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Austral journal of veterinary sciences

versión impresa ISSN 0719-8000versión On-line ISSN 0719-8132

Austral j. vet. sci. vol.54 no.3 Valdivia set. 2022

http://dx.doi.org/10.4067/S0719-81322022000300093 

REVIEW ARTICLE

Adhesion mechanisms of Actinobacillus pleuropneumoniae to the porcine respiratory system and biofilm formation

Eduardo Hernández-Cuellara 

Alma L. Guerrero-Barreraa 

Francisco J. Avelar-Gonzálezb 

Juan M. Díazc 

Jesús Chávez-Reyesd 

Alfredo Salazar de Santiagoe 

1aLaboratorio de Biología Celular y Tisular, Departamento de Morfología, Universidad Autónoma de Aguascalientes, Aguascalientes, México.

2b Laboratorio de Ciencias Ambientales, Departamento de Fisiología y Farmacología, Universidad Autónoma de Aguascalientes, Aguascalientes, México.

3c Unidad Médico Didáctica, Centro de Ciencias de la Salud, Universidad Autónoma de Aguascalientes, Aguascalientes, México.

4d Laboratorio de Farmacología y Terapéutica Experimental, Departamento de Fisiología y Farmacología, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico.

5e Unidad Académica de Odontología, Área de Ciencias de la Salud, Universidad Autónoma de Zacatecas, Zacatecas, México

ABSTRACT:

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the causative agent of porcine pleuropneumonia, a highly contagious disease of pigs characterised by fibrinohaemorrhagic necrotising pneumonia. Although it has been well controlled in some developed countries, outbreaks can occur in pigs of all ages in contact with asymptomatic carriers, leading to significant economic losses to the swine industry due to the high morbidity and mortality rates. Adhesion is a critical step in the colonisation of the swine respiratory tract and the pathogenesis of the porcine pleuropneumonia; however, a literature review of this process is not available to date. Therefore, this review aims to provide information regarding the molecules that have been described in the adhesion of A. pleuropneumoniae to cells and tissues of the porcine respiratory tract. Since adhesion is the first step in biofilm formation, we included a section to describe the genes involved in this process; some of these genes could participate directly or indirectly in the adhesion of A. pleuropneumoniae to the porcine respiratory system. Although the role of biofilms in porcine pleuropneumonia is still not clear, these molecules could be considered in the future as candidates for vaccine development.

Keywords:  Actinobacillus pleuropneumoniae; porcine pleuropneumonia; adhesion mechanisms; biofilm formation

INTRODUCTION

Porcine pleuropneumonia is a highly contagious disease in pigs of worldwide distribution. Actinobacillus pleuropneumoniae (AP) is a small encapsulated, gram negative rod and the etiological agent of this disease, which is characterised by fibrinohaemorrhagic and necrotising pneumonia that often follows a fatal course during acute presentations (Chiers et al., 2010). AP can be found in the nostrils, tonsils and lungs of infected pigs, and it can also be found in asymptomatic carriers previously infected or with a subclinical infection (Sidibé et al., 1993; Chiers et al., 2001). It is also known that pigs with chronic infection have deficient feed conversion and weight gain (Sassu et al., 2018). In addition, research on naturally and experimentally infected animals suggests that the natural course of infection starts with the presence of AP in the upper respiratory tract, progressing all the way from the nasal cavities to the lungs; here the bacterium induces lesions and the production of neutralising antibodies. Also, tonsils seem to act as a reservoir of AP (Chiers et al., 2001). Interestingly, it has been shown that there is no detection of neutralising antibodies in the serum of pigs that were positive for the presence of AP in the nasal cavity and/or tonsils, but negative for the presence of infected lung lesions, indicating a subclinical infection of pigs carrying the bacterium (Chiers et al., 2002). Based on the requirement of nicotinamide adenine dinucleotide (NAD), two biotypes of AP have been described. In addition, there are 19 serotypes of AP based on differences in the antigenic properties of the capsular polysaccharides (Stringer et al., 2021). It is known that there is a predominant serotype in herds endemically infected, however, more than one serotype has been isolated in some herds (Sidibé et al., 1993). In addition, some of the described virulence factors are AP involved in adhesion, nutrient acquisition, induction of lung lesions, evasion of the immune system and persistence in the host (Chiers et al., 2010). Furthermore, the severity of the disease is not only influenced by the bacterium, but also due to intrinsic factors such as the nutritional status and the immune system of the host and extrinsic factors related to environmental stress (Chiers et al., 2010). There are some variations in the virulence among serotypes, and this could be in part attributed to the production of different combinations of Apx exotoxins, which differ among them in their cytotoxic and hemolytic activities (Hernández-Cuellar et al., 2021). In this regard, ApxI expressed in serotypes 1, 5a, 5b, 9, 10, 11, 14, and 16 is highly haemolytic and cytotoxic, ApxII expressed in all serotypes but 10 and 14 are slightly haemolytic and moderately cytotoxic, and ApxIII expressed in serotypes 2, 3, 4, 6, 8, and 15 is non-haemolytic but highly cytotoxic (Sassu et al., 2018). Also, it has been recently described that AP can internalise not only to phagocytic cells but to non-phagocytic endothelial cells (Plasencia-Muñoz et al., 2021). However, the first step for the bacteria to colonise the porcine respiratory system is the adhesion to epithelial cells or extracellular matrix components. There is limited information available in the literature related to the adhesion mechanisms of AP even though this event represents the initial step in the establishment of the infection (Jacques & Paradis 1991). Therefore, the next section aims to present the most relevant findings regarding the adhesion molecules in AP described to date.

