Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Novel PhoH-encoding vibriophages with lytic activity against environmental Vibrio strains.

Cholera is a devastating diarrheal disease that accounts for more than 10% of children's lives worldwide, but its treatment is hampered by a rise in antibiotic resistance. One promising alternative to antibiotic therapy is the use of bacteriophages to treat antibiotic-resistant cholera infections, and control Vibrio cholera in clinical cases and in the environment, respectively. Here, we report four novel, closely related environmental myoviruses, VP4, VP6, VP18, and VP24, which we isolated from two environmental toxigenic Vibrio cholerae strains from river Kuja and Usenge beach in Kenya. High-throughput sequencing followed by bioinformatics analysis indicated that the genomes of the four bacteriophages have closely related sequences, with sizes of 148,180 bp, 148,181 bp, 148,179 bp, and 148,179 bp, and a G + C content of 36.4%. The four genomes carry the phoH gene, which is overrepresented in marine cyanophages. The isolated phages displayed a lytic activity against 15 environmental, as well as one clinical, Vibrio cholerae strains. Thus, these novel lytic vibriophages represent potential biocontrol candidates for water decontamination against pathogenic Vibrio cholerae and ought to be considered for future studies of phage therapy.

Pathogen Carriage by Peri-Domestic Fleas in Western Kenya.

Fleas are carriers for many largely understudied zoonotic, endemic, emerging, and re-emerging infectious disease agents, but little is known about their prevalence and role as a vector in Africa. The aim of this study was to determine the diversity of fleas and the prevalence of infectious agents in them collected from human dwellings in western Kenya. A total of 306 fleas were collected using light traps from 33 human dwellings; 170 (55.56%) were identified as Ctenocephalides spp., 121 (39.54%) as Echidnophaga gallinacea, 13 (4.25%) as Pulex irritans, and 2 (0.65%) as Xenopsylla cheopis. Of the 306 individual fleas tested, 168 (54.9%) tested positive for rickettsial DNA by a genus-specific quantitative real-time PCR (qPCR) assay based on the 17-kDa antigen gene. Species-specific qPCR assays and sequencing revealed presence of Rickettsia asembonensis in 166 (54.2%) and Rickettsia felis in 2 (0.7%) fleas. Borrelia burgdorferi, normally known to be carried by ticks, was detected in four (1.3%) flea DNA preparations. We found no evidence of Yersinia pestis, Bartonella spp., or Orientia spp. Not only were Ctenocephalides spp. the most predominant flea species in the human dwellings, but also almost all of them were harboring R. asembonensis.

A Review of Scrub Typhus (Orientia tsutsugamushi and Related Organisms): Then, Now, and Tomorrow.

Scrub typhus and the rickettsial diseases represent some of the oldest recognized vector-transmitted diseases, fraught with a rich historical aspect, particularly as applied to military/wartime situations. The vectors of Orientia tsutsugamushi were once thought to be confined to an area designated as the Tsutsugamushi Triangle. However, recent reports of scrub typhus caused by Orientia species other than O. tsutsugamushi well beyond the limits of the Tsutsugamushi Triangle have triggered concerns about the worldwide presence of scrub typhus. It is not known whether the vectors of O. tsutsugamushi will be the same for the new Orientia species, and this should be a consideration during outbreak/surveillance investigations. Additionally, concerns surrounding the antibiotic resistance of O. tsutsugamushi have led to considerations for the amendment of treatment protocols, and the need for enhanced public health awareness in both the civilian and medical professional communities. In this review, we discuss the history, outbreaks, antibiotic resistance, and burgeoning genomic advances associated with one of the world's oldest recognized vector-borne pathogens, O. tsutsugamushi.

Rickettsial Infections among Cats and Cat Fleas in Riverside County, California.

Presently, few studies have investigated the role of domestic cats (Felis catus) in the recrudescence of flea-borne rickettsioses in California and the southern United States. In this study, we aimed to investigate the presence of Rickettsia typhi or Rickettisa felis in domestic cats (F. catus) and the fleas (primarily Ctenocephalides felis, the cat flea) associated with these cats in Riverside County, California. Thirty cats and 64 pools of fleas collected from these cats were investigated for rickettsial infections. Three cats and 17 flea pools (from 10 cats) tested positive for rickettsial infections. polymerase chain reaction and DNA sequencing indicated that one of the cats was positive for R. felis infections, whereas two were positive for Candidatus Rickettsia senegalensis infection. In addition, 12 of the flea pools were positive for R. felis, whereas five were positive for Ca. R. senegalensis. By contrast, no cats or their associated fleas tested positive for R. typhi. Finally, eight sera from these cats contained spotted fever group Rickettsia (SFGR) antibodies. The detection of R. felis and SFGR antibodies and the lack of R. typhi and TGR antibodies support R. felis as the main rickettsial species infecting cat fleas. The detection of Ca. R. senegalensis in both fleas and cats also provides additional evidence that cats and their associated fleas are infected with other R. felis-like organisms highlighting the potential risk for human infections with R. felis or R. felis-like organisms.

