A multi-epitope DNA vaccine co-administered with monophosphoryl lipid A adjuvant provides protection against tick transmitted Ehrlichia ruminantium in sheep.

Previously, a heartwater experimental DNA vaccine provided 100% protection following laboratory challenge with Ehrlichia ruminantium administered by needle but not against an E. ruminantium tick challenge in the field. A multi-epitope DNA vaccine incorporating both CD4+ and CD8+ cytotoxic T lymphocytes epitopes could provide a better alternative. In this study, we investigated the use of multi-epitope DNA vaccines against an E. ruminantium experimental tick challenge in sheep. The multi-epitope DNA vaccines were delivered via the intramuscular route and intradermal route using the gene gun in the presence of monophosphoryl lipid A (MPL) adjuvant, which was either applied topically to the gene gun inoculation site or co-administered with the vaccine via the intramuscular route. Initially two constructs namely, pSignal plus and pLamp were tested with MPL applied topically only and no protection was obtained in this formulation. However, when pLamp was co-administered with MPL via the intramuscular route in addition to topical application, its protective efficiency improved to protect 60% of the sheep against tick challenge. In this formulation, the vaccine induced enhanced activation of memory T cell responses both before and after challenge with variations amongst the different sheep possibly due to their different genetic backgrounds. In conclusion, this study showed that a heartwater multi-epitope DNA vaccine, co-administered with MPL adjuvant can protect sheep following a laboratory E. ruminantium tick challenge.

Survey of Ticks Collected from Tennessee Cattle and Their Pastures for Anaplasma and Ehrlichia Species.

Anaplasma marginale is the causative agent for bovine anaplasmosis (BA) and Ehrlichia ruminantium is the causative agent for heartwater, 2 devastating diseases of cattle. BA is common in the United States and frequently reported in western Tennessee cattle; however, cases of heartwater are not yet established in the continental United States. Because both pathogens are transmitted via the bites of infected ticks, the objective of this study was to survey cattle and pastures for ticks and for each pathogen. University of Tennessee AgResearch has 7 research and education centers (REC) located throughout the state at which they manage cattle. Ticks were collected from selected cattle (every fourth to sixth animal) and pastures (via dragging) associated with the herd from each REC during the summer of 2013. A total of 512 ticks were collected from cattle (n = 386) and pastures (n = 126) and were PCR-screened for Anaplasma and Ehrlichia using genus-specific primers. Collections consisted of 398 (77.7%) Amblyomma americanum, 84 (16.4%) Amblyomma maculatum, and 30 (5.9%) Dermacentor variabilis. Ticks were not recovered from pastures or cattle east of the Tennessee Plateau. The North American vectors for An. marginale and E. ruminantium were identified (D. variabilis and A. maculatum, respectively), but neither pathogen was recovered. A large proportion of ticks were collected from cattle and, of these, a majority were attached to their host (compared to questing on their host or engorged on the host). Four A. americanum were positive for Ehrlichia spp. (Ehrlichia ewingii, Ehrlichia chaffeensis, and Panola Mountain Ehrlichia), all in western Tennessee. With the identification of a few Ehrlichia infections in cattle-associated ticks and current A. marginale rates in Tennessee beef cattle nearing 11%, additional research is needed to establish baseline tick, Anaplasma, and Ehrlichia data for future management studies.

Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens.

Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen-pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence.

Prevalence of Ehrlichia ruminantium in adult Amblyomma variegatum collected from cattle in Cameroon.

Ehrlichia ruminantium, the etiologic agent of the economically important disease heartwater, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, particularly A. hebraeum and A. variegatum. Although serologic and microscopic evidence of the presence of heartwater have been reported in ruminants in Cameroon, knowledge of E. ruminantium infection in the tick vector, A. variegatum, is lacking. In order to determine the infectivity of A. variegatum ticks by E. ruminantium, we analysed 500 un-engorged A. variegatum ticks collected by hand-picking from predilection sites from 182 cattle [115 ticks from 82 cattle at Société de Développement et d'Exploitation des Productions Animales (SODEPA) Dumbo ranch (SDR) and 385 ticks from 100 cattle at the Upper Farms ranch (UFR)] by amplification of the open reading frame (ORF) 2 of the pCS20 region of E. ruminantium. PCR amplification of the 279 bp fragment of the pCS20 region detected E. ruminantium DNA in 142 (28.4 %) of the 500 ticks with a higher infection rate (47/115; 40.9 %) observed in ticks from SDR and 24.7 % (95/385) of ticks collected from cattle at UFR. Twenty five randomly selected PCR products were sequenced and results indicated that some of the isolates shared homology with one another and to sequences of E. ruminantium in the GenBank. This report represents the first molecular evidence of E. ruminantium infection in A. variegatum ticks in Cameroon and suggests possible exposure of cattle to this pathogen in our environment.

Seroprevalence of tick-borne diseases in communal cattle reared on sweet and sour rangelands in a semi-arid area of South Africa.

