lyme disease after infection and are at risk of tick bite, protection via vaccination may be the ideal choice. The experience gained using these vaccinations may also be useful when vaccination for humans is eventually introduced. Other, more direct, measures should not be omitted and will be discussed in this chapter. Tick avoidance is possible but will restrict the dog s free movement. Therefore repellents and acaricides which can be applied directly to the dog s skin, or orally administered are most frequently used. The role of carefully applied antibiotics in veterinary medicine, taking the suspicion of arthropod borne Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _28, Wageningen Academic Publishers 2016

2 K. Emil Hovius infection into account for a dog with appropriate symptoms, cannot be omitted in the discussion on protective measures. With all the evaluations of risk it is logical that the comparison of city dogs versus outdoor dogs is accounted for (Mather et al. 1988). Dogs are more at risk than their owners and there are no signs that having a dog in the household puts the human occupants at any greater risk for acquiring Lyme disease (Eng et al. 1988, Goossens et al. 2001). Risk of tick borne diseases in dogs Many mammals and birds, including pet animals, are host species for ticks. The dog accompanies man into woods and other tick habitats, and is thereby put at far greater risk of contracting tickborne infections than man himself. Horses may be exposed if pastured in the grassland to wood interface. Free roaming cats can be exposed to ticks, but do not easily develop borreliosis, and their role in the ecology of ticks and Borrelia needs investigation. Dogs are frequently infected, and re-infected, by the spirochaetes, and in some cases (around 5%) develop disease (Hovius 1990, 2005, Hovius and Houwers 2007, Hovius et al. 2000, Kornblatt et al. 1985, Littman 2003, Magnarelli et al. 1985, 1987, Straubinger 2000a, Summers et al. 2005). The dog, therefore, has been selected as the companion animal for which any possible protective measures against Borrelia infection may be appropriately illustrated. Infection and symptomatic manifestations of an array of other tick-borne agents have also been described in dogs. The most important of these other tick-borne agents are the (bacterial and protozoan) genera (Neo)ehrlichia, Anaplasma, Rickettsia (including the recently discovered Neorickettsia), and Babesia moreover dogs are also prone to viral tickborne encephalitis. Some protective measures against Borrelia infections, those preventing tick infestations, for instance, will also offer protection against the other tick borne disease agents. However, not all infectious agents are harboured by the same tick species, Ehrlichia canis in Europe reside in the brown dog tick Rhipicephalus sanguineus, for example, while Babesia canis infecting dogs is transmitted by Dermacentor reticulatus and R. sanguineus. The two Ixodes species, I. ricinus in western Europe and I. persulcatus in the East, transmit several Borrelia species as well as Anaplasma phagocytophilum that propagates in dog granulocytes. Anaplasma platys, which infects dog thrombocytes, is mainly transmitted by R. sanguineus. In some European countries, antibodies against Anaplasma phagocytophilum may be far more prevalent in dog serum than antibodies directed Borrelia and the other tick borne agents and canine anaplasmosis is regularly diagnosed especially in Sweden (Egenvall 1997, Krupka et al. 2007, Kybicova et al. 2009). Preventive measures against tick infestations will also reduce the acquisition of the other disease agents. Symptoms and diagnosis of borreliosis in dogs In dogs the symptoms of disease caused by the array of tick borne zoonotic infectious agents can be similar in appearance especially when the first manifestations of disease develop. In the later stages of disease, symptoms may more clearly define one particular agents but for a diagnosis to be made serological and molecular laboratory investigations usually need to be made at several time intervals after infection. Moreover, coinfection or successive infections can occur which may hamper the interpretation of clinical and laboratory investigations. Thus it appears that, in clinical appearance, symptomology, and diagnostic workup, the dog is as complicated as the human Lyme disease patient. This also concerns borreliosis caused by a single Borrelia infection for which there are no pathognomonic symptoms, such as erythema migrans in man. However, unexplained fever and lameness may be seen as not specific but typical for canine borreliosis (Kornblatt et al. 1985, Littman et al. 2006). Chronic manifestations in the dog may be more easily observable than in man, due to the shorter lifetime of the dog. Also the apparent differences in breed susceptibility seem to indicate a genetic disposition more easily than is observable in man (Gerber et al. 2007, 410 Ecology and prevention of Lyme borreliosis

