Serotype distribution of Actinobacillus pleuropneumoniae isolated from diseased pigs in Argentina (2020–2024)

Abstract

This study aimed to monitor the serotype distribution of Actinobacillus pleuropneumoniae (App) in Argentina from 2020 to 2024. A total of 105 App strains, isolated from pigs that died of porcine pleuropneumonia (PP), were serotyped using multiplex PCR. The results showed a predominance of serotype 1 (66.7%), followed by serotypes 4 (12.4%), 5 (7.6%), 7 (6.7%), 8 (4.7%), and 12 (1.9%). This study confirms the continued dominance of serotype 1 in Argentina and highlights the importance of ongoing surveillance to improve disease control strategies.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, one of the most significant respiratory diseases in the porcine respiratory disease complex. Actinobacillus pleuropneumoniae is a small bacillus or coccobacillus, Gram-negative, encapsulated, classified based on its dependence on nicotinamide adenine dinucleotide (NAD) for in vitro growth into biotype I (NAD-dependent) and biotype II (NAD-independent). The most virulent strains produce fibrinous, hemorrhagic, and necrotizing pleuropneumonia with fatal outcomes, resulting in economic losses due to mortality, growth retardation in affected pigs, treatment and vaccination costs, and slaughterhouse penalties (1).

To date, 19 serotypes of A. pleuropneumoniae have been described, most of which belong to biotype I (serotypes 1–12 and 15–19), whereas the rest, serotypes 13 and 14, belong to biotype II (2,3). The association between serotypes and biotypes is predominant but not exclusive: serotypes 2, 4, 7, 9, 11, and 17 of biotype II have been reported, as well as serotype 13 strains that were identified as biotype I (1,2).

The virulence factors of A. pleuropneumoniae are numerous and are related to host adherence (lipopolysaccharides or LPS, pili, fibronectin-binding proteins, trimeric autotransporter proteins), acquisition of essential nutrients, persistence of infection (biofilm formation), and lesion induction (exotoxins) (4,5). Exotoxins constitute the main mechanism of damage: ApxI, ApxII, and ApxIII have hemolytic and cytolytic properties and can be secreted by different serotypes in various combinations. ApxI is expressed by serotypes 1, 5, 9, 10, 11, and 14; ApxII is expressed by all serotypes except 10 and 14; and ApxIII is present in serotypes 2, 4, 6, 8, and 15. Finally, ApxIV toxin, secreted only in vivo by all serotypes, is specific to A. pleuropneumoniae and is widely used for species identification (6). The virulence exhibited by strains (high, intermediate, or low) is a characteristic shaped by multiple factors. These include the specific toxin profile of the bacterium, concurrent coinfections with other pathogens (such as Mycoplasma hyopneumoniae, porcine circovirus type 2, and swine influenza virus), and the host having pre-existing immunity to particular serotypes (7).

Toxin production profiles within a single serotype can vary geographically. For example, serotype 2 is highly virulent in Europe and Asia because it secretes ApxII and ApxIII cytotoxins; however, in North America, serotype 2 secretes only ApxII and is less virulent. Biotype II strains, commonly isolated in European countries and rarely reported in North America, are traditionally considered to be of lower virulence but can occasionally cause fatal pleuropneumonia cases (1).

Historically, the typing of A. pleuropneumoniae serotypes has been based on serological techniques using the specificity of capsular polysaccharides (CPS), generally associated with a given LPS type. For instance, CPS type 1 was previously associated with LPS type 1. However, strains with divergent CPS and LPS combinations have been identified (1), leading to cross-reactions between serotypes such as 1, 9, and 11; 3, 6, 8, 15, and 17; and 4 and 7, complicating differentiation. These limitations have led to the decline in the use of traditional serological techniques. Currently, the genes encoding CPS have been identified and classified into 4 main types (CPS types I to IV) (8). This advance has allowed the implementation of molecular methods, such as polymerase chain reaction (PCR) for the precise identification of serotypes. Multiplex PCRs have been developed to simultaneously detect CPS-encoding genes specific to each serovar and the ApxIV exotoxin gene that defines the species (1,3). Despite these advances, challenges in molecular typing persist. For instance, the CPS loci of serovars 9 and 11 are nearly identical, differing by only one base in the cpsF gene. Moreover, both share the same O-Ag locus and a similar toxigenic profile, making differentiation difficult. As a result, serovars 9 and 11 can be considered a hybrid serovar 9/11 (3). Recently a new qPCR based on locked nucleic acid (LNA) was developed. An analysis of the cpsF gene is conducted to accurately determine whether the capsule of an isolate corresponds to serotype 9 or 11 (9). Although such analyses are feasible with the advent of third-generation sequencing technologies, their accessibility, cost, and time to obtain results limit their use in routine diagnostic applications.

