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| Figure 1. Comparison of T. parva antibody responses (PP) under two tick control regimes |
| Livestock Research for Rural Development 27 (6) 2015 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
A cross-sectional study of T. parva antibody and parasite prevalence was conducted on smallholder farms in Tanzania to compare the impact of two tick control regimes. A total of 182 cattle were sampled, whereby 104 were under constant exposure to ticks with minimal or sporadic tick control and 78 animals were kept in a regular weekly or bi-weekly tick control.
Across the two sites, 179 cattle were positive for T. parva antibodies determined by a PIM ELISA corresponding to an overall seroprevalence of 98.4%. Using a nested p104 PCR, prevalence of T. parva parasites was shown to be 37.1% across the two sites. Cattle under the sporadic tick control had significantly higher antibody levels compared to those managed under regular tick control (p=0.0014). Highest T. parva antibody percent positivity was 62.1 for cattle under regular tick control, but this parameter was 87.3 in cattle under sporadic tick control. On the contrary, T. parva PCR results revealed a significantly higher parasite prevalence in cattle under regular tick control (p<0.0001). The detection of low T. parva parasite prevalence in combination with high antibody responses points to a carrier state which may not support clinical disease but be advantageous for the establishment of endemic stability in the populations. We discuss the importance of these findings in the understanding of the epidemiology of ECF in endemic settings where tick control regimes vary and in the context of development of more effective control strategies for smallholder farming systems in endemic areas.
Keywords: East Coast fever, endemic stability, Rhipicephalus appendiculatus
In Tanzania, East Coast fever (ECF) is a significant constraint to improved productivity of cattle. The disease accounts to up to 70% of deaths in 6 – 8 month-old calves in pastoral herds and is a serious threat to smallholder dairy farmers for whom the death of a single dairy cow can cause a measurable economic setback. ECF continues to cause extensive economic losses to farmers in endemic areas of 11 countries in eastern, central and southern Africa. The disease is caused by Theileria parva, transmitted by Rhipicephalus appendiculatus, a 3-host ixodid tick species. East Coast Fever is transmitted between infected cattle and susceptible cattle (Bishop et al 1992; Thompson et al 2008). Buffalo are natural reservoirs of T. parva and cattle may become infected with T. parva strains from buffalo if suitable vector tick species are present (Norval et al 1992). A characteristic feature of T. parva infection is that cattle that recover from a primary infection become long-lasting carriers of the parasite, and cannot be clinically differentiated from uninfected cattle. Carrier animals, whether buffalo or cattle, remain a source of infection to susceptible animals that share the same habitat. These carriers may also arise following the infection-and-treatment method of immunization (Di Giulio et al 2003; 2009) or after treatment of a natural infection with anti-theilerial drugs (Matovelo et al 2003). Carrier animals have an important role in the transmission of the infection to ticks, which in turn transmit the parasites to a susceptible host (Odongo et al 2003), thus sustaining the disease in cattle populations.
The traditional smallholder livestock system in Tanzania is characterized by agro-pastoralism and pastoralism and thus resulting into uncontrolled mobility. Tick control regimes under this system vary widely with applications being regular, when animals are treated with acaricides at defined frequency, strategic targeting seasonal peaks of tick abundance or sporadic normally practiced only on calves or in few animals carrying heavy tick burdens. It is believed that, cattle in the traditional smallholder livestock sector, which comprise mostly zebu (Bos indicus) survive in ECF endemic areas due to their endemic stability, a situation where majority of animals become immune after recovery from ECF by 6 months and usually do not succumb to secondary infection. Previous studies elsewhere have reported factors which support development of endemic stability. Such factors include low but continuous tick challenge with low infection rates leading to an equilibrium between host, vector and the environment with no clinical disease (Norval et al 1992; Marcotty et al 2002; Kivaria et al 2004, Rubaire-Akiiki et al 2004). Our own field observations have demonstrated a similar status of zebu cattle in smallholder farming systems. No comprehensive studies have been conducted in Tanzania on seroprevalence and carrier state to TBDs. Therefore the objective of this study was to determine prevalence of T. parva antibodies and parasites in zebu cattle under smallholder farming systems. Our interest was to compare a regular tick control regime, where cattle are dipped once or bi-weekly and a sporadic regime, where tick control is absent or applied seldom at undefined frequency.
