Disease ecology and host range of Cyprinid herpesvirus 3 (CyHV-3) in CyHV-3 endemic lakes of North America.

Cyprinid herpesvirus-3 (CyHV-3) is an important pathogen of common carp (Cyprinus carpio, carp) causing significant economic and ecological impacts worldwide. The recent emergence of CyHV-3 in the Upper Midwest region of the United States has raised questions related to the disease ecology and host specificity of CyHV-3 in wild carp populations. To determine the prevalence of CyHV-3 in wild populations of fishes in Minnesota, we surveyed five lakes in 2019 in which the virus was known to have caused mass mortality events in carp from 2017 to 2018. A total of 28 species (n = 756 total fish) of native fishes and 730 carp were screened for the presence of CyHV-3 DNA using specific qPCR. None of the native fish tissues tested positive for CyHV-3 although the prevalence of CyHV-3 in carp was 10%-50% in the five lakes. A single lake (Lake Elysian) with a 50% DNA detection rate and evidence of ongoing transmission and CyHV-3-associated mortality was surveyed again in 2020 from April to September. During this period, none of the tissues from 24 species (n = 607 total fish) tested positive for CyHV-3 though CyHV-3 DNA and mRNA (indicating viral replication) was detected in carp tissues during the sampling period. CyHV-3 DNA was detected most often in brain samples without evidence of replication, potentially indicating that brain tissue is a site for CyHV-3 latency. Paired qPCR and ELISA testing for Lake Elysian in 2019-2020 identified young carp (especially males) to be the primary group impacted by CyHV-3-associated mortality and acute infections, but with no positive detections in juvenile carp. Seroprevalence of carp from Lake Elysian was 57% in 2019, 92% in April of 2020 and 97% in September 2020. These results further corroborate the host specificity of CyHV-3 to carp in mixed wild populations of fishes in Minnesota and provide additional insights into the ecological niche of CyHV-3 in shallow lake populations of carp in North America.

Influence of Environmental Stressors on the Microbiota of Zebra Mussels (Dreissena polymorpha).

Host-associated microbiota play a critical role in host fitness by providing nutrition, enhancing digestion capabilities, and by providing protection from pathogens. Here, we investigated the effects of two environmental stressors, temperature, and salinity, on the microbiota associated with zebra mussels (ZMs), a highly invasive bivalve in North America. To examine this in detail, lake-collected ZMs were acclimated to laboratory conditions, and subjected to temperature and salinity stress conditions. The impact of these stressors on the diversity, composition, and dynamics of ZM-associated microbiota were assessed by using amplicon- and shotgun-based sequencing, and qPCR-based approaches. Elevated temperature was found to be the primary driver of ZM mortality, although salinity alone also increased its likelihood. Stressor-induced ZM mortality, which ranged between 53 and 100%, was concomitant with significant increases in the relative abundance of several genera of putative opportunistic pathogens including Aeromonas. These genera were only present in low relative abundance in ZMs obtained from the control tank with 0% mortality. Shotgun sequencing and qPCR analyses indicated that the relative and absolute abundances of pathogenic Aeromonas species (particularly A. veronii) were significantly greater in temperature-induced dead ZMs. Taken together, our results show that environmental stress, especially elevated temperature (> 25 °C), is associated with the rapid mortality of ZMs as well as the proliferation of putative opportunistic bacterial pathogens.

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States.

Koi herpesvirus (KHV; cyprinid herpesvirus-3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real-time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.

Do Staphylococcus epidermidis Genetic Clusters Predict Isolation Sources?

