Background

Actinomyces (A.) hyovaginalis, formally proposed to be reclassified into the genus Schaalia (Alssahen et al. 2020), is a polymorphic, catalase-negative, rod-shaped, slow-growing, non-acid-fast, Gram-positive bacterium (Reichel and Wragg 2007; Broes et al. 2019). This agent was first isolated from purulent vaginal discharges of sows, aborted fetuses, and multiple tissue samples of domestic pigs (Sus scrofa domesticus) (Collins et al. 1993; Hommez et al. 1991; Wübbelmann et al. 2016). In domestic pigs, this infection is most commonly associated with sporadic disseminated pyogranulomatous lung lesions with extensive necrotic centers (Aalbæk et al. 2003; Reichel and Wragg 2007). In addition to pulmonary lesions in slaughtered domestic pigs, this agent has been also associated with caseous lymphadenitis (Foster et al. 2012), mastitis (Murakami et al. 1998; Christensen et al. 2007), abortion with necrotizing and suppurative placentitis in sows (Storms et al. 2002; Hogg et al. 2012), and suppurative bronchopneumonia with intralesional bacteria in aborted swine fetuses (Hogg et al. 2012). Moreover, osteomyelitis of the mandibular bone after tusk extraction was reported in a pot-bellied pig (Savard et al. 2021). In other animal species, A. hyovaginalis was isolated from caseous lymphadenitis and multiple other systemic pyemic lesions of sheep, goats, and moufflon (Schumacher et al. 2009; Foster et al. 2012; De La Fuente et al. 2017; Alssahen et al. 2020; Marinoff et al. 2020; Sting et al. 2020), abscesses in alpacas and llamas (Sting et al. 2020), and a case of suppurative arthritis in a captive zoo giraffe (Wickhorst et al. 2017).

In wild boars (Sus scrofa), A. hyovaginalis has previously been isolated from lung samples of an animal exhibiting severe lungworm infestation, fibrinopurulent bronchopneumonia, and pulmonary haemorrhage (Alssahen et al. 2020); however, in that case, Escherichia coli, Staphylococcus chromogenes, and α-hemolytic streptococci were also isolated from the same samples (Alssahen et al. 2020). Besides that, pyogranulomatous pneumonia caused directly by A. hyovaginalis infection is not described in this suid species. The main agents associated with bacterial pneumonia in wild boars are the same as those in domestic pigs, including Mycoplasma hyopneumoniae, Pasteurella multocida, Streptococcus suis serotype 2, Actinobacillus pleuropneumoniae, Glaeserella parasuis, and M. hyorhinis (Biondo et al. 2021; Souza et al. 2021; Silva Andrade et al. 2022; Ninkovic et al. 2024). However, none of these agents classically induces a pyogranulomatous or granulomatous reaction. In turn, this type of inflammation is occasionally induced by Mycobacterium bovis infection in both wild boars and domestic pigs (Zanella et al. 2008; Lopes et al. 2021a). Therefore, we report the gross, histological, and bacteriological findings in a case of pyogranulomatous pneumonia associated with A. hyovaginalis infection in a free-ranging wild boar as a differential diagnosis for bovine tuberculosis in this suid species.

Case presentation

Sections of the lungs, heart, and an unspecified intrathoracic lymph node from a free-ranging adult wild boar were submitted by wild boar hunters to the Official Veterinary Service of Rio Grande do Sul state, Brazil, due to suspected tuberculosis. The tissue samples were sent to the Veterinary Pathology Section of the Universidade Federal do Rio Grande do Sul for further evaluation. Grossly, the lung samples contained multiple, often coalescing, 0.1 to 0.8 cm-diameter nodules filled with light-yellow, opaque, friable material (Fig. 1A). The remaining lung parenchyma was multifocally pale and firm, and failed to collapse. There were no gross lesions in the lymph node and heart, and, according to the hunters, no other lesions were observed in the other organs. Sections of the lungs, heart, and lymph node were collected and fixed in 10% neutral-buffered formalin. After fixation, the tissue samples were routinely processed and embedded in paraffin for histopathological evaluation. Three-µm-thick sections were stained with hematoxylin and eosin (HE), Brown-Hopps (modified Gram), Ziehl-Neelsen, and Grocott-Gomori methenamine silver (GMS) stains. Additionally, a lung sample stored at 4 °C for 4 h was submitted to bacteriological culture. The sample was plated on 5% sheep blood agar and MacConkey agar, and incubated at 37 °C under aerobic and microaerophilic conditions for 48 h. Bacterial identification was performed by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

Fig. 1: Pyogranulomatous pneumonia caused by Actinomyces hyovaginalis infection, wild boar. A In the lung sections, there were multiple 0.1–0.8 cm white nodules with light-yellow, friable necrotic centers. Bar, 2 cm. B Lung. A central pyogranuloma surrounded by a marked neutrophilic inflammatory infiltrate within the alveolar spaces and bronchiolar lumen. Hematoxylin and eosin (HE). Bar, 500 μm. C Lung. Close-up view of the pyogranuloma. A moderate inflammatory infiltrate composed of lymphocytes, plasma cells (black asterisk), and macrophages (green asterisk), and mild fibrous tissue deposition surrounded a central necrotic area with degenerate neutrophils (red asterisk). HE. Bar, 150 μm. D Lung. Multiple intralesional bacterial colonies within the necrotic center. Brown-Hopps (Gram) stain. Bar, 50 μm

Histologically, in the lung parenchyma, there were multiple areas of coagulative necrosis (Fig. 1B), with abundant amorphous to granular eosinophilic material, multifocal inflammatory infiltrate composed of degenerate neutrophils, cellular debris, multifocal deposition of coarsely granular, intensely basophilic material (mineralization), and numerous intralesional 2–3 μm coccobacillary basophilic bacterial aggregates. Surrounding these areas, there was a marked concentric inflammatory infiltrate of lymphocytes, plasma cells, macrophages, epithelioid macrophages, and neutrophils (Fig. 1C). Occasionally, the macrophages palisade at the periphery. These necrotic areas were encircled by a moderate fibrous tissue proliferation. No Splendore-Hoeppli reaction was seen. The adjacent lung parenchyma showed numerous alveolar macrophages, marked multifocal neutrophilic inflammatory infiltrate, and fibrin-rich alveolar edema within the alveoli. No significant microscopic lesions were seen in the lymph node and heart sections. Brown-Hopps staining identified the intralesional coccobacilli as Gram-positive (Fig. 1D). Moreover, no acid-fast bacilli nor fungal structures were identified within the lungs, lymph nodes, or heart by the Ziehl-Neelsen and GMS stains, respectively.

After 48 h of bacteriological incubation in aerobiosis and microaerobiosis, small, white, partial-hemolytic pure colonies were visualized in 5% sheep blood agar (Fig. 2), in both atmospheres. No growth was detected in MacConkey agar. Gram-positive pleomorphic coccobacilli were identified at the Gram stain. The isolated bacterium was identified as A. hyovaginalis by MALDI-TOF MS with an identity score of 2.6.

Fig. 2: Bacterial culture of the Actinomyces hyovaginalis strain isolated in the research. White colonies with partial hemolysis after 48 h of incubation