A total of 182 clinically healthy adult cattle, males and females, were used in this study, whereby 104 were sampled from Simanjiro district in Manyara region (sporadic tick control regime) and 78 were from a livestock research institute farm in Mabuki in Mwanza region (regular tick control regime), Tanzania. All cattle were of Tanzania shorthorn zebu types and grazed year round in tick infested areas. Rainfall pattern in both districts is bimodal comprising short rains between September and December and the long rains between March and May. Upon receipt of consent from local authorities and individual farmers, blood was collected from the cattle once just at the end of the rainy season (May 2014). Information collected from animals included age, sex, animal history and ECF vaccination status. Inclusion criteria were age (above 8 months), vaccination status (only unvaccinated cattle were sampled) and animal history (only animals that were raised in local areas in the last one year). Simanjiro is a wildlife interface area and it was selected as a suitable site for year round tick infestation. Mabuki is research station herd, where cattle are dipped weekly (high peak) or each two weeks (low peak) depending on tick density, thus interrupting tick infestation cycles. In Mabuki, cattle are dipped using the synthetic pyrethroid, Alphacypermethrin (Dominex 100 EC). Usually livestock attendants dilute the acaricides and replenish dip tanks as per approved product instructions (1:2,000). In contrast to the tick control regime in Mabuki research station, tick control practices in Simanjiro are led by livestock owners themselves, who are mostly agro-pastoralists. The preferred mode of acaricide application on pastoral cattle is mostly by spraying and a range of acaricides (Paratick, Dominex) are used.
Whole blood samples were collected by jugular venipuncture using 10-ml vacutainer tubes (Becton Dickson Vacutainer Systems, England). At the time of blood sampling none of the animals showed any clinical ECF signs (temperature, blood smear results). The samples were labeled and stored in a cool box with ice packs while in the field and later put into a refrigerator until when they were transported to NM-AIST laboratory. The blood for serum collection was centrifuged at 3000 g for 20 min and serum aliquots were stored in a freezer at -20°C until ELISA tests were done. Blood for DNA extraction was kept frozen at -20°C until day of analysis.
Genomic DNA was extracted from 175 whole blood samples using the protocol as described in Thermo Scientific GeneJET Genomic DNA Purification Kit (#0721). (Seven samples clotted before DNA extraction and they were therefore omitted). Extracted DNA was stored at -200C until further analysis.
The nested p104 PCR was used to screen cattle DNA samples for the presence of T. parva. Primers derived from the T. parva-specific 104-kDa antigen (p104) gene were used in the PCR amplification as previously described by Odongo et al (2010) and Iams et al (1990). The sequences of the forward and reverse primers were 5′ATT TAA GGA ACC TGA CGT GAC TGC 3′ and 5′ TAA GAT GCC GAC TAT TAATGACAC C 3’, respectively, for first round and 5′ GGC CAA GGT CTC CTT CAG AAT ACG3′ and 5′TGG GTG TGT TTC CTC GTC ATC TGC 3′, respectively, for the second round. The nested polymerase chain reaction (nPCR) amplifications were performed in a total volume of 20 μl containing 14 μl nuclease-free water, 0.5 μl (10 pmol) of each of forward and reverse primers and 5 μl of genomic DNA (20 ng/μl) template added into the lyophilized pellet (Bioneer PCR Premix—Korea), followed by vortexing and brief spin down to dissolve the pellet. For the second round, the amount of water was 18.5 μl, and 0.5 μl of the primary PCR product was used as a template. Reaction conditions for the primary PCR included initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 60 s, annealing at 60 °C for 60 s and extension at 72 °C for 60 s, and the amplification was done in 30 cycles. The cycling profile condition for the second PCR was the same as the primary amplification, except for the annealing temperature which was 50 °C. The nPCR reactions were carried out in a thermocycler (VeritiTM, Applied Biosystems, USA). The nPCR products were separated on 1.5 % agarose gel and images visualized and documented on a Gel DocTM (Bio Rad, USA). Positive nPCR products were identified as 277 bp DNA fragments.
