Indirect Fluorescent Antibody Technique Research Articles

Vaccination of chickens with DNA vaccine encoding Eimeria acervulina 3-1E and chicken IL-15 offers protection against homologous challenge

Eimeria acervulina 3-1E antigen gene and mature chicken interleukin 15 (mChIL-15) gene were cloned into expression vector pcDNA3.1(+) in different forms, produced DNA vaccine pcDNA3.1-3-1E, and pcDNA3.1-3-1E-linker-mChIL-15 co-expressing E. acervulina 3-1E gene and mChIL-15 gene, respectively. The expression of objective gene in vitro was detected by indirect fluorescent antibody technique and immunohistochemistry. The two DNA vaccines were administered by intramuscular leg injection. An animal challenge experiment was carried out to evaluate the immune protective efficacy of the vaccines. The results indicated that DNA vaccines were successfully constructed and the expression of objective gene could be detected in vitro. The animal experimental results showed that both DNA vaccines could provide partial protection against homologous challenge in chickens. The chimeric DNA vaccine, pcDNA3.1-3-1E-linker-mChIL-15, could significantly increase oocyst decrease ratio, reduce the average lesion score in the duodenum, improve body weight gain, and increase anti-coccidial index (ACI) compared to the DNA vaccine pcDNA3.1-3-1E. Taken together, these results demonstrate ChIL-15 enhance the immunogenicity of 3-1E DNA vaccine, and co-expression of cytokine and optimized surface antigen of Eimeria may be a promising method to enhance immunogenicity of DNA vaccines in poultry.

Serological discrimination of several kinds of microsporidian spores isolated from the silkworm, Bombyx mori, by an indirect fluorescent antibody technique

Serological discrimination of several kinds of microsporidian spores isolated from the silkworm, Bombyx mori, by an indirect fluorescent antibody technique

The Indirect Fluorescent Antibody Technique as a Method for Detecting Serum Antibodies against Hog Cholera*

The Indirect Fluorescent Antibody Technique as a Method for Detecting Serum Antibodies against Hog Cholera*

Prenylcoumarin with Rev-export inhibitory activity from Cnidii Monnieris Fructus

By use of the fission yeast expressing the model fusion protein comprised of GST, SV40 T antigen NLS, GFP, and Rev-NES in the bioassay, the prenylcoumarin osthol (1) was disclosed as the new Rev-export inhibitor from the MeOH extract of Cnidii Monnieris Fructus. Furthermore, 1 was also found to inhibit export the genuine Rev in HeLa cells by indirect fluorescent antibody technique. By the competitive experiment using the biotinylated probe 3, osthol (1) was revealed to inhibit nuclear export of Rev through a NES non-antagonistic mode. Structure–activity relationship analysis of several analogs of 1 clarified that both prenyl side chain and double bond adjacent to the lactone carbonyl residue play an important role in the Rev-export inhibitory potency of 1.

Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa

The viral haemorrhagic septicaemia virus (VHSV) comprises 4 major genotypes and a number of subtypes with, in most cases, distinct geographical distribution. A quick and simple detection method that can discriminate the different genotypes is desirable for a quick and more efficient prevention of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected with each of the viral protein genes, it was shown that the MAb VHS-10 recognizes a nonlinear genotype IVa-specific epitope on the VHSV N-protein.

Serologic survey for antibodies to Rickettsia among domestic and wild animal populations in Brazil

