Resilience of swine nasal microbiota to influenza A virus challenge in a longitudinal study.

Influenza A virus (IAV) is an important contributing pathogen of porcine respiratory disease complex (PRDC) infections. Evidence in humans has shown that IAV can disturb the nasal microbiota and increase host susceptibility to bacterial secondary infections. Few, small-scale studies have examined the impact of IAV infection on the swine nasal microbiota. To better understand the effects of IAV infection on the nasal microbiota and its potential indirect impacts on the respiratory health of the host, a larger, longitudinal study was undertaken to characterize the diversity and community composition of the nasal microbiota of pigs challenged with an H3N2 IAV. The microbiome of challenged pigs was compared with non-challenged animals over a 6-week period using 16S rRNA gene sequencing and analysis workflows to characterize the microbiota. Minimal changes to microbial diversity and community structure were seen between the IAV infected and control animals the first 10 days post-IAV infection. However, on days 14 and 21, the microbial populations were significantly different between the two groups. Compared to the control, there were several genera showing significant increases in abundance in the IAV group during acute infection, such as Actinobacillus and Streptococcus. The results here highlight areas for future investigation, including the implications of these changes post-infection on host susceptibility to secondary bacterial respiratory infections.

Generation and Evaluation of a Glaesserella (Haemophilus) parasuis Capsular Mutant.

Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer's disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.

Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs.

BACKGROUND: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. RESULTS: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. CONCLUSIONS: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.

Methicillin-Resistant Staphylococcus aureus Sequence Type (ST) 5 Isolates from Health Care and Agricultural Sources Adhere Equivalently to Human Keratinocytes.

Staphylococcus aureus is part of the nasal microbiome of many humans and has become a significant public health burden due to infections with antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA) strains. Several lineages of S. aureus, including MRSA, are found in livestock species and can be acquired by humans through contact with animals. These livestock-associated MRSA (LA-MRSA) isolates raise public health concerns because of the potential for livestock to act as reservoirs for MRSA outside the hospital setting. In the United States, swine harbor a mixed population of LA-MRSA isolates, with the sequence type 398 (ST398), ST9, and ST5 lineages being detected. LA-MRSA ST5 isolates are particularly concerning to the public health community because, unlike the isolates in the ST398 and ST9 lineages, isolates in the ST5 lineage are a significant cause of human disease in both the hospital and community settings globally. The ability of swine-associated LA-MRSA ST5 isolates to adhere to human keratinocytes in vitro was investigated, and the adherence genes harbored by these isolates were evaluated and compared to those in clinical MRSA ST5 isolates from humans with no swine contact. The two subsets of isolates adhered equivalently to human keratinocytes in vitro and contained an indistinguishable complement of adherence genes that possessed a high degree of sequence identity. Collectively, our data indicate that, unlike LA-MRSA ST398 isolates, LA-MRSA ST5 isolates do not exhibit a reduced genotypic or phenotypic capacity to adhere to human keratinocytes.IMPORTANCE Our data indicate that swine-associated livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 isolates are as capable of adhering to human skin and have the same genetic potential to adhere as clinical MRSA ST5 isolates from humans. This suggests that humans in contact with livestock have the potential to become colonized with LA-MRSA ST5 isolates; however, the genes that contribute to the persistence of S. aureus on human skin were absent in LA-MRSA ST5 isolates. The data presented here are important evidence in evaluating the potential risks that LA-MRSA ST5 isolates pose to humans who come into contact with livestock.

Zinc Resistance within Swine-Associated Methicillin-Resistant Staphylococcus aureus Isolates in the United States Is Associated with Multilocus Sequence Type Lineage.

Zinc resistance in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 398 (ST398) is primarily mediated by the czrC gene colocated with the mecA gene, encoding methicillin resistance, within the type V staphylococcal cassette chromosome mec (SCCmec) element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of zinc in feed as an antidiarrheal agent has the potential to contribute to the emergence and spread of methicillin-resistant S. aureus (MRSA) in swine, through increased selection pressure to maintain the SCCmec element in isolates obtained from pigs. In this study, we report the prevalence of the czrC gene and phenotypic zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and U.S. swine-associated LA-MRSA ST398 isolates. We demonstrated that the prevalence of zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact and swine-associated LA-MRSA ST398 isolates, as well as prevalences from previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively, our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance determinants.IMPORTANCE Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is more associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance markers. This information is important for public health professionals, veterinarians, producers, and consumers.

