Genomic properties of a Bartonella quintana strain from Japanese macaque (Macaca fuscata) revealed by genome comparison with human and rhesus macaque strains.

Bartonella quintana, the causative agent of trench fever, is an intracellular bacterium that infects human erythrocytes and vascular endothelial cells. For many years, humans were considered the only natural hosts for B. quintana; however, it was recently discovered that wild Japanese macaques (Macaca fuscata) also serve as hosts for B. quintana. To elucidate the genetic characteristics of the B. quintana strain MF1-1 isolated from a Japanese macaque, we determined the complete genome sequence of the strain and compared it with those of strain Toulouse from a human and strain RM-11 from a rhesus macaque. General genomic features and orthologous gene cluster profiles are similar among the three strains, and strain MF1-1 is genetically closer to strain RM-11 than strain Toulouse based on the average nucleotide identity values; however, a significant inversion of approximately 0.68 Mb was detected in the chromosome of strain MF1-1. Moreover, the Japanese macaque strains lacked the bepA gene, which is responsible for anti-apoptotic function, and the trwL2, trwL4, and trwL6 genes, which may be involved in adhesion to erythrocytes of rhesus macaque and human. These features likely represent the genomic traits acquired by Japanese macaque strains in their host-associated evolution.

Interstitial Lung Disease With Respiratory Failure After COVID-19 mRNA Vaccination.

A septuagenarian woman developed dyspnea on the day following a fifth vaccination. Just before vaccination, a chest X-ray showed no abnormalities, but after the fifth vaccination, bilateral diffuse ground-glass opacities were detected. Bronchoalveolar lavage revealed a lymphocyte predominance and transbronchial lung biopsy revealed growth of the alveolar epithelium, along with organized polypoid granulation tissues in the alveolar ducts and bronchioles. Despite the administration of corticosteroids, imaging revealed persistent fibrosis, and she required long-term oxygen therapy. Although recent reports indicated that corticosteroids are effective for drug-induced interstitial lung disease related to COVID-19 mRNA vaccination, this case presented a somewhat different clinical manifestation.

Pulmonary Artery Aneurysm in a Patient with Nontuberculous Mycobacteria Infection.

Nontuberculous mycobacterial (NTM) infection sometimes leads to the development of pulmonary artery aneurysm (PAA), a rare but life-threatening complication. We herein report a 64-year-old woman with a history of NTM infection who presented with severe hemoptysis. Computed tomography revealed a ruptured PAA, which was treated successfully with pulmonary artery embolization. Subsequent right total pneumonectomy was performed to control infection. This case emphasizes the need to consider PAA in patients with NTM infection who present with hemoptysis. Early detection and appropriate management are critical for preventing this fatal complication.

Molecular epidemiology of Bartonella quintana endocarditis in patients from Israel and Eastern Africa.

BACKGROUND: Bartonella quintana is an important cause of culture-negative endocarditis. Although humans have been considered as its only reservoir, recent studies showed that macaque species are also reservoirs of B. quintana. Based on multi-locus sequence typing (MLST) B. quintana strains have been classified into 22 sequence types (STs), with 7 STs exclusively found in humans. Data regarding the molecular epidemiology of B. quintana endocarditis is limited to only 3 STs identified in 4 patients from Europe and Australia. We studied B. quintana endocarditis acquired in Eastern Africa or Israel to investigate the genetic diversity and clinical relatedness of B. quintana from distinct geographic regions. METHODS: Eleven patients with B. quintana endocarditis, 6 from Eastern Africa and 5 from Israel, were studied. DNA was extracted from cardiac tissue or blood specimens and analyzed by MLST based on 9 genetic loci. An evolutionary relationship between STs was visualized by a minimum spanning tree. A phylogenetic tree was constructed with the concatenated sequences (4271 bp) of the 9 loci using the maximum-likelihood method. RESULTS: Six strains were classified into previously described STs while 5 strains were identified for the first time and classified into new STs 23-27 which clustered with the previously reported STs 1-7 from human strains found in Australia, France, Germany, the USA, Russia, and the former Yugoslavia, without indication of geographical structuring. ST2 was the most prevalent ST, found in 5 of 15 patients with endocarditis (33.3%). ST26 appears to be a primary founder of the human lineage. CONCLUSIONS: The new and previously reported human STs form a single human lineage, clearly separated from the other 3 B. quintana lineages of cynomolgus, rhesus, and Japanese macaques. From evolutionary perspectives, these findings support the assumption that B. quintana has co-evolved with host species to form a host-speciation pattern. ST26 is suggested herein as a primary founder of the human lineage and may be key to explore where B. quintana had first originated; ST2 is a dominant genetic type associated with B. quintana endocarditis. To confirm these findings, additional worldwide molecular epidemiological studies are required.

