Swine Pathogen Research Articles

Discovery of Brassica Yellows Virus and Porcine Reproductive and Respiratory Syndrome Virus in Diaphorina citri and Changes in Virome Due to Infection with 'Ca. L. asiaticus'.

Detection of new viruses or new virus hosts is essential for the protection of economically important agroecosystems and human health. Increasingly, metatranscriptomic data are being used to facilitate this process. Such data were obtained from adult Asian citrus psyllids (ACP) (Diaphorina citri Kuwayama) that fed solely on mandarin (Citrus ×aurantium L.) plants grafted with buds infected with 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium associated with the severe Asian variant of huanglongbing (HLB), the most destructive disease of citrus. Brassica yellows virus (BrYV), the causative agent of yellowing or leafroll symptoms in brassicaceous plants, and its associated RNA (named as BrYVaRNA) were detected in ACP. In addition, the porcine reproductive and respiratory syndrome virus (PRRSV), which affects pigs and is economically important to pig production, was also found in ACP. These viruses were not detected in insects feeding on plants grafted with CLas-free buds. Changes in the concentrations of insect-specific viruses within the psyllid were caused by coinfection with CLas. IMPORTANCE The cross transmission of pathogenic viruses between different farming systems or plant communities is a major threat to plants and animals and, potentially, human health. The use of metagenomics is an effective approach to discover viruses and vectors. Here, we collected buds from the CLas-infected and CLas-free mandarin (Citrus ×aurantium L. [Rutaceae: Aurantioideae: Aurantieae]) trees from a commercial orchard and grafted them onto CLas-free mandarin plants under laboratory conditions. Through metatranscriptome sequencing, we first identified the Asian citrus psyllids feeding on plants grafted with CLas-infected buds carried the plant pathogen, brassica yellows virus and its associated RNA, and the swine pathogen, porcine reproductive and respiratory syndrome virus. These discoveries indicate that both viruses can be transmitted by grafting and acquired by ACP from CLas+ mandarin seedlings.

Immune Molecules' mRNA Expression in Porcine Alveolar Macrophages Co-Infected with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are economically important pathogens in swine, and pigs with dual infections of PCV2 and PRRSV consistently have more severe clinical symptoms and interstitial pneumonia. However, the synergistic pathogenesis mechanism induced by PRRSV and PCV2 co-infection has not yet been illuminated. Therefore, the aim of this study was to characterize the kinetic changes of immune regulatory molecules, inflammatory factors and immune checkpoint molecules in porcine alveolar macrophages (PAMs) in individuals infected or co-infected with PRRSV and/or PCV2. The experiment was divided into six groups: a negative control group (mock, no infected virus), a group infected with PCV2 alone (PCV2), a group infected with PRRSV alone (PRRSV), a PCV2-PRRSV co-infected group (PCV2-PRRSV inoculated with PCV2, followed by PRRSV 12 h later), a PRRSV-PCV2 co-infected group (PRRSV-PCV2 inoculated with PRRSV, followed by PCV2 12 h later) and a PCV2 + PRRSV co-infected group (PCV2 + PRRSV, inoculated with PCV2 and PRRSV at the same time). Then, PAM samples from the different infection groups and the mock group were collected at 6, 12, 24, 36 and 48 h post-infection (hpi) to detect the viral loads of PCV2 and PRRSV and the relative quantification of immune regulatory molecules, inflammatory factors and immune checkpoint molecules. The results indicated that PCV2 and PRRSV co-infection, regardless of the order of infection, had no effect on promoting PCV2 replication, while PRRSV and PCV2 co-infection was able to promote PRRSV replication. The immune regulatory molecules (IFN-α and IFN-γ) were significantly down-regulated, while inflammatory factors (TNF-α, IL-1β, IL-10 and TGF-β) and immune checkpoint molecules (PD-1, LAG-3, CTLA-4 and TIM-3) were significantly up-regulated in the PRRSV and PCV2 co-infection groups, especially in PAMs with PCV2 inoculation first followed by PRRSV. The dynamic changes in the aforementioned immune molecules were associated with a high viral load, immunosuppression and cell exhaustion, which may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions by dual infection with PCV2 and PRRSV in PAMs.

