Major Merozoite Surface Antigen Research Articles

Molecular Evidence on Theileria annulata Infection and Ixodid Ticks Infestation in the Cattle of Kurdistan Province, West of Iran

Background: Bovine theileriosis is an important disease in Iran and throughout the world with economic losses in Iranian cattle husbandry. The aim of the current study was to determine prevalence and geographic distribution of Theileria annulata infection in cattle and ixodid ticks species diversity in Kurdistan Province, West of Iran. Methods: A total number of 193 blood samples were randomly taken from jugular vein. Ixodid ticks were also collected from body surface of examined cattle in three sub-areas of the region, i.e. north, center and south. The genomic DNA was extracted and PCR was performed to amplify a 721-bp-long fragment of the 30 Kilo Dalton major merozoite surface antigen of T. annulata. Results: The overall prevalence was 50.2% (97/193) with lymphadenopathy (54.4%) and petechia in mucosal membrane (95%) of cross-breed cattle (24.9%) aged <3 year in north part of the region (82%). Of all cattle infected with T. annulata, 9.3% (18/193) were infested with a total of 147 unfed ixodid ticks. The ixodid ticks indices was 8.17. Eight species of ixodid ticks of two genus, i.e. Hyalomma (52.9%) and Rhipicephalus (23.3%) were identified. The predominant infesting tick in all examined cattle was R. sanguineus (12%, 23/193) in south area of the region. Conclusions: The results revealed that T. annulata infection was prevalent and ixodid ticks abundance, geographic distribution and the variety of species were wide in this part of Iran.

Classification of Babesia canis strains in Europe based on polymorphism of the Bc28.1-gene from the Babesia canis Bc28 multigene family

The vast majority of clinical babesiosis cases in dogs in Europe is caused by Babesia canis. Although dogs can be vaccinated, the level of protection is highly variable, which might be due to genetic diversity of B. canis strains. One of the major merozoite surface antigens of B. canis is a protein with a Mr of 28kDa that belongs to the Bc28 multigene family, that comprises at least two genes, Bc28.1 and a homologous Bc28.2 gene. The two genes are relatively conserved but they are very distinct in their 3′ ends, enabling the design of specific primers. Sequencing of the Bc28.1 genes from 4 genetically distinct B. canis laboratory strains (A8, B, 34.01 and G) revealed 20 mutations at conserved positions of which three allowed the classification of B. canis strains into three main groups (A, B and 34.01/G) by RFLP. This assay was subsequently used to analyze blood samples of 394 dogs suspected of clinical babesiosis from nine countries in Europe. All blood samples were first analyzed with a previously described assay that allowed detection of the different Babesia species that infect dogs. Sixty one percent of the samples contained detectable levels of Babesia DNA. Of these, 98.3% were positive for B. canis, the remaining cases were positive for B. vogeli. Analysis of the Bc28.1 gene, performed on 178 of the B. canis samples, revealed an overall dominance of genotype B (62.4%), followed by genotypes A (37.1%) and 34 (11.8%). Interestingly, a great variation in the geographical distribution and prevalence of the three B. canis genotypes was observed; in the North–East genotype A predominated (72.1% A against 27.9% B), in contrast to the South–West where genotype B predominated (10.3% A against 89.7% B). In the central part of Europe intermediate levels were found (26.0-42.9% A against 74.0-57.1% B, from West to East). Genotype 34 was only identified in France (26.9% among 78 samples) and mostly as co-infection with genotypes A or B (61.9%). A comparative analysis of the classification of 35 B. canis strains in genotypes A and B using a previously described 18SrDNA-derived PCR-RFLP test revealed a partial but no direct correlation with the classification based on polymorphism of the Bc28.1-gene described here.

