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Current knowledge on porcine circovirus diseases (PCVD) caused by Porcine circovirus 2 (PCV-2) includes the subclinical infection (PCV-2-SI), systemic (PCV-2-SD) and reproductive (PCV-2-RD) diseases, and porcine dermatitis and nephropathy syndrome (PDNS). Criteria to establish the diagnosis of these conditions have not changed over the years; thus, the triad composed by clinical signs, lesions and viral detection in lesions are still the hallmark for PCV-2-SD and PCV-2-RD. In contrast, PCV-2-SI diagnosis is not usually performed since this condition is perceived to be controlled by default through vaccination. PDNS is diagnosed by gross and histopathological findings, and PCV-2 detection is not recognized as a diagnostic criterion. Molecular biology methods as a proxy for PCVD diagnoses have been extensively used in the last decade, although these techniques should be mainly considered as monitoring tools rather than diagnostic ones. What has changed over the years is the epidemiological picture of PCV-2 through the massive use of vaccination, which allowed the decrease in infectious pressure paralleled with a decrease in overall herd immunity. Consequently, the need for establishing the diagnosis of PCVD has increased lately, especially in cases with a PCV-2-SD-like condition despite vaccination. Therefore, the objective of the present review is to update the current knowledge on diagnostic criteria for PCVDs and to contextualize the interest of using molecular biology methods in the overall picture of these diseases within variable epidemiological scenarios of PCV-2 infection.

Glaesserella parasuis is the etiological agent of Glässer’s disease, a common pathology in the pork industry with higher prevalence in the postweaning period. Vaccination is one of the strategies to control this disease. Here, we investigated the effect that sow vaccination against virulent strains of G. parasuis had in the nasal microbiota of their offspring. Nasal swabs from fifteen days-old piglets from vaccinated (vs-P, n = 11) and unvaccinated sows (cs-P, n = 11) were obtained and DNA was extracted for 16S amplicon sequencing. Microbiota composition was different, with lower diversity in vs-P, and a strong clustering of the groups in beta diversity analysis. Among the 1509 sequences associated to either study group, all the sequences classified as G. parasuis (10 ASVs) had lower relative abundance in the vs-P group. A list of 32 inferred metabolic pathways were statistically different between groups. A distinctive structure of the two microbial networks was detected, with modules in the cs-P not conserved in the vs-P network. In conclusion, vaccination of the sows had a large effect in the microbiota composition of their offspring that went beyond the effect on the targeted pathogen. The mechanisms underneath these changes may include alteration of the microbiota network due to the elimination of the targeted pathogen and/or immunological changes.

Porcine respiratory disease is one of the most important health problems in pig production worldwide. Cranioventral pulmonary consolidation (CVPC) and pleurisy are the two most common lesions in the respiratory tract of slaughtered pigs. The present review paper discusses pathogens involved in the lesions, lesion prevalence, scoring systems, advantages and disadvantages of slaughterhouse examination, and the impact of CVPC and pleurisy on performance, carcass, and meat quality. Cranioventral pulmonary consolidation and pleurisy in slaughter pigs are characteristic for infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae , respectively, although other pathogens may cause similar lesions and/or be involved in their development. The overall prevalence of CVPC and pleurisy in slaughter pigs are still high, being the prevalence of CVPC generally higher than that of chronic pleurisy. The advantages and disadvantages of slaughterhouse examination are discussed in relation to practical aspects, the assessment of lesions, the number and representativeness of the examined animals and the interpretation and value of the results for the stakeholders. The main scoring methods for CVPC and pleurisy are shortly reviewed. In general, scoring methods can be applied rapidly and easily, although significant variation due to abattoir and observer remains. Artificial intelligence-based technologies that automatically score lesions and facilitate processing of data may aid solving these problems. Cranioventral pulmonary consolidation and pleurisy have a major negative impact on pig performance, and the effects increase the extension of the lesions and/or presence of multiple lesions. The performance losses caused by these lesions, however, vary significantly between studies and farms, possibly due to differences in study population and used methodology. Both lesions also have a negative impact on different carcass and meat quality parameters, leading to increased risk for poor processing and storage of the carcasses. Monitoring lung lesions of slaughter pigs should be optimized and implemented routinely; however, it is recommended to complement this information with farm data and laboratory results for specific pathogens.