During the last 20 years, Brazilian spotted fever (BSF) has been the most common rickettsiosis and had the highest case–fatality ratio of any rickettsiosis in Brazil [1Lemos ERS Alvarenga FBF Cintra M et al.Spotted fever in Brazil: a seroepidemiological study and description of clinical cases in a endemic área in the state of São Paulo.Am J Trop Med Hyg. 2001; 65: 329-334PubMed Google Scholar]. In Minas Gerais, many cases were described in the 1930s and 1940s, but no reports of BSF cases appeared in the medical literature during the decades of the 1950s, 1960s and 1970s. In the 1980s an epidemic form appeared in rural and peri-urban areas, predominantly in the valleys of Jequitinhonha, Mucuri and Rio Doce [2Galvão MAM Febre Maculosa em Minas Gerais: um estudo sobre a distribuição da doença no Estado e seu comportamento em área de foco peri-urbano. Ph.D.Thesis, Tropical Medicine, Belo Horizonte. Faculdade de Medicina da UFMG, Brasil1996Google Scholar]. One of the objectives of this study was to understand the status of the BSF vectors and hosts in an area of low endemicity. The city of Santa Cruz do Escalvado, located in the Piranga Valley, Minas Gerais, Brazil (Table. 1), which is considered to be an old focus for BSF, was chosen. The first cases in this area occurred in 1985, with few cases after this date [3Galvão MAM, Ribeiro JGL, Sevalho G. Febre maculosa Brasileira. Grupo de Vigilância Epidemiológica e Zoonoses da Secretaria Estadual de Saúde de Minas Gerais. 1989;1–10.Google Scholar].TABLE 1Percentage of seroreactivity to rickettsiae by IFA at a titre of 1:64 in animals collected in Santa Cruz do Escalvado, Minas Gerais, BrazilSerumN1Total number of samples of sera collected and tested.IFA2Number of samples reactive by IFA at the titre of 1:64.Total (%)Dogs5323.8%Equines3738.1%Opossums14428.6%RodentsN. squamipes3300%O. subflavus200%R. rattus322681.3%1 Total number of samples of sera collected and tested.2 Number of samples reactive by IFA at the titre of 1:64. Open table in a new tab The capture of wild animals and the serological survey were carried out in Santa Cruz do Escalvado every 3 months during the period from May 2005 to March 2006 in partnership with professionals of the National Foundation of Health (FUNASA). During the visits, traps to catch rodents (a total of 100 traps/collection) and opossums (a total of 20 traps/collection) were placed in nearby dwellings, including garages, sheds, stockpiles of food, plantations of corn, bamboo thickets, waste deposits and near the homes. Those samples were evaluated by indirect immunofluorescence reaction (IFA) using antigen obtained in São Paulo University for spotted fever group (SFG) rickettsiae (Rickettsia rickettsii, R. parkeri, R. felis and R. amblyommii) and Rickettsia bellii. Serum samples were tested at a dilution of 1:64 using the CDC protocol [4CDC Division of Viral and Rickettsial Diseases Indirect Fluorescent Antibody Technique for the Detection of Rickettsial Antibodies:11. National Center for Infectious Diseases, Atlanta, Georgia, USA2001Google Scholar]. We captured wild vertebrates including rodents Rattus rattus (33), Nectomys squamipes (35) and Oryzomys subfalus (12) and opossums Didelphis aurita (14). From the 184 animals captured, we collected blood samples from 166 animals, as well as from 37 horses and 53 dogs. The rodents were caught around their common habitats. The species R. rattus was captured mainly in bunkers and warehouses. The species N. squamipes and O. subflavus were captured mainly in cultivated fields and woods. The 14 D. aurita opossums were captured in areas close to houses and also in thickets of bamboo. The data showed 81.25% seroreactivity to SFG rickettsiae among 32 sera of R. rattus examined. The rest of the captured rodents did not have antibodies. There was also a relatively prevalent serologic reactivity to SFG rickettsiae among 14 opossums captured, with 14.3% having antibodies to R. rickettsii and 14.3% to R. parkeri. The equines showed 5.4% seroreactivity to R. bellii and 2.7% to SFG rickettsiae, while canines showed 2% prevalence of antibodies to R. rickettsii and 2% to R. parkeri. This study demonstrated the presence of a high percentage of seroreactivity to rickettsiosis in synanthropic animals’ sera. This observation confirms the opinion that we must pay attention to the possibility of occurrence of new human cases in this area, caused by the mechanisms of natural focus invasion by man or by the dispersion of potential reservoirs and vectors of the rickettsiosis into new areas. The low percentage of seroreactivity in sentinel animals such as dogs and horses confirms that the studied area is an area of low endemicity for rickettsiosis. The next step for this study will be the confirmation of the results found by serology using molecular biological methods in the same tested animals.