Prior infection with Bordetella bronchiseptica enhanced colonization but not disease with Streptococcus suis.

Bordetella bronchiseptica and Streptococcus suis are widely distributed swine pathogens. B. bronchiseptica is a primary pathogen and causes atrophic rhinitis and bronchopneumonia. S. suis is a contributing agent to porcine respiratory disease complex and causes systemic diseases including arthritis, meningitis, polyserositis, and septicemia. Colonization with B. bronchiseptica has been associated with increased colonization by other pathogenic bacteria and increased disease severity with viral and bacterial pathogens. It has also been reported to predispose cesarean derived, colostrum deprived (CDCD) piglets to S. suis systemic disease. Here, we evaluated the role of B. bronchiseptica colonization on S. suis colonization, dissemination, and disease in one study using conventional pigs and another using CDCD pigs. Pigs were challenged with S. suis, B. bronchiseptica, or B. bronchiseptica followed by S. suis. Incidence of S. suis disease was not increased in either study for animals pre-inoculated with B. bronchiseptica. Nasal colonization with S. suis was increased in coinfected animals, while B. bronchiseptica was similar between mono- and co-infected animals. Although increased S. suis disease was not seen in coinfected pigs, there is evidence that B. bronchiseptica can increase colonization with S. suis, which may contribute to enhanced disease when animals are stressed or immunocompromised.

Shifts in the swine nasal microbiota following Bordetella bronchiseptica challenge in a longitudinal study.

Bordetella bronchiseptica is a widespread, highly infectious bacterial pathogen that causes respiratory disease in swine and increases the severity of respiratory infections caused by other viral or bacterial pathogens. However, the impact of B. bronchiseptica infection on the swine respiratory microbiota has not been thoroughly investigated. Here, we aim to assess the influence of B. bronchiseptica infection on the community structure and abundance of members of the swine nasal microbiota. To do so, the nasal microbiota of a non-infected control group and a group infected with B. bronchiseptica (BB group) were characterized prior to B. bronchiseptica strain KM22 challenge (day 0) and on selected days in the weeks following B. bronchiseptica challenge (days 1, 3, 7, 10, 14, 21, 36, and 42). Bordetella bronchiseptica was cultured from nasal samples of the BB group to assess nasal colonization. The results showed that B. bronchiseptica colonization did not persistently affect the nasal bacterial diversity of either of the treatment groups (alpha diversity). However, the bacterial community structures (beta diversity) of the two treatment groups significantly diverged on day 7 when peak colonization levels of B. bronchiseptica were detected. This divergence continued through the last sampling time point. In addition, Pasteurella, Pasteurellaceae (unclassified), Mycoplasma, Actinobacillus, Streptococcus, Escherichia-Shigella, and Prevotellaceae (unclassified) showed increased abundances in the BB group relative to the control group at various time points. This study revealed that B. bronchiseptica colonization can disturb the upper respiratory tract microbiota, and further research is warranted to assess how these disturbances can impact susceptibility to secondary infections by other respiratory pathogens.

Bacterin Vaccination Provides Insufficient Protection Against Streptococcus equi Subspecies zooepidemicus Infection in Pigs.

Streptococcus equi subspecies zooepidemicus (SEZ) is a zoonotic pathogen capable of causing severe disease in many mammalian species. Historically, SEZ has not been a common cause of disease in pigs in North America; however, in 2019, SEZ caused mortality events leading to severe illness and 30-50% mortality in exposed animal groups. Because of the rapid progression of disease, it is important to investigate intervention strategies to prevent disease development. In this study, pigs were divided into four groups: (1) vaccinated with an inactivated SEZ vaccine generated from a highly mucoid 2019 mortality event isolate; (2) vaccinated with an inactivated SEZ vaccine generated from a genetically similar, non-mucoid isolate from a guinea pig; (3) and (4) sham vaccinated. Following boost vaccination, groups 1-3 were challenged with a 2019 mortality event isolate and group 4 were non-challenged controls. Antibody titers were higher for SEZ vaccinated animals than sham vaccinated animals; however, no anamnestic response was observed, and titers were lower than typically seen following the use of inactivated vaccines. Vaccination did not provide protection from disease development or mortality following challenge, which could be associated with the comparatively low antibody titers generated by vaccination. Surviving pigs also remained colonized and transmitted SEZ to naïve contact pigs 3 weeks following challenge, indicating that healthy animals can act as a source of SEZ exposure. Future investigation should evaluate different vaccine formulations, such as increased antigen load or an alternative adjuvant, that could induce a more robust adaptive immune response.