Porcine circovirus 2 (PCV2) is a single stranded DNA virus with one of the highest mutation rates among DNA viruses. This ability allows it to generate a cloud of mutants constantly providing new opportunities to adapt and evade the immune system. This pig pathogen is associated to many diseases, globally called porcine circovirus diseases (PCVD) and has been a threat to pig industry since its discovery in the early 90’s. Although 11 ORFs have been predicted from its genome, only two main proteins have been deeply characterized, i.e. Rep and Cap. The structural Cap protein possesses the majority of the epitopic determinants of this non-enveloped virus. The evolution of PCV2 is affected by both natural and vaccine-induced immune responses, which enhances the genetic variability, especially in the most immunogenic Cap region. Intra-host variability has been also demonstrated in infected animals where long-lasting infections can take place. However, the association between this intra-host variability and pathogenesis has never been studied for this virus. Here, the within-host PCV2 variability was monitored over time by next generation sequencing during an experimental infection, demonstrating the presence of large heterogeneity. Remarkably, the level of quasispecies diversity, affecting particularly the Cap coding region, was statistically different depending on viremia levels and clinical signs detected after infection. Moreover, we proved the existence of hyper mutant subjects harboring a remarkably higher number of genetic variants. Altogether, these results suggest an interaction between genetic diversity, host immune system and disease severity.

Porcine dermatitis and nephropathy syndrome (PDNS) is a severe condition that affects mainly growing pigs and is considered to be caused by a type III hypersensitivity reaction. Although porcine circovirus 2 (PCV-2) is the antigen linked to this condition, porcine circovirus 3 (PCV-3) has also been proposed to be causally associated with PDNS. Moreover, the initial description of porcine circovirus 4 (PCV-4) also related this novel agent to this clinicopathological entity. Therefore, this retrospective study included a large number of PDNS cases ( n  = 102) fulfilling specific histologic criteria in search of known porcine circoviruses (PCV-1 to PCV-4) by conventional and/or quantitative PCR (qPCR). All the samples were subjected to PCV-2 immunohistochemistry (IHC) or conventional in situ hybridization (C-ISH), and RNAscope ® (R-ISH) was used to study PCV-2 and PCV-3 localization in a subset of the samples. All PDNS cases were PCV-2 positive by qPCR, while 30 of them (29.4%) yielded PCV-3 qPCR positivity; PCV-2 viral loads were significantly greater than PCV-3 viral loads. All animals were negative for PCV-1 and PCV-4. By C-ISH/IHC, 63 cases (61.8%) were positive for PCV-2, with low to moderate amounts of antigen. R-ISH demonstrated higher sensitivity, as all studied cases were positive; however, neither PCV-2 nor PCV-3 were consistently found within characteristic PDNS lesions. These results indicate that all PDNS-affected pigs were infected with PCV-2, emphasizing the likelihood that this viral antigen is causally linked to this condition. In contrast, no evidence of the association of PCV-1, PCV-3 or PCV-4 with PDNS was found.

Vaccination stands as one of the most sustainable and promising strategies to control infectious diseases in animal production. Nevertheless, the causes for antibody response variation among individuals are poorly understood. The animal microbiota has been shown to be involved in the correct development and function of the host immunity, including the antibody response. Here, we studied the nasal and rectal microbiota composition in association with the antibody response against the pathobiont Glaesserella parasuis . The nasal and rectal microbiotas of 24 piglets were sampled in two farms before vaccination and in one unvaccinated farm (naturally exposed to the pathobiont) at similar time. Microbiota composition was inferred by V3V4 16S rRNA gene sequencing and bioinformatics analysis, and the antibody response was quantified using the variation between the levels before and after vaccination (normalized per farm). Piglets with higher antibody responses showed more diverse nasal and rectal microbial communities compared to piglets with lower responses. Moreover, swine nasal core microbiota colonizers were associated with higher antibody levels, such as several members from Bacteroidales and Clostridiales orders and genera including Moraxella , Staphylococcus , Fusobacterium and Neisseria . Regarding taxa found in the rectal microbiota, associations with antibody responses were detected only at order level, pointing towards a positive role for Clostridiales while negative for Enterobacteriales . Altogether, these results suggest that the microbiota is associated with the antibody response to G. parasuis (and probably to other pathogens) and serves as starting point to understand the factors that contribute to immunization in pigs.

Porcine circovirus 3 (PCV-3) is a recently described virus belonging to the family Circoviridae . It represents the third member of genus Circovirus able to infect swine, together with PCV-1, considered non-pathogenic, and PCV-2, one of the most economically relevant viruses for the swine worldwide industry. PCV-3 was originally found by metagenomics analyses in 2015 in tissues of pigs suffering from porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis and multisystemic inflammation. The lack of other common pathogens as potential infectious agents of these conditions prompted the suspicion that PCV-3 might etiologically be involved in disease occurrence. Subsequently, viral genome was detected in apparently healthy pigs, and retrospective studies indicated that PCV-3 was already present in pigs by early 1990s. In fact, current evidence suggests that PCV-3 is a rather widespread virus worldwide. Recently, the virus DNA has also been found in wild boar, expanding the scope of infection susceptibility among the Suidae family; also, the potential reservoir role of this species for the domestic pig has been proposed. Phylogenetic studies with available PCV-3 partial and complete sequences from around the world have revealed high nucleotide identity (>96%), although two main groups and several subclusters have been described as well. Moreover, it has been proposed the existence of a most common ancestor dated around 50 years ago. Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years. Therefore, the present review will update the current knowledge and future trends of research on PCV-3.

Porcine circovirus 3 (PCV-3) has been widely detected in healthy and diseased pigs; among different pathologic conditions, the strongest evidence of association comes from reproductive disease cases. However, simple viral detection does not imply the causality of the clinical conditions. Detection of PCV-3 within lesions may provide stronger evidence of causality. Thus, this study aimed to assess the frequency of PCV-3 detection in tissues from fetuses/stillborn piglets in cases of reproductive problems in domestic swine, as well as the histopathologic assessment of fetal tissues. Fetuses or stillborn piglets from 53 cases of reproductive failure were collected and analyzed by PCV-3 qPCR. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV-2), and porcine parvovirus 1 (PPV1) was also checked. PCV-3 qPCR positive samples with a high viral load were tested by PCV-3 in situ hybridization (ISH), sequenced, and phylogenetically analyzed. PCV-3 DNA was detected in 18/53 (33.9%) reproductive failure cases and in 16 of them PCV-3 was the only pathogen found. PCV-2 DNA was found in 5/53 (9.4%), PRRSV RNA in 4/53 (7.5%) and PPV1 was not detected. Four out of the six PCV-3 qPCR-positive cases with Ct value <30 were positive when tested by ISH. In these samples, PCV-3 was detected within mild histopathologic lesions, such as arteritis and periarteritis in multiple tissues. The present work emphasizes the need to include PCV-3 as a potential causative agent of reproductive failure in swine.

The nasal microbiota is a key contributor to animal health, and characterizing the nasal microbiota composition is an important step towards elucidating the role of its different members. Efforts to characterize the nasal microbiota composition of domestic pigs and other farm animals frequently report the presence of bacteria that are typically found in the gut, including many anaerobes from the Bacteroidales and Clostridiales orders. However, the in vivo role of these gut-microbiota associated taxa is currently unclear. Here, we tackled this issue by examining the prevalence, origin, and activity of these taxa in the nasal microbiota of piglets. First, analysis of the nasal microbiota of farm piglets sampled in this study, as well as various publicly available data sets, revealed that gut-microbiota associated taxa indeed constitute a substantial fraction of the pig nasal microbiota that is highly variable across individual animals. Second, comparison of herd-matched nasal and rectal samples at amplicon sequencing variant (ASV) level showed that these taxa are largely shared in the nasal and rectal microbiota, suggesting a common origin driven presumably by the transfer of fecal matter. Third, surgical sampling of the inner nasal tract showed that gut-microbiota associated taxa are found throughout the nasal cavity, indicating that these taxa do not stem from contaminations introduced during sampling with conventional nasal swabs. Finally, analysis of cDNA from the 16S rRNA gene in these nasal samples indicated that gut-microbiota associated taxa are indeed active in the pig nasal cavity. This study shows that gut-microbiota associated taxa are not only present, but also active, in the nasal cavity of domestic pigs, and paves the way for future efforts to elucidate the function of these taxa within the nasal microbiota.

The nasal microbiota plays an important role in animal health and the use of antibiotics is a major factor that influences its composition. Here, we studied the consequences of an intensive antibiotic treatment, applied to sows and/or their offspring, on the piglets’ nasal microbiota. Four pregnant sows were treated with crystalline ceftiofur and tulathromycin (CT sows ) while two other sows received only crystalline ceftiofur (C sows ). Sow treatments were performed at D-4 (four days pre-farrowing), D3, D10 and D17 for ceftiofur and D-3, D4 and D11 for tulathromycin. Half of the piglets born to CT sows were treated at D1 with ceftiofur. Nasal swabs were taken from piglets at 22–24 days of age and bacterial load and nasal microbiota composition were defined by 16 s rRNA gene qPCR and amplicon sequencing. Antibiotic treatment of sows reduced their nasal bacterial load, as well as in their offspring, indicating a reduced bacterial transmission from the dams. In addition, nasal microbiota composition of the piglets exhibited signs of dysbiosis, showing unusual taxa. The addition of tulathromycin to the ceftiofur treatment seemed to enhance the deleterious effect on the microbiota diversity by diminishing some bacteria commonly found in the piglets’ nasal cavity, such as Glaesserella , Streptococcus , Prevotella , Staphylococcus and several members of the Ruminococcaceae and Lachnospiraceae families. On the other hand, the additional treatment of piglets with ceftiofur resulted in no further effect beyond the treatment of the sows. Altogether, these results suggest that intensive antibiotic treatments of sows, especially the double antibiotic treatment, disrupt the nasal microbiota of their offspring and highlight the importance of sow-to-piglet microbiota transmission.