Discovery of a new volcanic soil material, "Akahoya," as an adsorbent for bacterial and viral pathogens and its application to environmental purification.

Akahoya is a volcanic soil rich in alumina, primarily deposited in Kyushu, Japan. We have found that Akahoya adsorbs bacteria in the water surrounding cattle grazing areas, suggesting a potential for environmental purification. This study investigated the spectrum of microorganisms adsorbed by Akahoya using a column filled with Akahoya through which a suspension of microorganisms was passed. Shirasu soil, another volcanic soil with a different chemical composition, was used as a control. Akahoya effectively adsorbed a diverse range of microorganisms including Escherichia coli, Campylobacter jejuni, Vibrio parahaemolyticus, Salmonella Enteritidis, Staphylococcus aureus, Clostridium perfringens, spores of Bacillus subtilis and Bacillus anthracis, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), murine norovirus, and avian influenza virus (H3N2), whereas Shirasu soil did not adsorb any of the organisms examined. Moreover, bacteria naturally present in river water, such as aerobic bacteria, total coliforms, and Enterobacteriaceae as indicators of river contamination, as well as E. coli added artificially to sterilized river water, were reduced to below the detection limit (<1 CFU/mL) after being passed through Akahoya. Additionally, the number of viable E. coli continued to decrease after contact with Akahoya for 1 month, suggesting bactericidal effects. Notably, the adsorption of E. coli to Akahoya was influenced by the concentration of phosphate and the pH of the suspension due to the interaction between the surface phosphorylation of organisms and Al2O3, the major chemical component of Akahoya. The present results demonstrate the remarkable ability of Akahoya to remove phosphate and microbes, suggesting that Akahoya could be used for water purification processes.IMPORTANCEAlthough a safe and sufficient water supply is essential for the maintenance of hygienic conditions, a major challenge is to develop a comprehensive effective, sustainable, and cost-effective technological approach for the treatment and purification of contaminated water. In this study, we demonstrated that a novel volcanic soil, Akahoya, which has unlimited availability, is a highly effective adsorbent for a wide range of bacterial and viral pathogens, suggesting its potential as a sustainable resource for this purpose. It was suggested that the adsorption of microorganisms on Akahoya was mediated by phosphate groups present on the surface structures of microorganisms, which bind to the alumina component of Akahoya according to the phosphate concentration and pH of the liquid phase. The present findings highlight the exceptional ability of Akahoya to eliminate or reduce phosphate and microorganisms effectively in water purification processes, thus contributing to the development of efficient and sustainable solutions for addressing water pollution challenges.

The growth-stimulating factor of Treponema phagedenis from bovine digital dermatitis lesions.

Bovine digital dermatitis (BDD) is an infectious skin disease of the hoof characterized by painful ulcerations that cause lameness in dairy cattle. Cell-free supernatants (CFS) of Falsiporphyromonas endometrii predominantly isolated from BDD lesions had the highest growth-stimulating effect on Treponema phagedenis among BDD-associated bacteria. Butyric acid was detected at a concentration of 45.4 mM in CFS of F. endometrii, and the growth of T. phagedenis was significantly promoted by butyric acid supplementation.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Multiple genotypes of enterovirus G carrying a papain-like cysteine protease (PL-CP) sequence circulating on two pig farms in Japan: first identification of enterovirus G10 carrying a PL-CP sequence.

Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.

16S rRNA-based amplicon analysis of changes in the bacterial population in the lesions of papillomatous digital dermatitis in dairy cattle after topical treatment with allyl isothiocyanate.

Papillomatous digital dermatitis (PDD) is a foot disease causing lameness in dairy cattle. It is regarded as a polymicrobial infection, although its etiology is not fully understood. PDD is treated by the topical or systemic administration of antibiotics such as lincomycin (LCM); however, the milk of the cows cannot be marketed during the treatment and withdrawal period due to the residual antibiotics in milk. Allyl isothiocyanate (AITC), an extract of Wasabia japonica (known as wasabi or Japanese horseradish) widely employed as a food additive, can be used as an alternative antimicrobial agent that overcomes this problem. We previously showed that AITC is as effective as LCM in PDD treatment. Here, using the samples obtained in the previous clinical study, we analyzed changes in the bacterial population in the PDD-associated microbiota after AITC treatment and compared those with that following LCM treatment by 16S ribosomal RNA (rRNA)-based amplicon analysis. Both treatments induced major changes in the bacterial population, and Treponema species, which have been regarded as the major causative agents of PDD, were efficiently eliminated by both agents. However, the AITC-treated samples exhibited higher diversity compared with pretreatment samples, but this trend was not observed for LCM treatment, probably reflecting different antibacterial activities of the two agents. Importantly, this analysis detected population changes before morphological changes in PDD lesions (clinical signs of healing) became evident, indicating that 16S rRNA-based amplicon analysis represents an efficient strategy for analyzing and monitoring the treatment efficiency of PDD as well as other polymicrobial diseases.

