Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes.

AIMS: We investigated whether Listeria monocytogenes strains differ in their ability to escape from the primary phagosome after internalization into human intestinal epithelial cells. METHODS AND RESULTS: Food and clinical strains were used to study specific alleles; the activities of listeriolysin O (LLO) and phospholipases PlcA and PlcB, which promote rupture of the phagocytic vacuole; and initial intracellular bacterial growth in Caco-2 cells. Results showed no difference in LLO activities between food and clinical strains or among serotypes. In contrast, the LLO truncation mutant lacked detectable haemolytic activity and intracellular growth. PlcA and PlcB produced by the strains of serotypes 4b/4e and 1/2b exhibited significantly lower activities than those of serotypes 1/2a and 1/2c. In contrast, the strains of serotype 1/2b grew significantly faster than those of serotypes 4b/4e and 1/2a. Moreover, the PrfA truncation mutants lacked LLO and phospholipases activities and did not show intracellular growth. CONCLUSIONS: We determined that LLO and PrfA mutants exert a significant effect on intracellular growth, although it was unclear from this study whether PlcA and PlcB alleles affect escape from vacuoles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study estimates that low-virulence L. monocytogenes strains associated with escape ability from the primary vacuoles are not widely distributed among food strains.

Simple and rapid detection of Campylobacter spp. in naturally contaminated chicken-meat samples by combination of a two-step enrichment method with an immunochromatographic assay.

A simple and rapid method to detect Campylobacter spp. in chicken-meat samples was established. This method consisted of a combination of a two-step enrichment method with a commercially available immunochromatographic assay, named NH Immunochromato Campylobacter (NH IC Campy, Nippon Meat Packers, Ibaraki, Japan), which is able to detect Campylobacter antigen in an enrichment culture within 15 min. The enrichment method did not require much blood or a particular system of generating a microaerobic atmosphere, in contrast to the standard method of enriching Campylobacter spp. in chicken-meat samples. The sensitivity of a combination of the two-step enrichment method with NH IC Campy for detection of non- and freeze-stressed Campylobacter spp. in spiked chicken meat was determined using bacterial cells of Campylobacter jejuni and Campylobacter coli. The detection sensitivities for non-stressed C. jejuni and C. coli were found to range from 5.5 to 1.3x10(1) CFU per 25 g of chicken meat, and those for freeze-stressed C. jejuni and C. coli were found to range from 9.2x10(1) to 1.5x10(2) CFU per 25 g of chicken meat. When a total of 68 chicken-meat samples were tested, the combination method determined that 61 samples were positive for Campylobacter spp. This method was more sensitive than a bacterial culture test, which consists of standard enrichment culturing and plating onto selective agars. Because the combination could be conducted in approximately 48 h, from the beginning of the enrichment culture to final determination, it was more rapid than the bacterial culture test, which requires four to five days. Moreover, the combination was simple to perform. These results suggest that combining the two-step enrichment method with NH IC Campy is useful as a simple and rapid alternative to the conventional bacterial culture test for detecting Campylobacter spp. in naturally contaminated chicken meat samples.

Localization of tetrodotoxin in the skin of a brackishwater puffer Tetraodon steindachneri on the basis of immunohistological study.

A monoclonal antibody-based immunoassay for localization of tetrodotoxin (TTX) in the skin of a brackishwater puffer Tetraodon steindachneri is described in this paper. TTX was recognized in the undifferentiated basal cells and succiform cells in the skin under light microscope. Malpighian cells of the skin did not exhibit any TTX antigen. Neither gland nor enclosed gland-like apparatus possessing TTX was apparent in the skin.

IL-12 and IL-18 synergistically induce the bactericidal activity of murine peritoneal cells against M. leprae.

We examined the effect of IL-12 and IL-18 on bactericidal activities of mouse peritoneal cell (PC) against Mycobacterium leprae (M. leprae). We demonstrated that IL-12 and IL-18 synergistically induced the NO-dependent bactericidal activity of PC by stimulating Natural Killer (NK) cells and T-cells through IFN-gamma production. IL-12 and IL-18 induced host cell death through NK-cells and T-cells. Therefore. IL-12 and IL-18 play an important role on direct killing of intracellular M. leprae and on indirect killing of them through inducing host cell death.

Determination of domoic acid in Japanese mussels by enzyme immunoassay.