Alternatives to zinc oxide in pig production.

Zinc oxide (ZnO) has been applied for many years in the production of pigs to reduce the number of diarrhoea in weaned piglets. In June 2022, the European Union banned the use of zinc oxide (ZnO) in pig feed. According to scientific reports, the may reason was the accumulation of this microelement in the environment of pig production. It has been shown that frequent application of ZnO can lead to increased antibiotic resistance in pathogenic swine microflora. The main alternatives to ZnO are probiotics, prebiotics, organic acids, essential oils, and liquid feeding systems. Alternatives to ZnO can be successfully used in pig production to reduce the number of diarrhoea among piglets during the postweaning period. Additional reports indicated that bacteriophage supplementation has a positive effect on the health of pigs. The article provides an overview of current ZnO substitutes that can be used in pig farming.

Gallocatechin Gallate Inhibits the Replication of Pseudorabies Virus via Suppressing the Entry and Release Stages in Its Replication Cycle.

The pseudorabies virus is a widespread swine pathogen that has caused significant economic losses to the global pig industry. Due to the emergence of PRV variant strains in recent years, vaccines cannot provide complete protection against the infection of PRV. Therefore, the research on antiviral compounds is of great importance for PRV treatment. In this study, an EGFP-labeled PRV was used to screen anti-PRV compounds from 86 natural product extracts. Gallocatechin gallate was found to efficiently inhibit the replication of PRV with a half-maximal inhibitory concentration (IC50) of 0.41 μM. In addition, it was found that gallocatechin gallate was unable to directly inactivate PRV and had no effect on the attachment stage of PRV. However, it was found that gallocatechin gallate significantly suppressed the viral entry stage. Furthermore, it was found that the release stage of PRV was also significantly suppressed by gallocatechin gallate. Together, this study found that gallocatechin gallate could efficiently inhibit the replication of PRV by suppressing the entry and release stages of PRV, which will contribute to the development of a new therapeutic strategy against PRV infection.

Dynamics of Serological and Mucosal Antibody Responses against African Swine Fever Viruses in Experimentally Infected Pigs

African swine fever virus (ASFV) is a lethal swine pathogen, and there is no effective vaccine or treatment available for ASFV infection. Recently, the occurrence of ASFV genotype I and genotype II natural mutants that manifest as subacute, longer-incubation, or persistent infections poses threats to preventing ASFV infection. The dynamics of antibody responses to ASFV are still completely unrevealed, especially the secretion of mucosal antibodies in oral fluid. Here, a systematic analysis was performed of serological and mucosal antibody secretion against 6 ASFV antigens after direct or indirect infection with four different ASFV strains or genotypes, namely, the field virulent genotype II isolate ASFV HLJ/18, the artificially attenuated genotype II strain HLJ/18-7GD, the naturally attenuated genotype II isolate HLJ/HRB1/20, and genotype I isolate SD/DY-I/21. Severe clinical signs of HLJ/18 infection were observed in pigs from 4 days postinoculation. However, no clinical signs were observed in HLJ/18-7GD-infected pigs. The contact pigs cohoused with the pigs intramuscularly infected with the isolate SD/DY-I/21 or HLJ/HRB1/20 only showed chronic clinical signs. Interestingly, the oral fluid sIgA responses to all the selected antigens were significantly stronger and earlier than the serum IgG responses in both HLJ/18- and HLJ/18-7GD-challenged pigs. Although significant fluctuations and individual differences appeared in oral swab sIgA responses in the contact transmission group, they were earlier than the corresponding serological IgG responses. Moreover, according to the comparative analysis of the three infection groups, P54 was proposed to be a dominant target for serological IgG diagnosis, while P30, CD2v, P54, P22, and P10 were more advantageous as mucosal sIgA diagnosis targets. These results highlight the important role of mucosal antibodies in the early diagnosis of ASFV infection and can provide references to screen appropriate targets for ASFV detection.

Microfluidic-LAMP chip for the point-of-care detection of gene-deleted and wild-type African swine fever viruses and other four swine pathogens.

