Since the first description of an intracellular blood parasite, Haematococcus bovis, later to be included in the genus Babesia, a number of piroplasms belonging to the genus Theileria and Babesia have been described. They infect a wide range of domestic and wild animals, several bird species and even man. Of all the piroplasmoses, theileriosis of cattle in Africa, particularly Theileria parva, had a greater impact on the development of beef and dairy cattle industries, on veterinary research and infrastructure as well as legislation and policies than any other livestock disease complex. Piroplasmosis is still today one of the greatest obstacles to the development of the livestock industries in tropical and sub-tropical countries of the world. Cattle and horses seems to be affected the most with approximately one billion cattle and one hundred million equids worldwide at risk of contracting piroplasmosis. The real cost of tick-borne diseases, including piroplasmoses, is extremely difficult to estimate. However, despite the lack of reliable data, the costs are generally accepted to be substantial. Piroplasmoses result in direct losses as a result of poor growth, poor milk production, poor performance and mortality in infected animals and indirectly contribute enormously to the high costs involved in controlling the tick vectors and in limiting the exportation of many animals. Eradication of either the tick vector or the disease would at first appear to be the ideal approach to control piroplasmosis, however, there have been relatively few attempts with limited success over the years. Today, eradication of piroplasmosis is no longer considered economically justifiable and other means of control such as regular acaricide application, often in combination with vaccination and chemoprophylaxis, are used. Despite extensive research efforts, no molecularly engineered vaccine is as yet available for either cattle or horse piroplasmosis and in many countries live attenuated organisms are still being used for the immunization of cattle.
The 120 kDa serine-rich antigen (SERA) of Plasmodium falciparum and the high molecular weight rhoptry protein complex (Rhop-H) bind to erythrocytes and are co-eluted by high salt. Their interaction with the host erythrocyte may be of crucial importance to merozoite invasion and parasitophorous vacuole membrane (PVM) formation. We investigated the timing of protein release into the SCS. The release of Rhop-H and SERA into schizont SCS was detected two hours after removal of biosynthetic label with maximum levels detected at twenty-five hours. Optimal protein binding to erythrocytes was obtained with SCS collected at twenty-five hours. Both proteins were detected in the SCS of schizont stages by immunoprecipitation, but not in ring and trophozoite stages. The identity of SERA was confirmed using different antibodies specific to SERA and no immunological cross-reactivity was detected between SERA and Rhop-H. Affi-blue gel chromatography of SCS for enrichment of both proteins for erythrocyte binding and analysis by immunoblotting showed SERA and Rhop-H in the same fractions. The topological distribution of the proteins on the erythrocyte surface was investigated by chemical cross-linking analysis using DTSSP. SERA and Rhop-H proteins bound at a distance of approximately 12Å on the mouse erythrocyte surface with no other associated proteins. Similar results were obtained using cross-linked schizont extracts precipitated by specific antibodies and analyzed on two-dimensional (2-D) SDS-PAGE gels. Sedimentation analysis demonstrated that SERA and Rhop-H do not form a complex in vivo, and both types of proteins possess different sedimentation rates. We conclude that prior association of SERA with Rhop-H is not required for erythrocyte binding.
Several series of bench scale experiments were performed to evaluate the usefulness of well established sucrose flotation techniques for separation of Cryptosporidium oocysts from water samples under the various conditions.
Firstly, fresh (one month old) and aged (36 month old) oocysts of C. parvum were spiked into distilled water and separated by the modified sucrose flotation technique or by the discontinuous sucrose gradients. The mean recovery rate of fresh oocysts by the modified sucrose flotation technique was higher (87.2%) than by the discontinuous sucrose gradients (68.4%). In contrast, the mean recovery rates of aged oocysts by two methods were significantly lower. The recovery rate using different initial concentration of oocysts showed no significant differences in recovery between each of them. The usefulness of the modified sucrose flotation technique for the separation of oocysts from raw water samples was also evaluated. The recovery rate of oocysts from low and moderate turbidity water (ntu=0.8 and 15.4) was nearly 80%, however the recovery rate from high turbidity water (ntu=350) was significantly lower (60.7%).
From these results we assume that the modified sucrose flotation technique is still useful as a fast one-step, simple to perform and inexpensive method for the separation of Cryptosporidium oocysts in environmental water samples.