A survey was conducted to determine the seroprevalence of tick-borne diseases (TBD) in Nguni and non-descript cattle on the sweet and sour communal rangelands across seasons. Body condition scores, bodyweights, packed cell volume (PCV) and antibodies to B. bovis, B. bigemina, Ehrlichiaruminatium and Anaplasma marginale were determined seasonally in 144 cattle raised on communal rangelands from August 2007 to April 2008. Approximately 45% of the cattle were seropositive for B. bovis, 46% for B. bigemina and 26% for A. marginale. All animals were seronegative for E. ruminantium. Nguni cattle had lower (P<0.05) seroprevalence for A. marginale and B. bigemina in the cool-dry and hot-wet seasons. Cattle in the sweet rangeland had significantly lower seroprevalence of B. bovis and B. bigemina in all the seasons. Infection with B. bovis and B. bigemina negatively affected (P<0.05) bodyweight and body condition scores whilst B. bovis and A. marginale infections significantly affected PCV. The seroprevalence of TBD was lower in the Nguni breed. More work is required to understand the mechanisms of the adaptation and possible resistance of Nguni cattle to TBD.

Heartwater: an abridged historical account.

Heartwater was first recognised and recorded in South Africa as early as 1838. Since then the disease has been described from almost all the countries in Africa south of the Sahara, from certain islands around Africa and from a number of islands in the Caribbean. Most of the research on the disease, at least until fairly recently, was conducted in South Africa. Progress in research on the disease has been slow but a few important findings are highlighted in this paper.

In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures.

Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10% foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.

Amblyomma variegatum in cattle in Marie Galante, French Antilles: prevalence, control measures, and infection by Ehrlichia ruminantium.

We report Marie Galante as one of the Caribbean islands most heavily infested by the tropical bont tick (TBT) Amblyomma variegatum which is associated with two major diseases of ruminants: heartwater and dermatophilosis. In 2005, a survey was undertaken to assess the prevalence of TBT infestation in cattle, the prevalence of Ehrlichia ruminantium infection in TBTs, and the tick control measures implemented by livestock owners. A random sample of 195 cattle herds out of 1885 recorded on the island was investigated by thoroughly counting adult ticks on each animal and filling a questionnaire. A randomly collected sample of 136 TBTs was tested for infection by E. ruminantium by pCS20 nested PCR. Cattle herd prevalence (hp) was 73.8% for infestation by at least one TBT, 17.9% for infestation by at least one engorged female TBT, and 8.2% for clinical dermatophilosis. Cattle individual prevalence was 42.3% for infestation by at least one TBT, 6.6% for infestation by at least one engorged female TBT, and 2.2% for clinical dermatophilosis. The minimum, maximum and average numbers of TBTs per infested animal were, respectively 1, 108 and 11.5. Prevalence of TBT infection by E. ruminantium was 19.1%. No significant difference in herd prevalence was found among parishes or among ecological zones. For cattle owners treating against ticks (97.9% of all owners), all used aspersion of amitraz and herd prevalence was significantly different among those treating every 1-2-week (hp=69.6%, n=148), and less often than every 2-week (hp=88.6%, n=35) (P=0.031). Of the 42 herd subunits treated less than 4 days before the survey, 27 (64%) were infested with at least one TBT, and 6 (14%) with at least one engorged female TBT. These results indicate a high level of TBT infestation in Marie Galante, the inefficacy of tick treatments currently performed, and the need for an improved tick control strategy. Persisting high levels of infestation in Marie Galante threaten the success of on-going TBT eradication programs in the Caribbean because TBT can spread through migrating birds and trade of animals or of animal hides to other islands and potentially the American continent.

Climate changes and suitability for the ticks Amblyomma hebraeum and Amblyomma variegatum (Ixodidae) in Zimbabwe (1974-1999).

The spread of Amblyomma hebraeum has been reported in Zimbabwe. At the same time there was little or no spread in the distribution of Amblyomma variegatum. This paper examines the climatic cycles and their trends in the period 1974-1999 with a view to explaining the abiotic causes of this spread, and of forecasting the likely tendency in climate suitability for both tick species. An annual data-set of rainfall and air temperature was used as a source for climate, together with a habitat-modeling algorithm to estimate climate suitability for both ticks. Long-term suitable habitat for A. hebraeum exists mainly in the south and southeast of the country. Areas of adequate habitat for A. variegatum exist across the country, between approximately 17 degrees S and 18.5 degrees S, and are most suitable in regions of the northwest. The climate niches of the two species differ, and account for their almost allopatric distributions, as observed in the duration and intensity of the dry period and in total annual rainfall. Cyclic changes in both temperature and rainfall drive the periodic modifications in the distributions of the ticks. More intense periods of drought in the highveld, drive the expansion of A. hebraeum in this region. Temperature does not have any effect on the tendency in this area. Areas in south and southeast show a trend towards an increase in climatic suitability because of an increase in temperature. Zones in which habitat suitability is increasing for A. variegatum are restricted to the northwestern parts of the country, because warmer temperatures and a slight decrease in the intensity of the dry season. The progressive increase in temperatures seems to be forcing the dispersion of A. variegatum towards areas outside of zones that have a prolonged dry period. On the other hand A. hebraeum is compelled to spread northwards, following areas with adequate rainfall patterns, but halted by temperature limits and perhaps competition with A. variegatum. Without adequate control measures, invasive waves of A. hebraeum may occur over a background swell of northward expansion.