Different pathogens causing mixed infection are now threatening the pig industry in the context of the African Swine Fever (ASF) circulating especially in China, and it is crucial to achieving the early diagnosis of these pathogens for disease control and prevention. Here we report the development of a rapid, portable, sensitive, high-throughput, and accurate microfluidic-LAMP chip detection system for simultaneous detection and differentiation of gene-deleted type and wild-type African swine fever virus (ASFV), pseudorabie virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRSV). The newly developed system was shown to be sensitive with detection limits of 101 copies/μl for ASFV-MGF505-2R/P72, PPV, and PCV2, 102 copies/μl for ASFV-CD2v, PRV, and PRRSV. The system was highly specific (100%) and stable (C.V.s < 5%) in its ability to detect different pathogens. A total 213 clinical samples and 15 ASFV nucleic acid samples were collected to assess the performance of the detection system, showing highly effective diagnosis. Altogether, the developed microfluidic-LAMP chip system provides a rapid, sensitive, high-throughput and portable diagnostic tool for the accurate detection of multiple swine pathogens.

Streptococcus suis outbreak caused by an emerging zoonotic strain with acquired multi-drug resistance in Thailand.

Streptococcus suis is an emerging zoonotic swine pathogen which can cause severe infections in humans. In March 2021, an outbreak of S. suis infections with 19 confirmed cases of septicemia and meningitis leading to two deaths, occurred in Nakhon Ratchasima province, Thailand. We characterized the outbreak through an epidemiological investigation combined with Illumina and Nanopore whole genome sequencing (WGS). The source of the outbreak was traced back to a raw pork dish prepared from a single pig during a Buddhist ceremony attended by 241 people. WGS analysis revealed that a single S. suis serotype 2 strain belonging to a novel sequence type (ST) of the emergent Thai zoonotic clade CC233/379, was responsible for the infections. The outbreak clone grouped together with other Thai zoonotic strains from CC233/379 and CC104 in a global S. suis phylogeny and capsule switching events between serotype 2 zoonotic strains and serotype 7 porcine strains were identified. The outbreak strain showed reduced susceptibility to penicillin corresponding with mutations in key residues in the penicillin binding proteins (PBPs). Furthermore, the outbreak strain was resistant to tetracycline, erythromycin, clindamycin, linezolid and chloramphenicol, having acquired an integrative and conjugative element (ICE) carrying resistance genes tetO and ermB, as well as a transposon from the IS1216 family carrying optrA and ermA. This investigation demonstrates that multi-drug resistant zoonotic lineages of S. suis which pose a threat to human health continue to emerge.

Efficient detection of African Swine Fever Virus using minimal equipment through a LAMP PCR method.

African swine fever virus (ASFV) currently represents the biggest threat to the porcine industry worldwide, with high economic impact and severe animal health and welfare concerns. Outbreaks have occurred in Europe and Asia since ASFV was reintroduced into the continent in 2007 and, in 2021, ASFV was detected in the Caribbean, raising alarm about the reemergence of the virus in the Americas. Given the lack of vaccines against ASFV, control of the virus relies on molecular surveillance, which can be delayed due to the need for sample shipment to specialized laboratories. Isothermal PCR techniques, such as LAMP, have become increasingly attractive as point-of-care diagnostic tools given the minimal material expense, equipment, and training required. The present study aimed to develop a LAMP assay for the detection of ASFV. Four LAMP primer sets were designed, based on a consensus sequence for the ASFV p72 gene, and were tested using a synthetic plasmid containing the cloned ASFV p72 target gene as a positive control. Two primer sets, were selected for further validation, given their very short time for amplification. Both primer sets showed thermal stability, amplifying the ASFV DNA at temperatures between 60-70°C and proved to have an analytical limit of detection as low as one ASFV-plasmid DNA copy/µL, using both fluorometric and colorimetric methods. The selected primers did not yield false positive or cross reactive results with other common swine pathogens, showing high specificity. Testing of DNA-spiked samples showed that LAMP amplification was not affected by the nature of the matrices, including oral fluids, tonsils, blood, or rectal swabs. The primer sets were able to detect the two more prevalent ASFV genotypes in the field. Taken together, the results show that ASFV-LAMP-BG2 and ASFV-LAMP-BG3 would be a useful tool for rapid, highly sensitive on-site diagnostic testing.

