Cross Agglutination Research Articles

Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts.

The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.

Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters

To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters. The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT. The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels. The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Analysis of Leptospira interrogans ompA gene and immunological identification of its recombinant expression product

Objective To investigate the distribution of ompA gnne in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogate, and to express recombinant OmpA ( rOmpA ) and to identify immunogenicity and immunoprotection of rOmpA. Methods Genomic DNAs from different leptospiral strains were extracted by phenol-chloroform method. Entire ompA gene fragments from the strains were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of ompA gene from L. interrogans strain 56601 was constructed, and the expression and yield of rOmpA were determined by SOS-PAGE plus Bio-Rad Agarose Image Analyser. Rabbits were immunized with rOmpA for obtaining antiserum, and immunodiffusion test was used to measure the antiserum's titer. Western blot assay was performed to determine the immunoreaetivity of rOmpA with the antiserum against rOmpA and antiserum against whole cell of L. interrogans strain 56601, while mi-croscopic agglutination test (MAT) was applied to detect the cross agglutination to the 15 L. interrogans strains. A leptospire adhering cell model and a leptospire infecting guinea pigs model were used to determine the adhesion-bloc-king effect of rOmpA antiserum and immunoprotection of rOmpA. Results All the 15 L. interrogans strains, but not L. biflexa strain Patoe Ⅰ , had sequence conserved ompA genes. The yield of rOmpA was approximate 20% of the total bacterial proteins, rOmpA could induce rabbits to produce antibody and immunodiffusion titer of the anti-serum was 1:4. Both antisera against rOmpA and against whole cell of L. interrogans strain 56601 were able to pro-duce positive Western blot signs to rOmpA, and the former offered 1 : 20-1 : 320 MAT titers to the 15 L. interrogans strains. 1: 10-1:160 dilutions of rOmpA antiserum could efficiently block L. interrogans strain 56601 adhering to J774A. 1 cells, and 100 μg and 200 μg rOmpA displayed 50.0% and 75.0% immunoprotective rates in the infee-ted guinea pigs. Conclusion ompA gene only exists in genomes of different pathogenic L. interrogans serogroups. rOmpA has relatively stronger antigenicity, cross immunoreactivity and certain immunoprotection, implying that this recombinant protein may be used as a candidate antigen for developing universal genetic engineering vaccine of L. interrogans. Key words: Leptospira interrogans; ompA gene; Recombinant expression; Immunogenicity; Immunopro-teetion

Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil.

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers ≥ 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona.

Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product

To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

Isolation of Leptospira santarosai, serovar guaricura from buffaloes (Bubalus bubalis) in Vale do Ribeira, São Paulo, Brazil

In April 1998 urine samples from adult female buffaloes were collected in a farm located in Registro, Vale do Ribeira, Sao Paulo State, Brazil. The urine samples obtained after furosemide injection were immediately transported to the laboratory in liquid modified EMJH medium and seeded, by the serial dilution technique, into Fletcher's or modified EMJH-0.2% agar, both of them with 5-fluorouracil 100mg/mL. The intraperitoneoum inoculation of 0.5 mL was also performed with each urine sample in young, adult hamsters (Mesocricetus auratus). All samples seeded directly in culture medium were contaminated. The hamsters did not show any sign of disease and were killed at the 21st post inoculation day. At this time kidney cultures of these animals were performed and from one of them, one leptospira strain (M04-98) was isolated, identified as belonging to serogroup Sejroe by Microscopic Agglutination Test (MAT) with a panel of 36 rabbit sera against serovars representative for the pathogenic serogroups. Subsequently, MAT was carried out with antisera against the 19 reference strains of serogroup Sejroe, revealing a close relationship with serovar guaricura. Afterwards the MAT was done with a panel of 18 monoclonal antibodies representative for serovars of serogroup Sejroe. The histogram closely resembled that of serovar guaricura. So Cross Agglutination Absorption Test (CAAT) was carried out with the buffalo isolate and serovar guaricura, supporting the relationship between the buffalo isolate and serovar guaricura.

Antigenic Profile and Protective Role of a 37kDa Major Outer Membrane Protein of Edwardsiella tarda.

A 37 kDa major outer membrane protein (MOMP) was purified from the outer membrane fraction ofEdwardsiella tardaEF-1 strain. The protein had a glycoprotein profile and showed immunogenicity to Japanese eelAnguilla japonica. Immunoblot analysis by using an eel antiserum raised against this MOMP revealed no antigenic cross-reaction with other MOMPs of this bacterium. Eel antisera against 37 kDa MOMP extracted from EF-1 and V1 strains showed cross agglutination to formalin-killed cells and heated cells between the two strains. The results indicate that the heat-resistant antigen, 37 kDa, MOMP is located on the cell surface and has a common antigenic determinant. Immunization with the purified 37 kDa MOMP of EF-1 and V1 strains increased resistance in eel against challenge by intraperitoneal injection with live EF-1 strain. The results show that this 37 kDa MOMP is one of the protective antigens ofE. tarda. As the MOMP was found in two strains of different serotypes, it is probably contributed as a promising vaccine antigen against edwardsiellosis caused byE. tarda.

The isolation and identification of Leptospira interrogans serovar bratislava from a pig in Germany.

During a study on the improvement of selective media for the isolation of leptospires from clinical material, leptospires were isolated from the kidneys of a pig. Test material was cultured in EMJH-medium containing 0.4% rabbit serum and 3 inhibitory substances (Rifampicin 10 micrograms/ml, Amphotericin B 2 micrograms/ml, 5-Fluorouracil 100 micrograms/ml). The cultured leptospira-strain (Berlin 406) was identified to serogroup level using the cross agglutination method and it was further typed to serovar level by MAB's, factor analysis and restriction endonuclease analysis (REA). MAB typing of Berlin 406 gave an agglutination pattern profile typical of serovar bratislava. Factor serum identification clearly identified this strain as serovar bratislava. On REA examination, Berlin 406 gave a profile identical to that of the serovar bratislava type strain Jez Bratislava i.e. genotype B1. This is the first report of a confirmed isolate of Leptospira interrogans serovar bratislava from domestic animals in Germany. There exist a number of possibly important implications as this agent is being incriminated in the cause of clinical disease not only in pigs, but also in horses and dogs in other countries.

Serological follow-up of patients involved in a localized outbreak of leptospirosis

Eighteen patients involved in a localized outbreak of leptospirosis were subjected to a serological follow-up study over a 5-year period. Four distinct sets of sera from all patients and a fifth sample obtained from 10 of them were examined by the microscopic agglutination test (MAT) for demonstration of leptospiral antibodies. The test was carried out by using live leptospires from reference strains of 17 Leptospira interrogans serovars known to occur in Italy. In all cases, the highest titers of agglutinins were recorded against one or more of the three Australis group serovars tested (australis, bratislava, and lora). The highest antibody levels were reached soon after the acute phase of infection in some patients but only after some months in others. Titers then tended to recede with varying rapidity, but titers against the Australis group serovars were still detectable in some patients after 5 years. Coagglutinins against serovars of other serogroups were detected, generally at low levels, in the early sets of sera of most patients, but tended to disappear in the late-set sera. Specific immunoglobulin M (IgM) and IgG against the three Australis group serovars were determined in most serum samples from 16 patients by solid-phase enzyme immunoassay (EIA). In general, EIA titers were considerably lower than MAT titers, but there was a certain patient-to-patient variability in both the IgM/IgG ratio and the evolution and persistence of the two immunoglobulin classes. Since all the evidence indicated that the initial outbreak from a single source, the observed patient-to-patient variability in the progress of both MAT and EIA titers appeared to be attributable to factors inherent in the individual patients. Cross agglutination absorption tests, aimed at retrospectively determining to which of the Australis group serovars the outbreak-specific infecting strain belonged, were performed with six serum samples from different patients. Most absorbed sera seemed to originate from an australis or lora infection, but it was not possible to discriminate conclusively between the two serovars.

Detection of specific antigens for ten serogroups of Clostridium difficile

We previously described a serogrouping technique for Clostridium difficile based on slide agglutination with rabbit antisera raised against formol-treated cells. It allows the differentiation of ten serogroups, namely A, B, C, D, F, G, H, I, K and X. Each serogroup displays a specific profile with several distinctive bands by polyacrylamide gel electrophoresis (PAGE). In this study we investigated the common and specific antigenic determinants of the ten serogroups by immunoblotting. In a first experiment, whole cell proteins of the ten reference strains were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted against their homologous and heterologous antisera. Each serogroup was characterized by several common bands and one or two specific antigens which were proven to correspond to the lowest molecular weight distinctive band observed on PAGE profiles. New rabbit antisera were subsequently raised against the purified specific antigen obtained by electro-elution from polyacrylamide gels. Immunoblots were repeated with these new antisera: all reactions were serogroup specific except one minor cross reaction between C and F. The antisera still agglutinated the homologous strain without any cross agglutination, suggesting that the serogroup specific determinant is a surface antigen responsible for agglutination.

First human isolate of Leptospira interrogans as serovar bratislava in Italy.

A strain of Leptospira interrogans was isolated from a patient suffering from leptospirosis and was typed by the Cross Agglutination Absorption test using monoclonal antibodies prepared against different serovars of the Australis serogroup. This newly isolated strain belonged to serovar bratislava. It is the first reported isolation from man, in Italy, of Leptospira bratislava, thus supporting the emerging role of this serovar in human leptospirosis.

Antigenic analysis of three strains of Mycoplasma meleagridis of varying pathogenicity

Three strains of Mycoplasma meleagridis of varying pathogenicity were characterized by conventional serologic methods and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The three strains (RY-39A, MC-25B, and RY-39C) did not differ by cross agglutination, immunofluorescence and immunodiffusion tests. However, the strains differed in their protein patterns as determined by SDS-PAGE.

Difference of infectivity between two similar soybean rhizobial strains and immunohistochemical evidence of double infection

Abstract Two strains of soybean rhizobia (Bradyrhizobium japonicum OUG 115 and OUG 117), which were very similar in their microbiological properties but not in their imrnunological response, were tested for nodulation of Glycine max. Strain-specific antisera were obtained by adsorbing the antisera with the cross-reactive cellular antigen. The strains in the nodules formed were identified by cross agglutination and fluorescent immunohistochemical (FIHC) methods using the antibodies labeled with fluorescein isothiocyanate (FITC). Microscopic observation revealed clearly the difference of infectivity between the two strains. About 80 and 7.5% of the nodules tested were found to contain one strain, OUG 117 or OUG 115, respectively, and doubly infected nodules were estimated at 13%, when soybean was inoculated with the same concentration of the two strains.

Serological studies on British isolates of the Sejroe serogroup of leptospira. II. An evaluation of the factor analysis method of identifying leptospires using strains belonging to the Sejroe serogroup.

Twelve British isolates of leptospira belonging to the Sejroe serogroup were examined using a series of six factor sera prepared by a number of different absorption methods. Ten of the isolates were identified as Leptospira interrogans serovar hardjo and two as L. interrogans serovar saxkoebing. These isolates had previously been identified using the cross agglutination absorption method.

<I>Staphylococcus hyicus</I>subsp.<I>hyicus</I>による新生豚の化膿性関節炎および死産の発生

Staphylococcus hyicussubsp. hyicus was isolated in pure culture from the joints of two newborn piglets with arthritis and the lung and the stomach of a stillbirth in Torrori Prefecture during October, 1982 to January, 1983.Arthritis broke out in piglets 2 or 3 days old of the same litter on two farms. Stillbirth occurred in a sow on another farm.On these farms, S. hyicuswas also isolated from 17 of 21 swabs collected from the skin of dams.In the cross agglutination test, three strains ofS. hyicusisolated from the three farms showed a remarkable cross reaction with one another, but a one-sided reaction to the reference strain of ATCC 11249. On the contrary, all the 23 strains isolated from these farms and the reference strain produced an identical precipitation line in the agar gel diffusion test.

Serovar identification of leptospires of the Australis serogroup isolated from free-living and domestic species in the United Kingdom

Eighteen isolates from the Australis serogroup from free-living and domestic animals were identified using the cross agglutination absorption test. Serovar muenchen was found only in England and Wales in wood mice, short tailed and bank voles, a grey squirrel and a pig. Serovar bratislava was found in hedgehogs in England, Wales and Northern Ireland and also in a brown rat from Northern Ireland. Serovar bratislava was isolated from sheep in both England and Northern Ireland and from horses in Northern Ireland. The distribution of these serovars in relation to possible maintenance hosts is discussed.

Identification of a Reservoir of Leptospira interrogans Serovar muenchen in Voles (Microtus agrestis and Clethrionomys glareolus) in England

Infection with leptospires of the Australis serogroup is widespread in voles in Southern England and the serovar identification of five randomly-selected isolates was determined by cross agglutination absorption. Two isolates from short-tailed voles (Microtus agrestis) and three isolates from bank voles (Clethrionomys glareolus) were all identified as serovar muenchen. The only previous isolation of this serovar is that from a human in Germany.

A case of cholelithiasis, in which Listeria monocytogenes type 3 was isolated from the patient's bile

Most strains of Listeria monocytogenes isolated from human listeriosis in Japan so far have been identified as type 1 or lb and 4b.One case from which type 4d strain isolated was reported in 1974, other case from which type 2 strain isolated was reported in 1977 by T. Nagai, one of the authors. A strain was isolated from bile of cholelithiasis patient (23 years old, female) in Sapporo Medical College Hospital, May, 1980, and this strain was named as “Echizen strain”. Its morphological findings and biochemical properties were identical with those of Listeria monocytogenes.By serological tests i.e. by cross agglutination test and absorption test using immune serum of standard strains (type 1, 2, 3 and 4b) and Echizen strain, it was identified as Listeria monocytogenes type 3.This interest case is found to suggest that Listeria monocytogenes latently localize in bile.This is the first report of Listeria monocytogenes type 3 isolated from human in Japan. The total number of human listeriosis cases in Japan counts 335 (from 1958 to 1981).

The O Antigenic Relationships Amongst Dekkera, Hanseniaspora, Escherichia coli O:75, Salmonella cholerae-suis and Salmonella aberdeen

The immune sera for Dekkera bruxellensis, D. intermedia and Hanseniaspora osmophila agglutinated Salmonella cholerae-suis (O:62,7) and vice versa. The immune serum for H. osmophila also agglutinated E. coli (O:75), S. aberdeen (O:11) and vice versa. Cross agglutination and absorption tests demonstrated common antigenic factor(s) between above mentioned yeasts and Salmonella O:62, O:7 antigens. H. osmophila also showed a common antigenic factor(s) with E. coli (O:75) and S. aberdeen (O:11).

リステリア菌4d型による髄膜炎の一症例

All strains of Listeria monocytogenes isolated from human listeriosis in Japan so far have been identified as type 1 or type 4b.A strain was isolated from the cerebrospinal fluid of a meningitis baby (1 year and 4 months old, male) in Ishikawa Prefecture, December of 1971, and this strain was named as “Tsuchiyama strain”. Its morphological findings and biochemical properties are identical with those of Listeria monocytogenes.By serological tests, i.e. by cross agglutination test and absorption test using immune serum of standard strains (type 4a, 4b, 4c, 4d, 4e) and Tsuchiyama strain, it was identified as Listeria monoc ytogenes type 4d.It is the first case of listeriosis due to type 4d strain in Japan.