Prevalence of SARS-CoV-2 Contamination on Food Plant Surfaces as Determined by Environmental Monitoring.

ABSTRACT: The SARS-CoV-2 pandemic has presented new challenges to food manufacturers. During the early phase of the pandemic, several large outbreaks of coronavirus disease 2019 (COVID-19) occurred in food manufacturing plants resulting in deaths and economic loss, with approximately 15% of personnel diagnosed as asymptomatic for COVID-19. Spread by asymptomatic and presymptomatic individuals has been implicated in large outbreaks of COVID-19. In March 2020, we assisted in implementation of environmental monitoring programs for SARS-CoV-2 in zones 3 and 4 of 116 food production facilities. All participating facilities had already implemented measures to prevent symptomatic personnel from coming to work. During the study period, from 17 March to 3 September 2020, 1.23% of the 22,643 environmental samples tested positive for SARS-CoV-2, suggesting that infected individuals were actively shedding virus. Virus contamination was commonly found on frequently touched surfaces such as doorknobs, handles, table surfaces, and sanitizer dispensers. Most processing plants managed to control their environmental contamination when they became aware of the positive findings. Comparisons of positive test results for plant personnel and environmental surfaces in one plant revealed a close correlation. Our work illustrates that environmental monitoring for SARS-CoV-2 can be used as a surrogate for identifying the presence of asymptomatic and presymptomatic personnel in workplaces and may aid in controlling infection spread.

Comparison of ELISA and DNA Lateral Flow Assays for Detection of Pork, Horse, Beef, Chicken, Turkey, and Goat Contamination in Meat Products.

Background: Concerns about the contamination of meat products with undeclared meats and new regulations for the declaration of meat adulterants have established the need for a sensitive test to detect meat adulteration. To address this need, Microbiologique, Inc. has developed ELISA assays that can detect the presence of pork, horse, beef, chicken, turkey, and goat meat adulterants to 0.1% (w/w) and a deoxyribonucleic acid (DNA) lateral flow assay for pork, horse, beef, chicken, turkey, goat, and lamb adulterants to 0.1% (w/w). Objective: We compared the results of the DNA lateral flow assay to the ELISA assays. Methods: ELISA and DNA lateral flow assays were performed on the same spiked meat samples, prepared meats, and pet foods. Results: Both the DNA lateral flow and the ELISA assays were sensitive to 0.1% meat adulterant, and the agreement between the DNA lateral flow and ELISA assays for spiked samples, prepared meat, and pet foods was 100%. Conclusions: Based on the 100% concordance between the two assay formats, the choice between the two is dependent on whether quantitation is desired, which assay is more familiar to the particular laboratory, availability of the required equipment, and time restrictions. Highlights: The ELISA assays are less time consuming, taking about 1.5 h, compared with about 2.5 h for the DNA lateral flow assay. Because the DNA lateral flow test detects seven species in one test, it can be more cost effective when the potential adulterant is not known, while the ELISA may be better for quantification.

A Rapid, Sensitive Assay for Ralstonia solanacearum Race 3 Biovar 2 in Plant and Soil Samples Using Magnetic Beads and Real-Time PCR.

The Ralstonia solanacearum species complex causes economically significant diseases in many plant families worldwide. Although generally limited to the tropics and subtropics, strains designated race 3 biovar 2 (R3Bv2) cause disease in cooler tropical highlands and temperate regions. R3Bv2 has not become established in North America but, due to concerns that it could devastate the U.S. potato industry, it has been designated a Select Agent, and is subject to strict quarantine regulations. Quarantine screening for R3Bv2 requires rapid and robust assays applicable to small populations present in plant tissues or soil, and must distinguish R3Bv2 from the multiple other R. solanacearum subgroups. We developed a 100%-accurate real-time polymerase chain reaction (RT-PCR) assay that can detect R3Bv2 populations >1,000 cells ml-1. However, detection by RT-PCR was inhibited by compounds present in some plant and soil samples. Therefore, we developed simple immunomagnetic separation (IMS) and magnetic capture hybridization (MCH) methods to purify R. solanacearum cells or DNA from PCR inhibitors. When coupled with RT-PCR, these tools permitted detection of R3Bv2 at levels >500 cells ml-1 in stem, tuber, and soil samples when direct RT-PCR failed, and reduced detection time from days to hours. IMS-RT-PCR was usually more sensitive than MCH-RT-PCR, especially at lower population levels.

A septin from the filamentous fungus A. nidulans induces atypical pseudohyphae in the budding yeast S. cerevisiae.

BACKGROUND: Septins, novel cytoskeletal proteins, form rings at the bases of emerging round buds in yeasts and at the bases of emerging elongated hyphal initials in filamentous fungi. METHODOLOGY/PRINCIPAL FINDINGS: When introduced into the yeast Saccharomyces cerevisiae, the septin AspC from the filamentous fungus Aspergillus nidulans induced highly elongated atypical pseudohyphae and spore-producing structures similar to those of hyphal fungi. AspC induced atypical pseudohyphae when S. cerevisiae pseudohyphal or haploid invasive genes were deleted, but not when the CDC10 septin gene was deleted. AspC also induced atypical pseudohyphae when S. cerevisiae genes encoding Cdc12-interacting proteins Bem4, Cla4, Gic1 and Gic2 were deleted, but not when BNI1, a Cdc12-interacting formin gene, was deleted. AspC localized to bud and pseudohypha necks, while its S. cerevisiae ortholog, Cdc12, localized only to bud necks. CONCLUSIONS/SIGNIFICANCE: Our results suggest that AspC competes with Cdc12 for incorporation into the yeast septin scaffold and once there alters cell shape by altering interactions with the formin Bni1. That introduction of the A. nidulans septin AspC into S. cerevisiae induces a shift from formation of buds to formation of atypical pseudohyphae suggests that septins play an important role in the morphological plasticity of fungi.

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White.

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.

FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani.

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.

Cdc42 is required for proper growth and development in the fungal pathogen Colletotrichum trifolii.

Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.

Functional analysis of Ras in Colletotrichum trifolii.

Ras is a small monomeric GTP binding protein that transduces signals for growth and differentiation of eukaryotic organisms. Previously, a unique ras gene, designated Ct-ras, was cloned from the alfalfa fungal phytopathogen, Colletotrichum trifolii. Expression of Ct-Ras in mouse fibroblast cells (NIH3T3) demonstrated that Ct-ras is functionally similar to the mammalian ras genes since activating mutations of Ct-ras caused oncogenic phenotypes in nu/nu mice, including tumors. In C. trifolii, activated 'oncogenic' Ras (Val2) induced abnormal hyphal proliferation, defects in polarized growth and significantly reduced differentiation such as conidiation and appressorium formation in a nutrient dependent manner. Gene disruption of ct-ras was lethal. To further evaluate the function of Ct-Ras in C. trifolii, three different approaches were used: overexpression of cytosolic Ras by CAAX box deletion; expression of dominant negative Ct-RasT22N; and antisense ct-ras expression. Results showed that suppression of Ct-Ras activity significantly decreases fungal germination frequencies and hyphal growth rates. Taken together, these data suggest involvement of Ct-Ras in regulation of fungal cell growth and differentiation.

Proline reverses the abnormal phenotypes of Colletotrichum trifolii associated with expression of endogenous constitutively active Ras.

Colletotrichum trifolii is the causative organism of alfalfa anthracnose. We previously cloned and characterized the small prototypical G protein, Ras, of C. trifolii, which is involved in the signaling pathways that mediate interaction between the pathogen and its host. Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes. In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type. However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria. Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s). We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.