Systemic CD4 cytotoxic T cells improve protection against PRRSV-1 transplacental infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major swine pathogens causing reproductive failure in sows. Although modified-live virus (MLV) vaccines are available, only partial protection against heterologous strains is produced, thus vaccinated sows can be infected and cause transplacental infection. The immune effector mechanisms involved are largely unknown. The present study investigated the role of cytotoxic lymphocytes, including cytotoxic T cells (CTL), NKT, and NK cells, from blood in preventing PRRSV-1 transplacental infection in vaccinated primiparous sows (two doses vaccinated). Sows from a PRRSV-1 unstable farm were bled just before the last month of gestation (critical period for transplacental infection), then followed to determine whether sows delivered PRRSV-1-infected (n=8) or healthy (n=10) piglets. After that, functions of CTL, NKT, and NK cells in the two groups of sows were compared. No difference was found through cell surface staining. But upon in vitro re-stimulation with the circulating field virus, sows that delivered healthy piglets displayed a higher frequency of virus-specific CD107a+ IFN-γ-producing T cells, which accumulated in the CD4+ compartment including CD4 single-positive (CD4 SP) and CD4/CD8α double-positive (CD4/CD8α DP) subsets. The same group of sows also harbored a higher proportion of CD107a+ TNF-α-producing T cells that predominantly accumulated in CD4/CD8α double-negative (CD4/CD8α DN) subset. Consistently, CD4 SP and CD4/CD8α DN T cells from sows delivering healthy piglets had a higher virus-specific proliferative response. Additionally, in sows that delivered PRRSV-1-infected piglets, a positive correlation of virus-specific IFN-γ response with average Ct values of umbilical cords of newborn piglets per litter was observed. Our data strongly suggest that CTL responses correlate with protection against PRRSV-1 transplacental infection, being executed by CD4 T cells (IFN-γ related) and/or CD4/CD8α DN T cells (TNF-α related).

Antiviral activity of dandelion aqueous extract against pseudorabies virus both in vitro and in vivo.

Pseudorabies virus (PRV) is one of the most significant pathogens of swine. In recent years, the continual emergence of novel PRV variants has caused substantial economic losses in the global pig industry. PRV can infect humans leading to symptoms of acute encephalitis with implications for public health. Thus, new measures are urgently needed to prevent PRV infection. This study evaluated the anti-PRV capability of dandelion aqueous extract (DAE) in vitro and in vivo. DAE was found to inhibit the multiplication of the PRV TJ strain in PK15 cells in a concentration-dependent manner, with a 50% inhibitory concentration (IC50) of 0.2559 mg/mL and a selectivity index (SI) of 14.4. DAE inhibited the adsorption and replication stages of the PRV life cycle in vitro, and the expression of IE180, EP0, UL29, UL44, and UL52 was inhibited in the presence of DAE. In vivo experiment results of mice show that a 0.5 g/kg dose of DAE injected intraperitoneally protected 28.6% of the mice from the lethal challenge; decreased the viral load in the liver, lung, brain, heart, and kidney of PRV-infected mice; and attenuated brain damage caused by PRV infection. Furthermore, DAE could also ameliorate viral infection through regulation of the levels of cytokines (IFN-γ, TNF-α, and IL-4) in PRV-infected mouse serum. These results demonstrated that DAE exhibited potent inhibitory capability against PRV infection in vitro and in vivo; DAE is therefore expected to be a candidate TCM herb for use against PRV infection.