ADHESION MECHANISMS OF AP TO THE PORCINE RESPIRATORY TRACT

BINDING TO EXTRACELLULAR MATRIX COMPONENTS

Adhesion to porcine respiratory tract mucus was initially evaluated for 17 AP isolates. It was observed that ~70% of the isolates showed affinity to the mucus and this feature was independent of the serotype. It was also found that the presence of a capsule or a high capsular thickness decreased the adherence to the mucus (Bélanger et al., 1992). In a similar study, AP serotype 1 was able to bind in vitro to swine-lung collagen in a Ca2+-dependent manner. By using an overlay assay, it was shown that an unknown 60 kDa outer-membrane protein was able to bind to collagen and fibrinogen, but not to fibronectin and laminin (Enriquez-Verdugo et al., 2004). Hammer-Barrera et al. (2004) showed that adhesion by AP serotype 1 was higher to swine buccal epithelial cells (BEC) in comparison with their cell counterparts of human or rat origin. Treatment with proteolytic enzymes and periodate highly decreased the adherence to swine BEC, suggesting the participation of cell-surface glycoproteins in the adhesion of AP to these cells. Interaction with fibrinogen or fibronectin resulted in reduced adherence to swine BEC, suggesting also the adhesion of bacteria to these extracellular matrix components (Hamer-Barrera et al., 2004). We suggest that these extracellular matrix surfaces may be helpful for AP, an extracellular bacterium, to attach and progress from the upper to the lower porcine respiratory tract.

ROLE OF LPS AND CAPSULE IN THE ADHESION

Using porcine tracheal rings with ciliated epithelial cells maintained in culture, it was found that isolated AP serotypes 1, 2, 5, and 7 had variation in the adhesion capacity among serotypes or even within isolates of the same serotype. To analyse the role of the capsule on the adhesion, two capsulated isolates and their unencapsulated variants were tested and no differences were found in the adhesion index, suggesting that capsule was not involved in this process. There were differences in the lipopolysaccharide pattern of the bacterial isolates when a whole-cell lysate was subjected to a treatment with proteinase K. Based on this, isolates with a smooth-type lipopolysaccharide (75% of isolates with serotype 2 and 7) adhered in large number to porcine tracheal rings while isolates with a semi rough-type lipopolysaccharide (serotypes 1 and 5) adhered poorly (Bélanger et al., 1990). It is worth mentioning that lipopolysaccharide varies in structure among bacteria but possessed three different regions attached covalently, the lipid A, an oligosaccharide core, and the O-antigen. Lipopolysaccharide with O-antigen is referred to as smooth type, while rough-type lipopolysaccharide does not contain O-antigen (Steimle et al., 2016). Interestingly, purified lipopolysaccharides from homologous AP reference strains inhibited the bacterial adhesion to the porcine tracheal rings. Therefore, lipopolysaccharides were proposed for the first time as molecules important for the adhesion of AP to ciliated epithelial cells of the trachea (Bélanger et al., 1990).

Similar research related to the adhesion capacity of lipopolysaccharides in AP showed by flow cytometry and electron microscopy that these molecules were well exposed at the surface of the encapsulated AP analysed. In addition, immunostaining showed that the lipopolysaccharide extracted from AP serotype 1 and 2 adhered to lung vascular endothelium and tracheal epithelium when incubated with porcine lung or tracheal frozen sections, respectively. To know which part of the lipopolysaccharides had the adhesion capacity, an extract of lipopolysaccharides from AP was obtained and hydrolysed. Through an adhesion- inhibition assay, it was found that the polysaccharide moiety was responsible for the adhesion of AP, while the lipid A was dispensable in this process (Paradis et al., 1994). In addition, it was shown in an experiment trying to simulate the adhesion to cell membranes that AP serotype 1, 5b, and 7 were able to bind to phosphatidylethanolamine (PE) but not to other phospholipids. It was suggested that the lipopolysaccharide O-antigen was responsible for the binding to PE (Jeannotte et al., 2003).

Contrary to the findings showing that lipopolysaccharides play an important role in the adhesion of AP to tracheal and lung epithelial cells, a different workgroup analysed the adhesion of several strains of AP to primary cultures of porcine lung epithelial cells (LEC). It was found that adhesion of AP was faster and up to 30-fold more efficient for LEC than for swine kidney cells. However, adhesion to LEC did not change for a transposon mutant with a modification in the lipid A moiety of the lipopolysaccharide or even resulted in a three-fold more adhesion for a mutant lacking O antigen compared to the parent strains. Furthermore, lipopolysaccharides purified from AP serotype 1, 3, 7, and 8 did not alter the adhesion of AP serotype 8 to LEC (Boekema et al., 2003). These results clearly show that the mechanisms of adhesion for AP could be different depending on the surface of the porcine respiratory system.

In another study, 23 AP isolates were evaluated on their ability to adhere in vitro to porcine tracheal epithelial cells or frozen lung sections. Different to the frozen lung sections, adherence to the tracheal epithelial cells was very poor and, in both cases, there was no correlation of the adherence with the serotype of the AP isolates. However, contrary to the aforementioned study, two unencapsulated variants adhered in greater numbers to the lung sections compared to the capsulated parent strains (Jacques et al., 1991). Similarly, using a transposon mutagenesis system to generate an AP serotype 1 capsule-deficient mutant, it was found that the mutant strain showed more adhesion to porcine tracheal frozen sections than the parent strain. However, it was less virulent in pigs and it did not induce mortality. It was described that the product of mutation was the protein CpxC involved in polysaccharide transport across the cytoplasmic membrane during the biosynthesis of capsular polysaccharides. It was also concluded that the capsule was not important for adherence and may even mask an outer membrane protein important for adhesion (Rioux et al., 2000).

OUTER MEMBRANE PROTEINS INVOLVED IN ADHESION

Adhesion of AP serotypes 2, 5a, 9, and 10 to alveolar epithelial cells showed that optimal adherence was obtained in NAD-restricted medium for strains 5a, 9, and 10. Interestingly, under this condition, it was expressed an outer membrane protein of 55 kDa and the presence of fimbriae was observed by electron microscopy. However, the sequence of the N-terminal of this outer membrane protein did not correspond to any known protein. Bacterial adhesion was significantly reduced when treated with proteolytic enzymes. This finding suggested that besides lipopolysaccharides, proteins are also important for the adhesion of AP. Furthermore, treatment of AP with a combination of pronase and sodium metaperiodate produced a higher inhibition of the adherence to alveolar epithelial cells compared to reagents being used separately. Therefore, glycoproteins could also be involved in the adhesion of AP to these cells (Overbeke et al., 2002).

