Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1-2 x 10(3) parasites per ml of blood could be detected-a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.     

Artificial feeding of ixodid ticks

Ixodid ticks are economically important as they cause direct damage to livestock and are vectors of several pathogens that cause diseases in humans and animals. Some of the important tick-borne pathogens of livestock are Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdria ruminantium. These pathogens are responsible for causing enormous losses in livestock. Identification of factors that influence transmission and development of these pathogens in ticks will greatly facilitate development of rational strategies for control of tick-borne diseases. This research has been hampered by the lack of suitable artificial feeding methods. In this paper, Sam Waladde, Aian Young and Subhash Morzaria review recent developments in the artificial feeding of ixodid ticks and evaluate how this method can potentially be exploited. They use an example the transmission of an important livestock pathogen, T. parva, by Rhipicephalus appendiculatus.     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

Identification of Theileria buffeli/orientalis and Babesia bigemina in Apulian cattle using molecular techniques and study of changes in blood parameters

Recently several cases of theileriosis due to the haemoprotozoan Theileria buffeli/orientalis have been recorded in the Apulian region, Italy. In this area other tick-borne pathogens were usually identified such as Anaplasma marginale and Babesia bigemina. Outbreaks were recorded showing that these pathogens can be observed separately or in mixed infections. Sub-clinical cases and carrier animals were also previously identified. A lack of specific techniques could not rule out the presence of other haemoparasites such as T. annulata, B. divergens, B. bovis, Ehrlichia phagocytophila and E. bovis. Moreover little is known about the tick species involved in the dissemination of these diseases. Therefore more powerful techniques to specifically identify Theileria or Babesia species have been recently developed. A PCR technique and reverse line blotting (RLB) system to specifically identify six Theileria species and three Babesia species were used. T. buffeli/orientalis and B. bigemina were the only pathogens observed in the targeted animals. The authors also present some changes in blood parameters for the animals followed during this study.     

Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic.     

Seasonal prevalence of ticks and tick-transmitted haemoparasites in traditionally managed N'Dama cattle with reference to strategic tick control in the Gambia

Abstract A survey of tick spatial and seasonal distribution in traditional managed N' Dama cattle over 1 year old was carried out in The Gambia over 16 months. Presence of Anaplasma marginale and Babesia spp. in the blood smears and their antibodies in the serum of same animals were also examined. Tick species, in decreasing order of abundance, were: Boophilus geigy, Rhipicephalus senegalensis, B.decoloratus, Hyalomma truncatum, H. marginatum rufipes and Amblyomma variegatum. All tick species peaked during the rainy season. Additional B.geigy peaks occurred towards the end of the dry season. The most infested animal body areas were anogenital, udder and abdomen. A.variegatum, Hyalomma spp. and, to a lesser extent, Boophilus spp. showed strong preferences for these anatomical regions. Overall prevalence of A.marginale, B.bigemina and B.bovis haemoparasites were 3.2%, 0.9% and 0.1 %, respectively. Peaks of A.marginale and B.bigemina occurred at the end of the rainy season-beginning of the dry season; an additional A.marginale peak was observed at mid-end dry season. Overall antibody seroprevalence were 29.6% for A.marginale , 44.7% for B.bigemina and 5.2% for B.bovis; monthly fluctuations in percentage of seroreactors were limited. Endemic stability for A.marginale and B.bigemina is postulated. Implications of the outcomes are discussed in relation to tick-control strategy in The Gambia.     

First Evaluation of an Outbreak of Bovine Babesiosis and Anaplasmosis in Southern Brazil Using Multiplex PCR

Outbreaks of tick-borne disease cases in Santa Catarina, Brazil are known, but the presence of the pathogen DNA has never been determined. In this study, the first survey of Anaplasma marginale, Babesia bigemina, and Babesia bovis DNA on blood samples of 33 cattle from an outbreak in Ponte Alta Municipality, Santa Catarina, Brazil, has been carried out. A multiplex PCR detected 54.5% of animals were co-infected with 2 or 3 parasites, while 24.2% were infected with only 1 species. The most prevalent agent was B. bigemina (63.6%) followed by A. marginale (60.6%). This is the first report of tick-borne disease pathogens obtained by DNA analysis in Southern Brazil.     