Rapid detection and serovar identification of common Salmonella enterica serovars in Canada using a new pyrosequencing assay.

Serotyping of Salmonella enterica subsp. enterica is a critical step for foodborne salmonellosis investigation. To identify Salmonella enterica subsp. enterica serovars, we have developed a new assay based on a triplex polymerase chain reaction (PCR) with pyrosequencing for amplicon confirmation and phylogenetic discrimination of strains. The top 54 most prevalent serovars of S. enterica in Canada were examined with a total of 23 single-nucleotide polymorphisms (SNPs) and (or) single-nucleotide variations (SNVs) located on 3 genes (fliD, sopE2, and spaO). Seven of the most common serovars, Newport, Typhi, Javiana, Infantis, Thompson, Heidelberg, and Enteritidis, were successfully distinguished from the other serovars based on their unique SNP-SNV combinations. The remaining serovars, including Typhimurium, ssp I:4,[5],12:i:-, and Saintpaul, were further divided into 47 subgroups that demonstrate the relatedness to phylogenetic classifications of each serovar. This pyrosequencing assay is not only cost-effective, rapid, and user-friendly, but also provides phylogenetic information by analyzing 23 selected SNPs. With the added layer of confidence in the PCR results and the accuracy and speed of pyrosequencing, this novel method would benefit the food industry and provides a tool for rapid outbreak investigation through quick detection and identification of common S. enterica serovars in Canada.

Extraintestinal infection of Helicobacter cinaedi induced by oral administration to Balb/c mice.

Although Helicobacter cinaedi was initially considered an opportunistic pathogen in immunocompromised patients, it was later shown to also infect immunocompetent and healthy individuals. Sporadic bacteremia due to H. cinaedi has frequently been reported; however, whether the bacterium can be translocated after passage through the intestinal mucosa remains unclear. In the present study, a preclinical small animal model that faithfully reproduces H. cinaedi infection in humans was developed. Balb/c male mice were orally inoculated with a single dose of 6.8 × 107 CFU of a human clinical H. cinaedi strain. The organism persistently colonized the intestinal tract of the mice, particularly the cecum and colon, for at least 56 days, and the bacteria were excreted in the feces. Although inoculated bacteria were recovered from the spleen, liver, kidney, lung, bladder and mesenteric lymph nodes during the first 2 weeks of bacteremia, the organism was not isolated from these organs after 4 weeks, suggesting that complement- and antibody-mediated serum sensitivity account for the relatively low frequency of systemic infection. However, H. cinaedi was isolated from the biceps femoris, triceps branchii, latissimus dorsi, and trapezius muscles beyond 2 weeks after infection and after production of specific anti-H. cinaedi IgM and IgG antibodies. The present findings suggest that experimental infection of Balb/c mice with H. cinaedi may be a useful model for further studies of H. cinaedi pathogenesis, prophylaxis or therapeutic interventions in vivo.

Epithelial Coculture and l-Lactate Promote Growth of Helicobacter cinaedi under H2-Free Aerobic Conditions.

Helicobacter cinaedi is an emerging opportunistic pathogen associated with infections of diverse anatomic sites. Nevertheless, the species demonstrates fastidious axenic growth; it has been described as requiring a microaerobic atmosphere, along with a strong preference for supplemental H2 gas. In this context, we examined the hypothesis that in vitro growth of H. cinaedi could be enhanced by coculture with human epithelial cells. When inoculated (in Ham's F12 medium) over Caco-2 monolayers, the type strain (ATCC BAA-847) gained the ability to proliferate under H2-free aerobic conditions. Identical results were observed during coculture with several other monolayer types (LS-174T, AGS, and HeLa). Under chemically defined conditions, 40 amino acids and carboxylates were screened for their effect on the organism's atmospheric requirements. Several molecules promoted H2-free aerobic proliferation, although it occurred most prominently with millimolar concentrations of l-lactate. The growth response of H. cinaedi to Caco-2 cells and l-lactate was confirmed with a collection of 12 human-derived clinical strains. mRNA sequencing was next performed on the type strain under various growth conditions. In addition to providing a whole-transcriptome profile of H. cinaedi, this analysis demonstrated strong constitutive expression of the l-lactate utilization locus, as well as differential transcription of terminal respiratory proteins as a function of Caco-2 coculture and l-lactate supplementation. Overall, these findings challenge traditional views of H. cinaedi as an obligate microaerophile. IMPORTANCE: H. cinaedi is an increasingly recognized pathogen in people with compromised immune systems. Atypical among other members of its bacterial class, H. cinaedi has been associated with infections of diverse anatomic sites. Growing H. cineadi in the laboratory is quite difficult, due in large part to the need for a specialized atmosphere. The suboptimal growth of H. cinaedi is an obstacle to clinical diagnosis, and it also limits investigation into the organism's biology. The current work shows that H. cinaedi has more flexible atmospheric requirements in the presence of host cells and a common host-derived molecule. This nutritional interplay raises new questions about how the organism behaves during human infections and provides insights for how to optimize its laboratory cultivation.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

