Comparative pathogenomic analysis reveals a highly tetanus toxin-producing clade of Clostridium tetani isolates in Japan.

IMPORTANCE: C. tetani is a spore-forming, anaerobic bacterium that produces a toxin causing muscle stiffness and paralysis. Tetanus is preventable with the toxoid vaccine, but it remains a significant public health threat in regions with low vaccine coverage. However, there are relatively few isolates and limited genomic information available worldwide. In Japan, about 100 cases are reported each year, but there have been no nationwide surveys of isolates, and no genomic information from Japanese isolates has been published. In our study, we analyzed the genomes of 151 strains from a limited survey of soil in Kumamoto, Japan. Our findings revealed a high degree of genetic diversity, and we also identified a subset of strains that produced significantly more toxin, which provides new insights into the pathogenesis of tetanus. Our findings lay the foundation for future studies to investigate the distribution and evolution of C. tetani in Japan and neighboring countries.

Clinical Characteristics of Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

Rhabdophis tigrinus (Yamakagashi) Bites in Japan Over the Last 50 Years: A Retrospective Survey.

Introduction:Rhabdophis snakes, which include 27 species, are rear-fanged venomous snakes that are widely distributed from India to East Asia and Russia. Severe envenomation by R. tigrinus (Yamakagashi snake) in Japan and R. subminiatus in Southeast Asia has been reported. The epidemiology of R. tigrinus bites, such as geographical features, the incidence, and changes in the number of bites over time have not been comprehensively examined. Hence, we intended to clarify the epidemiological features of R. tigrinus bites through a careful review of scientific data over the last 50 years in Japan. Methods: Patient records of R. tigrinus bites between 1971 and 2020 at the Japan Snake Institute were examined retrospectively. The following were ascertained: patient characteristics, clinical symptoms, laboratory data, treatment-related factors, and hospital mortality. These variables were compared in the antivenom and the without-antivenom groups. Results: Over the 50-year study period, 43 R. tigrinus bites, including five fatal cases, were encountered. Severe cases of R. tigrinus bites have been treated with antivenom since 1985; however, fatalities occurred in 2006 and 2020. R. tigrinus bite cases have been well-distributed in the western part of Japan since 2000. The mortality rate in the antivenom group was significantly lower in the patient group that was not administered the antivenom (0 vs. 23.8%, p = 0.048). Conclusion: This study clarified the epidemiology of R. tigrinus bites in Japan over a 50-year period. Almost all severe cases of R. tigrinus bites have been treated with the antivenom in the current situation, and fatalities occurred in cases not treated with the antivenom. It is important to diagnose R. tigrinus bites in the early phase of the clinical course. The antivenom, the definitive treatment for R. tigrinus bites, is an unapproved drug. Hence, approval needs to be obtained for the drug.

Freeze-dried equine-derived redback spider antivenom: a local irritation study by intramuscular injection in rabbits and a repeated-dose toxicity study in rats.

The redback spider (Latrodectus hasseltii) is nonindigenous to Japan but has now spread throughout the country. Bites to humans are rare but can be fatal. We prepared freeze-dried redback spider antivenom for therapeutic use against bites in Japan by immunization of horse plasma. This study included two nonclinical tests of the antivenom: a local irritation study involving a single intramuscular administration to rabbits (with injections of physiological saline and an existing freeze-dried diphtheria antitoxin as control and comparison substances, respectively) and a 2-week repeated intermittent intravenous-dose toxicity study in rats. The irritation study showed the antivenom's irritancy to be comparable with that of the saline and the existing antitoxin preparations under the test conditions. In a repeated-dose toxicity study, no toxicity change was found in male or female rats, and the no-observed-adverse-effect level (NOAEL) was judged to be a dose volume of 20 mL/kg (1082 units/kg antivenom activity) in both male and female rats. In addition, there was no toxicological difference between proteinaceous diphtheria antitoxin and redback spider antivenom prepared to have the same protein content and the same additive composition. Based on these findings, we will further advance our research towards clinical application of the redback spider antivenom. This research was supported by the Research Program on Emerging and Re-emerging Infectious Disease of the Japan Agency for Medical Research and Development.

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part I. Venom Extraction, Preparation, and Laboratory Testing.