PCR-RELP for detecting of Theileria annulata infection in cattle and Hyalomma species in Kermanshah Province, Iran

Bovine theileriosis is important disease in tropical and subtropical areas with great economic losses in livestock husbandry in Iran. The aim of study was to assess the prevalence of Theileria annulata infection in cattle and Hyalomma species of Kermanshah Province, Iran. A number of 138 blood samples were randomly taken from examined cattle. The genomic DNA was extracted and PCR was performed to specifically amplify a 721-bp-long fragment of the 30 Kilo Dalton major merozoite surface antigen (30 KDa msa) of T. annulata. The amplified products were digested with TaqI, Rasl, and AluI restriction enzymes. Overall prevalence was 9.44% (13/138) with lymphadnopathy (1.17%) and pale mucosal membrane (1.9%) in Holstein cattle aged <1 year and more than 5 years-old, respectively. Five species of genus Hyalomma (18.8%, 141/750) including Hyalomma anatolicum anatolicum (30.4%), H. anatolicum excavatum (31.2%), H. marginatum (36.8%), H. asiaticum asiaticum (0.7%), and H. detritum (0.7%) were identified. The tick indices for each Hyalomma species were ranged from 0.01 to 0.36. PCR findings indicated that 3 out of 138 blood samples (2.17%) and 19 out of 141 Hyalomma ticks (4.13%) were infected with T. annulata. Amplified PCR products from blood samples generated similar RFLP patterns, but different RFLP pattern for T. annulata from H. anatolicum anatolicum (9.21%) and H. anatolicum excavatum (4.2%). The RFLP patterns of the amplified fragment of the 30 KDa msa of T. annulata indicated the circulation of four different T. annulata isolates of H. anatolicum anatolicum and H. anatolicum excavatum in the region.

Detection of Theileria annulata carriers in Holstein-Friesian (Bos taurus taurus) and Sistani (Bos taurus indicus) cattle breeds by polymerase chain reaction in Sistan region, Iran.

Theileria annulata is common in tropical and subtropical regions especially in Iran and causes great economic losses in cattle industry. In Iran the epidemiological aspects of bovine theileriosis in different breeds of cattle is poorly understood. The aim of present study is comparison of the number of T. annulata carriers in the two major cattle breeds (Holstein-Friesian and Sistani) in Sistan of Iran by giemsa and polymerase chain reaction (PCR) methods. During winter 2013, 160 native cattle, from the two major breeds in Sistan, with the mean age of more than one year and without typical clinical symptoms of theileriosis were selected. At first, a thin layer smear was held from their ear sublime vein blood for Giemsa staining method. In order to do PCR assay, jugular vein blood sample of each cow was taken. The PCR employs primers specific for the 721-bp gene fragment encoding the 30-kDa major merozoite surface antigen of T. annulata. By PCR method, 38 (47.5%) Holstein blood samples and 22 (27.5%) Sistani blood samples had DNA of T. annulata and considered positive (The correlation was significant at values of P<0.05). By checking 160 blood smears with light microscope and lens×100, only 10 samples (6.25%) were positive for T. annulata. Statistical comparison between PCR and smear method showed that the PCR method is more sensitive and accurate in comparison to Giemsa staining method to diagnose the asymptomatic carriers of T. annulata.

Hematobiochemical alterations and direct blood polymerase chain reaction detection of Theileria annulata in naturally infected crossbred cows.

