Journal of Vector Ecology 224. Vol. 31, no. 2

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1 Journal of Vector Ecology 224 Factors influencing the distribution of questing ticks and the prevalence estimation of T. parva infection in brown ear ticks in the Tanga region, Tanzania E.S. Swai 1, E.D. Karimuribo 2, E.A. Rugaimukamu 2, and D.M. Kambarage 2 1 Veterinary Investigation Centre, P.O. Box 168, Arusha, Tanzania 2 Faculty of Veterinary Medicine, Sokoine University of Agriculture, P.O.Box 21, Chuo Kikuu, Morogoro, Tanzania Received 23 January 26; Accepted 21 March 26 ABSTRACT: Questing ticks from various districts and agro-ecological zones (AEZ) in the Tanga Region of Tanzania were studied for a two-year period between September 1999 and July 21. Collections of both nymphal and adult ticks occurred at 29 sites using a blanket or white cloth dragging technique. The species recorded in the order of decreasing abundance were Rhipicephalus appendiculatus, Boophilus spp., and Ambylomma variegatum. Rhipicephalus appendiculatus field infestation levels varied across administrative districts and seasons, with Amani and the cool-to-dry season having lower tick counts ( = -2.9, SE =.71, P <.1 and = -1.54, SE =.56, P =.6 for Amani and cool to dry season, respectively). Based on the polymerase chain reaction technique, Theileria parva infection prevalence in adult R. appendiculatus was estimated to be 2.6%. Implications of these findings in light of the spatial and temporal distribution of ticks are discussed for the accurate diagnosis of multiple tick-borne diseases, the infected vector tick, the endemic status of T. parva in the region, and the implemention of control strategies. Journal of Vector Ecology 31 (2): Keyword Index: Questing ticks, brown ear ticks, seasonal distribution, Theileria parva-prevalence estimation, Tanzania. INTRODUCTION Studies of the seasonal occurrence of different development stages of ticks are of great significance in the epidemiology of tick-borne diseases and the planning of appropriate tick control strategies (Norval et al. 1992). Knowledge of tick numbers in pastures provides useful information on tick population dynamics, dynamics of disease transmission, and estimates of the resistance of different hosts (Norval et al. 1992). Tick abundances are known to vary with time (season to season and year to year) and space (between habitats and ecological zones) due to interactions of numerous factors, such as host diversity and climate (Norval and Lightfoot 1982), the levels of resistance of hosts, absence of control measures, and management practices that affect host behavior (Punyua and Hassan 1992). Generally, the seasonal activities of ticks are known to vary from species to species and country to country (Dipeolu 1989) due to variation in photoperiod (Rechev 1982, Dipeolu 1989), which therefore necessitates their study in various countries and different agro-ecological zones. The accurate estimation of the prevalence of Theileria parva infection in host-seeking ticks would allow an estimation of contact rates of vertebrate hosts with infected ticks and of transmission coefficients between ticks and hosts. In freshly field-collected infected questing ticks the numbers of sporozoites in the salivary glands are frequently low (Moll et al. 1986), and to detect infection the ticks must first be allowed to feed on laboratory animals to stimulate sporozoite multiplication in the tick salivary glands (Young and Leitch 1982). Structured studies aimed at estimating parasite infection rates in field-questing ticks (host-seeking), identifying and characterization of factors influencing vector tick counts, and distribution in cattle grazing areas in the Tanga region of the Tanzania remain largely unknown to date. The objectives of this study were three-fold: first, we explored the information on the relative abundance and distribution of ticks serving as vectors of major tick-borne diseases (TBDs). Second, we characterized the relationship between questing tick distribution and geographical/ ecological factors. Third, we employed a PCR that specifically targets the multi-copy TPR gene of T. parva (Bishop et al. 1992, Watt et al. 1997) to detect infection in host-seeking R. appendiculatus ticks in Tanga, Tanzania. Such information on the ecological relationship between questing ticks and T. parva infection rates is needed to build tick population models and to devise tick control strategies for the prevention and treatment of debilitating tick-borne diseases such as East Coast Fever (ECF). MATERIALS AND METHODS Study sites The study was conducted on 29 open communal grazing areas in the Tanga region located between latitude 4 o 21 and 6 o 14 S, and longitude 36 o 11 and 38 o 26 E, in northeast Tanzania. The study area included the humid coastal, hinterland, and highland zones of the region at an altitude of between 2 and 2, m above sea level. Characteristics of the region are described in detail by Swai et al. (25). The region included eight administrative districts (Tanga, Muheza, Pangani, Lushoto, Handeni, Kilindi, Mkinga, and

