Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns

Abstract Environmental isolates of fluorescent pseudomonads grown to early stationary phase in glucose-enriched Luria broth were treated with proteinase K in sodium dodecylsulphate (SDS) lysis buffer and subsequently analyzed by polyacrylamide gel electrophoresis (PAGE). Four silver-staining protein-fragment bands could be used for rapid identification at the species level. Pseudomonas aeruginosa isolates were easily recognized by a unique banding pattern. Isolates considered to be P. fluorescen from biochemical and physiological tests (classical biotypes I, II, III, IV and V) also had a characteristic banding pattern, which in turn was different from that of P. putida isolates (classical biotype A). A residual group representing intermediate isolates of P. fluorescens (new biotype VI of Barrett et al., J. Gen. Microbiol. 132, 1986) or P. putida (biotype B) had a banding pattern similar to that of classical P. fluorescens biotypes. On the other hand, a group representing other intermediate isolates of P. putida (new biotype C of Barrett et al., J. Gen. Microbiol. 132, 1986) had a unique banding pattern resembling that of classical P. putida biotype A. A small number of protein fragment bands appearing in SDS-PAGE analysis of whole-cell lysates seems adequate for a rapid identification at the species level of P. aeruginosa, P. fluorescens and P. putida isolated from natural environments.     

Streptococcus iniae: serological differences,presence of capsule and resistance to immune serum killing

The biochemical profiles, presence of capsule, outer membrane protein profiles and serological interactions of isolates of Streptococcus iniae obtained from different geographical and fish host origins were examined. The isolates had very similar biochemical profiles using API 20 Strep but varied as to whether they were arginine dihydrolase-negative, -positive or -intermediate (AD-ve, AD+ve, AD+/-ve, respectively). Representatives of each AD type were compared in subsequent experiments. All types possessed a polysaccharide capsule. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of outer membrane proteins or whole cells revealed no difference in banding patterns between isolates. All isolates were resistant to trout normal and specific immune serum and grew well in the presence of added fresh normal serum. Serological analyses of the isolates revealed antigenic differences. Trout antiserum against the AD+ve isolate did not agglutinate the AD-ve or AD+/-ve isolates, while antisera against the latter 2 types showed low agglutinating activity with all 3 isolates. When whole live cells of AD-ve and AD+ve isolates were dot-blotted, antiserum to the AD+ve isolate did not stain the AD-ve isolate, but antiserum to the AD-ve isolate stained both AD types. However, if the cells were pre-treated with Proteinase K (to remove surface-exposed protein antigens), the AD+ve isolate was stained only by its homologous antiserum. These results suggest that while certain protein antigens of the different AD type strains are immunologically cross-reactive, the capsular antigens appear to be AD type-specific. Furthermore, the results suggest that the cross-reactive antigens on the AD-ve isolate are effectively hidden by the strain-specific capsule, while they are partially exposed on the AD+ve isolate.     

A technique for typing Cryptosporidium isolates.

Antigens extracted from Cryptosporidium oocysts, which had been purified from faeces or chick egg culture, were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels, and blotted onto nitrocellulose membranes. A Cryptosporidium genus-specific monoclonal antibody MAb-C1 bound to multiple bands using several detection techniques, and these corresponded to bands detected using immune rabbit antisera. Using a detection system with fluorescein isothiocyanate (FITC)-labelled MAb-C1 and alkaline phosphatase-labelled anti-FITC, bands were detected between 50 and 300 kDa. Blots were examined directly and by using a laser scanner. The system was shown to be specific for Cryptosporidium spp., giving no staining with a variety of other pathogens, and with negative samples. The oocyst antigen which bound MAb-C1 was stable, and banding patterns were not significantly affected by pretreatment of oocysts with proteinase K, trypsin, formalin, or sodium hypochlorite, methods commonly used during preparation and storage of C. parvum oocysts. However, banding was reduced with potassium dichromate. Of 76 samples containing Cryptosporidium oocysts, 53 showed one or more MAb-C1 staining bands. Cryptosporidium baileyi and C. parvum could be clearly differentiated by their banding patterns, indicating that the system will distinguish between species. Some isolates, including a single isolate of C. muris, produced weak bands which made interpretation difficult. The technique showed differences between isolates of C. parvum, with two different banding types found in human isolates, and other banding types seen in calf and lamb isolates. This method provides a useful way of characterising isolates which may be new species.     

Molecular characterization of European and Egyptian isolates of Sclerotium cepivorum,the incitant of onion white rot

Abstract

The tested European and Egyptian isolates of Sclerotium cepivorum were able to infect Giza 6 onion cultivar causing white rot disease with a different degrees of disease severity (ranging from sever to weak). The pattern of esterase isozymes produced by the tested isolates of the pathogen showed two main bands (arrows) which were different in density. Such differences in density of bands were present in every run and therefore appear to be indicators for differences among the tested isolates. Analysis of the protein pattern of the tested isolates of the pathogen indicated that the tested isolates had major proteins of a molecular weight of 52, 36, 23 and 16 kDa. Variation between isolates was detected by presence of bands of low molecular weight. Isolate Nos. 1, 4, 5, 7, 8, 9, 10 and 13 had a band at 17 kDa, whereas isolate Nos. 2, 3, 6, 11, 12, 14, and 15 had a band at 20 kDa. Using RAPD analysis to evaluate the genetic diversity of the tested isolates indicated that the tested field population of the pathogen was genetically heterogeneous but shared a number of common bands with molecular weights ranging from 650 to 2500 bp. Based on the DNA banding pattern the tested isolates can be assigned to seven genetically different groups. All tested isolates produced a band at 2500 bp except isolate No. 7. No correlation was exibited between patterns esterase isozmes, protein and DNA patterns of S. cepivorum isolates and their virulence or geographical origin.     

Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3.

Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized. The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V. anguillarum serovars was also evaluated. The three serological assays used to establish the serogroups within V. anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected. Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera. Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V. anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns. On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V. anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay. From these results, it can be concluded that V. anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist.     

Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.     

Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000-daltons (53-kDa) and 72-kDa surface antigenic proteins and a 38-kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38-kDa axial flageller protein.     

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.     

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu,SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 °C (yeast form) and as cottonous colonies at 25 °C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp 43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population. This revised version was published online in August 2006 with corrections to the Cover Date.     

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.     

Serological investigation of the fish pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish.

The serological relationships among 32 isolates of Edwardsiella ictaluri obtained from fish were studied. The strains were extremely homogeneous in protein and lipopolysaccharide preparations as observed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Only minor variations were observed in the structural O-side chain subunits in three isolates; however, such variation did not preclude antigenic recognition by two E. ictaluri antisera in either microagglutination or Western blot immunoassays. The antigenic homogeneity of E. ictaluri was further demonstrated by microagglutination assays with both formalin-killed and heat inactivated cellular antigens. The minimal degree of antigenic variability observed suggested that most isolates of E. ictaluri compose a single antigenic serotype.     

Nucleic acids and protein content in relation to growth and regression of buffalo (Bubalus bubalis) corpora lutea

Information on nucleic acids and protein content of buffalo corpus luteum (CL) in relation to growth, development and regression is not available. An experiment was thus conducted to investigate the variation and relationship between nucleic acids and protein content in CL during different developmental stages and to determine the qualitative differences in protein constituents in any of these stages. Buffalo corpora lutea of different developmental stages viz., developing (day 5-10, n = 16), developed (day 11-17, n = 12) and regressed (day 18-21, n = 10) stages were collected from non-pregnant and -pathological genitalia (n = 38). The DNA, RNA and protein content in tissue extracts were determined and the proteins in pooled samples were analyzed by polyacrylamide gel electrophoresis. Developing stage CL had more total and per gram tissue level of DNA and RNA with significant positive relationship with total and per gram RNA and protein contents. Although there was no significant difference in total weight, a significant decrease in total DNA as well as per gram level of DNA and RNA was observed in developed stage compared to developing stage CL. The total protein content in developed stage CL was compared to developing and regressed stage CL. Non-denaturing PAGE analysis of CL proteins of different stages showed five protein bands of 210, 190, 82, 68 and 66 kDa and one that migrated with the dye front in all the stages however, not shown any differences in banding pattern. Denaturing PAGE showed 15 bands viz., 205, 66, 53, 42, 35, 27, 24, 22, 20, 18, 17, 14, 9, 7.5 and 6.5 kDa. Out of these 66 and 53 kDa bands appeared with maximum intensity in all the three stages of CL. Comparison of bands between the three stages revealed five 57, 31, 27, 19 and 16 kDa stage-specific bands in regressed stage CL. The present study indicated that the DNA, RNA and protein content of buffalo CL varied with the stages of development and regressed stage CL contained some unique protein bands which were not observed either in developed or developing stage CL.     

