Obradovic et al. Vet Res (2021) 52:49 https://doi.org/10.1186/s13567-021-00918-w REVIEW Open Access Review of the speculative role of co‑infections in Streptococcus suis‑associated diseases in pigs Milan R. Obradovic1, Mariela Segura1, Joaquim Segalés2,3,4 and Marcelo Gottschalk1* Abstract Streptococcus suis is one of the most important bacterial swine pathogens affecting post-weaned piglets, causing mainly meningitis, arthritis and sudden death. It not only results in severe economic losses but also raises concerns over animal welfare and antimicrobial resistance and remains an important zoonotic agent in some countries. The definition and diagnosis of S. suis-associated diseases can be complex. Should S. suis be considered a primary or secondary pathogen? The situation is further complicated when referring to respiratory disease, since the pathogen has historically been considered as a secondary pathogen within the porcine respiratory disease complex (PRDC). Is S. suis a respiratory or strictly systemic pathogen? S. suis is a normal inhabitant of the upper respiratory tract, and the presence of potentially virulent strains alone does not guarantee the appearance of clinical signs. Within this unclear context, it has been largely proposed that co-infection with some viral and bacterial pathogens can significantly influ- ence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field. In this review, we critically addressed studies reporting an epidemiological link (mixed infections or presence of more than one pathogen at the same time), as well as in vitro and in vivo studies of co-infection of S. suis with other pathogens and discussed their limitations and possibilities for improvement and proposed recommendations for future studies. Keywords: Streptococcus suis, Co-infections in pigs, Mixed infections, Porcine reproductive and respiratory syndrome virus, Swine influenza virus, Porcine circovirus 2, Bacterial mixed and co-infections, Porcine respiratory disease complex Table of Contents S. suis and PRRSV mixed and co‑infections 4.1  1 Introduction 4.1.1 PRRSV‑S. suis mixed infections 2 Complexity of the transition from infection (or coloni‑ 4.1.2  In vitro studies on the interactions zation) to clinical disease caused by S. suis between S. suis and PRRSV 3 Should S. suis be considered a respiratory or systemic 4.1.3  In vivo studies on the interactions pathogen? Or both? between S. suis and PRRSV 4 S. suis and viral mixed and/or co‑infections S. suis and swine influenza type A virus (SIV) 4.2  mixed and co‑infections *Correspondence: marcelo.gottschalk@umontreal.ca 4.2.1 SIV‑S. suis mixed infections 1 Groupe de Recherche Sur Les Maladies Infectieuses en Production 4.2.2 In vitro studies on the interaction between S. Animale (GREMIP), Centre de Recherche en Infectiologie Porcine et Aviaire (CRIPA), Faculty of Veterinary Medicine, University of Montreal, suis and SIV 3200 Sicotte, Saint‑Hyacinthe, QC J2S 2M2, Canada 4.2.3 In vivo studies on the interaction between S. Full list of author information is available at the end of the article suis and SIV © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​mmons.​org/​publi​cdoma​in/​ zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Obradovic et al. Vet Res (2021) 52:49 Page 2 of 22 4.3 S. suis and porcine circovirus 2 mixed macrolides/lincosamides and tetracyclines are observed and co‑infections and attributed to the intensive use of antimicrobials in 4.3.1 PCV‑2‑S. suis mixed infections pigs [3]. S. suis is considered a niche for antimicrobial resistance and represents a high risk of transmission of 4.3.2 In vitro studies on the interaction between S. such resistance to other veterinary and human pathogens suis and PCV‑2 due to the presence of mobile genetic elements carrying 4.3.3 In vivo studies on the interaction between S. resistance genes transferable at high frequency within the suis and PCV‑2 species and, even more alarmingly, toward other bacte‑ 4.4 S. suis and other virus co‑infections rial species [3, 4]. S. suis is also considered an important zoonotic infection. The target populations are mainly 5 S. suis mixed and/or co‑infections with other bacterial workers in the swine and pork industry (Western coun‑ pathogens tries), the general population due to close contact with 5.1 S. suis mixed infections with other bacterial pigs (China and other Asian countries) and individuals pathogens who consume raw or undercooked pork or pork by-prod‑ 5.2 In vitro and in vivo studies on the interaction ucts (Vietnam, Thailand and Laos) [5, 6]. between S. suis and other bacterial pathogens S. suis had originally been classified into 35 serotypes based on the antigenicity of the capsular polysaccharide, 6 Limitations of S. suis co‑infection studies which is suggested to be a major virulent factor [7]. How‑ 7 Conclusion and future perspectives ever, six of the serotypes (serotypes 20, 22, 26, 32, 33 and References 34) have been reclassified as belonging to other bacterial species [8, 9]. The distribution of serotypes recovered 1 Introduction from diseased pigs in different geographical regions var‑ Streptococcus suis is an important bacterial pathogen of ies, although serotypes 2 and 9 are the most prevalent in swine with a worldwide distribution [1]. The most com‑ several European countries [1]. Serotype 2 is, by far, the mon clinical and pathological outcomes of the infection most common serotype affecting humans, followed by are meningitis, arthritis, endocarditis, septicemia and serotype 14 [1]. The distribution of serotypes affecting sudden death [2]. The definition of Streptococcus suis- pigs in North America is different, with no clear preva‑ associated diseases should be based on the combination lence of serotype 2 [10, 11]. Highly, intermediately and of the presence of clinical signs, gross and/or micro‑ low virulent serotype 2 strains have been characterized scopic lesions and bacterial isolation (in either predomi‑ [7, 12]. Further studies using multilocus sequence typ‑ nance or pure culture) in affected organs and/or tissues. ing (MLST) showed that sequence type (ST) 1 strains However, even after bacterial isolation, interpretation (with other clonal complex 1 strains) normally found in of the role of S. suis as a primary pathogen is not always Europe and Asia present higher virulence potential and easy. Furthermore, since diagnosis in the field may some‑ are mainly isolated from diseased pigs and humans [6]. times be based solely on clinical observations, differentia‑ S. suis is normally present in the tonsils and naso‑ tion with infections caused by other pathogens (such as pharynx of most healthy pigs [13, 14] and some authors Glaesserella parasuis) is difficult. The more specific role therefore classify the bacterium as “pathobionts” [15]. of S. suis in pneumonia is also a topic of debate. Clinical The conditions under which certain serotypes/strains of signs and mortality are mainly observed in weaned and S. suis become pathogenic and cross the mucosal barrier (rarely) in suckling piglets and much less commonly in into the blood causing a systemic infection are not fully grower-finisher pigs. The infection caused by the patho‑ understood [16]. A plethora of potential virulent factors gen not only results in severe economic losses but also has been described, although there is still a debate sur‑ raises animal welfare concerns. In addition, S. suis-asso‑ rounding their significance to the pathogenesis of dis‑ ciated diseases are difficult to control [2]. Even when the eases linked to S. suis infection [7]. It is believed that, carrier rate of S. suis is high, the incidence of the disease under some circumstances, S. suis does not act alone and varies from period to period and is usually less than 5% takes advantage of concomitant or previous infections [2]. However, this is usually the case when antimicrobi‑ with other pathogens. Indeed, since co-infections have als (if allowed) are used as prophylactic/metaphylactic been largely been associated with the increase in clini‑ measures. One of the main problems is that antimicrobi‑ cal disease caused by S. suis, we will critically discuss the als that have efficacy are those the industry is trying to data available in the literature that directly or indirectly reduce given their importance in both human and veteri‑ address the issue. The definitions of co-infection, super- nary medicine [2]. Recent data on antimicrobial suscep‑ infection and mixed infections are not always clear but tibility of S. suis are alarming. High rates of resistance to were plainly explained in a recent review [17]. We will Obradovic et al. Vet Res (2021) 52:49 Page 3 of 22 use the term mixed infection when there is an epidemio‑ of virulence for a given strain of S. suis is not simple, a logical link only (detection of more than one pathogen in fact that is even truer for serotypes other than serotype 2, organs/tissues) and co-infection for in vitro and in vivo which have been less studied and for which animal mod‑ studies that specifically address the interaction between els to reproduce the disease are almost inexistent. For S. suis and other pathogens. example, serotype 9 virulent strains must be adminis‑ trated intravenously to susceptible pigs to reproduce 2 Complexity of the transition from infection (or disease [23], even though this serotype is the most preva‑ colonization) to clinical disease caused by S. suis lent among diseased pigs in several European countries Should S. suis be considered a primary or secondary/ [1]. The situation in North America is even more com‑ opportunistic pathogen? As mentioned, S. suis is a nor‑ plicated since serotypes 2 and 9 strains are very different mal inhabitant of the upper respiratory tract [2]. The from predominant virulent strains in Europe and display presence of potentially virulent strains alone does not lower virulence [2, 24]. Indeed, serotype 1/2, a poorly guarantee the appearance of clinical signs, the latter studied non-zoonotic serotype, is the predominant sero‑ observed sometimes in the absence of such strains, as type recovered from clinical cases in the USA and Can‑ occurs in North America where highly virulent Eura‑ ada [10 and unpublished data]. It has been proposed sian strains are seldom isolated [2]. Indeed, high doses that, in addition to the potential virulence of the strain(s) of inoculum (mostly with virulent serotype 2 strains) present in the herd, several factors may influence the and aggressive non-natural inoculation routes (intra‑ appearance of clinical signs (Figure 1): (a) environmen‑ peritoneal or intravenous) or sometimes two simultane‑ tal factors; (b) management factors; (c) host factors and/ ous routes of infection (intranasal and intramuscular) or (d) the presence