The association between gingivitis and oral spirochetes in young cats and dogs.

Although gingivitis frequently occurs in young cats, spirochetes are often found in the early stages of periodontal disease. This study was conducted to determine the association between gingivitis and oral spirochetes in young cats and dogs. The degree of gingivitis was evaluated in a total of 68 cats and 31 dogs under one year of age, and plaques were collected from each carnassial. To detect spirochetes or Porphyromonas gulae in plaque samples, 16S rRNA gene was amplified by polymerase chain reaction (PCR) using specific primers. All data were analyzed using Fisher's exact probability test and odds ratio (OR) with a 95% confidence interval (95% CI). The prevalence of gingivitis was significantly higher in young cats (92.6%) than in young dogs (45.2%). The positive rate of spirochetes by PCR in gingivitis cases was 85.4% in young cats and 15.4% in young dogs, and the positive rate of P. gulae was 66.7% in young cats and 15.4% in young dogs. Both results were significantly higher in young cats than in young dogs. In young cats, spirochetes were significantly associated with gingivitis (OR = 7.95; 95% CI = 1.17, 53.83; P < 0.05), but P. gulae was not (OR = 2.44; 95% CI = 0.38, 15.66; P = 0.23). These results suggest that spirochetes may be associated with the early stages of periodontal disease in cats.

Identification of pyrC gene as an immunosuppressive factor in Francisella novicida infection.

Francisella tularensis, a bacterial causative agent of the zoonosis tularemia, is highly pathogenic to humans. The pathogenicity of this bacterium is characterized by intracellular growth in immune cells, like macrophages, and host immune suppression. However, the detailed mechanism of immune suppression by F. tularensis is still unclear. To identify the key factors causing Francisella-mediated immunosuppression, large-scale screening using a transposon random mutant library containing 3552 mutant strains of F. tularensis subsp. novicida (F. novicida) was performed. Thirteen mutants that caused stronger tumor necrosis factor (TNF)-α production in infected U937 human macrophage cells than the wild-type F. novicida strain were isolated. Sequencing analysis of transposon insertion sites revealed 10 genes, including six novel genes, as immunosuppressive factors of Francisella. Among these, the relationship of the pyrC gene, which encodes dihydroorotase in the pyrimidine biosynthesis pathway, with Francisella-mediated immunosuppression was investigated. The pyrC deletion mutant strain (ΔpyrC) induced higher TNF-α production in U937 host cells than the wild-type F. novicida strain. The ΔpyrC mutant strain was also found to enhance host interleukin-1ß and interferon (IFN)-ß production. The heat-inactivated ΔpyrC mutant strain could not induce host TNF-α production. Moreover, the production of IFN-ß resulting from ΔpyrC infection in U937 cells was repressed upon treatment with the stimulator of interferon genes (STING)-specific inhibitor, H-151. These results suggest that pyrC is related to the immunosuppressive activity and pathogenicity of Francisella via the STING pathway.

The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation.

Intracellular pathogens lose many metabolic genes during their evolution from free-living bacteria, but the pathogenic consequences of their altered metabolic programs on host immunity are poorly understood. Here, we show that a pathogenic strain of Francisella tularensis subsp. tularensis (FT) has five amino acid substitutions in RibD, a converting enzyme of the riboflavin synthetic pathway responsible for generating metabolites recognized by mucosal-associated invariant T (MAIT) cells. Metabolites from a free-living strain, F. tularensis subsp. novicida (FN), activated MAIT cells in a T-cell receptor (TCR)-dependent manner, whereas introduction of FT-type ribD to the free-living strain was sufficient to attenuate this activation in both human and mouse MAIT cells. Intranasal infection in mice showed that the ribD FT-expressing FN strain induced impaired Th1-type MAIT cell expansion and resulted in reduced bacterial clearance and worsened survival compared with the wild-type free-living strain FN. These results demonstrate that F. tularensis can acquire immune evasion capacity by alteration of metabolic programs during evolution.

