The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.     

Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.     

Guinea pig resistance to infection by the spores of the tetanus causative agent at various levels of tetanus antitoxin and delayed hypersensitivity

Comparative experimental investigations on guinea pigs immunized against tetanus, carried out with the use of different methods for the determination of immunity to tetanus, have demonstrated the existence of a relationship between the results of the tests used in these investigations (the neutralization test, the passive hemagglutination test and the allergic skin test) and the resistance of the animals to challenge with C. tetani spores in Dcl and Dlm50. The mechanisms of not only humoral, but also cell-mediated immunity play an important role in protection from tetanus infection. The possibility of using, on principle, the allergic skin test with tetanin for the evaluation of immunity to tetanus in individual patients with the aim of differential approach to protection against tetanus in traumas has been shown.     

Experience with the expanded WHO program on immunization against tetanus

According to (WHO) statistics, over 1 million infants in the developing countries die each year from tetanus. The estimated annual occurrence of tetanus in the 3rd World exceeds 2.5 million cases, including approximately 1.3 million newborn infants. In 1974, WHO began an expanded program for the systematic immunization of infants against tetanus and certain other diseases. The program uses 2 approaches for preventing tetanus: 1) immunization of infants under 1 year of age with the AKDS vaccine; and 2) immunization of pregnant women or, if possible, all women, with tetanus anatoxin. The 2nd approach is more effective, especially when 2 doses of tetanus anatoxin are administered within a minimum interval of 4 weeks. The anatoxin has no harmful effects on the fetus and can be used during any stage of pregnancy. The program strives to reduce infant mortality caused by tetanus to less than 1 case in 1000 by 1990, and to 0 by 2000. To attain these goals, systematic immunization should be combined with drastic improvements in delivery techniques and hygiene in developing countries. Specialized surveys indicate that initial steps toward implementation of the program resulted in a significant reduction of infant mortality caused by tetanus. Experience with the expanded WHO program shows that elimination of tetanus in infants is a realistic and attainable goal.     

双佐剂包被的破伤风类毒素马匹免疫试验

为提高破伤风免疫马匹的血浆抗体效价,应用不同佐剂配制TT抗原,进行马匹超免疫比较研究;采用FIA和植物油双佐剂包被与单佐剂包被的TT抗原,注射马匹进行超免疫,比较三组血浆的效价;结果显示,双佐剂抗原较单佐剂的免疫效果好,但可能对马匹刺激较强,有待调整注射剂量和免疫程序。     

Mechanism of paradoxical resistance of the digastric muscle to the action of tetanus toxin

Excitability parameters of m. masseter and m. digastricus fibers (resting minute potential--MP, action potential--AP, critical level of depolarization--CLD and rheobase currents) were determined in anesthetized rats (nembutal, 40 mg/kg) on days 3-4 and 10-15 after local intramuscular tetanus toxin injection (20 and 40 mouse Dlm, respectively). The development of local tetanus in m. masseter was characterized by typical excitability changes--a decrease in MP and AP. Local spasm in m. digastricus under analogous conditions of tetanus toxin injection was absent, while excitability tests demonstrated MP, AP and CLD augmentation. As m. digastricus motoneurons are devoid of inhibitory synaptic regions, these nervous cells are not affected by tetanus toxin.     

Absence of Naturally Acquired Tetanus Antitoxin in the Free-Ranging Cayo Santiago Rhesus Monkeys (Macaca mulatta)

Tetanus is enzootic in the free-ranging rhesus monkey colony on Cayo Santiago. The disease accounts for 25% of all mortalities in the population. The high prevalence of tetanus provided a unique opportunity to study the potential roles of geophagia, wounding, and clinical tetanus infections on the development of naturally acquired tetanus antitoxin in rhesus macaques. Eighty-six unvaccinated monkeys, including six that recovered from tetanus, were serosurveyed using a mouse toxin neutralization test. None of the animals had detectable antitoxin titers (<0.001 AU/ml), suggesting that natural immunity to tetanus is either rare or nonexistent in the Cayo Santiago colony.     