Fluorescent Antibody Technique For Detection Of Salmonella Typhimurium In The Intestine Of Experimentally Infected Calves

A systemic study on the pathogenesis of experimentally inducedSalmonella typhimurium infection was carried out in calves. Sixteen normal,colostrum-fed, friesian calves, ranging in age from 3 to 6 weeks were used. Thecalves were divided into two equal groups. Group I calves were inoculatedorally with 1.5 x 1011 CFU of Salmonella typhimurium and group II calvesserved as control.Using an indirect fluorescent antibody technique, it was found thatSalmonella typhimurium has an apparent predilection for the small intestineparticularly jejunum and ileum. The results of this study suggest that thistechnique can be considered as a rapid reliable technique for diagnosis ofsalmonellosis in endemic outbreaks.

Identification of overwintering vegetative cells of the bivalve-killing dinoflagellate Heterocapsa circularisquama in Uranouchi Inlet, Kochi Prefecture, Japan

Red tides of Heterocapsa circularisquama have led to serious damage of bivalve aquacultures in western coastal areas of Japan. To understand the whole picture regarding the ecology of this species, it is essential to clarify its overwintering mechanisms. In this study, the population dynamics of H. circularisquama were investigated from February 2004 to November 2005, and overwintering cells were identified for the first time in water columns of Uranouchi Inlet, Kochi Prefecture, Japan. Heterocapsa circularisquama cells were detected by the indirect fluorescent antibody technique using monoclonal antibodies that specifically recognize and react to this species. Vegetative cells were almost always detected from the first observation in February 2004 to November 2005 with temperatures of 10.5-30.6°C. During the period from winter to spring, this species survived in areas with a temperature higher than 10°C. The overwintering cells of H. circularisquama were isolated in March 2004, and identification was made via observation of the morphology and body scales of the cultured cells. These overwintering cells were identified as H. circularisquama and reacted to the monoclonal antibody. These results indicate that H. circularisquama can overwinter and survive throughout the year in a vegetative cell state in Uranouchi Inlet.

Annual dynamics of marine birnavirus (MABV) in cultured Japanese flounder Paralichthys olivaceus and sea water

Seasonal changes in the infection state of marine birnavirus (MABV) in Japanese flounder Paralichthys olivaceus and in rearing sea water are described. Sea water and 10–11 healthy fish were sampled monthly from April 2002 to February 2003. The MABV genome was detected throughout the year in >80% of the fish examined at each sampling. The virus was isolated from the liver, kidney, and spleen, but not from the brain. The detection rate in each organ increased from April to October, and then decreased. Detection of virus antigen by the indirect fluorescent antibody technique also showed that the virus was present from spring to autumn (Jun–September) in the liver, kidney, and spleen, but not the brain. Sequence analysis of the MABV genome at the VP2-NS region revealed two specific mutations compared to the standard yellowtail strain (Y-6). It is suggested that the infection state of MABV in Japanese flounder changes to a latent or persistent infection after autumn. MABV was detected in sea water between September and February, suggesting that virus particles in the environment are relatively higher during cool seasons.