It was found that 170 genes were differentially expressed in AP attached to St. Jude porcine lung cell line (SJPL) compared with detached bacteria in the medium (planktonic). Two genes called TadB and rcpA, potentially involved in adhesion and biofilm formation were upregulated. Also, a gene (APL_0443) with high homology to the Hsf autotransporter adhesin of Haemophilus influenzae was upregulated (Auger et al., 2009). This Hsf-like autotransporter called Apa1 (AP antigenic protein) was previously found to be expressed by AP in necrotic porcine lung tissue (Baltes et al., 2004). Sequence analysis of the C-terminal region of Apa1 showed a translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains among different strains of AP. Adhesion to SJPL cell monolayers was tested by confocal microscopy through a GST fusion protein methodology in which GST was bound to the six ApaBD (HsfBD-like) domains. GST-ApaBD3 showed strong fluorescence while the other five domains had only basal fluorescence. It was confirmed the adhesion ability of ApaBD3 to epithelial cells through an adherence inhibition assay with a recombinant E. coli-ApaBD3 that expresses the domain on the surface (Xiao et al., 2012). It was shown later an extra N-terminal domain (residues 124-612) of the trimeric autotransporter Apa1 called Adh that was required for adhesion, autoaggregation, and biofilm formation (Wang et al., 2015).

In another study, the outer membrane protein Lip40 was described to mediate adherence of AP to SJPL cells using a mutant strain, Alip40. Interestingly, the mutant strain had also a reduced ability to invade the lungs of infected mice. Also, in an infection assay with pigs, the mutant strain produced fewer clinical signs (dyspnea, lethargy, and fever) and lung invasion than the wild-type or complemented strain (Liu et al., 2018). These findings suggest a critical role of the adhesion process in the virulence of this bacterium.

GENES INVOLVED IN FIMBRIAE FORMATION

The presence of fimbriae in AP has been previously described (Utrera et al., 1991, Dom et al., 1994, Overbeke et al., 2002) with the identification of ApfA, a 17 kDa type 4 fimbrial subunit protein (Zhang et al., 2000). An operon (apfABCD) consisting of four genes involved in type 4 fimbrial biogenesis was also proposed (Stevenson et al., 2003, Boekema et al., 2004). ApfA was found to be highly conserved among the different serotypes of AP. Also, it was suggested as an adhesin since its expression was greatly upregulated upon contact of AP with the SJPL cell line. Adhesion to SJPL cell line and porcine iliac artery endothelial cell line (PIEC cells) decreased significantly for AP 4074AapfA, a mutant strain deficient in ApfA. Furthermore, recombinant ApfA blocked the adhesion of AP to those cell lines. Interestingly, it was shown that ApfA mediates colonisation of AP to the lungs of infected mice, as the mutant strain AP 4074 AapfA had reduced bacterial loads in lungs compared with mice infected with the wt AP strain 4074. Also, using a purified recombinant ApfA protein, it was found an elevated humoral immune response and protection against AP in an infection model in mice, proposing this fimbrial subunit as a promising vaccine candidate (Zhou et al., 2013).

Two component systems (TCS) play important roles in adaptation to changes in the environment. Through genomic analysis, it has been described that AP have five pairs of TCS: ArcA/ArcB, CpxR/CpxA, NarP/NarQ, PhoB/PhoR, and QseB/QseC. It was analysed through a microarray the changes in the gene expression profile between a QseB/ QseC deficient AP strain and the corresponding parent strain AP 4074. The expression of 44 genes was shown to be different, with 27 of them being up-regulated and 17 down-regulated. The expression levels of some of these genes, such as PilM were validated using qRT-PCR. Also, with an electrophoretic mobility shift assay (EMSA), it was shown that a phosphorylated recombinant QseB (rQseB-P) was able to bind to the promoter sequence of

PilM. An AP deficient in the expression of PilM showed a significant decrease in the adherence to SJPL cell line and was less virulent in pigs (Liu et al., 2015). It was later found that the apfABCD and PilMNOPQ gene clusters were operons conserved in all the AP serovars and their products (apfA, apfB, apfC, apfD, pilM, pilN, pilO, pilP, andpilQ) are required for Tfp (a type IV pili) biogenesis, biofilm formation, and adhesion to SJPL cells (Liu et al., 2018).

The presence of the flp operon consisting of 14 genes (flp1-flp2-tadV-rcpCAB-tad-ZABCDEFG) was described in AP. In reference strains with serotypes 1, 4, 5, 7, 12, and 13, the complete operon was identified. However, the flp promoter was absent in serotypes 2, 3, 6, 9, and 11, and for serotypes 10 and 15, the flp1 gene was truncated resulting in the absence of pilus as observed by transmission electron microscopy. Adherence to SJPL cells resulted to be higher for piliated strains (Li et al., 2012). Later, it was shown that the genes flp1 and tadD were essential for Flp pilus biosynthesis using AP mutants in which biofilm formation and adherence to SJPL and porcine iliac artery endothelial (PIEC) cell lines was reduced. Also, those mutants lacking flp1 and tadD resulted in deficient colonisation with reduced bacterial loads in the lungs of infected mice and pigs (Li et al., 2019).

GLYCOSYLATION SYSTEMS

It was reported in AP the crystal structure of HMW1C, a glycosyltransferase of the GT41 family that was previously described in Haemophilus influenzae. HMW1C creates N-glycosidic linkages on HMW1, an adhesin that mediates adherence to respiratory epithelial cells (Kawai et al., 2011). A recent study described the role of the cytoplasmic N-linked glycosylation system of AP (NGT) in the adhesion to A549 cells, human adenocarcinoma lung epithelial cells. A putative NGT locus consisting of rimO (methylthiotransferase) was proposed and the glycosyltransferases ngt and agt. Using AP strain HS143 to generate mutants deficient in agt and ngt, it was shown that the adhesion to cells was almost abrogated for the mutant strains HS143Aagt and HS143Angt (Cuccui et al., 2017). Table 1 summarises all the molecules that have been described in the adhesion of AP to surfaces related to the porcine respiratory system.

