Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi

Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLex-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLex sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLex but express the structurally similar glycan, 6-sulfo-sLex, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLex antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLex antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilumbinding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection

The structure of a conserved telomeric region associated with variant antigen loci in the blood parasite Trypanosoma congolense

African trypanosomiasis is a vector-borne disease of humans and livestock caused by African trypanosomes (Trypanosoma spp.). Survival in the vertebrate bloodstream depends on antigenic variation of Variant Surface Glycoproteins (VSGs) coating the parasite surface. In T. brucei, a model for antigenic variation, monoallelic VSG expression originates from dedicated VSG expression sites (VES). Trypanosoma brucei VES have a conserved structure consisting of a telomeric VSG locus downstream of unique, repeat sequences, and an independent promoter. Additional protein-coding sequences, known as “Expression Site Associated Genes (ESAGs)”, are also often present and are implicated in diverse, bloodstream-stage functions. Trypanosoma congolense is a related veterinary pathogen, also displaying VSG-mediated antigenic variation. A T. congolense VES has not been described, making it unclear if regulation of VSG expression is conserved between species. Here, we describe a conserved telomeric region associated with VSG loci from long-read DNA sequencing of two T. congolense strains, which consists of a distal repeat, conserved noncoding elements and other genes besides the VSG; although these are not orthologous to T. brucei ESAGs. Most conserved telomeric regions are associated with accessory minichromosomes, but the same structure may also be associated with megabase chromosomes. We propose that this region represents the T. congolense VES, and through comparison with T. brucei, we discuss the parallel evolution of antigenic switching mechanisms, and unique adaptation of the T. brucei VES for developmental regulation of bloodstream-stage genes. Hence, we provide a basis for understanding antigenic switching in T. congolense and the origins of the African trypanosome VES

Comparative Genomics Identifies a Potential Marker of Human-Virulent Anaplasma phagocytophilum

We have previously described a comparative genome analysis of nine strains of Anaplasma phagocytophilum that showed similarity between strains infecting humans and U.S. dogs and a more distant relationship with horse and ruminant strains. This suggested that it may be possible to distinguish human-infective strains using simple DNA sequence-based diagnostic tests. This would be of epidemiologic significance in identifying and tracking the presence of virulent strains in tick vector populations. Further analysis identified a gene that was present in several strains, including U.S. Ap-variant 1 (ruminant), MRK (horse), and European sheep, but was deleted in strains infecting U.S. humans and dogs, suggesting that it could be a useful marker of human virulence. A simple PCR test was developed to identify the presence/absence of this gene. The PCR test discriminated A. phagocytophilum strains from clinically affected humans and U.S. dogs from the strains more distantly related in genome sequence. This warrants further testing of globally diverse A. phagocytophilum strains to examine world-wide conservation of this gene

Multistrain genome analysis identifies candidate vaccine antigens of Anaplasma marginale

Anaplasmosis in domestic livestock is an impediment to animal health and production worldwide, especially in developing countries in Africa, Asia, and South America. Vaccines have been developed and marketed against the causative organism, Anaplasma marginale; however, these have not been widely used because of breakthrough infections caused by heterologous strains and because of the risk of disease induced by live vaccine strains themselves. Recently, molecular studies have enabled progress to be made in understanding the causes for breakthrough infections and in defining new vaccine targets. A. marginale has a system for antigenic variation of the MSP2 and MSP3 outer membrane proteins which are members of the pfam01617 gene superfamily. In this study, we used high throughput genome sequencing to define conservation of different superfamily members in ten U.S. strains of A. marginale and also in the related live vaccine strain A. marginale subspecies centrale. The comparisons included the pseudogenes that contribute to antigenic variation and other superfamily-encoded outer membrane proteins. Additionally, we examined conservation of other proteins proposed previously as vaccine candidates. These data showed significantly increased numbers of SNPs in A. marginale subspecies centrale when compared to all U.S. A. marginale strains. We defined a catalog of 19 conserved candidate vaccine antigens that may be suitable for development of a multi-component recombinant vaccine. The methods described are rapid and may be suitable for other prokaryotes where repeats comprise a substantial portion of their genomes

An Emerging Tick-Borne Disease of Humans Is Caused by a Subset of Strains with Conserved Genome Structure

The prevalence of tick-borne diseases is increasing worldwide. One such emerging disease is human anaplasmosis. The causative organism, Anaplasma phagocytophilum, is known to infect multiple animal species and cause human fatalities in the U.S., Europe and Asia. Although long known to infect ruminants, it is unclear why there are increasing numbers of human infections. We analyzed the genome sequences of strains infecting humans, animals and ticks from diverse geographic locations. Despite extensive variability amongst these strains, those infecting humans had conserved genome structure including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen. These data provide potential targets to identify human-infective strains and have significance for understanding the selective pressures that lead to emergence of disease in new species

Plasmodium vivax merozoite surface proteins-3 beta and-3 gamma share structural similarities with P. vivax merozoite surface protein-3 alpha and define a new gene family

The National Institutes of Allergy and Infectious Diseases of the National Institutes of Health (AI24710-14), the UNDP/WORLD BANK/WHO Special Programme for Research and in Tropical Diseases (TDR), and the US Agency for International Development Malaria Vaccine Development ProgramEmory University. Department of Medicine. Emory Vaccine Research Center. Yerkes Primate Research Center. Atlanta, GA, USA.New York University. School of Medicine. New York, NY, USA.Emory University. Department of Medicine. Emory Vaccine Research Center. Yerkes Primate Research Center. Atlanta, GA, USA.Emory University. Department of Medicine. Emory Vaccine Research Center. Yerkes Primate Research Center. Atlanta, GA, USA.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Centers for Disease Control and Prevention. Division of Parasitic Diseases. Chamblee, GA, USA.The genes encoding two merozoite surface proteins of Plasmodium i ax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. i ax gt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. i ax DASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75 677 Da, and 3 and 5 RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. i ax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. i ax genes have putative signal peptides, large central domains that contain 20% alanine residues bound by charged regions, are predicted to form -helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3, Pvmsp-3 , and Pvmsp-3. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparu