G. Douglas Inglis

Chapter V-3 – Fungi: Hyphomycetes

Publisher Summary This chapter discusses isolation, culture, and production of order Hyphomycetes. Hyphomycetes are filamentous fungi that reproduce by conidia generally formed aerially on conidiophores arising from the substrate. Most entomopathogenic Hyphomycetes are facultative pathogens and are relatively easily grown in pure culture on defined or semidefined media. Entomopathogenic Hyphomycetes may be harvested directly from insect cadavers on which the fungus has already sporulated. Another isolation method requires the homogenization of cadavers followed by dilution plating of the homogenate on an appropriate selective medium. Selective media are frequently used for the isolation of entomopathogenic Hyphomycetes. Inhibition of contaminant fungi is more problematic than bacteria, and fungal contaminants are invariably a problem when attempting to isolate entomopathogenic Hyphomycetes from soil. It is found that although most entomopathogenic Hyphomycetes will produce blastospores in submerged culture, specific parameters need to be evaluated and adjusted for every strain studied for optimum blastospore production. An approach for bioassay of entomopathogenic fungi against fourth instar nymphs of the silverleaf whitefly is also elaborated.

Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

ABSTRACT This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at ≈104 CFU g−1, and 50 to 83% of the samples inoculated at ≈103 CFU g−1 were positive. At ≈102 CFU g−1, C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (≤2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers, Campylobacter DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers, C. jejuni and C. lanienae were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either C. coli, C. fetus, or C. hyointestinalis detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting C. lanienae (17%) but not C. jejuni. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of Campylobacter species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.

Direct quantification of Campylobacter jejuni and Campylobacter lanienae in feces of cattle by real-time quantitative PCR

ABSTRACT Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies (≈3 × 103 CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (≈250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.

Epidermal Growth Factor Inhibits Campylobacter jejuni-Induced Claudin-4 Disruption, Loss of Epithelial Barrier Function, and Escherichia coli Translocation

ABSTRACT Campylobacter jejuni is a leading cause of acute bacterial enteritis in humans. Poultry serves as a major reservoir of C. jejuni and is thought to act as a principal vehicle of transmission to humans. Epidermal growth factor (EGF) is a small amino acid peptide that exerts a broad range of activities on the intestinal epithelium. The aims of this study were to determine the effect of EGF on C. jejuni intestinal colonization in newly hatched chicks and to characterize its effects on C. jejuni-induced intestinal epithelial barrier disruption. White Leghorn chicks were treated with EGF daily, starting 1 day prior to C. jejuni infection, and were compared to control and C. jejuni-infected, EGF-treated chicks. Infected chicks shed C. jejuni in their feces throughout the study period. C. jejuni colonized the small intestine and cecum, disseminated to extraintestinal organs, and caused jejunal villus atrophy. EGF reduced jejunal colonization and dissemination of C. jejuni to the liver and spleen. In EGF-treated C. jejuni-infected chicks, villus height was not significantly different from that in untreated C. jejuni-infected chicks or controls. In vitro, C. jejuni attached to and invaded intestinal epithelial cells, disrupted tight junctional claudin-4, and increased transepithelial permeability. C. jejuni also promoted the translocation of noninvasive Escherichia coli C25. These C. jejuni-induced epithelial abnormalities were abolished by pretreatment with EGF, and the effect was dependent upon activation of the EGF receptor. These findings highlight EGFs ability to alter colonization of C. jejuni in the intestinal tract and to protect against pathogen-induced barrier defects.

Comparative genotypic and pathogenic examination of Campylobacter concisus isolates from diarrheic and non-diarrheic humans

