Capnocytophaga catalasegens sp. nov., isolated from feline oral cavities.

Three bacterial strains, KC07075, KC07079 and KC07084T, were isolated from the oral cavity of cats in 2007 in Japan. These strains were Gram-negative rods, exhibited gliding motility, grew in air with 5 % CO2, and showed oxidase activity, but not catalase activity. The 16S rRNA gene sequences of the three strains were 100 % identical. The 16S rRNA gene sequence of strain KC07084T showed 92.1 and 91.9% identity to the type strains of Capnocytophaga canis and Capnocytophaga felis, respectively, and showed 89.3-91.6% identity to other Capnocytophaga species. The major cellular fatty acids of strain KC07084T were iso-C15 : 0 (58.4 %) and summed feature 11 (13.1 %). The G+C content of DNA from strain KC07084T was 33.7 mol%, and the genome size was 2.92 Mbp. Strains KC07075, KC07079 and KC07084T showed digital DNA-DNA hybridization values (dDDH) values of 99.9 % and average nucleotide identity (ANI) values of 99.98 % with each other, strain KC07084T had dDDH values of 18.7-28.2 % and ANI values of 67.12-72.30 % to the type strains of other Capnocytophaga species. All known species of the genus Capnocytophaga inhabiting the oral cavity of dogs and cats have catalase activity, but the three strains, including type strain KC07084T, lacked catalase activity. These results of the phylogenetic analysis of the 16S rRNA gene sequence, biochemical characteristics, and dDDH and ANI values suggest that strain KC07084T represents a novel species. We propose the name Capnocytophaga catalasegens sp. nov., with KC07084T as the type strain (=JCM 32682T=DSM 107252T).

Capnocytophaga felis sp. nov. isolated from the feline oral cavity.

Four strains, KC07070T, KC07105, 11 025B-8C and 11 026B-8-C, were isolated from the oral cavity of cats in 2007 or 2011 in Japan. These strains were Gram-stain-negative rods, exhibited gliding motility, grew in air with 5 % CO2 and showed catalase and oxidase activity. The sequences of 16S rRNA genes of the four strains were 100 % identical. Additionally, the sequences of 16S rRNA genes of KC07070T had identity to those of the type strains of Capnocytophaga canimorsus (97.7 %), Capnocytophaga cynodegmi (97.8 %) and Capnocytophaga canis (97.4 %) and 91.2-93.8% identity to those of other species of the genus Capnocytophaga. The major cellular fatty acids of KC07070T were iso-C15 : 0 (56.2 %) and summed feature 11 (14.9 %). The G+C content of the DNA from KC07070T was 35.6 mol%, and the genome size was 2.88 Mbp. KC07070T had digital DNA-DNA hybridization (dDDH) values of 26.2-27.6% and average nucleotide identity (ANI) values of 75.4-83.3 % to the type strains of the closest relatives, C. canimorsus, C. cynodegmi and C. canis. These results of phylogenetic analysis of 16S rRNA gene sequence, cellular fatty acids compositions and dDDH and ANI values indicate that strain KC07070T represents a novel species, for which we propose the name Capnocytophaga felis sp. nov., with type strain KC07070T (=JCM 32681T=DSM 107251T).

Characterization of three strains of Capnocytophaga canis isolated from patients with sepsis.

Capnocytophaga canimorsus and Capnocytophaga cynodegmi, both commensal bacteria in the oral cavities of dogs and cats, are zoonotic pathogens. In particular, C. canimorsus causes sepsis and fatal septic shock. Recently, a novel Capnocytophaga species, C. canis, was isolated from the oral cavities of healthy dogs. It is reportedly oxidase-negative and therefore considered avirulent in humans. In the present study, three strains of C. canis were isolated from Japanese patients with sepsis. All three strains, HP20001, HP33001 and HP40001, were oxidase-positive. Nucleotide sequence identities of the 16S rRNA gene of the three strains to the C. canimorsus type strain ATCC35979, C. cynodegmi type strain ATCC49044 and C. canis type strain LMG29146 were 96.9-97.0%, 96.9-97.0% and 99.7-99.8%, respectively. Multi-locus sequence analysis based on seven house-keeping genes, dnaJ, fumC, glyA, gyrB, murG, trpB and tuf, revealed that the oxidase-positive C. canis strains isolated in Japan and oxidase-negative strains of C. canis from canine oral cavities in Switzerland were clustered in different genetic subgroups. These results indicate that the virulence of C. canis strains in humans is associated with oxidase activity.