Rapid identification and subtyping of Helicobacter cinaedi strains by intact-cell mass spectrometry profiling with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Helicobacter cinaedi infection is recognized as an increasingly important emerging disease in humans. Although H. cinaedi-like strains have been isolated from a variety of animals, it is difficult to identify particular isolates due to their unusual phenotypic profiles and the limited number of biochemical tests for detecting helicobacters. Moreover, analyses of the 16S rRNA gene sequences are also limited due to the high levels of similarity among closely related helicobacters. This study was conducted to evaluate intact-cell mass spectrometry (ICMS) profiling using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a tool for the identification of H. cinaedi. A total of 68 strains of H. cinaedi isolated from humans, dogs, a cat, and hamsters were examined in addition to other Helicobacter species. The major ICMS profiles of H. cinaedi were identical and differed from those of Helicobacter bilis, which show >98% sequence similarity at the 16S rRNA sequence level. A phyloproteomic analysis of the H. cinaedi strains examined in this work revealed that human isolates formed a single cluster that was distinct from that of the animal isolates, with the exception of two strains from dogs. These phyloproteomic results agreed with those of the phylogenetic analysis based on the nucleotide sequences of the hsp60 gene. Because they formed a distinct cluster in both analyses, our data suggest that animal strains may not be a major source of infection in humans. In conclusion, the ICMS profiles obtained using a MALDI-TOF MS approach may be useful for the identification and subtyping of H. cinaedi.

Comparative analysis of LTR and structural genes in an equine infectious anemia virus strain isolated from a feral horse in Japan.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

Characterization of methicillin-resistant Staphylococcus pseudintermedius isolated from dogs and cats.

The aim of this study was to explore the presence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in a collection of S. pseudintermedius strains isolated from dogs and cats with dermatitis in Japan and to compare their genotypic and phenotypic characteristics. Clonal relationships were determined by pulse field gel electrophoresis (PFGE), staphylococcal chromosomal cassette mec (SCCmec) typing, and multilocus sequence typing (MLST). Biofilm formation assay was performed using safranin staining in microplates. Three virulence genes coding for S. intermedius exfoliative toxin and Panton-Valentine leukocidin (siet, lukS-PV and lukF-PV) were searched for in a collection of strains. Antimicrobial resistance against 15 antibiotics was studied by a disc diffusion method. Twenty-seven MRSP were isolated. According to PFGE results the isolates were not closely related except for a few strains. MLST showed that the strains belonged to five groups, ST71 and ST26 being the two most prevalent. Three types of SCCmec (II, II-III and V) were identified. All isolates were siet-positive but PVL-negative. Most strains (except for two) produced strong biofilm in tryptic soy broth with glucose. Seventy-eight percent of the isolates were resistant or intermediate to twelve or more antibiotics. Our study demonstrates that the ST71 lineage is widespread in Japan and that ST26 could represent an emerging lineage. Moreover, most of our strains are capable of forming strong biofilm and possess siet gene, two virulence characteristics that probably help the bacteria to persist and spread. Finally, our MRSP strains show a strong resistance profile to antibiotics commonly used in veterinary medicine.

Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi.

The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.

Seroprevalence of severe fever with thrombocytopenia syndrome virus in medium-sized wild mammals in Miyazaki, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). SFTSV infection in humans and companion animals is a matter of concern in endemic areas. Various wild animals are involved in the transmission cycle of SFTSV with vector ticks. Because the home range of medium-sized wild mammals commonly overlaps with humans' living spheres, this study aimed to reveal the endemicity of SFTSV in such mammals. This study investigated the prevalence of antibodies against SFTSV and viral RNA in medium-sized wild mammals in Miyazaki Prefecture, Japan where human cases have been most frequently reported in Japan and performed a phylogenetic analysis to compare the detected SFTSV with those previously reported. Forty-three of 63 (68%) Japanese badgers (Meles anakuma) and 12 of 53 (23%) Japanese raccoon dogs (Nyctereutes procyonoides viverrinus) had antibodies against SFTSV. Japanese marten (n = 1), weasels (n = 4), and Japanese red fox (n = 1) were negative. Two of 63 (3%) badgers tested positive for SFTSV RNA, whereas the other species were negative. Phylogenetic analysis of the partial nucleotide sequence of SFTSV revealed that viral RNA detected from badgers exhibited 99.8% to 100% similarity to SFTSV, as previously reported in humans, cat, and ticks in the study area. This study demonstrated high seropositivity of antibodies in medium-sized wild mammals and suggested that SFTSV could be shared among these mammals, humans, and companion animals in endemic areas.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100.