Aim:The aim was to determine hemato-biochemical changes and rapid diagnosis of Theileria annulata in naturally infected crossbred cows.Materials and Methods:Blood samples from lactating crossbred cows (n=40) between 3 and 7 years of age and showing clinical signs of tropical theileriosis were collected, with or without anticoagulant, and analyzed for tropical theileriosis by direct smear, direct blood polymerase chain reaction (PCR) detection of merozoite-piroplasm surface antigen (Tams1) gene specific amplicon, estimation of hematological and biochemical parameters. Healthy crossbred cows (n=6), examined free from hemoprotozoan infections were included as control.Results:The infected crossbred cows revealed significantly (p<0.001) lower values of total erythrocytic counts (4.46±0.2 × 106/µL), hemoglobin (Hb 6.025±0.39 g%), packed cell volume (17.05±1.1%), mean corpuscular volume (37.94±1.70 fL) and mean corpuscular Hb (13.5±0.48 pg; p<0.002) compared with healthy control. The serum samples of infected cows revealed profound (p<0.05) hyponatremia (Na 133.21±2.36 mEq/l) and hypocalcemia (Ca 8.39±0.34 mg%). Infected crossbred cows showed a significant increase (p<0.05) of mean serum activity of alanine aminotransferase (61.45±13.36 U/L), aspartate aminotransferase (146.1±20.97 U/L), blood urea nitrogen (28.26±3.90 mg%), creatinine (1.55±0.13 mg%), direct bilirubin (0.33±0.04 mg%; p<0.001) and lactate dehydrogenase (3001.32±167.0 U/L; p<001). Blood direct PCR revealed a 721-bp fragment amplified from the target gene encoding 30-kDa major merozoite surface antigen of T. annulata using specific primer pairs. This assay was positive for all the infected animals.Conclusion:The assessments of hemato-biochemical parameters in T. annulata infected crossbred cows may be useful in understanding disease pathogenesis, prognosis and corrective measures for supportive therapy. Moreover, blood direct PCR can reliably be used for rapid detection of T. annulata in conjunction with microscopic examination.

Genetic diversity and phylogenetic analysis of Tams1 of Theileria annulata isolates from three continents between 2000 and 2012.

Theileria annulata, which is part of the Theileria sergenti/Theileria buffeli/Theileria orientalis group, preferentially infects cattle and results in high mortality and morbidity in the Mediterranean, Middle East, and Central Asia. The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of T. annulata that could be used as a marker for epidemiological studies and phylogenetic analysis. In the present study, a total of 155 Tams1 sequences were investigated for genetic diversity and phylogenetic relationships through phylogenetic analysis. Results showed that the Tams1 sequences were divided into two major groups and that distribution for some isolates also exhibited geographic specificity. As targeting polymorphic genes for parasite detection may result in underestimation of infection, polymerase chain reaction (PCR) assay using two different probes targeting tams-1 genes of these two groups can be more credible. In addition, the direction of the spread of the disease was discovered to be from the Mediterranean or the tropical zone to the Eurasian peninsula, Middle East, Southern Asia, and Africa, particularly for Group 2. A similar occurrence was also found between the Ms1 gene of Theileria lestoquardi and the Tams1 gene of T. annulata, which explains cross-immunogenicity to a certain extent. However, no potential glycosylation site in the Tams1 of T. annulata was found in this study, which illustrated that instead of N-glycosylation, other modifications have more significant effects on the immunogenicity of the Tams1 protein.

Detection of tropical bovine theileriosis by polymerase chain reaction in cattle

Tropical bovine theileriosis, a tick borne disease, caused by, Theileria annulata with marked clinical signs of pyrexia (102-105°F), enlargement of lymphnodes etc., causes heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of theileriosis is mainly based on clinical symptoms and microscopic examination of stained blood smears and lymph node biopsy smears but limitations of these methods against Theileria sp. limits the specificity. Hence, to overcome the limitations, the present study reports the detection of T. annulata in blood samples of cattle by polymerase chain reaction. The study was conducted on 155 cattle having typical clinical symptoms and blood smear after staining with Giemsa stain was examined for the presence of T. annulata in RBC. The Primer sequences were used as per d'Oliveira et al. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata and the amplification of 721bp was done. Out of the total 155 animals, 34 were positive for T. annulata by blood smear method whereas 134 samples were positive by PCR. So diagnosis of blood samples by PCR is found to be the most sensitive and specific methodology as compared to cytological blood smear examination. The sensitivity was 23.88% and specificity was 90.47% of blood smear method considering PCR as gold standard and it was found that PCR is more sensitive than the conventional method of examination.

