A spatiotemporal analysis of bovine brucellosis cases in Minas Gerais state, Brazil, from 2011 to 2018.

Our study explored the patterns of bovine brucellosis dissemination in Minas Gerais state, Brazil, by examining data on passive surveillance of bovine brucellosis cases from the Instituto Mineiro de Agropecuaria (IMA) (Animal Health Authority), as well as cattle population and bovine brucellosis testing, from 2011 to 2018 by means of a spatiotemporal analysis. We plotted cases, populations and testing distributions and performed spatial autocorrelation (Moran's I test) and local indicators of spatial autocorrelation (LISA) analyses. Moreover, we assessed the correlation of the spatial distribution and the compiled data (brucellosis cases, cattle populations, and brucellosis testing) by Lee's test. Our results showed that bovine brucellosis cases occurred mainly in the Triângulo Mineiro, Alto Paranaíba and Northwest regions, which reported cases in all analyzed years (2011 to 2018). The cattle population of Minas Gerais was concentrated in the same regions as bovine brucellosis cases, and the performed tests through the analyzed years (2011 to 2018). Moran's I test results of the case data showed significant spatial autocorrelation in 2011, 2015 and 2018 (p value < 0.05), and from 2011 to 2018, the population and testing data were also significant in Moran's I test (p value < 0.01). The results of cluster analysis (LISA) of cases showed clusters mainly in the Triângulo Mineiro, Alto Paranaíba, Northwest and South regions in 2011, 2015 and 2018. The local clusters for cattle populations and brucellosis testing were also observed in the same regions as bovine brucellosis cases in all years (2011 to 2018). The correlation results between clusters (Lee's test) were 0.22 (p value < 0.01) in 2011, 0.15 (p value < 0.01) in 2015 and 0.43 (p value <0.01) in 2018 between cases and populations, and 0.25 (p value <0.01) in 2011, 0.14 (p value <0.01) in 2015 and 0.38 (p value < 0.01) in 2018 for testing and cases. Therefore, our results showed that brucellosis cases were distributed together with cattle populations and brucellosis testing data, indicating that brucellosis in cattle in Minas Gerais state is being identified where there are more animals and where more tests are performed.

RT-LAMP as Diagnostic Tool for Influenza-A Virus Detection in Swine.

Point-of-care diagnostic technologies are becoming more widely available for production species. Here, we describe the application of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect the matrix (M) gene of influenza A virus in swine (IAV-S). M-specific LAMP primers were designed based on M gene sequences from IAV-S isolated in the USA between 2017 and 2020. The LAMP assay was incubated at 65 °C for 30 min, with the fluorescent signal read every 20 s. The assay's limit of detection (LOD) was 20 M gene copies for direct LAMP of the matrix gene standard, and 100 M gene copies when using spiked extraction kits. The LOD was 1000 M genes when using cell culture samples. Detection in clinical samples showed a sensitivity of 94.3% and a specificity of 94.9%. These results show that the influenza M gene RT-LAMP assay can detect the presence of IAV in research laboratory conditions. With the appropriate fluorescent reader and heat block, the assay could be quickly validated as a low-cost, rapid, IAV-S screening tool for use on farms or in clinical diagnostic labs.

Molecular characterization of emerging variants of PRRSV in the United States: new features of the -2/-1 programmed ribosomal frameshifting signal in the nsp2 region.

In this study, we characterized an emerging porcine reproductive and respiratory syndrome virus (PRRSV) isolate UIL21-0712, which is a lineage 1C variant with ORF5 restriction fragment length polymorphism (RFLP) cutting pattern of 1-4-4. The UIL21-0712 genome sequence has 85.3% nucleotide identity with the prototypic PRRSV-2 strain VR2332. The nsp2 region is the most variable, and the -2/-1 programmed ribosome frameshifting (PRF) signal therein is distinct from historical PRRSV strains. Analysis of PRRSV sequences in GenBank revealed that the majority of the emerging PRRSV variants contain substitutions that disrupt the -1 PRF stop codon to generate a nsp2N protein with a C-terminal extension. Two of the -1 PRF stop codon variant patterns were identified to be predominantly circulating in the field. They demonstrated higher growth kinetics than the other variants, suggesting that the most dominant -1 PRF stop codon variant patterns may provide enhanced growth fitness for the virus.

Active environmental surveillance of SARS-CoV-2 in Midwestern United States meatpacking plants.