Distinction of Paramecium strains by a combination method of RAPD analysis and multiplex PCR.

Paramecium is employed as a valuable model organism in various research fields since a large number of strains with different characteristics of size, morphology, degree of aging, and type of conjugation can be obtained. It is necessary to determine a method for the classification and simple identification of strains to increase their utility as a research tool. This study attempted to establish a polymerase chain reaction (PCR)-based method to differentiate strains of the same species. Genomic DNA was purified from several strains of P. caudatum, P. tetraurelia, and P. bursaria used for comparison by the random amplified polymorphic DNA (RAPD)-PCR method. In P. tetraurelia and P. bursaria, it was sufficiently possible to distinguish specific strains depending on the pattern of random primers and amplification characteristics. For the classification of P. caudatum, based on the sequence data obtained by RAPD-PCR analysis, 5 specific primer sets were designed and a multiplex PCR method was developed. The comparative analysis of 2 standard strains, 12 recommended strains, and 12 other strains of P. caudatum provided by the National BioResource Project was conducted, and specific strains were identified. This multiplex PCR method would be an effective tool for the simple identification of environmental isolates or the management of Paramecium strains.

Francisella novicida can utilize Paramecium bursaria as its potential host.

Francisella novicida is a facultative intracellular pathogen and the causative agent of tularemia. Although cases of infection caused by exposure to contaminated water have been reported, its natural host and ecology in the environment remain unclear. In this study, we investigated in vitro the possibility that Paramecium bursaria may be a useful tool as a protist host model of F. novicida. Experimental infection with F. novicida resulted in a stable intracellular relationship within P. bursaria. This symbiotic intracellular relationship was not observed in experimental infections with other Francisella species and Legionella pneumophila. We found that F. novicida showed similar behaviour to that of the eukaryotic endosymbiont of P. bursaria, the green algae Chlorella, in the internalization process. In addition, stable intracellular localization of F. novicida was possible only when Chlorella was not present. Although we investigated the type VI secretion system of F. novicida as a candidate for the bacterial factor, we found that it was not involved in the establishment of an intracellular relationship with P. bursaria. These results suggested that P. bursaria is potentially a protist host model for F. novicida and may be a useful tool for understanding the relationship between protist hosts and their symbionts.

Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method.

Paramecium spp. are a genus of free-living protists that live mainly in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium's unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

Peculiar Paramecium Hosts Fail to Establish a Stable Intracellular Relationship With Legionella pneumophila.

Legionella pneumophila, an intracellular human pathogen, establishes intracellular relationships with several protist hosts, including Paramecium caudatum. L. pneumophila can escape the normal digestion process and establish intracellular relationships in Paramecium. In this study, we identify new Paramecium strains that significantly reduce the number of L. pneumophila during infection. As a result, stable intracellular relationships between L. pneumophila and these Paramecium strains were not observed. These digestion-type Paramecium also showed high efficiency for Escherichia coli elimination compared to other strains of Paramecium. These results suggest that the digestion-type strains identified have high non-specific digestion activity. Although we evaluated the maturation process of Legionella-containing vacuoles (LCVs) in the Paramecium strains using LysoTracker, there were no discriminative changes in these LCVs compared to other Paramecium strains. Detailed understanding of the mechanisms of high digestion efficiency in these strains could be applied to water purification technologies and L. pneumophila elimination from environmental water.

Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model.