BIOFILM FORMATION

Biofilms are defined as communities of microbes embedded in an extracellular matrix, conferring them protection against environmental stress, host defence, and antibiotics (Hathroubi et al., 2018). Most of the experimental studies in biofilm formation have been on abiotic surfaces such as polystyrene microplates. Although the ability to form biofilms has been associated with the virulence of AP, it is still not clear how this process contributes in vivo to the pathogenesis of the porcine pleuropneumoniae (Hathroubi et al., 2018). In this respect, the presence of AP aggregates in the lungs of pigs naturally infected has been reported (Tremblay et al., 2017). In addition, it was described the ability of AP to form biofilms on a biotic surface, using a monolayer culture of SJPL cells in which the bacterium formed biofilms at later times (~24h) in comparison with the highest biofilm formation in microplates at 4h. This biofilm formation was associated with an increase in the adhesion number of bacteria to the cells, and PNAG (a polymer of N-acetyl-D-glucosamine residues in beta (1,6) linkage) was shown to be an important component necessary for biofilm formation (Tremblay et al., 2013). In addition, it was shown that medium replenishment was important to increase the biofilm biomass and delay bacterial dispersion. Using a drip flow system with constant nutrient supplementation, it was found that AP forms larger and more stable biofilms. In case of biofilm formation in microplates under static conditions, genes involved in energy metabolism were downregulated while genes involved in transport were upregulated in biofilm cells compared with planktonic cells, suggesting the need for an active nutrient supplementation of AP in biofilms. Also, it seems to be that the dispersion of AP in biofilms after 4h is driven by stress-related genes while at a growing phase, the bacterium expressed genes involved in transport and energy metabolism. For bacteria in biofilms coming from the drip flow system, genes involved in protein synthesis were upregulated in comparison with effluent bacteria (Tremblay et al., 2013). In a different approach looking for genes involved in biofilm formation by AP, it was found 16 genes from a transposon library with around 1200 mutants. The genes associated with an increase in the biofilm formation were of unknown function while those associated with a deficient biofilm formation encoded proteins involved in transport, protein and nucleic acid synthesis (Grasteau et al., 2011). Interestingly, it was found that sub-minimum inhibitory concentrations of penicillin G, an antibiotic used to control AP outbreaks, induced biofilm formation on polystyrene microplates in 9 out of 13 AP field isolates. These biofilms contained more PNAG, extracellular DNA and proteins compared with the control biofilms. Also, the expression of pgaA and genes of the envelope-stress two-component system CpxRA were up-regulated in AP under the presence of sub-minimum inhibitory concentrations of penicillin G, suggesting that the stress induced by the antibiotic on the cell wall of AP is associated with increased production of PNAG and the biofilm formation (Hathroubi et al., 2015).

On the other hand, we have described in this review the adherence of AP to cells and tissues of the porcine respiratory system; however, adhesion is also the first step in biofilm formation. In this regard, fimbriae assembly in AP through the operons apfABCD, pilMNOPQ, and flp were required for cell adhesion, biofilm formation, and to confer virulence in vivo (Liu et al., 2018; Li et al., 2019).

Table 1 Adhesion mechanisms of Actinobacillus pleuropneumoniae to the porcine respiratory system. 

Also, O-antigen, a key component of lipopolysaccharides was not only important for cell adhesion but also to form biofilms (Hathroubi et al., 2015). Finally, the trimeric autotransporter adhesin Apal participated in cell adhesion and biofilm formation (Wang et al., 2015). Table 2 summarises more genes involved in biofilm formation for AP and their biological role; however, for most of those genes, it is not known whether they could be involved

directly or indirectly in the process of adhesion of AP to cells and tissues of the porcine respiratory system. It is worth mentioning that many of the genes described in table 2 belong to stress-responding genes whose products may be important in the adaptation of AP to different environmental changes, for example, the two component systems (TCS) proteins CpxA, CpxR, and ArcA (Li et al., 2018, Buettner et al 2008), the quorum sensing LuxS/

Table 2 Genes involved in biofilm formation by Actinobacillus pleuropneumoniae. 

AI-2 system (Li et al., 2008), the stress tolerance proteases LonA and ClpP (Xie et al., 2013, Xie et al., 2016a), the multidrug efflux channel protein TolC (Lie et al., 2016), the RNA chaperone and posttranscriptional regulator Hfq (Subashchandrabose et al 2013), rseA, a regulator of

sigma E (Bossé et al., 2010), and RelA, an enzyme that participates in the (p)ppGpp-mediated stringent response (Li et al., 2015). PNAG was shown to be an important component of the AP biofilm matrix, and AP strains able to form biofilms failed in this process when treated with dispersin B, a PNAG-hydrolysing enzyme (Izano et al., 2007). Dispersin B is an enzyme that induces the release of adherent cells from mature biofilms through catalysing the hydrolysis of linear polymers of N-acetyl-D-glucosamines (Kaplan et al., 2004). Interestingly, the pgaABC operon genes encoding for PNAG were regulated by the TCS CpxA/CpxR (Li et al 2018), TolC (Li et al., 2016), Hfq (Subashchandrabose et al., 2013), LuxS (Li et al., 2008), rseA, and the histone-like nucleoid structuring protein H-NS (Bossé et al., 2010). Although the mutation of most of these genes in AP resulted in a deficient in vitro biofilm formation and an in vivo attenuated virulence, for LuxS and H-NS, however, it was an increased biofilm formation with attenuated virulence (Bossé et al., 2010, Li et al., 2008). These differences challenge the notion of the association of the in vitro biofilm formation on abiotic surfaces with the pathogenicity in an infection model. It is worth mentioning that some of these differences may be due to the animal model used, the experimental infection route, and the role of the genes in the pathogenicity independently of the contribution to form biofilms. Figure 1 describes the molecules involved in biofilm formation and/or adhesion to the porcine respiratory system, as well as the regulation of the pgaABCD operon.

