Michael J. Burridge

INTRODUCTION OF POTENTIAL HEARTWATER VECTORS AND OTHER EXOTIC TICKS INTO FLORIDA ON IMPORTED REPTILES

Following the discovery of establishment of the African tortoise tick Amblyomma marmoreum in Florida, the present study was undertaken to determine the extent of introduction of exotic ticks into Florida on imported reptiles. Exotic ticks were identified on 29 (91%) of 32 reptile premises in 18 counties of Florida. The ticks, found on a variety of imported tortoises, snakes, and monitor lizards, belonged to 4 Amblyomma species (A. marmoreum, Amblyomma nuttalli, Amblyomma sabanerae, and Amblyomma sparsum) and 4 Aponomma species (Aponomma exornatum, Aponomma flavomaculatum, Aponomma latum, and Aponomma varanensis). The most commonly encountered ticks were A. latum and A. marmoreum. The identifications of A. marmoreum on 8 premises in 7 counties, and of A. sparsum on 1 premises, are of great concern because both species are vectors of heartwater, a lethal disease of cattle, sheep, goats, and deer.

Heartwater in the Caribbean

Abstract Cowdria ruminantium , causal rickettsia of heartwater in ruminants, was until recently only known to occur in Africa. it is transmitted by Amblyomma ticks. Amblyomma variegatum , one of its African vectors, was introduced into the Caribbean area with cattle from Senegal about 1830. Before 1900 only Guadeloupe and Antigua were known to be infested. Martinique was invaded as late as 1948. The spread of the tick accelerated afterwards, especially during the last decade. It has now been found on Guadeloupe (Grande Terre and Basse Terre), Antigua, Martinique, St. Croix, Puerto Rico, St. Lucia, Nevis, St. Kitts, St. Maarten/St. Martin, Vieques, Anguilla, Marie-Galante and La Desirade. All of these islands are probably still infested today, except possibly St. Croix and La Desirade. The existence of heartwater on Guadeloupe has been suspected since 1966, and was at last confirmed in 1980, having almost certainly been present for a century and a half. A strain of C. ruminantium was isolated in 1982. Retrospectively, a bovine disease with symptoms like those of heartwater has been known on the island since 1932. Two other African infections of which A. variegatum is a vector, Theileria mutans and T. velifera , also occur in cattle on Guadeloupe. Heartwater has so far been diagnosed only in Guadeloupe and in near-by Marie-Galante. An investigation for its presence has been started on other islands where A.variegatum occurs. However, it has been shown that the American tick Amblyomma maculatum is a good experimental vector, and that A.cajennense can also transmit C.ruminantium , but probably not very efficiently. If the agent can adapt to these and other American Amblyomma species, it could spread to large areas in the western hemisphere, well outside those where the strictly tropical A.variegatum would find a suitable habitat. An assessment of the extent of this serious threat to the livestock industry of the Americas should be followed by urgent action while there is still hope of removing the danger as long as the disease remains limited to a few of the smaller islands. Apart from a survey of the distribution of the disease and Amblyomma ticks, information is needed on animal movements between islands, on migration routes of birds (hosts of immature stages), animal husbandry methods, and species and numbers of domestic animals, as well as wild hosts which may interfere with tick eradication. Severe dermatophilosis (cutaneous streptothricosis) of cattle appears to be closely linked with the presence of A.variegatum and causes high losses on the infested islands. This will make it easier to obtain the collaboration of livestock owners and authorities for eradication campaigns, even on islands where heartwater does not (yet) occur.

Immunisation of sheep against heartwater with inactivated Cowdria ruminantium

The immunisation of sheep with inactivated Cowdria ruminantium organisms (the causative agent of heartwater) emulsified in Freunds adjuvant induced protective immunity against a homologous challenge with virulent cell culture-derived C ruminantium organisms. This protective immunity was associated with the development of high titres of C ruminantium-specific antibodies, a low level of rickettsiosis in brain endothelium and no mortalities. In contrast, when unimmunised sheep were challenged, they developed higher levels of rickettsiosis in brain endothelium, and some of them died. This method of immunisation against heartwater has the potential to replace the infection and treatment method that is in current use.

Responses of the ticksAmblyomma hebraeum andA. variegatum to known or potential components of the aggregation-attachment pheromone. I. Long-range attraction

Ten known or potential components of the aggregation-attachment pheromone (AAP) of the ticksAmblyomma hebraeum andA. variegatum, as well as mixtures of these components, extracts of prefed males and live prefed males, were tested as aggregation stimulants. In field assays, laboratory-reared unfed male and female ticks were released 20 cm downwind of CO2/pheromone release sites; the numbers of ticks that aggregated at the release sites were recorded after 30 min. InA. variegatum, aggregation was induced by methyl salicylate,o-nitrophenol, 2,6-dichlorophenol, phenylacetaldehyde and some mixtures containing these compounds; a strong aggregation response was induced by an extract of five prefed malesA. variegatum and a weak response was induced by an extract of 50 prefed males ofA. hebraeum. InA. hebraeum, aggregation was induced by phenylacetaldehyde, mixtures of compounds that included phenylacetaldehyde, extracts of 50 prefed males ofA. hebraeum orA. variegatum and 50 live prefed males ofA. hebraeum. InA. variegatum, aggregation was inhibited if compounds that do not occur naturally in the AAP of the species were included in mixtures. InA. hebraeum, phenylacetaldehyde appeared to act as an arrestant for ticks that had been attracted to release sites by other compounds.

