Jean H. Juba

A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex

We constructed a two-locus database, comprising partial translation elongation factor (EF-1alpha) gene sequences and nearly full-length sequences of the nuclear ribosomal intergenic spacer region (IGS rDNA) for 850 isolates spanning the phylogenetic breadth of the Fusarium oxysporum species complex (FOSC). Of the 850 isolates typed, 101 EF-1alpha, 203 IGS rDNA, and 256 two-locus sequence types (STs) were differentiated. Analysis of the combined dataset suggests that two-thirds of the STs might be associated with a single host plant. This analysis also revealed that the 26 STs associated with human mycoses were genetically diverse, including several which appear to be nosocomial in origin. A congruence analysis, comparing partial EF-1alpha and IGS rDNA bootstrap consensus, identified a significant number of conflicting relationships dispersed throughout the bipartitions, suggesting that some of the IGS rDNA sequences may be non-orthologous. We also evaluated enniatin, fumonisin and moniliformin mycotoxin production in vitro within a phylogenetic framework.

Genetic Diversity of Human Pathogenic Members of the Fusarium oxysporum Complex Inferred from Multilocus DNA Sequence Data and Amplified Fragment Length Polymorphism Analyses: Evidence for the Recent Dispersion of a Geographically Widespread Clonal Lineage and Nosocomial Origin

ABSTRACT Fusarium oxysporum is a phylogenetically diverse monophyletic complex of filamentous ascomycetous fungi that are responsible for localized and disseminated life-threatening opportunistic infections in immunocompetent and severely neutropenic patients, respectively. Although members of this complex were isolated from patients during a pseudoepidemic in San Antonio, Tex., and from patients and the water system in a Houston, Tex., hospital during the 1990s, little is known about their genetic relatedness and population structure. This study was conducted to investigate the global genetic diversity and population biology of a comprehensive set of clinically important members of the F. oxysporum complex, focusing on the 33 isolates from patients at the San Antonio hospital and on strains isolated in the United States from the water systems of geographically distant hospitals in Texas, Maryland, and Washington, which were suspected as reservoirs of nosocomial fusariosis. In all, 18 environmental isolates and 88 isolates from patients spanning four continents were genotyped. The major finding of this study, based on concordant results from phylogenetic analyses of multilocus DNA sequence data and amplified fragment length polymorphisms, is that a recently dispersed, geographically widespread clonal lineage is responsible for over 70% of all clinical isolates investigated, including all of those associated with the pseudoepidemic in San Antonio. Moreover, strains of the clonal lineage recovered from patients were conclusively shown to genetically match those isolated from the hospital water systems of three U.S. hospitals, providing support for the hypothesis that hospitals may serve as a reservoir for nosocomial fusarial infections.

Widespread Occurrence of Diverse Human Pathogenic Types of the Fungus Fusarium Detected in Plumbing Drains

ABSTRACT It has been proposed that plumbing systems might serve as a significant environmental reservoir of human-pathogenic isolates of Fusarium. We tested this hypothesis by performing the first extensive multilocus sequence typing (MLST) survey of plumbing drain-associated Fusarium isolates and comparing the diversity observed to the known diversity of clinical Fusarium isolates. We sampled 471 drains, mostly in bathroom sinks, from 131 buildings in the United States using a swabbing method. We found that 66% of sinks and 80% of buildings surveyed yielded at least one Fusarium culture. A total of 297 isolates of Fusarium collected were subjected to MLST to identify the phylogenetic species and sequence types (STs) of these isolates. Our survey revealed that the six most common STs in sinks were identical to the six most frequently associated with human infections. We speculate that the most prevalent STs, by virtue of their ability to form and grow in biofilms, are well adapted to plumbing systems. Six major Fusarium STs were frequently isolated from plumbing drains within a broad geographic area and were identical to STs frequently associated with human infections.

Fusarium hostae sp.nov., a relative of F. redolens with a Gibberella teleomorph

Fusarium hostae sp. nov. causes Fusarium root and crown rot of container-grown hosta plants. Morphology, pathogenicity and molecular analyses based on sequences from portions of beta-tubulin and translation elongation factor 1-or genes indicate that this species is closely related to but distinct from F redolens Wollenw. Fusarium hostae can be distin-

Fusarium species of the Gibberella fujikuroi complex and fumonisin contamination of pearl millet and corn in Georgia, USA.