3 28. Protection of pets Lindenmayer et al. 1991) (Figure 1). Therefore, lately it has been suggested that the dog might not only be used as a sentinel for the infection in humans but could also serve as a model for the pathogenesis of disease in man (Elsner et al. 2015). Re-infection is more easily determined in dogs than in humans, if the appropriate time frames and particular antibodies are studied (Hovius et al. 1999a). These reinfections (probably with more than one Borrelia species; see below) do not usually lead to disease. Apparently, some of the seroconverting antibodies, along with other reactions of the adaptive immune-system clear the spirochaetes. When attributable symptoms (not specific) occur this is accompanied by rising and high titres, which usually leads to a diagnosis when several serum samples are taken throughout one tick season (Hovius et al. 2000). To date, four Borrelia species have been detected in dog tissues and tissue tropism for the three pathogenic species seems to be apparent. However, after limited investigation, it seems that this tropism in the dog is not directed in completely the same way as it is in humans. However, in dogs as in man, Borrelia burgdorferi sensu stricto is mostly detected in tissues which are rich in collagen such as skin and peripheral joint tissues (Straubinger 2000b, Summers et al. 2005, Van Dam et al. 1993). Borrelia garinii is frequently observed in man in the tissue of the nervous system. In the dog it was mainly detected in liver tissue (Hovius et al. 1999b, Van Dam et al. 1993). Borrelia afzelii and Borrelia valaisiana were only found in liver tissue in dogs (Hovius et al. 1999b).The dog s liver may function as a sink in which spirochaetes are collected and killed by liver macrophages, as Sambri has shown in rat livers (Sambri et al. 1996). In a Dutch study on tick infested seropositive dogs, liver samples containing Borrelia DNA were mainly found in symptomatic dogs, putatively diagnosed for Lyme disease. Most of the positive samples contained DNA of more than one Borrelia species and nearly always included B. garinii DNA. Healthy dog livers much less frequently contained Borrelia DNA (Hovius et al. 1999b). Figure 1. The Bernese mountain dog, is frequently found to be seropositive for the Borrelia IR6 antigen, measured with the C6 SNAP test (by Idexx, see text). It may also show episodes of fever and lameness. The diagnosis of an episode of active Lyme disease, is complicated by possible osteological problems or immunological aberrations that may occur in this breed. In general a thorough diagnostic workup with exclusion of differential diagnosis and multiple serological tests is needed to confirm or discard the diagnosis of Lyme disease (copyright photo: Tijdschrift voor Diergeneeskunde 2007). Ecology and prevention of Lyme borreliosis 411

4 K. Emil Hovius The dog is imperfectly protected against Borrelia by its innate immunity Borrelia species circumvent the innate immunity of their hosts by expressing host specific factor H binding proteins that inhibit factor H regulatory proteins in their function as first step in the cleavage of the C3b complement factor (Bhide et al. 2009, Van Burgel et al. 2010). The differing complement resistance of Borrelia species determines the evolutionary coupled reservoir competence of certain animals (mammals, birds, reptiles) (Kurtenbach et al. 1998, 2002). In this chapter it is interesting to explore the known putative reservoir function of canids (and other mammals) for the different Borrelia species, derived from complement mediated killing experiments, (Bhide et al. 2005, Hovius et al. 2000,Van Dam et al. 1997) or factor H binding experiments (Bhide et al. 2009, Van Burgel et al. 2010) (Table 1). Several Borrelia species are resistant to dog serum such as B. burgdorferi s.s, B afzelii, Borrelia bavariensis (formerly B. garinii serotype 4-Pb1) and B. valaisiana. While most B. garinii strains are not resistant and thus killed by the dog and mammalian complement system, in line with B. garinii being hosted by birds. Remarkably, B. afzelii and B. valaisiana are killed by wolf complement and not by dog complement. A factor H binding protein for dog complement was detected for B. afzelii and B. valaisiana, thus preventing bactericidal activity by dog serum (Bhide et al. 2009). In line with these results 4 Borrelia species were found in organ tissues of dogs (Hovius et al. 1999b). It is hypothesised that evolutionary time has been too short for dog and man to develop innate immunity against the 4 species (Skotarczak 2014). B. afzelii is the most frequently occurring species in ticks. The Western European wolf Canis lupus lupus opposes this threat with an evolutionary developed defence strategy of the complement system. The dog Canis lupus familiaris seems to have lost this ability in the domestication process. B. burgdorferi s.s. appears to have made transatlantic colonisation vice versa and some strains, such as B31 used in the assays, have North-American origins (Castillo-Ramirez et al. 2016). Therefore, it is not surprising that the European wolf and its domestic pedigree is maladapted to withstand certain non-european B. burgdorferi s.s. strains (Bhide et al. 2009). This implies that dog and the wolf are a putative reservoir for B. burgdorferi s.s.. The Beagle breed has been experimentally determined to be a competent reservoir animal for B. burgdorferi s.s., which is passed to ticks (Mather et al. 1994). Table 1 also shows the capacities of feline predators to fence off different Borrelia species. The reservoir function of the cat has not been investigated. But, if the cat were to transmit Borrelia to ticks like the Beagle dog does, the feral cats, collecting and releasing ticks in the woods, could contribute to the ecology of the Borrelia