Identification of novel vaccine candidates against cryptosporidiosis of neonatal bovines by reverse vaccinology.

The apicomplexan protozoan Cryptosporidium parvum is an important causative agent of diarrhea of neonatal bovines. Vaccination has been proposed as an advantageous strategy against cryptosporidiosis of calves since besides protection against disease it has also the potential to prevent dissemination of infective oocysts into the environment. Antigens anchored to the parasite surface via glycosylphosphatidylinositol (GPI) are implicated in host cell attachment and invasion and represent promising vaccine candidates. A reverse vaccinology approach was employed to (i) identify the GPI-anchored proteome of C. parvum using available web-based bioinformatic tools and (ii) characterize previously unrecognized novel vaccine antigens. Altogether, 14 putative GPI-anchored proteins could be determined of which CpH1 and CpSUB2 as well as GP60 were further characterized. Sequencing and comparison of GP60, CpH1, and CpSUB1 alleles amplified from different geographic isolates showed a high degree of conservation. All three antigens were recombinant expressed and immunoblotted using sera of 12 Cryptosporidium-infected calves sampled at age periods 1-11 and 12-28 days after birth. Specific antibody reactions against the studied antigens were detected in all analyzed calves, demonstrating their immunreactivity and expression, and recognition in vivo at an early stage of host infection. Besides the acknowledged GP60 vaccinogen, the presented reverse vaccinology approach reveals the additional vaccine candidates CpH1 and CpSUB1 for inclusion into a subunit vaccine formulation.

Isolation of Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans from rumen of Creole goats fed native forage diet.

We isolated and identified functional groups of bacteria in the rumen of Creole goats involved in ruminal fermentation of native forage shrubs. The functional bacterial groups were evaluated by comparing the total viable, total anaerobic, cellulolytic, hemicellulolytic, and amylolytic bacterial counts in the samples taken from fistulated goats fed native forage diet (Atriplex lampa and Prosopis flexuosa). Alfalfa hay and corn were used as control diet. The roll tubes method increased the possibility of isolating and 16S rDNA gene sequencing allowed definitive identification of bacterial species involved in the ruminal fermentation. The starch and fiber contents of the diets influenced the number of total anaerobic bacteria and fibrolytic and amylolytic functional groups. Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans were the main species isolated and identified. The identification of bacterial strains involved in the rumen fermentation helps to explain the ability of these animals to digest fiber plant cell wall contained in native forage species.

Evidence for extensive genetic diversity and substructuring of the Babesia bovis metapopulation.

Babesia bovis is a tick-transmitted haemoprotozoan and a causative agent of bovine babesiosis, a cattle disease that causes significant economic loss in tropical and subtropical regions. A panel of nineteen micro- and minisatellite markers was used to estimate population genetic parameters of eighteen parasite isolates originating from different continents, countries and geographic regions including North America (Mexico, USA), South America (Argentina, Brazil), the Middle East (Israel) and Australia. For eleven of the eighteen isolates, a unique haplotype was inferred suggesting selection of a single genotype by either in vitro cultivation or amplification in splenectomized calves. Furthermore, a high genetic diversity (H = 0.780) over all marker loci was estimated. Linkage disequilibrium was observed in the total study group but also in sample subgroups from the Americas, Brazil, and Israel and Australia. In contrast, corresponding to their more confined geographic origin, samples from Israel and Argentina were each found to be in equilibrium suggestive of random mating and frequent genetic exchange. The genetic differentiation (F(ST)) of the total study group over all nineteen loci was estimated by analysis of variance (Θ) and Nei's estimation of heterozygosity (G(ST')) as 0.296 and 0.312, respectively. Thus, about 30% of the genetic diversity of the parasite population is associated with genetic differences between parasite isolates sampled from the different geographic regions. The pairwise similarity of multilocus genotypes (MLGs) was assessed and a neighbour-joining dendrogram generated. MLGs were found to cluster according to the country/continent of origin of isolates, but did not distinguish the attenuated from the pathogenic parasite state. The distant geographic origin of the isolates studied allows an initial glimpse into the large extent of genetic diversity and differentiation of the B. bovis population on a global scale.

Search for Babesia bovis vaccine candidates.