Prevalence and Genetic Diversity of Bartonella Spp. in Northern Bats (Eptesicus nilssonii) and Their Blood-Sucking Ectoparasites in Hokkaido, Japan.

We investigated the prevalence of Bartonella in 123 northern bats (Eptesicus nilssonii) and their ectoparasites from Hokkaido, Japan. A total of 174 bat fleas (Ischnopsyllus needhami) and two bat bugs (Cimex japonicus) were collected from the bats. Bartonella bacteria were isolated from 32 (26.0%) of 123 bats. Though Bartonella DNA was detected in 79 (45.4%) of the bat fleas, the bacterium was isolated from only one bat flea (0.6%). The gltA sequences of the isolates were categorized into genotypes I, II, and III, which were found in both bats and their fleas. The gltA sequences of genotypes I and II showed 97.6% similarity with Bartonella strains from a Finnish E. nilssonii and a bat flea from a E. serotinus in the Netherlands. The rpoB sequences of the genotypes showed 98.9% similarity with Bartonella strain 44722 from E. serotinus in Republic of Georgia. The gltA and rpoB sequences of genotype III showed 95.9% and 96.7% similarity with Bartonella strains detected in shrews in Kenya and France, respectively. Phylogenetic analysis revealed that Bartonella isolates of genotypes I and II clustered with Bartonella strains from Eptesicus bats in Republic of Georgia and Finland, Myotis bats in Romania and the UK, and a bat flea from an Eptesicus bat in Finland. In contrast, genotype III formed a clade with B. florencae, B. acomydis, and B. birtlesii. These data suggest that northern bats in Japan harbor two Bartonella species and the bat flea serves as a potential vector of Bartonella transmission among the bats.

Genetics of randomly bred cats support the cradle of cat domestication being in the Near East.

Cat domestication likely initiated as a symbiotic relationship between wildcats (Felis silvestris subspecies) and the peoples of developing agrarian societies in the Fertile Crescent. As humans transitioned from hunter-gatherers to farmers ~12,000 years ago, bold wildcats likely capitalized on increased prey density (i.e., rodents). Humans benefited from the cats' predation on these vermin. To refine the site(s) of cat domestication, over 1000 random-bred cats of primarily Eurasian descent were genotyped for single-nucleotide variants and short tandem repeats. The overall cat population structure suggested a single worldwide population with significant isolation by the distance of peripheral subpopulations. The cat population heterozygosity decreased as genetic distance from the proposed cat progenitor's (F.s. lybica) natural habitat increased. Domestic cat origins are focused in the eastern Mediterranean Basin, spreading to nearby islands, and southernly via the Levantine coast into the Nile Valley. Cat population diversity supports the migration patterns of humans and other symbiotic species.

Prevalence and whole-genome sequence analysis of Campylobacter spp. strains isolated from wild deer and boar in Japan.

As a part of risk analysis for consumption of meat from wild animals, the prevalence of Campylobacter spp. in wild deer and boar in Japan was investigated. C. hyointestinalis subsp. hyointestinalis (C. hyointestinalis) was isolated from 2.8% (7/253) of the wild deer and 22.1% (71/321) of the wild boar examined. All 23 wild deer isolates and 141 (72.7%) wild boar isolates carried both chcdt-I and chcdt-II genes. The remaining 53 (27.3%) wild boar isolates had only the chcdt-II gene. By whole-genome sequence analysis, we detected 38-40 virulence- and survival-associated genes (motility, chemotactic, adhesion, invasion, toxin, glycosylation, iron uptake, drug resistance, and stress response), which had been identified in C. jejuni and C. coli. In conclusion, our study highlights C. hyointestinalis as a possible cause of food-borne disease in humans and emphasizes the importance of food hygiene in the processing of wild meats for human consumption.

Whole-genome sequence analysis of Shiga toxin-producing Escherichia coli O157 strains isolated from wild deer and boar in Japan.