Francisella tularensis, the causative agent of tularemia, is transmitted by arthropod vectors within mammalian hosts. The detailed mechanisms contributing to growth and survival of Francisella within arthropod remain poorly understood. To identify novel factors supporting growth and survival of Francisella within arthropods, a transposon mutant library of F. tularensis subsp. novicida (F. novicida) was screened using an F. novicida-silkworm infection model. Among 750 transposon mutants screened, the mltA-encoding membrane-bound lytic murein transglycosylase A (MltA) was identified as a novel growth factor of F. novicida in silkworms. Silkworms infection with an mltA deletion mutant (ΔmltA) resulted in a reduction in the number of bacteria and prolonged survival. The ΔmltA strain exhibited limited intracellular growth and cytotoxicity in BmN4 silkworm ovary cells. Moreover, the ΔmltA strain induced higher expression of the antimicrobial peptide in silkworms compared to the wild-type strain. These results suggest that F. novicida MltA contributes to the survival of F. novicida in silkworms via immune suppression-related mechanisms. Intracellular growth of the ΔmltA strain was also reduced in human monocyte THP-1 cells. These results also suggest the contribution of MltA to pathogenicity in humans and utility of the F. novicida-silkworm infection model to explore Francisella infection.

Soluble lytic transglycosylase SLT of Francisella novicida is involved in intracellular growth and immune suppression.

Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells.

Attenuated Legionella pneumophila Survives for a Long Period in an Environmental Water Site.

Legionella pneumophila is known as a human pathogen and is ubiquitous in natural and artificial aquatic environments. Many studies have revealed the virulence traits of L. pneumophila using clinical strains and a number of studies for characterizing environmental strains are also reported. However, the association between the virulence and survivability in the environment is unclear. In the present study, L. pneumophila was isolated from environmental water sites (Ashiyu foot spa, water fountain, and public bath), and the serogroups of isolated strains were determined by serological tests. Isolated strains were found to belong to serogroups SG1, SG2, SG3, SG4, SG5, SG8, SG9, and SG13. Untypeable strains were also obtained. Isolated strains were used for intracellular growth assay in a human monocytic cell line, THP-1. Among these strains, only an untypeable strain, named AY3, failed to replicate in THP-1. In addition, AY3 was maintained for a long period in an environmental water site, Ashiyu foot spa 2. Further, we compared the characteristics of several strains isolated from Ashiyu foot spa 2 and a clinical strain, Togus-1. AY3 failed to replicate in THP-1 cells but replicated in an amoeba model, Dictyostelium discoideum. Compared with Togus-1, the culturable cell number of environmental strains under stress conditions was higher. Moreover, biofilm formation was assessed, and AY3 showed the same degree of biofilm formation as Togus-1. Biofilm formation, replication in amoebae, and resistance against stress factors would explain the predominance of AY3 at one environmental site. Although the mechanism underlying the difference in the ability of AY3 to replicate in THP-1 cells or amoebae is still unclear, AY3 may abandon the ability to replicate in THP-1 cells to survive in one environment for a long period. Understanding the mechanisms of L. pneumophila in replication within different hosts should help in the control of Legionnaires' disease, but further study is necessary.

Expression of Francisella pathogenicity island protein intracellular growth locus E (IglE) in mammalian cells is involved in intracellular trafficking, possibly through microtubule organizing center.

Francisella tularensis is the causative agent of the infectious disease tularemia and is designated a category A bioterrorism agent. The type VI secretion system encoded by the Francisella pathogenicity island (FPI) is necessary for intracellular growth; however, the functions of FPI proteins are largely unknown. In this study, we found that the FPI protein intracellular growth locus E (IglE) showed a unique localization pattern compared to other FPI proteins. Deleting iglE from Francisella tularensis subsp. novicida (F. novicida) decreased intracellular growth. Immunoprecipitation and pull-down assays revealed that IglE was associated with ß-tubulin. Additionally, GFP-fused IglE colocalized with microtubule organizing centers (MTOCs) in 293T cells. The iglE deletion mutant was transferred with dynein toward MTOCs and packed into lysosome-localizing areas. Conversely, the wild-type F. novicida exhibited intracellular growth distant from MTOCs. In addition, IglE expressed in 293T cells colocalized with dynein. These results suggest that IglE helps to prevent dynein- and MTOC-mediated intracellular trafficking in host cells to inhibit the transport of F. novicida toward lysosomes.

Identification of novel Legionella genes required for endosymbiosis in Paramecium based on comparative genome analysis with Holospora spp.