Exotic ticks introduced into the United States on imported reptiles from 1962 to 2001 and their potential roles in international dissemination of diseases

Since 1962, a total of 29 species of exotic ticks have been introduced into the United States on imported reptiles, with 17 species from the genus Amblyomma, 11 from the genus Aponomma and one from the genus Hyalomma. In the absence of measures to control introduction of these importations, some exotic tick species will develop breeding colonies and become established as indigenous species and some tickborne diseases may be introduced to wreak havoc among susceptible native populations. However, formulation of risk assessments and rational control measures have been hampered by a lack of knowledge of these exotic ticks, with much of the available data published in older and relatively obscure publications. This report is an attempt to collate information for all 29 exotic tick species, including previously unpublished data from our laboratory, with particular reference to their geographical distribution, hosts, life cycles and vector potential, and to review methods to minimize their global dissemination.

DNA vaccination with map1 gene followed by protein boost augments protection against challenge with Cowdria ruminantium, the agent of heartwater

A DNA vaccine encoding the immunodominant MAP1 protein of Cowdria ruminantium (Crystal Springs (CS) strain) was shown to partially protect DBA/2 mice against homologous lethal challenge. To enhance the protective capacity of this DNA vaccine, the effects of length of interval between vaccinations and of prime-boost regimes were investigated. Increasing the interval between vaccinations from 2 to 12 weeks did not result in better protection (P=0.900). However, boosting DNA vaccine-primed mice with recombinant MAP1 protein significantly augmented protection on homologous challenge in various trials from 13-27 to 53-67% (P<0.050). The augmented protection by the prime-boost regimen correlated with augmented T(H1) type immune responses that were induced by the DNA vaccine. These responses were characterized by production of IFN-gamma, IL-2 and anti-MAP1 antibodies of predominantly IgG2a isotype, and were critical for protection against C. ruminantium infection. Cytokine analyses were done at 48h after in vitro stimulation of splenocytes with C. ruminantium or control antigens. In contrast, splenocytes of DNA vector control mice produced no cytokines and these mice were fully susceptible to challenge. In addition, DBA/2 mice immunized with the recombinant MAP1 protein without DNA vaccine priming produced non-protective T(H2) type immune responses which were characterized by production of IL-4, IL-5, IL-10 and IgG1 anti-MAP1 antibodies. A second DNA vaccine containing map1 gene from the Mbizi strain of C. ruminantium also delivered by a prime-boost regime, conferred less protection against heterologous challenge. Hence, in developing DNA vaccines against heartwater that contain map1 gene, a prime-boost regimen should be adopted and gene sequence heterogeneity of field isolates should also be considered.

COMPARISON OF EFFICACY OF AMERICAN AND AFRICAN AMBLYOMMA TICKS AS VECTORS OF HEARTWATER (COWDRIA RUMINANTIUM) INFECTION BY MOLECULAR ANALYSES AND TRANSMISSION TRIALS

The ability of Amblyomma americanum, Amblyomma cajennense, Amblyomma maculatum, and Amblyomma variegatum to acquire and transmit Cowdria ruminantium infection was investigated. Uninfected nymphs were fed on clinically reacting C. ruminantium-infected sheep and then analyzed for infection by specific DNA detection assays and by tick transmission trials. By polymerase chain reaction (PCR), the mean infection prevalence of A. maculatum ticks (50.7%) was similar to that of A. variegatum, Elevage strain (43.5%; P = 0.83) and Petit Bourg strain (45.9%; P = 0.26) ticks. Though Amblyomma hebraeum were not tested by PCR, by DNA probe their infection prevalence was 94%. In contrast, A. americanum and A. cajennense ticks demonstrated very low susceptibility to C. ruminantium, and the prevalence of infection by PCR was approximately 1%. The higher susceptibility of A. maculatum and A. variegatum to C. ruminantium correlated with superior vector efficiency, depicted by similar prepatent periods and severity of disease transmissions to sheep. Amblyomma americanum and A. cajennense failed to transmit infection, confirming that low susceptibility to C. ruminantium correlates with the poor vector status of these species. These results highlight the importance of A. maculatum as a potential vector that is likely to play a major role in the establishment and maintenance of heartwater, if the disease were to be introduced to the U.S.A., Central, and South America.