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constrain for the development of cattle industries worldwide. Effective control can be achieved by vaccination with live attenuated forms of the parasite, but they have several drawbacks and thus the development of alternative subunit vaccines, either based in recombinant versions of full size proteins or in recombinant or synthetic peptides containing combinations of protective B-cell and T-cell epitopes is needed. Our current strategies for the identification of vaccine candidate antigens include the identification of functionally relevant antigens, bioinformatics, and comparative genomics using the recently sequenced B. bovis genome. These led us to the functional and immunological characterization of members of the VMSA gene family, a group of well conserved putative cysteine and serine proteases, and to the definition of a surface exposed B-cell epitope present in the Merozoite Surface Antigen-2c. Work in progress is focused in defining additional epitopes, and to determine whether they are neutralization-sensitive. These approaches might unravel useful vaccine candidates for B. bovis, and will increase our understanding of the pathogenicity mechanisms of these and related hemoparasites.

At least two genetically distinct large Babesia species infective to sheep and goats in China.

A fatal disease of sheep and goats in the northern part of China has been reported to be due to Babesia ovis. However, some characteristics of the causative agent in recent reports are not in accordance with the original attributes ascribed to this parasite. Therefore, the 18S small subunit ribosomal RNA (18S rRNA) genes of a number of Babesia isolates in China were sequenced and compared with that of other Babesia and Theileria species in an attempt to clarify their taxonomic position. In the present study, seven Babesia isolates were collected from distinct areas of northern China, and the 18S rRNA genes were amplified and sequenced. The phylogenetic trees were inferred based on 18S rRNA gene sequences of the Chinese ovine Babesia isolates and some of ovine Babesia and Theileria species available in GenBank. In the phylogenetic tree, Babesia sp. isolates from Madang, Tianzhu, Lintan, Ningxian, Hebei and Liaoning all grouped with B. motasi with 88.2-99.9% identity, while Babesia sp. Xinjiang grouped in a separate clade between B. ovis and B. crassa with 79.7-81.2% identity. The results indicated that there are at least two distinct Babesia species groups-B. motasi and Babesia sp. Xinjiang, the latter was distinctly different from other ovine Babesia isolates from China with less than 86.6% identity.

Application of the recombinant Theileria annulata surface protein in an indirect ELISA for the diagnosis of tropical theileriosis.

The recombinant surface protein of Theileria annulata (TaSP) was used in the standardization and validation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against tropical theileriosis. ELISA data were expressed as the percentage positivity (PP) of the reactivity of an internal positive control. A total of 50 sera samples from a disease-free area were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera samples. This was determined as the mean PP plus two standard deviations or the twice the mean PP of the results obtained with these negative samples. The obtained thresholds were 17.8% and 18.3%, respectively. Accordingly, the reactivity of 140 field sera samples collected at random from an area known to be endemic for tropical theileriosis in Sudan was determined as PP values which were then compared to the results obtained using the indirect fluorescence antibody test (IFAT) from the same samples. Both tests showed a high degree of correlation. The TaSP-ELISA had a sensitivity of 99.1% and specificity of 90.47% when taking the IFAT as a reference test. Our test has proved its suitability for the diagnosis of tropical theileriosis and could be used in serological surveys to map out the prevalence of the disease or to monitor vaccination efficiencies in disease-free populations.

Characterization of a polymorphic gene of T. lestoquardi and of a recently identified Theileria species pathogenic for small ruminants in China.

In the present study, we identified a gene from Theileria lestoquardi and from a recently described Theileria species which is highly pathogenic for small ruminants in China. Since the taxonomic position of the latter parasite is still not clear, we refer to it as Theileria (China) species. The gene described here comprises an open reading frame of about 948 bp which prospectively encodes a 35-kDa protein. Its sequence is most closely related to the polymorphic immunodominant membrane protein of T. parva (36% identity). A search for sequence patterns and motifs within the predicted amino acid sequence revealed that this gene possesses three membrane-spanning regions at its C-terminal part, suggesting that it is a membrane protein. Several allelic variants of this gene were found in each parasite species, demonstrating interspecies and intraspecies variation. The predicted amino acid sequence variants display a substantial size and sequence polymorphism in the central part of its presumed extracellular region, while the N-terminal and the transmembrane/intracellular regions are highly conserved.

Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70.

A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T. annulata-infected bovine lymphoblastoid cell line with an antibody raised against T. annulata schizonts. The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens). The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle. The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins. The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 degrees C in comparison with cells cultured at 37 degrees C. By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells. Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites. However, it was also found in small amounts in the cytoplasm of the parasite and host cell. This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm.

Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China.