Female specific proteins in Triatoma infestans haemolymph

The presence of female specific proteins in triatoma infestans haemolymph, as well as the relationship between the female specific proteins and egg proteins, were analysed. At the same time, the presence of specific female proteins in different instars was studied. Cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, and immunochemical methods were used.No differences between immature female and male haemolymph were established. Female haemolymph obtained from insects with ovary development revealed quantitative differences, with cellulose acetate, with respect to the control males. With polyacrylamide gel electrophoresis, one component that is not detected in control males was detected in mature female haemolymph. With immunochemical assays, at least two antigenic components that were not observed in male haemolymph were detected.Egg extract showed, with cellulose acetate, two bands with a mobility similar to that of the proteins increased in the haemolymph of the mature female; with polyacrylamide gel, two major bands with a mobility similar to that of the specific female haemolymph protein were detected. Egg extract contains at least two components demonstrated by double-diffusion assays and three components by immunoelectrophoresis, with immunological identity to specific mature female haemolymph proteins.The extract obtained from recently hatched insects revealed two components with immunological identity to specific female proteins. Haemolymph from first, second, third, fourth and fifth instars do not appear to contain any femalespecific haemolymph protein.     

Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-molecular-weight precursors

Crystals were purified from spore-crystal complexes of Bacillus sphaericus 2362 by disruption in a French pressure cell followed by centrifugation through 48% (wt/vol) NaBr. Crystals from such preparations had a 50% lethal concentration of 6 ng of protein per ml for the larvae of the mosquito Culex pipiens. When subjected to polyacrylamide gel electrophoresis under denaturing conditions, the proteins in B. sphaericus crystals migrated in positions corresponding to 43, 63, 98, 110, and 125 kilodaltons (kDa); solubilization of the crystal at pH 12 with NaOH eliminated all but the bands at 43 and 63 kDa. Since NaOH-solubilized preparations were toxic to mosquito larvae, these proteins were purified to electrophoretic homogeneity and antiserum was obtained to each. Analysis of the two purified proteins indicated that the 43-kDa protein was toxic to mosquito larvae (50% lethal concentration, 35 ng of protein per ml), whereas the 63-kDa protein was not. Further differences between them were their amino acid compositions, their lack of immunological cross-reactivity, their opposite net charges at pH 7.5, and their susceptibility to digestion by larval midgut proteases (the 63-kDa protein was highly susceptible, whereas the 43-kDa protein was not). The sequence of the 40 N-terminal residues of the 43-kDa protein was determined and found to contain a high percentage of hydrophobic amino acids. The sequence of the 63-kDa protein could not be determined, since it had multiple N termini. By electrophoretically separating the crystal proteins and then electroblotting onto nitrocellulose paper and visualizing the bands with antisera to the 43- and 63-kDa proteins in conjunction with an immunoblot assay, it was found that the high-molecular-mass crystal proteins (98 to 125 kDa) contained antigenic determinants of both proteins. These results suggested that the lower-molecular-weight crystal proteins detected in polyacrylamide gels after electrophoresis under denaturing conditions were derivatives of one or more of the higher-molecular-weight crystal proteins. In vivo studies of the products of crystal degradation by larvae of Culex pipiens indicated that the high-molecular-weight proteins and the 63-kDa antigenic determinants were rapidly degraded and that a 40-kDa protein related to the 43-kDa toxin persisted for the duration of the experiment (4 h). Some of the studies performed with B.sphaericus 2362 were extended to strains 1593, 1691, and 2297 of this species with results which indicated a high degree of similarity between the crystal proteins of all these larvicidal strains.     

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.     

