Actinobacillus suis-associated iliac thromboembolism caused acute posterior paralysis in a young boar

Abstract

A 5-month-old imported boar showed posterior paralysis upon arrival in Japan. Both limbs had coldness, algesia, and thigmesthesia. A thrombus was detected from the abdominal aorta to the left external iliac artery. Irregular vegetation was observed on the left atrioventricular valve. The skeletal muscles in the hind limb appeared like boiled meat. The histological diagnoses were endocarditis, glomerulonephritis, and meningoencephalitis with Gram-negative bacilli as well as diffuse intravascular coagulation and diffuse alveolar damage. The bacilli were identified as Actinobacillus suis via immunohistochemistry and 16S rRNA gene sequencing. This is the first case report of acute posterior paralysis due to iliac thromboembolism in swine with A. suis infection. Iliac thromboembolism should be considered if posterior paralysis is observed in swine.

Actinobacillus suis is a Gram-negative bacillus and an opportunistic pathogen on farms with a high health status [23]. The clinical signs of its infection in young and adult pigs differ. Fattening pigs, gilts, and sows present with dermatitis and abortion [9, 10, 24], whereas sudden death and increased mortality rates are observed among suckling and weaned pigs [11, 16].

Iliac thromboembolism occasionally occurs in domestic animals due to congenital cardiac defects and intracardiac thrombi [14]. In cats with cardiomyopathy, iliac thromboembolism originates from thrombi in the dilated left atrium in cases of cardiomyopathy [14]. Ohkubo et al. reported that cardiac defects were found in one of 4 calves of sudden posterior paralysis due to thromboembolism [12]. Pigs are used as an animal model of iliocaval deep venous thrombosis [7, 17]. However, no reports of naturally occurring iliac thromboembolism with posterior paralysis have been provided. This is the first report of acute posterior paralysis due to iliac thromboembolism in swine with A. suis infection.

Twenty-six boars (5 and 6-month-old) were raised commercially on a porcine reproductive and respiratory syndrome (PRRS)-free farm in the United States of America (USA) in 2025. No pigs were introduced into the exported herd in the USA for 11 days of export inspection before shipment to Japan. The boars showed no evidence of any infectious disease based on careful clinical inspection conducted by the USA governmental authorities immediately before shipment to Japan. None of the boars showed abnormal clinical signs or evidence of disease upon transportation to Japan. One boar had posterior paralysis upon arrival at Narita Airport; however, no clinical or hematological abnormalities were observed in the other boars during the subsequent 15 days of import quarantine. The boar with posterior paralysis showed polypnea, tachycardia, and fever (41.2°C) upon arrival at the isolation facility. Physical examination revealed that both posterior limbs of the boar presented coldness, algesia, and thigmesthesia. Closely aggregated or fused scars were observed on the right thigh, and the hoof of the left hind limb was dehisced. The boar was found dead the morning after its body temperature was measured as 42.1°C. A large red thrombus extending from the abdominal aorta to the left external iliac artery were detected (Fig. 1a). Irregular vegetation was observed on the left atrioventricular valve (Fig. 1b). All lobes of the lungs heightened the degree of hardness were diffusely dark red and had a patchy appearance. The skeletal muscles in both hind limbs appeared like boiled meat. In the right limb, the skeletal muscles were discolored, and encapsulated abscesses less than 1 cm in diameter were found under the thigh scars. The pylorus of the stomach had red-brown mucosa. No gross lesions were found in any other organ, including the spinal cord.

Fig. 1.

(a) Gross findings of the abdominal artery. A large red thrombus was found from the abdominal artery to the left iliac artery. (b) Gross findings of the heart. Irregular vegetation (arrowheads) was observed on the left atrioventricular valve.

The liver, spleen, tonsils, inguinal lymph nodes, kidneys, heart, lungs, stomach, brain, spinal cord (from 2nd thoracic vertebra to 6th lumbar vertebra), and skin were sampled and fixed in 10% neutral-buffered formalin. Fixed tissues were embedded in paraffin wax, sectioned (4-μm thick), and stained with hematoxylin and eosin (HE) and Gram (Brown-Hopps) for histological examination.

Histologically, bacterial endocarditis characterized by Gram-negative bacilli was observed in the left atrioventricular valve, along with scattered lesions of coagulative necrosis of the myocardium with severe neutrophil aggregation (Fig. 2a). Diffuse segmental necrosis with neutrophil infiltration, fibrin thrombi, and a few Gram-negative bacterial emboli were observed in the glomeruli of the kidneys (Fig. 2b). Fibrin thrombi were also observed in the arterioles, muscular arteries, veins, and capillaries of the heart, lungs, liver, stomach, brain, and spinal cord. Coarse neuropils, neutrophil aggregation, and hemorrhage surrounded the Gram-negative bacterial emboli in the brain. In the spinal cord, only one small area of ​​neutrophil aggregate was found in the white matter, and no neuronal necrosis was observed. Exudation of edema fluid and formation of hyaline membranes in alveolar spaces were found in all the lobes of the lungs, and they corresponded with the characteristic lesions of diffuse alveolar damage. Encapsulated abscesses with Gram-positive cocci were found in the subcutaneous tissue of the right thigh with scars. Severe lymphoid depletion and macrophage proliferation were also observed in the spleen (Fig. 2c). Crypt abscesses with Gram-negative bacilli were observed in the tonsils. The inguinal lymph nodes had no noticeable changes.