Figure 1 Molecules involved in biofilm formation and/or adhesion to the porcine respiratory system by Actinobacillus pleuropneu moniae. PNAG (Polymer of N-acetyl-D-glucosamine residues in beta (1,6) linkage) is a key component of biofilms. pgaABCD operon is necessary for PNAG synthesis and its expression is regulated by the chaperone Hfq, the global gene regulator H-NS, the two-component system (TCS) CpxA/CpxR, and RseA (the repressor of RpoE/oE). Other molecules involved in biofilm formation are the proteases ClpP, LonA, and Aasp, the outer membrane proteins VacJ and TolC, the relA hydrolase, the TCS ArcA, and the LuxS/ Al-2 quorum-sensing system. The lipoprotein Lip40 and the glycosyltransferase system NGT participate in the adhesion to porcine respiratory cells. It is suggested that HMW1C, another glycosyltransferase may be involved in this process. Flp and type IV fimbriae, the trimeric autotransporter Apa1, and the lipopolysaccharide (o-antigen) were shown to participate in biofilm formation and adhe sion to the porcine respiratory epithelial cells. OM, outer membrane; IM, inner membrane; CW, cell wall. This figure was created by BioRender software (https://www.biorender.com). 

In this review, we have described the adhesion features of AP to the porcine extracellular matrix components such as mucus, collagen, fibronectin, and fibrinogen. These surfaces may be the initial contact of AP (an extracellular bacterium) to progress throughout the porcine respiratory system till reaching the lungs. Lipopolysaccharides were also initially proposed as molecules involved in adhesion to cells of the upper porcine respiratory tract. However, as we mentioned before, these molecules were dispensable for adhesion in a different cell model. We suggest that this difference could be explained not only because of a different model but the former protocols of lipopolysaccharides extraction that were contaminated with proteins and other cellular products. We consider that new studies with purified lipopolysaccharides or mutant strains deficient in the synthesis of these molecules will be helpful to analyse their exact role in the adherence of AP. Interestingly, unencapsulated AP strains and capsule- deficient mutants showed higher adhesion capacity to lung and tracheal epithelial tissue, thus, the capsule is not only dispensable in cell adhesion, but it seems that it masks other molecules important for adhesion such as

outer membrane proteins. However, how these molecules are exposed in AP with an intact capsule is not clear. In this case, the adhesion of AP must be through the type IV pilus and Flp pilus. It is worth mentioning that most of the studies were performed in vitro. Furthermore, most of the research related to the adhesion of AP employed the SJPL cell line which was mistakenly classified as being of simian instead of pig origin, as previously thought. Therefore, a more appropriate cell line model must be considered in the future. On the other hand, biofilm formation, a sessile mode of growth, has been associated with the virulence of bacteria. Although it is still not clear the role of biofilms during infection by AP, it was shown that AP can grow as aggregates on porcine respiratory tissue and biotic surfaces such as cell monolayers. We have presented information related to the genes involved in biofilm formation by AP at different stages, under different conditions, and comparing with the planktonic bacteria. For those genes, many of them with unknown functions, it is not known whether they are involved in adhesion to the porcine respiratory tract or in a different biological process affecting biofilm formation. Finally, most of the commercially available vaccines for porcine pleuropneumonia are based on the use of whole-cell bacterins (first-generation vaccines), an attenuated form of a specific AP serovar or a combination of serovars. One of the limitations of these vaccines is a partial protection against heterologous serovars and the lack of important virulence factors produced in live bacteria such as the Apx toxins. Although lipopolysaccharides of AP are known to participate in the adhesion to the porcine respiratory system, the potential as antigens to generate vaccines was not as expected due to the high heterogeneity of LPS among serotypes and the same was true for capsular polysaccharides. Because of this, it seems to be that the most promising antigens to generate vaccines in AP are outer membrane proteins and lipoproteins with conserved sequences among serotypes. In this regard, this review may be helpful to find conserved molecules with antigenic properties involved directly or indirectly in the adhesion of AP to the porcine respiratory tract or in biofilm formation to develop new vaccines that may confer protection against porcine pleuropneumonia.

ACKNOWLEDGEMENTS

This review was part of a more complete research supported by the Autonomous University of Aguascalientes with internal project number PIB17-6 and PIBB18-7.

REFERENCES

Auger, E., Deslandes, V., Ramjeet, M., Contreras, I., Nash, J. H. E., Harel, J., Gottschalk, M., Olivier, M., & Jacques, M. (2009). Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells. Infection and Immunity, 77(4), 1426 -1441. https://doi.org/10.1128/IAI.00297-08Links ]

Baltes, N., & Gerlach, G. F. (2004). Identification of genes transcribed by Actinobacillus pleuropneumoniae in necrotic porcine lung tissue by using selective capture of transcribed sequences. Infection and Immunity , 72(11), 6711-6716. https://doi.org/10.1128/ IAI.72.11.6711-6716.2004 [ Links ]

Bélanger, M., Debreuil, D., Harel, J., Girard, C., & Jacques, M. (1990). Role of lipopolysaccharides in adherence of Actinobacillus pleuropneumoniae to porcine tracheal rings. Infection and Immunity , 58(11), 3523-3530. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC313692/Links ]

Bélanger, M., Rioux, S., Foiry, B., & Jacques, M. (1992). Affinity for porcine respiratory tract mucus is found in some isolates of Actinobacillus pleuropneumoniae. FEMS Microbiology Letters, 97(1-2), 119-125. https://doi.org/10.1111/j.1574-6968.1992.tb05450.xLinks ]

Boekema, B. K. H. L., Stockhofe-Zurwieden, N., Smith, H. E., Kamp, E. M., van Putten, J. P., & Verheijden, J. H. (2003). Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells. Veterinary Microbiology, 93(2), 133-144. https://doi. org/10.1016/S0378-1135(03)00020-8 [ Links ]

Boekema, B. K. H. L., Van Putten, J. P. M., Stockhofe-Zurwieden, N., & Smith, H. E. (2004). Host cell contact-induced transcription of the type IV fimbria gene cluster of Actinobacillus pleuropneumoniae. Infection and Immunity , 72(2), 691-700. https://doi.org/10.1128/ IAI.72.2.691-700.2004 [ Links ]

Bossé, J. T., Sinha, S., Li, M. S., O’Dwyer, C. A., Nash, J. H. E., Rycroft, A. N., Kroll, J. S., & Langford, P. R. (2010). Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS. Journal of Bacteriology, 192(9), 2414-2423. https://doi.org/10.1128/JB.01513-09Links ]

