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The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/[mu]l (25[mu]l volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

We found serologic evidence of spotted fever group Rickettsia in humans and dogs and typhus group Rickettsia in dogs in Reynosa, Mexico. Our investigation revealed serologic samples reactive to spotted fever group Rickettsia in 5 community members, which highlights a potential rickettsial transmission scenario in this region.

Key Points Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular bacterial pathogens that reside in distinct compartments in host cells during infection. Successful infection by both pathogens requires a functionally exchangeable type IV secretion system called Dot/Icm, which translocates hundreds of virulence factors, termed effectors, into host cells. The majority of Legionella spp. and Coxiella spp. effectors are unique to these pathogens, and functional redundancy exists among many of them. Functional domains that are associated with most of these effectors are enigmatic and cannot be readily predicted by currently available bioinformatics tools. Legionella spp. and Coxiella spp. promote intracellular bacterial replication by interfering with host gene expression through effectors that impose epigenetic modifications on host chromatin by different mechanisms. L. pneumophila extensively manipulates the early phases of the secretory branch of the host vesicle trafficking pathway by hijacking the activity of key regulatory proteins such as RAB small GTPases via multiple effectors. L. pneumophila effectors function coordinately to alter the composition of lipids, such as phosphoinositides, on the vacuole that contains the bacterium and other organelles to facilitate its intracellular growth. L. pneumophila co-opts the ubiquitin network of host cells by effectors that function through diverse biochemical mechanisms, including the SidE family effectors, which catalyse ubiquitylation by an E1 enzyme and E2 enzyme-independent mechanism, which represents a paradigm shift in our understanding of this important post-translational modification. The intracellular pathogens Legionella pneumophila and Coxiella burnetii use the Dot/Icm type IV secretion system to translocate effectors into host cells. Qiu and Luo explore the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence. Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular pathogens that use the Dot/Icm type IV secretion system to translocate effectors into host cells. These effectors are essential for the establishment of membrane-bound compartments known as replication vacuoles, which enable the survival and replication of bacteria inside host cells. The effectors interfere with diverse signalling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, to promote pathogen survival. In this Review, we explore Dot/Icm effectors from L. pneumophila and C. burnetii as key virulence factors, and we examine the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence.

Background Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide. Methods A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n  = 3,995; Jeollabuk, n  = 680; Chungcheongnam, n  = 1,478; and Chungcheongbuk, n  = 331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17 kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR). Results Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larvae), and Ixodes nipponensis (431; 25 adults, 5 nymphs and 401 larvae). The minimum field infection rate/100 ticks (assuming 1 positive tick/pool) was 0.93% for the 17 kDa antigen gene and 0.82% for the ompA nPCR assays. The partial 17 kDa antigen and ompA gene sequences from positive pools of H. longicornis were similar to: Rickettsia sp. HI550 (99.4–100%), Rickettsia sp. FUJ98 (99.3–100%), Rickettsia sp. HIR/D91 (99.3–100%), and R. japonica (99.7%). One sequence of the partial 17 kDa antigen gene for H. flava was similar to Rickettsia sp. 17kd-005 (99.7%), while seven sequences of the 17 kDa antigen gene obtained from I. nipponensis ticks were similar to R. monacensis IrR/Munich (98.7–100%) and Rickettsia sp. IRS3 (98.9%). Conclusions SFG rickettsiae were detected in three species of ixodid ticks collected in the southwestern provinces of the ROK during 2013. A number of rickettsiae have been recently reported from ticks in Korea, some of which were identified as medically important. Results from this study and previous reports demonstrate the need to conduct longitudinal investigations to identify tick-borne rickettsiae and better understand their geographical distributions and potential impact on medical and veterinary health, in addition to risk communication and development of rickettsial disease prevention strategies.

The work has uncovered a breathtaking amount of microbial diversity in samples ranging from soil to permafrost, marine sponges, hydrothermal vents and the crevices of the human body. Since adopting the new 'axenic' or host-cell-free culture technique, the C. burnetii field has expanded.