This pilot project investigated environmental SARS-CoV-2 presence in seven Midwestern meatpacking plants from May 2020 to January 2021. This study investigated social distancing and infection control practices and incorporated environmental sampling of surfaces and air in employee common areas. All plants increased their social distancing efforts, increased the frequency of cleaning and disinfecting worker areas, and screened for symptomatic people to prevent entry into the workplace. 575 samples from common areas were collected and evaluated with RT-qPCR for the presence of SARS-CoV-2. 42/367 surface samples were positive, while no virus was detected in air samples. Case positive data from the counties surrounding each plant showed peak positive SARS-CoV-2 cases from 12-55 days before the virus was detected in the plant, indicating that environmental sampling is likely a lagging indicator of community and plant infection.

Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals.

To optimize the public health response to coronavirus disease 2019 (COVID-19), we must first understand the antibody response to individual proteins on the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the antibody's cross reactivity to other coronaviruses. Using a panel of 37 convalescent COVID-19 human serum samples, we showed that the magnitude and specificity of responses varied across individuals, independent of their reactivity to seasonal human coronaviruses (HCoVs). These data suggest that COVID-19 vaccines will elicit primary humoral immune responses in naïve individuals and variable responses in those previously exposed to SARS-CoV-2. Unlike the limited cross-coronavirus reactivities in humans, serum samples from 96 dogs and 10 cats showed SARS-CoV-2 protein-specific responses focused on non-S1 proteins. The correlation of this response with those to other coronaviruses suggests that the antibodies are cross-reactive and generated to endemic viruses within these hosts, which must be considered in seroepidemiologic studies. We conclude that substantial variation in antibody generation against coronavirus proteins will influence interpretations of serologic data in the clinical and veterinary settings.

Effect of Single Dose of Antimicrobial Administration at Birth on Fecal Microbiota Development and Prevalence of Antimicrobial Resistance Genes in Piglets.

Optimization of antimicrobial use in swine management systems requires full understanding of antimicrobial-induced changes on the developmental dynamics of gut microbiota and the prevalence of antimicrobial resistance genes (ARGs). The purpose of this study was to evaluate the impacts of early life antimicrobial intervention on fecal microbiota development, and prevalence of selected ARGs (ermB, tetO, tetW, tetC, sulI, sulII, and blaC TX-M) in neonatal piglets. A total of 48 litters were randomly allocated into one of six treatment groups soon after birth. Treatments were as follows: control (CONT), ceftiofur crystalline free acid (CCFA), ceftiofur hydrochloride (CHC), oxytetracycline (OTC), procaine penicillin G (PPG), and tulathromycin (TUL). Fecal swabs were collected from piglets at days 0 (prior to treatment), 5, 10, 15, and 20 post treatment. Sequencing analysis of the V3-V4 hypervariable region of the 16S rRNA gene and selected ARGs were performed using the Illumina Miseq platform. Our results showed that, while early life antimicrobial prophylaxis had no effect on individual weight gain, or mortality, it was associated with minor shifts in the composition of fecal microbiota and noticeable changes in the abundance of selected ARGs. Unifrac distance metrics revealed that the microbial communities of the piglets that received different treatments (CCFA, CHC, OTC, PPG, and TUL) did not cluster distinctly from CONT piglets. Compared to CONT group, PPG-treated piglets exhibited a significant increase in the relative abundance of ermB and tetW at day 20 of life. Tulathromycin treatment also resulted in a significant increase in the abundance of tetW at days 10 and 20, and ermB at day 20. Collectively, these results demonstrate that the shifts in fecal microbiota structure caused by perinatal antimicrobial intervention are modest and limited to particular groups of microbial taxa. However, early life PPG and TUL intervention could promote the selection of ARGs in herds. While additional investigations are required to explore the consistency of these findings across larger populations, these results could open the door to new perspectives on the utility of early life antimicrobial administration to healthy neonates in swine management systems.

Surveillance for respiratory and diarrheal pathogens at the human-pig interface in Sarawak, Malaysia.

BACKGROUND: The large livestock operations and dense human population of Southeast Asia are considered a hot-spot for emerging viruses. OBJECTIVES: To determine if the pathogens adenovirus (ADV), coronavirus (CoV), encephalomyocarditis virus (EMCV), enterovirus (EV), influenza A-D (IAV, IBV, ICV, and IDV), porcine circovirus 2 (PCV2), and porcine rotaviruses A and C (RVA and RVC), are aerosolized at the animal-interface, and if humans working in these environments are carrying these viruses in their nasal airways. STUDY: This cross-sectional study took place in Sarawak, Malaysia among 11 pig farms, 2 abattoirs, and 3 animal markets in June and July of 2017. Pig feces, pig oral secretions, bioaerosols, and worker nasal wash samples were collected and analyzed via rPCR and rRT-PCR for respiratory and diarrheal viruses. RESULTS: In all, 55 pig fecal, 49 pig oral or water, 45 bioaerosol, and 78 worker nasal wash samples were collected across 16 sites. PCV2 was detected in 21 pig fecal, 43 pig oral or water, 3 bioaerosol, and 4 worker nasal wash samples. In addition, one or more bioaerosol or pig samples were positive for EV, IAV, and RVC, and one or more worker samples were positive for ADV, CoV, IBV, and IDV. CONCLUSIONS: This study demonstrates that nucleic acids from a number of targeted viruses were present in pig oral secretions and pig fecal samples, and that several viruses were detected in bioaerosol samples or in the nasal passages of humans with occupational exposure to pigs. These results demonstrate the need for future research in strengthening viral surveillance at the human-animal interface, specifically through expanded bioaerosol sampling efforts and a seroepidemiological study of individuals with exposure to pigs in this region for PCV2 infection.

Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle.

BACKGROUND: The importance of upper airway structure in the susceptibility of the lower respiratory tract to colonization with potential pathogens is well established. With the advent of rapid, high throughput, next generation sequencing, there is a growing appreciation of the importance of commensal microbial populations in maintaining mucosal health, and a realization that bacteria colonize anatomical locations that were previously considered to be sterile. While upper respiratory tract microbial populations have been described, there are currently no published studies describing the normal microbial populations of the bovine lower respiratory tract. Consequently, we have little understanding of the relationship between upper and lower respiratory tract microbiota in healthy cattle. The primary objective of our study was to characterize the composition, structure and relationship of the lower and upper respiratory microbial communities in clinically healthy feedlot cattle. Nasopharyngeal swabs (NPS), and bronchoalveolar lavage (BAL) fluid, were collected from clinically healthy feedlot calves (n = 8). Genomic DNA from each sample was extracted, and the V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced using Illumina Miseq platform. RESULTS: Across all samples, the most predominant phyla were Proteobacteria, Actinobacteria and Firmicutes. The most common genera were Rathayibacter, Mycoplasma, Bibersteinia and Corynebacterium. The microbial community structure was distinct between these two biogeographical sites. Most of the bacterial genera identified in the BAL samples were also present in the NPS, but biogeographical-specific genera were enriched in both the NPS (Rathayibacter) and BAL (Bibersteinia) samples. There were strong associations between the presence of certain taxa at each specific location, and strong correlations between the presence of specific taxa in both the NPS and BAL samples. CONCLUSIONS: The correlation between the presence of specific taxa in both the NPS and BAL samples, supports the notion of a mutualistic interrelationship between these microbial communities. Future studies, in large cohorts of animals, are needed to determine the role and clinical importance of the relationships of respiratory tract microbial communities with health, productivity, and susceptibility to the development of respiratory disease, in growing cattle.

Influenza Virus Surveillance in Coordinated Swine Production Systems, United States.

To clarify the epidemiology of influenza A viruses in coordinated swine production systems to which no animals from outside the system are introduced, we conducted virologic surveillance during September 2012-September 2013. Animal age, geographic location, and farm type were found to affect the prevalence of these viruses.

Influenza A viruses of swine circulating in the United States during 2009-2014 are susceptible to neuraminidase inhibitors but show lineage-dependent resistance to adamantanes.

Antiviral drug susceptibility is one of the evaluation criteria of pandemic potential posed by an influenza virus. Influenza A viruses of swine (IAV-S) can play an important role in generating novel variants, yet limited information is available on the drug resistance profiles of IAV-S circulating in the U.S. Phenotypic analysis of the IAV-S isolated in the U.S. (2009-2011) (n=105) revealed normal inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir, zanamivir, and peramivir. Screening NA sequences from IAV-S collected in the U.S. (1930-2014) showed 0.03% (1/3396) sequences with clinically relevant H274Y-NA substitution. Phenotypic analysis of IAV-S isolated in the U.S. (2009-2011) confirmed amantadine resistance caused by the S31N-M2 and revealed an intermediate level of resistance caused by the I27T-M2. The majority (96.7%, 589/609) of IAV-S with the I27T-M2 in the influenza database were isolated from pigs in the U.S. The frequency of amantadine-resistant markers among IAV-S in the U.S. was high (71%), and their distribution was M-lineage dependent. All IAV-S of the Eurasian avian M lineage were amantadine-resistant and possessed either a single S31N-M2 substitution (78%, 585/747) or its combination with the V27A-M2 (22%, 162/747). The I27T-M2 substitution accounted for 43% (429/993) of amantadine resistance in classic swine M lineage. Phylogenetic analysis showed that both S31N-M2 and I27T-M2 emerged stochastically but appeared to be fixed in the U.S. IAV-S population. This study defines a drug-susceptibility profile, identifies the frequency of drug-resistant markers, and establishes a phylogenetic approach for continued antiviral-susceptibility monitoring of IAV-S in the U.S.