A 2015 outbreak of flea-borne rickettsiosis in San Gabriel Valley, Los Angeles County, California.

Although flea-borne rickettsiosis is endemic in Los Angeles County, outbreaks are rare. In the spring of 2015 three human cases of flea-borne rickettsiosis among residents of a mobile home community (MHC) prompted an investigation. Fleas were ubiquitous in common areas due to presence of flea-infested opossums and overabundant outdoor cats and dogs. The MHC was summarily abated in June 2015, and within five months, flea control and removal of animals significantly reduced the flea population. Two additional epidemiologically-linked human cases of flea-borne rickettsiosis detected at the MHC were suspected to have occurred before control efforts began. Molecular testing of 106 individual and 85 pooled cat fleas, blood and ear tissue samples from three opossums and thirteen feral cats using PCR amplification and DNA sequencing detected rickettsial DNA in 18.8% of the fleas. Seventeen percent of these cat fleas tested positive for R. felis-specific DNA compared to under two (<2) percent for Candidatus R. senegalensis-specific DNA. In addition, serological testing of 13 cats using a group-specific IgG-ELISA detected antibodies against typhus group rickettsiae and spotted fever group rickettsiae in six (46.2%) and one (7.7%) cat, respectively. These results indicate that cats and their fleas may have played an active role in the epidemiology of the typhus group and/or spotted fever group rickettsial disease(s) in this outbreak.

Rickettsia asembonensis Characterization by Multilocus Sequence Typing of Complete Genes, Peru.

While studying rickettsial infections in Peru, we detected Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions (17kDa, gltA, ompA, ompB, and sca4) and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.

Worldwide Presence and Features of Flea-Borne Rickettsia asembonensis.

Rickettsia asembonensis, the most well-characterized rickettsia of the Rickettsia felis-like organisms (RFLO), is relatively unknown within the vector-borne diseases research community. The agent was initially identified in peri-domestic fleas from Asembo, Kenya in an area in which R. felis was associated with fever patients. Local fleas collected from domestic animals and within homes were predominately infected with R. asembonensis with < 10% infected with R. felis. Since the identification of R. asembonensis in Kenya, it has been reported in other locations within Africa, Asia, the Middle East, Europe, North America, and South America. With the description of R. asembonensis-like genotypes across the globe, a need exists to isolate these R. asembonensis genotypes in cell culture, conduct microscopic, and biological analysis, as well as whole genome sequencing to ascertain whether they are the same species. Additionally, interest has been building on the potential of R. asembonensis in infecting vertebrate hosts including humans, non-human primates, dogs, and other animals. The current knowledge of the presence, prevalence, and distribution of R. asembonensis worldwide, as well as its arthropod hosts and potential as a pathogen are discussed in this manuscript.

Molecular characterization of novel mosquito-borne Rickettsia spp. from mosquitoes collected at the Demilitarized Zone of the Republic of Korea.

Rickettsiae are associated with a diverse range of invertebrate hosts. Of these, mosquitoes could emerge as one of the most important vectors because of their ability to transmit significant numbers of pathogens and parasites throughout the world. Recent studies have implicated Anopheles gambiae as a potential vector of Rickettsia felis. Herein we report that a metagenome sequencing study identified rickettsial sequence reads in culicine mosquitoes from the Republic of Korea. The detected rickettsiae were characterized by a genus-specific quantitative real-time PCR assay and sequencing of rrs, gltA, 17kDa, ompB, and sca4 genes. Three novel rickettsial genotypes were detected (Rickettsia sp. A12.2646, Rickettsia sp. A12.2638 and Rickettsia sp. A12.3271), from Mansonia uniformis, Culex pipiens, and Aedes esoensis, respectively. The results underscore the need to determine the Rickettsia species diversity associated with mosquitoes, their evolution, distribution and pathogenic potential.

Molecular and serological evidence of flea-associated typhus group and spotted fever group rickettsial infections in Madagascar.