Seasonal distribution of the shellfish-killing dinoflagellate Heterocapsa circularisquama in Ago Bay monitored by an indirect fluorescent antibody technique using monoclonal antibodies

Heterocapsa circularisquama is the most noxious dinoflagellate to bivalves such as oysters, short-necked clams and pearl oysters because its red tides have caused major mass mortalities. In order to reduce the negative impacts of H. circularisquama, predictions of red tide occurrences are essential, and hence it is important to grasp its population dynamics. The population dynamics of H. circularisquama, however, have not yet been clarified throughout the year. To precisely monitor population dynamics, the fluorescent antibody technique is effective. We here report on H. circularisquama population dynamics monitored with the indirect fluorescent antibody technique (IFAT) using monoclonal antibodies. Samplings were carried out once a week in summer and twice a month in other seasons at 4 points in Ago Bay, Mie Prefecture, Japan, from April 2001 to March 2005. Direct counting of the cells was also performed using a normal optical microscope. Vegetative cells of H. circularisquama monitored with IFAT were generally detected from late spring (May) to late autumn, and the cell density increased (maximum 2.33×106 cells L-1) during summer. The lowest detection level was 1.33 cells L-1 with IFAT. In contrast, monitoring by counting with a common optical microscope often failed to detect H. circularisquama cells even at a density of 103 cells L-1 or more. Water temperature was significantly correlated to the abundances of H. circularisquama, and the cells were almost always observed at a temperature of 25°C or higher (summer season), and never detected at 10°C or lower (winter and early spring) in Ago Bay. In the waters of the bottom layer, H. circularisquama cells tended to be more abundant with lower dissolved oxygen (DO) values during summer. Thus, the seasonal distribution of H. circularisquama was clarified almost perfectly together with environmental factors in Ago Bay between spring and early winter.

In vitro antiprotozoal activity of organic waste materials against Cryptosporidium parvum

Plant residues and by-products from pharmaceutical or food production may be cost-efficient sources of functional feed ingredients in animal nutrition. In the course of the EU-funded project SAFEWASTES numerous materials that usually are treated as wastes are evaluated for their potential for further usage. The present study investigated in vitro antiprotozoal effects against Cryptosporidium parvum, an intestinal parasite (Apicomplexa) that infects a wide range of hosts including humans. Especially young or immunocompromised animals are susceptible to infection and mass propagation of the parasite which is often accompanied by loss of weight and water, retarded development and increased susceptibility to other diseases. Among several methods a cell culture assay with HCT-8 host cells was chosen for determination of anti-cryptosporidial effects of test substances. Intracellular parasite development was evaluated microscopically after labelling by indirect fluorescent antibody technique (IFAT). Cell vitality assays were conducted simultaneously to assess eventual deleterious effects on the host cells, since it is important to discriminate unspecific cytotoxic effects from direct effects against the parasites. 53 extracts prepared from 18 different organic waste materials were screened in this assay. An ethanol extract from olive press-cake effectively inhibited C. parvum development while being tolerated by the host cells in the applied concentrations. Monensin as a positive control inhibited parasite development in a highly reproducible way. Bioassay-guided fractionation was done to identify the active principles of the materials.

Epidemiological study of cryptosporidiosis in immunocompetent & immunocompromised children, prepared parasitic antigen and used it in IFAT

An epidemiological study was done on 2 groups of children infectedwith watery diarrhea in some hospitals of Baghdad (group 1: immunocompetentoutpatients, group 2: immunosuppressed inpatients).Modified cold Ziehl Neelsen stain was used in diagnosis. The wholeoocysts antigen prepared after conduction of experimental infection in 2 calvesby using isolated oocysts from children stool, and this prepared antigen wasused in Indirect Fluorescent Antibody Technique (IFAT).The percentage of infection in the 2 groups of outpatients and inpatientswere 21.2% and 32% respectively, and there was a difference in percentage ofinfection between the 3 groups of children, the highest was 36.4% in group withleukemia and received chemical and radiotherapy.There was no significant difference between the two sexes.The clinical signs of experimental infection began after 4 days and continued for10 days. The oocysts appeared in IFAT as spherical in shape with clear wallsand bright yellowish green color. There was no significant difference betweenthe 2 methods used for diagnosis (stain and immunological test).