Interaction between Porcine Alveolar Macrophage-Tang Cells and Streptococcus suis Strains of Different Virulence: Phagocytosis and Apoptosis

Streptococcus suis is an important swine bacterial pathogen that activates macrophages to secrete inflammatory cytokines. Primary porcine alveolar macrophages (PAMs) are inconvenient to obtain, but it is unknown whether immortalized PAM-Tang cells can replace them as a better cell model for the study of the interaction between S. suis and macrophages. In this study, the phagocytic integrity, polarization, and pro-inflammatory cytokine secretion of PAM-Tang cells were confirmed by live-cell imaging, electron microscopy, confocal microscopy, and ELISA. Interestingly, the S. suis serotype 9 avirulent strain W7119 induced higher levels of adhesion and pro-inflammatory cytokines in PAM-Tang cells than the S. suis serotype 2 virulent strain 700794. Prolonged incubation with S. suis caused more cytotoxic cell damage, and the virulent strain induced higher levels of cytotoxicity to PAM-Tang cells. The virulent strain also induced higher levels of apoptosis in PAM-Tang cells, as shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. In addition, it is the first report of virulent and avirulent S. suis inducing PAM-Tang polarization towards pro-inflammatory M1 macrophages and p53- and caspase-dependent apoptosis in PAMs. Taken together, this study contributes to a better understand of interactions between macrophages and S. suis isolates of different virulence, and confirms that PAM-Tang cells provide a long-term, renewable resource for investigating macrophage infections with bacteria.

Laminin-binding protein of Streptococcus suis serotype 2 influences zinc acquisition and cytokine responses

Streptococcus suis serotype 2 is an important bacterial pathogen of swine, responsible for substantial economic losses to the swine industry worldwide. The knowledge on the pathogenesis of the infection caused by S. suis is still poorly known. It has been previously described that S. suis possesses at least one lipoprotein with double laminin and zinc (Zn)-binding properties, which was described in the literature as either laminin-binding protein (Lmb, as in the current study), lipoprotein 103, CDS 0330 or AdcAII. In the present study, the role of the Lmb in the pathogenesis of the infection caused by S. suis serotype 2 was dissected. Using isogenic mutants, results showed that Lmb does not play an important role in the laminin-binding activity of S. suis, even when clearly exposed at the bacterial surface. In addition, the presence of this lipoprotein does not influence bacterial adhesion to and invasion of porcine respiratory epithelial and brain endothelial cells and it does not increase the susceptibility of S. suis to phagocytosis. On the other hand, the Lmb was shown to play an important role as cytokine activator when tested in vitro with dendritic cells. Finally, this lipoprotein plays a critical role in Zn acquisition from the host environment allowing bacteria to grow in vivo. The significant lower virulence of the Lmb defective mutant may be related to a combination of a lower bacterial survival due to the incapacity to acquire Zn from their surrounding milieu and a reduced cytokine activation.

Multiple-probe-assisted DNA capture and amplification for high-throughput African swine fever virus detection.

African swine fever (ASF) is one of the most devastating infectious diseases affecting domestic pigs and wild boar. The grave socio-economic impact of African swine fever infection at a global level makes large-scale rapid and robust diagnosis a critical step towards effective control. Here, we describe multiple-probe-assisted DNA capture and amplification technology (MADCAT) - a novel, sensitive, simple, and high-throughput method for detecting ASFV directly from whole blood or other complex matrices. Through a unique DNA capture approach which specifically captures the target DNA onto 96-well plate for subsequent amplification, MADCAT abandons the complicated extraction protocol and achieves ultrafast and high-throughput detection. The sample-to-result time for 96 samples is about 90min, as compared with the 3-4h time of the conventional real-time qPCR method. The limit of detection (LOD) of MADCAT is 0.5 copies/μL blood and is 5 times more sensitive than an extraction-based qPCR assay when testing serially diluted whole blood samples. The assay is 100% specific against other common swine pathogens. In the clinical diagnosis of 96 field samples, all 22 positive samples were correctly identified with lower Ct values than extraction-based qPCR, confirming its high diagnostic sensitivity (100%). Owing to its high-throughput, specific high sensitivity, and direct detection features, MADCAT shows great potential for use in large-scale ASFV surveillance and monitoring for effective disease control. KEY POINTS: • No nucleic acid extraction, 100% capture efficiency, and high-throughput • Ultra-high sensitivity of 0.5 DNA copies/μL or 6 DNA copies/reaction • The sample-to-answer time for 96 samples is about 90min.

