Retrospective anti-tetanus antibody responses of zoo-based Asian elephants (Elephas maximus) and rhinoceros (Rhinocerotidae).

Tetanus toxoids (TT) commercially available for use in horses and livestock are commonly used to vaccinate elephants and rhinoceros that are in human care. Although recommendations for booster intervals have changed in human and horse protocols to reduce the risks associated with hyper-immunity (i.e. B-cell anergy and hypersensitivity reactions) these have generally not been adopted in zoo protocols. Additionally, there is no evidence to demonstrate commercial TT immunogenicity in rhinoceros. In this study, a preliminary analysis of rhinoceros antibody responses to TT was conducted, in addition to an exploration of the impact of various booster frequencies on antibody responses in elephant. Retrospective analysis of archived serum samples was conducted for 9 Asian elephants (Elephas maximus), 7 southern black (Diceros bicornis minor), one southern white (Ceratotherium simum simum), and two greater one-horned (Rhinoceros unicornis) rhinoceros. Pre-vaccination (baseline) samples and those following priming vaccination (rhinoceros only), annual and non-annual boosters were targeted. A commercially available competitive ELISA kit was used to quantify serum anti-TT antibodies. Average baseline and post-vaccination anti-tetanus antibody concentrations were greater in elephant (92 mg/L ± 42, n = 3, N = 3; 125 ± 76, n = 82, N = 9) than in rhinoceros (47 mg/L ± 39, n = 8, N = 8; 44 mg/L ± 37, n = 16, N = 7). Rhinoceros antibody concentrations did not differ markedly following vaccinations from their naturally acquired high pre-vaccination concentrations. Eight elephants demonstrated antibody maintenance for 3-5 years without a tetanus booster. Additionally, although five out of nine elephants developed local reactions consistent with delayed type IV hypersensitivity following some boosters, there was no association between high antibody concentrations and increased incidence of adverse reactions. In addition, no decrease in antibody concentrations was detected as a result of annual vaccination in elephants, though this does not entirely rule out potential for B-cell anergy.

Tetanus in animals.

Tetanus is a neurologic disease of humans and animals characterized by spastic paralysis. Tetanus is caused by tetanus toxin (TeNT) produced by Clostridium tetani, an environmental soilborne, gram-positive, sporulating bacterium. The disease most often results from wound contamination by soil containing C. tetani spores. Horses, sheep, and humans are highly sensitive to TeNT, whereas cattle, dogs, and cats are more resistant. The diagnosis of tetanus is mainly based on the characteristic clinical signs. Identification of C. tetani at the wound site is often difficult.

Sharpening Host Defenses during Infection: Proteases Cut to the Chase.

The human immune system consists of an intricate network of tightly controlled pathways, where proteases are essential instigators and executioners at multiple levels. Invading microbial pathogens also encode proteases that have evolved to manipulate and dysregulate host proteins, including host proteases during the course of disease. The identification of pathogen proteases as well as their substrates and mechanisms of action have empowered significant developments in therapeutics for infectious diseases. Yet for many pathogens, there remains a great deal to be discovered. Recently, proteomic techniques have been developed that can identify proteolytically processed proteins across the proteome. These "degradomics" approaches can identify human substrates of microbial proteases during infection in vivo and expose the molecular-level changes that occur in the human proteome during infection as an operational network to develop hypotheses for further research as well as new therapeutics. This Perspective Article reviews how proteases are utilized during infection by both the human host and invading bacterial pathogens, including archetypal virulence-associated microbial proteases, such as the Clostridia spp. botulinum and tetanus neurotoxins. We highlight the potential knowledge that degradomics studies of host-pathogen interactions would uncover, as well as how degradomics has been successfully applied in similar contexts, including use with a viral protease. We review how microbial proteases have been targeted in current therapeutic approaches and how microbial proteases have shaped and even contributed to human therapeutics beyond infectious disease. Finally, we discuss how, moving forward, degradomics research can greatly contribute to our understanding of how microbial pathogens cause disease in vivo and lead to the identification of novel substrates in vivo, and the development of improved therapeutics to counter these pathogens.

Generalized Tetanus in a Gyrfalcon ( Falco rusticolus ) with Pododermatitis.

A 2-yr-old male gyrfalcon ( Falco rusticolus ) was presented for severe and generalized muscle spasticity and pododermatitis. The falcon had been treated for pododermatitis over the previous 4 mo. Muscle rigidity and spasms involved the entire bird but were more severe on the right leg. The bird was also tachypneic and hyperthermic at 45 C. While the plantar pododermatitis lesions had healed, there was still a small abscess on the lateral aspect of the right foot. Clinical signs were consistent with tetanus. Several bacteria were isolated from the abscess including Clostridium tetani . The isolate was confirmed to be toxigenic by PCR. Attempts to detect tetanus toxin in the bird's plasma were unsuccessful. The abscess was debrided. The gyrfalcon received equine tetanus antitoxin, intravenous metronidazole, methocarbamol, midazolam, a constant-rate infusion of Fentanyl, active cooling, and supportive care. Inhalant anesthesia with isoflurane was the only treatment that would lower the body temperature and reduce the clinical signs. The gyrfalcon died a few hours after admission. The characteristic clinical signs and isolation of toxigenic C. tetani from a wound were strong supportive evidence for a diagnosis of tetanus. This case constitutes the first reported natural occurrence of tetanus in an avian species. Further information is needed to determine whether gyrfalcons are more susceptible to tetanus than are other avian species and whether pododermatitis lesions may be risk factors.

Structural and functional analysis of hypothetical and conserved proteins of Clostridium tetani.