Staphylococcus epidermidis is a ubiquitous colonizer of human skin and a common cause of medical device-associated infections. The extent to which the population genetic structure of S. epidermidis distinguishes commensal from pathogenic isolates is unclear. Previously, Bayesian clustering of 437 multilocus sequence types (STs) in the international database revealed a population structure of six genetic clusters (GCs) that may reflect the species' ecology. Here, we first verified the presence of six GCs, including two (GC3 and GC5) with significant admixture, in an updated database of 578 STs. Next, a single nucleotide polymorphism (SNP) assay was developed that accurately assigned 545 (94%) of 578 STs to GCs. Finally, the hypothesis that GCs could distinguish isolation sources was tested by SNP typing and GC assignment of 154 isolates from hospital patients with bacteremia and those with blood culture contaminants and from nonhospital carriage. GC5 was isolated almost exclusively from hospital sources. GC1 and GC6 were isolated from all sources but were overrepresented in isolates from nonhospital and infection sources, respectively. GC2, GC3, and GC4 were relatively rare in this collection. No association was detected between fdh-positive isolates (GC2 and GC4) and nonhospital sources. Using a machine learning algorithm, GCs predicted hospital and nonhospital sources with 80% accuracy and predicted infection and contaminant sources with 45% accuracy, which was comparable to the results seen with a combination of five genetic markers (icaA, IS256, sesD [bhp], mecA, and arginine catabolic mobile element [ACME]). Thus, analysis of population structure with subgenomic data shows the distinction of hospital and nonhospital sources and the near-inseparability of sources within a hospital.

Susceptibility of Pimephales promelas and Carassius auratus to a strain of koi herpesvirus isolated from wild Cyprinus carpio in North America.

Cyprinid herpesvirus-3 (CyHV-3, syn. koi herpesvirus) is an important pathogen worldwide and a common cause of mass mortality events of wild common carp (Cyprinus carpio) in North America, however, reference strains and genomes obtained from wild carp are not available. Additionally, it is unclear if fishes in North America are susceptible to CyHV-3 infection due to incomplete susceptibility testing. Here we present the first North American type strain and whole-genome sequence of CyHV-3 isolated from wild carp collected from a lake with a history and recent incidence of carp mortality. Additionally, the strain was used in an in-vivo infection model to test the susceptibility of a common native minnow (Pimephales promelas) and goldfish (Carrasius auratus) which is invasive in North America. Detection of CyHV-3 DNA was confirmed in the tissues of a single fathead minnow but the same tissues were negative for CyHV-3 mRNA and samples from exposed fathead minnows were negative on cell culture. There was no detection of CyHV-3 DNA or mRNA in goldfish throughout the experiment. CyHV-3 DNA in carp tissues was reproducibly accompanied by the detection of CyHV-3 mRNA and isolation on cell culture. Additionally, environmental CyHV-3 DNA was detected on all tank filters during the study. These findings suggest that fathead minnows and goldfish are not susceptible to CyHV-3 infection and that detection of CyHV-3 DNA alone in host susceptibility trials should be interpreted with caution.

Host Range of Carp Edema Virus (CEV) during a Natural Mortality Event in a Minnesota Lake and Update of CEV Associated Mortality Events in the USA.

Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America.

Investigation of Cyprinid Herpesvirus 3 (CyHV-3) Disease Periods and Factors Influencing CyHV-3 Transmission in A Low Stocking Density Infection Trial.

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpesvirus disease (KHVD) and important pathogen of aquaculture and wild populations of common carp worldwide. Understanding the relative contributions of direct and indirect transmission of CyHV-3 as well as the factors that drive CyHV-3 transmission can clarify the importance of environmental disease vectors and is valuable for informing disease modeling efforts. To study the mechanisms and factors driving CyHV-3 transmission we conducted infection trials that determined the kinetics of KHVD and the contributions of direct and indirect forms of CyHV-3 transmission, as well as the contributions of contact rate, viral load, pathogenicity and contact type. The incubation period of KHVD was 5.88 + 1.75 days and the symptomatic period was 5.31 + 0.87 days. Direct transmission was determined to be the primary mechanism of CyHV-3 transmission (OR = 25.08, 95%CI = 10.73-99.99, p = 4.29 × 10-18) and transmission primarily occurred during the incubation period of KHVD. Direct transmission decreased in the symptomatic period of disease. Transmissibility of CyHV-3 and indirect transmission increased during the symptomatic period of disease, correlating with increased viral loads. Additionally, potential virulence-transmission tradeoffs and disease avoidance behaviors relevant to CyHV-3 transmission were identified.