Rat-bite fever due to Streptobacillus notomytis isolated from a human specimen.

Rat-bite fever (RBF) is a disease that usually presents with fever, arthralgia and skin rash. Streptobacillus moniliformis was considered the main cause of RBF among the genus Streptobacillus. Although with similar clinical presentation, RBF due to Streptobacillus notomytis is unusual in humans. To the best of our knowledge, we present a case involving the first isolate of S. notomytis in humans. A 63-year-old woman was admitted to our hospital with fever, rash and polyarthritis. She recalled being bitten by a rat on her finger 5 days before presentation. Clinical manifestations were compatible with rat-bite fever (RBF) and the diagnosis was confirmed by the detection of Streptobacillus species from both blood and pustule samples. Initial polymerase chain reaction tests revealed that the organism was S. moniliformis. However, thorough genetic analysis revealed the organism to be S. notomytis. The condition was successfully treated with ampicillin.

Yip1A, a novel host factor for the activation of the IRE1 pathway of the unfolded protein response during Brucella infection.

Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments. 　On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation.

Streptobacillus ratti sp. nov., isolated from a black rat (Rattus rattus).

An indole-, oxidase- and catalase-negative, non-motile bacterium, strain OGS16T, was isolated from an oral swab of a feral black rat (Rattus rattus) in 2007 in Japan. It stained Gram-negative and had pleomorphic, rod-shaped, non-spore-forming cells. Based on 16S rRNA gene sequence analyses, strain OGS16T was assigned to the genus Streptobacillus, with 16S rRNA gene sequence similarities of 99.3, 99.0, 98.6 and 95.5% to the type strains of Streptobacillus moniliformis, Streptobacillus notomytis, Streptobacillus felis and Streptobacillus hongkongensis, respectively. Strain OGS16T could also be differentiated clearly from other species of the genus Streptobacillus by rpoB, groEL and recA nucleotide and deduced amino acid sequence analysis. DNA-DNA relatedness as obtained by average nucleotide identity was 89.10% between strain OGS16T and Streptobacillus moniliformis DSM 12112T. Chemotaxonomic and physiological data for strain OGS16T were congruent with results for other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in discriminating strain OGS16T unequivocally from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus ratti sp. nov., with the type strain OGS16T (=JCM 31098T=DSM 101843T). The G+C content of the DNA of the type strain is 25.9 mol% and the genome size is 1.50 Mbp.

Epidemiological and pathological study of feline morbillivirus infection in domestic cats in Japan.

BACKGROUND: Feline morbillivirus (FmoPV) is a novel paramyxovirus found to infect domestic cats. FmoPV has been isolated in several countries in Asia and Europe and is considered to have genetic diversity. Also, it is suspected to be associated with feline renal diseases including tubulointerstitial nephritis (TIN), which affects domestic cats with a high incidence rate. RESULTS: To clarify the state of FmoPV infection among domestic cats in Japan, an epidemiological survey was conducted. Twenty-one out of 100 cats were found to have serum antibodies (Ab) against FmoPV-N protein by indirect immunofluorescence assay (IF) using FmoPV-N protein-expressing HeLa cells. Twenty-two of the cats were positive for FmoPV RNA in the urine and/or renal tissues. In total, 29 cats were positive for Ab and/or viral RNA. These FmoPV-infected cats were classified into three different phases of infection: RNA+/Ab + (14 cats), RNA+/Ab- (8 cats) and RNA-/Ab + (7 cats). In immunohistochemistry (IHC), 19 out of 29 cats were positive for FmoPV-N protein in kidney tissues; however, the FmoPV-N protein was located in the inflammatory lesions with severe grade in only four out of the 19 cats. Since 15 out of 29 infected cats were positive for viral RNA and Ab, approximately half of the infected cats were persistently infected with FmoPV. CONCLUSIONS: A statistically significant difference was observed between infection of FmoPV and the presence of inflammatory changes in renal lesions, indicating a relationship between FmoPV infection and feline renal diseases. However, we could not obtain histopathological evidence of a relationship between FmoPV infection and TIN.