Ten samples of commercial blue mussels (Mytilus edulis) from Japan were analyzed for domoic acid by an indirect competitive enzyme immunoassay (idc-EIA) based on an anti-domoic acid monoclonal antibody. Domoic acid was found in all samples at low concentrations (0.11-1.81 ng/g mussel tissue). The presence of domoic acid was confirmed by liquid chromatography coupled with immunoaffinity chromatography using an anti-domoic acid monoclonal antibody as ligand. To our knowledge, this is the first reported detection of domoic acid, a causative agent of amnesic shellfish poisoning, in Japanese mussels.

Comparison of polymerase chain reaction, immunohistochemistry and conventional histopathology in the diagnosis of early leprosy in Sichuan Province of China.

46 formalin-fixed, paraffin-embedded skin biopsy specimens, which were clinically suspected or diagnosed as early leprosy, were retrieved from the files of Sichuan, China from 1997 to 1999. All of them were examined by polymerase chain reaction (PCR) using the primers amplifying the 130 base-pair fragment of the gene from the 16S ribosomal RNA of Mycobacterium leprae, hematoxylin and eosin (H&E) staining, modified Fite-Faraco technique for M. leprae and immunostaining with the antiserum against the PGL-1, LAM-B, S-100 protein using ABC method. PCR was positive for 27 (58.7%) of 46 specimens. In 13 (28.3%) among them, only PCR signals were positive for M. leprae and all other test were negative. AFB was positive for 7 (15.2%) of 46, PGL-1 was positive for 17 (36.9%) of 46, LAM-B was positive for 10 (21.7%) of 46. Early epithelioid cells granuloma was detected in 4 (8.7%) patients (TT 3, BT 1), macrophage granuloma was detected in 1 (2.2%) patient (BL), S-100 protein staining showed early nerve granuloma for 4 (8.7%) of 46, peripheral nerve inflammatory infiltration for 11 (23.9%) of 46. Comparison PCR with other method showed statistically significant difference. PCR have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli.

Production and characterization of a monoclonal antibody against domoic acid and its application to enzyme immunoassay.

For production of monoclonal antibodies against domoic acid, a causative agent of amnesic shellfish poisoning, three immunogens, domoic acid conjugated with bovine serum albumin (BSA), ovalbumin (OVA) and human gamma globulin (HGG), were prepared. The antiserum obtained from BALB/c mice immunized with domoic acid-BSA showed the highest affinity for domoic acid. The monoclonal antibody, DA-3, obtained from the mice was highly specific for domoic acid and showed a minor cross-reactivity with the isomers of domoic acid (isodomoic acids B, E, F, G and H), except for isodomoic acid A. Using DA-3 antibody, an indirect competitive enzyme immunoassay (idc-EIA) was developed for measurement of domoic acid. The working range for quantitative measurement of domoic acid and the quantification limit for domoic acid in shellfish were estimated to be 0.15-10 ng/ml and less than 0.04 microg/g, respectively. The mean recovery of domoic acid added to extracts of shellfish at toxin levels of 0.02 to 0.2 microg/ml was 103% with a coefficient of variation of 4.5%. The newly developed idc-EIA seems to be a useful method for monitoring domoic acid in shellfish.

An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique.

One of the most important unsolved questions in epidemiology of leprosy is the highly uneven geographic distribution of the disease. There are many hyperendemic "pockets" in endemic countries. Little is known about the reasons why leprosy is hyperendemic in these areas. We conducted, therefore, a series of epidemiological studies on Mycobacterium leprae infection and prevalence of leprosy in North Maluku district, Maluku Province, Indonesia where leprosy is highly endemic. It was found that considerable number of general inhabitants are seropositive to various mycobacterial antigens and 27% of the villagers were carrying leprosy bacilli on their surface of nasal cavity. These results suggested the importance of M. leprae in the residential environment in infection of the leprosy bacillus and the resulting transmission of the disease. Based on these observations, we conclude that new preventive measures are essential for global elimination of leprosy in addition to early diagnosis and multidrug therapy (MDT).

Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients.

Immunoaffinity chromatography using the monoclonal antibody (Tl-1) specific for tetrodotoxin (TTX) has been developed for isolating TTX from urine samples. By combining immunoaffinity chromatography with fluorometric high performance liquid chromatography (HPLC), it has become possible to detect a small amount of TTX in urine samples. The detection limit of TTX in urine was 2 ng/ml. By this combined method, TTX was detected in all the urine samples that were collected from poisoned patients during the week following TTX ingestion. The combination of immunoaffinity chromatography with HPLC was very useful in detecting TTX from the urine samples of poisoned patients for diagnosis of TTX-food poisoning.

[Present situation of leprosy in highly endemic area of tropical Asia--a seroepidemiological study of Mycobacterium leprae infection in general inhabitants].