Buettner, F. F. R., Maas, A., & Gerlach, G. F. (2008). An Actinobacillus pleuropneumoniae arcA deletion mutant is attenuated and deficient in biofilm formation. Veterinary Microbiology , 127(1), 106-115. https://doi.org/10.1016/j.vetmic.2007.08.005Links ]

Chiers, K., Van Overbeke, I., Donné, E., Baele, M., Ducatelle, R., De Baere, T., & Haesebrouck, F. (2001). Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene. Veterinary Microbiology , 83(2), 147-159. https://doi.org/10.1016/ S0378-1135(01)00414-X [ Links ]

Chiers, K., Donné, E., Van Overbeke, I., Ducatelle, R., & Haesebrouck, F. (2002). Actinobacillus pleuropneumoniae infections in closed swine herds: infection patterns and serological profiles. Veterinary Microbiology , 85(4), 343-52. https://doi.org/10.1016/S0378-1135(01)00518-1 [ Links ]

Chiers, K., De Waele, T., Pasmans, F., Ducatelle, R., & Haesebrouck, F. (2010). Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host. Veterinary Research, 41(5). https://doi.org/10.1051/vetres/2010037 [ Links ]

Cuccui, J., Terra, V S., Abouelhadid, S., Vohra, P., Wren, B. W., Bossé, J. T., Li, Y., Langford, P R., Naegeli, A., Lin, C.-W., Aebi, M., Tucker, A. W., Maskell, D. J., & Rycroft, A. N. (2017). The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering. Open Biology, 7(1). https://doi.org/10.1098/rsob.160212Links ]

Dalai, B., Zhou, R., Wan, Y., Kang, M., Li, L., Li, T., Zhang, S., & Chen, H. (2009). Histone-like protein H-NS regulates biofilm formation and virulence of Actinobacillus pleuropneumoniae. Microbial Pathogenesis, 46(3), 128-134. https://doi.org/10.1016Zj.micpath.2008.11.005 [ Links ]

Dom, P., Haesebrouck, F., Ducatelle, R., & Charlier, G. (1994). In vivo association of Actinobacillus pleuropneumoniae serotype 2 with the respiratory epithelium of pigs. Infection and Immunity , 62(4), 1262-1267. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC186267/Links ]

Enriquez-Verdugo, I., Guerrero, A. L., Serrano, J. J., Godinez, D., Rosales, J. L., Tenorio, V., & de la Garza, M. (2004). Adherence of Actinobacillus pleuropneumoniae to swine-lung collagen. Microbiology, 150(7), 2391-2400. https://doi.org/10.1099/mic.0.27053-0Links ]

Grasteau, A., Tremblay, Y.D.N., Labrie, J., & Jacques, M. (2011). Novel genes associated with biofilm formation of Actinobacillus pleuropneumoniae. Veterinary Microbiology, 153(1), 134-143. https://doi.org/10.1016/j.vetmic.2011.03.029Links ]

Hamer-Barrera, R., Godínez, D., Enríquez, V. I., Vaca-Pacheco, S., Martínez-Zúñiga, R., Talamás-Rohana, P., Suárez-Güemez, F., & de la Garza, M. (2004). Adherence of Actinobacillus pleuropneumoniae serotype 1 to swine buccal epithelial cells involves fibronectin. Canadian Journal of Veterinary Research , 68(1), 33-41. https://pubmed.ncbi.nlm.nih.gov/14979433/Links ]

Hathroubi, S., Fontaine-Gosselin, S.-E., Tremblay, Y.D.N., Labrie, J., & Jacques, M. (2015). Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae. Veterinary Microbiology, 179(3-4), 277-286. https://doi.org/10.1016/j.vetmic.2015.06.011Links ]

Hathroubi, S., Tremblay, Y D. N., Labrie, J., Jacques, M., Hancock, M. A., Bossé, J. T., & Langford, P. R. (2015). Surface polysaccharide mutants reveal that absence of O antigen reduces biofilm formation of Actinobacillus pleuropneumoniae. Infection and Immunity , 84(1), 127-137. https://doi.org/10.1128/IAI.00912-15Links ]

Hathroubi, S., Loera-Muro, A., Guerrero-Barrera, A., Tremblay, Y., & Jacques, M. (2018). Actinobacillus pleuropneumoniae biofilms: Role in pathogenicity and potential impact for vaccination development. Animal Health Research Reviews, 19 (1), 17-30. https://doi.org/10.1017/S146625231700010X [ Links ]

Hernández-Cuellar, E., Guerrero-Barrera, A. L., Avelar-Gonzalez, F. J., Díaz, J. M., Chávez-Reyes, J., & Salazar de Santiago, A. (2021). An in vitro study of ApxI from Actinobacillus pleuropneumoniae serotype 10 and induction of NLRP3 inflammasome-dependent cell death. Veterinary Record Open, 8(1), e20. https://doi.org/10.1002/vro2.20Links ]

Izano, E. A., Sadovskaya, I., Vinogradov, E., Mulks, M. H., Velliyagounder, K., Ragunath, C., Kher, W. B., Ramasubbu, N., Jabbouri, S., Perry, M. B., & Kaplan, J. B. (2007). Poly- N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae. Microbial Pathogenesis , 43(1), 1-9. https://doi. org/10.1016/j.micpath.2007.02.004 [ Links ]

Jacques, M., Bélanger, M., Roy, G., & Foiry, B. (1991). Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections. Veterinary Microbiology , 27(2), 133-143. https://doi.org/10.1016/0378-1135(91)90004-YLinks ]

Jacques, M., & Paradis, S. E. (1998). Adhesin-receptor interactions in Pasteurellaceae. FEMS Microbiology Reviews, 22(1), 45-59. https:// doi.org/10.1111/j.1574-6976.1998.tb00360.x [ Links ]

Jeannotte, M. E., Abul-Milh, M., Dubreuil, J. D., & Jacques, M. (2003). Binding of Actinobacillus pleuropneumoniae to phosphatidylethanolamine. Infection and Immunity , 71(8), 4657 -4663. https://doi.org/10.1128/IAI.7E8.4657-4663.2003Links ]