Genotype and enterotoxigenicity of Staphylococcus epidermidis isolate from ready to eat meat products.

We have previously shown that potentially pathogenic isolates of Staphylococcus epidermidis occur at high incidence in ready-to-eat food. Now, within 164 samples of ready-to-eat meat products we identified 32 S. epidermidis isolates. In 8 isolates we detected the genes encoding for staphylococcal enterotoxins, but in 7 S. epidermidis isolates these genes were not stable over passages. One isolate designated 4S was shown to stably harbour sec and sel genes. In the genome sequence of S. epidermidis 4S we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis FRI909. Alignment of S. epidermidis 4S and S. epidermidis FRI909 SePIs revealed 6 nucleotide mismatches located in 5 of the total of 29 ORFs. Genomic location of S. epidermidis 4S SePI was the same as in FRI909. S. epidermidis 4S is a single locus variant of ST561, being genetically different from FRI909. SECepi was secreted by S. epidermidis 4S to BHI broth ranging from 14 to almost 36µg/mL, to milk ranging from 6 to 9ng/mL, to beef meat juice from 2 to 3µg/mL and to pork meat juice from 1 to 2µg/mL after 24 and 48h of cultivation, respectively. We provide the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to Staphylococcus aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety.

Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress.

Staphylococcus epidermidis is a leading cause of hospital-associated infections, including those of intravascular catheters, cerebrospinal fluid shunts, and orthopedic implants. Multiple biofilm matrix molecules with heterogeneous characteristics have been identified, including proteinaceous, polysaccharide, and nucleic acid factors. Two of the best-studied components in S. epidermidis include accumulation-associated protein (Aap) and polysaccharide intercellular adhesin (PIA), produced by the enzymatic products of the icaADBC operon. Biofilm composition varies by strain as well as environmental conditions, and strains producing PIA-mediated biofilms are more robust. Clinically, biofilm-mediated infections occur in a variety of anatomical sites with diverse physiological properties. To test the hypothesis that matrix composition exhibits niche specificity, biofilm-related genetic and physical properties were compared between S. epidermidis strains isolated from high-shear and low-shear environments. Among a collection of 105 clinical strains, significantly more isolates from high-shear environments carried the icaADBC operon than did those from low-shear settings (43.9% versus 22.9%, P < 0.05), while there was no significant difference in the presence of aap (77.2% versus 75.0%, P > 0.05). Additionally, a significantly greater number of high-shear isolates were capable of forming biofilm in vitro in a microtiter assay (82.5% versus 45.8%, P < 0.0001). However, even among high-shear clinical isolates, less than half contained the icaADBC locus; therefore, we selected for ica-negative variants with increased attachment to abiotic surfaces to examine PIA-independent biofilm mechanisms. Sequencing of selected variants identified substitutions capable of enhancing biofilm formation in multiple genes, further highlighting the heterogeneity of S. epidermidis biofilm molecules and mechanisms. IMPORTANCEStaphylococcus epidermidis is a leading cause of infections related to biomaterials, mostly due to their ability to form biofilm. Biofilm accumulation mechanisms vary, including those that are dependent on specific proteins, environmental DNA (eDNA), or polysaccharide intercellular adhesin (PIA). We found that those isolates obtained from high-shear environments, such as the lumen of a catheter, are more likely to produce PIA-mediated biofilms than those isolates obtained from a low-shear biomaterial-related infection. This suggests that PIA functions as a mechanism that is protective against shear flow. Finally, we performed selection experiments documenting the heterogeneity of biofilm accumulation molecules that function in the absence of PIA, further documenting the biofilm-forming potential of S. epidermidis.

Modulation of immune signaling, bacterial clearance, and corneal integrity by toll-like receptors during streptococcus pneumoniae keratitis.

PURPOSE: Bacterial keratitis, without effective antimicrobial treatment, leads to poor patient prognosis. Even after bacterial clearance, the host inflammatory response can contribute to corneal damage. Though Streptococcus pneumoniae (pneumococcus) is a common cause of bacterial keratitis, the role of host innate immunity during pneumococcal keratitis is not well characterized. This study investigated the role of Toll-like receptors (TLRs) during pneumococcal keratitis. MATERIALS AND METHODS: C57BL/6, as well as TLR2(-/-) and TLR4(-/-) mice, were infected with S. pneumoniae, and infected corneas were examined for 21 days. Quantitative real-time reverse-transcriptase polymerase chain reaction was performed using primers for genes involved in the inflammatory response and TLR signaling. Bacterial survival and leukocyte invasion were examined over a 72-h period. RESULTS: The corneal expression of TLR2, TLR4, and other inflammatory genes was increased at 72 h post-infection (p.i.) compared to uninfected C57BL/6 scratch controls. TLR2(-/-) mice showed a significant increase in bacterial survival at 24 h p.i. likely due to decreased neutrophil infiltration; however, after Day 5 p.i. observed clinical scores of TLR2(-/-) and C57BL/6 mice were not significantly different. In contrast, permanent corneal damage was observed for TLR4(-/-) mice over 21 days. Initially, both TLR(-/-) mouse strains exhibited lower expression levels in many immune genes, but returned to similar or elevated levels compared to C57BL/6 mice by 72 h p.i. CONCLUSIONS: TLR2 and TLR4 are involved in the response to pneumococcal keratitis and TLR2 may aid in bacterial clearance by recruitment of neutrophils to the cornea, whereas TLR4 may be necessary to modulate the immune response to limit cellular damage.

Moxifloxacin and cholesterol combined treatment of pneumococcal keratitis.

PURPOSE: Compare the efficacy of treatment of pneumococcal keratitis with cholesterol, moxifloxacin, or a mixture of the two (moxifloxacin/cholesterol). MATERIALS AND METHODS: New Zealand white rabbits were injected intrastromally with 10(6) colony-forming units (CFU) of a clinical keratitis strain of Streptococcus pneumoniae. Eyes were examined before and after treatment of topical drops every 2 hr from 25 to 47 hr post-infection (PI). Corneas were harvested to quantitate bacterial CFU, and myeloperoxidase (MPO) activity was measured at 48 hr PI. Eyes were extracted for histology. Minimal inhibitory concentrations (MICs) were determined for each compound. RESULTS: Eyes treated with moxifloxacin/cholesterol had a significantly lower mean slit lamp examination (SLE) score than eyes treated with phosphate-buffered saline (PBS), moxifloxacin alone, or cholesterol alone (P ≤ 0.02). A significantly lower log(10) CFU was recovered from corneas treated with moxifloxacin/cholesterol and moxifloxacin alone as compared to corneas of eyes treated with PBS or cholesterol alone (P < 0.01). At 48 hr PI, significantly lower MPO activity was quantitated from eyes treated with moxifloxacin/cholesterol as compared to eyes treated with cholesterol or moxifloxacin alone (P ≤ 0.046). Eyes treated with moxifloxacin/cholesterol had fewer immune cells and less corneal destruction than eyes from all other treatment groups. The MIC for moxifloxacin alone was 0.125 µg/mL, and cholesterol alone was unable to inhibit growth at any of the concentrations tested. The MIC for moxifloxacin when combined with 1% cholesterol was 0.0625 µg/mL. CONCLUSIONS: Treatment with a mixture of moxifloxacin and cholesterol significantly lowers the severity of infection caused by pneumococcal keratitis as compared to treatment with moxifloxacin alone, cholesterol alone, or PBS. This treatment mixture eradicates the bacteria in the cornea, unlike treatment with PBS or cholesterol alone. Using cholesterol with moxifloxacin as a treatment for bacterial keratitis could help lower the clinical severity of the infection.