Structural and Functional Characterization of Bc28.1, Major Erythrocyte-binding Protein from Babesia canis Merozoite Surface

Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of protozoa parasites from the genus Babesia. Like Plasmodium falciparum, the agent of malaria, or Toxoplasma gondii, responsible for human toxoplasmosis, Babesia belongs to the Apicomplexa family. Babesia canis is the agent of the canine babesiosis in Europe. Clinical manifestations of this disease range from mild to severe and possibly lead to death by multiple organ failure. The identification and characterization of parasite surface proteins represent major goals, both for the understanding of the Apicomplexa invasion process and for the vaccine potential of such antigens. Indeed, we have already shown that Bd37, the major antigenic adhesion protein from Babesia divergens, the agent of bovine babesiosis, was able to induce complete protection against various parasite strains. The major merozoite surface antigens of Babesia canis have been described as a 28-kDa membrane protein family, anchored at the surface of the merozoite. Here, we demonstrate that Bc28.1, a major member of this multigenic family, is expressed at high levels at the surface of the merozoite. This protein is also found in the parasite in vitro culture supernatants, which are the basis of effective vaccines against canine babesiosis. We defined the erythrocyte binding function of Bc28.1 and determined its high resolution solution structure using NMR spectroscopy. Surprisingly, although these proteins are thought to play a similar role in the adhesion process, the structure of Bc28.1 from B. canis appears unrelated to the previously published structure of Bd37 from B. divergens. Site-directed mutagenesis experiments also suggest that the mechanism of the interaction with the erythrocyte membrane could be different for the two proteins. The resolution of the structure of Bc28 represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.

Molecular and phylogenetic analysis of the partial tams1 gene sequence of a vaccine strain of Theileria annulata

The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata and is highly variable. In this study, the partial nucleotide (nt) sequence of the Tams1 (522 nt) gene of Iranian vaccine strain (Vaccine-ir68) recovered from an outbreak of disease in Iran was determined and compared with the corresponding sequences of eighteen previously published Tams1 genes. According to sequencing result, a novel amino acid substitution at the Tams1 region (K→Q) was found exclusively in isolate Vaccine-ir68. Genetic distance values, estimated from the sequence data, revealed striking sequence homology (approximately 99%) between Vaccine-ir68 isolate and Tunisian isolates, showing that they were same isolates of T. annulata which were spread in these areas. The phylogenetic tree constructed based on the sequence alignment of 19 Tams1 coding regions was distinctly divided into five lineages. There might be some unknown tick carrier birds immigrating to the different geographical regions. These birds have an effective role to distribute the T. annulata species in North Africa, Palestine and Iran.

Babesia divergens experimental infection of spleen-intact sheep results in long-lasting parasitemia despite a strong humoral response: Preliminary results

Babesia divergens is an intraerythrocytic Apicomplexa and the main agent of bovine babesiosis in Europe. The infection in cattle develops in 2 phases: an acute phase with hemolytic anemia and a chronic phase with asymptomatic persistence of the parasite for several years. The acute phase of B. divergens infection can be studied using the gerbil ( Meriones unguiculatus) as a laboratory model but unlike cattle, this animal rapidly eliminates the parasite. An experimental model to study the chronic phase of infection was therefore developed by our laboratory. Spleen-intact sheep, with a potential full immune response, were inoculated with infected red blood cells (iRBC) or with free merozoites, by several routes (intraperitoneal, intravenous or subcutaneously). No clinical signs were ever observed but the installation of a persistent low level infection was shown in sheep with susceptible erythrocytes (able to sustain B. divergens growth in vitro). Neither feature was observed in sheep with non-susceptible erythrocytes. IgG production, involving both IgG1 and IgG2, was mainly directed against the major merozoite surface antigen Bd37, similar to the humoral immune response described in naturally infected cattle. The use of spleen-intact sheep to study the immune response to B. divergens is discussed.

