NOTE Pathology

Aborted Lesions of a Pig Associated with abortisuis and the Immunohistochemical Features

Satoshi MURAKAMI1)*, Akihiro OGAWA2), Ryozo AZUMA1), Takemi OHBA3) and Ryo MURATA1)

1)Department of Animal Science, Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa 243–0034, 2)Chuo Animal Health and Hygiene Service Center, 497 Iwatomi, Sakura, Chiba 285–0072 and 3)Toyama Prefectural Meat Inspection Center, 28–4 Shinbori Imizu, Toyama 934–0035, Japan

(Received 20 October 2010/Accepted 23 December 2010/Published online in J-STAGE 6 January 2011)

ABSTRACT. A microorganism isolated from an aborted sow’s placenta has been proposed as a novel species, Arcanobacterium abortisuis. The lesions in the placenta were suppurative and necrotic placentitis, whereas lesions of three fetuses were suppurative bronchopneumo- nia. Gram and Grocott-positive organisms in these lesions were immunolabeled with anti- Arcanobacterium abortisuis antibody. This is the first case report, which showed the association of Arcanobacterium abortisuis with the aborted lesions of a pig. KEY WORDS: Arcanobacterium abortisuis, immunohistochemistory, porcine abortion. J. Vet. Med. Sci. 73(6): 797–799, 2011

Recently, we isolated a diphtheroid-shaped organism neys and superficial lymph nodes were removed and fixed from an aborted sow’s placenta, and have proposed as a in 20% neutral buffered formalin. Fixed tissues were then novel species, Arcanobacterium abortisuis sp. nov. (acces- embedded in paraffin, sectioned 3 to 4 µm thick, and stained sion no. DDJB AB305159; ATCC BAA-1522, DSM 19515 with haematoxylin and eosin (HE). The sections were and JCM 14813) [1]. To date, isolated stained by Gram’s and Grocott’s methods to show the from pigs has been known to have three genera and ten spe- microorganism. cies. In the genus , [5], Act- In order to examine immunohistochemical cross-reactivi- inomyces hyovaginalis [3], Actinomyces israelii [18], ties of rabbit hyperimmune serum against Arcanobacterium Actinomyces naeslundii [18], Actinomyces suimastitidis [8], abortisuis between Arcanobacterium abortisuis and ten Act- [4] and unpublished Actinomyces sp. inomycetaceae isolated from pigs mentioned above, anti- strain Chiba 101 (Actinomyces sp. Chiba 101: This strain gens were prepared from ten bacterial species listed below: has very similar morphological and microbiological proper- Seven species of Actinomyces were Actinomyces bovis ties to those of Actinomyces suis reported by Franke (1973) (ATCC 13683), Actinomyces hyovaginalis (BM 1192/5), and Grässer (1962) [15], and forms actinomycotic abscesses Actinomyces israelii (ATCC 43320), Actinomyces naeslun- in swine tonsillar crypts and mammary glands [13]) have dii (ATCC 12104), Actinomyces suimastitidis (CCUG been reported. The other species are Arcanobacterium pyo- 39276), Actinomyces viscosus (ATCC 15987) and Actino- genes [17], Arcanobacterium sp. strain (TO16177) (Arcano- myces sp. Chiba 101; two species of Arcanobacterium were bacterium sp. TO16177) [14] and Actinobaculum suis Arcanobacterium pyogenes (ATCC 19411) and Arcanobac- (former Actinomyces suis, Eubacterium suis) [9, 12]. terium sp. TO16177; and Actinobaculum suis (DMS Among them Actinomyces hyovaginalis [6], Actinomyces 20369). Arcanobacterium abortisuis (strain Murakami) [1] naeslundii [16] and Actinomyces suis [21] have been was used as a positive control. reported as infective agents, which cause porcine abortion. For preparation of the tissue samples, nine Actinomyceta- In the present study we report the first case of porcine abor- ceae mentioned above except for Arcanobacterium sp. tion associated with Arcanobacterium abortisuis, and it’s TO16177 were directly injected into the lung of sacrificed pathological and immunohistochemical features. mice and tissues were fixed in 10% neutral buffered forma- Porcine abortion had occurred off and on during six lin and embedded in paraffin. Arcanobacterium sp. months in one pig farm, raising approximately 600 sows, in TO16177 was directly injected into swine liver followed by Chiba Prefecture Japan. These sows were commonly the same histopathological process described above [14]. injected with live vaccine against Japanese encephalitis As the primary serum, rabbit hyperimmune serum against virus, Aujesky’s virus and porcine reproductive and respira- Arcanobacterium abortisuis was used and diluted stepwise tory syndrome virus. A placenta and three fetuses of a sow, in 0.01 M phosphate-buffered saline with 0.05% Tween 20. which aborted at day 87 of her fifth gestation, were exam- Immunohistochemical technique was performed with the ined for diagnosis. streptavidin-biotin conjugation method using a Histofine At autopsy, placenta, brain, heart, lung, liver, spleen, kid- SAB-PO(R) Kit (Nichirei, Inc., Tokyo, Japan) following the manufacturer’s instructions. The serial sections were *CORRESPONDENCE TO: MURAKAMI, S., Department of Animal Sci- ence, Tokyo University of Agriculture, 1737 Funako, Atsugi, stained without the primary antibody and used as negative Kanagawa 243–0034, Japan. controls. In addition, the sections from the fetuses and the e-mail: [email protected] placenta which contained the purulent lesions were also 798 S. MURAKAMI ET AL. immunostained using the same method. Three fetuses macroscopically revealed subcutaneous edema, congested lungs with/without hemorrhages, and sig- nificantly increased fluids in the body cavities. The placenta showed a cloudy surface accompanied by congestion. The major histopathological lesions in the all three fetuses were suppurative bronchopneumonia. The alveoli and bron- chial lumen were filled with a large number of degenerative immature neutrophils and bacterial clumps. Congestion and mild infiltration of macrophages were also observed around the lesions (Fig. 1a). In the placenta, although only a few neutrophils and macrophages could be seen in the embry- onic connective tissue on the maternal side, on the allantoic side the tissue was thickened with a large number of degen- erative neutrophils and macrophages showing necrotic and suppurative placentitis (Fig. 2a). Other lesions of these fetuses included mild infiltration of macrophages in the peri- cardium, congestion or hemorrhages in the cardiac paren- chyma. The microorganisms in the lesions were positive for Gram’s and Grocott’s staining and revealed to be rod-shaped or frizzled hair-like filament (Fig. 1c). In the Immunohistochemical analysis, the antigens of Arcanobacterium abortisuis were clearly immunolabeled with 1:51,200 diluted antibody (Fig. 1b and 2b) on the fila- mentous microorganisms in the pulmonary and the placental Fig. 1. a: Congestion around the bronchi and piled degenerative lesions. The bacterial antigen of Arcanobacterium sp. immature neutrophils in bronchi and alveoli of the fetus. HE. TO16177 was immunolabeled with 1:25,600 dilution. On Bar=50 m. b: Frizzled hair-like filaments or rod-shaped bacte- the contrary, the other were not stained even with a ria stained positively with Grocott’s method in the aborted fetal dilution of 1:800 or less. lung. Bar=10 m. c: Immunohistochmically labeled Arcanobac- Although the lesions linked to viral infections were not terium abortisuis antigen (*) in pulmonary lesions in a serial sec- found in the present case, it is not able to rule out the possi- tion of a by streptavidin-biotin conjugate method. Bar=50 m. ble association with virus infections because of no virologi- cal examinations performed. However, it is reasonable to very similar to the case we presented here. However the conclude that this may be the first report of porcine abortion previous reports were lack of the detailed description on the primarily associated with Arcanobacterium abortisuis, bacteriological investigations. In the present case, there which is elucidated by pathological and immunohistochem- were no cross-reactivities between the antigen against ical investigations. Arcanobacterium abortisuis and all other antigens exam- The genus Arcanobacterium has become independent ined except for Arcanobacterium sp. TO16177, which pre- from the genus Actinomyces, which had originally consisted sented a strong positive reaction against Arcanobacterium of eight species: Arcanobacterium bernardiae [17], Arcano- abortisuis antibody. Apart from the immunohistochemical bacterium bialowiezense [11], Arcanobacterium bonasi investigation, Arcanobacterium abortisuis (strain [11], Arcanobacterium haemolyticum [2], Arcanobacterium Murakami) showed 99.4% similarity to Arcanobacterium hippocoleae [7], Arcanobacterium phacae [17], Arcanobac- sp. TO16177 [14] in 16S ribosomal DNA gene sequencing terium pluranimalium [10] and Arcanobacterium pyogenes analysis based on a comparison of 677 bp (unpublished [17]. These bacteria were isolated from the inflammatory data). Therefore, it can be highly possible that Arcanobac- lesions of human and/or animals, and Arcanobacterium pyo- terium abortisuis is the same or closely-related to Arcano- genes has been known only as the causative agent of bacterium sp. OT16177. abscesses of pigs [19], but there has been no report about the In the present study, Arcanobacterium abortisuis was abortion caused by Arcanobacterium pyogenes. On the associated with the aborted lesions of a pig. On the other other hand, there have been three reports on porcine abor- hand, it has been indicated that Arcanobacterium sp. tions caused by Actinomyces hyovaginalis [6], Actinomyces TO16177 was associated with multiple organ failures naeslundii [16] and Actinomyces suis [21]. The typical accompanied by acute hemorrhagic necrotizing splenititis in lesions of these aborted fetuses infected with Actinomyces the pig [14]. Furthermore, in the very recent past, it has naeslundii and Actinomyces suis were suppurative bron- been confirmed that the microorganism isolated from the chopneumonia with intralesional bacterial clumps and pyo- urogenital tract of nine pigs with varied clinical symptoms granulomatous bronchopneumonia, although no was Arcanobacterium abortisuis, though it was unclear histopathology for Actinomyces hyovaginalis has been whether Arcanobacterium abortisuis was the exact caus- available so far. Those lesions in the previous reports are ative agent for the symptoms [20]. Thus, it may be not able ARCANOBACTERIUM ABORTISUIS INFECTION 799

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