2 225 Journal of Vector Ecology December 26 Korogwe) and the land is classified into five agro-ecological zones (AEZs) based on elevation, soil type, rainfall amount and pattern, water retention ability, and crops grown (National Coconut Development Council, NCDC 1981). For logistical reasons, the study was not conducted in Handeni, Kilindi, and Mkinga districts. Seasons were classified as rainy (April to June), cool to dry (July to October), or hot dry (November to March). Time of collection was classified as morning (8: to 1: h), afternoon (12: to 14: h), and evening (16: to 18: h). Tick collections The study was carried out between September 1999 and July 21. Study sites were visited every eight weeks when questing ticks were collected using blanket dragging techniques as described by Short and Norval (1981a). Collection usually started at 8: h and ended between 14: and 18: h and was conducted in transect walks in each study site. Ticks were morphologically identified on site (at the time of collection) as described by Hoogstraal (1979) and Horak et al. (22). Identified ticks were counted and recorded by date of collection, site number, district of origin, agro-ecological zone, time of collection, tick species, and sex. Transportation and storage of questing ticks After collection, the ticks were placed in 1 ml universal bottles with cotton wool dampened with sterile water. The ticks were dispatched to the laboratory by placing the bottles in a cool box maintained at approximately o C by ice packs to prevent death of the ticks and bacterial spoilage. Once at the laboratory, questing ticks were stored at 2 o C before further processing. Determination of Theileria parva infection prevalence in adult R. appendiculatus The DNA of questing brown ear ticks (R. appendiculatus) was extracted by Proteinase-K treatment of macerated ticks followed by phenol/chloroform treatment and ethanol precipitation (Maniatis et al. 199). DNA was pelleted, dried, and resuspended in 2 µl of sterile double distilled water. Table 1. Total ticks counted and distribution of the tick collection sites by district, September, July 21. Zone/ district Number Average number No. of ticks of sites of times each collected visited Amani* Korogwe Lushoto Muheza* Maramba** Pangani Tanga Total *Amani represents an Upland zone of Muheza district. **Maramba represents hinterland zone of Muheza district. DNA samples were then subjected to PCR as previously described (Watt et al. 1997) although the reactions volume used was 25 µl. Reaction mixtures comprised a half unit of Taq polymerase (AB, Epsom), 5mM KCl, 1mM Tris-HCl (ph 8.3), 1.5mM MgCl 2,.1mM of each dntp, 4pmol of each of the primers IL194 and IL197 (Bishop et al. 1992), and 5 µl template DNA and sterile double distilled water to a final volume of 25 µl. PCR conditions comprised cycles of 1-min denaturation at 94 o C, 1-min annealing at 65 o C, and 1-min extension at 72 o C. Four hundred and five base pair products were visualized in ethidium bromide stained agarose gels under ultraviolet (UV) light following electrophoresis. Statistical analysis Distribution of non-brown ear ticks (ticks other than R. appendiculatus) was highly aggregated. For this reason, only counts of R. appendiculatus were included in the statistical analysis. The presence of a brown ear ticks abundance was investigated as a binary variable (i.e., defined as found or not found) in logistic regression models using Egret for Windows (version 2., Seattle, WA). In these models, site ID was included as a random effect to account for clustering of ticks by site and repeated sampling of ticks (Diggle et al. 1994). Explanatory variables investigated were site district, season, time of collection, and AEZ. Substitution and elimination of these variables was performed to produce the most parsimonious multivariable model in which no variables could be removed without significantly altering model deviance. Two-way interaction between selected explanatory variables and with the fixed effect of level of site district was assessed and the relationship explored. The level of significance was set as P <.5. RESULTS Tick populations Three species of ixodid ticks were found in varying numbers from the study sites. The mean number of eight visits per site occurred over the whole period of study (Table 1). A total of 868 adult ticks were collected during 9,28 transects. These were, in order of abundance, Rhipicephalus appendiculatus (826), Boophilus spp (28), and Ambylomma varieagatum (14) (Figure 1). R. appendiculatus field infestation levels varied across farm districts and seasons. (Figures 1 and 2). Results of the final regression models are shown in Table 2. R. appendiculatus was significantly less abundant in Amani zone compared to other zones ( = -2.9, SE =.71, P <.1). Of the three seasons covered, cool to dry (July to October) had the lowest tick counts ( = -1.54, SE =.56, P =.61). Of the brown ear ticks collected, 7% (578/826) were males and % (248/826) were females. Time of sampling and agro-ecological zone had no detectable variation on the number of tick counts (P >.5) (Figures 2 and 3, Table 2). Time of sampling and site district interaction did not reveal any variation in the number of tick counts (P >.5).