Science and popular participation in the investigation of heartwater in South Africa, c. 1870-1950.

During the late nineteenth century, settler farmers in southern Africa identified heartwater as a damaging disease of small stock and cattle. They advanced various explanations of the disease, including the theory that it was caused by the bite of ticks. Around 1900, the American entomologist C.P. Lousbury demonstrated that heartwater was transmitted by the bont tick. He also worked out the life cycle and life habits of the tick. Subsequently, farmers developed methods of controlling ticks by dipping animals in solutions of arsenic. By 1910, the practice of dipping cattle had become very widespread over much of southern Africa. The expansion of the practice was greatly stimulated by the coming of the deadly tick-borne disease, East Coast fever. At this time, veterinary scientists attempted to develop a vaccine against heartwater, but with little success. Little further progress was made until the 1920s, when the American scientist E.V. Cowdry identified a causal agent, Rickettsia ruminantium, while on a research secondment to South Africa. By the 1940s, South African veterinary scientists had devised methods of immunising stock against heartwater, but there remained considerable technical difficulties and their use remained limited. Dipping in arsenic solutions to attack the tick on the animal thus remained the most important means of controlling disease in the first half of the twentieth century.

Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA.

BACKGROUND: The epidemiology of E. ruminantium infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia. METHODS: We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting E. ruminantium infection was also assessed. RESULTS: The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable A. variegatum infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher E. ruminantium prevalence in the animals with increasing age and both the Spearman's rank test (rs = -0.1512; P = 0.003) and kappa statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays. CONCLUSION: The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.

Molecular detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in The Gambia.

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3-15.1%) than in the easterly divisions (Central River and Upper River; range 1.6-7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.

Differential transcription of the major antigenic protein 1 multigene family of Ehrlichia ruminantium in Amblyomma variegatum ticks.

The rickettsial pathogen Ehrlichia ruminantium causes heartwater in ruminants and is transmitted by ticks of the genus Amblyomma. The map1 gene, encoding the major surface protein MAP1, is a member of a multigene family containing 16 paralogs. In order to investigate differential transcription of genes of the map1 multigene family in vivo in unfed and feeding ticks, RNA was extracted from midguts and salivary glands of E. ruminantium-infected adult female Amblyomma variegatum ticks and analysed by RT-PCR using MAP1 paralog-specific primers. In unfed ticks, only transcripts from the map1-1 gene were observed in midguts and no transcripts were detected in salivary glands. In feeding ticks, map1-1 transcripts were more abundant in midguts whereas high levels of map1 transcripts were observed in salivary glands. Our results show that differential transcription of genes of the E. ruminantium map1 cluster occurs in vivo in different tissues of infected ticks before and during transmission feeding, indicating that this multigene family may be involved in functions of biological relevance in different stages of the life cycle of E. ruminantium.

Understanding the mechanisms of transmission of Ehrlichia ruminantium and its influence on the structure of pathogen populations in the field.

The understanding of the structure of Ehrlichia ruminantium stock population in the field was highlighted by experiments done in controlled conditions on the goat model. The mixture of strains observed in ticks seemed to be due to simultaneous infections rather than successive infections of the carrier. During a dual infection, the timing of Ehrlichia ruminantium circulation of the two stocks in hosts influenced their selection by ticks.

Dedication: Conrad Yunker.

This volume of the proceedings of STVM-03 is dedicated to Dr. Connie Yunker for his many contributions to tropical veterinary medicine and for being a good colleague and friend.

Characterization of the pCS20 region of different Ehrlichia ruminantium isolates.

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.

Increasing risks of introduction of heartwater onto the American mainland associated with animal movements.

Opportunities to introduce heartwater onto the American mainland through animal movements include importation from Africa of tick-infested reptiles and of subclinically infected wild ungulates and importation of livestock from islands in the Caribbean infested with Amblyomma variegatum ticks. Measures to control importation of heartwater vectors on reptiles include importation bans of infested species, treatment of imported reptiles, and eradication of established infestations on the American mainland. Measures to control importation of infected wildlife must focus on improved methods, such as the PCR assay, of screening animals to prevent the entry of carriers of Cowdria ruminantium. Measures to control importation of infected animals from the Caribbean must be based on knowledge of the islands that are infected with C. ruminantium so that the risk of dissemination of heartwater can be established.

Control and eradication of chelonian tick infestations, with particular reference to vectors of heartwater.

Studies using the African tortoise tick (Amblyomma marmoreum) and leopard tortoises (Geochelone pardalis) demonstrated that cyfluthrin and permethrin were safe and efficacious acaricides for control of Amblyomma ticks on tortoises. A protocol was developed that successfully eradicated an A. sparsum infestation from a tortoise breeding facility in Florida. It involved treatment of all tortoises with a permethrin formulation, followed by treatment of the premises with a cyfluthrin formulation. Sentinel tortoises were later placed on the treated premises to establish successful tick eradication.