Introduction

Trueperella pyogenes is a commensal bacterium in the mucous membrane of the respiratory and urogenital tracts of animals (Jarosz et al., 2014). It is an omnipresent opportunistic microorganism responsible for causing purulent infections in domestic animals and birds (Lin et al., 2010; Rzewuska et al., 2012). T. pyogenes was initially named Corynebacterium pyogenes, later Actinomyces pyogenes and subsequently Arcanobacterium pyogenes (Ramos et al., 1997). T. pyogenes is the most commonly isolated species from various suppurative cases in pigs (Azuma et al., 2009). Being a commensal oppurtunistic pathogen its emergence in different animals is unpredictable. In recent times, T. pyogenes infections in pigs have become a growing epidemiological problem in many farms worldwide (Jarosz et al., 2014). Most important risk factor for the occurrence of T. pyogenes infection includes immunosuppression of host animals by stress, various therapeutic agents and infections (Pejsak et al., 2006). Abscess formation occurs in those tissues which undergone any injury previously (Meyer et al., 2005). In this study we report the pathology in heart and lungs of a pig with T. pyogenes infection.

Case Description and Methods

One month old crossbred female pig with a history of sudden death was presented for necropsy examination and disease diagnosis. Systematic necropsy examination was done on dead animal and gross lesions were recorded. Pieces of spleen, lungs, intestines, heart, brain, kidney etc were collected on ice for microbiological examination and in 10 % NBF for histopathology.

The heart blood and abscess from lung tissue were streaked in 5% sheep blood agar and incubated at 37º C aerobically. The growth was examined daily for 4 days. Gram’s staining was done according to standard procedure. Colony PCR was done using 16SrRNA primers (16SF: GTGCCAGCMGCCGCGG and 16SR: TACGYTACCTTGTTACGACT) to identify the bacteria (Lane, 1991). The reaction was carried out using 2.5 μl 5x DreamTaq MasterMix, 0.5 μl 2mM dNTP mix, 0.5 μl each of 10 pmole/ μl forward and reverse primers, 3 μl of DNA and nuclease free water upto 25 μl. Amplification was performed for 35 cycles consisting of initial denaturation at 95ºC for 120 sec, followed by denaturation at 95º C for 30 sec and annealing at 55 º C for 30 sec. The PCR product was visualized by agarose gel electrophoresis stained with ethidium bromide. The PCR product was purified by GenJet Gel extraction kit according to manufacture protocol and sequenced in both directions. The sequence obtained was blasted in NCBI and analyzed to identify the species of bacteria.

Results and Discussion

On external examination, general body condition and nutritional status was good. Necropsy examination revealed enlarged, oedematous and congested inguinal and cervical lymph nodes. Multiple abscesses of varying sizes from few millimeters to 4cm diameter were noticed on different lobes of left lung with moderate pleural wall thickening (Fig. 1). Left parenchyma was fully studded with different sized nodules filled with pus, a few large nodules measuring 4 cm diameter were found projecting outside the lung parenchyma. Local adhesion of left lung with chest wall was also noticed. Pericardium was moderately thickened and pericardial sac contained viscous suppurative exudates (Fig. 2). Ecchymosis was noticed on endocardial wall. Tracheobronchial lymph nodes were highly congested, oedematous and enlarged. Prominent lesions were noticed only in pleural cavity.

Fig. 1: Lung parenchyma was fully studded with variable sized nodules filled with pus. Abscesses were encapsulated by thick or thin fibrous capsules

Fig. 2: Pericardium was moderately thickened and pericardial sac contained viscous suppurative exudates

On histopathological examination, in lung multiple suppurative necrotic areas encapsulated by fibrous tissue capsule with numerous inflammatory cell infiltrations were noticed. Adjacent alveoli were thickened due to proliferation of pneumocytes and numerous macrophages, plasma cells and a few neutrophils infiltrated the alveolar lumen (Fig. 3). Bronchi and bronchioles showed epithelial desquamation along with infiltration of a few macrophages in lumen. Abscesses were encapsulated by thick or thin fibrous capsules that were surrounded by numerous macrophages, plasma cells, lymphocytes and a few polymorphonuclear neutrophils. Lymph nodes were severely congested with multiple areas of extensive haemorrages in the medullary sinuses. In brain, perivascular haemorrages were noticed with capillary congestion. In heart, multiple petechial hemorrhages were noticed along with capillary engorgement, mild oedema and focal areas of myocardial fiber degeneration.

On blood agar, very small pinpoint white opaque colonies with circular edges were seen after 24 hour of incubation at 37°C. The sizes of the colonies were increased after 48 hour incubation with narrow zone of complete hemolysis.