species which have serum resistance. Hoofed mammals are dead end hosts for all the Borrelia species and strains investigated and have no reservoir function. The ecological impact of these animals is discussed in earlier chapters of this book. Man is similarly maladapted to withstand the different Borrelia species as is the dog and in this sense the dog may be an animal model for Lyme disease. Do dogs have a role in the transmission cycle of some Borrelia species? The possibility of vectors and their transmissible disease agents being distributed in the wild by wild canines and felids to their domesticated or feral counterparts has been reviewed by Otranto (Otranto et al. 2015). Wolfs are expanding and roam together with feral dogs and dog hybrids in certain parts of Europe. These animals can function as reservoirs for Borrelia and maintain infections in the region that may be picked up by their tame counterparts. The Beagle dog breed has been determined to be a competent reservoir animal for B. burgdorferi s.s., it acquires and delivers the spirochaetes to ticks (Mather et al. 1994). The wolf, however, is a dead end host for more Borrelia species than the dog, see Table 1. Feral dogs and the wolf in the European ecosystem may differ in their reservoir function to propagate different Borrelia species (Bhide et al. 2005).The preliminary bactericidal assays have to be repeated with several dog breeds, with wolves from 412 Ecology and prevention of Lyme borreliosis

5 28. Protection of pets Table 1. Resistance (yes/no) of the most occurring West European Borrelia species and strains to serum of several mammalian species including dog and man. Borrelia garinii is heterogeneous and distinguished by the Osp A serotype as determined by Wilske et al. (1995, 1996). 1,2 Species (OspA serotype) Strain Horse Cow Lynx Cat Wolf Dog Man B. burgdorferi s.s. B31 yes: 2 yes: 1, 5 (serotype 1) SKT2 no: 4 no: 3, 4 yes/no: 3 yes: 3; yes: 3 yes: 3, 4 yes: 3, 4 no: 4 B. afzelii SKT4-5 no: 4 no: 3, 4 yes/no: 3 yes: 3, 4 no: 3 yes: 3,4 yes: 3, 4 (serotype 2) pko yes: 2 yes: 1, 2 B. garinii (serotype 3) Rio 2 no: 4 no: 3, 4 yes: 3 yes: 3; no: 4 yes: 3 yes: 3; no: 4 yes/no: 3; no: 4 B. garinii G117 no: 4 no: 4 no: 4 no: 4 no: 4 (serotype 5) PV6 no: 3 no: 3 yes/no: 3 no: 3 no: 3 no: 3 A87S no: 2 no: 1 B. garinii SKT3 no: 4 no: 3, 4 yes: 3 yes: 3; no: 3 no: 3, 4 no: 3, 4 (serotype 6) no: 4 VSBP no: 1, 5 B. garinii T25 no: 4 no: 4 no: 4 no: 4 yes: 4 (serotype 7) B. garinii (serotype 8) CLI no: 4 no: 3,4 yes: 3 yes: 3; no: 4 yes: 3 yes: 3; no: 4 no: 3,4 B. bavariensis (B. garinii serotype 4) PBi no: 4; yes: 5 no: 3, 4, 5 yes: 3 yes: 3, no: 4, 5 yes: 3 yes: 3, 5; no: 4 yes: 1, 4, 5; yes/no: 3 B. valaisiana (not serotyped) VS116 no: 4 no: 3, 4 yes: 3 yes: 3; no: 4 no: 3 yes: 4; yes/no: 3 yes: 1, 4; yes/no: 3 1 yes = serum does not kill the Borrelia strain; no = serum kills the Borrelia strain. 2 Numbers represent the authors (in the references) that have employed different assays: 1, 2, 3 explored the bactericidal activity of serum against live spirochaetes (yes, is less than 40% killed; yes/no is between 40 and 60% killed; no is 60% or more spirochaetes killed); authors 4 and 5 determined the factor H binding protein on the spirochaetes (yes is detected; no is not detected). References: 1 = Van Dam 1997; 2 = Hovius 2000; 3 = Bhide 2005; 4 = Bhide, 2009; 5 = Van Burgel different countries and with several strains per Borrelia species. However, it may be tentatively expected that the dog will be a reservoir animal for B. burgdorferi s.s. B. afzelii, B. bavariensis and B. valaisiana and the European wolf for B. burgdorferi s.s. and B. bavariensis. Therefore, after expected reintroductions, the wolf may have a direct influence on tick cycles and the ecology of Borrelia systems, besides possibly influencing these cycles by changing the spectrum of reservoir biodiversity. Spanish wolves were found to be seropositive (14.7%), but the infecting species was not determined (Sobrino and Gortazar 2008). In the urbanised western countries of Europe, the influence of feral dogs in the patchy woody ecosystems can be neglected. When dogs are walked by their owners through these environments they contract many ticks, which drop off elsewhere. In gardens, large enough to contain some species of mice, rats, squirrels, hedgehogs and birds, a local cycle of tick and Borrelia may develop Ecology and prevention of Lyme borreliosis 413

6 K. Emil Hovius which could pose an extra threat of tick infestation and Borrelia transmission to the owners (Bhide et al. 2004). But a greater infection rate in dog owners than in non-dog owners has never been documented (Eng et al. 1988, Little et al. 2010). However, the possibility exists that unattached ticks in the dog s coat can walk onto the owner s skin (personal observation). For the above reasons, the protection of dogs against ticks is a rewardable precaution. Tick avoidance is possible but will restrict the dog s free movement. It is, therefore mostly repellents and acaricides that are applied to the dog s skin, or orally administered. In future it may be possible to vaccinate against ticks (Klouwens et al. 2016). Anti-Borrelia vaccines are available for dog breeds sensitive to the development of Lyme disease. Added protection of dogs Protection by avoidance strategies Not roaming the woods and avoiding shrubby areas will destroy the dog s