Replication of Streptococcus equi subspecies zooepidemicus infection in swine.

Streptococcus equi subspecies zooepidemicus (SEZ) is a commensal bacterium of horses and causes infections in mammalian species, including humans. Historically, virulent strains of SEZ caused high mortality in pigs in China and Indonesia, while disease in the U.S. was infrequent. More recently, high mortality events in sows were attributed to SEZ in North America. The SEZ isolates from these mortality events have high genetic similarity to an isolate from an outbreak in China. Taken together, this may indicate SEZ is an emerging threat to swine health. To generate a disease model and evaluate the susceptibility of healthy, conventionally raised pigs to SEZ, we challenged sows and five-month-old pigs with an isolate from a 2019 mortality event. Pigs were challenged with a genetically similar guinea pig isolate or genetically distinct horse isolate to evaluate comparative virulence. The swine isolate caused severe systemic disease in challenged pigs with 100 % mortality. Disease manifestation in sows was similar to field reports: lethargy/depression, fever, reluctance to rise, and high mortality. The guinea pig isolate also caused severe systemic disease; however, most five-month-old pigs recovered. In contrast, the horse isolate did not cause disease and was readily cleared from the respiratory tract. In conclusion, we were able to replicate disease reported in the field. The results indicate differences in virulence between isolates, with the highest virulence associated with the swine isolate. Additionally, we generated a challenge model that can be used in future research to evaluate virulence factors and disease prevention strategies.

Importance of strain selection in the generation of heterologous immunity to Glaesserella (Haemophilus) parasuis.

Glaesserella (Haemophilus) parasuis is a part of the microbiota of healthy pigs and also causes the systemic condition called Glässer's disease. G. parasuis is categorized by it capsular polysaccharide into 15 serovars. Because of the serovar and strain specific immunity generated by whole cell vaccines and the rapid onset of disease, G. parasuis has been difficult to control in the swine industry. This report investigated the protection afforded by the use of two serovar 5 isolates (Nagasaki and HS069) as whole cell, killed bacterins against homologous challenge and heterologous challenge with the serovar 1 strain 12939 to better understand bacterin generated immunity. Both bacterins induced a high antibody titer to the vaccine strain and the heterologous challenge strain. Protection was seen with both bacterins against homologous challenge; however, after heterologous challenge, the HS069 bacterin provided complete protection and all Nagasaki bacterin vaccinated animals succumbed to disease. The difference in protection appears to be due to differences in antibody specificity and the capacity of induced antibody to fix complement and opsonize G. parasuis, as shown by Western blotting and functional assays. This report shows the importance of strain selection when developing bacterin vaccines, as some strains are better able to generate heterologous protection. The difference in protection seen here can also be utilized to detect proteins of interest for subunit vaccine development.

Comparative Virulence and Genomic Analysis of Streptococcus suis Isolates.

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry. Mechanisms used by S. suis to colonize and cause disease remain unknown and vaccines and/or intervention strategies currently do not exist. Studies addressing virulence mechanisms used by S. suis have been complicated because different isolates can cause a spectrum of disease outcomes ranging from lethal systemic disease to asymptomatic carriage. The objectives of this study were to evaluate the virulence capacity of nine United States S. suis isolates following intranasal challenge in swine and then perform comparative genomic analyses to identify genomic attributes associated with swine-virulent phenotypes. No correlation was found between the capacity to cause disease in swine and the functional characteristics of genome size, serotype, sequence type (ST), or in vitro virulence-associated phenotypes. A search for orthologs found in highly virulent isolates and not found in non-virulent isolates revealed numerous predicted protein coding sequences specific to each category. While none of these predicted protein coding sequences have been previously characterized as potential virulence factors, this analysis does provide a reliable one-to-one assignment of specific genes of interest that could prove useful in future allelic replacement and/or functional genomic studies. Collectively, this report provides a framework for future allelic replacement and/or functional genomic studies investigating genetic characteristics underlying the spectrum of disease outcomes caused by S. suis isolates.