The PIM-based enzyme-linked-immunosorbent assay (ELISA) described by Katende et al (1998) was used to measure specific antibodies to T. parva (sensitivity > 99%, specificity 94–98%). Optical density (OD) of each sample was measured at 405nm on an Erba Lisascan II ELISA reader (ERBA diagnostics, Mannheim GmbH, Germany). The OD readings were used to compute percent positivity (PP) for each sample using the formula: Mean OD (sample or negative control) divided by mean OD of positive control multiplied by 100. PP of 20% or higher was considered positive.
Data were entered, checked and cleaned using Microsoft excel. Descriptive statistics and graphical representation were also done in Microsoft Excel for Windows (Version 4.0). Analytical statistical procedures (ANOVA and Chi-square) were carried out using StatView (Version 5.0.1). Statistical significance was tested at the 95% confidence level.
A total of 182 cattle were sampled from two districts in northern Tanzania, where different tick control regimes are practiced. One hundred and four cattle were under minimal or sporadic tick control (Simanjiro district) and hence were constantly exposed to ticks including R. appendiculatus, the vector of T. parva. The other 78 animals were de-ticked weekly or bi-weekly (Mabuki station). Out of the 182 cattle from the two study sites, 179 were positive for T. parva antibodies as shown by the ELISA test with a 20% cut-off point. This corresponds to an overall seroprevalence of 98.4% across the two study sites. Only 175 cattle samples were available for PCR detection of T. parva parasites, after removal of 7 samples, which did not give quality DNA yield. The PCR test revealed a T. parva parasite prevalence of 37.1% across the two sites (Table 1). Summing up the results of antibody and parasite prevalence, the results indicate presence of T. parva infection in both areas, where two different tick control regimes are practiced.
| Table 1. Overall frequency distribution of ELISA and PCR results in the study sites | |||
| Parameter | Positive (N, %) | Negative (N, %) | Total (N, %) |
| T. parva ELISA | 179 (98.4) | 3 (1.65) | 182 (100) |
| T. parva PCR | 65 (37.1) | 110 (62.9) | 175 (100) |
In order to gain a deeper understanding of the distribution pattern of specific antibodies in the populations, results of antibody percent positivity were clustered in 5 categories shown in Table 2. Only 2.75% of the animals (5/182) had low to negative result for T. parva antibodies (PP>20). The majority of cattle clustered to PP categories 20-40 (106/182, 58.2%) and 40-60 (49/182, 26.9%). Further, only 4.95% (9/182) of cattle had highest antibody responses (PP>80). Divergent seroconversion states to T. parva were observed among the cattle, with 17.7 and 96 representing minimum and maximum antibody PP levels, signifying different clinical states of the cattle (infected, recovered, immune) in the study population.
| Table 2. Frequency distribution of antibody percent positivity | ||||||
| Antibody Percent Positivity | ||||||
| Categories | <20 | 20-40 | 40-60 | 60-80 | >80 | Total |
| Counts, N | 5 | 106 | 49 | 13 | 9 | 182 |
| Percent, % | 2.75 | 58.2 | 26.9 | 7.14 | 4.95 | 100 |
| Antibody PP | 18.9±0.86 | 29.9±5.53 | 48.9±5.76 | 70.3±8.28 | 87.3±4.67 | |
| Mean Ab PP± SD 40.5±17.4; Minimum 17.7; Maximum 96 | ||||||
Frequency distribution of ELISA results of T. parva antibodies was not significant between the two tick control regimes but rather seroprevalence was shown to be high in each group. Thus, seroprevalence was 98.7% (77/78 cattle) in the regular tick control group and 98% (102/104 cattle) in the sporadic tick control group (Table 3). With regard to T. parva PCR results, the prevalence rate of positives was significantly higher in cattle under regular tick control (43/71 cattle; 60.1%). With respect to the sporadic tick control, 78.8% of the cattle (82/104) did not carry PCR-detectable T. parva parasites. Differences of parasite prevalence between the two tick control regimes was highly significant (p<0.0001).