Attenuation of inhibitory processes in the central nervous system by tetanus toxin: an in vitro study on rat hippocampal slices

The influence of tetanus toxin on the efficiency of recurrent inhibition in the rat hippocampal slice was tested. The efficiency of the recurrent inhibition diminished in a dose-dependent manner following incubation of the slices with tetanus toxin. The effect was not observed in the slices preincubated for 3 hours with neuraminidase from Vibrio cholerae. This treatment reduces markedly the level of polysialogangliosides (receptor for tetanus toxin). It is concluded that tetanus toxin influences the efficiency of some inhibitory synapses in the central nervous system and that a certain level of polysialogangliosides is necessary for tetanus toxin to exert its action.     

Immunity to diphtheria and tetanus in some of the administrative territories of the RSFSR

The article deals with the state of immunity to diphtheria and tetanus among the adult population in some administrative regions of the RSFSR. Of the children and adults covered by the survey, 91.3-96.7% were found to have protective antibody titers against diphtheria and 98.7-100%, against tetanus. An essential drop in the level of immunity to diphtheria in persons over 18 years of age was revealed: 71.7% of them were nonimmune, which correlated with the high morbidity rate among these persons. At the same time the percentage of adults nonimmune to tetanus was considerably lower than that of adults nonimmune to diphtheria, reaching only 27.1%. The state of immunity to tetanus in adults was found to depend on the seasons.     

Antidiphtheria and antitetanus antitoxic immunity indices in the population of the Estonian SSR (an assessment of the effectiveness of a planned vaccine prophylaxis)]

In 1972-1974 and 1977 in the Estonian SSR children and adults were surveyed for the presence of antibodies against tetanus and diphtheria toxins (toxoids) by means of the passive hemagglutination test. The level of protection against tetanus was revealed to correspond to the proportion of child population covered by vaccination: in 1977, with 98.8% covered by vaccination, the level of protection among children aged 7 to 14 years and adolescents of 15-19 years exceeded 98%; with the increase of age (every 10-15 years) the level of protection against tetanus regularly decreased. This dynamics correlated with the existing terms of postvaccinal immunity and the epidemiological independence of tetanus as infectiion. The level of protection in child population against diphtheria in 1972-1974 and 1977 lagged behind the level of protection against tetanus and the coverage by vaccination. The diphtheria component of adsorbed DPT vaccine seemed to be unable to ensure the sufficient level and intensity of immunity under conditions of a sharply decreased risk of encounter with the infective agent. In persons aged 40 years and over the indices of immunity against diphtheria were higher than against tetanus. These indices resulted from diphtheria infection at the prevaccination period and could serve as an objective sign in following up the decrease of the process of diphtheria epidemics.     

Effect of regional (wound) revaccination with tetanus anatoxin on the functional activity of immunocompetent cells in the region]

The study made with the use of complex methods established that the local (wound) application of tetanus toxoid rapidly made the manifestation of the lysosomal apparatus more pronounced, increased the oxidizing activity (determined in the nitro blue tetrazolium test) and phagocytic activity of the mononuclear phagocytizing system in the wound and in the regional lymph nodes. The wound application of tetanus toxoid significantly increased blast transformation of T lymphocytes in guinea pigs simultaneously with tetanus wound infection. The study confirmed the pathogenetic expediency of the proposed method for the stimulation of anti-tetanus immunity by the application of tetanus toxoid on the wound which specifically inhibited the primary stage of the infectious process.     