of co-infections. Environmental fac‑ have been used in experimental trials using conventional tors that may influence the appearance of S. suis-related pigs to reproduce clinical disease [18–21]. On the other diseases include poor ventilation, high humidity, inad‑ hand, mortalities up to 20% with non-serotype 2 strains equate sanitation, high levels of dust and ammonia and may be observed in the natural infection in the field if large temperature variations between night and day [2]. no medication is used [22]. As mentioned, the definition Management factors such as high level of cross-fostering, Figure 1 Factors influencing the appearance of clinical signs of Streptococcus suis infection. Obradovic et al. Vet Res (2021) 52:49 Page 4 of 22 overcrowding, teeth clipping and tail docking, ear notch‑ few cases in which respiratory signs were observed are ing, mixing pigs of different ages, poor adaptation to solid frequently associated with heart lesions (endocarditis or feed in the nursery and low levels of vitamin E have also pericarditis) with no pulmonary involvement (unpub‑ been cited as playing important roles [2]. Host factors lished observations). Intranasal experimental infections such as high levels of stress and the presence/absence of may, under some circumstances (previous irritation of anti-S. suis antibodies may also influence the appearance nasal mucosa), induce mostly meningitis and arthritis of clinical disease. Indeed, it has been shown that clini‑ with no lung lesions associated with respiratory prob‑ cal signs appear when the level of maternal antibodies is lems ([33] and unpublished data). Intranasal infection of low [22, 25]; antibodies slowly increase at the end of the highly susceptible caesarian-derived, colostrum-deprived post-weaning period, and animals become more resistant piglets also led to systemic disease [34]. More recent co- to infection [26]. These antibodies would not necessarily infection studies in conventional pigs (see below) showed be all specific to the virulent strains of S. suis present in some lung lesions following S. suis infection, although no the herd (unpublished observations) but probably also to indication of the presence of other opportunistic bacte‑ other S. suis or even other streptococci that are normally rial pathogens in these studies is discussed [20]. Indeed, present in tonsils [25]. Finally, co-infections have been when evaluating isolates of S. suis for its potential viru‑ suggested to play a major role in the development of S. lence (pathotypes), those recovered from lungs are either suis disease. Indeed, co-infections/mixed infections of not included in the study or often considered as possibly different swine pathogens with S. suis have been reported opportunistic [10, 35]. S. suis may also reach lungs as and suggested in the literature, although there is still a post-mortem invasion, and that is one of the causes that certain lack of scientific support for many of them. This is disqualify the use of isolates recovered from lungs to pro‑ the main reason why we address the topic in this review. duce autogenous vaccines [26]. Finally, S. suis recovered from the lungs of young ani‑ 3 Should S. suis be considered a respiratory mals with respiratory signs and pulmonary lesions should or systemic pathogen? Or both? be differentiated from those recovered from lungs at Although many studies addressed the role of S. suis in slaughter. S. suis rarely induce disease in finisher ani‑ respiratory diseases, a pathogen’s route of entry should mals due to the high levels of antibodies present in be differentiated from the induced pathology. Although older animals [25], and the detection of the pathogen in there is a proposed hypothesis of intestinal translocation such mixed infections at slaughter should not be taken (still to be confirmed) [27], it is widely accepted that the into consideration. Indeed, S. suis may be isolated from main route of infection for systemic S. suis disease is the healthy lungs without lesions, probably due to the fact respiratory route [2], and airborne transmission of the that some carrier isolates may go from the upper to the infection has clearly been demonstrated [28]. Within this lower respiratory tract during agony [2, 36]. context, it may be argued whether S. suis should be con‑ sidered as a true respiratory pathogen. Infectious agents involved in porcine respiratory disease complex (PRDC) 4 S. suis and viral mixed and/or co‑infections are classified into primary or secondary (or opportunis‑ Mixed infections of S. suis with swine viruses are a fre‑ tic) pathogens [17]. Primary pathogens are defined as the quent finding in swine herds. These interactions are ones that can infect the animal as the first unique patho‑ believed to have a synergistic effect, resulting in the gen and then facilitate secondary or opportunistic co- aggravation of clinical signs and increased mortality. infection(s). S. suis is usually considered as a secondary/ However, few controlled studies on real co-infections opportunistic pathogen [17]. However, the role of S. suis have been conducted. The most important viral patho‑ in respiratory pathology, even as an opportunistic agent, gens that have been suggested to be associated with is still not clear. Studies based on natural infection cases S. suis disease are porcine reproductive and respiratory indicate that S. suis may cause pulmonary lesions gener‑ syndrome virus (PRRSV) and, to a lesser extent, swine ally described as suppurative bronchopneumonia and/or influenza virus (SIV) and porcine circovirus 2 (PCV-2). fibrino-hemorrhagic pleuropneumonia [29–31]. How‑ Studies that consider other viruses are rare. To better ever, in most cases of natural infections, other concomi‑ understand the importance of S. suis co-infections with tant bacteria were also isolated, and the specific role of S. swine viruses, we will discuss the main findings from the suis is difficult to evaluate [32]. Respiratory clinical signs reported mixed infections, as well as controlled in vitro are usually not observed after experimental infection and in vivo assays that may help elucidate the interac‑ with S. suis, and the clinical outcomes are mainly septice‑ tions. The important details and conditions of in vitro mia, meningitis and/or arthritis, which are also observed and in vivo experimental studies with S. suis and viruses in studied cases of airborne transmission [28, 33]. The are summarized in Tables 1 and 2. Table 1 Summary of the experimental conditions of S. suis co-infection with viral pathogens using in vitro models S. suis Type of cells Virus strain S. suis Virus/bacterial infection Incubation time (hours) Goal of the study Main observed effects of Refs. co-infection serotype 2 dose co-infection pathogen strain Obradovic et al. Vet Res Virus S. suis PRRSV Swine bone marrow Genotype 2 ST 1 0.1 MOI/1 MOI 2 0.5 to 5; 12 for Phagocytosis/ intracel- ↓ Bacterial phagocy- [56] dendritic cells (BMDC) gene expres- lular survival; gene tosis (BMDC); ↑ gene and monocytes sion expression (microarray) expression coding for pro-inflammatory (2021) 52:49 mediators Porcine alveolar mac- Genotype 2 ST 1 0.1 MOI/10 MOI 1 to 8 Phagocytosis/intracellular ↑ Bacterial phagocytosis; [20] rophages survival; inflammatory- ↑ intracellular survival related gene expression and ↑ gene expression (qRT-PCR) for pro-inflammatory mediators SIV Neonatal tracheal por- H1N1 ST 1 1 MOI/10 MOI 12 2 and 4 Adhesion and invasion; ↑ Bacterial adhesion [79] cine cell line (NTPr) inflammatory-related and invasion; ↑ gene expression (qRT- gene expression for PCR) pro-inflammatory mediators NTPr H1N1 ST 1 1 MOI/10 MOI 12 12 Gene expression (micro- ↑ Gene expression for [85] array) pro-inflammatory mediators Porcine precision-cut H1N1 and H3N2 ST 1 105 ­TCID50/107 CFU 1 24 to 72 Adhesion, invasion, and ↑ Bacterial adherence/ [81] lung slice colonization colonization/invasion Porcine bronchial epithe- H3N2 ST 1 5 × ­104 24 24 and 48 Adhesion, invasion, and ↑ Bacterial adherence/ [83] lial cells (PBEC) under ­TCID50/2.5 × ­107 CFU colonization colonization/invasion air–liquid interface PCV-2 Immortalized swine PCV-2 Chinese strain ST 7 1 MOI/10 MOI 24 and 48 2 Adhesion, invasion and ↑ Permeability and [99] tracheal epithelial cells bacterial translocation bacterial transloca- (STEC) in transwell tion through tracheal chambers epithelium STEC PCV-2 Chinese strain ST 7 1 MOI/30 MOI 24 and 48 1 to 4 Intracellular survival ↑ Bacterial intracellular [100] survival; ↓ gene expression for pro-inflammatory mediators Porcine monocytic PCV-2 Chinese strain ST 7 1 MOI/10 MOI 24 and 48 1 and 8 Inflammatory-related ↑ Gene expression for [21] 3D4/21 cell line gene expression (qRT- pro-inflammatory PCR) mediators; ↓ MHC-II expression PRSSV: Porcine Reproductive and Respiratory Syndrome Virus; SIV: Swine Influenza Virus; PCV-2: Porcine Circovirus type 2; ­TCID50: medium tissue culture infectious dose; CFU: colony-forming units; ST: sequence type. Page 5 of 22 Obradovic et al. Vet Res (2021) 52:49 Page 6 of 22 4.1 S. suis and PRRSV mixed and co‑infections farms (18%) compared to those that were not affected For over 30 years, PRRSV has been one of the leading by the virus infection (2%) [48]. Interestingly, a temporal viral diseases in pigs, causing significant losses in the and spatial association of occurrence of human meningi‑ swine industry, causing more than $600 million in losses tis caused by S. suis and PRRSV outbreaks in pig farms every year in the USA alone [37]. The clinical signs can was observed in that country [49]. It should be noted that range from reproductive failure in pregnant sows to the study addresses association and not causality. The severe respiratory clinical signs in weaned and growing study also has limitations (clearly stated by the authors): pigs. PRRSV strains have considerable genetic variabil‑ incomplete details on PRRSV outbreaks, differences in ity (approximately 60%) and are categorized as PRRSV1 sample collection, underestimation of the real prevalence (European genotype) and PRRSV2 (North American gen‑ and distribution of PRRSV outbreaks and lack of details otype) [38]. PRRSV can cause severe disease alone or act on individual patient data that may constitute confound‑ in concert with other viruses or bacteria to significantly ing factors. contribute to PRDC [40–42]. It has been reported that The few epidemiological studies and clinical obser‑ PRRSV infects pulmonary interstitial, alveolar and intra‑ vations by practitioners around the world point to the vascular macrophages with consequent decreased phago‑ importance of the S. suis-PRRSV association with regard cytic activity and altered innate immune response in the to the occurrence of severe disease in pigs. However, respiratory tract, opening the door to other opportunistic the mechanisms behind the observed synergistic effect viral and bacterial pathogens [43]. However, there are still of both pathogens have yet to be explained. To better some gaps with regard to the specific cell types the virus understand the cellular, immunological and molecu‑ infects [37]. lar implications behind this synergy, some in vitro and in vivo models have been developed. 