Constraints to estimating the prevalence of trypanosome infections in East African zebu cattle

Abstract

Background: In East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense) that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR) based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations.

Results: The Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%), T. congolense (42.9%) and T. vivax (22.9%).

Conclusions: These results show that, despite the convenience of Whatman FTA cards and specific PCR based detection tools, the chronically low parasitaemias in indigenous African zebu cattle make it difficult to establish true prevalence. Although this study specifically applies to FTA cards, a similar effect would be experienced with other approaches using blood samples containing low parasite densities. For example, using blood film microscopy or PCR detection from liquid samples where the probability of detecting a parasite or DNA molecule, in the required number of fields of view or PCR reaction, is less than one.

Figure 1

Success rate of detection of trypanosomes in the artificial dilution series of T. b. brucei. Cultured T. brucei brucei was diluted in cow blood at a concentration of 10⁶ trypanosomes per millilitre and placed onto Whatman FTA cards. Lanes 1 to 3 are negative controls, lane 4 is a positive control lane 5 is a DNA marker, lanes 6 to 13 show the results of repeated PCR of a 10^-7 dilution of the original stock (equivalent to 0.1 trypanosome per ml). Lanes 14 to 21 show the results of repeated PCR of a 10^-6 dilution of the original stock (equivalent to 1 trypanosome per ml). Lanes 22 to 29 show the results of repeated PCR of a 10^-5 dilution of the original stock (equivalent to 10 trypanosomes per ml). Lane 30 is a DNA marker.

Figure 2

Mapping of positive PCR punches on FTA cards. The figure shows three diagrammatic representations of the repeated PCR of blood samples from zebu cattle. Each small circle or shape represents a punch taken for PCR analysis. The positions of each punch were recorded and the results for that PCR were related back to the position on the original sample. Key; open circle, negative PCR result; closed circle, T. theileri; closed triangle, T. brucei; closed square, T. congolense. Examples of a low, medium and high parasitaemia result are shown.

Figure 3

Cumulative prevalence achieved at each round of screening of blood samples taken from thirty-five African zebu cattle. The figure shows the plot of the cumulative prevalence (upper curve) for all species of trypanosome at each round of screening of the thirty five blood samples. As the number of screenings increases the cumulative prevalence also continues to increase as new samples are found positive. The cross sectional prevalence at each round of screening is also shown (lower curve). The mean cross sectional prevalence across all screenings is shown by the dotted line (9.7%).