The prevalence of Shiga toxin-producing Escherichia coli O157 (STEC O157) strains in wild deer and boar in Japan was investigated. STEC O157 strains were isolated from 1.9% (9/474) of the wild deer and 0.7% (3/426) of the wild boar examined. Pulsed-field gel electrophoresis (PFGE) analysis classified the wild deer and boar strains into five and three PFGE patterns, respectively. The PFGE pattern of one wild boar strain was similar to that of a cattle strain that had been isolated from a farm in the same area the wild boar was caught, suggesting that a STEC O157 strain may have been transmitted between wild boar and cattle. Clade analysis indicated that, although most of the strains were classified in clade 12, two strains were classified in clade 7. Whole-genome sequence (WGS) analysis indicated that all the strains carried mdfA, a drug resistance gene for macrolide antibiotics, and also pathogenicity-related genes similar to those in the Sakai strain. In conclusion, our study emphasized the importance of food hygiene in processing meat from Japanese wild animals for human consumption.

Lipoptena fortisetosa as a vector of Bartonella bacteria in Japanese sika deer (Cervus nippon).

BACKGROUND: Two species of deer ked (Lipoptena cervi and L. mazamae) have been identified as vectors of Bartonella bacteria in cervids in Europe and the USA. In an earlier study we showed that Japanese sika deer (Cervus nippon) harbor three Bartonella species, namely B. capreoli (lineage A) and two novel Bartonella species (lineages B and C); however, there is currently no information on the vector of Bartonella bacteria in sika deer. The aim of this study was to clarify potential vectors of Bartonella in Japanese sika deer. METHODS: Thirty-eight wingless deer keds (L. fortisetosa) and 36 ticks (Haemaphysalis and Ixodes species) were collected from sika deer. The prevalence of Bartonella in the arthropods was evaluated by real-time PCR targeting the 16S-23S internal transcribed spacer (ITS) and by culture of the organisms. The total number of Bartonella bacteria were quantified using real-time PCR. The distribution of Bartonella bacteria in deer ked organs was examined by immunofluorescence analysis. The relationship of Bartonella strains isolated from sika deer and arthropods were examined by a phylogenetic analysis based on concatenated sequences of the gltA, rpoB, ftsZ, and ribC genes, followed by a BLAST search for gltA and rpoB. RESULTS: Bartonella prevalence in deer keds was 87.9% by real-time PCR and 51.5% in culture and that in the ticks was 8.3% by real-time PCR and 2.8% in culture. The mean number of Bartonella bacteria per ked was calculated to be 9.2 × 105 cells. Bartonella aggregates were localized in the midgut of the keds. The phylogenetic analysis and BLAST search showed that both the host deer and the keds harbored two Bartonella species (lineages B and C), while B. capreoli (lineage A) was not detected in the keds. Two novel Bartonella species (lineages D and E) were isolated from one ked. CONCLUSIONS: Lipoptena fortisetosa likely serves as a vector of at least two Bartonella species (lineages B and C), whereas ticks do not seem to play a significant role in the transmission of Bartonella between sika deer based on the lower detection rates of Bartonella in ticks compared to keds. Bartonella species in lineages D and E appear to be L. fortisetosa-specific strains.

Vector-Borne Pathogens in Ectoparasites Collected from High-Elevation Pika Populations.

The American pika, Ochotona princeps, is projected to decline throughout North America as climate change reduces its range, and pikas have already disappeared from several locations. In addition to climate, disease spillover from lower elevation mammalian species might affect pikas. We sampled pika fleas in Colorado and Montana across elevations ranging from 2896 to 3612 m and screened them for the presence of DNA from rodent-associated bacterial pathogens (Bartonella species and Yersinia pestis) to test the hypothesis that flea exchange between pikas and rodents may lead to occurrence of rodent-associated pathogens in pika ectoparasites. We collected 275 fleas from 74 individual pikas at 5 sites in Colorado and one site in Montana. We found that 5.5% of 275 pika fleas in this study tested positive for rodent-associated Bartonella DNA but that variation in Bartonella infection prevalence in fleas among sites was not driven by elevation. Specifically, we detected DNA sequences from two loci (gltA and rpoB) that are most similar to Bartonella grahamii isolates collected from rodents in Canada. We did not detect Y. pestis DNA in our survey. Our results demonstrate evidence of rodent-associated flea-borne bacteria in pika fleas. These findings are also consistent with the hypothesis that rodent-associated pathogens could be acquired by pikas. Flea-borne pathogen spillover from rodents to pikas has the potential to exacerbate the more direct effects of climate that have been suggested to drive pika declines.

Detection and phylogenetic analysis of Bartonella species from bat flies on eastern bent-wing bats (Miniopterus fuliginosus) in Japan.

