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Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

ABSTRACT: To determine potential intermediate hosts of Oxyspirura petrowi, a common nematode eyeworm of wild gallinaceous birds, various arthropod species including red harvester ants, beetles, wood cockroaches, crickets, grasshoppers, katydids, and desert termites were screened for the presence of O. petrowi using specific polymerase chain reaction (PCR) primers targeting the internal transcribed spacer 2 region (ITS2) of the eyeworm ribosomal deoxyribonucleic acid (rDNA). This is the first study to investigate the intermediate hosts of O. petrowi utilizing molecular techniques. We determined 38% (13/34) of the cockroaches, 27% (3/11) of the crickets, and 23% (68/289) of the grasshoppers which were positive for O. petrowi. Identifying potential intermediate hosts of O. petrowi is essential to better understanding the epizoology of the eyeworm's transmission mechanics and to controlling infections in wild gallinaceous birds.

SUBMITTER: Almas S

PROVIDER: S-EPMC5846834 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

Almas Sadia S Gibson Anna G AG Presley Steven M SM

Parasitology research 20180125 3

To determine potential intermediate hosts of Oxyspirura petrowi, a common nematode eyeworm of wild gallinaceous birds, various arthropod species including red harvester ants, beetles, wood cockroaches, crickets, grasshoppers, katydids, and desert termites were screened for the presence of O. petrowi using specific polymerase chain reaction (PCR) primers targeting the internal transcribed spacer 2 region (ITS2) of the eyeworm ribosomal deoxyribonucleic acid (rDNA). This is the first study to inve ...[more]

PMID: 29372319

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Project description:BACKGROUND: Oxyspirura petrowi appears to be emerging as a nematode parasite that could negatively impact Northern Bobwhite quail individuals and populations within Texas and other regions of the United States. Despite this eye worm's potential importance in the conservation of wild quail, little is known about the general biology and genome composition of O. petrowi. To fill the knowledge gap, we performed a small scale random genome sequence survey, sequenced its 18S rRNA and the intergenic region between the 18S and 28S rRNA genes, studied its phylogenetic affinity, and developed a PCR protocol for the detection of this eye worm. RESULTS: We have generated ~240 kb of genome sequence data derived from 348 clones by a random genome survey of an O. petrowi genomic library. The eye worm genome is AT-rich (i.e., 62.2% AT-content), and contains a high number of microsatellite sequences. The discovered genes encode a wide-range of proteins including hypothetical proteins, enzymes, nematode-specific proteins. Phylogenetic analysis based on 18S rRNA sequences indicate that the Spiruroidea is paraphyletic, in which Oxyspirura and its closely related species are sisters to the filarial nematodes. We have also developed a PCR protocol based on the ITS2 sequence that allows sensitive and specific detection of eye worm DNA in feces. Using this newly developed protocol, we have determined that ~28% to 33% of the fecal samples collected from Northern Bobwhites and Scaled Quail in Texas in the spring of 2013 are O. petrowi positive. CONCLUSIONS: The O. petrowi genome is rich in microsatellite sequences that may be used in future genotyping and molecular fingerprinting analysis. This eye worm is evolutionarily close to the filarial nematodes, implying that therapeutic strategies for filariasis such as Loa loa would be referential in developing treatments for the Thelazoidea parasites. Our qPCR-based survey has confirmed that O. petrowi infection is of potential concern to quail managers in Texas.

Project description:BackgroundSnails of the genus Biomphalaria are intermediate hosts of Schistosoma mansoni, the causative agent of the human intestinal schistosomiasis. Two Biomphalaria species (Biomphalaria pfeifferi and Biomphalaria camerunensis) are involved in the transmission in Cameroon, where the disease is present nationwide. However, difficulty in the identification of both vectors impedes proper assessment of the epidemiological burden caused by each species. To overcome this issue, we designed a PCR-based molecular diagnostic tool to improve the identification of these species.MethodsWe analyzed the internal transcribed spacer 2 (ITS2) region of Biomphalaria ribosomal DNA (rDNA) using polymerase chain reaction amplification (PCR) and restriction fragment length polymorphism (RFLP).ResultsThe amplification of the ITS2 region of Biomphalaria snails resulted in a 490 bp fragment and produced two profiles for each species after digestion with the restriction enzyme Hpa II. The profile 1 (Bc-HpaII-1: 212-bp and 139-bp bands) for B. camerunensis, was common in all the sampling points; the profile 2 (Bc-HpaII-2: 212-bp and 189-bp bands), was only observed in the Lake Monoun Njindoun sampling site. Biomphalaria pfeifferi profile 1 (Bpf-HpaII-1: 211-bp and 128-bp bands) was common in most of B. pfeifferi sampling points; the profile 2 (Bpf-HpaII-2: 289-bp and 128-bp bands) was only observed in Mokolo (Far North Cameroon).The second restriction enzyme TaqαI, revealed three band profiles, Bc-TaqαI-1 (243-bp, 136-bp and 118-bp bands) and Bc-TaqαI-2 (244-bp, 136-bp and 99-bp) for B. camerunensis and Bpf-TaqαI-1 (242-bp, 135-bp and 107-bp bands) for B. pfeifferi. Sequencing analysis revealed the occurrence of six haplotypes for B. camerunensis and three haplotypes for B. pfeifferi. The level of gene flow was low and the Biomphalaria populations were not in demographic expansion according to neutrality tests (Tajima's D and Fu's Fs).ConclusionsThe PCR-RFLP technique revealed genetic diversity in Biomphalaria snails, and the combination with the morphological method could improve the identification of B. pfeifferi and B. camerunensis in Cameroon. This could help focus on the infection to evaluate the transmission risk with respect of the different species and to develop efficient and cost-effective control measures.

Project description:Background Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. Methodology/Principal Findings The majority (~80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30–80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. Conclusions/Significance Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms.

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Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

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Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

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