BACKGROUND: Rickettsiae are obligate intracellular bacteria responsible for many febrile syndromes around the world, including in sub-Saharan Africa. Vectors of these pathogens include ticks, lice, mites and fleas. In order to assess exposure to flea-associated Rickettsia species in Madagascar, human and small mammal samples from an urban and a rural area, and their associated fleas were tested. RESULTS: Anti-typhus group (TGR)- and anti-spotted fever group rickettsiae (SFGR)-specific IgG were detected in 24 (39%) and 21 (34%) of 62 human serum samples, respectively, using indirect ELISAs, with six individuals seropositive for both. Only two (2%) Rattus rattus out of 86 small mammals presented antibodies against TGR. Out of 117 fleas collected from small mammals, Rickettsia typhi, a TGR, was detected in 26 Xenopsylla cheopis (24%) collected from rodents of an urban area (n = 107), while two of these urban X. cheopis (2%) were positive for Rickettsia felis, a SFGR. R. felis DNA was also detected in eight (31%) out of 26 Pulex irritans fleas. CONCLUSIONS: The general population in Madagascar are exposed to rickettsiae, and two flea-associated Rickettsia pathogens, R. typhi and R. felis, are present near or in homes. Although our results are from a single district, they demonstrate that rickettsiae should be considered as potential agents of undifferentiated fever in Madagascar.

Rickettsial Infections among Ctenocephalides felis and Host Animals during a Flea-Borne Rickettsioses Outbreak in Orange County, California.

Due to a resurgence of flea-borne rickettsioses in Orange County, California, we investigated the etiologies of rickettsial infections of Ctenocephalides felis, the predominant fleas species obtained from opossums (Didelphis virginiana) and domestic cats (Felis catus), collected from case exposure sites and other areas in Orange County. In addition, we assessed the prevalence of IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) rickettsiae in opossum sera. Of the 597 flea specimens collected from opossums and cats, 37.2% tested positive for Rickettsia. PCR and sequencing of rickettsial genes obtained from C. felis flea DNA preparations revealed the presence of R. typhi (1.3%), R. felis (28.0%) and R. felis-like organisms (7.5%). Sera from opossums contained TGR-specific (40.84%), but not SFGR-specific antibodies. The detection of R. felis and R. typhi in the C. felis fleas in Orange County highlights the potential risk for human infection with either of these pathogens, and underscores the need for further investigations incorporating specimens from humans, animal hosts, and invertebrate vectors in endemic areas. Such studies will be essential for establishing a link in the ongoing flea-borne rickettsioses outbreaks.

Isolation and characterization of a novel Rickettsia species (Rickettsia asembonensis sp. nov.) obtained from cat fleas (Ctenocephalides felis).

A novel rickettsial agent, 'Candidatus Rickettsia asembonensis' strain NMRCiiT, was isolated from cat fleas, Ctenocephalides felis, from Kenya. Genotypic characterization of the new isolate based on sequence analysis of five rickettsial genes, rrs, gltA, ompA, ompB and sca4, indicated that this isolate clustered with Rickettsia felis URRWXCal2. The degree of nucleotide similarity demonstrated that isolate NMRCiiT belongs within the genus Rickettsia and fulfils the criteria for classification as a representative of a novel species. The name Rickettsia asembonensis sp. nov. is proposed, with NMRCiiT (=DSM 100172T=CDC CRIRC RAS001T=ATCC VR-1827T) as the type strain.

Q Fever, Scrub Typhus, and Rickettsial Diseases in Children, Kenya, 2011-2012.

To increase knowledge of undifferentiated fevers in Kenya, we tested paired serum samples from febrile children in western Kenya for antibodies against pathogens increasingly recognized to cause febrile illness in Africa. Of patients assessed, 8.9%, 22.4%, 1.1%, and 3.6% had enhanced seroreactivity to Coxiella burnetii, spotted fever group rickettsiae, typhus group rickettsiae, and scrub typhus group orientiae, respectively.

Molecular Detection of Zoonotic Rickettsiae and Anaplasma spp. in Domestic Dogs and Their Ectoparasites in Bushbuckridge, South Africa.

Members of the order Rickettsiales are small, obligate intracellular bacteria that are vector-borne and can cause mild to fatal diseases in humans worldwide. There is little information on the zoonotic rickettsial pathogens that may be harbored by dogs from rural localities in South Africa. To characterize rickettsial pathogens infecting dogs, we screened 141 blood samples, 103 ticks, and 43 fleas collected from domestic dogs in Bushbuckridge Municipality, Mpumalanga Province of South Africa, between October 2011 and May 2012 using the reverse line blot (RLB) and Rickettsia genus and species-specific quantitative real-time PCR (qPCR) assays. Results from RLB showed that 49% of blood samples and 30% of tick pools were positive for the genus-specific probes for Ehrlichia/Anaplasma; 16% of the blood samples were positive for Ehrlichia canis. Hemoparasite DNA could not be detected in 36% of blood samples and 30% of tick pools screened. Seven (70%) tick pools and both flea pools were positive for Rickettsia spp; three (30%) tick pools were positive for Rickettsia africae; and both flea pools (100%) were positive for Rickettsia felis. Sequencing confirmed infection with R. africae and Candidatus Rickettsia asemboensis; an R. felis-like organism from one of the R. felis-positive flea pools. Anaplasma sp. South Africa dog strain (closely related to Anaplasma phagocytophilum), A. phagocytophilum, and an Orientia tsutsugamushi-like sequence were identified from blood samples. The detection of emerging zoonotic agents from domestic dogs and their ectoparasites in a rural community in South Africa highlights the potential risk of human infection that may occur with these pathogens.

Isolation of Candidatus Rickettsia asemboensis from Ctenocephalides Fleas.

Candidatus Rickettsia asemboensis was identified molecularly in fleas collected in 2009 from Asembo, Kenya. Multilocus sequence typing using the 17-kD antigen gene, rrs, gltA, ompA, ompB, and sca4 demonstrated that Candidatus R. asemboensis is closely related to Rickettsia felis but distinct enough to be considered for separate species classification. Following this molecular characterization of Candidatus R. asemboensis, the in vitro cultivation of this bacterium was then performed. We used Ctenocephalides canis and Ctenocephalides felis fleas removed from dogs in Kenya to initiate the in vitro isolation of Candidatus R. asemboensis. Successful cultures were obtained using Drosophila melanogaster S2 and Aedes albopictus C6/36 cell lines. Cytological staining and quantitative real-time PCR (qPCR) assays were used to visualize/confirm the culture of the bacteria in both cell lines. Sequencing of fragments of the 17-kD antigen gene, gltA, and ompB genes confirmed the identity of our Candidatus R. asemboensis isolates. To date, we have passaged Candidatus R. asemboensis 12 times through S2 and C6/36 cells, and active and frozen cultures are currently being maintained. This is the first time that a R. felis-like organism has been grown and maintained in culture and is therefore the first time that one of them, Candidatus R. asemboensis, has been characterized beyond molecular typing.

Whole-Genome Sequence of "Candidatus Rickettsia asemboensis" Strain NMRCii, Isolated from Fleas of Western Kenya.

Herein we present the draft genome sequence and annotation of "Candidatus Rickettsia asemboensis" strain NMRCii. "Ca. Rickettsia asemboensis" is phylogenetically related to but distinct from the flea-borne spotted fever pathogen Rickettsia felis. "Ca. Rickettsia asemboensis" was initially identified in and subsequently isolated from Ctenocephalides cat and dog fleas from Kenya.

High prevalence of Rickettsia africae variants in Amblyomma variegatum ticks from domestic mammals in rural western Kenya: implications for human health.

Tick-borne spotted fever group (SFG) rickettsioses are emerging human diseases caused by obligate intracellular Gram-negative bacteria of the genus Rickettsia. Despite being important causes of systemic febrile illnesses in travelers returning from sub-Saharan Africa, little is known about the reservoir hosts of these pathogens. We conducted surveys for rickettsiae in domestic animals and ticks in a rural setting in western Kenya. Of the 100 serum specimens tested from each species of domestic ruminant 43% of goats, 23% of sheep, and 1% of cattle had immunoglobulin G (IgG) antibodies to the SFG rickettsiae. None of these sera were positive for IgG against typhus group rickettsiae. We detected Rickettsia africae-genotype DNA in 92.6% of adult Amblyomma variegatum ticks collected from domestic ruminants, but found no evidence of the pathogen in blood specimens from cattle, goats, or sheep. Sequencing of a subset of 21 rickettsia-positive ticks revealed R. africae variants in 95.2% (20/21) of ticks tested. Our findings show a high prevalence of R. africae variants in A. variegatum ticks in western Kenya, which may represent a low disease risk for humans. This may provide a possible explanation for the lack of African tick-bite fever cases among febrile patients in Kenya.

Development and validation of a quantitative real-time polymerase chain reaction assay specific for the detection of Rickettsia felis and not Rickettsia felis-like organisms.