This study was designed to identify and compare the Fusarium species of the Gibberella fujikuroi complex on pearl millet (Pennisetum glaucum (L.) R. Br) and corn (Zea mays L.) crops grown in southern Georgia, and to determine their influence on potential fumonisin production. Pearl millet and corn samples were collected in Georgia in 1996, 1997 and 1998. Three percent of the pearl millet seeds had fungi similar to the Fusarium species of the G. fujikuroi species complex. One hundred and nineteen representative isolates visually similar to the G. fujikuroi species complex from pearl millet were paired with mating population A (Fusarium verticillioides (Sacc.) Nirenberg), mating population D (F. proliferatum (Matsushima) Nirenberg) and mating population F (F. thapsinum (Klittich, Leslie, Nelson and Marasas) tester strains. Successful crosses were obtained with 50.4%, 10.1% and 0.0% of these isolates with the A, D and F tester strains, while 39.5 of the isolates did not form perithecia with any tester strains. Two of the typical infertile isolates were characterized by DNA sequence comparisons and were identified as Fusarium pseudonygamai (Nirenberg and O’Donnell), which is the first known isolation of this species in the United States. Based on the pattern of cross-compatibility, conidiogenesis, colony characteristics and media pigmentation, a majority of the infertile isolates belong to this species. Fumonisins FB1 and FB2 were not detected in any of the 81 pearl millet samples analyzed. The species of the G. fujikuroi species complex were dominant in corn and were isolated from 84%, 74% and 65% of the seed in 1996, 1997 and 1998, respectively. Representative species of the G. fujikuroi species complex were isolated from 1996 to 1998 Georgia corn survey (162, 104 and 111 isolates, respectively) and tested for mating compatibility. The incidence of isolates belonging to mating population A (F. verticillioides) ranged from 70.2% to 89.5%. Corn survey samples were assayed for fumonisins, and 63% to 91% of the 1996, 1997 and 1998 samples were contaminated. The total amount of fumonisins in the corn samples ranged from 0.6 to 33.3 μg/g.

Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex

Fusisporium solani was described as the causal agent of a dry rot of potato in Germany in the mid 19th century. As Fusarium solani, the species became known as a plurivorous plant pathogen, endophyte, decomposer, and opportunistic pathogen of humans and nutritional symbiont of insects. In parallel, it became evident that the morphologically defined species F. solani represents a phylogenetically and biologically complex group of often morphologically cryptic species that has come to be known in part as the F. solani species complex (FSSC), accommodating several formae speciales and mating populations/biological species. The FSSC currently includes more than 60 phylogenetic species. Several of these have been named, but the majority remains unnamed and the identity of F. solani sensu stricto is unclear. To promote further taxonomic developments in the FSSC, lectoand epitypification is proposed for Fusisporium solani. Although no type material for F. solani is known to exist, the species was abundantly illustrated in the protologue. Thus, a relevant illustration provided by von Martius is selected as the lectotype. The epitype selected here originates from a rotting potato collected in a field in Slovenia. This strain causes a dry rot of artificially inoculated potatoes. It groups in the heretofore unnamed phylogenetic species 5, which is nested within clade 3 of the FSSC (FSSC 5). Members of this phylogenetic species have a wide geographic distribution and include soil saprotrophs and plant and opportunistic human pathogens. This typification is consistent with the original description of Fusisporium solani and the concept of F. solani as a widely distributed soil inhabitant and pathogen.