pleasure during field walks. Expecting dog owners to be able to judge the tick favourable habitats is unrealistic, so appropriate warning signs could be placed that owners and dogs enter at risk of contracting ticks. Apart from instructing people how best to avoid tick contact, advice for dog handling in this sense should also be put on the signs. When the tick season is at its most intense and the weather is favourable for questing ticks, with moderate to high temperatures and very high relative humidity, it is particularly important to avoid the undergrowth of woods and thickets. At such times the dog is best kept on a lead. Many people live in the countryside and for these dogs and for working dogs (guard dogs, herding dogs, hunting dogs) the avoidance of tick habitat is impossible. After field work, just as in man, these dogs should be checked for ticks immediately when returning home or to the pen. Particular attention should be paid to checking the areas above the eye, behind the ears, the front of the neck, around the front legs (axil and shoulder) and in the groin and the ticks removed with a special instrument or a pair of pliers (Figure 2). The result of these inspections depends on the texture and colour of the dog s coat and will never be perfect. It is therefore wise to include other preventive measures. It has been shown that in an area which has about 1 in 10 ticks infected, the first contact with an infected tick occurs on the first or second walk. This single attachment of an infected tick in all cases leads to the transmission of Borrelia to the dog (Leschnik et al. 2010). There may be 3 strategies to prevent this transmission. 1. Preventing ticks from clinging to the dog s coat by applying effective repellents. Or, if the repellents do not succeed in preventing attachment and fixation to the skin, by applying acaricides which have immediate efficacy. 2. If the first strategy does not work, ticks can be killed when attached, preferably in the very first 12 hours of attachment when (in the majority of ticks) the Borrelia have not yet reached the salivary gland to be transported by the saliva to the vertebrate host (Cook 2015, Piesman et al. 1987). 3. If the acaricides do not work properly because of the composition of the dog s skin, for instance, Borrelia infection may then be prevented by vaccination. Protection by prevention of ticks clinging onto the coat: repellents With regard to ticks, the repellent effect cannot be viewed the same as with flying vectors where the repellent prevents the host from being landed on. It is hard to discourage ticks from grabbing onto the fur or skin of the host when they come in accidental contact with it, and repellency is defined as the reluctance to attach (insert the mouthparts) or leave altogether (Halos et al. 2012). 414 Ecology and prevention of Lyme borreliosis

7 28. Protection of pets Figure 2. Shown is a dogs ear with on the pinna attached adult ticks at several stages of engorgement. All three developmental stadia of Ixodes ricinus ticks may infest dogs. Deeper in the ear nymphal tick are attached, also engorged. The larval stage may also be found on dogs, especially with high numbers on the ears, when the dog has passed a recent hatched egg cluster. Repellent ointments used by man to fend off mosquitos and ticks do not work sufficiently for furred animals and should not be applied; plant products such as citronella and lemon eucalyptus oil are equally ineffective. Also the synthetic made DEET (N,N-diethyl-m-toluamide) should not be used and can even be toxic for dogs (Schoenig et al. 1999). The only substance having a repellent effect on several tick species is permethrin, and this too is not absolute. Permethrin is one of the synthetic pyrethroidic substances (natural product: pyrethrin, derived from the plant Tanacetum cinerariifolium) whose actions are enhanced with certain solvents like piperonyl butoxide. The liquid, contained per dose in small pipettes (for instance Pulvex spot on; MSD, New York, NY, USA), is applied topically onto the skin between the shoulder blades, so that the dog cannot lick the site if it itches. From the application site, the substance diffuses through the fat layer and exerts its acaricidal effect killing ticks evenly from nose to tail (Lussenhop et al. 2011). Other synthetic pyrethroids that are applied in acaricidal products are deltamethrin (also used in powders and shampoos) and flumethrin, usually used in combination with other products (see also Van Wieren et al. 2016). Protection by inhibiting or killing ticks shortly after attachment: acaricides Amitraz, a strong acaricidal, is a product widely used and applied onto the skin of farm animals (Van Wieren et al. 2016) but can have adverse effects when applied all over the skin of dogs. Also in small concentrations when combined with other products and topically applied it may show unwanted effects. More recently the acaricidal properties of substances like fipronil (a fenylpyrazolon) and imidacloprid (a nicotine derivate) have been utilised with no adverse effects. These substances can be combined with permethrin to enhance the bactericidal effects and are marketed as Frontect (Merial, Lyon, France), Effitix (Virbac, Carros, France) and Advantix (Bayer, Leverkusen, Germany), all of which have been shown to have almost 100% efficacy in experimental settings (Bonneau et al. 2015, Dumont et al. 2015, Otranto et al. 2005). Under natural conditions these combinations Ecology and prevention of Lyme borreliosis 415