Temporal and spatial dynamics of porcine reproductive and respiratory syndrome virus infection in the United States.

OBJECTIVE: To measure incidence and estimate temporal and spatial dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) infection in US sow herds. ANIMALS: 371 sow herds in the United States from 14 production companies. PROCEDURES: The exponentially weighted moving average was used to monitor incident PRRSV infections for onset of an epidemic. The spatial scan statistic was used to identify areas at significantly high risk of PRRS epidemics. A χ(2) test was used to estimate whether there were significant differences in the quarterly and annual PRRS incidence among time periods, and a bivariable logistic regression model was used to estimate whether PRRSV infection during a given year increased the odds of that herd being infected in the following year. RESULTS: During the 4-year period of this study, 29% (91/319; 2009 to 2010), 33% (106/325; 2010 to 2011), 38% (135/355; 2011 to 2012), and 32% (117/371; 2012 to 2013) of the herds reported new infections. Weekly incidence was low during spring and summer and high during fall and winter. The exponentially weighted moving average signaled the onset of a PRRSV epidemic during the middle 2 weeks of October each year. Disease incidence was spatially clustered. Infection in the previous year increased the odds of infection in 2010 to 2011 and 2011 to 2012. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated a striking repeatability in annual PRRSV temporal and spatial patterns across 4 years of data among herds from 14 production companies, which suggested that efforts to control PRRSV at a regional level should continue to be supported.

Active surveillance for influenza A virus among swine, midwestern United States, 2009-2011.

Veterinary diagnostic laboratories identify and characterize influenza A viruses primarily through passive surveillance. However, additional surveillance programs are needed. To meet this need, an active surveillance program was conducted at pig farms throughout the midwestern United States. From June 2009 through December 2011, nasal swab samples were collected monthly from among 540 groups of growing pigs and tested for influenza A virus by real-time reverse transcription PCR. Of 16,170 samples, 746 were positive for influenza A virus; of these, 18.0% were subtype H1N1, 16.0% H1N2, 7.6% H3N2, and 14.5% (H1N1)pdm09. An influenza (H3N2) and (H1N1)pdm09 virus were identified simultaneously in 8 groups. This active influenza A virus surveillance program provided quality data and increased the understanding of the current situation of circulating viruses in the midwestern US pig population.

Immunologic responses and reproductive outcomes following exposure to wild-type or attenuated porcine reproductive and respiratory syndrome virus in swine under field conditions.

OBJECTIVE: To compare immunologic responses and reproductive outcomes in sows housed under field conditions following controlled exposure to a wild-type strain of porcine reproductive and respiratory syndrome virus (PRRSV strain WTV) or vaccination with a modified-live virus (MLV) vaccine. DESIGN: Randomized controlled trial. ANIMALS: 30 PRRSV-naïve 10-week-old female pigs. PROCEDURE: Humoral and cell-mediated immune responses were monitored while pigs were held in isolation for 84 days after inoculation with the WTV strain (n = 10), inoculation with the WTV strain and 42 days later vaccination with a killed-virus vaccine (10), or vaccination with an MLV vaccine (10). Reproductive outcomes were measured after pigs were released into the farm herd. RESULTS: Inoculation with the WTV strain, regardless of whether a killed-virus vaccine was subsequently administered, elicited faster and more substantial production of strain-specific neutralizing antibodies, as well as a more rapid generation of interferon-gamma secreting cells, than did vaccination with the MLV vaccine. Despite the enhanced immune responses in pigs inoculated with the WTV strain, animals vaccinated with the MLV vaccine produced a mean of 2.45 more pigs than did sows exposed to the WTV strain, mainly because of a lower rate for failure to conceive. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that current assays of immunity to PRRSV correlate only imperfectly with degree of clinical protection and that the practice of controlled exposure of sows to a circulating PRRSV strain should be reconsidered in light of negative clinical outcomes.

Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) displays notorious genetic, antigenic, and clinical variability. Little is known, however, about the nature and extent of viral variation present within naturally infected animals. By amplifying and cloning the open reading frame 5 gene from tonsils of naturally infected swine, and by sequencing individual clones, we characterized viral diversity in nine animals from two farms. All animals harbored multiple PRRSV variants at both the nucleic and the amino acid levels. Structural variation and rates of synonymous and nonsynonymous nucleotide substitution were no different within known epitopes than elsewhere. Analysis of molecular variance indicated that differences between farms, among animals within farms, and within individual animals accounted for 92.94, 3.84, and 3.22% of the total viral genetic variability observed, respectively. PRRSV exists during natural infection as a quasispecies distribution of related genotypes. Positive natural selection for immune evasiveness does not appear to maintain this diversity.