The relationship between Legionella and protist hosts has a huge impact when considering the infectious risk in humans because it facilitates the long-term replication and survival of Legionella in the environment. The ciliate Paramecium is considered to be a protist host for Legionella in natural environments, but the details of their endosymbiosis are largely unknown. In this study, we determined candidate Legionella pneumophila genes that are likely to be involved in the establishment of endosymbiosis in Paramecium caudatum by comparing the genomes of Legionella spp. and Holospora spp. that are obligate endosymbiotic bacteria in Paramecium spp. Among the candidate genes, each single deletion mutant for five genes (lpg0492, lpg0522, lpg0523, lpg2141 and lpg2398) failed to establish endosymbiosis in P. caudatum despite showing intracellular growth in human macrophages. The mutants exhibited no characteristic changes in terms of their morphology, multiplication rate or capacity for modulating the phagosomes in which they were contained, but their resistance to lysozyme decreased significantly. This study provides insights into novel factors required by L. pneumophila for endosymbiosis in P. caudatum, and suggests that endosymbiotic organisms within conspecific hosts may have shared genes related to effective endosymbiosis establishment.

RtxA like protein contributes to infection of Francisella novicida in silkworm and human macrophage THP-1.

Tularemia is a zoonosis caused by CDC-declared Tier 1 threat agent Francisella tularensis. F. tularensis subsp. novicida (F. novicida) is virulent in mice but non-pathogenic in immunocompetent humans and serves as a potential surrogate organism. In a recent study, we established a silkworm (Bombyx mori) model of infection for F. novicida. Francisella secretes its virulence factors through various mechanisms that modify the intracellular environment to ensure its replication and survival. To identify new pathogenic factors, we focused on the type I secretory system (T1SS) of Francisella. In silico analysis revealed a RtxA (Repeats-in-toxin) like protein in the Francisella genome. The characteristics of RtxA like protein were investigated using mutant analysis. Firstly, the role of rtxA in silkworms was investigated by infecting them with F. novicida strains into the hemocoel. The rtxA mutant failed to kill the silkworms, whereas F. novicida wild-type (WT) strain killed silkworms within 3-7 days post infection. The arrested growth of the mutant strain in silkworms was observed using a whole-body CFU count assay. We also investigated the growth characteristics of the rtxA mutant in hemocytes, one of the primary multiplication sites of Francisella within silkworms. Interrupted growth of the rtxA mutant with significantly reduced cytotoxicity was observed in hemocytes via confocal microscopy. Next, we analyzed the effect of rtxA in human monocyte cell line THP-1. The mutant strain showed significantly decreased growth and reduced cytotoxicity compared with its parental strain in THP-1 cells. This study newly identified RtxA like protein of F. novicida as an important lethal pathogenic factor in silkworm and mammalian cells.

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires' disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries' disease, understanding the ecology of L. pneumophila in the environment is essential.

Species distribution, virulence factors, and antimicrobial resistance of Acinetobacter spp. isolates from dogs and cats: a preliminary study.

We investigated the prevalence of virulence factors and antimicrobial resistance among 67 Acinetobacter spp. isolates, consisting of 21 Acinetobacter baumannii and 46 non-baumannii Acinetobacter from companion animals. The PCR analysis showed that the most prevalent virulence gene was afa/draBC (29.9%), followed by papC (22.4%) and cvaC (20.9%). Antimicrobial susceptibility testing revealed that resistance to gentamicin (14.9%) and ciprofloxacin (11.9%) was relatively prevalent. Five gentamicin- and/or ciprofloxacin-resistant A. baumannii strains were assigned to ST25, ST149, ST164, ST203, and ST1198. All ciprofloxacin-resistant isolates harbored point mutations in gyrA and/or parC. This is the first preliminary monitoring of animal-origin Acinetobacter spp. in Japan.

Silkworm model for Francisella novicida infection.

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida, which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm (Bombyx mori) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida. We found that silkworms died within 3-7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella.