An All-in-One Concept of a Mobile System for On-Farm Swine Depopulation, Pathogen Inactivation, Off-Site Carcass Disposal, and Biosecure Cleanup

Infectious animal diseases can cause severe mortality on infected farms. An outbreak challenges the system and forces difficult decisions to stop the disease progression. We propose an ‘all-in-one’ concept of a mobile system for on-farm swine depopulation and pathogen inactivation. The system uses vaporized CO2 followed by heat treatment, broadening options for off-site carcass disposal and cleanup. A direct-fired heater supplies heat into the insulated trailer to reach and maintain the inactivation temperature for targeted pathogens. We developed a user-friendly model based on engineering principles for estimating site- and scenario-specific CO2 amounts and times required to inactivate targeted pathogens. Multipoint CO2 injection and improved distribution to animals follow the plug-flow reactor air replacement model. The model illustrates the depopulation and inactivation of two diseases, African swine fever (ASF) and the porcine reproductive and respiratory syndrome (PRRS) viruses. The model allows for dump trailer size, pig number, weights, and environmental conditions input. Model outputs provide users with practical information about the required CO2 injection rate, temperature setpoints, and times to effectively depopulate and inactivate pathogens in carcasses. The concept could be adopted for a routine or a mass depopulation/treatment/disposal with a single or fleet of ‘all-in-one’ units.

Pathogen exposure influences immune parameters around weaning in pigs reared in commercial farms.

Multiple antigenic stimulations are crucial to immune system training during earlypost-natal life. These stimulations can be either due to commensals, which accounts for the acquisition and maintenance of tolerance, or to pathogens, which triggers immunity. In pig, only few works previously explored the influence of natural exposition to pathogens upon immune competence. We propose herein the results of a multicentric, field study, conducted on 265 piglets exposed to contrasted pathogen levels in their living environment. Piglets were housed in 15 different commercial farms, sorted in two groups, low (HSLOW)- and high (HSHIGH)-health status farms, depending on their recurrent exposition to five common swine pathogens. Using animal-based measures, we compared the immune competence and growth performances of HSLOW and HSHIGH pigs around weaning. As expected, we observed a rise in the number of circulating leucocytes with age, which affected different cell populations. Monocyte, antigen-experienced and cytotoxic lymphocyte subpopulation counts were higher in piglets reared in HSLOW farms as compared to their HSHIGH homologs. Also, the age-dependent evolution in γδ T cell and neutrophil counts was significantly affected by the health status. With age, circulating IFNα level decreased and IgM level increased while being greater in HSLOW piglets at any time. After weaning, LPS-stimulated blood cells derived from HSLOW piglets were more prone to secrete IL-8 than those derived from HSHIGH pigs did. Monocytes and granulocytes issued from HSLOW pigs also exhibited comparable phagocytosis capacity. Altogether our data emphasize the more robust immunophenotype of HSLOW piglets. Finally, piglets raised under higher pathogen pressure grew less than HSHIGH piglets did and exhibited a different metabolic profile. The higher cost of the immune responses associated with the low farm health status may account for lower HSLOW piglet performances. Altogether, our data, obtained in field conditions, provide evidence that early exposure to pathogens shapes the immune competence of piglets. They also document the negative impact of an overstimulation of the immune system on piglets' growth.

Mutations in troABCD against Copper Overload in a copA Mutant of Streptococcus suis.

Streptococcus suis is a major swine pathogen that is increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role during the process of bacterial infection. In this study, RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular copper homeostasis. CopA was identified as the primary copper exporter in S. suis. The copA deletion mutant strain was found to be more sensitive to copper and accumulated more intracellular copper than the wild-type (WT) parent strain. In addition, adding manganese increased the ability of S. suis to resist copper, and the manganese transporter, TroABCD, was involved in tolerance to copper. The copA deletion mutant strain accumulated less copper when supplemented with manganese. Furthermore, when cultured with copper, the double deletion mutant (ΔcopAΔtroA) exhibited improved growth compared to the copA deletion mutant strain. In addition, the double deletion mutant (ΔcopAΔtroA) accumulated less copper than the copA deletion mutant strain. These data were consistent with a model wherein defective TroABCD resulted in decreased cellular copper accumulation and protected the strain against copper poisoning. IMPORTANCE Metal homeostasis plays a critical role during the process of bacterial infection. We identified three important potential candidate genes involved in maintenance of intracellular copper homeostasis. CopA was demonstrated to be the main copper exporter in Streptococcus suis, and manganese increased the tolerance of S. suis to copper. The double deletion mutant (ΔcopAΔtroA) improved growth ability over the copA deletion mutant strain in the presence of high concentrations of copper and accumulated less copper. These findings are consistent with a model wherein defective TroABCD resulted in decreased cellular accumulation of copper and protected the strain against copper poisoning.

