Mouse Footpad Test Research Articles

COMPARISON OF RADIORESPIROMETRIC BUDDEMEYER ASSAY WITH ATP ASSAY AND MOUSE FOOT PAD TEST IN DETECTING VIABLE MYCOBACTERIUM LEPRAE FROM CLINICAL SAMPLES

Purpose: This study compares the results of radiorespirometric Buddemeyer assay with adenosine triphosphate (ATP) assay and mouse foot pad (MFP) test to validate the sensitivity of Buddemeyer assay in detecting viable M. leprae in clinical samples. Methods: Viability was assessed using all the three methods in 60 skin biopsy specimens, including 20 untreated lepromatous leprosy (BL-LL), 13 treated BL-LL, 12 untreated borderline tuberculoid to mid borderline (BT-BB) and 15 treated BT-BB cases. Results: Of the 20 untreated BL-LL cases tested, positivity indicating the presence of viable M. leprae was detected in 85, 60 and 85% with Buddemeyer, ATP and MFP test, respectively. Among the 13 treated BL-LL cases, scores were 61, 54 and 0%; among the 12 untreated BT-BB cases, the scores were 58, 16 and 16% and among the 15 treated BT-BB cases, the scores were 46, 20, 0%, respectively. Conclusion: The detection sensitivity (positive scores) with three tests were closely comparable in the two untreated groups of cases. On the other hand, in the two treated groups, a good proportion of cases scored positive in the in vitro tests but none in the MFP test. Among the two in vitro methods, the Buddemeyer assay emerged as a better test, in terms of sensitivity and specificity.

Comparison of radiorespirometric budemeyer assay with ATP assay and mouse foot pad test in detecting viable <i>Mycobacterium leprae </i>from clinical samples

This study compares the results of radiorespirometric Buddemeyer assay with adenosine triphosphate (ATP) assay and mouse foot pad (MFP) test to validate the sensitivity of Buddemeyer assay in detecting viable M. leprae in clinical samples. Viability was assessed using all the three methods in 60 skin biopsy specimens, including 20 untreated lepromatous leprosy (BL-LL), 13 treated BL-LL, 12 untreated borderline tuberculoid to mid borderline (BT-BB) and 15 treated BT-BB cases. Of the 20 untreated BL-LL cases tested, positivity indicating the presence of viable M. leprae was detected in 85, 60 and 85% with Buddemeyer, ATP and MFP test, respectively. Among the 13 treated BL-LL cases, scores were 61, 54 and 0%; among the 12 untreated BT-BB cases, the scores were 58, 16 and 16% and among the 15 treated BT-BB cases, the scores were 46, 20, 0%, respectively. The detection sensitivity (positive scores) with three tests were closely comparable in the two untreated groups of cases. On the other hand, in the two treated groups, a good proportion of cases scored positive in the in vitro tests but none in the MFP test. Among the two in vitro methods, the Buddemeyer assay emerged as a better test, in terms of sensitivity and specificity.

Comparison of the mouse foot pad test with a Buddemeyer type radiorespirometric assay in detecting viableMycobacterium lepraein human lesional biopsies

This study assesses the utility of a Buddemeyer type radiorespirometric (RR) assay in detecting viable Mycobacterium leprae in clinical samples taking the mouse foot pad (MFP) test as gold standard. A total of 131 skin biopsy specimens comprising of 56 untreated, smear-positive, borderline lepromatous and lepromatous leprosy (BL-LL) and 75 untreated, smear-negative, borderline tuberculoid and mid-borderline (BT-BB) specimens were processed by both the methods. The cut-off value (in counts per minute, cpm) for test samples in the RR assay was determined using nonleprous (normal) skin biopsy specimens. In the untreated BL-LL and BT-BB groups, 86 and 56% of the cases tested positive in the RR assay respectively, which was comparable to the results of the MFP test (80 and 45% respectively). The overall concordance between the two tests was 74.79%, whereas the sensitivity and specificity were 75 and 74.3% respectively. A Kappa value of 0.459 indicated a fairly good agreement between the two methods. There was no linear relationship observed between the bacteriological index (BI) and the morphological index (MI) with the counts per minute (cpm) output. The results indicate the ability of this RR assay to detect viability; however a comparison with another sensitive method would further validate the assay system.