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

Fine Particle Adsorption Capacity of Volcanic Soil from Southern Kyushu, Japan.

"Akahoya" is a volcanic soil classified as a special soil deposited in Kyushu, Japan. Many of its properties are not yet clearly understood. We found that Akahoya had the potential to adsorb bacteria in cattle feces, which prompted us to investigate its material properties and perform experiments to comprehensively evaluate its adsorption performance for various fine particles such as acidic and basic dyes, NOx/SOx gas, and phosphoric acid ions, in addition to bacteria. Akahoya had a very high specific surface area owing to the large number of nanometer-sized pores in its structure; it exhibited a high adsorption capacity for both NO2 and SO2. Regarding the zeta potential of Akahoya, the point of zero charge was approximately pH 7.0. The surface potential had a significant effect on the adsorption of acidic and basic dyes. Akahoya had a very high cation exchange capacity when the sample surface was negatively charged and a high anion exchange capacity when the sample surface was positively charged. Akahoya also exhibited a relatively high adsorption capacity for phosphoric acid because of its high level of Al2O3, and the immersion liquid had a very high Al ion concentration. Finally, filtration tests were performed on Escherichia coli suspension using a column filled with Akahoya or another volcanic soil sample. The results confirmed that the Escherichia coli adhered on the Akahoya sample. The results of the Escherichia coli release test, after the filtration test, suggested that this adhesion to Akahoya could be phosphorus-mediated.

Survival capability of Campylobacter upsaliensis under environmental stresses.

OBJECTIVE: Campylobacter upsaliensis has been recognized as an emerging pathogen. However, little is known about its survival in the environment. To evaluate its survival capability, we estimated the reduction in viable counts of C. upsaliensis after aerobic exposure to starvation in phosphate-buffered saline (PBS), acidity (pH = 4.3), high osmolarity (4% NaCl), and dryness in wet pulp disks at different temperatures. Also, survival in dog feces and dog food at variable temperate was assessed. RESULTS: Campylobacter upsaliensis remained culturable under starvation for 4 days at 25 °C and for 10 weeks at 4 °C. C. upsaliensis was also recoverable after exposure to high osmolality for 9 days, dryness for 5 days, and acidity for 2 days, respectively. Similarly, C. upsaliensis survived in dog feces and dog food for several days at 25 °C and weeks at 4 °C. The survival capability of the organism was dependent on the water content, and also temperature. Notably, the tested C. upsaliensis strain was less resilient under all tested conditions than a C. jejuni strain used as a control. The findings showed that C. upsaliensis is able to survive under various environmental stresses, suggesting that it could pose a potential threat to public health.

Comprehensive Analyses of the Bacterial Population in Non-Healing Claw Lesions of Dairy Cattle.

Non-healing claw lesions (NHCLs) are a newly characterized disorder affecting the deep dermis of the hoof in dairy cattle. Although NHCLs are thought to be associated with bovine digital dermatitis (BDD), their precise etiology is not yet understood. To investigate the bacterial populations present in each type of NHCL (toe necrosis: TN, non-healing white line disease: nhWLD, and a non-healing sole ulcer: nhSU), and the newly added entity non-healing verrucous-like lesions (nhVLL), 16S rRNA-based metagenomic analysis with next-generation sequencing (NGS) was employed. Twelve cases of NHCLs (3 TN, 3 nhWLD, 4 nhSU, and 2 nhVLL) were collected from five dairy farms in two prefectures in Japan. Three samples of healthy hoof dermis collected from two farms and a slaughterhouse were used as controls. Furthermore, culture-dependent and -independent approaches were conducted for detecting Treponema species and Fusobacterium necrophorum. As reported in BDD, Treponema species and F. necrophorum were detected frequently from NHCLs by PCR and immunohistochemistry, but NGS showed that these bacterial genera were not predominant in NHCLs. The predominant bacterial genera in NHCLs differed among the lesions examined, suggesting that Treponema species present predominantly in BDD were not predominant in NHCLs and that the bacterial population in NHCLs may vary among individual cattle and/or farms.

Helicobacter cinaedi is a human-adapted lineage in the Helicobacter cinaedi/canicola/'magdeburgensis' complex.

Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.

Monitoring IgG against Mycobacterium tuberculosis proteins in an Asian elephant cured of tuberculosis that developed from long-term latency.

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.