Baroreflexes of the rat. V. Tetanus-induced potentiation of ADN A-fiber responses at the NTS

In a long-term neuromuscular blocked (NMB) rat preparation, tetanic stimulation of the aortic depressor nerve (ADN) enhanced the A-fiber evoked responses (ERs) in the cardiovascular region, the nucleus of the solitary tract (dmNTS). The potentiation persisted for at least several hours and may be a mechanism for adaptive adjustment of the gain of the baroreflex, with functional implications for blood pressure regulation. Using a capacitance electrode, we selectively stimulated A-fibers and acquired a stable 10-h "A-fiber only" ER baseline at the dmNTS. Following baseline, an A+C-fiber activating tetanus was applied to the ADN. The tetanus consisted of 1,000 "high current" pulses (10 trains; 300 mus, 100 Hz, 1 s), with intertrain interval of 9 s. A 10-h A-fiber only posttetanic test phase repeated the stimulus pattern of the baseline. Fourteen tetanus experiments were done in 12 rats. Compared with the baseline before tetanus, the A-fiber ER magnitudes of posttetanus hours were larger [F(13, 247) = 3.407, P < .001]; additionally, the 10-h posttetanus magnitude slopes were more positive than during 10 h before tetanus (df = 13; t = -3.47; P < 0.005); thus, an ADN A+C fiber-activating tetanus produced increases in the magnitude of the A-fiber ERs in the dmNTS that persisted for several hours. In an additional rat, application of an NMDA receptor antagonist, prior to the tetanus, blocked the potentiation effect. The stimulus protocols, magnitude and duration of the effect, and pharmacology resemble associative long-term potentiation (LTP).     

Prevention of tetanus in the wounded.

Recommendations for the prevention of tetanus in the wounded have been revised to incorporate the use of human tetanus immunoglobulin, which is now available in the United Kingdom. Surgical toilet is of prime importance for all wounds, and is usually sufficient for tetanus prophylaxis in patients with wounds that are less than six hours old, clean, non-penetrating, and with negligible tissue damage. Human tetanus immunoglobulin should be given to patients with more serious wounds sho have had toxoid injections over 10 years earlier, had an incomplete course, or do not know their immunity status. The importance of active immunization is emphasized. The recommendations should be regarded as guidelines as the circumstances in individual cases will differ.     

Analysis of the unfused tetanus course in fast motor units of the rat medial gastrocnemius muscle

The course of unfused tetani with the sag effect in fast motor units of rat medial gastrocnemius was studied. The analysis of the course of successive contractions within these tetani showed that the high peak force at the beginning of tetanus before the sag resulted from temporary, very efficient sum mation of contractions at this phase, both in FF (fast fatigable) and FR (fast resistant to fatigue) units. The process of summation developed in spite of parallel shortening of the contraction and relaxation. The peak of tetanus force was visible on the average at the 2nd contraction in FF units and at the 5th contraction in FR units. After the tetanus peak the process of the efficient summation was completed and the force decreased what was visible as a sag. In the following part of the tetanus, mainly in FF units, the potentiation occurred and the force of successive contractions increased. The rise of force was visible in spite of shortening of the contraction time and was due to prolongation of the relaxation in this part of the tetanus. These observations indicated that the processes of the summation of successive contractions before sag and during the potentiation underwent different mechanisms discussed in this paper. Considerable release of Ca2+ ions from the sarcoplasmic reticulum was proposed as a possible mechanism responsible for a very efficient summation at the beginning of the tetanus whereas phosphorylation of regulatory light chain of myosin (RLC) in muscle fibers was considered as the reason of potentiation. Moreover, the present analysis revealed that previously found differences in tetani profiles between FF and FR units resulted from faster development of described changes in the course of contractions summating into the tetanus in FF motor units.     

Characteristics of the immune response of inoculated and intact animals to the administration of toxigenic and nontoxigenic strains of Clostridium tetani

In experiments on guinea pigs the immune reactions of the animals immunized and not immunized against tetanus in response to the injection of C. tetani spores were studied. In the immunized animals an increase in the production of tetanus antitoxin, the development of delayed hypersensitivity and the activation of the mechanisms of cell-mediated immunity were observed. The nonimmunized animals showed specific changes in the T-system of immunity without the appearance of the clinical symptoms of tetanus, which is, probably, one of the mechanisms of natural immunity.     

Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.     

The titration of tetanus antitoxin IV. Studies on the sensitivity and reproducibility of the toxin neutralization test

The quality of tetanus toxin affected the sensitivity of the toxin neutralization (TN) test greatly. By using purified toxin a minimum level of 0.001 IU/ml of tetanus antitoxin could be detected whereas with crude toxin a level of 0.025 IU/ml only could be detected. The TN test described in this report permitted titration of tetanus antitoxin in twofold dilution steps from levels as low as 0.001 IU/ml using 0.6 ml of serum only at the L+/5000 level of purified tetanus toxin. Treatment of the sera with 2-mercaptoethanol (2-ME) did not affect the TN titres showing that the TN test detects the neutralizing antibodies (IgG) which are not affected by 2-ME. The TN test was found to be a highly sensitive and reproducible test.     

Correlated reduction of velocity of shortening and the rate of energy utilization in mouse fast-twitch muscle during a continuous tetanus

Isometric tetani of slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles of the mouse were studied at 20 degrees C. The total energy cost for 3- and 9-s isometric tetani was measured as a function of length above L0 and partitioned into a filament overlap-dependent fraction and a smaller filament overlap-independent fraction. In both muscles, the rate of filament overlap-independent energy cost did not change with tetanic duration. In the EDL, but not in the soleus, the rate of filament overlap-dependent energy utilization was greater in a 3-s tetanus than in a 9-s tetanus. The force-velocity relationships were studied after 3 and 9 s of isometric tetanus. In the soleus, Vmax was 2 fiber lengths/s and was not dependent on the duration of isometric tetanus. In contrast, in the EDL, Vmas decreased from 5.9 fiber lengths/s at 3 s to 3.9 fiber lengths/s at 9 s. The velocity of unloaded shortening (Vus) was examined by the slack test method as a function of the duration of isometric tetanus duration over the range of 1-15 s. In the soleus, Vus did not change, whereas in the EDL, Vus declined progressively from 6.4 to 3.2 fiber lengths/s after an isometric tetanus of increasing duration from 1 to 15 s. These results cannot exclude the hypothesis that in a maintained tetanus there is a decrease in the intrinsic cross- bridge turnover rate in the fast-twitch EDL, but not in the slow-twitch soleus muscle.     

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.     

Tetanus antibody titers and duration of immunity to clinical tetanus infections in free-ranging rhesus monkeys (Macaca mulatta)

Prior to 1985 tetanus was a major cause of mortality in the free-ranging colony of rhesus monkeys on Cayo Santiago, accounting for almost a quarter of annual deaths. In 1985 and 1986 all animals (except infants) received primary and booster doses, respectively, of tetanus toxoid. In subsequent years primary immunizations were given to all yearlings, and boosters were administered to all 2-year-old animals during the annual capture of the colony. The main objectives of the tetanus immunization program were to reduce the pain and suffering caused by tetanus infections and to decrease mortality in the colony. Other objectives were to evaluate the efficacy of the two-dose tetanus toxoid immunization protocol and to determine whether additional boosters might be required to provide adequate long-term protection against tetanus infections. The immediate effect of the mass immunization program was the elimination of clinical tetanus infections in the population and a 42.2% reduction in the overall mortality rate. Since the immunization program began, no cases of tetanus have been observed in the colony, except in two unimmunized infants, and it has not been necessary to give tertiary injections of tetanus toxoid to maintain protection against infection. A sample collected in 2004 of the original cohort of monkeys immunized in 1985 and 1986 showed that 93.3% (14/15) had protective tetanus antibody titers (>0.01 IU/ml) at the ages of 20-23 years, which is close to the life expectancy of the Cayo Santiago rhesus macaques. Two intramuscular doses of tetanus toxoid provided long-term, if not lifelong, protection against tetanus for rhesus monkeys living in a tropical clime where tetanus is enzootic and the risk of infection is great.