Transcriptomic differences noted in Glaesserella parasuis between growth in broth and on agar.

Glaesserella parasuis is the cause of Glӓsser's disease in pigs and is a significant contributor to post-weaning mortality in the swine industry. Prevention of G. parasuis disease relies primarily on bacterin vaccines, which have shown good homologous protection and variable heterologous protection. Bacterin production involves large scale growth of the bacteria and proteins produced during the proliferation phase of production become important antigens that stimulate the immune response. In order to evaluate genes activated during G. parasuis growth on different media substrates, the transcriptome of broth and agar grown G. parasuis strain 29755 were sequenced and compared. The transcription of most purported virulence genes were comparable between broth and agar grown G. parasuis; however, four virulence-associated genes, including ompA and vapD, had elevated expression under agar growth, while six virulence-associate genes had elevated expression during broth growth, including several protease genes. Additionally, there were metabolic shifts toward increased protein and lipid production and increased cellular division in broth grown G. parasuis. The results contribute to the understanding of how growth substrate alters gene transcription and protein expression, which may impact vaccine efficacy if immunogens important to the protective immune response are not produced under specific in vitro conditions. While the results of this work are unable to fully elucidate which growth medium presents a transcriptome more representative of in vivo samples or best suited for bacterin production, it forms a foundation that can be used for future comparisons and provides a better understanding of the metabolic differences in broth and agar grown bacteria.

Administration of granulocyte-colony stimulating factor (G-CSF) to pigs results in a longer mean survival time after exposure to Streptococcus suis.

The use of immunomodulators is a promising alternative to the use of antibiotics for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease. Previously we demonstrated a replication-defective adenovirus vector that expresses porcine granulocyte colony-stimulating factor (G-CSF) elicited a sustained neutrophilia, lasting nearly 3 weeks, which may be beneficial to prevent bacterial diseases during times of peak incidence. In a pilot study using the vectored G-CSF with a Caesarian-derived, colostrum-deprived (CDCD) pig model of Streptococcus suis disease, only 1 of 4 pigs given G-CSF developed disease, while 3 of 4 non-treated pigs developed Streptococcal disease. In a subsequent study using a larger number of pigs, although there was no difference in overall survival, there was a longer mean survival time in G-CSF treated pigs. S. suis infection is more severe in CDCD pigs than conventionally raised pigs, consequently results in the field may be superior to the ones reported in this study. Although there were positive effects from the use of G-CSF in this study, further research is needed to determine if improved clinical outcomes could be achieved under field conditions and whether the use of G-CSF in pigs to induce a sustained increase in circulating neutrophil numbers may be useful as an adjunct to antibiotics to diminish the severity of Streptococcal disease, especially during times of stress and pathogen exposure such as post-weaning.

Shifts in the nasal microbiota of swine in response to different dosing regimens of oxytetracycline administration.

The impacts of antibiotic treatment and dosing regimen of an antibiotic on the swine respiratory microbiota are poorly defined. To begin to address this, this study characterized the impact of oxytetracycline administration, given either parenterally or in feed, on the diversity of the nasal and tonsil microbiotas of post-weaned pigs over a two-week period. One group received a single intramuscular injection (IM) of oxytetracycline, the second was treated with oxytetracycline mixed in feed (IF), and the control group received non-medicated (NON) feed. Nasal samples were collected on days 0 (before start of treatment), 4, 7, 11, and 14. Tonsil tissue samples were collected from a subset of pigs selected for necropsy on days 4, 7, and 14. The results showed that the tonsil microbiota was stable regardless of antibiotic treatment. In contrast, the nasal bacterial diversity decreased for both oxytetracycline-treated groups compared to NON. The IF group also exhibited decreased diversity on more days than the IM group. The nasal bacterial community structures of the antibiotic treatment groups were significantly different from the NON group that persisted from day 4 until day 7 for the IM group, and up until day 11 for the IF group. This included relative increased abundances of Actinobacillus and Streptococcus, and relative decreased abundances of multiple commensal genera. The microbiota of the IF group was also more disturbed than the microbiota of the IM group, relative to NON. This study revealed that short-term exposure to broad-spectrum antibiotics like oxytetracycline can disturb the upper respiratory microbiota, and the dosing regimen has differential effects on the microbiota.