The progress in biological technologies has led to rapid accumulation of microbial genomic sequences with a vast number of uncharacterized genes. Proteins encoded by these genes are usually uncharacterized, hypothetical, and/or conserved. In Clostridium tetani (C. tetani), these proteins constitute up to 50% of the expressed proteins. In this regard, understanding the functions and the structures of these proteins is crucially important, particularly in C. tetani, which is a medically important pathogen. Here, we used a variety of bioinformatics tools and databases to analyze 10 hypothetical and conserved proteins in C. tetani. We were able to provide a detailed overview of the functional contributions of some of these proteins in several cellular functions, including (1) evolving antibiotic resistance, (2) interaction with enzymes pathways, and (3) involvement in drug transportation. Among these candidates, we postulated the involvement of one of these hypothetical proteins in the pathogenic activity of tetanus. The structural and functional prediction of these proteins should serve in uncovering and better understanding the function of C. tetani cells to ultimately discover new possible drug targets.

Infections with spore-forming bacteria in persons who inject drugs, 2000-2009.

Since 2000 in the United Kingdom, infections caused by spore-forming bacteria have been associated with increasing illness and death among persons who inject drugs (PWID). To assess temporal and geographic trends in these illnesses (botulism, tetanus, Clostridium novyi infection, and anthrax), we compared rates across England and Scotland for 2000-2009. Overall, 295 infections were reported: 1.45 per 1,000 PWID in England and 4.01 per 1,000 PWID in Scotland. The higher rate in Scotland was mainly attributable to C. novyi infection and anthrax; rates of botulism and tetanus were comparable in both countries. The temporal and geographic clustering of cases of C. novyi and anthrax into outbreaks suggests possible contamination of specific heroin batches; in contrast, the more sporadic nature of tetanus and botulism cases suggests that these spores might more commonly exist in the drug supply or local environment although at varying levels. PWID should be advised about treatment programs, injecting hygiene, risks, and vaccinations.

BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani.

Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the sigma70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.

Colonization of the intestinal tract of mice with Clostridium tetani.

Colonization of intragastrically inoculated Clostridium tetani in intestinal tract of mice was investigated by cultivation of daily stool samples and quantification of the bacilli over a period of 3 weeks. We found that most of the inoculum was excreted in the stool during the first 24 hr. The amount of C. tetani recovered on the second day after inoculation was very small. C. tetani apparently thrived in the intestinal tract when more than 6 log colony forming units (CFU) of tetanus spores had been inoculated. Otherwise, C. tetani colonized poorly in intestinal tract. Tetanus toxin was not detected in the stool nor in the contents of the small and large intestines 24 hr after 6.23 log CFU of tetanus spores had been inoculated. We concluded that the intestinal cavity of mouse is not a favorable environment for colonization of C. tetani under natural conditions.

[Fine morphology of the anomalies of sporulation in Clostridium tetani]. / Tonkaia morfologiia anomalii formirovaniia spor u Clostridium tetani.

The electron microscopic study of the cultures of Cl. tetani strains Nos. 154 and 741 has been carried out, starting from the late stationary phase of their development in a liquid culture medium prepared on the basis of casein hydrolysate. Under experimental conditions sporogenesis is observed in 17-33% of the organisms in the population of strain No. 154 and in 7-14% of the organisms in the population of strain No. 471. In most of the sporulating cells sporogenesis occurs in consecutive stages commonly observed in clostridia. Anomalies in the formation of pores are manifested by the ectopic initial formation of protospores, the disordered excess growth of osmiophil structures of the exosporic type, the early lysis of the cytoplasmic structures of the sporangium cell or the spore itself. Such changes resulting in the death of the cell are regarded as the pathomorphology of the bacterial cell.

The effect of oxidation-reduction potential on spore germination, outgrowth, and vegetative growth of Clostridium tetani, Clostridium butyricum, and Bacillus subtilis.

Spores of Clostridium tetani germinated in liver broth with +580 mV as the starting Eh value, and those of Clostridium butyricum germinated in liver broth with an initial Eh of +400 mV regardless of the presence or absence of a liquid paraffin covering. Spores of Bacillus subtilis germinated in liver broth with--100 mV as the starting Eh value. Also, it was found that there are two ranges of starting Eh values for germination and vegetative growth of Cl. tetani, Cl. butyricum, and B. subtilis. In the first range these spores germinated and grew, but in the second range they only germinated and then died without outgrowth.

Tetanus--review of the literature and report of case.

This case presents many of the difficulties of management that are inherent in a severe case of tetanus. Also it shows a seldom-considered therapeutic modality, that of dental extraction, gingival debridement, and gingivectomy with a confirmed case of tetanus without an established portal of entry. It is well known that periodontal and periapical locations can easily give rise to an anaerobic focus. In this case, anaerobic culture of C tetani was unsuccessful, possibly because of the inherent difficulty of anaerobic transfer from an oral locus and the extreme fastidiousness of the organism. As a rule, all patients who recover from tetanus do so completely, without any residual deficit. This was true in the case discussed here. Conclusions that can be drawn from this case are the following: a diagnosis of tetanus should be considered in any case of unexplained trismus; aggressive treatment of the immunologic deficit by antitoxin should be instituted along with a course of active immunization; and aggressive symptomatic treatment should be started. The bases of the treatment of this case were management of airways, sedation, treatment of autonomic crisis, and eradication of the causative agent. The result was that a severely ill patient reponded to treatment and completely recovered from a devastating disease.

[Phage-like particles produced by Clostridium tetani]. / Particules phagiques produites par Clostridium tetani.

Two C. tetani strains used for toxin production spontaneously produce two varieties of phage-like particles with isometric heads. Type A has a contractile tail, whereas type B shows a non-contractile tail with a long, wavy tail fiber. No relationship between the presence of these particles and the amount of toxin produced was found.