We examined Bartonella prevalence in 281 bat flies collected from 114 eastern bent-wing bats (Miniopterus fuliginosus) in Japan and phylogenetically analyzed with other bat fly and bat strains. The bat flies were identified as Penicilidia jenynsii (PJ; n = 45), Nycteribia allotopa (NA; n = 157), and novel Nycteribia species (NS; n = 79). Bartonella DNAs were detected in 31.7 % (89/281) of bat flies by PCR targeting the citrate synthase (gltA) gene. The prevalence of Bartonella DNA among the bat flies was 47.1 % (74/157) in NA, 15.2 % (12/79) in NS, and 6.7 % (3/45) in PJ. Bartonella bacteria were also isolated from two NA and one NS. A phylogenetic analysis of the gltA sequences revealed that bat fly-associated strains were classified into three lineages and the same lineages of Bartonella were commonly detected from both Nycteribia bat flies and Miniopterus bats. These results suggest that Nycteribia bat flies are potential vectors for transmitting Bartonella among Miniopterus bats.

Identification and characterization of a novel bat polyomavirus in Japan.

A novel polyomavirus (PyV) was identified in the intestinal contents of Japanese eastern bent-wing bats (Miniopterus fuliginosus) via metagenomic analysis. We subsequently sequenced the full genome of the virus, which has been tentatively named Miniopterus fuliginosus polyomavirus (MfPyV). The nucleotide sequence identity of the genome with those of other bat PyVs was less than 80%. Phylogenetic analysis revealed that MfPyV belonged to the same cluster as PyVs detected in Miniopterus schreibersii. This study has identified the presence of a novel PyV in Japanese bats and provided genetic information about the virus.

Isolation and genetic properties of Bartonella in eastern bent-wing bats (Miniopterus fuliginosus) in Japan.

The prevalence and genetic characteristics of Bartonella species in eastern bent-wing bats (Miniopterus fuliginosus) from Japan were investigated. Bartonella bacteria were isolated from 12/50 (24%) of bats examined. Analyses of sequence similarities of the citrate synthase gene (gltA) and RNA polymerase beta-subunit-encoding (rpoB) gene indicated that the isolates from M. fuliginosus were distinct from those present in known Bartonella species as the levels of similarity for both of the genes were lower than the cut-off values for species identification in Bartonella. A phylogenetic analysis of the gltA sequences revealed that the Miniopterus bat-associated strains fell into five genotypes (I to V). Though genotypes I to IV formed a clade with Bartonella from Miniopterus bats from Taiwan, genotype V made a monophyletic clade separate from other bat isolates. In a phylogenetic analysis with the concatenated sequences of the 16S rRNA, gltA, rpoB, cell division protein (ftsZ) gene, and riboflavin synthase gene (ribC), isolates belonging to genotypes I to IV clustered with Bartonella strains from Taiwanese Miniopterus bats, similar to the outcome of the phylogenetic analysis with gltA, whereas genotype V also made a monophyletic clade separate from other bat-associated Bartonella strains. The present study showed that M. fuliginosus in Japan harbor both genus Miniopterus-specific Bartonella suggesting to be specific to the bats in Japan.

Detection of Zoonotic Bartonella Pathogens in Rabbit Fleas, Colorado, USA.

We detected 3 Bartonella species in wild rabbit fleas from Colorado, USA: B. vinsonii subsp. berkhoffii (n = 16), B. alsatica (n = 5), and B. rochalimae (n = 1). Our results support the establishment of the zoonotic agent B. alsatica in North America.

MOLECULAR SURVEY OF BARTONELLA ROCHALIMAE IN JAPANESE RACCOON DOGS (NYCTEREUTES PROCYONOIDES VIVERRINUS).

Wild carnivores serve as reservoirs of several zoonotic Bartonella species such as Bartonella henselae, Bartonella vinsonii subsp. berkhoffii, and Bartonella rochalimae. The raccoon dog (Nyctereutes procyonoides viverrinus) is the most common native carnivore in Japan, but epidemiologic studies of Bartonella infections have not been performed in this animal species yet. Here, we report a molecular survey of B. rochalimae prevalence in 619 wild raccoon dogs captured from 2009 to 2017 in western Japan. Bartonella rochalimae DNA was detected in 7.1% (44/619) of the raccoon dogs examined by PCR targeting the rpoB and ssrA genes. All of the sequences obtained were identical in each of the genes. The prevalence of B. rochalimae by sex of the animals was 6.1% (21/344) in male and 8.4% (23/275) in female. The prevalence by year varied from 2% (1/45) in 2011 to 14% (4/28) in 2016. The prevalence (7.9%) of B. rochalimae in the raccoon dogs with sarcoptic mange tended to be higher than the prevalence (4.0%) in the animals without the infestation of mites, although the differences were not significant. Sequence analysis indicated that Japanese raccoon dogs in the area examined were infected with B. rochalimae carrying identical sequences in the rpoB and ssrA genes. In addition, the raccoon dog strain had few sequence variations in both genes compared to other known B. rochalimae strains detected in other parts of the world.

