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.

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.

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.

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.

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.

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.