Mouldy feed, mycotoxins and Shiga toxin - producing Escherichia coli colonization associated with Jejunal Hemorrhage Syndrome in beef cattle

BackgroundBoth O157 and non-O157 Shiga toxin - producing Escherichia coli (STECs) cause serious human disease outbreaks through the consumption of contaminated foods. Cattle are considered the main reservoir but it is unclear how STECs affect mature animals. Neonatal calves are the susceptible age class for STEC infections causing severe enteritis. In an earlier study, we determined that mycotoxins and STECs were part of the disease complex for dairy cattle with Jejunal Hemorrhage Syndrome (JHS). For STECs to play a role in the development of JHS, we hypothesized that STEC colonization should also be evident in beef cattle with JHS. Aggressive medical and surgical therapies are effective for JHS, but rely on early recognition of clinical signs for optimal outcomes suggesting that novel approaches must be developed for managing this disease. The main objective of this study was to confirm that mouldy feeds, mycotoxins and STEC colonization were associated with the development of JHS in beef cattle.ResultsBeef cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium poae, F. verticillioides, F. sporotrichioides, Penicillium roqueforti and Aspergillus fumigatus. Mixtures of STECs colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood collected from the lumen of the hemorrhaged jejunum. Feed extracts containing mycotoxins were toxic to enterocytes and 0.1% of a prebiotic, Celmanax Trademark, removed the cytotoxicity in vitro. The inclusion of a prebiotic in the care program for symptomatic beef calves was associated with 69% recovery.ConclusionsThe current study confirmed that STECs and mycotoxins are part of the disease complex for JHS in beef cattle. Mycotoxigenic fungi are only relevant in that they produce the mycotoxins deposited in the feed. A prebiotic, Celmanax Trademark, acted as a mycotoxin binder in vitro and interfered with the progression of disease.

A prebiotic, Celmanax™, decreases Escherichia coli O157:H7 colonization of bovine cells and feed-associated cytotoxicity in vitro

BackgroundEscherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax™, or probiotic, Dairymans Choice™ paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.FindingsDairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillusflavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax™ removed the cytotoxicity in vitro. There was no effect of Dairymans Choice™ paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax™ removed the cytotoxicity in vitro. Celmanax™ also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairymans Choice™ paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.ConclusionThe current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax™, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.

Fusarium nelsonii and F. musarum: two new species in section Arthrosporiella related to F. camptoceras

An emended description of Fusarium camptoceras is provided to include the presence of pedicellate macroconidia produced in sporodochia and mesoconidia produced on polyblastic conidi- ogenous cells in the aerial mycelium. Two new Fu- sarium species morphologically related to F campto- ceras in Section Arthrosporiella are described and il- lustrated. Fusarium nelsonii sp. nov. was isolated mainly from plant debris in soil and some other sources in South Africa, and differs from F campto- ceras primarily in the presence of red pigment on PDA, shorter macroconidia and mesoconidia with fewer septa and in the production of characteristic terminal pairs of chlamydospores. Fusarium musarum sp. nov. was isolated from banana fruit in Central America and differs from F camptoceras primarily in the presence of red pigment on PDA, longer meso-

Aflatoxin, Fumonisin and Shiga Toxin-Producing Escherichia coli Infections in Calves and the Effectiveness of Celmanax®/Dairyman’s Choice™ Applications to Eliminate Morbidity and Mortality Losses

Mycotoxin mixtures are associated with Shiga toxin-producing Escherichia coli (STEC) infections in mature cattle. STEC are considered commensal bacteria in mature cattle suggesting that mycotoxins provide a mechanism that converts this bacterium to an opportunistic pathogen. In this study, we assessed the mycotoxin content of hemorrhaged mucosa in dairy calves during natural disease outbreaks, compared the virulence genes of the STECs, evaluated the effect of the mucosal mycotoxins on STEC toxin expression and evaluated a Celmanax®/Dairyman’s Choice™ application to alleviate disease. As for human infections, the OI-122 encoded nleB gene was common to STEC genotypes eliciting serious disease. Low levels of aflatoxin (1–3 ppb) and fumonisin (50–350 ppb) were detected in the hemorrhaged mucosa. Growth of the STECs with the mycotoxins altered the secreted protein concentration with a corresponding increase in cytotoxicity. Changes in intracellular calcium indicated that the mycotoxins increased enterotoxin and pore-forming toxin activity. A prebiotic/probiotic application eliminated the morbidity and mortality losses associated with the STEC infections. Our study demonstrates: the same STEC disease complex exists for immature and mature cattle; the significance of the OI-122 pathogenicity island to virulence; the significance of mycotoxins to STEC toxin activity; and, finally, provides further evidence that prebiotic/probiotic applications alleviate STEC shedding and mycotoxin/STEC interactions that lead to disease.