Fig. 3: Abscess encapsulated by fibrous tissue capsule with numerous inflammatory cell infiltrations was noticed. Adjacent alveoli were thickened due to proliferation of pneumocytes and numerous macrophages, plasma cells and a few neutrophils infiltrated the alveolar lumen

Gram’s stained smears from culture showed Gram-positive, pleomorphic coccobacilli of varying length. Catalase and oxidase test were negative. Colony PCR of the culture yielded a 994 bp product of expected size. Purified PCR product was sequenced (Accession no:  KY694370) and the obtained sequence when blasted in NCBI showed highest similarity with the T. pyogenes isolated from China and USA.

T. pyogenes is commensal opportunistic pathogen in domestic animals. In this case, immune suppression may be occurred due to severe cold as death of animal occurred in the month of December. Gross pathology noticed were osteoartrithis, polyarthritis, mastitis, endocarditis, myocardial abscesses, lung abscesses, abscesses in subcutaneous tissue and various organs (Jarosz et al., 2014). The histopathology of infected sites included inflammatory reaction, marked suppurative reaction, abscess formation, and necrosis, (Kotrajaras and Tagami, 1987). Clinical signs, gross and histo-pathological lesions, culture characteristics and result of colony PCR and sequencing suggested the disease to be suppurative condition associated with T. pyogenes. Clinical signs and gross lesions found in this case were unique and pronounced. Pyopericardium is less reported lesion of heart in Trueperella infection in pigs. In heart mainly petechial and ecchymosis of pericardium and endocardium, endocarditis and myocardial abscesses, haemopericardium were mostly reported manifestation (Reddy et al., 1997). In lungs, multiple abscesses were noticed with severe pleural thickening. Histologically, chronic suppurative bronchopneumonia followed by abcess formation was a common finding. There was diffuse lymphoid hyperplasia in the tracheobronchial lymph node and granulomas were reported (Hariharan et al., 2015). Overall, published information on lung and heart lesions caused by T. pyogenes in pig is sparse.

Based on gross pathology, histpathological examination, colony characteristic, microscopic morphology Gram stained organism and sequence results, the organism was identified as T. pyogenes. Case was diagnosed as suppurative pneumonia and pyopericaricardium associated with T. pyogenes.

Acknowledgement

The authors thank the Director, Indian Veterinary Research Institute for providing facilities and fund to conduct this research work and acknowledge ICMR for their financial support.

References

1. Hariharan H, Bhaiyat, MI, Tiwari KP and Chikweto A. 2015. Multifocal pulmonary abscesses due to trueperella pyogenes in a sheep. EC Veterinary Science 2.2: 98-102.
2. Jarosz LS, Grądzki Z and Kalinowski M. 2014. Trueperella pyogenes infections in swine:clinical course and pathology. Polish Journal of Veterinary Sciences. 17(2): 395–404.
3. Kotrajaras R and Tagami H.  1987. Corynebacterium pyogenes. Its pathogenic mechanism in leg ulcers in Thailand. International Journal of Dermatology. 26:45–50.
4. Lane D J. 1991.16S/23S rRNA sequencing in Nucleic acid techniques in bacterial systematics eds Stackebrandt E, Goodfellow M, John Wiley and Sons, pp. 115–175.
5. Lin CC, Chen TH, Shyu CL, Su NY and Chan JP. 2010. Disseminated abscessation complicated with bone marrow abscess caused by Arcanobacterium pyogenes in a goat. Journal of Veterinary Medical Science. 72: 1089-1092.
6. Meyer DK and Reboli AC. 2005. Other Corynebacteria and Rhodococcus. In: Mandell GL, Bennet JE, Dolin R (eds) Principles and Practice of Infectious Diseases. New York, Churchill-Livingstone, pp. 2465-2478.
7. Pejsak Z, Markowska-Daniel I, Samorek M, Truszczyński M. 2006. Wykrywanie i ocena właściwości krajowych izolatów Arcanobacterium pyogenes wyosobnionych od świn. Medycyna Weterynaryjna .62: 781-784.
8. Ramos CP, Foster G and Collins MD. 1997. Phylogenetic analysis of the genus Actinomyces based on 16S rRNA gene sequences: description of Arcanobacterium phocae sp. nov., Arcanobacterium bernardiae comb. nov.,and Arcanobacterium pyogenes comb. International Journal of Systematic Bacteriology. 47: 46–53.
9. Reddy I, Donald A, Ferguson Jr and Sarubbi FA. 1997. Endocarditis due to Actinomyces pyogenes. Clinical Infectious Diseases. 25:1477–8.
10. Rzewuska M, Stefańska I, Osińska B, Kizerwetter-Świda M, Chrobak D, Kaba J and Bielecki W. 2012 Phenotypic characteristics and virulence genotypes of Trueperella (Arcanobacterium) pyogenes strains isolated from European bison (Bison bonasus). Veterinary Microbiology.160: 69-76.