Host community diversity interacts with soil biota and water availability to predict foliar pathogen damage and diversity on tallgrass prairie plants.

The provisioning of ecosystem services in tallgrass prairies is mediated by interactions between the plants, their associated above- and belowground microbiota, and the abiotic environment, but global change alters these interactions through a variety of pathways. This study investigates the interactions between belowground microbiota, foliar pathogens, and their host plants in order to better understand how ecosystem functioning may change in a tallgrass prairie system under an altered watering regime. The project focuses on North American tallgrass prairie ecosystems and asks whether host plant diversity and abundance, soil biota, and water availability interactively affect plant susceptibility to damage by a wide range of aboveground pathogens and herbivores in a large outdoor experimental garden. Surveyed plant communities include monocultures of each of seventeen focal plant species and six-species polycultures containing one or more of the focal species. Each community is planted in either the presence or absence of live soil biota, and plant communities are watered to replicate natural soil moisture conditions in a prairie ecosystem or are subjected to drought conditions. Individual plants were assessed for foliar damage by each of eight different groups of foliar pathogens and herbivores, quantified as percent of the leaf surface damaged. Plants grown in monoculture communities experienced more foliar damage from more diverse sources than plants grown in polyculture communities, suggesting that low diversity plant communities accumulate more foliar pathogens than high diversity plant communities, and that plants in low-diversity communities are more susceptible to damage from foliar pathogens than plants in high-diversity communities. Additionally, host plant community diversity, soil community composition, and water availability interacted to influence foliar pathogen damage but not diversity of damage sources. These interactive effects suggest that the identities of the soil biota, host plants, and foliar pathogens influence the host plant’s susceptibility to foliar infection. Ultimately, our ability to manage ecosystems in a sustainable way which maintains the provisioning of valuable ecosystem services is dependent on a nuanced understanding of the underlying interactions which drive these ecosystems. This study attempts to disentangle the interactions between plants and their associated microbiota under a changing climate in order to provide insight to an understudied facet of tallgrass prairie management

First Complete Genome Sequence of a Genotype A2, Subgroup 4 Small Ruminant Lentivirus

Genetic variation in the ovine TMEM154 gene associates with susceptibility to small ruminant lentivirus (SRLV) infection. We report here the first complete genome sequence for a genotype A2, subgroup 4 SRLV isolated from a Hampshire ewe with two copies of a TMEM154 frameshift mutation predicted to abolish protein function

First Complete Genome Sequence of a Genotype A2, Subgroup 4 Small Ruminant Lentivirus

Genetic variation in the ovine TMEM154 gene associates with susceptibility to small ruminant lentivirus (SRLV) infection. We report here the first complete genome sequence for a genotype A2, subgroup 4 SRLV isolated from a Hampshire ewe with two copies of a TMEM154 frameshift mutation predicted to abolish protein function

Complete Genome Sequence of Highly Virulent Porcine Reproductive and Respiratory Syndrome Virus Variants That Recently Emerged in the United States

A recent outbreak of particularly virulent disease caused by porcine reproductive and respiratory syndrome virus has occurred in swine herds across the United States. We report here the complete genome sequence of eight viral isolates from four Nebraska herds experiencing an outbreak of severe disease in 2016

Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus

Modified-live virus (MLV) vaccines are widely used to protect pigs against porcine reproductive and respiratory syndrome virus (PRRSV). However, current MLV vaccines do not confer adequate levels of heterologous protection, presumably due to the substantial genetic diversity of PRRSV isolates circulating in the field. To overcome this genetic variation challenge, we recently generated a synthetic PRRSV strain containing a consensus genomic sequence of PRRSV-2. We demonstrated that our synthetic PRRSV strain confers unprecedented levels of heterologous protection. However, the synthetic PRRSV strain at passage 1 (hereafter designated CON-P1) is highly virulent and therefore, is not suitable to be used as a vaccine in pigs. In the present study, we attenuated CON-P1 by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Using a young pig model, we demonstrated that the synthetic virus at passages 90 and 122 (designated as CON-P90 and CON-P122, respectively) were fully attenuated, as evidenced by the significantly reduced viral loads in serum and tissues and the absence of lung lesion in the infected pigs. Most importantly, CON-P90 confers similar levels of heterologous protection as its parental strain CON-P1. Taken together, the results indicate that CON-P90 is an excellent candidate for the formulation of next generation of PRRSV MLV vaccines with improved levels of heterologous protection

Complete Genome Sequences of Two Genotype A2 Small Ruminant Lentiviruses Isolated from Infected U.S. Sheep

Two distinct subgroups of genotype A2 small ruminant lentiviruses (SRLVs) have been identified in the United States that infect sheep with specific host trans- membrane protein 154 (TMEM154) diplotypes. Here, we report the first two com- plete genome sequences of SRLV strains infecting U.S. sheep belonging to genotype A2, subgroups 1 and 2

Longitudinal study of humoral immunity to bovine coronavirus, virus shedding, and treatment for bovine respiratory disease in pre-weaned beef calves