3 Journal of Vector Ecology Frequency (%) Figure 1. Frequency distribution of questing ticks by districts/zone, September 1999 to July 21 (n = 186 number of observations). Amani Korogwe Lushoto Maramba Pangani Tanga Muheza District/ Zone % all tick spp % brown ear tick Frequency (%) Figure 2. Frequency distribution of questing ticks by AEZ, September 1999 to August 21 (n = 186 number of observations). V V1 V11 X11 Agro-ecological zone %all tick spp % brown ear tick % Figure 3. Time of collection of questing ticks (n = 186 number of observations). Afternoon Evening Morning Time of the day

4 227 Journal of Vector Ecology December 26 Table 2. Factors found to be significantly associated with the likelihood of picking one or more adult R. appendiculatus ticks during tick sampling in minimal multivariable logistic regression models. Factor Coefficient SE Wald P value Minimal multivariable model Zone: Korogwe* vs. Amani Lushoto Muheza Pangani Tanga Time of collection: Morning* vs. Afternoon Evening Season: Rain season* vs. Cool to dry season Hot dry season Constant * Reference variable. T. parva infection in ticks Twenty-two of 826 questing adult R. appendiculatus (2.7%; 95% CI= ) were PCR positive producing 45 bp products consistent with T. parva infection. No PCR products greater than 45bp in length were detected. DISCUSSION Likelihood ratio P value < The ecology of R. appendiculatus has been studied extensively in the past and most aspects of their biology have been described in detail including host preference, resistance to desiccation, and disease transmission (Norval % Rain season Cool to dry Hot dry season season Figure 4. Seasonal variation in the abundance of R. appendiculatus in the study area (29 sites were sampled, 4 transects per site, each transect 1 paces -1 paces x 4 transects x 29 x 8) between September 1999 to July 21. et al. 1992, Short and Norval 1981a, Short and Norval 1981b). Consistent with other studies (Ndamukong 1993, Mulei et al. 1989), large numbers of ticks, mainly R. appendiculatus, were observed during the rainy to hot dry seasons. The amount of rainfall is the principal stimulus to R. appendiculatus activity (Yeoman 1966). Indeed, the behavior of active unfed ticks (questing) has been reported to be affected by many external factors of the environment and the physiological state of the ticks (Punyua et al. 1991). It has been reported that season alone is not a determinant source of variation for R. appendiculatus activity, but hydration status of the tick is considered to be the most important determinant factor (Punyua et al. 1991). Broad indices of AEZ were poorly associated with tick distribution as revealed in this study. The geographical and administrative location of the tick site was the principal confounder. The low number of tick counts in Amani could be related to the low number of traditional cattle stock, often thought to be a reservoir of ticks (personal observation). In light of this observation, traditional local grazing cattle may be the main source of ticks in these areas. The infection prevalence in field-collected questing adult R. appendiculatus was low but comparable to that found in other studies that used salivary gland staining to detect infection in questing ticks collected from endemically stable foci of T. parva infection (Walker et al. 1981, Moll et al. 1986). It is likely that most infections in the fieldcollected ticks in our study would have been acquired from traditionally managed pastoral cattle (with some from grazed smallholder dairy cattle) because wild reservoirs are thought to be absent from almost all the study sites in the Tanga region, and because most smallholder dairy cattle in the region are zero-grazed (Food and Agriculture Organization 1997). The low prevalence of infection in the ticks in the present study would be consistent with a state of endemic stability of T. parva that serological surveys suggest to occur in the grazed cattle in the Tanga region (Norval et al. 1992, Food and Agriculture Organization 1997). Under conditions of endemic stability, most infected ticks would have acquired their infections from carrier animals and hostto-tick transmission coefficients are thought to be low from carrier cattle (Medley et al. 1993). Grazed goats and sheep that occur in the region are not competent reservoirs of T. parva (Norval et al. 1992) but are likely to have carried a proportion of nymphal R. appendiculatus, thus diluting the infection prevalence in questing adult ticks arising from traditionally managed cattle. Absence of PCR products greater than 45bp (which corresponds to Theileria taurotragi) in length suggests that T. taurotragi is not likely to be prevalent in the study area within the Tanga region (Watt et al. 1997). Acknowledgments We thank the government of the UK through the Department for International Development (DFID/NRRD) Animal Health Research Programme for financial support of this work. Laboratory technical assistance at the Sokoine

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