Kaplan, J. B., Velliyagounder, K., Ragunath, C., Rohde, H., Mack, D., Knobloch, J. K. M., & Ramasubbu, N. (2004). Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms. Journal of Bacteriology , 186(24), 8213-8220. [ Links ]

Kawai, F., Grass, S., Kim, Y., Choi, K. J., St. Geme, I. J. W., & Yeo, H. J. (2011). Structural insights into the glycosyltransferase activity of the Actinobacillus pleuropneumoniae HMW1C-like protein. Journal of Biological Chemistry, 286(44), 38546-38557. https:// doi.org/10.1074/jbc.M111.237602 [ Links ]

Li, G., Xie, F., Zhang, Y., Bossé, J. T., Langford, P. R., & Wang, C. (2015). Role of (p)ppGpp in viability and biofilm formation of Actinobacillus pleuropneumoniae S8. PLoS ONE, 10(10), 1-17. https://doi.org/10.1371/journal.pone.0141501Links ]

Li, H., Liu, F., Peng, W., Yan, K., Zhao, H., Liu, T., Cheng, H., Chang, P,, Chen, H., Bei, W., & Yuan, F. (2018). The CpxA/CpxR two-component system affects biofilm formation and virulence in Actinobacillus pleuropneumoniae. Frontiers in Cellular and Infection Microbiology 8(72). https://doi.org/10.3389/fcimb.2018.00072Links ]

Li, L., Zhou, R., Li, T., Kang, M., Wan, Y., Xu, Z., & Chen, H. (2008). Enhanced biofilm formation and reduced virulence of Actinobacillus pleuropneumoniae luxS mutant. Microbial Pathogenesis , 45(3), 192-200. https://doi.org/10.1016/j.micpath.2008.05.008Links ]

Li, T., Xu, Z., Zhang, T., Li, L., Chen, H., & Zhou, R. (2012). The genetic analysis of the flp locus of Actinobacillus pleuropneumoniae. Archives of Microbiology , 194(3), 167-176. 10.1007/s00203-011-0741-6 [ Links ]

Li, T., Zhang, Q., Wang, R., Zhang, S., Pei, J., Li, Y., Li, L., & Zhou, R. (2019). The roles of flp1 and tadD in Actinobacillus pleuropneumoniae pilus biosynthesis and pathogenicity. Microbial Pathogenesis , 126, 310-317. https://doi.org/10.1016/j.micpath.2018.11.010Links ]

Li, Y., Cao, S., Zhang, L., Lau, G. W., Wen, Y., Wu, R., Zhao, Q., Huang, X., Yan, Q., Huang, Y., & Wen, X. (2016). A TolC-like protein of Actinobacillus pleuropneumoniae is involved in antibiotic resistance and biofilm formation. Frontiers in Microbiology , 7. https://doi. org/10.3389/fmicb.2016.01618 [ Links ]

Li, Y., Cao, S., Zhang, L., Yuan, J., Lau, G. W., Wen, Y., Wu, R., Zhao, Q., Huang, X., Yan, Q., Huang, Y., & Wen, X. (2016). Absence of TolC impairs biofilm formation in Actinobacillus pleuropneumoniae by reducing initial attachment. PLoS ONE , 11(9), 1-14. https://doi. org/10.1371/journal.pone.0163364 [ Links ]

Liu, F., Peng, W., Liu, T., Zhao, H., Yan, K., Yuan, F., Chen, H., & Bei, W. (2018). Biological role of Actinobacillus pleuropneumoniae type IV pilus proteins encoded by the apf and pil operons. Veterinary Microbiology , 224 , 17-22. https://doi.org/10.1016/j.vetmic.2018.08.006Links ]

Liu, J., Hu, L., Xu, Z., Tan, C., Yuan, F., Fu, S., Cheng, H., Chen, H., & Bei, W. (2015). Actinobacillus pleuropneumoniae two-component system QseB/QseC regulates the transcription of PilM, an important determinant of bacterial adherence and virulence. Veterinary Microbiology , 177(1-2), 184-192. https://doi.org/10.1016/j.vetmic.2015.02.033 [ Links ]

Liu, J., Cao, Y., Gao, L., Zhang, L., Gong, S., Yang, J., Zhao, H., Zhao, J., Meng, J., Qi, C., Yang, D., & Gao, Q. (2018). Outer membrane lipoprotein Lip40 modulates adherence, colonization, and virulence of Actinobacillus pleuropneumoniae . Frontiers in Microbiology , 9(1472). https://doi.org/10.3389/fmicb.2018.01472Links ]

Overbeke, I. V., Chiers, K., Charlier, G., Vandenberghe, I., Beeumen, J. V., Ducatelle, R., & Haesebrouck, F. (2002). Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells. Veterinary Microbiology , 88(1), 59-74. https://doi.org/10.1016/S0378-1135(02)00080-9Links ]

Paradis, S. E., Dubreuil, D., Rioux, S., Gottschalk, M., & Jacques, M. (1994). High-molecular-mass lipopolysaccharides are involved in Actinobacillus pleuropneumoniae adherence to porcine respiratory tract cells. Infection and Immunity , 62(8), 3311-3319. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC302961/Links ]

Plasencia-Muñoz, B., Avelar-González, FJ., De la Garza, M., Jacques, M., Moreno-Flores, A., & Guerrero-Barrera, A. L. (2020). Actinobacillus pleuropneumoniae interaction with swine endothelial cells. Frontiers in Veterinary Science, 7, 569370. https://doi.org/10.3389/fvets.2020.569370 [ Links ]

Qin, W., Wang, L., Zhai, R., Ma, Q., Liu, J., Bao, C., Zhang, H., Sun, C., Feng, X., & Gu, J. (2016). Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages. Antonie Van Leeuwenhoek, 109(1), 51-70. https://doi.org/10.1007/s10482-015-0609-xLinks ]

Rioux, S., Galarneau, C., Harel, J., Kobisch, M., Frey, J., Gottschalk, M., & Jacques, M. (2000). Isolation and characterization of a capsule- deficient mutant of Actinobacillus pleuropneumoniae serotype 1. Microbial Pathogenesis , 28(5), 279-289. https://doi.org/10.1006/mpat.1999.0347 [ Links ]