4.1.1 PRRSV‑S. suis mixed infections There is strong unwritten evidence of a clear synergic 4.1.2 In vitro studies on the interactions between S. suis association between PRRSV and S. suis from the clini‑ and PRRSV cal perspective, and most swine practitioners strongly Owing to the characteristics of PRRSV, S. suis co-infec‑ believe that positive unstable (Category I) herds [44] tion studies mainly addressed the interactions with cells are more prone to serious problems of S. suis disease of the innate immune system. Although the pathogenesis than PRRSV stable or free herds. Some examples in the of the infection caused by S. suis is not yet completely literature describe the simultaneous detection of both understood [7], it is believed that its interaction with res‑ pathogens in the same farm (mixed infections). Although piratory phagocytic cells may be one of the initial steps of PRRSV seroprevalence is a good indicator of swine herd the infection [16]. In this first line of defense, pulmonary exposure to the virus, RT-PCR analysis is required to alveolar macrophages (PAMs) play an essential role in the detect active infections and confirm mixed infections of innate immune response against pathogens through bac‑ PRRSV with other swine pathogens [45]. For example, terial phagocytosis and elimination [42]. Since these cells RT-PCR analysis of oral fluids from pig farms in Korea are also among PRRSV’s most important targets, they showed that PRRSV and S. suis were frequently detected may represent an interesting model to study co-infec‑ together (around 50%) in 3 to 7 week-old piglets [46]. tion effects [43]. The hypothesis behind the PAM cells’ Studies performed at slaughter in Canada showed that use in co-infection studies is that a prior PRRSV infec‑ RT-PCR analysis of swine pathogens present on tonsils tion would reduce S. suis phagocytosis either by reducing detected S. suis and PRRSV in 53.7% and 22% of samples, phagocyte activity or inducing cell apoptosis. However, respectively [13]. Similarly, another study at slaughter under normal conditions, S. suis possesses a thick cap‑ showed that lungs that were RT-PCR positive for PRRSV sule that usually prevents bacterial phagocytosis—a have significantly higher odds of being positive for S. fact confirmed by different research groups using non- suis [47]. These data (from both abattoirs and non-clin‑ encapsulated mutants [7]. It is important to note that the ical samples) do not reveal the nature of the pathogens’ bacteria that are internalized do not survive intracellu‑ interaction and their possible synergistic effect due to the larly [50]. So, it may also be hypothesized that although endemic characteristics of PRRSV and ubiquitous pres‑ the internalization of S. suis is a rare event, a previous ence of S. suis. Indeed, it is important to state that both viral infection may affect the intracellular killing of bac‑ pathogens may be present in herds without any associ‑ teria, which may influence the outcome of the disease. ated clinical signs of disease. Another important aspect of the interaction of S. suis In Vietnam, Hoa et al. showed an increased isolation with phagocytes is the capacity to induce inflammation. rate of highly virulent S. suis serotype 2 in the blood and Indeed, it has been shown that an excess of inflammation internal tissues of diseased pigs from PRRSV-affected is a hallmark of S. suis disease [7]. Hence, two pathogens Obradovic et al. Vet Res (2021) 52:49 Page 7 of 22 acting together would increase the inflammatory reaction to S. suis secondary infection has been hypothesized, no through an increase in the secretion of pro-inflammatory study has specifically addressed this interaction. How‑ cytokines. Still, immunosuppression has been demon‑ ever, an interesting feature of PRRSV is that it may affect strated in some PRRSV infection studies and thus may the thymus and its ability to carry out its normal func‑ lead to a higher susceptibility to bacterial co-infection in tions [52, 59]. In this way, pigs would be less able to resist affected pigs [51, 52]. Interestingly, the two processes are and/or eliminate secondary infections. It has also been not necessarily in contradiction, since the dual activity of reported that S. suis infection can cause atrophy of the PRRSV (immunosuppression but increased inflamma‑ thymus and induce apoptosis of thymocytes, thus likely tion) has been observed in cases of PRRSV and porcine suppressing host immunity [60]. How these two patho‑ respiratory coronavirus co-infection studies in pigs [53]. gens interact with this important immune organ is still Several studies addressed the interaction between unknown. S. suis and PAMs [50, 54, 55], but only one reported Finally, co-infection studies with non-immune cells how a previous PRRSV infection of these cells affects have not been conducted. Since PRRSV induces nonsup‑ S. suis phagocytosis and inflammation (Table 1) [20]. purative rhinitis and metaplasia of the turbinate epithe‑ PAMs infected with a highly pathogenic PRRSV geno‑ lium, it has been suggested that it may predispose pigs to type 2 strain and a poorly characterized S. suis sero‑ the colonization of the respiratory tract by S. suis sero‑ type 2 strain showed increased bacterial phagocytosis type 2, potentially creating a portal of entry for S. suis and survival [20]. In addition, co-infection significantly [61]. However, the hypothesis of virus predisposition to increased the mRNA expression of most pro-inflamma‑ bacterial colonization has never been confirmed. tory cytokines tested, including interleukin (IL)-1β, IL-6, In summary, although it is widely accepted that a IL-8, chemokine (C–C motif ) ligand 4 (CCL4), tumor PRRSV infection increases the susceptibility to S. suis co- necrosis factor-α (TNF-α) and interferon-β (INF-β). infection (see results in vivo below), it seems evident that Authors concluded that the deleterious effect of the co- there is a serious lack of scientific evidence that clearly infection was mainly due to excessive inflammation [20]. explains the specific mechanisms involved in such inter‑ Surprisingly, no other study addressed the effect of co- actions and more mechanistic studies are needed. infection on these cells. Another study addressed the effect of co-infection 4.1.3 In vivo studies on the interactions between S. suis using monocytes and swine bone marrow-derived den‑ and PRRSV dritic cells (BMDC) [56]. Both cell types were first Although a first report failed to demonstrate any influ‑ infected with a PRRSV genotype 2 strain and then ence of PRRSV infection on an S. suis secondary infec‑ co-infected with the European (ST1) virulent sero‑ tion [62], further in vivo experiments were carried out type 2 P1/7 strain of S. suis (used in virulence studies by to demonstrate the synergistic effect of these pathogens most laboratories) [56]. Unlike the previous study with on morbidity and the severity of clinical signs in pigs [20, PAMs, results showed that PRRSV significantly reduced 52, 61, 63, 64]. The most common type of co-infection the internalization of S. suis by the BMDC with no model was developed by infecting animals intranasally observed effect on bacterial intracellular survival [20]. No with PRRSV and then, after 5 to 7 days, inoculating S. differences were observed with monocytes, which hardly suis intranasally or intramuscularly (an overview of the allowed virus replication and poorly phagocytosed S. suis important study parameters is shown in Table 2) [20, 61, [56]. As shown with PAMs, microarray analysis revealed 63, 65]. Only one study addressed an intrauterine infec‑ significant up-regulation of pro-inflammatory genes in tion with PRRSV (sows) followed by S. suis infection in co-infected BMDC [56]. Previous S. suis research showed piglets [52] (see below). Different PRRSV types (low to the capacity of the bacteria alone to induce the secretion high virulent) and origins (North America or Asia), as of pro-inflammatory cytokines involved in disease patho‑ well as S. suis serotype 2 (sometimes poorly character‑ genesis [57]. Co-infection with PRRSV may exacerbate ized) strains, were assayed. There is only one study that the inflammation by increasing the secretion of cytokines used a S. suis serotype 7 strain [65]. The consensus of from immune cells, although this hypothesis should be these studies is that higher mortality and morbidity were confirmed in vivo. Unfortunately, there are no other stud‑ observed in groups in which piglets were co-infected by ies of co-infection of these two important pathogens with the two pathogens compared to those infected only with phagocytic cells. Indeed, it has been shown that pulmo‑ S. suis, independently of the virus genotype and S. suis nary intravascular macrophages may play an important serotype [20, 61, 65]. S. suis was cultured in higher num‑ role in PRRSV infection [58]. Although a possible asso‑ bers from tissues and blood (bacteremia) of co-infected ciation between a virus-dependent suppression of the animals compared to S. suis mono-infected piglets and functions of these cells and an increased susceptibility macroscopic and microscopic lesions in different internal Obradovic et al. Vet Res Table 2 Summary of the experimental conditions of S. suis co-infection with viral pathogens using in vivo models S. suis Age of pigs at the Route of infection Virus strain and dose S. suis strain and dose Main observed effects of co-infection Ref co-infection infection Virus / Bacteria (2021) 52:49 parhogen Virus / Bacteria PRRSV 19 days 26 days IN IN 2 × ­104.99 ­TCID50, high virulent PRRSV 2 × ­108.3 CFU; serotype 2 (no further ↑ Severity of clinical signs, mortality, [61] strain, genotype 2 details on the strain) and interstitial pneumonia lesions 2 × ­104.47 ­TCID50, low virulent PRRSV vac- (especially those infected with the ™ cine strain RespPRRS/Repro high virulent PRRSV strain) 98 days 5 days IN IN 103.5 ­TCID50, PRRSV, genotype 2 8 × ­107 CFU, serotype 2 (no further ↑ Susceptibility of piglets to bacterial [52] of sow details on the strain) infection gestation 21 days 28 days IN/IM IN/IM 2 × ­104.97 ­TCID50 PRRSV, genotype 2 109 CFU; serotype 7 (no further details ↑ Clinical signs and mortality [65] on the strain) 49 days 42 days* IN IN 2 × ­104 ­TCID50 106 CFU; serotype 2 (no further details ↑ Clinical severity related to PRRSV [64] Asian HP-PRRSV strains; US PRRSV strains, on the strain) virulence; genotype 2 ↓ levels of proinflammatory cytokines 28 days 21 days* IN IM 106 ­TCID50 2 × ­108 CFU; serotype 2 low virulence ↑ Clinical signs and mortality [67] PRRSV MLV-like strain strain (no further details on the strain) SIV 35 days 38 days IN IN 2 × ­107 egg infectious ­dose50/mL, H1N1 2 × ­106 CFU; serotype 2; ST7 ↑ Clinical signs, pathological changes, [76] strain and cell apoptosis; ↑ gene expression for pro-inflammatory mediators PCV-2 28 days 33 days IN/IM IN/IM 2 × ­106.5 ­TCID50 4.5 × ­109 CFU; serotype 2; ST7 ↑ Clinical signs; ↑ gene expression for [21] 2 mL/3 mL 2 mL/3 mL pro-inflammatory mediators; ↓ gene expression for CD4, CD8, and MHC-II PRV 63 days 63 days IN IN 5 × ­103 ­TCID50 high or low virulent PRV 109 CFU S. suis serotype 2 (no further ↑ Clinical signs [107] strains details on the strain) * Animals were infected first with S. suis and then infected with a virus. IM: intramuscular injection; IN: intranasal inoculation; PRSSV: Porcine Reproductive and Respiratory Syndrome Virus; SIV: Swine Influenza Virus; PCV-2: Porcine Circovirus type 2; PRV: pseudorabies virus; ­TCID50: medium tissue culture infectious dose; CFU: colony-forming units; ST: sequence type. Page 8 of 22 Obradovic et al. Vet Res (2021) 52:49 Page 9 of 22 organs were significantly exacerbated in animals in the these studies used 6-week-old PRRSV-negative pig‑ co-infected groups [20, 61, 65]. In particular, co-infected lets weaned before one week of age and kept free of piglets had significantly more severe gross and micro‑ pathogens. Animals were intranasally inoculated with scopic interstitial pneumonia lesions, suggesting a pos‑ a bacterial cocktail containing S. suis and two other sible secondary role of S. suis in PRDC [20, 42, 65]. The pathogens, mimicking sub-clinical bacterial infec‑ co-infection was able to reproduce similar morbidity and tions observed in the field. One week later, piglets were mortality to those observed in the high-fever outbreak challenged with PRRSV strains of different origins and caused by atypical PRRSV in China [39]. Interestingly, variable virulence [64]. Results showed low mortality a vaccine strain of PRRSV given intranasally prior to S. in general, although morbidity and lesions were more suis infection induced increased mortality compared to severe in piglets inoculated with a highly virulent Asian the single-infected controls, although the mortality was PRRSV strain [64]. The frequency of secondary bacte‑ lower than in the cases of co-infection with a virulent rial pneumonia was directly associated with the clini‑ PRRSV strain [61]. It must be noted that this was an off- cal severity induced by the PRRSV strains evaluated. label use of the PRRSV live attenuated vaccine, and it is Levels of pro-inflammatory cytokines were lower or at unlikely that the vaccine would be administered intrana‑ the same level in single or co-infected animals, which sally under field conditions. contradict somehow previous in vitro co-infection PRRSV is a causative agent of complex disease, attack‑ studies [20, 56]. Another study used three-week-old ing multiple organs. The infection of pregnant sows with conventional piglets that were inoculated intramus‑ PRRSV results in late abortion, early farrowing and still‑ cularly with S. suis serotype 2 strain and seven days born piglets [66]. Piglets from PRRSV infected sows are later with a highly pathogenic PRRSV MLV-like isolate PRRSV positive and, in theory, would have a weakened [67]. Results showed that while virus or S. suis infec‑ immune system that predisposes them to other oppor‑ tion caused transitional fever and moderate clinical tunistic pathogens [66]. One animal model was developed signs, the co-infection induced higher fever, anorexia to examine the effect of intrauterine PRRSV infection in and respiratory distress, leading to 60% mortality [67]. piglets on susceptibility to further intranasal infection It should be noted that the route of animal infection with S. suis serotype 2 [52]. To the authors’ knowledge, with S. suis does not reflect natural conditions, and the this is the only published study on adult PRRSV infection results should be interpreted carefully. followed by S. suis piglet co-infection. Pregnant gilts were The synergy between PRRSV and S. suis in vivo can‑ either inoculated or not with PRRSV at 98 days of ges‑ not be denied, and experimental infection studies gen‑ tation (see Table 2) [52]. Five day-old piglets born from erally confirm this fact. However, there are still many these sows were inoculated intranasally with an S. suis variables in previous co-infection studies to identify the serotype 2 strain [52]. The mortality rate, clinical signs, mechanisms involved. The pathogenicity of the PRRSV organ lesions and S. suis isolation from joints and brain strain is just one part of the puzzle, while other factors in the co-infected group were significantly higher com‑ like stress, environment, host susceptibility and bacte‑ pared to control non-S. suis infected piglets and those rial burden are some of the variables that may influence infected with S. suis alone [52]. Increased susceptibility the clinical and pathological outcomes. One important was explained as being linked to the immuno-suppres‑ point is also the strain of S. suis serotype 2 used: in sion caused by the effect of PRRSV on innate immune many studies, strains are poorly characterized although cells. Indeed, PRRSV-positive piglets had lesions in their it is well known that the virulence of serotype 2 strains thymus and bone marrow and significantly reduced is highly variable [7]. One study showed either no effect numbers of leukocytes, including lymphocytes and of co-infection using one S. suis strain (with a North monocytes [52]. In conclusion, this study showed that American phenotype) or a strong synergy when a sec‑ intrauterine PRRSV infection has significant effects on ond and different strain (with a Eurasian phenotype) immune response and may increase the susceptibility of was used [2, 63]. It is important to note that co-infec‑ piglets to intranasal S. suis serotype 2 infections. Even so, tions of PRRSV and typical North American serotype 2 the presence of confounding factors, especially concern‑ (ST28 or ST25) S. suis strains are frequently reported ing the experimental model used, must be considered. in Canada and the USA. Even though the virulence of Piglets were deprived of sow’s colostrum, early-weaned these S. suis strains is probably lower than that of Eur‑ and challenged with bacteria at five days of life, which is asian strains [7], a combination of other factors may rare in the field, where mostly post-weaned animals are have an influence on the effects of the co-infection (Fig‑ affected by S. suis [25, 52]. ure 1). Interestingly, other than the study mentioned Other studies addressed the role of a pre-infection above, no co-infection trials have been performed with with S. suis with subsequent PRRSV infection. One of typical North American S. suis strains. Obradovic et al. Vet Res (2021) 52:49 Page 10 of 22 4.2 S. suis and swine influenza type A virus (SIV) mixed mechanism of host–pathogen interactions during co- and co‑infections infection (Table 1). Most in vitro studies address the Swine influenza viruses (mainly type A) are able to cause influence of a previous SIV infection on secondary S. suis respiratory disease in pigs worldwide, that can lead to infection. However, an earlier study using the Madin- 10–15% mortality [68]. Influenza disease in pigs is highly Darby canine kidney (MDCK) cell line model showed contagious with no evident clinical signs or mild to mod‑ that the supernatant from a S. suis serotype 2 strain can erate ones characterized by runny nose and coughing increase the infection ability of SIV H3N2 for these cells [69]. Swine influenza causes significant economic losses [78]. The results may suggest that S. suis has secretory primarily due to weight loss, though some cases may be factors that facilitate SIV entrance even in the absence much more severe in co-infection with other pathogens, of bacteria. It should be noted that MDCK cells do not such as M. hyopneumoniae, PRRSV and Actinobacillus resemble swine respiratory mucosal cells and, in this pleuropneumoniae [68]. The three main SIV subtypes study, the effect of the supernatant was evaluated in a encountered in pigs are H1N1, H1N2, and H3N2, and closed environment (a well) in which extracellular fac‑ since 2009, the pandemic H1N1 (pH1N1) has also been tors of S. suis are concentrated. The results have not been circulating in domestic pigs worldwide [70]. Influenza confirmed or invalidated by other research groups. viruses cause respiratory disease by infecting the epi‑ The first study that demonstrated the effect of a previ‑ thelial cells leading to cell apoptosis and the destruction ous SIV infection in cells followed by a subsequent S. suis of the mucosal barriers [71]. In addition, cell death is infection was carried out using neonatal tracheal por‑ enhanced by the effect of cytokines and innate immune cine epithelial (NTPr) cells [79]. SIV H1N1 pre-infected cells, which causes bronchitis and interstitial pneumonia cells enabled bacterial adhesion and invasion levels that [71]. were over 100 times higher compared to those of control cells. Inhibition studies confirmed that bacterial capsu‑ 4.2.1 SIV‑S. suis mixed infections lar sialic acid moiety is responsible for the binding to the It is well known that influenza virus infections in humans viral hemagglutinin expressed on the NTPr cell surface are usually complicated by secondary bacterial infections, [79]. Also, pre-incubation of S. suis with SIV H1N1 sig‑ especially Streptococcus pneumoniae [72]. The capac‑ nificantly increased bacterial adhesion to epithelial cells ity of SIV to aggravate respiratory bacterial infections and epithelial cell invasion. Similar results were obtained in pigs has also been documented [73–75]. However, with other S. suis sialic-acid positive serotypes (such as the potential of SIV and S. suis mixed or co-infections serotypes 1 and 14), but not with serotypes that lack the to cause serious pulmonary disease has been much less presence of such sugar moiety in their capsular polysac‑ studied and there are only a few published reports. Sero‑ charides [79]. The results were confirmed by an inde‑ logical data indicate a link between SIV and S. suis in pendent research group using the same cell type and both swine farms in China, although detailed information on SIV H1N1 and H3N2 subtypes [80]. The latter study also the correlation of antibody titers and isolation of these revealed that viruses bound to bacteria retained infectiv‑ two pathogens from healthy or diseased animals is miss‑ ity but induced only tiny plaques compared to the control ing [76]. The report on SIV H1N1 virus infections in pigs virus. In contrast, bacterial co-infection had a negative in England provided more information, since data show effect on SIV replication. The real cause of this effect is that clinical signs were mostly mild with low mortality unknown, but the authors hypothesized that the reduc‑ in older animals infected with SIV alone. Increased mor‑ tion in the amount of released virus may be caused by the tality was observed in nursery pigs, in which influenza direct binding of virions to bacteria and/or a detrimental infection was complicated by environmental stress and/ effect caused by bacteria bound to the surface of infected or co-infections, with S. suis being the most prevalent cells [80]. Further studies are needed to elucidate this pathogen isolated in these cases [77]. S. suis serotypes effect. 