| Table 3. Frequency distribution of antibody ELISA and parasite PCR counts | ||||
| Parameter | Result | Tick control regimes | p-value | |
| Regular | Sporadic | |||
| T. parva ELISA | Positive | 77 | 102 | 0.74 |
| Negative | 1 | 2 | ||
| T. parva PCR | Positive | 43 | 22 | <0.0001 |
| Negative | 28 | 82 | ||
Cattle under the sporadic tick control had significantly higher antibody levels compared to those managed under regular tick control (Table 4). This was also further evident when the antibody levels of animals were clustered into PP categories. Thus, cattle under sporadic tick control showed higher antibody levels at all PP categories, except at PP>20. (p=0.0014). Cattle under the regular tick control group attained peak mean antibody PP level at 62.1, whereas this level was much higher (87.3) in cattle under sporadic tick control. Figure 1 further compares the range of antibody responses between the two tick control regimes and illustrates that higher antibody PP values were attained by cattle in the sporadic tick control group.
| Table 4. Comparison of T. parva antibody levels under two tick control regimes | ||||
| Category | Subcategory | Tick control regimes | P value | |
|
Regular N=78 |
Sporadic N=104 |
|||
| Mean antibody PP | 35.8±1.25 | 43.9±1.98 | 0.0014 | |
| Antibody PP categories | >20 | 19.8±0 | 18.7±0.81 | |
| 20-40 | 29.8±5.30 | 30.2±5.79 | ||
| 40-60 | 48.6±6.31 | 49.2±5.38 | ||
| 60-80 | 62.1±2.67 | 71.8±8.10 | ||
| >80 | nil | 87.3±4.67 | ||
|
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| Figure 1. Comparison of T. parva antibody responses (PP) under two tick control regimes |
This is the first report comparing the impact of tick control regimes, regular and sporadic, on T. parva antibody and parasite prevalence in Tanzania. Our study provides an insight on the epidemiological assessment of T. parva infection in endemic areas. The results presented here indicate high overall seroprevalence (98.4%) and T. parva parasite prevalence (37.1%) across the two study sites, implying presence of T. parva and exposure of cattle to the parasite both in Mabuki and Simanjiro. In fact, high seroprevalence was also shown for each group separately, when we compared cattle maintained under the two tick control regimes. Moreover, in each group we found only 1-2 seronegative animals. The high seroprevalence to ECF probably aligns with factors, such as constant exposure of cattle to ticks, indigenous type of zebu cattle, as well as ecological and climatic suitability for tick vector habitats, which have also been previously reported to influence seroprevalence and development of endemic stability (Norval et al 1992).
Our findings on prevalence of T. parva parasites in both study sites is a reflection of presence of vector tick species R. appendiculatus (Gitau et al 1997; Rubaire-Akiiki et al 2006) in Mabuki as well as Simanjiro sites. Furthermore, T. parva prevalence is biologically associated with T. parva seroprevalence (Norval et al 1992). Other studies have reported that associations between T. parva prevalence and T. parva antibodies may vary depending on frequency of acaricide application and age of the animals (Gachohi et al 2010), as well as resistance of the hosts to the parasites and their tick vectors (Bakheit and Latif, 2002). Our results agree with reports by Bakheit and Latif (2002) and Gachohi et al (2010) in that the association of prevalence of parasites and antibodies was variable between the two study sites. High parasite prevalence in cattle under regular acaricide treatments may also partly be attributed to management of dipping practices. However high serological prevalence in relation to low parasite prevalence shown here may imply that majority of the sampled animals had recovered from previous T. parva infection, hence establishing a carrier state in the populations.