In the United States and Canada, with some exceptions (10), severe outbreaks of A. pleuropneumoniae are rare. Clinical problem persists in Asia, Europe, and Latin America, including Argentina.

In Argentina, reports on serotype distribution are available up to 2010, with no recent information. Studies conducted between 1990 and 2010 chronologically identified the presence of serotypes 1 and 5 (up to 1990), followed by serotype 8 (in 1996), serotype 12 (in 1997), serotype 7 (in 1999), serotype 3 (in 2004), and serotype 15 (in 2005) (11). By 2010, serotypes 4 and 6 had also been identified (12). Biotype II strains have never been isolated so far.

Characterizing A. pleuropneumoniae strains involved in disease outbreaks is essential for understanding epidemiology, preparing vaccines (bacterins) needed for disease control (there is no cross-protection between serotypes), and adjusting serological monitoring in farms. This work aimed to monitor A. pleuropneumoniae serotypes associated with clinical cases of porcine pleuropneumonia in Argentina from 2020 to 2024.

Strains isolated between July 2020 and December 2024 were analyzed. These strains were obtained from intensive pig farming operations, mostly located in the primary pig production region of the country (provinces of Santa Fe, Buenos Aires, and Córdoba), except for 4 farms in Entre Ríos, 1 in La Rioja, and 1 in San Juan. All isolates were derived from pigs that had died of porcine pleuropneumonia during the fattening phase. Clinically, all cases presented peracute deaths, and necropsies revealed fibrinous pleuropneumonia with necrohemorrhagic foci.

Samples from lung lesions compatible with A. pleuropneumoniae were cultured on tryptic soy agar with 5% sheep blood supplemented with NAD-impregnated discs (Rosco), incubated in a 5% CO₂ atmosphere for 24 h at 37°C and identified by biochemical tests. The A. pleuropneumoniae strains obtained from primary isolation were subcultured in brain-heart broth (Oxoid) supplemented with 0.004 g/mL NAD (Sigma) and then impregnated onto FTA cards (QIAcard FTA Micro), which were labeled and stored at room temperature. The determination of the serotypes was carried out using a PCR multiplex technique (3) at the University of Montreal. A total of 105 A. pleuropneumoniae strains were recovered, all of which exhibited NAD-dependent growth (satellitism). Polymerase chain reaction confirmed the identity and biotype of the isolates. Serotyping of the strains revealed the presence of serotypes 1, 4, 5, 7, 8, and 12 in farms across Argentina.

When considering the detection frequency of each serotype, serotype 1 was predominant, representing 66.7% of the isolates. Serotype 4 ranked second, identified in 12.4% of the isolates, followed by serotype 5 at 7.6%, and serotype 7 at 6.7%. Serotype 8 was detected in 4.7% of the samples. Serotype 12, representing 1.9%, was the least prevalent (Table I).

Table I.

Serotype distribution of Actinobacillus pleuropneumoniae in Argentina (July 2020 to 2024).

Serotype	Number of isolates	Percentage
1	70	66.7%
4	13	12.4%
5	8	7.6%
7	7	6.7%
8	5	4.7%
12	2	1.9%

Regarding the region of the country from which the strains originated, serotype 1 was predominant in Santa Fe, Córdoba, and Buenos Aires, accounting for 83%, 45.5%, and 61.1% of the isolates from each province, respectively. Samples from farms located outside the main pig production area shared the presence of serotype 7, which was the only serotype isolated in the cases from La Rioja and San Juan (Table II).

Table II.

Serotype distribution of Actinobacillus pleuropneumoniae in different provinces of Argentina.