Background: Emergent vector-borne diseases have gained significant attention in recent years due to their increasing prevalence and impact on public health. With its vast geographic and ecological diversity, Algeria has limited available data on the distribution and prevalence of neglected vector-borne diseases. This study aimed to inventory hematopha­gous ectoparasites, including ticks and fleas, collected from domestic and wild animals such as dogs, hedgehogs, cattle, and rodents across diverse biotopes in northwestern Algeria (Mascara, Oran, Tlemcen, Sidi Bel Abbes, Mostaganem, Tiaret, and Ain Temouchent) and southern Algeria (Laghouat). Methods: A total of 984 arthropods, comprising 609 ticks and 375 fleas, were collected from domestic and wild an­i­mals. Among these, 193 ticks and 105 fleas underwent molecular screening for Rickettsia spp. using gltA and ompA gene-specific primers. Results: The minimum infection rate (MIR) for Rickettsia spp. was estimated at 6.37%, assuming one positive individ­ual per pool. Quantitative PCR revealed the presence of Rickettsia massiliae in 1/68 (1.47%) of Rhipicephalus san­guineus ticks and Rickettsia felis in 7/48 (14.58%) of Ctenocephalides felis fleas. Additionally, a novel strain of Rickett­sia sp. was identified in Rhipicephalus sanguineus and Rhipicephalus turanicus. Conclusion: This study expands the understanding of tick- and flea-borne Rickettsia species in Algeria, highlighting the diverse range of ectoparasite-borne pathogens associated with domestic and wild animals. The findings underscore the importance of continued surveillance and molecular characterization to address the public health risks posed by these pathogens.  

Ticks and other arthropods often are hosts to nutrient providing bacterial endosymbionts, which contribute to their host's fitness by supplying nutrients such as vitamins and amino acids. It has been detected, in our lab, that Ixodes pacificus is host to Rickettsia species phylotype G021. This endosymbiont is predominantly present, and 100% maternally transmitted in I. pacificus. To study roles of phylotype G021 in I. pacificus, bioinformatic and molecular approaches were carried out. MUMmer genome alignments of whole genome sequence of I. scapularis, a close relative to I. pacificus, against completely sequenced genomes of R. bellii OSU85-389, R. conorii, and R. felis, identified 8,190 unique sequences that are homologous to Rickettsia sequences in the NCBI Trace Archive. MetaCyc metabolic reconstructions revealed that all folate gene orthologues (folA, folC, folE, folKP, ptpS) required for de novo folate biosynthesis are present in the genome of Rickettsia buchneri in I. scapularis. To examine the metabolic capability of phylotype G021 in I. pacificus, genes of the folate biosynthesis pathway of the bacterium were PCR amplified using degenerate primers. BLAST searches identified that nucleotide sequences of the folA, folC, folE, folKP, and ptpS genes possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to the corresponding genes of Rickettsia buchneri. Phylogenetic tree constructions show that the folate genes of phylotype G021 and homologous genes from various Rickettsia species are monophyletic. This study has shown that all folate genes exist in the genome of Rickettsia species phylotype G021 and that this bacterium has the genetic capability for de novo folate synthesis.

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Fragmentation of the Golgi apparatus is observed during a number of physiological processes including mitosis and apoptosis, but also occurs in pathological states such as neurodegenerative diseases and some infectious diseases. Here we show that highly virulent strains of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, induce selective fragmentation of the trans-Golgi network (TGN) soon after infection of host cells by secretion of the effector protein Rickettsial Ankyrin Repeat Protein 2 (RARP2). Remarkably, this fragmentation is pronounced for the trans-Golgi network but the cis-Golgi remains largely intact and appropriately localized. Thus R. rickettsii targets specifically the TGN and not the entire Golgi apparatus. Dispersal of the TGN is mediated by the secreted effector protein RARP2, a recently identified type IV secreted effector that is a member of the clan CD cysteine proteases. Site-directed mutagenesis of a predicted cysteine protease active site in RARP2 prevents TGN disruption. General protein transport to the cell surface is severely impacted in cells infected with virulent strains of R. rickettsii. These findings suggest a novel manipulation of cellular organization by an obligate intracellular bacterium to determine interactions with the host cell.

In 2015, we evaluated 221 patients with undifferentiated fever and tick bite or animal exposure in Xinyang, China, for Rickettsia infection. Three with mild disease were infected with Candidatus R. xinyangensis, which clustered with R. fournieri and R. vini in phylogenetic analyses. Field investigations suggest Haemaphysalis longicornis ticks might be involved in transmission.