8 K. Emil Hovius showed that they were able to prevent the transmission of arthropod transmissible infections in I. ricinus and other tick species (Jongejan et al. 2015, Otranto et al. 2008, 2010). Topical applications are also employed by way of impregnated collars which slowly release the acaricidals and have the advantage of a half year activity. Seresto collars (Bayer), a combination of imidacloprid and flumethrin, and Scalibor collars (MSD), impregnated with deltamethrin, both appear to have enduring action and are approximately 98% effective with one application providing months of protection (Brianti et al. 2010, Stanneck et al. 2012, Van den Bos and Curtis 2002). The effect of these collars in preventing the transmission of tick borne disease agents by killing the vector before transmission occurs, has been shown for E. canis and its vector R. sanguineus (Stanneck and Fourie 2013). Just recently Simparica (Zoetis, Madison, NJ, USA) and Bravecto (MSD) chewable tablets were licensed on the European market. These tablets are given to dogs every 2 months when R. sanguineus is the target or may be given every 3 months when the action is directed at Ixodes or Dermacentor (Williams et al. 2015). The pharmaceutical compounds Sarolaner and Fluranaler belong to isoxazoline organic chemicals. These compounds, as others, exert their action by the selective inhibition of arthropod GABA-gated chloride nervous system channels (Gassel et al. 2014). By blocking these channels the arthropod nervous system is inhibited which results in the death of the insect or tick (McTier et al. 2016). The vertebrate chloride nervous system channels are much less sensitive to these products and are safe for dogs up to several times the prescribed dose (Walther et al. 2014a). The product did not show any adverse effects when administered together with an anthelmintic or the deltamethrin collar (Walther et al. 2014b). The most advantageous property of these drugs is the speed of their action in killing ticks before transmission of disease agents occurs. When applied: after 2 days 100%, and after 12 weeks 98% of ticks are killed within 12 hours (Wengenmayer et al. 2014). Prevention of transmission of B. canis by D. reticulatus has also been shown (Taenzler et al. 2015). Protection by vaccination directed at killing attached ticks Theoretically, the quick killing of a tick could be accomplished by vaccinating with tick proteins functional in the salivary gland or the tick gut. For cattle the standard practice is vaccinating with the Bm86 gut lining protein of Rhipicephalus microplus, (Klouwens et al. 2016). A homolog of this protein from I. ricinus did not work in a laboratory setting (Coumou et al. 2014). In the future we may probably see vaccination of dogs with tick proteins. Protection by vaccination directed at killing Borrelia just before or after infection Antigenic shifts of Borrelia in the vector-host interface and the possibilities for vaccination In nature, Borrelia infection is established with help of tick salivary proteins (Ramamoorthi et al. 2005). The different salivary proteins exert different immunomodulatory actions, enabling the tick to survive the host s immune system (Hovius et al. 2008). Moreover, the Ixodes scapularis protein Salp 15, enables B. burgdorferi s.s. to surpass the immune system by cloaking outer surface protein C (Osp C) (Ramamoorthi et al. 2005). Osp C is thought to be essential for the transfer from the midgut to the salivary glands of the tick and subsequently to the vertebrate host (Pal et al. 2004b). If Salp 15 is inhibited experimentally or if anti-salp 15 has developed after some initial infections, the second blockade against infection becomes apparent when bactericidal antibodies against Osp C, destroy the spirochaete in a complement dependent process (Callister 1999). Osp 416 Ecology and prevention of Lyme borreliosis

9 28. Protection of pets C is expressed on the spirochaete outer membrane after the tick has attached to the host. The temperature and the first vertebrate blood ingestion induces a change of expression (switch) of the outer service proteins. The spirochaete reside in the tick gut connected with Outer Surface Proteins A (Osp A) to TRospA ligand protein (Pal et al. 2004a). A Vaccine directed at Osp A has the remarkable effect of preventing the switch from Osp A to Osp C by killing the spirochaetes in the gut of the tick and thus preventing entrance into the vertebrate host (Coughlin et al. 1995). Anti-Osp A induced by Osp A, that does not seem to have a function when in the vertebrate host, may be detected only in prolonged infection when it may prevent reinfections in the way described above. In some naturally infected dogs Osp A was detected (Gauthier and Mansfield 1999, Hovius et al. 2000). It may be hypothesised that this is an eventual reaction of the body against reinfections. The vaccination with Osp A then seems a natural phenomenon, artificially introduced earlier in the infective process. Upon vaccination with whole cell lysates of spirochaete (bacterins), the most prevalent antibody induced is anti-osp A (Barthold et al. 1995, Gauthier and Mansfield 1999). Other antibodies may be produced after several revaccinations but anti-osp A remains prevalent in high