BackgroundCampylobacter concisus is an emerging enteric pathogen, yet it is commonly isolated from feces and the oral cavities of healthy individuals. This genetically complex species is comprised of several distinct genomospecies which may vary in pathogenic potential.ResultsWe compared pathogenic and genotypic properties of C. concisus fecal isolates from diarrheic and healthy humans residing in the same geographic region. Analysis of amplified fragment length polymorphism (AFLP) profiles delineated two main clusters. Isolates assigned to AFLP cluster 1 belonged to genomospecies A (based on genomospecies-specific differences in the 23S rRNA gene) and were predominantly isolated from healthy individuals. This cluster also contained a reference oral strain. Isolates assigned to this cluster induced greater expression of epithelial IL-8 mRNA and more frequently contained genes coding for the zonnula occludins toxin and the S-layer RTX. Furthermore, isolates from healthy individuals induced greater apoptotic DNA fragmentation and increased metabolic activity than those from diarrheic individuals, and isolates assigned to genomospecies A (of which the majority were from healthy individuals) exhibited higher haemolytic activity compared to genomospecies B isolates. In contrast, AFLP cluster 2 was predominated by isolates belonging to genomospecies B and those from diarrheic individuals. Isolates from this cluster displayed greater mean epithelial invasion and translocation than cluster 1 isolates.ConclusionTwo main genetically distinct clusters (i.e., genomospecies) were identified among C. concisus fecal isolates from healthy and diarrheic individuals. Strains within these clusters differed with respect to clinical presentation and pathogenic properties, supporting the hypothesis that pathogenic potential varies between genomospecies. ALFP cluster 2 isolates were predominantly from diarrheic patients, and exhibited higher levels of epithelial invasion and translocation, consistent with known roles for these factors in diarrhoeal disease. Conversely, isolates from healthy humans and AFLP cluster 1 or genomospecies A (which were predominantly isolated from healthy humans) exhibited increased haemolytic ability, apoptotic DNA fragmentation, IL-8 induction, and/or carriage of toxin genes. Given that this cluster contains an oral reference strain, it is possible that some of the AFLP cluster 1 isolates are periodontal pathogens and may cause disease, albeit via a different mechanism than those from AFLP cluster 2.

A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.

During outbreaks of infectious animal diseases, composting may be an effective method of disposing of mortalities and potentially contaminated manure. Duplicate biosecure structures containing 16 cattle (Bos taurus) mortalities (343 kg average weight) were constructed with carcasses placed on a 40-cm straw layer and overlaid with 160 cm of feedlot manure. At a depth of 80 cm (P80), compost heated rapidly, exceeding 55 degrees C after 8 d and maintained temperatures of 55 to 65 degrees C for > 35 d. Temperatures at 160 cm (P160) failed to exceed 55 degrees C, but remained above 40 degrees C for >4 mo. To investigate rates of microbial inactivation, Escherichia coli O157:H7, Campylobacter jejuni, and Newcastle disease virus (NDV) were inoculated in manure (E. coli O157:H7 and C. jejuni approximately 10(8) CFU g(-1); NDV, approximately 10(6) EID(50) g(-1)), embedded at P80 and P160 and retrieved at intervals during composting. Escherichia coli O157:H7 and NDV were undetectable after 7 d at both depths. The C. jejuni DNA was detected up to 84 d at P80 and >147 d at P160. To estimate degradation of recalcitrant substrates, bovine brain, hoof, and rib bones were also embedded at P80 and P160 and retrieved at intervals. Residues of soft tissues remained in carcasses after opening at 147 d and bovine tissue decomposition ranked as brain > hoof > bone. More than 90% dry matter (DM) of brain disappeared after 7 d and 80% DM of hoof decomposed after 56 d. High degradation of cattle carcasses, rapid suppression of E. coli O157:H7 and NDV and reduction in viable cell densities of >6 logs for C. jejuni demonstrates that the biosecure composting system can dispose of cattle carcasses and manure in an infectious disease outbreak.

Effect of Bait Substrate and Formulation on Infection of Grasshopper Nymphs by Beauveria bassiana

The effects of two formulations (oil and water) and two bait substrates (lettuce and bran) on infection of grasshopper nymphs (Melanoplus sanguinipes) by Beauveria bassiana were investigated. More nymphs died of mycosis after they had ingested substrates inoculated with conidia in oil than in water, but there was no difference between the lettuce and bran substrates. Of the conidia recovered in frass, most (95%) were recovered within 24 h of ingestion of the lettuce and bran substrates by nymphs. Significantly more conidia averaged over time were recovered in frass from the water than from the oil formulation. A higher incidence (33-82%) and more rapid onset of mycosis was observed in nymphs that were surface-sterilized before ingestion, compared with those surface-sterilized after ingestion of lettuce and bran substrates inoculated with B. bassiana in both formulations. A similar trend was observed in nymphs receiving the sterilization treatment before, rather than after, ingestion of wheat leaves spraye...

Prolonged survival of Campylobacter species in bovine manure compost.