Recombinant Viral Vaccines Expressing Merozoite Surface Protein-1 Induce Antibody- and T Cell-Mediated Multistage Protection against Malaria

SummaryProtecting against both liver and blood stages of infection is a long-sought goal of malaria vaccine design. Recently, we described the use of replication-defective viral vaccine vectors expressing the malaria antigen merozoite surface protein-1 (MSP-1) as an antimalarial vaccine strategy that elicits potent and protective antibody responses against blood-stage parasites. Here, we show that vaccine-induced MSP-1-specific CD4+ T cells provide essential help for protective B cell responses, and CD8+ T cells mediate significant antiparasitic activity against liver-stage parasites. Enhanced survival is subsequently seen in immunized mice following challenge with sporozoites, which mimics the natural route of infection more closely than when using infected red blood cells. This effect is evident both in the presence and absence of protective antibodies and is associated with decreased parasite burden in the liver followed by enhanced induction of the cytokine IFN-γ in the serum. Multistage immunity against malaria can thus be achieved by using viral vectors recombinant for MSP-1.

Generation of a mosaic pattern of diversity in the major merozoite-piroplasm surface antigen of Theileria annulata

The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata. Generation and selection of divergent antigenic types has implications for the inclusion of the Tams1 antigen in a subunit recombinant vaccine or use in the development of a diagnostic ELISA. In this study a total of 129 Tams1 sequences from parasites isolated in Bahrain, India, Italy, Mauritania, Portugal, Spain, Sudan, Tunisia and Turkey were obtained to estimate the extent of Tams1 diversity throughout a wide geographical range. Significant sequence diversity was found both within and between isolates and many of the sequences were unique. No geographical specificity of sequence types was observed and almost identical sequences occurred in different geographical areas and a panmictic population structure is suggested by our results. A sliding window analysis identified sub-regions of the molecule where selection for amino acid changes may operate. Evidence is also presented for the generation of diversity through intragenic recombination with switching of corresponding variable domains between alleles. Recombination to exchange variable domains appears to occur throughout the length of the gene sequence, and has the potential to generate a mosaic pattern of diversity.

Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences.

A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 microl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at -20 degrees C.

Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems

Allelic forms ( Tams1-1 and Tams1–2) of the major merozoite surface antigen gene of Theileria annulata have recently been expressed in Escherichia coli and in Salmonella typhimurium aroA vaccine strain SL3261. To test the potential of subunit vaccines against T. annulata infection, we immunized four groups of three calves with either recombinant (re-) (Tams1-1 and Tams1–2) proteins or naked DNA encoding these antigens. Group I was immunized intramuscularly with both re-proteins incorporated into immunostimulating complexes (ISCOMs). Group II was inoculated intramuscularly with naked plasmid DNA encoding Tams1-1 and Tams1–2. Groups III and IV received S. typhimurium SL3261 [pSTams1-1][pIP5] and SL3261 [pSTams1–2][pIP5] subcutaneously and orally, respectively. A final group of three animals (Group V) served as an unimmunized control group. Four weeks after the last immunization all calves were challenged with a T. annulata stabilate generated from blood of an infected animal with 30% piroplasm parasitaemia. All calves vaccinated with ISCOMs proved to be protected from T. annulata infection and had generated antibodies against both re-(Tams1-1 and Tams1–2) at the time of challenge. In two of these animals the antibody had a surface binding profile by IFAT. Two of three calves immunized with naked DNA also proved to be protected, but none of the animals had generated any detectable antibodies against the recombinants. Salmonella-based delivery of the recombinants did not induce any protection; two of six animals died of theileriosis and there was no difference between subcutaneous or oral administration. These preliminary results show that re-(Tams1-1 and/or Tams1–2) may elicit protective immune responses in cattle, depending on the antigen delivery system.

Detection of Theileria annulata by the PCR in ticks (Acari:Ixodidae) collected from cattle in Mauritania.