Region	Serotype
	1	4	5	7	8	12
Santa Fe	49	6	3	1	0	0
Córdoba	10	5	2	2	3	0
Buenos Aires	11	0	3	0	2	2
Entre Ríos	0	2	0	2	0	0
San Juan	0	0	0	1	0	0
La Rioja	0	0	0	1	0	0

In Argentina, up to the year 2010, the presence of serotypes 1, 3, 4, 5, 6, 7, 8, 12, and 15 was reported in the lungs of pigs that had died from the disease, with serotype 1 being the most prevalent. According to these results, serotype 1 continues to dominate. Comparing the percentages observed with those reported in 2010, the presence of serotype 1 increased from 45 to 66.7% of the total isolates. Strains of this serotype are considered highly virulent.

Secondly, serotype 4 was identified, showing a considerable increase in frequency compared to 2010, rising from 2.3 to 12.4% (this second position was previously occupied by serotype 15). Serotype 4 was one of the most frequently virulent serotypes reported in Spain in 2009 (1), but in a recent publication (2024) its detection from disease pigs has dropped to 5th place in frequency, surpassed by serotypes 9/11, 2, 13, and 17 (7). Serotype 4 has not been isolated from clinical case material in North America but has been detected in asymptomatic pigs in Canada (1).

Serotype 5, considered highly virulent, was the 3rd most frequently isolated serotype in this study, increasing its presence from 1.5 to 7.6% compared to findings in 2010. Detection of serotype 7 decreased from 12.2% (3rd most frequently reported in the past) to 6.7% in this study. It was the only one detected in the provinces of La Rioja and San Juan. Due to the small number of samples analyzed from these regions, it cannot be stated that this serotype is predominant over others.

Serotypes 8 and 12 were detected in 4.7% and 1.9% of the isolates, respectively. These serotypes had not been noted in 2010, although they had been reported in previous studies. Serotype 8 belongs to an antigenic group (LPS) along with serotypes 3, 6, 15, and 17; however, it was the only serotype from this group identified in the present analysis. These results align with those obtained using modern techniques in a study of Argentine strains classified within this group during the period from 2001 to 2010 (13). Serotype 8 is predominant in the United Kingdom and is occasionally isolated in North America and Japan (1). In Argentina, it was isolated at a low frequency from clinical cases, contrasting with recent reports from Brazil and UK, where it was the most prevalent (14,15). In addition, it has been recently reported as the cause of an important outbreak in the USA (16). Serotype 12, which exhibits low virulence in most countries, was the least prevalent in this study, although it was consistently associated with severe disease outbreaks.

The distribution of serotypes involved in outbreaks of the disease is very different in North America, South America, and Europe. In a report carried out in Quebec, Canada during a similar period as this work (2020–2023), serotype 7 predominated (61.5%), followed by serotype 5 (12.8%), and then serotypes 2 and 8 (7.7% in each case) (17). Serotypes 2 and 9/11 are the most common in Europe (1). In South America, reports from countries bordering Argentina, such as Chile, confirmed the circulation of serotypes 4, 6, and 7, with a predominance of the latter, in isolations carried out during 2007 to 2009. In previous works, serotypes 1 and 5 had also been detected (18). In Brazil, to date, the presence of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 14 has been reported (19). A. pleuropneumoniae strains isolated between 2014 to 2018 from states in the center and south of the country, indicated that the most relevant serotypes in these regions were 8 (43%), 7 (25%) and 5 (18%), and the presence of serotype 14 was recorded for the first time (14).

It has been described that the relative prevalence of serotypes in a country can change dramatically over the years (20). Due to the lack of cross-immunological reaction, the introduction of serotypes different from those circulating in a given area can trigger outbreaks, highlighting the importance of periodic monitoring. Identifying the active serotypes allows for improve control and prevention of porcine pleuropneumonia.

In Argentina, serotype 1 remains predominant. Although serotypes 2, 3, 6, 8, 12, and 15 are considered to have low to intermediate virulence, in this study, serotypes 8 and 12 were recovered from severe disease outbreaks. Serotypes identified in the disease outbreaks caused no observable differences in clinical signs or lesions.

In conclusion, the serotypes involved in porcine pleuronpneumonia outbreaks in Argentina between July 2020 and December 2024 were primarily serotype 1 (66.7%), followed by serotypes 4 (12.4%), serotype 5 and 7 in a similar proportion (7.6% and 6.7%, respectively), then serotypes 8 (4.7%), and finally serotype 12 (1.9%).