General review of tick species present in Portugal

At present, 24 species are known to occur in Portugal: Argas vespertilionis, Ornithodoros maritimus and Ornithodoros erraticus in Argasidae; Ixodes acuminatus, Ixodes bivari, Ixodes canisuga, Ixodes frontalis, Ixodes hexagonus, Ixodes ricinus, Ixodes simplex simplex, Ixodes ventalloi, Ixodes vespertilionis, Dermacentor marginatus, Dermacentor pictus', Haemaphysalis hispanica, Haemaphysalis inermis, Haemaphysalis punctata, Rhipicephalus bursa, Rhipicephalus pusillus, Rhipicephalus sanguineus, Rhipicephalus turanicus, Hyalomma lusitanicum, Hyalomma marginatum marginatum and Boophilus annulatus in Ixodidae. The more relevant diseases transmitted to cattle by ticks, particulary in Ribatejo and Alentejo regions, are the babesiosis due to Babesia bigemina and Babesia bovis, the theileriosis by Theileria annulata and the anaplasmosis due to Anaplasma marginale; the theileriosis by Theileria mutans2 may not be considered significant. The sheep and goats parasitoses transmitted by ticks are of less importance than the cattle diseases. However the babesiosis due to Babesia motasi and Babesia ovis and also theileriosis by Theileria hirci3 are present in some districts of the country.     

Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for differentiation of Theileria species and characterisation of Theileria parva stocks have been developed. These tests have been derived through physical mapping and sequencing of key elements of the T. parva genome, which include repetitive and telomeric regions, minisatellite sequences, antigen genes and a number of random DNA sequences. These tools are currently being deployed in conjunction with field immunisation programmes to determine the biological impact of introducing live vaccines of T. parva on population dynamics.     

Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP)

Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1 pg DNA for the T. sergenti PCR and 1 pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.     

Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain

The genetic diversity and prevalence of virtually all Theileria and Babesia species in a sheep population were studied using a specifically designed reverse line blot macroarray. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against generic and species-specific probes. In a first screening (Study I), 320 apparently healthy animals corresponding to 32 flocks located in the Basque Country (Northern Spain) were analysed. The survey demonstrated a high prevalence of subclinical infections (64.7%). Three Theileria genotypes were identified, sharing 96.7-97.0% similarity between their 18S rRNA gene sequences: Theileria ovis, Theileria sp. OT1 (99.6% similarity with the recently described pathogenic piroplasm Theileria sp. China 1), and Theileria sp. OT3. Two Babesia species sharing 91.5% similarity were also detected: Babesia ovis and Babesia motasi. The complete 18S rRNA gene sequences of these and other piroplasm species were phylogenetically analysed. Prevalence of piroplasms was also investigated in a second group of 80 sheep from 16 flocks reared in mountain areas that had been heavily exposed to ticks and had suffered a recent abortion episode (Study II). The screening revealed a significantly higher (P < 0.05) prevalence (78.7%) of piroplasm infections compared to Study I. Although the prevalence rates for some piroplasm species were significantly related to abortion (e.g. Theileria sp. OT3), decreases in the red cell parameters were not significant. The widespread distribution of Theileria spp. in the studied sheep population suggests that the parasites involved are of relatively low pathogenicity, in contrast to what has been reported for Theileria sp. China 1 in other countries.     

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Pathogen prevalence in ticks collected from the vegetation and livestock in Nigeria

Ticks are important disease vectors that can cause considerable economic losses by affecting animal health and productivity, especially in tropical and subtropical regions. In this study, we investigated the prevalence and diversity of bacterial and protozoan tick-borne pathogens in ticks collected from the vegetation and cattle in Nigeria by PCR. The infection rates of questing ticks were 3.1% for Rickettsia species, 0.1% for Coxiella burnetii and 0.4% for Borrelia species. Other pathogens, such as Babesia, Theileria, Anaplasma, and Ehrlichia species, were not detected in ticks from the vegetation. Feeding ticks collected from cattle displayed infection rates of 12.5% for Rickettsia species, 14% for Coxiella burnetii, 5.9% for Anaplasma species, 5.1% for Ehrlichia species, and 2.9% for Theileria mutans. Babesia and Borrelia species were not detected in ticks collected from cattle. Mixed infections were found only in feeding ticks and mainly Rickettsia species and Coxiella burnetii were involved. The diversity of tick-borne pathogens in Nigeria was higher in feeding than in questing ticks, suggesting that cattle serve as reservoirs for at least some of the pathogens studied, in particular C. burnetii. The total estimated herd infection rates of 20.6% for a Rickettsia africae-like species, 27% for Coxiella burnetii, and 8.5% for Anaplasma marginale/centrale suggest that these pathogens may have considerable implications for human and animal health.     