titers. Thus, vaccination with a bacterin induces the same protection after vaccination as with a (recombinant) Osp A vaccine. The high anti-osp A titre makes the differentiation of serum of vaccinated or naturally infected dogs possible (Gauthier and Mansfield 1999). Some recent experiments with vaccinated and naturally infected dogs yielded some re-evaluation on this clear differentiation. The time effect on the intensities of certain antibodies needs to be taken into account to make the distinction (Leschnik et al. 2010). Vaccination of puppies with Osp C proteins or with whole cell lysates containing Osp C induce high levels of bactericidal Osp C antibodies killing the spirochaetes upon entrance with the first infective tick bite (Callister 1999). Anti-Osp C in natural infection is detected as one of the first induced antibodies, which may be explained by the requirement of Osp C for entering the vertebrate host (see above). A dog survey in the Netherlands among naturally infected dogs revealed anti-osp C bactericidal antibodies against three different Borrelia species (Hovius et al. 2000). The implications of this finding for vaccine development need further investigation since the Osp C protein is very variable and regionally fixed (see below). History of vaccination in dogs The Osp A vaccine strategy was developed at first in human vaccination schemes (De Silva et al. 1996), but the manufacturer has withdrawn Lymerix (recombinant Osp A) from the market because it was not generally accepted by the public (Embers and Narasimhan 2013). Several vaccines for dogs have been marketed and vaccine development has recently expanded to a third stage with new vaccines employing different geno-species and antigens exerting more crossprotection (Table 2). The first vaccines contain whole cell lysates of the spirochaete, so-called bacterins, whilst the second stage contains recombinant Osp A and Osp C as dominant antigen. The newest recently developed third stage vaccines are: (1) bacterins composed of a broad array of cross-protective antigens where several Borrelia geno-species are employed together; and (2) recombinant Osp A combined with chimeric Osp C which combines the most essential epitopes in one molecule. These third stage vaccines have only been on the market a few months or years and no field trials are yet available. It is expected that more cross-protection will be gained by vaccinating with these new vaccines. Ecology and prevention of Lyme borreliosis 417

10 K. Emil Hovius Table 2. Vaccines employed for protection of dogs against the Lyme disease Borrelia. First stage vaccines (1) have been employed for decades. The second stage (2) of development appeared somewhat later on the market. And the recent developed vaccines (3) were just licensed and the effect in the field with clinical trials have not yet been determined or published. The first and second stage contain only one strain of Borrelia burgdorferi s.s. (Bb s.s.). The last developed vaccines contain strains of several species in combination including Bb s.s., Borrelia garinii (Bg) and Borrelia afzelii (Ba) or only Bb s.s. with a combination of several Osp C serotypes in one so called chimeric molecule. The references describing experiments or clinical trials with the respective vaccines are discussed in the text. Name vaccin Company Fabrication B. species employed Specific antigens Most induced antibodies Used in Stage Reference lymevax Zoetis lysate and adjuvans Bb s.s. 2 strains mulitple antigens anti-ospa, anti-ospc America, Europe Duramun Lyme Boehringer lysate and adjuvans Bb s.s. multiple antigens anti-ospc America, Europe Merilym Merial lysate and adjuvans Bb s.s. multiple antigens anti-ospa, anti-ospc, anti-66kda Nobivac Lyme Recombitek Lyme America, Europe MSD 2 lysates, adjuvans Bb s.s. 2 strains OspA bivalent OspC anti-ospa, anti-ospc America, Europe 1 Chu et al. (1992), Gauthier and Mansfield (1999), Levy (2010), Levy et al. (1993, 2002), Topfer and Straubinger (2007) 1 Levy (2010) 1 Leschnik et al. (2010), Topfer and Straubinger (2007) 2 LaFleur et al. (2009, 2010) Merial recombinant Bb s.s. OspA anti-ospa America 2 Conlon et al. (2000), Eschner and Mugnai (2015), Levy et al. (2005), Topfer and Straubinger (2007) Biocan B Bioveta 2 lysates and adjuvans Bb s.s.; Bg OspA and multiple anti-flagellin (41 kda) anti 45 and 58 kda Europe 3.1 Topfer and Straubinger (2007) Merilym3 Merial 3 lysates and adjuvans Bb s.s.; Bg; Ba multiple antigens anti-ospa and others Europe 3.1 information by Merial Vanguard rc Zoetis recombinants Bb s.s. OspA chimeric OspC anti-ospa, anti-ospc America 3.2 Earnhart and Marconi(2007) Lyme 418 Ecology and prevention of Lyme borreliosis