Retrospective assessment of porcine circovirus 3 (PCV-3) in formalin-fixed, paraffin-embedded tissues from pigs affected by different clinical-pathological conditions

BackgroundPorcine circovirus 3 (PCV-3) is a recently discovered pathogen of swine that has been associated with several conditions. However, many questions remain unanswered regarding its infection, especially in terms of pathogenesis and disease impact. The aim of the present study was to retrospectively investigate the presence of PCV-3 genome by real time quantitative PCR (qPCR) and in situ hybridization (ISH) on selected formalin-fixed paraffin-embedded tissues of pigs affected by different clinical conditions and histological lesions.Materials and methodsConditions investigated included porcine dermatitis and nephropathy syndrome (PDNS), periweaning failure-to-thrive syndrome (PFTS), congenital tremors type AII, reproductive disorders, and pigs affected by systemic periarteritis/arteritis, myocarditis, or encephalitis. Studied cases (n = 587) were investigated from a diagnostic database (n = 4162) that comprised samples collected within the period 1998–2021. From each condition/lesion, 10 to 12 cases were subsequently selected and tested by qPCR and ISH (72 cases total).ResultsA total of 587 cases fulfilled inclusion criteria of the different studied conditions and were distributed among the seven groups. For the further selected cases, PCV-3 genome was found by qPCR in 12/12 periarteritis, 5/10 reproductive disease, 5/10 PFTS, 3/10 myocarditis, 1/10 encephalitis and 1/10 congenital tremor cases. PCV-3 was not found in any of the PDNS cases assessed. In periarteritis cases, tissues more commonly affected were mesenteric arteries and kidney. Reproductive disease cases associated to PCV-3 genome consistently displayed myocarditis. The lesions and labelling distribution of PFTS cases with presence of PCV-3 genome were comparable to those of the periarteritis group. qPCR and ISH yielded similar results within each studied case and were statistically comparable.ConclusionOur results suggest that periarteritis is the hallmark lesion of PCV-3-SD, and that mesenteric lymph node and kidney appeared to be the most reliable organs to confirm the presence of PCV-3 genome in cases with periarteritis.

Porcine respiratory disease complex: Dynamics of polymicrobial infections and management strategies after the introduction of the African swine fever.

A few decades ago, porcine respiratory disease complex (PRDC) exerted a major economic impact on the global swine industry, particularly due to the adoption of intensive farming by the latter during the 1980's. Since then, the emerging of porcine reproductive and respiratory syndrome virus (PRRSV) and of porcine circovirus type 2 (PCV2) as major immunosuppressive viruses led to an interaction with other endemic pathogens (e.g., Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Streptococcus suis, etc.) in swine farms, thereby exacerbating the endemic clinical diseases. We herein, review and discuss various dynamic polymicrobial infections among selected swine pathogens. Traditional biosecurity management strategies through multisite production, parity segregation, batch production, the adoption of all-in all-out production systems, specific vaccination and medication protocols for the prevention and control (or even eradication) of swine diseases are also recommended. After the introduction of the African swine fever (ASF), particularly in Asian countries, new normal management strategies minimizing pig contact by employing automatic feeding systems, artificial intelligence, and robotic farming and reducing the numbers of vaccines are suggested. Re-emergence of existing swine pathogens such as PRRSV or PCV2, or elimination of some pathogens may occur after the ASF-induced depopulation. ASF-associated repopulating strategies are, therefore, essential for the establishment of food security. The "repopulate swine farm" policy and the strict biosecurity management (without the use of ASF vaccines) are, herein, discussed for the sustainable management of small-to-medium pig farms, as these happen to be the most potential sources of an ASF re-occurrence. Finally, the ASF disruption has caused the swine industry to rapidly transform itself. Artificial intelligence and smart farming have gained tremendous attention as promising tools capable of resolving challenges in intensive swine farming and enhancing the farms' productivity and efficiency without compromising the strict biosecurity required during the ongoing ASF era.