Fig. 2.

(a) Numerous bacilli and inflammatory cell infiltration were observed on the left atrioventricular valve. (inset, higher magnification of the border between bacilli and the valve, Bar=50 μm). Hematoxylin-eosin staining (HE). Bar=200 μm. (b) Segmental necrosis with neutrophil infiltration, fibrin thrombi (white arrowhead), and bacterial embolus (black arrowhead) were observed in the glomeruli of the kidneys. HE. Bar=50 μm. (c) Significant lymphoid depletion and replacement by macrophages were observed in white pulp of the spleen. Positive reactions against anti-porcine circovirus type 2 antibody were observed in the cytoplasm of the macrophages (inset, higher magnification of the macrophage aggregation, Bar=20 μm). HE. Bar=500 μm. (d) Immunohistochemical analysis demonstrated that the Gram-negative bacilli reacted with the anti-Actinobacillus suis rabbit antibody at the same site shown in (a) (inset, higher magnification of the border between the bacilli and valve, Bar=50 μm). Bar=200 μm.

Formalin-fixed paraffin-embedded (FFPE) tissue samples of the heart, kidneys, brain, spleen, and tonsils, which were cut into 4-μm-thick sections, were subjected to immunohistochemical examination to detect A. suis and porcine circovirus type 2 (PCV2) antigens. The primary antibodies used were anti-A. suis rabbit polyclonal antibody (National Institute of Animal Health, Tsukuba, Japan) and anti-PCV2 rabbit polyclonal antibody (National Institute of Animal Health) diluted 1:8,000 and 1:500, respectively [8]. The antibodies were used in a commercially available antibody diluent (Enzo Life Sciences Inc., Farmingdale, NY, USA). The procedure was performed with commercial kits according to the manufacturer’s instructions (Histofine Simple Stain MAX-PO (MULTI) and Histofine Simple Stain AEC solution; Nichirei, Tokyo, Japan). Primary antibodies were omitted as negative controls. Several positive reactions to the anti-A. suis antibody were observed in Gram-negative bacilli of the heart, kidneys, brain, and tonsils (Fig. 2d). In the spleen, positive reactions against the anti-PCV2 antibody were observed in the cytoplasm of macrophages (Fig. 2c, inset).

For bacterial isolation, the liver, spleen, kidneys, heart, lungs, right thigh abscess, and blood were spread onto sheep blood agar plates (Nissui Pharmaceutical Co., Tokyo, Japan). These plates were incubated at 37°C in the presence of 5% carbon dioxide under aerobic or anaerobic conditions for 18 hr. Gram-negative bacilli were isolated from the heart and kidneys of the swine on blood agar. In addition, Gram-positive cocci were isolated from the abscesses of the right thigh.

The isolated colonies were analyzed using 16S ribosomal RNA gene (16S rDNA) sequencing. DNA was extracted from bacterial colonies using a DNA extraction kit (InstaGene Matrix; Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. A variable region of 16S rDNA was amplified and sequenced using a MicroSeq^TM 500 16S rDNA PCR/Sequencing Kit (Applied Biosystems Life Technologies, Carlsbad, CA, USA) [22]. Sequence data were collated in the EzBioCloud database (https://www.ezbiocloud.net/) [25]. Gram-negative bacilli matched A. suis ATCC 33415 (99.32% identity). Gram-positive cocci matched Streptococcus porcinus, ATCC 43138 (99.54% identity) and Staphylococcus hyicus, Private JH70 (99.92% identity).

To detect the PCV2 gene, the spleen, and tonsils of diseased pig and serum samples from the other twenty-five boars were subjected to nested polymerase chain reaction (PCR) [6]. All serum samples were collected on the date of death. The QIAamp DNA Kit for DNA Extraction (Qiagen, Redwood City, CA, USA) and High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland) were used for DNA extraction, following the manufacturers’ instructions. The primer set of 1st PCR were 5′-CAGAACAATCCACGGAGGAA and 5′-ATGTTGCTGCTGAGGTGCTGC. The primer set of 2nd PCR were 5′-CACTTCGTAATGGTTTTTATTATT and 5′-CAGCGCACTTCTTTCGTTTT. A specific band of approximately 753 bp of PCV2 was amplified and visualized by agarose gel electrophoresis using GelRed (Biotium, Fremont, CA, USA) (data not shown). The nucleotide sequences of the 2nd PCR products were compared with those available in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST). The nucleotide sequences of the 2nd PCR products derived from the spleen and tonsils of the diseased boar and the serum samples from two live boars were consistent with that of PCV2.