The redback spider (Latrodectus hasseltii Thorell) reportedly invaded Japan in September 1995. To date, 84 redback spider bite cases have been reported; 7 of these cases employed the antivenom. Antivenom has been imported from Australia in the past, but because of restrictions on exportation it was evident that nearly all of the antivenom present in Japan would expire during 2014. In 2014, a plan was proposed to experimentally manufacture and stockpile a horse antiserum for ourselves, using redback spiders indigenous to Japan. A total of 11,403 female spiders were captured alive: 1,217 from the vicinity of Nishinomiya City, Hyogo prefecture, and 10,186 from Osaka prefecture. Of these, 10,007 females were dissected, and the venom was extracted from the venom glands of individuals and subjected to crude purification to yield 4 lots, of which the majority was α-latrotoxin. Among them, a large amount of single lots with an estimated protein content of 236 mg is subsequently scheduled to be used for immunizing horses. We also determined lethal toxicity of the venom (LD50: 9.17 µg per mouse), and established the assay for the determination of an anti-lethal titer of antivenom in mice.

Combined therapy with gas gangrene antitoxin and recombinant human soluble thrombomodulin for Clostridium perfringens sepsis in a rat model.

Cases of Clostridium perfringens septicemia, such as liver abscess, often develop a rapidly progressive intravascular hemolysis and coagulation; the mortality rate with current standard care including antibiotics and surgery is high. Herein, we firstly investigated the effects of gas gangrene antitoxin (GGA) (antitoxin against C. perfringens) and recombinant human soluble thrombomodulin (rTM) on the hemolysis, coagulation status, inflammatory process, and mortality in α-toxin-treated rats. Male 11-week-old Sprague Dawley rats were randomly divided into five groups: control group, α-toxin group, GGA group, rTM group, and combined GGA and rTM (combination group). After α-toxin injection, mortality and platelet counts, and hemolysis were observed for 6 h. The fibrin/fibrinogen degradation products (FDP), and plasma high-mobility group box 1 (HMGB1) were also measured at 6 h. The combination group demonstrated 100% survival compared with 50% survival in the α-toxin group and demonstrated significantly improved hemolysis, platelet counts, and lactate levels compared with those in the α-toxin group (p < .01). The FDP and HMGB1 levels in the combination therapy group were significantly lower than those in the α-toxin group (p < .05). Combination therapy with GGA and rTM administration is applicable as adjunct therapy for fatal C. perfringens sepsis.

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part II. Experimental Production of Equine Antivenom against the Redback Spider.

This is the first report on large-scale experimental production of an equine antivenom against the redback spider (Latrodectus hasseltii) lived in Japan. We captured 10,000 redback spiders in Japan and prepared the toxoids of crude venom extract, mixed the toxoids with a mineral oil adjuvant, and immunized healthy horses repeatedly over a period of several weeks. Thereafter, we separated the horse plasma, purified the γ-globulin fraction, and stocked it as a purified antivenom concentrate. Consequently, we manufactured approximately 6,500 vials of a single-dose freeze-dried test lot from a portion of the purified γ-globulin fraction, equivalent to the extract derived from 520 spiders. This test lot had an antitoxin titer comparable to that of a similar drug commercially available overseas (a liquid preparation), and the other quality met all quality reference specifications based on the Minimum Requirements for Biological Products and other guidelines relevant to existing antivenom drug products in Japan.

Clinical Study of New Tetravalent (Type A, B, E, and F) Botulinum Toxoid Vaccine Derived from M Toxin in Japan.

Botulinum toxin is the most poisonous substance known, and is believed to be a highly lethal as a biological weapon; researchers of the toxin are exposed to this hazard. Botulinum toxoid vaccines have been produced and used in Japan. However, since clinical studies involving these vaccines were conducted before establishment of the Ethical Guidelines for Clinical Research in Japan, their immunogenicity and safety were not systematically assessed. In this study, we produced a new tetravalent (type A, B, E, and F) botulinum toxoid vaccine, the first ever to be derived from M toxin, and conducted quality control tests with reference to the Minimum Requirements in Japan for adsorbed tetanus toxoid vaccine. Subsequently, a clinical study using the new vaccine in 48 healthy adult volunteers was conducted according to the guidelines in Japan. No clinically serious adverse event was noted. Neutralizing antibody titers for each type of toxin in the participants' sera, 1 month after the 4th injection were more than 0.25 IU/mL, indicating sufficient protection. This study demonstrated that the vaccine has marked immunogenicity and is safe for use in humans.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Retrospective survey to evaluate the safety and efficacy of Japanese botulinum antitoxin therapy in Japan.