ABSTRACT The persistence of naturally occurring campylobacteria in aerobic compost constructed of manure from beef cattle that were administered chlortetracycline and sulfamethazine (AS700) or from cattle not administered antibiotics (control) was examined. Although there were no differences in population sizes of heterotrophic bacteria, the temperature of AS700 compost was more variable and did not become as high as that of control compost. There were significant differences in water content, total carbon (C), total nitrogen (N), and electrical conductivity but not in the C/N ratio or pH between the two compost treatments. Campylobacteria were readily isolated from pen manure, for up to day 15 from control compost, and throughout the active phase of AS700 compost. Campylobacter DNA (including Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, and Campylobacter jejuni) was detected over the ca. 10-month composting period, and no reductions in quantities of C. jejuni DNA were observed over the duration of the active phase. The utilization of centrifugation in combination with ethidium monoazide (EMA) significantly reduced (>90%) the amplification of C. jejuni DNA that did not originate from cells with intact cell membranes. No differences were observed in the frequency of Campylobacter DNA detection between EMA- and non-EMA-treated samples, suggesting that Campylobacter DNA amplified from compost was extracted from cells with intact cell membranes (i.e., from viable cells). The findings of this study indicate that campylobacteria excreted in cattle feces persist for long periods in compost and call into question the common belief that these bacteria do not persist in manure.

Genetic diversity of Metarhizium anisopliae var. anisopliae in southwestern British Columbia.

The abundance and genetic diversity of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, in southwestern British Columbia (BC) and southern Alberta was examined. The fungus was found to be widespread in soil throughout southwestern BC, and was recovered from 56% of 85 sample sites. In contrast to southwestern BC, no M. anisopliae isolates were recovered in southern Alberta. An automated fluorescent amplified fragment length polymorphism (AFLP) method was used to examine genetic diversity. In excess of 200 isolates were characterized. The method identified 211 polymorphic amplicons, ranging in size from approximately 92 to 400 base pairs, and it was found to be reproducible with a resolution limit of 86.2% similarity. The AFLP method distinguished Metarhizium from other entomopathogenic fungal genera, and demonstrated considerable genetic diversity (25 genotypes) among the reference strains of M. anisopliae isolates examined (i.e. recovered from various substrates and geographical locations). Although 13 genotypes of M. anisopliae var. anisopliae were recovered from southwestern BC soils, the vast majority of isolates (91%) belonged to one of two closely-related genotypes. Furthermore, these two genotypes predominated in urban, agricultural and forest soils. The reasons for the limited diversity of M. anisopliae var. anisopliae in southwestern BC are uncertain. However, findings of this study are consistent with island biogeography theory, and have significant implications for the development of this fungus for microbial control of pest insects.

Animal models to study acute and chronic intestinal inflammation in mammals.

Acute and chronic inflammatory diseases of the intestine impart a significant and negative impact on the health and well-being of human and non-human mammalian animals. Understanding the underlying mechanisms of inflammatory disease is mandatory to develop effective treatment and prevention strategies. As inflammatory disease etiologies are multifactorial, the use of appropriate animal models and associated metrics of disease are essential. In this regard, animal models used alone or in combination to study acute and chronic inflammatory disease of the mammalian intestine paired with commonly used inflammation-inducing agents are reviewed. This includes both chemical and biological incitants of inflammation, and both non-mammalian (i.e. nematodes, insects, and fish) and mammalian (i.e. rodents, rabbits, pigs, ruminants, dogs, and non-human primates) models of intestinal inflammation including germ-free, gnotobiotic, as well as surgical, and genetically modified animals. Importantly, chemical and biological incitants induce inflammation via a multitude of mechanisms, and intestinal inflammation and injury can vary greatly according to the incitant and animal model used, allowing studies to ascertain both long-term and short-term effects of inflammation. Thus, researchers and clinicians should be aware of the relative strengths and limitations of the various animal models used to study acute and chronic inflammatory diseases of the mammalian intestine, and the scope and relevance of outcomes achievable based on this knowledge. The ability to induce inflammation to mimic common human diseases is an important factor of a successful animal model, however other mechanisms of disease such as the amount of infective agent to induce disease, invasion mechanisms, and the effect various physiologic changes can have on inducing damage are also important features. In many cases, the use of multiple animal models in combination with both chemical and biological incitants is necessary to answer the specific question being addressed regarding intestinal disease. Some incitants can induce acute responses in certain animal models while others can be used to induce chronic responses; this review aims to illustrate the strengths and weaknesses in each animal model and to guide the choice of an appropriate acute or chronic incitant to facilitate intestinal disease.