One of the most important unsolved problems in epidemiology of leprosy is the heterogeneous geographic distribution of the disease. There are highly endemic area called "Pocket" in the endemic countries. Little is known why leprosy is so endemic in the area. We conducted, therefore, an epidemiological study on M. leprae infection and distribution of leprosy bacilli in the environment by using serological and molecular biological techniques. It was found that considerable number of general inhabitants in the pocket are infected with leprosy bacilli and more than 20% of the villagers are carrying M. leprae on the surface of the nasal cavity; suggesting that leprosy bacilli in the residential environment play an important role in high prevalence of leprosy in the endemic area. New preventive measures such as chemoprophylaxis, in addition to MDT, will be needed for global elimination of the disease.

Transepidermal elimination of lepromatous granuloma: a mechanism for mass transport of viable bacilli.

A 35-year-old male with lepromatous leprosy showed significant progression of the disease on initial examination. Along with typical lepromatous skin lesions, many scar-forming lesions were present, mainly on his extremities. Some lesions showed erosive surfaces. From clinicopathological findings, these lesions were suspected to be due to the partial excretion of intradermal lepromatous granulomata by 'transepidermal elimination'. Increased local volume, which might be due mainly to rapidly growing lepromatous infiltration before chemotherapy, is suspected of triggering this phenomenon. There is no doubt that many fresh Mycobacterium leprae were included in these excretions. After the initiation of chemotherapy, no new scar-forming lesions were observed.

Rapid and highly sensitive enzyme immunoassay for quantitative determination of tetrodotoxin.

A monoclonal antibody against tetrodotoxin (TTX) was obtained from Balb/c mice immunized with TTX-bovine serum albumin (BSA) conjugate. The monoclonal antibody was highly specific for TTX and had no cross-reaction to tetrodonic acid, which is a TTX derivative, or gonyautoxins, although a minor cross-reaction to anhydro-tetrodotoxin was observed. The monoclonal antibody neutralized the lethal activity of TTX. By using the monoclonal antibody, a rapid and highly sensitive competitive enzyme immunoassay (EIA) for quantitative analysis of TTX was developed. By the competitive EIA system, TTX can be determined quantitatively in about 30 min (90 min are required if the time for preparation of the solid-phase antigen was included), and the working range for quantitative analysis of TTX was 2-100 ng/ml. In recovery tests and examinations of TTX samples, results of the mouse bioassay and EIA analyses correlated well (r = 0.987). Moreover, it was demonstrated that low concentrations of TTX, which could not be detected by the mouse bioassay, could be determined quantitatively by the competitive EIA.

Pathological investigation of armadillos infected with Mycobacterium leprae.

An infection experiment with M. leprae was carried out using 20 nine-banded armadillos. As a result, the development of leprous lesions and a marked multiplication of AFB were confirmed in a high rate of 13 out of 15 cases (86.8%) in the inoculated groups. These changes were found to be progressing at post mortem of one case even with the shortest life period for 7.5 months and were very serious in one case with the longest life period for 33 months, suggesting the continuation of symptoms, though it is an expression neglecting the individual difference in susceptibility to leprosy. Among infected viscera with AFB, the most conspicuous lesions were found in the liver and spleen. The developed lesions were found in the lung, stomach and kidney which had been never seen in HD in human cases, and so, which may characterize armadillos' leprosy. The change in the peripheral nerve was not so severe when compared with that in HD in human cases. This difference will remain as a future pathological problem to be solved.

A serologic Mycobacteria leprae gelatin particle agglutination (MLPA) in the diagnosis of leprosy: comparison with conventional enzyme-linked immunoassay and bacterial index.

A gelatin particle agglutination test (MLPA) for the detection of anti-phenolic glycolipid-1 (PGL-1) antibodies was compared with the slit skin smear method in the diagnosis of leprosy. MLPA and BI tests showed a good agreement rate of 88.1% and MLPA and ELISA tests showed an excellent agreement rate 96.2%. This MLPA test is simple and reliable, it will be very convenient for the medical practitioners, it would be of great benefit for leprosy patient as well because this test would look like a routine blood examination compared with slit skin smear method which is widely known diagnostic tool for leprosy.

Cloning and sequencing of the chaperonin-encoding Cctd gene from Fugu rubripes rubripes.

CCT, a chaperonin containing t-complex polypeptide 1 (TCP-1), is a cytosolic molecular chaperone involved in the folding of proteins. We have isolated the Cctd gene from a Fugu rubripes rubripes (Frr) genomic library using a rat Ccta cDNA as a probe, and cloned its cDNA by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of oligodeoxyribonucleotides corresponding to the 5' and 3' non-coding regions of Frr Cctd. Cctd spans a region of 4.7 kb and consists of at least 13 exons with small introns of about 144 bp on average. The Cctd cDNA sequence revealed a deduced polypeptide of 536 amino acids sharing a high degree of homology with that of the mouse Cctd cDNA (88%). Cctd is present as a single-copy gene, as shown by genomic Southern blot analysis, and can be used for evolutionary and classification analyses of Fugu species.

[Uveitis in leprosy patients].

We examined 24 dermatologically cured leprosy patients with ongoing uveitis (UV+) and 22 age and type matched controls (UV-) to study the late phase leprous UV. All patients have been skin smear negative for more than 10 years. The history of chemotherapy, 5 years before and after a accomplishing bacterial negativity, was evaluated and represented by "SCORE". It was found that anti-PGL-I and anti-LAM-B antibodies were significantly higher in UV+ group compared to the controls. The mean SCORE of chemotherapy in UV+ group was significantly lower than in the controls. Iris pearls were seen in 10 cases or 42% out of 24 UV+ patients. No iris pearls were seen in control group. These results suggest that insufficient chemotherapy and consequent incomplete elimination of bacilli are the risk factors for leprous UV in the quiescent stage of the disease.

Immunopathological stain of lipoarabinomannan-B (LAM-B) for diagnosis of leprosy.

We developed an immunopathological staining of LAM-B antigen in formalin-fixed paraffin-embedded tissues, and compared it with, PGL-I immunostaining, Fite Faraco's stain and periodic acid carbol pararosaniline (PACPR) stain. Out of the total 28 leprosy cases, 27 were positive to LAM-B immunostaining while 23 were positive to PGL-I stain. Fite's stain was positive in 21 cases while PACPR stain was positive in 24 cases. In scrofuloderma, LAM-B antigen was observed only in the granuloma while no other positive findings were noted with other stains. Normal skin did not give any positive findings with any of the stains. Other dermatoses showed no positive findings to any of the stains tested. LAM-B staining was observed in the nerve even in the absence of bacilli in leprosy tissues. Presence of LAM-B in the cutaneous nerves is helpful in discriminating leprosy from other mycobacterioses. Considering the high sensitivity of LAM-B and the predilection of M. leprae for the nerves, we concluded that LAM-B staining can be a useful new tool in the prompt diagnosis of leprosy, especially in suspected or early cases.

Demonstration of PGL-I & LAM-B antigens in paraffin sections of leprosy skin lesions.

An investigation on the demonstration of PGL-I and LAM-B antigens in thirty-four paraffin embedded skin biopsies taken from leprosy patients who covered the whole spectrum of the disease and in four control specimens was carried out. Neither the PGL-I antigen nor the LAM-B antigen was demonstrated in the normal skin specimens that were used as negative control; and only the LAM-B antigen appeared in the tuberculosis specimens in which the PGL-I antigen was negative. The PGL-I antigen was demonstrated on thirty-three leprosy samples except one TT sample and the LAM-B antigen, on all samples by immunochemical staining technique. The antigens were identified as intracytoplasmic bacillary staining, in solitary, granular as well as debris patterns; and as soluble antigenic staining, in vacuolar or amorphous pattern. In LL and BL cases, the antigens were detected predominantly from macrophages and peripheral nerves in all five staining patterns; in BB cases, from macrophages mostly in the granular as well as debris patterns, from the nerves in the vacuolar pattern; while in TT and the majority of BT cases, they were mainly from nerve remnants inside the granuloma in the vacuolar or amorphous staining pattern. In addition, it is interesting to note that the immunochemical staining was able to differentiate the foamy change from the hydropic degeneration. We also found that the antigens distributed in arrector pili muscles and the walls of muscular vessels were obviously related to the unmyelinated nerve fibers innervating the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[Modification of Harada's method for rapid staining of mycobacteria].

Harada employed periodic acid-carbol pararosaniline and periodic acid-methenamine silver stain for demonstrating chromophobic bacilli which do not get stained with conventional carbol fuchsin or counter stain. This staining method takes considerable time for complete oxidation with periodic acid. We have succeeded in reducing the oxidation time by using hydrogen peroxide treatment prior to periodic acid and with the use of acidified sodium hydrogen sulfite treatment before carbol pararosaniline stain. We also found that in methenamine silver stain, combined use of semicarbazide and microwave treatment can shorten the whole staining time up to four hours without losing ito sensitivity.