NUCLEAR PROTEIN PATTERNS IN DEVELOPING AND ADULT BRAIN AND IN ETHYLNITROSOUREA-INDUCED NEUROECTODERMAL TUMOURS OF THE RAT1,2

In a comparative study, the patterns of histones and non-histone proteins were analysed in the chromatin of foetal (18th day of gestation), 10-day-old, and adult BD IX-rat brain, as well as in the chromatin of two ethylnitrosourea-induced neuroectodermal tumours (TV1A1 and GV1A1) and the corresponding malignant cell culture lines TV1C1 and GV1C1. Separation of nuclear proteins at high resolution was obtained by electrophoresis in 15% and 10% polyacrylamide gels containing urea (2·5 m or 6·25 m ). In spite of an overall similarity, significant quantitative and qualitative differences were observed between the respective non-histone proteins banding patterns of normal brain and the neoplastic cells analysed. The non-histone protein banding patterns of brain (∼40 different bands) at different stages of development revealed both quantitative differences and the presence of particular bands characteristic of foetal or adult brain, respectively. Both the‘foetal’and‘adult’non-histone protein bands also appeared in the electrophoretograms of the neoplastic neuroectodermal cells.     

嗜肺巴氏杆菌蛋白及抗原图谱初步分析

对不同来源的 1 1株嗜肺巴氏杆菌进行了全菌可溶性蛋白及抗原图谱分析。使用SDS -聚丙烯酰胺凝胶电泳 (SDS -PAGE) ,梯度凝胶电泳结果显示 ,嗜肺巴氏杆菌蛋白主要分布于相对分子质量 ( 1 4 4～ 97 4)× 1 0 3,且带型基本一致。 6株菌与参考菌株有完全相同的蛋白带 ,另外 5株则缺乏 40× 1 0 3带。分别用嗜肺巴氏杆菌免疫血清和自然感染嗜肺巴氏杆菌小鼠血清对 1 1株菌进行了免疫印迹 (Westernblots)试验。与免疫小鼠血清的反应显示 ,1 1株受试菌主要有相对分子质量大约 ( 1 7、31 )× 1 0 3两条反应带 ;在与自然感染血清的反应中主要的反应带在 1 7× 1 0 3处 ,缺乏 40× 1 0 3蛋白的 5株菌有 31× 1 0 3反应带 ,其余 7株均有大约 ( 2 0、2 8)× 1 0 3的反应带 ,故可以认为该菌主要抗原大约为 ( 1 7、31 )× 1 0 3;1 0个流行株根据 40× 1 0 3蛋白和 ( 2 0、2 8)× 1 0 3抗原的有无可被分为两型。本研究为精制血清学方法的诊断抗原 ,以及将SDS -PAGE和Westernblot法用于嗜肺巴氏杆菌检测奠定了基础。     

Cleavage of VP1 and modification of antigenic site 1 of type 2 polioviruses by intestinal trypsin.

We have exposed 22 independent type 2 poliovirus isolates to human intestinal fluid and purified trypsin. In all cases the virus retained its infectivity, while polyacrylamide gel electrophoresis of viral proteins showed disappearance of the VP1 bands. Concomitantly, the viruses became resistant to antigenic site 1-specific monoclonal antibodies, indicating that the cleavage took place at the antigenic site 1. Sera from persons immunized solely with the inactivated poliovirus vaccine (IPV) neutralized intact type 2 polioviruses more readily than the corresponding trypsin-cleaved virus preparations. The ratio between the neutralization indices for the intact and trypsin-cleaved type 2 polioviruses was not significantly changed by a dose of trivalent oral poliovirus vaccine given to children previously immunized with IPV. These results indicate that while the antigenic site 1 of type 2 poliovirus is immunogenic in humans when IPV is used, the relative role of this antigenic site in human immunity appears to be less critical than that in the case of type 3 polioviruses. Before we obtained these results, only antigenic site 1 had been shown to be immunogenic in type 2 polioviruses.     

Analysis of SDS-dissociated proteins of pathogenic and nonpathogenicFusarium species

SDS — dissociated proteins from eight isolates belonging to threeFusarium species were assessed and compared using polyacrylamide gel electrophoresis. Intra and inter specific variation in banding patterns were analyzed both qualitatively and quantitatively. Special emphasis was placed on variability of protein banding pattern between two nonpathogenic strains ofF. oxysporum (C5 and C14). Protein profiles from host-associated isolates were distinct, and each isolate showed a uniquely characteristic profile. Attempts were made to provide information on the genetic system controlling pathogenicity, compared to nonpathogenicity, at the protein level. The data obtained from electrophoresis support the potential use of this experimental approach to help distinguish between differentFusarium isolates.     

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.