1, 2, 14, and 24 as well as some untypable isolates were The presence of differentiated epithelial cells and detected in SIV-infected pigs with severe signs of cough, mucus in animal respiratory mucosa creates additional meningitis, lameness and sudden death [77]. These complexity that could not be replicated with epithelial results suggest that SIV may complicate S. suis infections cell lines. To overcome this limitation, Meng et al., estab‑ observed in the field. lished a porcine precision-cut lung slice (PCLS) co-infec‑ tion model [81]. PCLSs use a procedure that preserves 4.2.2 In vitro studies on the interaction between S. suis multicellular tissue consisting of ciliated cells, mucus- and SIV producing cells and pneumocytes [81]. Hence, the pres‑ Different in vitro and ex vivo tissue cell culture mod‑ ence of differentiated epithelial cells with preserved els were therefore developed to examine the complex functions in this model made it possible to study S. suis Obradovic et al. Vet Res (2021) 52:49 Page 11 of 22 interactions in an environment that more closely resem‑ during the mono-infection. When SIV H3N2 was present bles an in vivo respiratory mucosal surface [81]. PLCSs in a co-infection study using both wild-type and suilysin- were pre-infected with SIV subtypes H1N1 or H2N3 and negative mutant strains, the latter was also able to adhere subsequently co-infected with an S. suis serotype 2 strain and invade deeper layers of differentiated epithelial cells [81]. As shown in previous studies [79, 80], the adhesion [83]. Indeed, the authors propose that suilysin-negative of S. suis to epithelial cells was significantly increased by S. suis strains, which are common in North America [2], SIV in a bacterial capsule-dependant manner, especially can become invasive in a co-infection scenario with influ‑ in the early stage [81]. However, at a later stage of co- enza A viruses. infection, results were virus strain-dependent, since only The role of inflammation through an increase of the SIV H3N2 induced the increased attachment of not only expression of pro-inflammatory mediators has been the encapsulated S. suis but also of its non-encapsulated described for both S. suis and SIV [84]. A preliminary mutant [81]. Indeed, confocal microscopy of cryosections study showed that co-infection significantly increased showed the destruction of ciliated epithelial cells and an the expression of proinflammatory genes [79]. A more increased presence of both encapsulated and non-encap‑ detailed study on gene expression in NTPr cells dur‑ sulated S. suis in the sub-epithelium of PLCSs [81]. These ing S. suis and SIV H1N1 co-infection using a microar‑ results imply that SIV promotes the bacterial infection ray assay confirmed that the infection of cells with SIV of respiratory epithelial cells in two phases, depending H1N1 alone or co-infection with both pathogens induced on viral subtype. The first phase is at the beginning of higher mRNA expression of genes in different biological the infection, when adherence of bacteria is enhanced by categories than cells infected by S. suis alone, with genes viral hemagglutinin (from both virus subtypes) expressed involved in immune response and inflammation being on the surface of the epithelial cells that bind S. suis cap‑ particularly overexpressed [85]. This synergy may be the sular polysaccharide sialic acid moiety. The second phase consequence, at least in part, of an increased bacterial occurs later when SIV H3N2 damages the epithelial cells adhesion/invasion of epithelial cells previously infected and opens the path for the bacterial colonization of the by SIV. subepithelial tissue in a capsule (and sialic acid)-inde‑ pendent manner [81]. 4.2.3 In vivo studies on the interaction between S. suis To better resemble the luminal respiratory environ‑ and SIV ment in airway epithelium, an air–liquid interface (ALI) Because S. suis and SIV co-infection became of interest culture system for differentiated porcine airway epithelial just recently, there is only one published animal model cells was developed [82]. Primary porcine tracheal and (Table 2). Lin et al. developed an in vivo model in which bronchial epithelial cells were cultured in a Transwell five-week-old piglets were intranasally infected with SIV filter system that enables the differentiation of epithelial H1N1 and then intranasally co-infected with a (suilysin cells under air–liquid interface conditions [82]. Indeed, positive) S. suis serotype 2 strain three days later [76]. the cell monolayer acquired cilia, pseudostratified epi‑ The co-infected group showed more severe clinical signs thelium and tight junctions that resemble the cells of air‑ and viral-induced pneumonia compared to the virus or S. way epithelium in a live animal [82]. The model was first suis single-infected groups [76]. The results confirmed, used to evaluate the adhesion and cytotoxic properties at least in part, the clinical and epidemiological field of S. suis serotype 2, showing that the hemolysin pro‑ data from England in which clinical signs seemed to be duced by S. suis (suilysin) contributes to the loss of cili‑ aggravated in co-infected animals [77]. Interestingly, the ated cells and cell apoptosis, as well as bacterial adhesion viral loads in the lungs were significantly higher in the co- and invasion (76). The same system was further devel‑ infected group compared to the control group infected oped to examine the interactions of SIV H3N2 and S. suis with either bacteria or SIV H1N1 alone, thus contradict‑ serotype 2 [83]. The previous infection of cells with the ing previous in vitro observations on a possible inhibi‑ virus increased S. suis sialic acid-dependent adherence tory role of S. suis on virus replication [80]. Moreover, and colonization, once more confirming previous studies the bacterial load was not increased in the co-infected [79–81]. There was a prominent difference in cytopatho‑ group compared to the S. suis-infected group [76]. genicity observed during the single infection with the Results also showed significant upregulation changes bacteria or SIV in this study. S. suis infection resulted in genes involved in pro-inflammatory (TLR4, MyD88, in early destruction of the differentiated cells, while IL-17D, IL-6, IL-8 and CCL2) and apoptosis (CASP2, SIV H3N2 induced apoptosis in the later stage of infec‑ CASP3, BCL2L11, FASLG and TNFRSF8) in the co- tion. It was suggested that S. suis cytopathogenicity may infected group compared to the control mono-infected be due to the suilysin, since a mutant defective in the pro‑ groups [76]. These results confirm previous observations duction of the toxin adhered but did not damage the cells in vitro using tracheal epithelial cells [79]. In sum, the Obradovic et al. Vet Res (2021) 52:49 Page 12 of 22 study results confirm a synergic clinical and pathological but no clear correlation with disease could be established. effect of SIV H1N1 and S. suis serotype 2 in pigs. How‑ Another study detected the presence of both pathogens ever, from the mechanistic perspective, this study did not by qPCR in oral fluid samples [46] but, similarly, no asso‑ characterize the immune cells involved in the generation ciation with the disease was observed. However, the per‑ of mucosal and systemic immune responses. centage of PCV-2 PCR positive results increased with The characterization of molecular and immunological age, whereas positive results for S. suis showed the oppo‑ aspects of co-infections in pigs may also contribute to a site, indicating that perhaps both pathogens are not nec‑ better understanding of disease in human medicine [86]. essarily prevalent at the same time [46]. Taken together, Indeed, as mentioned, human influenza disease is often although epidemiological results might suggest a possible complicated by secondary pneumonia caused by S. pneu- role of S. suis as a complication of PCV-2-associated dis‑ moniae, which leads to significant morbidity and mor‑ eases, no clear data confirm this hypothesis. tality around the world [72]. However, the mechanisms involved in this co-infection are also partially known [72]. 4.3.2 In vitro studies on the interaction between S. suis and PCV‑2 4.3 S. suis and porcine circovirus 2 mixed and co‑infections Only three in vitro co-infection studies have been per‑ Porcine circoviruses (PCVs) single-stranded DNA viruses formed using different cell types to study S. suis and that belong to the genus Circovirus under the family Cir- PCV-2 co-infection (Table 1) [21, 99, 100]. The results coviridae [87]. Four types of PCVs have been described: of the first study, which used poorly characterized PCV-1, PCV-2, PCV-3 and PCV-4 [88]. PCV-2 is, by far, immortalized swine tracheal epithelial cells, showed the most important PCV for the swine industry and is that although no increased adhesion or invasion of S. found to be associated with multiple clinical manifesta‑ suis serotype 2 was observed, the virus pre-infection tions of the disease in pigs referred to as porcine circovi‑ decreased the expression levels of ZO-1 and occlu‑ rus diseases (PCVD) in Europe and as porcine circovirus din, increased cell permeability and contributed to the associated disease (PCVAD) in North America [89, 90]. translocation of S. suis serotype 2 across the tracheal The clinical signs of PCVD/PCVAD were first described epithelium [99]. Although this in vitro model suggests as a postweaning multisystemic wasting syndrome a possible role of PCV-2 during the initial steps of co- (PMWS, currently known as PCV-2-systemic disease, infection by S. suis, the level of replication of PCV-2 in PCV-2-SD) in Canada [91]. PCV-2 was also associated tracheal and other respiratory epithelia in pigs is limited, with the porcine dermatitis and