Human infections with Rickettsia felis have been reported worldwide. Recent studies have revealed the presence of many closely related but unique rickettsiae, referred to as Rickettsia felis-like organisms (RFLO), identified in various arthropods. Due to the recent discovery of the lack of specificity of earlier R. felis-specific assays, there has become a need to develop a new generation of R. felis-specific molecular assays that will differentiate R. felis not only from other rickettsiae but more importantly from other members of the R. felis genogroup that may not be pathogenic to humans. This new generation of assays is essential for determining the true risk for flea-borne spotted fever (FBSF) by surveying arthropod vectors/hosts. Because of the lack of specificity of previous assays developed to detect R. felis infections, prior surveys may have overestimated the prevalence of R. felis in arthropod vectors and thus the perceived risk of FBSF. We have developed a specific quantitative real-time polymerase chain reaction (qPCR) assay to detect R. felis (RfelB). Specificity of the assay was determined by testing it with a panel of 17 related Rickettsia species and 12 nonrickettsial bacterial DNA preparations. The RfelB qPCR assay was positive for R. felis DNA and negative for all of the 17 related Rickettsia species and 12 nonrickettsia bacterial DNA preparations. The limit of detection of the RfelB qPCR assay was determined to be two copies (two genoequivalents) per microliter of R. felis target ompB fragment-containing plasmid. Validation of the RfelB qPCR assay was accomplished by testing 83 previously sequence-confirmed R. felis and RFLOs containing DNA preparations from human and flea samples collected from different geographical locations around the world. This assay will be useful for rapid detection, identification, and enumeration of R. felis, an emerging human pathogen of worldwide importance, from both clinical and environmental samples.

Molecular detection of Rickettsia felis and Candidatus Rickettsia asemboensis in fleas from human habitats, Asembo, Kenya.

The flea-borne rickettsioses murine typhus (Rickettsia typhi) and flea-borne spotted fever (FBSF) (Rickettsia felis) are febrile diseases distributed among humans worldwide. Murine typhus has been known to be endemic to Kenya since the 1950s, but FBSF was only recently documented in northeastern (2010) and western (2012) Kenya. To characterize the potential exposure of humans in Kenya to flea-borne rickettsioses, a total of 330 fleas (134 pools) including 5 species (Xenopsylla cheopis, Ctenocephalides felis, Ctenocephalides canis, Pulex irritans, and Echidnophaga gallinacea) were collected from domestic and peridomestic animals and from human dwellings within Asembo, western Kenya. DNA was extracted from the 134 pooled flea samples and 89 (66.4%) pools tested positively for rickettsial DNA by 2 genus-specific quantitative real-time PCR (qPCR) assays based upon the citrate synthase (gltA) and 17-kD antigen genes and the Rfelis qPCR assay. Sequences from the 17-kD antigen gene, the outer membrane protein (omp)B, and 2 R. felis plasmid genes (pRF and pRFd) of 12 selected rickettsia-positive samples revealed a unique Rickettsia sp. (n=11) and R. felis (n=1). Depiction of the new rickettsia by multilocus sequence typing (MLST) targeting the 16S rRNA (rrs), 17-kD antigen gene, gltA, ompA, ompB, and surface cell antigen 4 (sca4), shows that it is most closely related to R. felis but genetically dissimilar enough to be considered a separate species provisionally named Candidatus Rickettsia asemboensis. Subsequently, 81 of the 134 (60.4%) flea pools tested positively for Candidatus Rickettsia asemboensis by a newly developed agent-specific qPCR assay, Rasemb. R. felis was identified in 9 of the 134 (6.7%) flea pools, and R. typhi the causative agent of murine typhus was not detected in any of 78 rickettsia-positive pools assessed using a species-specific qPCR assay, Rtyph. Two pools were found to contain both R. felis and Candidatus Rickettsia asemboensis DNA and 1 pool contained an agent, which is potentially new.

Coxiella burnetii in humans, domestic ruminants, and ticks in rural western Kenya.

We conducted serological surveys for Coxiella burnetii in archived sera from patients that visited a rural clinic in western Kenya from 2007 to 2008 and in cattle, sheep, and goats from the same area in 2009. We also conducted serological and polymerase chain reaction-based surveillance for the pathogen in 2009-2010, in human patients with acute lower respiratory illness, in ruminants following parturition, and in ticks collected from ruminants and domestic dogs. Antibodies against C. burnetii were detected in 30.9% (N = 246) of archived patient sera and in 28.3% (N = 463) of cattle, 32.0% (N = 378) of goats, and 18.2% (N = 159) of sheep surveyed. Four of 135 (3%) patients with acute lower respiratory illness showed seroconversion to C. burnetii. The pathogen was detected by polymerase chain reaction in specimens collected from three of six small ruminants that gave birth within the preceding 24 hours, and in five of 10 pools (50%) of Haemaphysalis leachi ticks collected from domestic dogs.