Sassu, E. L., Bossé, J. T., Tobias, T.J., Gottschalk, M., Langford, P. R., & Hennig, P. I. (2018). Update on Actinobacillus pleuropneumoniae - knowledge, gaps and challenges. Transboundary and Emerging Diseases, 65, 72-90. https://dx.doi.org/10.1111/tbed.12739Links ]

Sidibé, M., Messier, S., Lariviere, S., Gottschalk, M., & Mittal, K. R. (1993). Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests. Canadian Journal of Veterinary Research , 57(3), 204-208. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1263624/Links ]

Steimle, A., Autenrieth, I. B., & Frick, J. S. (2016). Structure and function: Lipid A modifications in commensals and pathogens. International Journal of Medical Microbiology , 306(5), 290-301. https://doi. org/10.1016/j.ijmm.2016.03.001 [ Links ]

Stevenson, A., Macdonald, J., & Roberts, M. (2003). Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae. Veterinary Microbiology , 92(1), 121-134. https:// doi-org.dibpxy.uaa.mx/10.1016/S0378-1135(02)00351-6 [ Links ]

Stringer, O. W., Bossé, J. T., Lacouture, S., Gottschalk, M., Fodor, L., Angen, 0., Velazquez, E., Penny, P., Lei, L., Langford, P. R., & Li, Y. (2021). Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars. Veterinary Microbiology , 255(109021). https://doi.org/10.1016/j.vetmic.2021.109021Links ]

Subashchandrabose, S., Leveque, R. M., Kirkwood, R. N., Kiupel, M., & Mulks, M. H. (2013). The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia. Infection and Immunity , 81 (8), 2952-2961. https://doi.org/10.1128/IAI.00392-13 [ Links ]

Tegetmeyer, H. E., Fricke, K., & Baltes, N. (2009). An isogenic Actinobacillus pleuropneumoniae AasP mutant exhibits altered biofilm formation but retains virulence. Veterinary Microbiology , 137(3-4), 392-396. https://doi.org/10.1016/j.vetmic.2009.01.026Links ]

Tremblay, Y. D., Deslandes, V., & Jacques, M. (2013). Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus. BMC Genomics, 14, 364. https://doi.org/10.1186/1471-2164-14-364Links ]

Tremblay, Y. D., Lnvesque, C., Segers, R. P., & Jacques, M. (2013). Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface. BMC Veterinary Research , 9, 213. https://doi. org/10.1186/1746-6148-9-213. [ Links ]

Tremblay, Y. D., Labrie, J., Jacques, M., & Chénier, S. (2017). Actinobacillus pleuropneumoniae grows as aggregates in the lung of pigs: is it time to refine our in vitro biofilm assays? Microbial Biotechnology, 10(4), 756-760. https://doi.org/10.1111/1751-7915.12432Links ]

Utrera, V., & Pijoan, C. (1991). Fimbriae in A. pleuropneumoniae strains isolated from pig respiratory tracts. Veterinary Research, 128(15), 357-358. https://pubmed.ncbi.nlm.nih.gov/1676554/Links ]

Wang, L., Qin, W., Yang, S., Zhai, R., Zhou, L., Sun, C., Pan, F., Ji, Q., Wang, Y., Gu, J., Feng, X., Du, C., Han, W., Langford, P. R., & Lei, L. (2015). The Adh adhesin domain is required for trimeric autotransporter Apa1-mediated Actinobacillus pleuropneumoniae adhesion, autoaggregation, biofilm formation and pathogenicity. Veterinary Microbiology , 177(1-2), 175-183. https://doi.org/10.1016/j.vetmic.2015.02.026 [ Links ]

Xiao, L., Zhou, L., Sun, C., Feng, X., Du, C., Gao, Y., Ji, Q., Yang, S., Wang Y., Han, W., Langford, P. R., & Lei, L. (2012). Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence. Journal of Basic Microbiology , 52(5), 598-607. https://doi.org/10.1002/jobm.201100365 [ Links ]

Xie, F., Zhang, Y., Li, G., Zhou, L., Liu, S., & Wang, C. (2013). The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae. PLoS ONE , 8(1), 1-11. https:// doi.org/10.1371/journal.pone.0053600 [ Links ]

Xie, F., Li, G., Zhang, Y., Zhou, L., Liu, S., Liu, S., & Wang, C. (2016a). The Lon protease homologue LonA, not LonC, contributes to the stress tolerance and biofilm formation of Actinobacillus pleuropneumoniae . Microbial Pathogenesis , 93 , 38-43. https://doi. org/10.1016/j.micpath.2016.01.009 [ Links ]

Xie, F., Li, G., Zhang, W., Zhang, Y., Zhou, L., Liu, S., Liu, S., & Wang, C. (2016b). Outer membrane lipoprotein VacJ is required for the membrane integrity, serum resistance and biofilm formation of Actinobacillus pleuropneumoniae . Veterinary Microbiology , 183 , 1-8. https://doi.org/10.1016/j.vetmic.2015.11.021Links ]

Zhang, Y., Tennent, J. M., Ingham, A., Beddome, G., Prideaux, C., & Michalski, W. P. (2000). Identification of type 4 fimbriae in Actinobacillus pleuropneumoniae . FEMS Microbiology Letters , 189(1), 15-18. https://doi.org/10.1111/j.1574-6968.2000.tb09199.xLinks ]

Zhou, Y., Li, L., Chen, Z., Yuan, H., Chen, H., & Zhou, R. (2013). Adhesion protein ApfA of Actinobacillus pleuropneumoniae is required for pathogenesis and is a potential target for vaccine development. Clinical and Vaccine Immunology, 20(2), 287-294. https://doi.org/10.1128/CVI.00616-12Links ]

Recibido: 22 de Marzo de 2022; Aprobado: 15 de Junio de 2022

Corresponding author: E Hernández-Cuellar; Avenida Universidad #940, Ciudad Universitaria, C.P. 20131, Aguascalientes, Ags. México; ecuellar@correo.uaa.mx

CONFLICT OF INTEREST STATEMENT The authors declare that there is no conflict of interest.

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