nephropathy syndrome since PCV-2 infects mainly lymphoblast and monocytes and with enteritis [92–94]. However, the most prevalent (101). Therefore, the impact of the described viral effect form detected in the field is as PCV-2 subclinical infec‑ in this study is somehow controversial. tions [95]. In a complementary study by the same research group and using the same cells (with the same limitations), the 4.3.1 PCV‑2‑S. suis mixed infections authors suggested that a previous PCV-2 cell infection An analysis of 484 field cases of PCV-2-SD in the USA would enhance bacterial intracellular survival through revealed that most cases were in association with other a decrease in reactive oxygen species. Moreover, the co- pathogens and only 1.9% of investigated cases were infection of tracheal cells with PCV-2 and S. suis would caused by PCV-2 alone [96]. PCV-2 was most often be responsible for a downregulation of the expression of detected with PRRSV (51.9%) and M. hyopneumoniae pro-inflammatory cytokines, which would weaken the (35.5%) [96]. Bacterial septicemia and bacterial pneu‑ host’s defensive response [100]. The results, which have monia were detected in 14% and 7.6% of the PCV-2-SD yet to be confirmed, must be interpreted carefully since it cases, respectively [96]. The most prevalent bacteria has been largely reported that inflammation plays a major detected in bacterial septicemia cases was S. suis. A simi‑ role in the pathogenesis of PCV-2 infections. Again, these lar 2009 study on PCV-2 mixed infections from the same results should be carefully interpreted. region yielded similar results, with S. suis found in In the third in vitro study, a co-infection of the porcine around 35% of PCV-2-SD cases with bacterial septice‑ monocytic cell line 3D4/21 with PCV-2 and S. suis sero‑ mia [97]. Furthermore, a qPCR assay was applied in a type 2 induced a significant upregulation of the expres‑ retrospective epidemiological survey of 121 PCV-2-SD sion of pro-inflammatory cytokines as well as TLR2, cases from Canadian farms to examine the association compared to single-infected cells [21]. The upregulation between PCV-2 and other swine pathogens [98]. The of TLR4 was mainly induced by PCV-2, whereas the study revealed that high PCV-2 loads increased the odds downregulation of MHC-II was observed mostly with S. ratio of isolating A. pleuropneumoniae and S. suis sero‑ suis [21]. Results may indicate a possible role of the co- types 1/2, 1, 2, 3, 4 and 7 from respiratory samples [98], infection on increased inflammation and the modulation Obradovic et al. Vet Res (2021) 52:49 Page 13 of 22 of antigen presentation, although more studies are and neurological outcomes. In an experimental study, required to confirm the hypothesis. nine-week-old piglets were intranasally inoculated with S. suis and a high or low virulent PRV strain (Table 2). 4.3.3 In vivo studies on the interaction between S. suis Interestingly, both PRV strains induced more severe S. and PCV‑2 suis clinical signs in co-infected piglets [107]. The biolog‑ There is only one in vivo study using both pathogens ical relevance of this type of co-infection is still not clear (Table 2) [21]. In this model, four-week-old piglets were since there are no recent experimental or epidemiologi‑ infected with a PCV-2 strain through intranasal and cal data evaluating S. suis and PRV associations. PRV has intramuscular inoculation. Five days later, the animals been eradicated from many countries and, when present, were intranasally and intramuscularly challenged with vaccination has been used to successfully prevent Aujesz‑ a S. suis serotype 2 strain. Compared to single-infected ky’s disease in pigs. This may partly explain the reason pigs, the co-infected group showed higher temperatures why research into its association with other pathogens is and lower weight gains. The amounts of S. suis in the scarce [101]. internal organs of co-infected animals were inconsist‑ Nipah virus is a single-stranded non-segmented RNA ently and slightly higher than those in the S. suis-infected virus belonging to the family of Paramyxoviridae [108, group, although the biological significance of the results 109]. It causes disease in multiple animal species with has yet to be confirmed. Co-infected animals had higher fruit bats and flying foxes being the main reservoirs scores of clinical signs and lesions of pneumonia, myo‑ [108, 110, 111]. Pigs could be naturally infected, but the carditis and arthritis compared to single-infected animals infection is often asymptomatic with the virus present [21]. Peripheral blood mononuclear cells collected from in nasal cavities [112]. In experimental studies, young co-infected piglets showed higher expression levels of piglets displayed pronounced respiratory clinical signs, inflammatory cytokines, TLR2 and TLR4 and reduced while older pigs had predominantly neurological clinical levels of CD4, CD8 and MHC-II. It was also shown that signs [113, 114]. Nipah virus has significant public health PCV-2 disrupted the integrity and decreased mRNA and impact, since it causes disease in humans, especially in protein levels of tight junctions in the lungs of co-infected East and Southeast Asia [115], where human infection animals [99]. Although the authors showed a reduced caused by S. suis is also predominant [49]. In the case of antibody response against S. suis in virus-infected ani‑ experimental oronasal infection in six-week-old piglets mals, a poorly characterized commercial kit available in with Nipah virus, it was observed that the piglets with China only was used [21]. Currently, there is no widely aggravated respiratory clinical signs had S. suis isolated scientifically accepted test to measure antibodies against from the respiratory tissue [116]. This finding should be S. suis [2]. More studies using other strains and different carefully considered, since bacteria were already present animal models must be conducted to confirm the results. in infected pigs (healthy carriers) and the isolation of S. suis was merely accidental [116]. As discussed above, the 4.4 S. suis and other virus co‑infections presence of S. suis in the respiratory tissue does not prove The worldwide distribution and high prevalence of S. suis the involvement of the bacteria in respiratory disease. in pig herds increase the possible interactions with mul‑ Although further studies are required to elucidate the rel‑ tiple viruses. Co-infections of S. suis with pseudorabies evance of S. suis and Nipah virus co-infection, there are virus (PRV) and Nipah virus have been recorded in the technical and economic obstacles to develop pig experi‑ field and experimentally studied in pigs. The common mental co-infection models. The Nipah virus is a biose‑ characteristics of S. suis, PRV and Nipah virus are that curity level 4 pathogen that requires strict measures and they invade through the respiratory epithelium, have tro‑ special research facilities that are not widely available. pism for neurological tissue and are zoonotic pathogens. PRV, also known as Aujeszky’s disease virus, has a sig‑ 5 S. suis mixed and/or co‑infections with other nificant economic importance for the pig industry [102]. bacterial pathogens Infected pigs have respiratory or neurological clini‑ Described mixed swine bacterial infections concern cal signs with subsequent high mortality [103]. Pigs are mainly respiratory pathogens involved in the PRDC, natural hosts, although other domestic and wild animals such as M. hyopneumoniae, Mycoplasma hyorhinis, A. may also be infected [104], and the disease has zoonotic pleuropneumoniae, Actinobacillus suis, Glaesserella par- potential [105]. PRV may be associated with other viral asuis, Bordatella bronchiseptica, Pasteurella multocida pathogens, such as PCV-2, and may be detected together and S. suis [42]. As discussed previously, some are pri‑ using PCR in clinically ill pigs and aborted fetuses [106]. mary pathogens (mainly A. pleuropneumoniae and M. It was speculated that due to the prevalence of S. suis in hyopneumoniae), while others are considered secondary pig herds, PRV co-infection can aggravate respiratory pathogens (S. suis and G. parasuis) [17, 42]. There are Obradovic et al. Vet Res (2021) 52:49 Page 14 of 22 very few real co-infection studies of S. suis with other caballi, S. suis and Streptococcus zooepidemicus were iso‑ bacteria, and most reports address mixed infections. lated from the lungs, indicating that the cause of pneu‑ monia and subsequent death was due to the bacterial 5.1 S. suis mixed infections with other bacterial pathogens mixed infection of the lungs [128]. However, although One of the main problems of most studies is that mixed these bacteria were isolated in high loads from lung and infections are described at the abattoir [32, 117]. For tracheo-bronchial lymph nodes, it was not possible to primary respiratory pathogens that induce fairly typi‑ determine if they were the primary cause or secondary cal lesions and affect grower-finisher animals (such as invaders [128]. Furthermore, the transport of the horses A. pleuropneumoniae), the studies are appropriate [118]. was lengthy and during winter months—two factors When available, the combination of serological surveys that may have created major environmental stress that based on validated antibody detection techniques (for enhanced bacterial growth and susceptibility to infection example against A. pleuropneumoniae and M. hyopneu- [128]. moniae) at the herd level along with the presence of The isolation of S. suis with other bacterial pathogens lesions at slaughter may indicate the pathogens’ involve‑ in tissues other than lungs has also been reported. S. ment in the pulmonary pathology of pigs [119]. However, suis was isolated with M. hyorhinis or M. hyopneumo- for a secondary respiratory pathogen such as S. suis that niae from the pericardium [129]. Another study reported mainly affect nursery piglets and for which validated mixed infections of G. parasuis and S. suis in cases of serological tests are not available, the studies are far less polyserositis [130]. Both pathogens were also detected relevant. As mentioned above, S. suis sometimes colo‑ together in more than 30% of samples, although the tis‑ nizes the lungs pre- or ante-mortem without playing any sue/organ of origin (lungs versus internal organs) was not major pathological role in pneumonia [2, 119]. reported [131]. Finally, a case of abortion with dual iso‑ Early studies with organs recovered from diseased lation of S. suis and Arcobacter spp was described [132]. pigs showed that S. suis was frequently isolated in con‑ Mixed infections caused by different serotypes of S. suis junction with A. pleuropneumoniae, P. multocida, E. coli should also be considered, as