The newly determined sequences derived from A. suis, S. hyicus, S. porcinus, and PCV2 were registered in the DNA Data Bank of Japan (accession numbers: LC880381, LC880382, LC880383, and LC890020, respectively).

Posterior paralysis is generally caused by an insult to the spinal cord, and its etiology is infectious or other. The clinical signs of posterior paralysis with iliac thromboembolism are pain, pulselessness, poikilothermy, and pallor [14]. In swine, it occurs in several infectious diseases such as classical swine fever, pseudorabies (Aujeszky’s disease), porcine enterovirus polioencephalomyelitis, and vertebral abscess [1, 3]. It can also occur in non-infectious diseases such as fibrocartilaginous embolism, vitamin D deficiency, and vertebral fracture [5, 15, 21]. In our investigation, we found no reports of posterior paralysis caused by naturally occurring iliac thromboembolisms in pigs. In the present imported lot, one boar showed posterior paralysis upon arrival at Narita Airport. However, none of the imported boars showed abnormal clinical signs or evidence of disease from the export inspection upon transport to Japan. In the physical examination upon arrival at the isolation facility, both posterior limbs of the boar presented coldness, algesia, and thigmesthesia. In gross findings, a large red thrombus extending from the abdominal aorta to the left external iliac artery was detected. The skeletal muscles in the hind limb appeared like boiled meat. However, no pathological findings were found in the spinal cord that could cause posterior paralysis. Based on the etiological, clinical, and pathological findings, posterior paralysis in the boar was attributed to iliac thromboembolism.

Endocarditis is a life-threatening condition usually caused by bacterial infection, although infective endocarditis is occasionally caused by parasites or fungi [14]. Several bacterial species can cause acute valvular endocarditis in domestic animals, depending on the host [14]. In swine, streptococci and Erysipelothrix rhusiopathiae are the most common causative agents [14]. The prevalence of endocarditis was 0.21% (4/1,933 cases) among cases of diseases caused by A. suis at the Iowa State University Veterinary Diagnostic Laboratory from 2017 to 2022 [20]. In the present case, A. suis caused bacterial endocarditis and vegetation on the left atrioventricular valve. The inflammatory reaction to the bacterial colonies of the vegetation in the left atrioventricular valve was less than that in the myocardial necrosis and bacterial emboli in the brain and kidneys. Generally, septicemia may cause diffuse intravascular coagulation [14]. In addition, a portion of the fragmented bacterial colony and diffuse intravascular coagulation may cause iliac thromboembolism [14]. Considering the difference in the inflammatory reaction to the valvular bacterial colonies and necrotic lesions in organs, the death of the boar was attributed to septicemic A. suis infection with the acute course from vegetation formation to iliac thromboembolism.

Septicemic A. suis infection in adult pigs resembles acute swine erysipelas or A. pleuropneumoniae infection [10, 24]. In addition to dermatitis or severe fibrinohemorrhagic and necrotizing pneumonia, bacterial colonies and fibrin thrombi are occasionally observed within vessels in organs [10, 24]. In the present case, no findings suggestive of a route of infection were observed in the gastrointestinal tract, respiratory system, or skin; however, A. suis antigens were detected in crypt abscesses of the tonsils. As A. suis is a ubiquitous opportunistic pathogen that colocalizes the upper respiratory tract, it might have invaded the vessels via the tonsils [4]. Immunosuppression caused by transport stress may have accelerated the disease progression from infection to death due to sepsis.

PCV2 infection is a well-known cause of immunosuppression in swine and can be detected in clinical healthy swine at all ages [2]. In experimental infections using 11-week-old pigs, no clinical disease was observed through 140 days post-inoculation and no gross lesions were observed at 140 days post-inoculation [13]. However, PCV2 detection in serum alternated between positive and negative [13]. In addition, lymphoid depletion, histiocytic replacement of follicles, and low amounts of PCV2 antigen in lymphoid tissues were rarely observed [13]. These experimental results were consistent with the individual diagnostic criteria for PCV2 subclinical infection (PCV2-SI) [18]. Due to the ubiquitous distribution of PCV2 among swine farms worldwide, the diagnostic value for PCV2-SI is considered poor [19]. In the present imported lot, the PCV2 gene was detected in the diseased boar and two healthy boars. In the diseased boar, significant lymphoid depletion and replacement by macrophages were observed in the white pulp of the spleen. Additionally, the PCV2 antigen was detected in the spleen. In two health boars, no clinical or hematological abnormalities were observed during the subsequent 15 days of import quarantine. Based on the clinical, pathological, and genetic findings, the three boars in which PCV2 was detected were diagnosis with PCV2-SI.

In conclusion, this is the first known report of acute posterior paralysis due to iliac thromboembolism in swine with A. suis infection. Iliac thromboembolism and spinal cord diseases should be considered if posterior paralysis is observed in swine.

CONFLICT OF INTEREST

The authors declare no conflicts of interest associated with this manuscript.