Antimicrobial Resistance Distribution Differs Among Methicillin Resistant Staphylococcus aureus Sequence Type (ST) 5 Isolates From Health Care and Agricultural Sources.

Antimicrobial resistance (AMR) is an expanding public health concern and methicillin resistant Staphylococcus aureus (MRSA) is a notable example. Since the discovery of livestock associated MRSA (LA-MRSA), public health concerns have arisen surrounding the potential of LA-MRSA isolates to serve as a reservoir for AMR determinants. In this study, we compare swine associated LA-MRSA ST5 and human clinical MRSA ST5 isolates for phenotypic antimicrobial susceptibilities determined via broth microdilution and genotypic determinants of AMR using whole genome sequencing and comparative genomic analysis to identify AMR elements. Swine associated LA-MRSA ST5 isolates exhibited phenotypic resistance to fewer antibiotics than clinical MRSA ST5 isolates from humans with no swine contact. Distinct genomic AMR elements were harbored by each subgroup, with little overlap in shared AMR genes between swine associated LA-MRSA ST5 and clinical MRSA ST5 isolates. Our results demonstrate that phenotypic antimicrobial susceptibilities and genotypic determinants of AMR among swine associated LA-MRSA ST5 and clinical MRSA ST5 isolates are separate and distinct.

Single Nucleotide Polymorphism Analysis Indicates Genetic Distinction and Reduced Diversity of Swine-Associated Methicillin Resistant Staphylococcus aureus (MRSA) ST5 Isolates Compared to Clinical MRSA ST5 Isolates.

Livestock associated methicillin resistant S. aureus (LA-MRSA) are lineages adapted to livestock species. LA-MRSA can be transmitted to humans and public health concerns exist because livestock may be the largest MRSA reservoir outside of hospital settings. Although the predominant European (ST398) and Asian (ST9) lineages of LA-MRSA are considered livestock adapted, North American swine also harbor ST5, a globally disseminated and highly pathogenic lineage. This study applied whole genome sequencing and single nucleotide polymorphism (SNP) typing to compare the population structure and genetic relatedness between swine associated and human clinical MRSA ST5 isolates. The established high-resolution phylogenomic framework revealed that LA-MRSA and human clinical MRSA ST5 are genetically distinct. LA-MRSA isolates were found to be clonal within farms, while greater genome diversity was observed among sampled clinical MRSA ST5. Analysis of the accessory genome demonstrated that LA-MRSA ST5 isolates and clinical MRSA ST5 isolates harbor different AMR genes and virulence factors, consistent with the SNP analysis. Collectively, our data indicate LA-MRSA and clinical MRSA ST5 isolates are distinct and the swine reservoir is likely of minimal significance as a source of clinical MRSA ST5 infections.

Draft Genome Sequences of One Methicillin-Sensitive and Seven Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Isolates Obtained in California.

Staphylococcus aureus is a commensal bacterium of humans that can cause a spectrum of diseases. An isolate's capacity to cause disease is partially attributed to the acquisition of novel mobile genetic elements. This report provides the draft genome sequence of one methicillin-susceptible and seven methicillin-resistant clinical human S. aureus isolates.

Draft Genome Sequences of 50 Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Isolates Obtained from a U.S. Hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) can be a commensal or pathogen in humans. Pathogenicity and disease are related to the acquisition of mobile genetic elements encoding virulence and antimicrobial resistance genes. Here, we report draft genome sequences for 50 clinical MRSA isolates from humans with MRSA-related disease.

Draft Genome Sequences of 14 Staphylococcus aureus Sequence Type 5 Isolates from California, USA.

Staphylococcus aureus is part of the human epithelial microbiota; however, it is also a pathogen. The acquisition of mobile genetic elements plays a role in the virulence of S. aureus isolates and contributes to treatment failures. This report details the draft genome sequences of 14 clinical S. aureus isolates.

Complete Genome Sequences of Two Staphylococcus aureus Sequence Type 5 Isolates from California, USA.

Staphylococcus aureus causes a variety of human diseases ranging in severity. The pathogenicity of S. aureus can be partially attributed to the acquisition of mobile genetic elements. In this report, we provide two complete genome sequences from human clinical S. aureus isolates.