there have been recorded and many other microorganisms, although it is not indi‑ cases of meningitis and septicemia in a seven-week-old cated exactly how many of these cases were from lungs piglet caused by S. suis serotype 3 cultured from the versus other internal organs/tissues [120, 121]. It has spleen and bursa and a S. suis serotype 7 recovered from been reported that S. suis was isolated with many other the cerebrospinal fluid [133]. It should be noted that all bacterial pathogens in more than 75% of lungs display‑ cases discussed in this section represent sporadic mixed ing fibrino-hemorrhagic pneumonia [122]. S. suis and P. bacterial infections without any clear indication of the multocida were isolated together in almost 50% of lungs specific role of co-infections in the development of from animals suffering from respiratory clinical signs in disease. China [123]. In addition, there was a reported case of concurrent lung infection of S. suis and P. multocida in 5.2 In vitro and in vivo studies on the interaction conjunction with Pneumocystis carinii [124]. Using more between S. suis and other bacterial pathogens sensitive PCR techniques, statistical associations were One in vitro study addressed a co-infection between S. found in samples from lung tissues between PCR-positive suis and G. parasuis using NTPr cells and PAMs (Table 3) results for P. multocida or A. pleuropneumoniae and S. [54]. G. parasuis is a causative agent of Glässer’s disease suis [32]. Interestingly, a case of pneumonia in a sow with in pigs. Similar to S. suis, low virulent strains colonize mixed infections of S. suis and B. bronchiseptica was also the upper respiratory epithelial surface of healthy ani‑ described, although S. suis causing disease in adult ani‑ mals, while high virulent strains can cross the epithelial mals is extremely rare [125]. Another study indicated that barrier and cause mainly systemic disease and (probably) almost 30% of affected lungs yielded common isolation of pneumonia (as a secondary invader) [134]. The disease is these two pathogens [126]. The association of both S. suis characterized by polyserositis and polyarthritis [134]. G. and B. bronchiseptica was further studied in in vitro and parasuis and S. suis infections also share similar clinical in vivo models (see below). Interesting, there is only one signs that frequently represent a challenge in the clini‑ report indicating a possible correlation between S. suis cal differential diagnosis. The in vitro model using NTPr and M. hyopneumoniae in the lungs [127]. cells showed that low and high virulent strains of S. suis The indication of the involvement of S. suis in mixed and G. parasuis have a different pattern of adhesion to infections as a cause of pulmonary disease may also be and invasion of epithelial cells as single infections [54]. found in other animal species. There was a reported However, co-infection studies show limited in vitro inter‑ case of acute death of a racehorse with clinical signs of action between the two bacterial species, which likely use pneumonia during transport in Japan [128]. Pasteurella different host receptors. Although it has been found that Obradovic et al. Vet Res Table 3 Summary of the experimental conditions of S. suis co-infection with bacterial pathogens using in vitro models Pathogen studied Type of cells or growth Co-infection bacterial S. suis serotype 2 strain Co-infection dose Goal of the study Conclusions Refs. medium strain(s) (2021) 52:49 Glaesserella parasuis Neonatal tracheal porcine G. parasuis serotype 6 (low ST1 (high virulence) and Adhesion/invasion assays: Adhesion and invasion; No effect [54] cell line (NTPr) cells virulence) and serotype ST25 (intermediate 5 MOI (both bacterial expression of inflamma- 5 (high virulence) virulence) species) tory genes (qRT-PCR) Gene expression: 10 MOI (both bacterial species) Pulmonary alveolar mac- G. parasuis serotype 6 (low ST1 (high virulence) and Adhesion/invasion assays: Adhesion/invasion, No effect [54] rophages virulence) and serotype ST25 (intermediate 5 MOI (both bacterial phagocytosis, inflam- 5 (high virulence) virulence) species) matory gene expres- Gene expression: sion, and expression of 10 MOI (both bacterial surface markers species) *Phagocytosis: 100 MOI (both bacterial species) Expression of surface markers: 500 MOI/ 50 MOI for G. parasuis/S. suis Actinobacillus pleuro- Medium for bacterial App (no further details on No details App and S. suis mixed in Biofilm formation, anti- ↑ Biofilm formation (App), [136] pneumoniae (App) culture (no cells) the strain) 1:1 ratio biotic resistance, and antibiotic resistance expression of genes for and genes coding for virulent factors different (putative) viru- lence factors for both pathogens Bordetella bronchiseptica Porcine precision-cut lung Field strain of B. bron- ST1 (high virulence) 104 and ­104 CFU/well for Effect of a pre-infection ↑ Bacterial adherence, [137] slice chiseptica B. bronchiseptica and S. with B. bronchiseptica on colonization and cell suis, respectively adhesion, colonization toxicity and toxicity of S. suis * Cells were infected with S. suis first and then with G. parasuis; ST: sequence type. Page 15 of 22 Obradovic et al. Vet Res (2021) 52:49 Page 16 of 22 the capsular polysaccharide of S. suis serotype 2 has anti- conditions where the pre-infection of B. bronchiseptica phagocytic properties against a heterologous bacterial induced reduction of ciliary activity in the absence of sig‑ species, pre-treatment of PAMs with S. suis did not have nificant toxicity. Under these conditions, it was shown a clear effect on the phagocytosis of G. parasuis. When that S. suis significantly increase adhesion and coloniza‑ inflammatory mediators produced by both epithelial and tion, although both pathogens seem to adhere and colo‑ PAM co-infected cells were studied, an additive (but not nize different areas of tissue. In addition, the increased synergistic) effect was observed. Virulence factors of G. adhesion of S. suis resulted in higher cytotoxicity due to parasuis and S. suis are not fully known and other condi‑ the presence of suilysin [137]. Although not confirmed, tions in respiratory mucosa may play an important role in in vivo experiments suggest that a pre-infection with B. G. parasuis and S. suis co-infections [7, 135]. bronchiseptica may promote not only adhesion but also A recent report suggested that co-infections between invasion into deeper tissues (see below). A. pleuropneumoniae and S. suis are frequently found in In vivo studies are also very limited (Table 4). There is China, although no data were presented [136]. However, one report in which S. suis and Aerococcus viridans were the authors hypothesized that there are synergistic inter‑ isolated together from a pig with meningitis in China actions between both pathogens. They reported that, [138]. A. viridans has been isolated mostly from swine when A. pleuropneumoniae was cultured with S. suis in and humans [139, 140]. However, because A. viridans is culture media, biofilm formation by A. pleuropneumo- a bacterium that is present in the environment, it may niae was significantly increased. Moreover, compared to simply be a contaminant in a sample. To examine the cultures with a single bacterium species, the antibiotic potential of S. suis and A. viridans interaction, Pan et al. resistance, as well as the expression of genes coding for used an intraperitoneal mouse model of co-infection different (putative) virulence factors of both pathogens, using both bacterial strains recovered from the above- were enhanced in the co-culture model. The authors sug‑ mentioned case of meningitis [138]. Results showed that gest that the interspecies interactions between S. suis and the co-infection significantly increased systemic infec‑ A. pleuropneumoniae may be cooperative under specific tion and acute meningitis [138]. Titers of S. suis were conditions and may play an important role in disease significantly increased in the lungs of co-infected mice, progression and persistent infection [136]. It should be while titers of A. viridans in lungs remained at a low level noted, however, that both pathogens affect piglets of dif‑ [138]. It should be noticed that colonization of the lungs ferent ages, with A. pleuropneumoniae mainly causing was probably the result of septicemia (due to the injec‑ disease in grower-finisher animals and S. suis affecting tion route) and does not indicate a respiratory infection. mostly nursery piglets [36]. Indeed, the implications of Further research is required to see if these results could the results obtained in the S. suis-A. pleuropneumoniae be reproduced in the swine model and examine the cel‑ co-infection study should be carefully interpreted, as they lular and molecular mechanisms involved in this type of probably do not reflect the situation in the field. co-infection. However, it is important to keep in mind A last in vitro study became available very recently that his type of co-infection is almost never observed [137], where the interaction of B. bronchiseptica and in the field and that the presence of A. viridans in the S. suis was analyzed using the PCLS system previously original clinical sample could have simply represented a reported (81). Interestingly, the authors established contamination. Table 4 Summary of the experimental conditions of S. suis co-infection with bacterial pathogens using in vivo models S. suis Age of animals Age of animals 1st bacteria 2nd bacteria 1st bacteria and 2nd bacteria Conclusions Refs. coinfection infected with infected with route of route of dose and dose pathogen 1st bacterial spp. 2nd bacterial infection infection spp. Aerococcus Five-week-old Five-week-old IP IP 103 – ­108 CFU 103 – ­108 CFU ↑ Mortality [137] viridans BABL/c mice BABL/c mice S. suis « novel A. viridians serotype» (no further details on the strain) Bordetella bron- Five-day old Seven-day-old IN IN 0.5 × ­107 CFU 0.7 × ­106 CFU; ↑ Clinical signs [141] chiseptica germ-free germ-free B. bronchiseptica serotype 2, piglets piglets ST1, different phenotypes IP: intraperitoneal injection; IN: intranasal inoculation; CFU: colony-forming units; ST: sequence type. Obradovic et al. Vet Res (2021) 52:49 Page 17 of 22 The most important in vivo study has been carried out the media may have a major impact on pathogen interac‑ with S. suis and B. bronchiseptica [141]. Interestingly, the tions, growth and survival. For example, the expression of original goal of the use of B. bronchiseptica in this study certain metabolic and virulent bacterial genes by S. suis was simply to predispose pigs to the intranasal infection may be different or reduced in nutritionally rich media with S. suis. Indeed, as mentioned above, it is difficult to