Streptobacillus notomytis sp. nov., isolated from a spinifex hopping mouse (Notomys alexis Thomas, 1922), and emended description of Streptobacillus Levaditi et al. 1925, Eisenberg et al. 2015 emend.

A pleomorphic, Gram-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium was isolated in 1979 from the heart of a spinifex hopping mouse (Notomys alexis Thomas, 1922) with septicaemia and stored as Streptobacillus moniliformis in the strain collection of the Animal Health Laboratory, South Perth, Western Australia (AHL 370-1), as well as under CCUG 12425. On the basis of 16SrRNA gene sequence analyses, the strain was assigned to the genus Streptobacillus, with 99.4 % sequence similarity to the type strain of Streptobacillus moniliformis, 95.6 %sequence similarity to the type strain of Streptobacillus hongkongensis and 99.0 %sequence similarity to the type strain of Streptobacillus felis. The clear differentiation of strain AHL 370-1T from Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis was also supported by rpoB, groEL and recA nucleotide and amino acid sequence analysis. Average nucleotide identity was 87.16 % between strain AHL 370-1T and Streptobacillus moniliformis DSM 12112T. Physiological data confirmed the allocation of strain AHL 370-1T to the family Leptotrichiaceae, considering the very similar profiles of enzyme activities and fatty acids compared to closely related species. Within the genus Streptobacillus,isolate AHL 370-1T could also be separated unambiguously from the type strains of Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis by MALDI-TOF mass spectrometry. Two further strains (KWG2 and KWG24) isolated from asymptomatic black rats in Japan were highly similar to AHL 370-1T. On the basis of these data, we propose the novel species Streptobacillus notomytis sp. nov., with the type strain AHL370-1T (=CCUG 12425T=DSM 100026T=CCM 8593T=EF 12425T).

Cutting Edge: Brucella abortus exploits a cellular prion protein on intestinal M cells as an invasive receptor.

Brucella abortus is a Gram-negative bacterium causing brucellosis. Although B. abortus is known to infect via the oral route, the entry site in the gastrointestinal tract has been unclear. We found that B. abortus was selectively internalized by microfold cells (M cells), a subset of epithelial cells specialized for mucosal Ag uptake. During this process, colocalization of cellular prion protein (PrP(C)) and B. abortus was evident on the apical surface as well as in subapical vacuolar structures in M cells. Internalization of B. abortus by M cells of PrP(C)-deficient (Prnp(-/-)) mice was greatly reduced compared with that in wild-type mice. Furthermore, an oral infection study revealed that translocation of B. abortus into the Peyer's patch was significantly lower in Prnp(-/-) than in wild-type mice. These observations suggest that orally infected B. abortus invades the host through M cells by using PrP(C) on the apical surface of M cells as an uptake receptor.

A Case of Uncomplicated Bacteremia Caused by Capnocytophaga canimorsus in an Immunocompetent Patient.

This report describes uncomplicated bacteremia caused by Capnocytophaga canimorsus in an immunocompetent woman who presented with rigor and fever. She was hemodynamically stable. Two blood samples were immediately cultured because rigor indicated bacteremia. Although her symptoms were relieved, Gram-negative rods grew from blood cultures. She noted that she had been bitten by her dog before the first examination. The bacterium was confirmed as C. canimorsus by gene analysis. Infection with C. canimorsus can be fatal when accompanied by sepsis in elderly or immunocompromised patients. However, this case was considered rare as the patient was 41 years old and immunocompetent.

Non-severe Serovar Type E Capnocytophaga canimorsus Infection in a Post-splenectomy Male: A Case Report.