Prevalence and characteristics of necrotoxigenic Escherichia coli (NTEC) strains isolated from diarrhoeic dairy calves

Fecal samples from 246, 1–90-days old diarrhoeic dairy calves in 72 herds were screened for the presence of cytotoxic necrotizing factors (CNF)-producing Escherichia coli (NTEC). NTEC were detected by tissue culture assays and PCR in 39 (15.8%) of the diarrheic calves, and the majority of these animals (34 of 39, ca. 87.2%) were infected by NTEC producing CNF2. Calves were grouped according to their age (1–7 days, 8–14 days, 15–21 days, 22–30 days and 31–90 days) and analyses of prevalence were done by the Mantel-Haenzsel χ 2-test for trend. A significant age-associated increase in the prevalence of NTEC producing CNF2 ( p < 0.0001) was found. Eighty-one (8.4%) of the 958 E. coli isolates from the 246 diarrheic calves were positive for CNF in the tissue culture assays. These strains were analyzed by PCR and this technique showed that three (3.7%) strains were CNF1-positive and 75 (92.6%) were CNF2-positive. Moreover, three of the strains positive in the tissue culture assays were negative by PCR. These strains were subsequently assayed in several biological tests (rabbit skin test, mouse intraperitoneal test and mouse footpad test) which showed that they were really NTEC, probably producing CNF2, but with some different properties to classical strains producing CNF2. NTEC strains producing CNF2 belonged to different serogroups (O2, O7, O9, O14, O15, O41, O43, O45, O55, O76, O86, O88, O109, O115, O123, O128, O153 and O159) than strains producing CNF1 (O11 and O32) or PCR-negative strains (O111). Moreover, a strong association between CNF2 and F17 fimbriae was found (78.6% of CNF2-positive strains were F17-positive, whereas only 22.9% of CNF2-negative strains were F17-positive).

An international randomized study with long-term follow-up of single versus combination chemotherapy of multibacillary leprosy

A total of 307 patients with lepromatous leprosy and borderline lepromatous leprosy were randomized to dapsone monotherapy or to one of two types of drug combinations. A 3-year treatment phase was followed by a 5-year observation phase. The evaluation included 233 patients for whom together there were 1,404 years of observation. A total of 1,956 blinded histopathological specimens were processed centrally. When entering the trial isolates from 13 patients (5.6%) showed dapsone resistance in the mouse footpad test, and these patients were evaluated separately. Dapsone monotherapy (68 patients) had the same frequency of cure as the combination of dapsone and rifampin (77 patients) or the four-drug regimen consisting of dapsone, rifampin, isoniazid, and prothionamide (75 patients). We did not find a significant difference in the clearance of bacteria either between the monotherapy and the two-drug combination or the monotherapy and the four-drug combination. Six months after the initiation of treatment, disease in 15% of the patients who received dapsone monotherapy but none of the patients who received combined treatment were clinically progressive. After another 1 to 9 months of treatment the disease in all patients was stable or regressive. There was no difference in the type or frequency of reactions. Only after the end of the scheduled observation phase three relapses were reported. All three treatment regimens well tolerated. Dapsone monotherapy is highly effective in the treatment of multibacillary leprosy under the conditions of well-controlled treatment. Combination regimens seem only to accelerate the regression of the active disease when they are compared with monotherapy with dapsone. The mouse footpad test does not reflect the clinical resistance and cannot be recommended for use in making therapeutic decisions.