Background: Bovine coronavirus (BCV) is associated with respiratory infections in cattle of all ages; however, a temporal study to evaluate the effect of BCV immunity on virus shedding and bovine respiratory disease (BRD) incidence in pre-weaned beef calves has not been reported. Thus, we report here a prospective study in three herds of crossbred beef calves (n = 817) with endemic BCV. Serial blood samples for measurement of serum anti-BCV antibody titers and nasal swabs for detection of BCV and other common viral and bacterial BRD pathogens were collected from all calves or subsets of calves at predetermined times from birth through weaning. The calves were monitored for BRD and those that developed signs of respiratory disease were sampled for diagnostic testing. To discover additional risk factors that could have influenced BRD development, sequence analysis of the BCV strain(s) circulating in each herd, and the prevalence of common opportunistic bacterial pathogens in the upper respiratory tract of sick and apparently healthy cattle were also evaluated. Results: Two hundred forty-eight of the 817 study calves (30.4%) were treated for BRD prior to weaning; 246 of those were from a single herd involved in two outbreaks of BRD leading to mass treatment of all calves in that group. Molecular diagnostic testing found BCV and Histophilus somni in nasal swabs taken at the time of BRD treatment. Between herd analyses revealed anti-BCV serum antibody abundance did not associate with the incidence of BRD or BCV shedding, though these measurements may have been hindered by the long periods between sample collections. Analysis of the BCV spike gene hypervariable region revealed four polymorphisms in 15 isolates from the three herds, making strain variation unlikely to account for differences in treatment rates between herds. Persistent or recurrent shedding episodes of BCV occurred in some animals treated for BRD. Conclusion: Co-detection of BCV and H. somni at the time of the disease outbreak suggests that these pathogens contributed to disease pathogenesis. Developing appropriate control measures for respiratory BCV infections may help decrease the incidence of pre-weaning BRD. The role of antibodies in protection must still be further defined

Longitudinal study of humoral immunity to bovine coronavirus, virus shedding, and treatment for bovine respiratory disease in pre-weaned beef calves

Background: Bovine coronavirus (BCV) is associated with respiratory infections in cattle of all ages; however, a temporal study to evaluate the effect of BCV immunity on virus shedding and bovine respiratory disease (BRD) incidence in pre-weaned beef calves has not been reported. Thus, we report here a prospective study in three herds of crossbred beef calves (n = 817) with endemic BCV. Serial blood samples for measurement of serum anti-BCV antibody titers and nasal swabs for detection of BCV and other common viral and bacterial BRD pathogens were collected from all calves or subsets of calves at predetermined times from birth through weaning. The calves were monitored for BRD and those that developed signs of respiratory disease were sampled for diagnostic testing. To discover additional risk factors that could have influenced BRD development, sequence analysis of the BCV strain(s) circulating in each herd, and the prevalence of common opportunistic bacterial pathogens in the upper respiratory tract of sick and apparently healthy cattle were also evaluated. Results: Two hundred forty-eight of the 817 study calves (30.4%) were treated for BRD prior to weaning; 246 of those were from a single herd involved in two outbreaks of BRD leading to mass treatment of all calves in that group. Molecular diagnostic testing found BCV and Histophilus somni in nasal swabs taken at the time of BRD treatment. Between herd analyses revealed anti-BCV serum antibody abundance did not associate with the incidence of BRD or BCV shedding, though these measurements may have been hindered by the long periods between sample collections. Analysis of the BCV spike gene hypervariable region revealed four polymorphisms in 15 isolates from the three herds, making strain variation unlikely to account for differences in treatment rates between herds. Persistent or recurrent shedding episodes of BCV occurred in some animals treated for BRD. Conclusion: Co-detection of BCV and H. somni at the time of the disease outbreak suggests that these pathogens contributed to disease pathogenesis. Developing appropriate control measures for respiratory BCV infections may help decrease the incidence of pre-weaning BRD. The role of antibodies in protection must still be further defined

Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR (n = 125) than were detected using culture alone (n = 25)

Classification of small ruminant lentivirus subtype A2, subgroups 1 and 2 based on whole genome comparisons and complex recombination patterns

Background: Small ruminant lentiviruses (SRLVs) cause a multisystemic chronic wasting disease in sheep across much of the world. SRLV subtype A2 is prevalent in North America and further classified into multiple subgroups based on variation in the group antigens gene (gag) and envelope (env) genes. In sheep, the ovine transmembrane protein 154 (TMEM154) gene is associated with SRLV susceptibility. Ewes with at least one copy of TMEM154 encoding a fulllength protein with glutamate at position 35 (E35; haplotypes 2 and 3), are highly susceptible to SRLV infection while ewes with any combination of TMEM154 haplotypes which encodes lysine (K35; haplotype 1), or truncated proteins (haplotypes 4 and 6) are several times less so. A2 subgroups 1 and 2 are associated with host TMEM154 genotypes; subgroup 1 with the K35/K35 genotype and subgroup 2 with the E35/E35 genotype. Methods: The goals of this study were to analyze sequence variation within and among SRLV subtype A2 subgroups 1 and 2 and to identify genome-scale recombination patterns. This was done using full-length assemblies of virus samples. Results: Consensus viral genomes were assembled for 23 infected sheep, including animals of assorted TMEM154 genotypes comprised of haplotypes 1, 2, or 3. Viral genome analysis identified viral subgroups 1 and 2 among the samples, and revealed additional substructure within subgroup 2 based on models predicting complex patterns of recombination between the two subgroups in several genomes. Animals with evidence of dual subgroup infection also possessed the most diverse quasi-species and the most highly recombined genomes. Conclusions: The viral subgroup framework developed to classify SRLV consensus genomes along a continuum of recombination suggests that animals with the TMEM154 E35/K35 genotype may represent a reservoir for producing viral genomes representing recombination between A2 subgroups 1 and 2