A fatal disease of sheep and goats in the northwestern part of China has been reported to be due to Theileria lestoquardi (syn. T. hirci). However, some characteristics of the causative agent are not in accordance with attributes ascribed to this parasite. We therefore determined the nucleotide sequence of the small-subunit ribosomal RNA (srRNA) gene of T. lestoquardi and the parasite identified in China and compared it with that of other Theileria and Babesia species. In the inferred phylogenetic tree the srRNA sequence of the Chinese parasite was found to be most closely related to T. buffeli and clearly divergent from T. lestoquardi, suggesting that it is an as yet unrecognized Theileria species. Extensive structural similarities were observed between the srRNA sequences of T. lestoquardi and T. annulata, revealing a close phylogenetic relationship between these two Theileria species. On the basis of the srRNA nucleotide sequence, polymerase chain reaction (PCR) primers were designed that specifically amplified genomic DNA of the Chinese Theileria species. These primers may be valuable tools in future epidemiology studies.

Phylogenetic analysis by rRNA comparison of the highly pathogenic sheep-infecting parasites Theileria lestoquardi and a Theileria species identified in China.

In the Northwestern part of China there have been reports of clinical cases in small ruminants of a haemoparasite with the characteristics of Theileria hirci (T. lestoquardi). However, some properties of this parasites argue against its classification as T. lestoquardi. In this paper, we present evidence that T. lestoquardi and the Chinese Theileria isolate are distinct parasite species. Phylogenetic analysis of determined nucleotide sequences of small subunit ribosomal RNA (srRNA) genes of T. lestoquardi and the Chinese Theileria parasite show that they belong to different clades within the phylogenetic tree of piroplasms. The srRNA sequence of the Chinese parasite was found to be most closely related to T. buffeli, which, with T. sergenti, belongs to an evolutionary lineage of non-lymphoproliferative Theileria species. On the other hand, it was clearly divergent to a lineage of lymphoproliferative Theileria species; T. annulata, T. parva, T. taurotragi, and T. lestoquardi, the latter being most closely related to T. annulata.

Lymphocyte stimulatory capacity of Theileria annulata-infected ovine lymphoblastoid cells.

T. annulata, the causative agent of tropical theileriosis in cattle, is transmitted by ticks of the genus Hyalomma. Sporozoites of this parasite invade their target cells, where they differentiate to macroschizonts. T. annulata additionally invades and transforms ovine and caprine leukocytes. T. annulata infection in the ovine system is poorly studied, thus we used a mixed lymphocyte culture (MLC) to analyze the capacity of these cells to activate naïve uninfected ovine cells. The peak response was observed on day three or four and the response could not be induced by lysates of infected cells or their supernatants. The stimulated cells expressed IL-2 and secreted an IL-2-like growth factor.

Generation of cytotoxic T lymphocytes and cytostatic acting cells in T. annulata-immune cattle.

Cattle immunized against Theileria annulata with schizont containing autologous cell lines are immune to challenge with a homologous parasite strain. Two cell types have been detected in the peripheral blood of the immunized animals: cytotoxic T lymphocytes (CTL) and cytostatic acting cells (CAC). Killing the target cells by CTL is infection associated and is MHC class I restricted. Hence, no cytotoxicity was observed against target cells that were treated with the theilericidal drug buparvaquone or autologous Con A-blasts. The growth inhibition of CAC is MHC unrestricted, and not mediated by cytokine interferon gamma (IFN-gamma).

Proliferation and cytokine profile of T. annulata-infected ovine, caprine, and bovine lymphoblastoid cells.

T. annulata, the causative agent of tropical theileriosis in cattle, can also infect ovine and caprine leukocytes in vitro. In vivo studies showed that this parasite causes a mild infection in both these animal species, and in sheep merozoite stage development seems to be inhibited. Since the nature of T. annulata infected caprine and ovine cells is not known, all three cell lines were karyotyped and phenotypically characterized by flow cytometry. They all express mRNA of cytokines IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha, but not of IFN-gamma, IL-2, and IL-4. In contrast, IL-6 mRNA was expressed in the cattle cell line only, while mRNA of IL-10 was exclusively produced by the sheep cell line. The observed differences in cytokine mRNA expression may be responsible for the different pathogenesis of T. annulata infection in cattle and sheep.

TNF-alpha and HLA-DR genotyping as potential prognostic markers in pulmonary sarcoidosis.

We have analyzed the HLA-DRB1 alleles and -308 TNF-alpha gene polymorphism in 78 sarcoidosis patients and 50 controls. The sarcoidosis group as a whole did not show any significant correlation with the TNF-A or the HLA-DR alleles compared to the control group. However, the patient subgroups of Löfgren and non-Löfgren sarcoidosis exhibited significant allele associations. In the Löfgren patient group, the TNF-A2 and the HLA-DR3 alleles were represented significantly higher, with a highly significant relative risk resulting from the presence of the TNF-A2 or the HLA-DR3 allele or both. In the non-Löfgren patient group, the phenotype expressing HLA-DR2 and lacking TNF-A2 was significantly higher than in the Löfgren patient group. Due to these significant genetic differences in the subgroups of Löfgren and non-Löfgren sarcoidosis patients, we conclude that the genotyping of these two loci (-308 TNF-alpha promoter polymorphism and HLA-DR) may be of prognostic value for the course of disease in sarcoidosis.

Review: Theileria schizonts induce fundamental alterations in their host cells.

The sporozoites of Theileria annulata and T. parva invade bovine leukocytes, where they differentiate into schizonts. The latter can immortalize and induce fundamental changes in their host cells. T. annulata infects mainly major histocompatibility complex class II cells, whereas T. parva preferentially transforms T-lymphocytes, which proliferate continuously without the need for exogenously added growth factors. Most of the cell lines appear to be independent of a growth factor but may express several cytokines that influence the outcome of the disease. The mechanisms underlying this transformation are not well understood. The infected cells show increased activity of casein kinase II and Jun NH2-terminal kinase (JNK), whereas extracellular signal-related kinase 1 and 2 and P38 are not activated. In addition, several transcriptional factors such as NFkB and AP-1 are activated. It has been postulated that parasite proteins either expressed on the surface of the schizonts or secreted into the host cell cytoplasm may interfere with the signal-transduction pathway of the host cells. A possible candidate may the casein kinase II homologue that was identified in schizonts of both T. annulata and T. parva.

HLA DPA1/DPB1 genotype and haplotype frequencies, and linkage disequilibria in Nigeria, Liberia, and Gabon.

The frequencies of DPA1 and DPB1 alleles and their occurrence in haplotypic linkage were assessed and compared in Nigerian, Liberian, and Gabonese individuals. Differences were seen in the distribution patterns; these differences were more pronounced between the Gabonese and the other two populations than between Liberians and Nigerians. Several haplotypic DPA1-DPB1 combinations could be verified by homozygosity. Linkage disequilibria of DPA1-DPB1 combinations, indicating further probable haplotypes, were estimated. Although different allele and haplotype frequencies were recognized in the three subgroups, the linkage disequilibria were mostly either positive or negative in all populations.

HLA-DP control of human Schistosoma haematobium infection.

The DPA1 and DPB1 alleles of the major histocompatibility complex (MHC) class II were determined in 110 patients and 120 healthy controls of a Gabonese population from an area endemic for Schistosoma haematobium infection. The MHC-DP alleles of the variable second exons and their human leukocyte antigen (HLA) epitopes were correlated with egg excretion, interleukin-4 and interferon-gamma patterns, and bladder abnormalities, as detected by ultrasonography. A methionine at position 11 of the DP alpha molecule (Met-11) and DPA1*0301 were associated with schistosomiasis when compared with controls (phenotypic gene frequencies = 0.791 versus 0.583 and 0.555 versus 0.375, respectively). Met-11 homozygosity occurred more often in patients, whereas healthy controls were more frequently homozygous for an alanine at position 11 (Ala-11). The combination of the DPB1-epitope DEAV (positions 84-87 of the DP beta molecule) and Met-11 positive DPA1 alleles was more frequent in patients than in controls (0.573 versus 0.316). Two years after antischistosomal treatment, the rate of reinfection as examined in 55 of the 110 former patients was higher in DPA1*0301-positive individuals than in those not possessing this allele (P < 0.001). Ala-11 positive individuals showed less frequently ultrasonographic signs of bladder pathology than Ala-11 negative individuals (P < 0.05). Our results suggest a role of MHC-DP elements in the manifestation of disease in S. haematobium infection.

HLA DRB1-DQA1-DQB1 haplotype diversity in two African populations.

HLA class II DRB1-DQA1-DQB1 haplotypic polymorphism was determined in 120 Liberian and 230 Gabonese individuals. In our study groups, the number of allelic variants observed for each locus was similar to that found in non-African populations. However, 39 novel haplotypes and several yet unrecognized DRB1-DQA1 and DQA1-DQB1 combinations were identified. The extent of HLA-haplotypic variability in Africans appears to result from the high degree of allele combinations rather than from allelic polymorphism.