We report on the detection of Theileria annulata in infected Hyalomma ticks by the PCR using primers derived from the gene encoding the 30 kDa major merozoite surface antigen (TamsI-1). No inhibition of the PCR was observed and as little as 0.1 pg of parasite DNA, corresponding to 12 sporozoites, could be detected in non-infected tick DNA samples, spiked with T. annulata genomic DNA. Hyalomma dromedarii ticks, fed on a calf experimentally infected with T. annulata, were used to validate the PCR further. The infection rate in the adult ticks, fed as nymphs during the febrile reaction, was high (62%), dropped to zero for 1 day in tick batches that engorged after treatment with Butalex and increased to 30% 2 days later and 38% of the ticks acquired the infection after feeding as nymphs during a carrier state piroplasm parasitaemia of less than 0.1%. As an internal control, 16S tick rDNA sequences could be amplified from T. annulata-negative tick samples. Finally, 202 adult ticks from Mauritania, collected from zebu cattle carrying low levels of Theileria piroplasms, were tested by the PCR. Thirty-eight out of 52 (73%) and 17 out of 30 (57%) H. dromedarii from the Gorgol and Trarza regions, respectively and two out of 30 (7%) Hyalomma marginatum rufipes from the Gorgol region were positive. Hyalomma marginatum rufipes, Rhipicephalus evertsi evertsi and Rhipicephalus guilhoni from the Trarza region were negative. These findings confirm that H. dromedarii is the main vector of T. annulata in Mauritania and that the PCR is a useful method of determining the infection rates in ticks collected from cattle carrying low levels of T. annulata piroplasms.

Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimurium aroA vaccine strain

The genes, Tams1-1 and Tams1-2, encoding the 30-and 32-kDa major merozoite surface antigens of Theileria annulata (Ta), have recently been cloned and characterized. Both genes encode a protein of 281 amino acids (aa) containing a putative hydrophobic N-terminal signal peptide. Another hydrophobic stretch is predicted at the C terminus which probably functions to anchor the protein in the membrane of the merozoite and piroplasm. Here, we report the successful expression of both Tamsl-1 and Tams1-2 in Escherichia coli (Ec) using gene fragments lacking both hydrophobic domains. Attempts to produce high amounts of the entire recombinant (re-) protein, or a fragment containing the N terminus only, were unsuccessful. This is presumably due to the toxicity of these re-proteins. The internal part of both genes was also expressed in Salmonella typhimurium (St) aroA vaccine strain SL3261. We employed a dual-plasmid expression system based on an invertible promoter and selected the most stable St construct in vitro using liquid cultures and a macrophage-like cell line. The re-Tamsl-1 protein produced in Ec, as well as in St, was recognized by monoclonal antibody (mAb) 5E1 specific to the 30-kDa protein. Both re-Tamsl-1 and re-Tamsl-2 were recognized by Ta immune calf serum.

Identification of an immunodominant 40 kDa merozoite antigen common to the Australian T and Dixie vaccine strains of Babesia bovis and the development of diagnostic tests specific for these strains

Antigenic differences among Australian vaccine and field strains of Babesia bovis were investigated in an attempt to identify strain specific antigens. Immunoblots revealed substantial differences between the current vaccine strains, designated T and Dixie, and previous vaccine strains and field isolates collected on properties where vaccination with the T or Dixie strains had failed to provide complete protection against tick-borne challenge. A major difference was an immunodominant 40 kDa antigen (T40) present in only the T and Dixie strains. The molecular weight and immunodominant nature of this antigen suggest that it may be the equivalent of the major merozoite surface antigen (MSA-1) described by others in North American strains of B. bovis. MSA-1 was shown to be conserved in north American isolates but not in an isolate from Israel or in the Australian S and L isolates. The work presented here suggests that merozoite surface antigen diversity exists among geographically different isolates of B. bovis within Australia. Monospecific antiserum to T40 was used to develop an indirect fluorescent antibody (IFA) test specific for T and Dixie strain parasites, and a blocking enzyme-linked immunosorbent assay (ELISA) specific for antibody to the T and Dixie strains. In cases of babesiosis in recently vaccinated cattle, the IFA test will be a useful tool for determining whether clinical symptoms are due to a severe vaccine reaction or to a concurrent tick-borne infection. In a preliminary assessment of potential of the ELISA for the serological identification of vaccinated cattle using a total of 160 sera, the test clearly differentiated between animals vaccinated with the T or Dixie strains and non-vaccinated animals, and was not affected by presence of antibodies to other B. bovis strains.

The epidemiology of malaria in the Wosera area, East Sepik Province, Papua New Guinea, in preparation for vaccine trials. I. Malariometric indices and immunity.