A modular and self-adjuvanted multivalent vaccine platform based on porcine circovirus virus-like nanoparticles

BackgroundVirus-like particles (VLPs) are supramolecular structures composed of multiple protein subunits and resemble natural virus particles in structure and size, making them highly immunogenic materials for the development of next-generation subunit vaccines. The orderly and repetitive display of antigenic epitopes on particle surface allows efficient recognition and cross-link by B cell receptors (BCRs), thereby inducing higher levels of neutralizing antibodies and cellular immune responses than regular subunit vaccines. Here, we present a novel multiple antigen delivery system using SpyCatcher/Spytag strategy and self-assembled VLPs formed by porcine circovirus type 2 (PCV2) Cap, a widely used swine vaccine in solo.ResultsCap-SC, recombinant Cap with a truncated SpyCatcher polypeptide at its C-terminal, self-assembled into 26-nm VLPs. Based on isopeptide bonds formed between SpyCatcher and SpyTag, classical swine fever virus (CSFV) E2, the antigen of interest, was linked to SpyTag and readily surface-displayed on SpyCatcher decorated Cap-SC via in vitro covalent conjugation. E2-conjugated Cap VLPs (Cap-E2 NPs) could be preferentially captured by antigen presenting cells (APCs) and effectively stimulate APC maturation and cytokine production. In vivo studies confirmed that Cap-E2 NPs elicited an enhanced E2 specific IgG response, which was significantly higher than soluble E2, or the admixture of Cap VLPs and E2. Moreover, E2 displayed on the surface did not mask the immunodominant epitopes of Cap-SC VLPs, and Cap-E2 NPs induced Cap-specific antibody levels and neutralizing antibody levels comparable to native Cap VLPs.ConclusionThese results demonstrate that this modularly assembled Cap-E2 NPs retains the immune potential of Cap VLP backbone, while the surface-displayed antigen significantly elevated E2-induced immune potency. This immune strategy provides distinctly improved efficacy than conventional vaccine combination. It can be further applied to the development of dual or multiple nanoparticle vaccines to prevent co-infection of PCV2 and other swine pathogens.

Comparative virulence and antimicrobial resistance distribution of Streptococcus suis isolates obtained from the United States.

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry worldwide. Most S. suis genome sequences available in public databases are from isolates obtained outside the United States. We sequenced the genomes of 106 S. suis isolates from the U.S. and analyzed them to identify their potential to function as zoonotic agents and/or reservoirs for antimicrobial resistance (AMR) dissemination. The objective of this study was to evaluate the genetic diversity of S. suis isolates obtained within the U.S., for the purpose of screening for genomic elements encoding AMR and any factors that could increase or contribute to the capacity of S. suis to transmit, colonize, and/or cause disease in humans. Forty-six sequence types (STs) were identified with ST28 observed as the most prevalent, followed by ST87. Of the 23 different serotypes identified, serotype 2 was the most prevalent, followed by serotype 8 and 3. Of the virulence genes analyzed, the highest nucleotide diversity was observed in sadP, mrp, and ofs. Tetracycline resistance was the most prevalent phenotypic antimicrobial resistance observed followed by macrolide-lincosamide-streptogramin B (MLSB) resistance. Numerous AMR elements were identified, many located within MGE sequences, with the highest frequency observed for ble, tetO and ermB. No genes encoding factors known to contribute to the transmission, colonization, and/or causation of disease in humans were identified in any of the S. suis genomes in this study. This includes the 89 K pathogenicity island carried by the virulent S. suis isolates responsible for human infections. Collectively, the data reported here provide a comprehensive evaluation of the genetic diversity among U.S. S. suis isolates. This study also serves as a baseline for determining any potential risks associated with occupational exposure to these bacteria, while also providing data needed to address public health concerns.