PREVALENCE OF YERSINIA AMONG WILD SIKA DEER (CERVUS NIPPON) AND BOARS (SUS SCROFA) IN JAPAN.

We examined the prevalence of Yersinia, including pathogenic species such as Yersinia enterocolitica and Yersinia pseudotuberculosis, among wild sika deer (Cervus nippon) and boars (Sus scrofa) captured in Japan. The prevalence of Yersinia in the wild deer was 75% (207/277) and in the boars was 74% (40/54). A total of 417 isolates of nine Yersinia species were isolated from the animals examined: the largest number of isolates (48%, 200/417) were Y. enterocolitica biotype 1A. Pathogenic Y. enterocolitica 1B/O:8 were also isolated from two deer, and Y. pseudotuberculosis serogroups 3 and 4 were isolated from two boars and a deer, respectively. The pathogenic Y. enterocolitica 1B/O:8 isolates carried four virulence genes (ail, ystA, yadA, and virF), and Y. pseudotuberculosis serogroups 3 and 4 isolates carried three virulence genes (inv, yadA, and lcrF). Although the Y. enterocolitica 1B/O:8 and Y. pseudotuberculosis isolates were sensitive to almost all the antimicrobials tested, the two Y. enterocolitica 1B/O:8 isolates were resistant to azithromycin and ampicillin, and the three Y. pseudotuberculosis isolates were resistant only to azithromycin. These findings suggested that wild deer and boars might be important reservoirs for the agent causing human yersiniosis.

Isolation and phylogenetic analysis of Bartonella species from Rusa deer (Rusa timorensis) in Thailand.

The aim of the present study is to investigate the prevalence of Bartonella infection in deer in Thailand and to characterize the isolates by biochemical, morphological and genetic analysis. A total of 247 blood samples were collected from Rusa deer (Rusa timorensis) in a livestock breeding facility in Thailand. Bartonella bacteria were isolated in 3.6% of the blood samples. Three out of 110 (2.7%) males and 6 of 137 (4.4%) females were positive for Bartonella. A higher prevalence of Bartonella was observed in young deer under 4 years of age compared to adults over 4 years of age, but no Bartonella was isolated from deer over 8 years of age. Phylogenetic analysis of concatenated sequences of seven loci of Bartonella indicated that all the isolates from Rusa deer in Thailand were identical and formed a distinct cluster from other known Bartonella species.

Evolutionary Dynamics of Pathoadaptation Revealed by Three Independent Acquisitions of the VirB/D4 Type IV Secretion System in Bartonella.

The α-proteobacterial genus Bartonella comprises a group of ubiquitous mammalian pathogens that are studied as a model for the evolution of bacterial pathogenesis. Vast abundance of two particular phylogenetic lineages of Bartonella had been linked to enhanced host adaptability enabled by lineage-specific acquisition of a VirB/D4 type IV secretion system (T4SS) and parallel evolution of complex effector repertoires. However, the limited availability of genome sequences from one of those lineages as well as other, remote branches of Bartonella has so far hampered comprehensive understanding of how the VirB/D4 T4SS and its effectors called Beps have shaped Bartonella evolution. Here, we report the discovery of a third repertoire of Beps associated with the VirB/D4 T4SS of B. ancashensis, a novel human pathogen that lacks any signs of host adaptability and is only distantly related to the two species-rich lineages encoding a VirB/D4 T4SS. Furthermore, sequencing of ten new Bartonella isolates from under-sampled lineages enabled combined in silico analyses and wet lab experiments that suggest several parallel layers of functional diversification during evolution of the three Bep repertoires from a single ancestral effector. Our analyses show that the Beps of B. ancashensis share many features with the two other repertoires, but may represent a more ancestral state that has not yet unleashed the adaptive potential of such an effector set. We anticipate that the effectors of B. ancashensis will enable future studies to dissect the evolutionary history of Bartonella effectors and help unraveling the evolutionary forces underlying bacterial host adaptation.

Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats.

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined.