Transcriptomics reveal potential vaccine antigens and a drastic increase of upregulated genes during Theileria parva development from arthropod to bovine infective stages

<div><p><i>Theileria parva</i> is a protozoan parasite transmitted by the brown ear tick <i>Rhipicephalus appendiculatus</i> that causes East Coast fever (ECF) in cattle, resulting in substantial economic losses in the regions of southern, eastern and central Africa. The schizont form of the parasite transforms the bovine host lymphocytes into actively proliferating cancer-like cells. However, how <i>T</i>. <i>parva</i> causes bovine host cells to proliferate and maintain a cancerous phenotype following infection is still poorly understood. On the other hand, current efforts to develop improved vaccines have identified only a few candidate antigens. In the present paper, we report the first comparative transcriptomic analysis throughout the course of <i>T</i>. <i>parva</i> infection. We observed that the development of sporoblast into sporozoite and then the establishment in the host cells as schizont is accompanied by a drastic increase of upregulated genes in the schizont stage of the parasite. In contrast, the ten highest gene expression values occurred in the arthropod vector stages. A comparative analysis showed that 2845 genes were upregulated in both sporozoite and schizont stages compared to the sporoblast. In addition, 647 were upregulated only in the sporozoite whereas 310 were only upregulated in the schizont. We detected low p67 expression in the schizont stage, an unexpected finding considering that p67 has been reported as a sporozoite stage-specific gene. In contrast, we found that transcription of p67 was 20 times higher in the sporoblast than in the sporozoite. Using the expression profiles of recently identified candidate vaccine antigens as a benchmark for selection for novel potential vaccine candidates, we identified three genes with expression similar to p67 and several other genes similar to Tp1—Tp10 schizont vaccine antigens. We propose that the antigenicity or chemotherapeutic potential of this panel of new candidate antigens be further investigated. Structural comparisons of the transcripts generated here with the existing gene models for the respective loci revealed indels. Our findings can be used to improve the structural annotation of the <i>T</i>. <i>parva</i> genome, and the identification of alternatively spliced transcripts.</p></div

Two Theileria parva CD8 T Cell Antigen Genes Are More Variable in Buffalo than Cattle Parasites, but Differ in Pattern of Sequence Diversity

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology/Principal Findings:&lt;/b&gt; Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (similar to 12%) in Tp1 and in 320 positions (similar to 61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions/Significance:&lt;/b&gt; The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.&lt;/p&gt

Two Theileria parva CD8 T Cell Antigen Genes Are More Variable in Buffalo than Cattle Parasites, but Differ in Pattern of Sequence Diversity

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology/Principal Findings:&lt;/b&gt; Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (similar to 12%) in Tp1 and in 320 positions (similar to 61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions/Significance:&lt;/b&gt; The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.&lt;/p&gt

Characterization of Salmonella isolates obtained from pigs slaughtered at Wambizzi Abattoir in Kampala, Uganda

Globally, non-typhoidal salmonellosis accounts for approximately 80.3 million cases of human infections annually. Estimates of salmonellosis due to consumption of pork or pork products is difficult to determine but, it ranges from < 1% to 25%. These invasive pathogens colonize intestinal mucosal surface but, they are self-limiting in health individuals due to a noble immunity. Utilization of antimicrobial agents in pig farming has been associated with the spread of resistant Salmonella species to man and the carrier status presents a major hazard to human health. This study examined 54 isolates for antimicrobial resistance, sequenced seven housekeeping genes, and performed Multi-Locus Sequence Type (MLST) analysis. We detected β-lactamase and tetA(B) genes in 100% and 80% of the isolates respectively. Data analysis using Codonbased Test of Neutrality analysis between sequences revealed P-value less than 0.05, an indication of strong forces of natural selection pressure acting at the sequence type level. Further data analysis using the Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution discovered frequencies of 0.177 (A), 0.244 (T/U), 0.263 (C), and 0.317 (G). The transition/transversion rate ratios were found to be k1 = 2.698 (purines) and k2 = 20.089 (pyrimidines) with an overall transition/transversion bias of R = 6.565, where R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)] further confirming that indeed the Salmonella isolates studied here were divergent. These results suggest that, Salmonella isolates of sequence types (STs) coexist in the intestine thereby providing for an efficient intestinal colonization and multiple adaptations. Our results offer general and rapid approaches for identifying genetic diversity of Salmonella serotypes in individual pig carcasses which can be adopted for molecular epidemiological surveys of important food contaminating bacterial pathogens

Prevalence and molecular characterization of human noroviruses and sapoviruses in Ethiopia

Viral gastroenteritis is a major public health problem worldwide. In Ethiopia, very limited studies have been done on the epidemiology of enteropathogenic viruses. The aim of this study was to detect and characterize noroviruses (NoVs) and sapoviruses (SaVs) from acute gastroenteritis patients of all ages. Fecal samples were collected from diarrheic patients (n = 213) in five different health centers in Addis Ababa during June-September 2013. The samples were screened for caliciviruses by reverse transcription polymerase chain reaction (RT-PCR) using universal and genogroup-specific primer pairs. Phylogenetic analyses were conducted using the sequences of the PCR products. Of the clinical samples, 25.3 % and 4.2 % were positive for NoV and SaV RNA, respectively. Among the norovirus positives, 22 were sequenced further, and diverse norovirus strains were identified: GI (n = 4), GII (n = 17) and GIV (n = 1). Most strains were GII (n = 17/22: 77.2 %), which were further divided into three different genotypes (GII.4, GII.12/GII.g recombinant-like and GII.17), with GII.17 being the dominant (7/17) strain detected. GI noroviruses, in particular GI.4 (n = 1), GI.5 (n = 2) and GI.8 (n = 1), were also detected and characterized. The GIV strain detected is the first from East Africa. The sapoviruses sequenced were also the first reported from Ethiopia. Collectively, this study showed the high burden and diversity of noroviruses and circulation of sapoviruses in diarrheic patients in Ethiopia. Continued surveillance to assess their association with diarrhea is needed to define their epidemiology, disease burden, and impact on public health

Cryptosporidium species detected in calves and cattle in Dagoretti, Nairobi, Kenya

A total of 1,734 cattle faecal samples from 296 dairy-keeping households were collected from urban settings in Nairobi, Kenya. Modified Ziehl–Neelsen staining method and an immunofluorescence assay were used to identify those samples with Cryptosporidium oocyst infection. Oocysts from positive faecal samples were isolated by Sheather's sucrose flotation method and picked from the concentrate using cover slips. Genomic DNA was extracted from 124 of the faecal samples that were positive for Cryptosporidium and was used as template for nested PCR of the 18S rRNA gene. Twenty-five samples (20 %) were PCR-positive for Cryptosporidium, and 24 of the PCR products were successfully cloned and sequenced. Sequence and phylogenetic analysis identified 17 samples (68 %) as Cryptosporidium parvum-like, four samples (16 %) as Cryptosporidium ryanae, three samples (12 %) as Cryptosporidium andersoni and one sample (4 %) as Cryptosporidium hominis. To the best of our knowledge, this is the first genotyping study to report C. parvum-like, C. andersoni and C. hominis in cattle from Kenya. The results of this study show Cryptosporidium infections in calves and cattle may be potential zoonotic reservoirs of the parasite that infects humans