Tick surveillance for Borrelia miyamotoi and phylogenetic analysis of isolates in Mongolia and Japan.

Borrelia miyamotoi, recently recognized as a human pathogenic spirochete, was isolated from Ixodes persulcatus and I. ovatus in northern Mongolia and Honshu Island, a major island in Japan. Although no human B. miyamotoi infections have been reported in Mongolia, the prevalence of B. miyamotoi in ticks from Mongolia is higher than that in ticks from Hokkaido, Japan, where human cases have been reported. Moreover, the multi-locus sequence analysis of cultured isolates revealed that B. miyamotoi isolates in Mongolia belong to the Siberian type, a sequence type that was originally reported from isolates from I. persulcatus in Hokkaido. Thus, there is a possibility of unrecognized human B. miyamotoi infections in Mongolia. Moreover our data support the hypothesis of clonal expansion of the Siberian type B. miyamotoi. In contrast, although the isolates were found to belong to the Siberian type B. miyamotoi, two isolates from I. persulcatus in Honshu Island were identified to be of a different sequence type. Furthermore, B. miyamotoi isolates from I. ovatus were distinguishable from those from I. ricinus complex ticks, according to genetic analysis. In this study, we show that there may be some genetic diversity among B. miyamotoi in ticks from Honshu Island.

Epidemiological study of relapsing fever borreliae detected in Haemaphysalis ticks and wild animals in the western part of Japan.

The genus Borrelia comprises arthropod-borne bacteria, which are infectious agents in vertebrates. They are mainly transmitted by ixodid or argasid ticks. In Hokkaido, Japan, Borrelia spp. were found in deer and Haemaphysalis ticks between 2011 and 2013; however, the study was limited to a particular area. Therefore, in the present study, we conducted large-scale surveillance of ticks and wild animals in the western part of the main island of Japan. We collected 6,407 host-seeking ticks from two regions and 1,598 larvae obtained from 32 engorged female ticks and examined them to elucidate transovarial transmission. In addition, we examined whole blood samples from 190 wild boars and 276 sika deer, as well as sera from 120 wild raccoons. We detected Borrelia spp. in Haemaphysalis flava, Haemaphysalis megaspinosa, Haemaphysalis kitaokai, Haemaphysalis longicornis, and Haemaphysalis formosensis. In addition, we isolated a strain from H. megaspinosa using Barbour-Stoenner-Kelly medium. The minimum infection rate of ticks was less than 5%. Transovarial transmission was observed in H. kitaokai. Phylogenetic analysis of the isolated strain and DNA fragments amplified from ticks identified at least four bacterial genotypes, which corresponded to the tick species detected. Bacteria were detected in 8.4%, 15%, and 0.8% of wild boars, sika deer, and raccoons, respectively. In this study, we found seasonal differences in the prevalence of bacterial genotypes in sika deer during the winter and summer. The tick activity season corresponds to the season with a high prevalence of animals. The present study suggests that a particular bacterial genotype detected in this study are defined by a particular tick species in which they are present.

Analysis of IMP-1 type metallo-ß-lactamase-producing Acinetobacter radioresistens isolated from companion animals.

IMP-1 type metallo-ß-lactamase-producing (MBL-producing) Acinetobacter radioresistens was isolated from a dog with cystitis and a cat with conjunctivitis. The MBL-producing A. radioresistens isolates were resistant to all of the ß-lactam antibiotics used in the sensitivity tests, but were susceptible to gentamicin, amikacin, and minocycline. Also, one of the two strains of A. radioresistens was susceptible to ciprofloxacin and levofloxacin. These two cases were cured by administration of tetracyclines and fluoroquinolones, which elicited a positive result in the sensitivity tests. This report of the isolation of MBL-producing A. radioresistens in companion animals is the first in the world. To prevent the proliferation of MBL-producing bacteria, veterinary hospitals need to be aware of the behavior of MBL-producing organisms.