Japanese botulinum antitoxins have been used for more than 50 years; however, their safety and therapeutic efficacy are not clear. In order to analyze the available data on botulinum antitoxin therapy in Japan, we surveyed published reports about botulism cases in which botulinum antitoxins were used, and retrospectively analyzed the safety and efficacy of the therapy. A total of 134 patients administered botulinum antitoxins were identified from published reports. Two cases of side effects (1.5%) were detected after antitoxin administration, both not fatal. The fatality rate was 9.4%, and more than 70% of the patients showed improvement in their symptoms and better clinical conditions than those not treated with antitoxins. These data suggest that the therapy with Japanese antitoxins is safe and highly effective.

Genetic characterization and comparison of Clostridium botulinum isolates from botulism cases in Japan between 2006 and 2011.

Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.

A national reference for inactivated polio vaccine derived from Sabin strains in Japan.

As one aspect of its campaign to eradicate poliomyelitis, the World Health Organization (WHO) has encouraged development of the inactivated polio vaccine (IPV) derived from the Sabin strains (sIPV) as an option for an affordable polio vaccine, especially in low-income countries. The Japan Poliomyelitis Research Institute (JPRI) inactivated three serotypes of the Sabin strains and made sIPV preparations, including serotypes 1, 2 and 3 D-antigens in the ratio of 3:100:100. The National Institute of Infectious Diseases, Japan, assessed the immunogenic stability of these sIPV preparations in a rat potency test, according to an evaluation method recommended by the WHO. The immunogenicity of the three serotypes was maintained for at least 4 years when properly stored under -70°C. Based on these data, the sIPV preparations made by JPRI have been approved as national reference vaccines by the Japanese national control authority and used for the quality control of the tetracomponent sIPV-containing diphtheria-tetanus-acellular pertussis combination vaccines that were licensed for a routine polio immunization in Japan.

Seroprevalence of tetanus toxoid antibody and booster vaccination efficacy in Japanese travelers.

Tetanus can be prevented by vaccination, which is especially important for overseas travelers. However, despite booster vaccination every 10 years being recommended, most Japanese adults do not receive it in the absence of physical injury or overseas travel. We aimed to investigate the level of protective immunity against tetanus among Japanese travelers, which may provide valuable information for formulating booster vaccination recommendations. 113 Japanese travelers given tetanus toxoid were recruited. The collected samples included paired samples prior to and 3-5 weeks after receiving the booster vaccination. Travelers who did not return and those lacking sample collection at the second visit were excluded. Finally, 96 paired blood samples were collected. History of immunization against tetanus, including DPT and DT vaccines, was determined from interviews or immunization records. The pre-vaccination geometric mean titer for the 96 participants was 1.07 IU/mL; 76% had a protective antitoxin level (>0.1 IU/mL), and 50% had a long-term protective antitoxin level (>1.0 IU/mL). Most participants <40 years old had protective immunity without receiving booster vaccination, whereas only 30.8% of those >50 years of age had protective immunity. Among the 23 participants without protective antitoxin levels (<0.1 IU/mL), booster vaccination was efficient in 100% of those <40 years but in only 28.6% of those >50 years of age. Although the tetanus antitoxin level decreases with age, booster vaccination helped to achieve an adequate protective antitoxin levels in Japanese travelers <40 years of age. Furthermore, the individuals who have never been vaccinated against tetanus especially in those >50 years old need to obtain protective immunity against tetanus according to a basic immunization schedule to prevent tetanus in travelers and residents of Japan.

Differences in immunological responses of polyclonal botulinum A1 and A2 antitoxin against A1 and A2 toxin.

Botulinum type A antitoxin in standard and therapeutic preparation is a polyclonal antibody purified from immunized sera with subtype A1 toxin. To investigate the difference between immunological responses of antitoxin against toxin among different subtypes, we examined the response of polyclonal A1 and A2 antitoxins with A1 and A2 toxins. In the mouse neutralization test, the A1 antitoxin had equivalent potency against both the A1 and A2 toxins. However, the neutralization titer of the A2 antitoxin was 4-9 fold higher against the A2 toxin than against the A1 toxin. Since the titers of the antitoxins were calculated relative to the standard antitoxin, we assumed that the difference between the antibody titers against the test toxins was due the standard antitoxin having different reactivities with the toxins. The binding volume of the A2 toxin against the standard and A1 antitoxins was 3-4% of the binding volume of the A1 toxin. The neutralization curve of the standard antitoxin was parallel against both the A1 and A2 toxins. However, the curve of the A2 antitoxin was not parallel against the A1 and A2 toxins. Furthermore, binding analysis comparing these antitoxins and toxins showed that the A1 antitoxin had a higher binding affinity and slower dissociation speed with the A1 toxin than with the A2 toxin. In contrast, the A2 antitoxin showed a higher binding affinity with the A2 toxin than with the A1 toxin. These findings indicated that antitoxin reacted strongly against the same subtype, and showed a weak response against the toxin of different subtypes. We propose that the existing standard type A antitoxin should be used for reaction with A1 toxins, and that it is necessary to establish a new standard antitoxin for each subtype of toxin.