The use of an amino acid as a precursor of a bioactive metabolite in combatting an infectious disease: The efficacy of a tryptophan-enriched diet in the treatment of leprosy

Deoxyfructosylserotonin (DFS), a stable derivative of serotonin, was shown to have an anti-leprosy effect (Mester and Antia). Therefore, a tryptophan-enriched food (NAL) was devised to increase the concentration of free tryptophan as serotonin precursor in blood.32 multibacillary lepromatous leprosy patients were divided in 3 groups receiving: 1) MDT (multi-drug therapy, control - 8 patients), 2) NAL (50 g/day - 13 patients) and 3) NAL + 1/2 MDT (11 patients). The clinical improvement (clinical score) was 24.2% for 1), 19.9% for 2) and 30.4% for 3). The loss of viability of theM. leprae bacilli in the mouse foot-pad test was 66% for 1), 75% for 2) and 70% for 3). The improvement by serodiagnosis (ELISA) was 19.4% for 1), 25.1% for 2) and 23.3% for 3).These preliminary results (6 months) suggest that the NAL-food is efficient against leprosy and apparently not very different from the control MDT in that respect.

Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli

We have characterized the in vitro and in vivo toxic properties of cell sonic extracts from 22 animal and human clinical isolates of Escherichia coli that caused both necrosis in the rabbit skin and multinucleation in tissue cultures, two toxic properties previously reported as being specific for E. coli cytotoxic necrotizing factor (CNF). Two distinct toxic phenotypes were observed. Type 1, which was displayed by originally described CNF strains, was characterized by extensive multinucleation and rounding of cells in HeLa cell culture assays, moderate necrosis in the rabbit skin test, and absence of necrosis in the mouse footpad test. Type 2, which has recently been shown to be associated with E. coli Vir plasmid, was characterized by moderate multinucleation, by polymorphism and elongation of HeLa cells, and by an intense necrotic response in both the rabbit skin test and the mouse footpad test. The distinction between the two cytotoxins accounting for these effects (CNF 1 and CNF 2), together with their partial relatedness, was confirmed by seroneutralization studies of both cytopathic effects and necrosis in the rabbit skin test. In addition, type 2 extracts were more lethal in the mouse intraperitoneal test and induced a moderate, although not totally repetitive, fluid accumulation in the ileal loop test. The original toxic properties of these recently recognized categories of E. coli strains, together with their association with enteritis and septicemia, suggest that these strains may play a significant role in pathology.

Leprosy.

Leprosy affects over 10 million people in the world. The disease is a model of graded cell-mediated immunity, in this case to the causative organism, Mycobacterium leprae. The clinical manifestations are due to (i) bacterial progression, (ii) immunologic responses of the host, (iii) peripheral nerve damage due to either or both bacterial progression and immunologic responses of the host, and (iv) preventable secondary deformities following nerve damage, which account for most of the stigma of the disease. Treatment modalities are now available to control or minimize the effects of bacterial progression, harmful immunologic responses of the host, peripheral nerve damage, and secondary deformities. Unique biochemical characteristics of M. leprae reside in the cell wall and associated macromolecules. Some of these molecules are potent immunogens in humans, while others constitute the structural integrity of the bacillus. Proteins of M. leprae are currently under intensive investigation as a result of deoxyribonucleic acid cloning of M. leprae genes. Structure-function and antigenic relationships of M. leprae proteins should become available by using recombinant deoxyribonucleic acid procedures coupled with T- and B-cell cloning to advance our understanding of the immunologic reactions encountered in Hansen's disease. Until recently, the study of the immunology of leprosy has been stymied by the lack of immunologically specific M. leprae antigens. The definition of specific antigens and production of recombinant and synthetic immunologic reagents have fostered state-of-the-art research efforts into new immunodiagnostic procedures and development of a leprosy vaccine. Also discussed is progress in understanding of the mechanism(s) underlying the M. leprae-specific immunodeficiency associated with lepromatous leprosy, including the role of suppressor T cells and defective macrophage function. Metabolic studies of M. leprae suggest intact catabolic pathways and energy generation with purine bases and catalase as possible growth factors. Special attention may also need to be given to biophysical parameters for eventual in vitro cultivation. Rapid in vitro systems, using quantitation of bacillary metabolic activity, may soon replace the lengthy mouse footpad test for determining the viability and drug susceptibility of the leprosy bacillus.