The epidemiological features of malaria were studied through seven community-based surveys in a population of 4000 in the Wosera area, East Sepik Province, Papua New Guinea. Prevalence of parasitaemia (all species, all ages) was 60%. Plasmodium falciparum was the predominant species in all surveys (55%), followed by P. vivax (25%) and P. malariae (20%). The highest prevalence for asexual forms of P. falciparum occurred in the 5-9-year age group, whereas P. falciparum gametocytaemia and P. vivax parasitaemia were observed most frequently in the 1-4-year age group and P. malariae in the 10-15-year age group. Mean densities of all species decreased with age except for that of P. malariae, which was lower in children aged < 1 year than in those aged 1-4 years. The prevalence of enlarged spleen was 57% in children and 10% in adults and closely matched the corresponding age-related parasite rate. Seroprevalence of antibody to the major merozoite surface antigen 2 rapidly increased with age, with > 90% of individuals older than 5 years being positive. Malariological indices showed irregular changes over time but there was no clear-cut seasonal pattern. The geographical distribution of these indices and immune responses was not uniform within the study area. Bednet use and drug consumption were negatively correlated with malariometric indices. Identification of significant temporal and local variations in malaria endemicity is important for the design and evaluation of intervention studies, including field trials of an antimalarial vaccine.

Cellular and humoral immune responses to well-defined blood stage antigens (major merozoite surface antigen) of Plasmodium falciparum in adults from an Indian zone where malaria is endemic.

Conserved and variant regions of two blood stage vaccine candidate antigens of Plasmodium falciparum, merozoite surface antigen (MSA-1) and ring-infected erythrocyte surface antigen (Pf155/RESA), have been shown to be immunogenic. However, the relative immunogenicity of these immunogens in different populations has not been studied. The conserved N-terminal region of MSA-1 was investigated for its immunogenicity by studying cellular (T cell) and humoral (B cell) immune responses in P. falciparum-primed individuals, living in malaria-hyperendemic areas (Orissa State, India), where malaria presents an alarming situation. MSA-1-derived synthetic peptides contained sequences that activated T cells to proliferate and release gamma interferon in vitro. There was considerable variation in the responses to different peptides. However, the highest responses (51% [18 of 35] by proliferation and 34% [12 of 35] by gamma interferon release) were obtained with a synthetic hybrid peptide containing sequences from conserved N- and C-terminal repeat regions of MSA-1 and Pf155/RESA, respectively. Antibody reactivities in an enzyme immunoassay of plasma samples from these donors to different peptides used for T-cell activation were heterogeneous. In general, there was poor correlation between DNA synthesis and either gamma interferon release or antibody responses in individual donors, underlining the importance of examining several parameters of T-cell activation to assess the total T-cell responsiveness of a study population to a given antigen. However, the results from our studies suggest that synthetic constructs containing sequences from the N- and C-terminal regions of MSA-1 and Pf155/RESA representing different erythrocytic stages of the P. falciparum parasite are more immunogenic in humans living in malaria-hyperendemic areas of India who have been primed by natural infection.

The use of untreated bednets and malaria infection, morbidity and immunity.

The effect of bednet use was investigated, without undertaking a specific intervention, in four cross-sectional community-based surveys in 10 villages of a highly endemic area of Papua New Guinea. Over half (55%) of the villagers interviewed reported that they had used a bednet on the previous night. In general and after adjustment for age, village and housing characteristics, bednet users, particularly children, had lower parasite prevalences and spleen rates and less enlarged spleens than non-users. However, users were similar to non-users in terms of fever reported for the previous week, axillary temperature, parasite density and haemoglobin level. The prevalence of antibody to the ring erythrocyte surface antigen and the major merozoite surface antigen 2 was lower in users than non-users. The association with malariometric indices and immune responses remained significant when bednet users were compared with non-users in houses without bednets. Thus, untreated bednets do not reduce malaria transmission sufficiently to decrease morbidity. They might paradoxically increase the risk of clinical malaria by lowering the development of humoral immunity.