11 28. Protection of pets First and second stage vaccines and the bacterin vaccine including B. garinii were comparatively investigated by Töpfer and Straubinger (2007) (Table 2). Ten seronegative dogs per vaccine, of all age groups and differing breeds, were vaccinated with the 5 vaccines using a double vaccination 3 weeks apart. For comparison, another 50 dogs were vaccinated with the same 5 vaccines 3 times at 2 and 3 week intervals and so received a double booster. The dogs, which were normal household dogs, were walked by their owners in Saxony, Germany and blood was taken for serological purposes every month. Titres of an automated whole cell Elisa reached their highest values for all dogs at 45 days after primo vaccination. But the 3 times vaccinated dogs peaked at about a 10% higher value. Thereafter titres more than halved in the course of one year and rose to the maximum level in the 20 days after revaccination on day 365. The gain of the second booster is the prolongation of higher titers during the one year decline to half the maximum value. Depending on which vaccine was used, the second booster resulted in between 0 to 30% difference in titre height. All the vaccines that were derived from B. burgdorferi s.s. performed with markedly lower titres when antibodies were tested against heterologous antigens (B. garinii and B. afzelii). Not so the European fabricated B burgdorferi s.s. and B garinii bacterin, which performed even better when tested with homologous B garinii. The differences between the heights of titers for the sera of dogs which had received one or two boosters became more pronounced in an ELISA that employs only Osp A as antigen. In Western blots of vaccinated dogs only antibodies against Osp A were apparent which made the distinction with the infected dogs that developed a whole array of antibodies against different protein weights in the Western blots at kda 39, 41 (flagellin), and 23 (Osp C). The Osp A antibody in vaccinated dogs is bactericidal in contrast with the serum of experimentally infected dogs without Osp A and will protect dogs from infection and disease in an experimental setting (Chang et al. 1995, Straubinger et al. 1995). Of the 100 vaccinated dogs only one showed signs of infection which means that, in 99% of dogs, infection was prevented by the bactericidal action of the vaccine induced antibodies. The same investigation with one of the vaccines (Merilyme-Merial) was performed in Austria and in this research all the vaccinated dogs remained uninfected while unvaccinated dogs became infected (Leschnik et al. 2010). Borrelia detected in the ticks infecting the dogs were B. gariniii and B. afzelii, not B. burgdorferi s.s., which is the species used in the vaccine, thus good cross protection was achieved. This cross protection was not foreseen from the lowered heterologous antibody titres in the trial performed by Töpfer and Straubinger (2007) (see above). In the Austrian trial more bands were in seen in Western blots of vaccinated dogs such as strong reacting Osp C, and even the p41flagellin B. garinii band reacted strongly which explains the apparent cross protection. In the USA, Recombitek Osp A by Merial is able to protect 12 week old, mixed breed dogs in a laboratory setting. Vaccinated dogs had negative skin biopsies and ticks could not acquire spirochaetes from them. In contrast to the tick infected non vaccinated dogs that had positive skin biopsies and could transmit the spirochaetes to ticks (xenodiagnoses) (Conlon et al. 2000). Interestingly the efficacy of the same Osp A vaccine Recombitek Lyme by Merial could be evaluated in a veterinary practice In Maine USA, where a thorough registration was kept of the 6,202 dogs which were vaccinated over a five year period. The 2 booster protocol recommended by Töpfer and Straubinger (2007) was adopted by the practice; the dogs received an initial injection, a booster after 3 weeks and a second booster after 6 months and subsequent vaccinations were then given annually. Of the 4,551 dogs which fully complied with the protocol, 1.01% became infected in the five year period apparently by being bitten naturally while walking with their owners. Surprisingly, of the 1,294 dogs that did not fully comply with the protocol, some 21.6% became infected during the 5 year period. Infection was measured as a positive reaction in the 4DX SNAP Ecology and prevention of Lyme borreliosis 419

12 K. Emil Hovius test (Idexx) measuring the C6 antibody. C6 is the synthetic counterpart of the IR6 antigen used in the SNAP test that is derived from the conserved part of the variable VlsE protein that changes antigenatically during the course of an active infection (Philipp et al. 2001). The C6 antibody is never induced by vaccination (Liang et al. 2000). The preventable fraction was calculated on a yearly basis and in the last study year (2013) was 98.08% which meant that a non-compliant dog was 52 times more likely to become infected than a fully compliant dog (Eschner and Mugnai 2015). In accordance, a smaller trial employing 60 dogs vaccinated with a single booster resulted in 25% infections (Levy et al. 2005). The vaccine by MSD, Nobivac Lyme, deliberately employs special strains one expressing Osp A combined with a second one which only expresses Osp C containing an epitope that is recognised by B. afzelii and B. garinii (LaFleur et al. 2009). The vaccine prevented Lyme disease development in laboratory dogs for a duration of one year but did not entirely prevent early infection in these dogs (LaFleur et al. 2010). The addition of the second strain not expressing Osp A but only Osp C has recently been shown to induce high bactericidal activity. But used singly (without the Osp A strain included) the vaccine reduced infectivity but in itself was not able to prevent establishment of spirochaetes in the joints of vaccinated laboratory dogs (LaFleur et al. 2015). This may be explained by the extreme variability of the Osp C strains in Lyme spirochaetes (Seinost et al. 1999). The Osp A sero-types, formerly determined by Wilske, broadly coincide with the now recognised Borrelia species (Table 1) (Wilske et al. 1996). Each of the Osp A sero-types includes several Osp C serotypes (Wilske et al. 1995). Broadly around 30 of these Osp C serotypes exist which appear to be geographically located and to have developed by recombination and not by selective pressure on mutations (Earnhart and Marconi 2007). The Osp C type specific epitopes induce bactericidal antibodies that do not cross-react with the other Osp C types. Importantly, Osp C types that are found in dogs are different from the ones found in man, which has to be accounted for when constructing vaccines for dogs (Rhodes et al. 2013). Recently such a third stage vaccine was licensed in the USA (VanguardcrLyme by Zoetis) based on the B. burgdorferi s.s. serotype with one recombinant Osp A protein combined with one chimeric Osp C protein including 7 type epitopes most commonly found in dogs. The first vaccine marketed, under the name of LymeVax and now distributed by Zooetis has been described by Chu, tested under experimental conditions and its cross protection for American strains of B. burgdorferi s.s. determined (Chu et al. 1992). In its current form, it contains two American Borrelia strains, which induces anti Osp A and anti Osp C antibodies. The LymeVax vaccine is widely used in the USA and the trials were performed under clinical conditions in veterinary practices in the USA where large numbers of vaccinated dogs (up to thousands of dogs) were compared with non-vaccinated dogs. Tens of millions of dogs have been vaccinated giving ample opportunity for the efficacy and safety of the vaccines marketed to be studied. Under field conditions the preventable fraction (or disease) was higher (88%) when seronegative dogs were vaccinated compared to seropositive vaccinated dogs (58%). It is important to test for antibodies in serum prior to vaccination since, in contrast to the experimental studies, vaccinated dogs have also been diagnosed as having Lyme disease (1% against 4.7% of non-vaccinated dogs) (Levy et al. 1993). The use of an in House Snap test determining C6 antibodies can easily identify dogs that have been infected before vaccination (Levy et al. 2002), as antibodies against the C6 antigen are not produced as a reaction to vaccination (Liang et al. 2000). On the other hand a reaction to the outer surface proteins Osp A and Osp C has been noted after vaccination with this vaccine (Levy 2010). 420 Ecology and prevention of Lyme borreliosis

13 28. Protection of pets Protection by clearance after infection, use of antibiotics Antibiotics have been used therapeutically in animals with Lyme disease after which improvement of the condition can be noted and antibody decline in serum is monitored usually with the quantitated C6 ELISA to confirm clearance of the spirochaete (Philipp et al. 2001). The argument as to whether antibiotics could be used in (heavily) tick infested humans to clear the spirochaetes before dissemination is disputed (Nadelman et al. 2001, Shapiro 2001). It is not recommended for dogs as new ticks can infest a few days later. However, in veterinary practice, antibiotics are easily administered to treat a fever of unknown origin which may have stopped further dissemination and in some cases may have prevented disease (Wagner et al. 2015). The tighter regulations governing antibiotic prescriptions will stimulate research on the diagnostic aspects of fevers of an unknown origin in general and Lyme borreliosis in particular. And at the same time, restricting antibiotic application, is an encouragement for the alternative employment of protective measures. Public health significance The dog may function as a sentinel for its owners, warning them for the risk of tick bite. Owners may check their dogs on a daily basis and when ticks are detected the chance of having acquired ticks themselves should not be neglected. In this way, owners will also gain knowledge about the whereabouts of tick habitats and may avoid these places. Ticks may be introduced to their owners gardens raising the risk for tick bite close at home. The dog appears to be a good animal model to study Lyme disease. It responds comparably as the human patient to an infection of B. burgdorferi sensu lato. Re-infection and recurrence of disease may be more easily observed in the dog than in man because of the relatively shorter live span. Some breeds may be more prone to the disease revealing a putative genetic predisposition, when further studied may reveal genetically coupled pathogenic mechanisms. Experiences with the different vaccines employed for dogs may pave the way to similar vaccines for man. Veterinarians as well as physicians should adapt the one-health way of thinking and communicate structurally, directed and stimulated by governmental facilities, for maximum benefit of the knowledge gained in dog studies. Public health relevance The susceptibility for the different Borrelia species is similar, which provides the opportunity in comparing human and canine clinics and diagnosis. Lifetime clinically monitoring of dogs and breed predilections provide insight in age and genetic related pathogenic mechanisms. The accelerating vaccine development for dogs, and the experience gained in the application, will provide valuable knowledge for human vaccine development. Ecology and prevention of Lyme borreliosis 421

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