Tick-borne diseases in ruminants of Central and Southern Italy: epidemiology and case reports

Sera and blood from cattle and sheep were examined for the presence of Babesia and Theileria spp by microscopy and serology at the Parasitology Department of the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise (IZSAM). Of the 47 bovine herds (323 animals) tested, 15 were found positive for Babesia bigemina and 1 for Babesia bovis. Two outbreaks occurred, one caused by B. bigemina and one by B. bovis. The B. bigemina outbreak occurred in Abruzzo and has been followed for two years. The isolate of B. bigemina was very pathogenic leading to the death of two cows out of 57. The vector responsible of the transmission appeared to be Rhipicephalus bursa. Parasites were observed in the erythrocytes for 30 days whereas sera were positive to indirect immunofluorescence (IFA) for at least one year. The B. bovis outbreak occurred in the province of Mantova (Northern Italy) in a group of 70 beef cattle imported from France. The infection resulted in the death of 5 animals and severe illness in another 6. In contrast with what occurred for Babesia infection, no clinical cases were recorded in cattle when species of Theileria were detected by microscopy. Of the 24 bovine herds (252 animals) tested for Theileria, 21 were found positive for the T. "sergenti"/buffeli/orientalis group. Single and mixed infection of T. "sergenti" and T. buffeli/orientalis were detected in herds of cross-bred cattle from Abruzzo and Marche. The parasites were identified by using a polymerase chain reaction which amplified DNA encoding p32/34. Most of the collected ticks (90%) were adults of R. bursa whereas the others were adults of Hyalomma detritum. During the period the animals have been observed (18 months), no clinical cases have been recorded and no associations have been found between blood abnormalities and animals found infected with Theileria. Prevalences of subclinically infected carriers increased from February till December (95.4%) even if the animals were indoors and no ticks were present. The prevalence then dropped dramatically six months later (76.7%). In calves less than 1 year old, the prevalence of infection significantly (p<0.05) increased with age, however intraerythrocytic stages of Theileria were found in the blood of three newborn calves (<7 days of age). Of the 18 ovine flocks tested for Babesia spp. (150 animals examined), 1 was positive for B. ovis and 2 for B. motasi. B. motasi infection was not associated with symptoms, while an outbreak of babesiosis caused by B. ovis occurred in Abruzzo. The infection resulted in the death of 3 animals (0.75% of the flock), two rams (20% of the total number) and a ewe, and severe illness in another 5 ewes (2% of the flock). Specimens of R. bursa and R. turanicus were collected from the infected animals. Of the 18 flocks (150 animals) examined, 12 were microscopically positive for Theileria spp. No clinical cases were recorded and identification at species level was not possible on the basis of morphological criteria. The prevalence distribution of infected herds and infected animals within herds and flocks have been calculated by a Monte Carlo simulation model, running 10,000 iterations. The most likely levels of prevalence of infected herds and infected animals within herds found for the species observed were as follows: 20% for B. bigemina with a prevalence within herd of 27%, 11% for B. bovis (18% within herd), 10% for Babesia ovis (19% within herd), 10% for B. motasi (17.5% within herd), 63% for Theileria in cattle (66% within herd) and 51% for Theileria in sheep (55% within herd).     

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Serological and immunological studies with a hexane extract of Babesia bovis-infected erythrocytes.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.     

Molecular investigation of tick-borne infections in cattle from Xinjiang Uygur Autonomous Region,China

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. However, information about the tick-borne infections in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. In this study, nested polymerase chain reaction (PCR) assays and gene sequencing were used to detect and analyze epidemiological features of Babesia bovis, B. bigemina, Coxiella burnetii and Anaplasma bovis infections in XUAR. Out of 195 samples tested, 24 (12.3%), 67 (34.4%), 40 (20.5%) and 10 (5.1%) were positive for B. bovis, B. bigemina, C. burnetii and A. bovis, respectively. Sequencing analysis indicated that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA genes from XUAR showed 99%–100% identity with documented isolates from other countries. Phylogenetic analyses revealed that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA gene sequences clustered in the same clade with isolates from other countries. To the best of our knowledge, this is the first report of C. burnetii infection of cattle in XUAR. Furthermore, this study provides important data for understanding the distribution of tick-borne pathogens, and is expected to improve the approach for prevention and control of tick-borne diseases in China.     

Titrating Theileria parva: single stocks against combination of stocks

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.