Capnocytophaga canimorsus (CP) causes severe infections in immunocompromised individuals. Three serovars (A, B, and C) are known to be responsible for more than 90% of infections associated with dog bites, although these three constitute only 8% of the serovars carried by dogs. We experienced a post-splenectomy non-severe case of CP withserovar type E, which has never been isolated in Japan. The prognosis of type E CP infections may be better than that of types A, B, and C infections because of the disproportion of serovars between clinical human isolates and dog oral isolates.

Meningoencephalomyelitis Caused by Brucella Canis: A Case Report and Literature Review.

Human brucellosis, one of the most common zoonoses worldwide, is rare in Japan. Brucella canis is the specific pathogen of human brucellosis carried by dogs. According to an epidemiological study of B. canis infection in Japan, B. canis is the specific pathogen of human brucellosis in dogs. We herein report a rare case of meningoencephalomyelitis caused by B. canis in a 68-year-old Japanese man. Neurobrucellosis was diagnosed based on a serum tube agglutination test and abnormal cerebrospinal fluid findings. The patient was started on targeted treatment with a combination of doxycycline and streptomycin. Although extremely rare, neurobrucellosis should be considered in patients with a fever of unknown origin and unexplained neurological symptoms.

Lethal severe fever with thrombocytopenia syndrome virus infection causes systemic germinal centre failure and massive T cell apoptosis in cats.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is a fatal viral disease characterized by high fever, thrombocytopenia, leukopenia, and multi-organ haemorrhage. Disruption of the humoral immune response and decreased lymphocyte numbers are thought to contribute to the disease severity. These findings have been obtained through the analysis of peripheral blood leukocytes in human patients, whereas analysis of lymph nodes has been limited. Thus, in this study, we characterized the germinal centre response and apoptosis in the lymph nodes of cats with fatal SFTS, because SFTS in cats well mimics the pathology of human SFTS. Methods: Lymph node tissue sections collected during necropsy from seven fatal SFTS patients and five non-SFTS cases were used for histopathological analysis. Additionally, lymph node tissue sections collected from cats with experimental infection of SFTS virus (SFTSV) were also analysed. Results: In the lymphoid follicles of cats with SFTS, a drastic decrease in Bcl6- and Ki67-positive germinal centre B cells was observed. Together, the number of T cells in the follicles was also decreased in SFTS cases. In the paracortex, a marked increase in cleaved-caspase3 positivity was observed in T cells. These changes were independent of the number of local SFTS virus-positive cell. Furthermore, the analysis of cats with experimental SFTSV infection revealed that the intrafollicular Bcl6- and CD3-positive cell numbers in cats with low anti-SFTSV antibody production were significantly lower than those in cats with high anti-SFTSV antibody production. Discussion: These results suggest that dysfunction of the humoral response in severe SFTS was caused by the loss of germinal centre formation and massive apoptosis of T cells in the lymph nodes due to systemically circulating viruses.

Putative Novel Species of Genus Capnocytophaga, Capnocytophaga stomatis Bacteremia in a Patient with Multiple Myeloma after Direct Contact with a Cat.

Capnocytophaga species are among the typical zoonotic pathogens causing infections following direct contact with animals. Recently, a putative novel species of zoonotic Capnocytophaga, Capnocytophaga stomatis, was reported. We herein report the first case of bacteremia caused by C. stomatis. A woman in her 80s with multiple myeloma who was receiving bortezomib and dexamethasone therapy was admitted to our hospital with a 2-day history of a fever and right calf redness. She was often licked by her cat. On a blood culture, thin, Gram-negative rods were detected, which were identified as C. stomatis by whole-genome sequencing. The patient was successfully treated with ampicillin-sulbactam treatment. Our case highlights the pathogenic potential of the putative novel Capnocytophaga, C. stomatis, in immunocompromised hosts.

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions. METHODOLOGY/PRINCIPLE FINDINGS: Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1-10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients. CONCLUSION/SIGNIFICANCE: This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.

Epidemiological Survey of Babesia divergens Asia Lineage in Wild Sika Deer (Cervus nippon) by Using Direct PCR in Japan.