compared to in vivo-like environments [143]. To repro‑ reproduce S. suis disease with conventional pigs using duce cell interactions in a more complex environment, this route of infection. In this study, a B. bronchiseptica the development of ALI and PCLS systems constitutes strain isolated from the nose of a pig with regressive a major improvement to study S. suis co-infections [81, atrophic rhinitis was used. In other words, the study was 82]. Enhanced in vitro systems such as three-dimensional oriented to a local infection (nose) affecting the mucosa organotypic tissue models that more closely resemble rather than a lower respiratory infection (lungs). Indeed, the 3D architecture, cellular composition and matrix in further studies, acetic acid was used to replace B. bron- complexity of the organ may be further developed. The chiseptica infection [33]. In the co-infection study, S. suis adaptability of these tissue-engineered models to multi‑ strains that were phenotypically different with regard to ple pathogens suggests significant potential for infectious virulence markers were used. Only S. suis strains that disease studies [86]. harbored the proposed virulence markers were able to In many cases, the innate immune system plays an induce disease. Interestingly, S. suis was detected in the important role in the outcome of the co-infection and, lungs with bronchopneumonia only when B. bronchisep- as such, many studies focus on the interaction with the tica (at higher concentrations) was also present. Indeed, immune cells. In comparison with mouse or human B. bronchiseptica was recovered from the lungs of 19 ani‑ immunology, there is still a lower number of immunolog‑ mals, whereas S. suis was present in 8 samples. None ical tools designed to detect and analyze swine immune of the samples contained S. suis only. The authors con‑ cells [144]. However, since the field of swine immunology cluded that S. suis may be considered a secondary agent is evolving rapidly, we can expect that more sophisticated of pneumonia [142]. In vitro results recently obtained immunological tools will be developed in the near future and discussed above may explain mechanisms involved and significantly expand our knowledge of the immune in the interaction of both bacterial species [137]. response induced during these co-infections. The abundance of bacterial pathogens found in epide‑ In general, for many infectious agents, in vivo mod‑ miological association with S. suis, especially in pneumo‑ els of co-infections are sometimes difficult to reproduce nia cases, is not followed by the appropriate number of [17]. This is even truer when referring to S. suis: co-infection studies to understand the role and mecha‑ nisms involved in such interactions. In vivo studies of a. Firstly, S. suis is a normal inhabitant and almost all co-infection of S. suis with other important bacterial conventional animals used in experimental infections pathogens are not available, and it is therefore difficult are already colonized. Since there are no sensitive to speculate on the significance of each pathogen in the and specific validated tests (neither PCR nor sero- inception of disease. logical tests) to evaluate whether animals are carri- ers of virulent strains, it is difficult to predict how the 6 Limitations of S. suis co‑infection studies animals will behave after experimental infection. We Generally speaking, the limitations of co-infection stud‑ have already recovered S. suis serotypes 1 and 14 iso- ies are well addressed by Saade et al. [17]. More specifi‑ lates from the internal organs (one case for each cally with regard to S. suis, although mixed infections serotype) of animals experimentally infected with studies may be taken into consideration, it is important a virulent serotype 2 strain, possibly due to a previ- to interpret the data on a real synergic effect in the field ous infection present in the animals’ herds of origin carefully, and reports from lungs recovered at the abat‑ (unpublished observations). On the other hand, the toir should be avoided. Indeed, the fact that S. suis is a use of caesarian-derived, colostrum-deprived piglets normal inhabitant of the upper respiratory tract should (highly susceptible to S. suis) constitutes a very use- not be neglected. Regarding in vitro models of co-infec‑ ful tool to generate knowledge but somehow remains tion, several have limitations and room for improvement. relatively far from the reality of the field. S. suis infection models using different immortalized or b. Another issue is that the routes of S. suis inocula- primary cells are still useful to study the mechanisms of tion (even with virulent serotype 2 strains) are rather gene expression and cellular pathways induced by viruses artificial (intravenous, intraperitoneal, intramuscu- and bacteria and may shed light on the molecular interac‑ lar) due to the difficulties to reproduce the disease in tions that occur during co-infections. It should be noted conventional animals [16]. A seeder-to-naïve model, that the choice of cell type and nutritional components of as developed for other infections such as A. pleuro- Obradovic et al. Vet Res (2021) 52:49 Page 18 of 22 pneumoniae [145], has yet to be established. In co- be adapted to S. suis co-infection studies: (i) authors infection studies, these procedures complicate the should clearly indicate and characterize the S. suis interpretation of the results, since the first pathogen strains used (e.g., serotype, sequence type, presence frequently infects animals through the respiratory of virulence markers and other phenotypic character‑ tract, and both pathogens should be evaluated under istics); (ii) well-characterized S. suis strains of sero‑ similar conditions. types other than type 2 should also be used, especially c. As mentioned, the virulence of serotype 2 strains in North American studies in which other serotypes from different geographical origins may be different (e.g., serotype 1/2) may be more prevalent than sero‑ [2], and almost no co-infection studies were con- type 2; (iii) in in vivo studies, the route of infection ducted with well-characterized North American should follow the natural infection of both pathogens, serotype 2 strains with lower virulence capacity when which should be able to interact in the same environ‑ compared to their Eurasian counterparts. ment; (iv) since S. suis mainly causes disease in nurs‑ d. There are almost no studies with serotypes other ery piglets, co-infection studies should mostly address than serotype 2, such as serotype 1/2 which is highly pathogens affecting animals at a similar age; (v) no test prevalent in North America. This is mainly due to the is specific or sensitive enough to monitor the status of fact that experimental infection models with non- S. suis infection in a herd. Authors should avoid refer‑ serotype 2 strains are rarely developed. ring to the use of S. suis serotype 2-free animals based e. Environmental and management factors may highly on serological (no scientific test available) and/or ton‑ influence the development of the disease by S. suis. sillar PCR evaluations, being the latter low sensitive. These factors are usually not present under experi- Although some genes involved in the susceptibility mental conditions in well-controlled research envi- or resistance to S. suis disease have been suggested in ronments. In this regard, recent experiments on S. mice [143], no study has addressed the issue in pigs, suis mucosal infection models tried to mimic, at least and it is therefore not possible to identify animals sus‑ in part, the weaning stress that piglets experience ceptible to the infection yet; (vi) authors should try to under field conditions by applying several stressors, use animals from farms with no or limited clinical cases like transport and social stresses [4]. (and serotype different from the one to be evaluated) of S. suis where no prophylactic/metaphylactic procedures are in place at the nursery; (vii) re-isolation of S. suis, 7 Conclusion and future perspectives serotyping and, if possible, pathotyping are mandatory Co-infections of S. suis with important swine patho‑ in experimental co-infection studies using conventional gens have been addressed in recent studies, and new animals, since S. suis other than the strain used during mechanisms of pathogen interactions of viral and bacte‑ the co-infection may be isolated from diseased animals, rial pathogens have been proposed. However, owing to especially the lungs. Improved and more standardized the importance given to co-infections in S. suis-associ‑ models will generate new data on the intricate process ated diseases, the number of studies (mostly addressing of S. suis interactions with major swine pathogens. mechanisms) is considerably low. In general, viral patho‑ Finally, there may be additional effects of a previous gens such as SIV may destroy the epithelial barrier and infection with a pathogen on a secondary S. suis infec‑ increase the permeability of the mucosal surface, while tion. For example, it has been reported that a previous PRSSV may have a negative impact on the functions of infection with PRRSV may alter the efficacy of cefti‑ pulmonary alveolar and intravascular macrophages and ofur treatment of S. suis co-infection [146]. Whether a PCV-2 has a strong tropism for lymphoblasts. However, previous virus infection affects the antibody and pro‑ how these mechanisms actually affect S. suis pathogenic‑ tective response of S. suis autogenous vaccines is also ity is still debatable. Similarly, an increased inflammation unknown [26]. (a hallmark of S. suis infections) caused by co-infection with other pathogens may also play an important role. Abbreviations However, most co-infection studies showing upregula‑ ALI: Air–liquid culture system; BMDC: Bone marrow-derived dendritic cells; DCs: Dendritic cells; MDCK: Madin-Darby canine kidney cells; NTPr: Neona- tions of inflammatory mediators were conducted in vitro tal tracheal epithelial cells; PAMs: Pulmonary alveolar macrophages; PCLS: and the data must still be validated in vivo within the Precision-cut lung slice; PCV-2: Porcine circovirus 2; PCVAD: Porcine circovirus natural host. Finally, data on co-infections (and mecha‑ associated diseases; PCVD: Porcine circovirus diseases; PMWS: Postweaning multisystemic wasting syndrome; PRDC: Porcine respiratory disease complex; nisms involved) of S. suis with other bacterial pathogens PRRSV: Porcine reproductive and respiratory syndrome virus; PRV: Pseudora- are mostly unavailable. bies virus; ST: Sequence type; SIV: Swine influenza virus. Saade et al. made general recommendations for future co-infection studies [17]. 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