Comparative study of the edema-forming activity of costa rican snake venoms and its neutralization by a polyvalent antivenom

1. 1. The edema-forming activity of eight Costa Rican crotaline snake venoms and its neutralization by a polyvalent antivenom were studied using the mouse footpad test. 2. 2. All of the venoms induced edema, the highest activity being present in the venoms of Bothrops lateralis and Bothrops picadoi. 3. 3. When experiments were performed with preincubation of venom and antivenom, neutralization of edema was poor. Moreover, it was observed that, with some venoms, edema increased when large doses of antivenom were used. This effect was also observed when some venoms were incubated with coral snake antivenom, suggesting that venoms may release some pharmacologically active component(s) from antivenom, since the latter contains traces of alpha-2 and beta globulins. 4. 4. Based on these findings, an alternative approach to the study of the neutralization of edema was used; in this new method, antivenom was injected i.v. before venom administration, thereby avoiding preincubation. With this technique, a much better neutralization of edema was observed, although with some venoms it was still poor. 5. 5. Venoms contain low molecular weight factors which induce edema, suggesting that lack of immunogenicity of some components may cause a poor neutralization. However, such components are responsible for only a minor portion of the edema induced by crude venoms. 6. 6. It is suggested that experiments in which venom and antivenom are preincubated in testing the neutralization of edema should be avoided, and that a more adequate approach may be an independent inoculation of venom and antivenom.

Rifampin in the treatment of leprosy.

The minimal inhibitory concentration of rifampin for Mycobacterium leprae is less than 1 microgram/ml. Therapy with rifampin has proved efficacious both in mice experimentally infected with M. leprae and in humans with leprosy. Rifampin kills M. leprae more rapidly than do other antileprosy drugs currently available. Consequently, M. leprae bacilli from patients with lepromatous disease are rendered noninfectious within three weeks after the institution of rifampin therapy, as determined in the mouse footpad test system. Administration of this antibiotic substantially reduces the quantities of M. leprae discharged in the nasal secretions of lepromatous patients within three weeks, thus rapidly decreasing the potential infectivity of these individuals. Intermittent rifampin therapy for leprosy has been successful, with a low incidence of adverse reactions to the drug. Worldwide, the prevalence of primary and secondary resistance of M. leprae to dapsone has increased markedly. Therefore, the World Health Organization recommends a multidrug regimen that includes intermittent administration of rifampin for the treatment of leprosy.

Response of hypersensitive mice to the footpad injection of living homologous or heterologous mycobacteria: preliminary report.

Mice sensitized by the injection of viable mycobacteria into one of the hind footpads responded to a second injection of mycobacteria (3 to 4 weeks later), introduced into the contralateral foot, with a degree of footpad swelling that was both accelerated and exaggerated beyond that observed after the first inoculation. The degree of specificity of this reaction (i.e., response to homologous versus heterologous mycobacteria) was comparable to that previously reported for dermal reactions of hypersensitive guinea pigs to tuberculin or tuberculin-like antigens from mycobacteria. In preliminary studies it was impossible to achieve this state of specific sensitization by vaccinating mice subcutaneously with water-in-oil emulsions of heat-killed mycobacteria; reasons for the failure are discussed. It is suggested that this tool could prove useful in both taxonomic and immunological investigations. Advantages and disadvantages of the mouse footpad test in relation to the dermal skin test in guinea pigs are discussed.

Response of Hypersensitive Mice to the Footpad Injection of Living Homologous or Heterologous Mycobacteria: Preliminary Report

Mice sensitized by the injection of viable mycobacteria into one of the hind footpads responded to a second injection of mycobacteria (3 to 4 weeks later), introduced into the contralateral foot, with a degree of footpad swelling that was both accelerated and exaggerated beyond that observed after the first inoculation. The degree of specificity of this reaction (i.e., response to homologous versus heterologous mycobacteria) was comparable to that previously reported for dermal reactions of hypersensitive guinea pigs to tuberculin or tuberculin-like antigens from mycobacteria. In preliminary studies it was impossible to achieve this state of specific sensitization by vaccinating mice subcutaneously with water-in-oil emulsions of heat-killed mycobacteria; reasons for the failure are discussed. It is suggested that this tool could prove useful in both taxonomic and immunological investigations. Advantages and disadvantages of the mouse footpad test in relation to the dermal skin test in guinea pigs are discussed.