Based on T. parva ELISA results, divergent levels of seroconversion were indicated among the cattle. Majority of the animals showed moderate antibody responses and the mean antibody percent positivity was 40.5 across the two sites. The low frequency of high antibody responders shown here may either mean that there is a high number of animals in our study which recently recovered from a primary ECF infection or that majority of the cattle may have carried T. parva parasites for a long time as happens with carrier animals. The ELISA test used in our study is capable to detect T. parva antibodies 10-14 days post-infection. With this test, high levels of antibodies can be detected 30-60 days after animals have recovered from a T. parva infection, but low antibody levels can be detected up to one year post recovery (OIE Terrestrial Manual 2008). Using ELISA and PCR tests together, we have demonstrated agreement between the antibody and parasite prevalence indicated by the two tests. All 65 PCR positive samples were also sero-positive. The seropositive but PCR negative samples therefore may be recently seroconverted animals carrying no or low level of parasites. These results reflect the real field situation with regards to seroprevalence and carrier state of T. parva in endemic settings.
When we compared the two tick control regimes separately, our results showed that cattle under the sporadic tick control regime had significantly higher antibody levels. This is supported by the presence of high responder animals in this group, which had antibody positivity above >80, which were apparently absent in the regular tick control group. Demonstration of T. parva antibodies in cattle serum indicates previous exposure and recovery from T. parva. According to Young et al (1986) antibodies acquired during ECF infection usually disappear within 6 months. However, under field settings, T. parva antibodies are persistently detected in cattle, provided that the animals are continuously exposed to infected ticks and tick control is minimal. Our own findings have revealed a similar pattern of results, since all sampled cattle were adults (>8 months) and were reported by farmers to have recovered from T. parva infection. Higher antibody responses were therefore expected under sporadic tick control, where small holder farmers in Simanjiro mostly practice agro-pastoralism. In comparison to data on antibody prevalence (sporadic tick control, seroprevalence=98.1; PP=44.0; regular tick control, seroprevalence=98.7; PP=35.8), we have shown that detection of T. parva parasite prevalence by PCR from the same animals was lower (sporadic tick control, 21.1%; regular tick control, 60.1%), possibly due to persistence of T. parva antibodies following infection and recovery.
Summing up our results, this study has demonstrated high seroprevalence in cattle under sporadic as well as regular tick control regimes. The study has provided evidence for T. parva presence in the areas and previous exposure of the cattle to the parasite. We have shown that higher T. parva antibody responses but lower T. parva parasite prevalence were associated with the sporadic tick control, as expected for endemically stable populations. The detection of T. parva parasites in combination with high antibody responses points to a carrier state which may not support clinical disease but contribute to the establishment of endemic stability to T. parva. Since all sampled animals were above 8 months of age, we rule out the possibility of maternal nature of the antibodies detected by the ELISA test. Typically, infection with T. parva varies greatly with age (Walker et al 2014), whereas more severe disease is likely to develop in calves below 6 months of age. Seroprevalence reported in this study therefore insinuates a measure of antibodies acquired by cattle as a result of infection and recovery in addition to a continuous low exposure to infective ticks. Our study findings are important to understand the epidemiology of ECF in endemic settings where tick control regimes vary and may be used to develop more effective control strategies for smallholder farming systems in ECF endemic areas.
We acknowledge the scholarship support from the Commission for Science and Technology in Tanzania through the Nelson Mandela African Institution for Science and Technology given to the first author. We are enormously grateful to the farmers in Simanjiro for allowing us to use their animals for the study. We are also grateful to the Prof. S. Chenyambuga, Dr. G. Msallya, Mr. E. Rugaimukamu and Mr. E. Laisser in Sokoine University of Agriculture, Morogoro for providing us with samples from Mabuki and the cooperation during field work.
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Received 1 October 2014; Accepted 20 March 2015; Published 3 June 2015