Rapid detection and identification of Streptococcus iniae using a monoclonal antibody-based indirect fluorescent antibody technique

Streptococcus iniae is among the major pathogens of a large number of fish species cultured in fresh and marine recirculating and net pen production systems. The traditional plate culture technique to detect and identify S. iniae is time consuming and may be problematic due to phenotypic variations of S. iniae isolates. The rapid transmission and severe economic impact of this disease necessitates the development of a rapid, reliable, specific, and sensitive technique to detect and identify S. iniae. We developed an indirect fluorescent antibody technique (IFAT) based on a highly specific mononclonal antibody for S. iniae. The IFAT was found to be suitable for the detection and identification of S. iniae from experimentally and naturally infected tilapia ( Oreochromis niloticus). The olfactory epithelium of the naris of experimentally and naturally infected tilapia was demonstrated to be a reliable, sensitive, and non-lethal sample site for the detection and identification of S. iniae. This IFAT was validated by the traditional plate culture techniques but found to be considerably less time consuming and problematic. These findings reveal that this IFAT is an improvement in the ability to detect and identify S. iniae in infected and carrier fish.

A Lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model

Transmission of Lawsonia intracellularis from experimentally inoculated pigs to naive swine was demonstrated in this study. The study was conducted using conventional pigs divided into three groups as follows: principles inoculated with L. intracellularis, sentinels, and controls. The pigs were inoculated and paired on 13 and 9 days post-inoculation with a sentinel pig for 7 days. Fecal samples and serum samples were collected throughout the study for polymerase chain reaction (PCR) and antibody testing by indirect fluorescent antibody techniques. After co-mingling, the inoculated group was necropsied; sentinel and control pigs were necropsied 7–14 days later. The intestinal tracts were evaluated grossly and microscopically for lesions. PCR was performed on intestinal mucosal scrapings and feces. Warthin–Starry and fluorescent antibody staining procedures were conducted to confirm colonization with L. intracellularis. Gross and microscopic lesions typical of porcine proliferative enteropathy (PPE) were observed in both the inoculated and sentinel groups. Transmission was demonstrated from inoculated principle pigs to sentinel pigs. PCR results detected cyclical shedding of L. intracellularis in the feces. Seroconversion occurred in pigs that were exposed to L. intracellularis. From this study, it was demonstrated that transmission of L. intracellularis can occur easily in an environment with experimentally infected pigs and that PCR can be a useful tool to monitor fecal shedding of the organism.

Antigenic characterization of Brazilian isolates of Anaplasma marginale.

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

Molecular diagnosis of leishmaniosis in dogs: Comparative application of traditional diagnostic methods and the proposed assay on clinical samples

Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis. In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use. The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis. The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre ≥200), and 19% by microscopic examination of lymph node aspirates.

Seroprevalence of Coxiella burnetii infections among cats in different living environments.

The seroprevalence of Coxiella burnetii infection among pet cats in Japan and Korea and stray cats in Japan was investigated by an indirect fluorescent antibody technique and PCR test. Forty-four (14.2%) of 310 pet cats in Japan were seropositive, as were 15 (41.7%) of 36 stray cats in Japan and 10 (8.6%) of 116 pet cats in Korea. The antibody positive rate in stray cats was significantly higher than that in pet cats, but there was no correlation between the rates in Japanese and Korean pet cats. In this study, the prevalence of C. burnetii infection among cats in different living environments was found and it is difficult to deny that stray cats would be one of the important sources of infection for human Q fever.