Isolation and characterization of toxigenic Corynebacterium ulcerans from 2 closed colonies of cynomolgus macaques (Macaca fascicularis) in Japan.

The aim of the present study was to determine the prevalence of infection by toxigenic Corynebacterium ulcerans in cynomolgus macaques (Macaca fascicularis) housed in an animal facility in Japan. Samples from the pharynges of animals from 2 closed colonies (colony A, n = 47; colony B, n = 21) were cultured. C. ulcerans grew from 43% and 47% of the samples from colonies A and B, respectively. The toxigenicity of these isolates was assessed by using PCR analysis for the diphtheria toxin gene and the Elek test and Vero cytotoxicity assay to detect diphtheria toxin. The proportion of macaques harboring toxigenic C. ulcerans was 6% in colony A and 29% in colony B. Analysis of diphtheria antitoxin neutralization titers in the sera revealed that 23% and 33% of macaques from colonies A and B, respectively, had a history of infection with toxigenic C. ulcerans. Pulsed-field gel electrophoresis of the toxigenic isolates showed that all of those recovered from macaques in colony B showed an identical genotype, suggesting that transmission of the organism occurred within the colony. However, isolates from colony A macaques showed 3 different genotypes, one of which was identical to the isolate from colony B. Additional studies evaluating the prevalence and transmission of toxigenic C. ulcerans within colonies of nonhuman primates are necessary to help control the spread of the infection. The current study is the first description of the isolation and characterization of toxigenic C. ulcerans from nonhuman primates in Japan.

Clinical efficacy of antivenom and cepharanthine for the treatment of Mamushi (Gloydius blomhoffii) bites in tertiary care centers in Japan.

To our knowledge, no one has conducted a multi-center trial evaluating the efficacy of antivenom and cepharanthine (CEP) for the treatment of mamushi (Gloydius blomhoffii) bites. Thus, we conducted a large-scale survey among tertiary care centers in Japan from November 2009 to October 2010 to evaluate the efficacy of antivenom and CEP for the treatment of mamushi bites. We divided the therapeutic interventions received by patients into 4 groups: CEP, antivenom, both CEP and antivenom, and neither CEP nor antivenom. We collected data on age, sex, comorbidities, laboratory measurements, length of hospital stay, and grades of mamushi bites (indication of bite severity ranged from I [mild] to V [severe]). We sent questionnaires to 219 tertiary care centers, of which 114 (52.1%) returned completed questionnaires. Two hundred and thirty-four cases of mamushi bites were reported. Among the severe cases (grades of mamushi bites III, IV, and V), patients administered antivenom had a significantly shorter length of hospital stay than those administered CEP (P = 0.024). In contrast, there was no significant difference in the length of hospital stay between mild cases (grades of mamushi bites I and II) (P = 0.77). Our results show that antivenom is effective in reducing the length of hospital stay in patients with severe mamushi bites.

Effects of simultaneous immunization of Haemophilus influenzae type b conjugate vaccine and diphtheria-tetanus-acellular pertussis vaccine on anti-tetanus potencies in mice, guinea pigs, and rats.

Haemophilus influenzae type b vaccine conjugated with tetanus toxoid (HibT) was licensed for use in childhood immunization in Japan in 2007. As adsorbed diphtheria-tetanus-acellular pertussis (DTaP) combined with HibT vaccine has not been introduced in Japan, DTaP and HibT vaccines are injected at separate sites with a similar immunization schedule. There are various interfering or stimulatory effects between components of combined vaccines contained in DTaP and HibT vaccines. In this study, we investigated the effect of HibT containing combination vaccines on anti-tetanus potencies by using animal models (mouse, guinea pig, and rat). HibT vaccine and HibT components of imported DTaP-HibT vaccine alone showed comparable or higher anti-tetanus potency than DTaP vaccine and DTaP-containing components of combination vaccines. Mixing these components before injection resulted in potencies greater than the sum of individual potencies. Injecting individual components at separate sites in animals resulted in potency roughly equivalent to the sum of the individual potencies. These results provide useful information regarding the use of HibT-containing multivalent vaccines in childhood immunization.

Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.