Application of immunofluorescence assay and nested polymerase chain reaction for query fever diagnosis in animal handlers of Puducherry, South India, and phylogenetic analysis based on IS1111 repetitive gene element

Background and Aim: Diagnosis of query fever (QF) is mostly done on the basis of serological/molecular tests, due to the stringent requirement of biosafety level-3 containment facilities for isolating Coxiella burnetii in culture. QF is an important zoonosis and is considered to be an occupational hazard to livestock handlers. This report describes our study on the serological as well as molecular evidence of QF in animal handlers from Puducherry and surrounding Tamil Nadu, from where, to the best of our knowledge, no such reports are available so far. Materials and Methods: Seventy-five animal handlers were recruited, comprising veterinarians, slaughterhouse workers, butchers, and animal attendants of various government veterinary clinics from Puducherry and surrounding areas of Tamil Nadu state. QF serology was performed to identify Phase I and Phase II immunoglobulin G antibodies to C. burnetii. Nested polymerase chain reaction (N-PCR) was carried out to detect C. burnetii DNA in buffy coat samples by targeting IS1111 gene element. N-PCR-positive samples were sequenced and phylogenetic analysis was performed using MEGA software version 10.0. Results: A total of 21 animal handlers (28.1%) were positive for either serology or PCR. PCR alone was positive in 10 (13.4%), only serology was positive in 8 (10.7%), and both serology and PCR were positive in three samples (4.0%). GenBank accession numbers were obtained for 13 N-PCR-positive samples (MG548608-MG548620). Six of our study sequences showed close similarity with the reference isolates from Bengaluru, Colombia, Brazil, France, and Iran. Conclusion: A significant percentage of QF positivity in animal handlers of this part of South India, Puducherry, warrants a prospective study with follow-up of a large number of this occupational group.

Correlation in Expression between LTR Retrotransposons and Potential Host Cis-Targets during Infection of Antherea pernyi with ApNPV Baculovirus

The published genome sequence of Antheraea yamamai (Saturnnidae) was used to construct a library of long terminal repeat (LTR)-retrotransposons that is representative of the wild silkmoth (Antherea) genus, and that includes 22,666 solo LTRs and 541 full-length LTRs. The LTR retrotransposons of Antheraea yamamai (AyLTRs) could be classified into the three canonical groups of Gypsy, Copia and Belpao. Eleven AyLTRs contained the env gene element, but the relationship with the env element of baculovirus, particularly A. yamamai and pernyi nucleopolyhedrovirus (AyNPV and ApNPV), was distant. A total of 251 “independent” full-length AyLTRs were identified that were located within 100 kb distance (downstream or upstream) of 406 neighboring genes in A. yamamai. Regulation of these genes might occur in cis by the AyLTRs, and the neighboring genes were found to be enriched in GO terms such as “response to stimulus”, and KEGG terms such as “mTOR signaling pathway” among others. Furthermore, the library of LTR-retrotransposons and the A. yamamai genome were used to identify and analyze the expression of LTR-retrotransposons and genes in ApNPV-infected and non-infected A. pernyi larval midguts, using raw data of a published transcriptome study. Our analysis demonstrates that 93 full-length LTR-retrotransposons are transcribed in the midgut of A. pernyi of which 12 significantly change their expression after ApNPV infection (differentially expressed LTR-retrotransposons or DELs). In addition, the expression of differentially expressed genes (DEGs) and neighboring DELs on the chromosome following ApNPV infection suggests the possibility of regulation of expression of DEGs by DELs through a cis mechanism, which will require experimental verification. When examined in more detail, it was found that genes involved in Notch signaling and stress granule (SG) formation were significantly up-regulated in ApNPV-infected A. pernyi larval midgut. Moreover, several DEGs in the Notch and SG pathways were found to be located in the neighborhood of particular DELs, indicating the possibility of DEG-DEL cross-regulation in cis for these two pathways.

Sequence Organization and Insertion Specificity of the Novel Chimeric ISHp609 Transposable Element of Helicobacter pylori

ABSTRACT Here we describe ISHp609 of Helicobacter pylori, a new member of the IS605 mobile element family that is novel and contains two genes whose functions are unknown, jhp960 and jhp961, in addition to homologs of two other H. pylori insertion sequence (IS) element genes, orfA, which encodes a putative serine recombinase-transposase, and orfB, whose homologs in other species are also often annotated as genes that encode transposases. The complete four-gene element was found in 10 to 40% of strains obtained from Africa, India, Europe, and the Americas but in only 1% of East Asian strains. Sequence comparison of 10 representative ISHp609 elements revealed higher levels of DNA sequence matches (99%) than those seen in normal chromosomal genes (88 to 98%) or in other IS elements (95 to 97% for IS605, IS606, and IS607) from the same H. pylori populations. Sequence analysis suggested that ISHp609 can insert at many genomic sites with its left end preferentially next to TAT, with no target specificity for its right end, and without duplicating or deleting target sequences. A deleted form of ISHp609, containing just jhp960 and jhp961 and 37 bp of orfA, found in reference strain J99, was at the same chromosomal site in 15 to 40% of the strains from many geographic regions but again in only 1% of the East Asian strains. The abundance and sequence homogeneity of ISHp609 and of this nonmobile remnant suggested a recent bottleneck and then rapid spread in H. pylori populations, possibly selected by the contributions of the elements to bacterial fitness.