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

A molecular epidemiological study was performed with Babesia bigemina isolates from five geographical regions of Brazil. The genetic analysis was done with random amplification of polymorphic DNA (RAPD), repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) and enterobacterial repetitive intergenic consensus sequences-polymerase chain reaction (ERIC-PCR) that showed genetic polymorphism between these isolates and generated fingerprinting. In RAPD, ILO872 and ILO876 primers were able to detect at least one fingerprinting for each B. bigemina isolate. The amplification of B. bigemina DNA fragments by REP-PCR and ERIC-PCR gave evidence for the presence in this haemoprotozoan of the sequences described previously in microorganisms of the bacterial kingdom. For the first time it was demonstrated that both techniques can be used for genetic analysis of a protozoan parasite, although the ERIC-PCR was more discriminatory than REP-PCR. The dendogram with similarity coefficient among isolates showed two clusters and one subcluster. The Northeastern and Mid-Western isolates showed the greatest genetic diversity, while the Southeastern and Southern isolates were the closest. The antigenic analysis was done through indirect fluorescent antibody technique and Western blotting using a panel of monoclonal antibodies directed against epitopes on the merozoite membrane surface, rhoptries and membrane of infected erythrocytes. As expected, the merozoite variable surface antigens, major surface antigen (MSA)-1 and MSA-2 showed antigenic diversity. However, B cell epitopes on rhoptries and infected erythrocytes were conserved among all isolates studied. In this study it was possible to identify variable and conserved antigens, which had already been described as potential immunogens. Considering that an attenuated Babesia clone used as immunogen selected populations capable of evading the immunity induced by this vaccine, it is necessary to evaluate more deeply the cross-protection conferred by genetically more distant Brazilian B. bigemina isolates and make an evaluation of the polymorphism degree of variable antigens such as MSA-1 and MSA-2.

The value of Chlamydia trachomatis antibody testing in predicting tubal factor infertility.

The objective of the present study was to compare the likelihood of abnormal Chlamydia trachomatis antibody test results with that of abnormal hysterosalpingography (HSG) test results in patients with tubal factor infertility. Anti-C. trachomatis immunoglobulin G antibodies were determined prospectively in 295 infertility patients by means of an indirect fluorescent antibody technique. In 48 of the 295 patients both HSG and laparoscopy with chromotubation were performed. The results of C. trachomatis antibody testing were compared with the results of HSG with respect to their predictive value of tubal factor infertility. Likelihood ratios for abnormal C. trachomatis antibody and HSG test results were determined in infertility patients, as assessed by laparoscopy. The positive likelihood ratio for C. trachomatis antibody testing was 1.8. This was comparable with the HSG, which had a positive likelihood ratio of 1.7. The predictive value of C. trachomatis antibody testing was equal to that of HSG, but ratios of 1.7 and 1.8 indicate a poor test, so both C. trachomatis antibody testing and HSG have a poor predictive value. C. trachomatis antibody testing causes minimal inconvenience to the patient, in contrast to HSG, and therefore should be maintained in infertility examinations.

Detailed analysis of rice chitinase gene expression in transgenic cucumber plants showing different levels of disease resistance to gray mold ( Botrytis cinerea)

The class I chitinase cDNA (RCC2) of rice driven by the CaMV 35S promoter was introduced into cucumber by Agrobacterium-mediated transformation. The transgenic cucumbers showed varying levels of disease resistance to gray mold. We selected three transgenic lines representing different resistance levels, namely the most resistant line (CR32), an intermediate-resistance line (CR3), and a susceptible line (CR20), and studied the correlation between RCC2 expression and disease resistance. ELISA analysis showed that the rice chitinase levels of CR32 and CR3 were higher than that of CR20. RCC2 expression was then examined in more detail using an in situ indirect fluorescent antibody technique and 3-D fluorescence microscopy (CELL Scan system). A homogeneous distribution of cells expressing RCC2 to a high degree was observed in the epidermal and mesophyll cells of CR32 leaves, whereas the intensity of RCC2 expression in CR3 leaves was lower. However, CR3 often showed high expression of RCC2 in parts of leaf tissue. Rice chitinase was localized within cells but was not detected between cells. Optical microscopy of Botrytis cinerea infection behavior revealed that none of the transgenic lines nor any of the non-transgenic lines inhibited the penetration of B. cinerea. However, fungal growth within leaf tissue was suppressed in the resistant lines but not in the non-transformed or CR20 leaf tissue. Thus, we speculate that high expression and intracellular localization of rice chitinase may be involved in enhancing the resistance of transgenic plants to gray mold.