Reduction in mortality from heartwater in cattle, sheep and goats exposed to field challenge using an inactivated vaccine

Inactivated vaccines for heartwater prepared with the commercially acceptable Montanide ISA 50 (ISA 50) adjuvant were field tested in Boer goats in Botswana, Angora goats in South Africa, and Merino sheep in Zambia and Zimbabwe. Two vaccines, one made using the Zimbabwean Mbizi isolate and the other using the respective local field isolate (Sunnyside in Botswana; Bathurst in South Africa; Lutale in Zambia), were tested at each site, except in Zimbabwe where only the Mbizi vaccine was tested. Compared with unvaccinated animals, the Mbizi vaccine significantly protected goats and sheep against field Amblyomma tick challenge in Botswana, Zambia and Zimbabwe (P = 0.018, 0.002 and 0.017, respectively), but failed to protect Angora goats in South Africa. However, in South Africa the vaccine prepared using the local field isolate Bathurst, induced significant protection (P=0.008). The vaccines containing the local isolates at all other sites were less protective than the Mbizi vaccine. The Mbizi inactivated vaccine also significantly protected 17 of 21 cattle (P = 0.05) against heartwater challenge from field ticks in Zimbabwe. Against the same challenge only 7 of 21 unvaccinated control cattle survived. This study demonstrates that heartwater is a major constraint to upgrading livestock in endemic areas, and caused an overall mortality of 77.6% in naive sheep and goats (97 of 125 died) and 67% in cattle (14 of 21 died). In contrast, the vaccine had a protective effect by reducing the overall mortality in sheep and goats to 54.3% (113 of 208 died) and to 19% in cattle (4 of 21 died).

Olfactory responses of adult Amblyomma hebraeum and A. variegatum (Acari : Ixodidae) to attractant chemicals in laboratory tests

Unfed adults of the African ticks,Amblyomma hebraeum Koch andA. variegatum (Fabricius), important vectors of human and animal diseases, were exposed to volatile compounds in an olfactometer in efforts to identify both tick-produced or synthetic chemicals capable of eliciting an attraction response. A formula, relative efficacy of attraction, was devised for comparison of responses between species and sexes to a particular test stimulus, or within a homogeneous population to different stimuli. Adults of both species responded strongly to known tick-pheromone constituents, nonanoic acid, methyl salicylate, 2.6-dichlorophenol and benzyl alcohol, as well as to a commercially produced antiseptic, TCP (Pfizer), and its major components, chlorinated and iodinated phenols. Benzaldehyde, a proposed tick-pheromone component, and heptadecane, not known from ticks, were markedly attractive to adults ofA. hebraeum but not to those ofA. variegatum. Males of the former species, but neither conspecific females not either sex of the latter species, responded significantly to salicylaldehyde (known from males of four species of ticks, includingA. variegatum).o-nitrophenol, a major component of the aggregation-attachment pheromone of males of bothA. variegatum andA hebraeum and a proven long-range attractant for them in the field, was only partially attractive to either species in the olfactometer. Neither species was attracted to 2-methylpropanoic acid, previously identified in volatile effluents form feeding maleA. hebraeum. It is concluded that these important disease vectors respond positively to a variety of volatile chemicals, which may conceivably be used to attract them to traps, animals or acaricides in efforts to control ticks or the diseases they transmit.

Efficacy of pheromone-acaricide-impregnated tail-tag decoys for controlling the bont tick,Amblyomma hebraeum (Acari: Ixodidae), on cattle in Zimbabwe

A large-scale field test using pheromone-acaricide-impregnated plastic tail-tag decoys demonstrated excellent efficacy of these devices for control of the bont tick,Amblyomma hebraeum, on cattle in Zimbabwe. The tail tags were impregnated with a mixture containingo-nitrophenol, methyl salicylate, 2,6-dichlorophenol and phenylacetaldehyde and one of three different acaricides (cyfluthrin, flumethrin or alphacypermethrin).o-Nitrophenol and methyl salicylate are components of theA. hebraeum attraction-aggregation-attachment pheromone, while 2,6-dichlorophenol and phenylacetaldehyde are proven attractants for this tick. Botho-nitrophenol and methyl salicylate were lost gradually from the tags over 12 and 14 week periods, respectively. In field trials, tick counts were compared between cattle that received tail tags either impregnated with pheromone mixture alone, cyfluthrin and pheromone mixture, flumethrin and pheromone mixture, alphacypermethrin and pheromone mixture or were left untreated. During the first 3 month trial period, control of adult bont ticks was 94.9% with cyfluthrin tail tags and 87.5% with flumethrin tail tags. In general, there was no significant difference in bont tick numbers on cattle without tags and those with tail tags containing pheromone only. When the trial was repeated for another 3 month period, control of bont ticks with tail tags containing cyfluthrin and flumethrin was 99.3 and 95.1%, respectively. However, control of bont ticks using alphacypermethrin was only 79.2%. Overall, retention of tail tags was excellent although some loss was encountered during the rainy season. In addition to controlling bont ticks, the tail tags provided moderate control of other tick species (Rhipicephalus evertsi evertsi, Rhipicephalus zambeziensis andHyalomma spp.) simultaneously infesting cattle in the trials.