Babesia divergens is the major causal agent of zoonotic human babesiosis across Europe. Previously, we reported the detection of a B. divergens Asia lineage in wild sika deer (Cervus nippon) in Japan which was genetically closely related to the European B. divergens. To further elucidate its etiology, we conducted a large epidemiological survey by combining lineage-specific PCR system and blood direct PCR. The infection rate of the Asia lineage was 6.6% (116/1,747) throughout Japan, where Hokkaido (45%), Nagano (17%), Iwate (12%), Gunma (11%), and Yamanashi (11%) were highly enzootic (> 10%) among the 30 prefectures examined. European B. divergens was not detected. A geographical information system (GIS) map revealed dense populations of PCR-positive deer in the mountains including the Japanese Alps in eastern Honshu, and Hokkaido. These areas markedly overlapped with the major habitats of Ixodes persulcatus, a principal tick vector responsible for the transmission of the Asia lineage. Other areas in southern Japan including Miyazaki, Kagoshima, and Shimane Prefectures, where positive sika deer were sporadically detected, may be habitats for other tick species involved in the enzootic cycle as I. persulcatus were scarce. The rise in human babesiosis cases is occasionally attributed to healthy blood donors who were unaware of tick bites and Babesia infection. Therefore, there is an urgent need to investigate whether infections in humans have occurred in Japan.

[An autopsy case involving severe sepsis due to Capnocytophaga canimorsus infection].

Capnocytophaga canimorsus, a commensal bacterium from the carine mouth, causes septicemia in human beings through bites or scratches. We report a case of a 60-year-old man contracting septicemia due to C. canimorsus infection after a dog bite who died less than 12 hours after admission. We observed neutrophils with intracytoplasmic bacilli in the peripheral blood smear. We discuss clinical presentation and autopsy findings.

Imported Brucellosis In The Era Of Dramatically Increasing Immigrants And Foreign Travelers From Endemic Areas: Occupational Hazards Of Secondary Infection Among Laboratory Technicians In A Nonendemic Country.

BACKGROUND: Brucellosis, an important zoonotic disease, is endemic in various parts of the world. Patients diagnosed with brucellosis in developed countries are often travelers and immigrants from endemic areas. This pathogen is listed as biosafety level 3, which means that it is highly contagious and therefore a risk to clinical laboratory technicians. CASE PRESENTATION: A 43-year-old Chinese man, who could not understand Japanese, visited our hospital because of an intermittent fever that had persisted for 5 months. Associated symptoms included muscle pain whenever he had a fever. He reported currently working as a welder in Japan. However, his previous employment working in animal husbandry in Heilongjiang, mainland China was not determined at the initial visit owing to language barriers. Two sets of blood culture showed nonfermenting gram-negative bacilli, initially misidentified as Ochrobactrum anthropi and subsequently identified as Brucella abortus. Six-week doxycycline and rifampicin were administered, with intravenous gentamicin for the initial 1 week. The patient recovered without relapse, confirmed by the negative result of a Brucella agglutination test. The patient's wife and three laboratory technicians were required to undergo blood examinations, which revealed no evidence of infection; however, they received prophylaxis with 3 weeks' doxycycline and rifampicin. CONCLUSION: In nonendemic countries, immigrants with imported brucellosis can be treated, to prevent secondary brucellosis infection, an occupational hazard among laboratory technicians. Greater attention is needed for positive findings of blood cultures, which may initially be misidentified as O. anthropi. When providing medical care for immigrants with fever of unknown origin, it is especially important for primary care physicians to overcome language barriers so as to assess pertinent information regarding their home country, such as previous employment, to prevent the spread the imported zoonoses in the era of a dramatically increasing number of immigrants and foreign travelers.

Chronic Brucellosis in Japan.

Human brucellosis, one of the most common zoonoses worldwide, rarely occurs in Japan, and only a few chronic cases have been reported. We herein report the case of a 39-year-old Japanese woman with chronic human brucellosis, considered a Brucella canis infection, that persisted for 19 years. Her medical history and fever pattern suggested chronic brucellosis, and the diagnosis was made based on the results of a serum tube agglutination test (SAT). After undergoing combination therapy with streptomycin and doxycycline, she achieved symptomatic relief and showed negative SAT results. Even in non-endemic areas, chronic brucellosis is an important differential diagnosis in patients with long-term persistent fatigue or a fever.