Non-suppurative and necrotizing testicular arteritis in the male reproductive organs of a heavy draft horse

ABSTRACT

Equine testicular arteritis commonly occurs as a consequence of the migration of nematode larvae or equine arteritis virus (EAV) infection. However, testicular arteritis without evidence of these infections has been reported, and the underlying pathogenesis remains unclear. We encountered testicular arteritis without evidence of nematode or EAV infection in a 3-year-old male heavy draft horse with scrotal enlargement. Grossly, the volume of the pampiniform plexus was markedly increased due to edema. Histologically, non-suppurative and necrotizing testicular arteritis, characterized by lymphocyte infiltration and fibrinoid necrosis of the arterial walls, was diffusely observed in the spermatic cord, pampiniform plexus (most severe), testis, and epididymis. We were unable to identify the cause of arteritis, such as a viral infection or autoimmune abnormality.

Vasculitis is the pathological term used to describe inflammation within and around blood vessel walls. The pathogenesis of vasculitis is mainly classified into two types, direct damage by infectious pathogens and non-infectious mechanisms [8]. The infectious pathogens inducing vasculitis include viruses, chlamydia, bacteria, fungi, protozoa, and helminths [9]. The pathogenesis of vasculitis due to non-infectious mechanisms can be further divided into two broad categories: immune-mediated and non-immune-mediated mechanisms. In the equine testicular artery, the migration of strongyle larvae and equine arteritis virus (EAV) infection are common causes of inflammation [4, 9, 11]. Inflammation of the testicular artery in horses without evidence of strongyle larvae or EAV infection has also been reported; however, detailed pathological investigations other than examinations for these pathogens have not been performed. Thus, its pathogenesis remains unclear [4, 6]. In the present study, we examined inflammation of the testicular artery in a heavy draft horse with no evidence of strongyle larvae or EAV infection and discussed the pathogenesis.

A 3-year-old male heavy draft horse (crossbreed between Percheron, Belgian, and Breton heavy draft horses) was presented to the Veterinary Medical Center of Obihiro University of Agriculture and Veterinary Medicine with an approximately 3-month history of bilateral scrotal enlargement (Fig. 1). Ultrasonography of the scrotum revealed the intrascrotal accumulation of a large amount of fluid (Fig. 2). The case showed no other apparent clinical signs and did not present any abnormalities in a hematological examination or serum biochemical analysis. The level of serum amyloid A was measured several times and was always under the detection limit. However, scrotal enlargement did not improve for several weeks, and castration was performed. In surgery, approximately 500 ml of translucent, reddish, serous fluid was found to have accumulated in the scrotum on each side. The fluid was subjected to bacterial culture examinations, and no bacteria were detected in aerobic or anaerobic cultures. The prognosis has been favorable to date (15 months after castration).

Fig. 1.

The scrotum before castration. The volume of the bilateral scrotum is markedly increased.

Fig. 2.

Ultrasonography of the right scrotum. A large amount of fluid accumulated in the scrotum (asterisk).

The resected tissue was subjected to pathological examinations. Grossly, the volume of the bilateral pampiniform plexus was markedly increased due to severe edema of the interstitial connective tissue (Fig. 3a). Moderate to severe edema was also present in the tunica albuginea and connective tissue around the spermatic cords. Edema was most severe in the pampiniform plexus. The collected tissue was fixed in 15% neutral buffered formalin and processed using routine methods, sectioned at a thickness of 3 µm, and stained with hematoxylin and eosin for histological examinations.

Fig. 3.

a) Cross section of the long axis of the testis and pampiniform plexus. The volume of the pampiniform plexus (surrounded by the blue dotted line) is increased due to severe interstitial edema. Edema is also observed in connective tissue around the spermatic cord (surrounded by the pink dotted line). There are no gross changes in the testicular parenchyma (surrounded by the green dotted line). Bar=10 cm. b) Arteritis in the pampiniform plexus. Vasculitis is observed in small- to medium-sized testicular arteries. Severe edema is observed in interstitial connective tissue. Hematoxylin and eosin (HE) stain. Bar=3,000 µm. c) Arteritis in the pampiniform plexus. Arteries, characterized by lymphocyte infiltration into the blood vessel walls, is observed in small-sized arteries. On the other hand, in the vein (arrow), edema and inflammation of the blood vessel wall are milder than those in the arteries. There are no lesions in the lymph duct (arrowhead). HE stain. Bar=100 µm. d) Arteritis in the spermatic cord. Non-suppurative arteritis with fibrinoid necrosis of the tunica media is observed. HE stain. Bar=50 µm. e) Arteritis in the tunica albuginea. Areas of fibrinoid necrosis are positive for the PAS reaction. Bar=50 µm. f) Arteritis in the testis and testicular degeneration. In addition to arteritis, testicular degeneration is observed. Seminiferous tubules consist only of Sertoli cells, and germ cells and spermatogenesis are absent. HE stain. Bar=100 µm. g, h) Arteritis in the pampiniform plexus. CD3-immunolabelled T lymphocytes (g) and CD20-immunolabelled B lymphocytes (h) both infiltrate the vascular walls, and the former is the more predominant subtype. Bar=50 µm.

Arteritis, which was characterized by inflammatory cell infiltration into the tunica media and tunica adventitia, was observed diffusely in the testicular arteries in the spermatic cord, pampiniform plexus, tunica albuginea, testis, and epididymis (Fig. 3b–f). In the following histopathological descriptions, which are based on the international classification of vasculitis in humans [5], the words large-, medium-, and small-sized arteries were used for the aorta and its major branches, the main visceral arteries and their main branches, and the intraparenchymal arteries and arterioles, respectively. Vasculitis in the present case was very severe in small- to medium-sized arteries and was absent or very mild in large-sized arteries and veins (Fig. 3b, 3c). The inflammatory cells infiltrating vascular walls were mainly lymphocytes, and a few macrophages were also observed. Arteritis was frequently accompanied by swelling of endothelial cells and fibrinoid necrosis of blood vessel walls (Fig. 3d). Fibrinoid necrosis was confirmed by a positive reaction in PAS staining (Fig. 3e) and staining red with Masson’s trichrome stain. Arteritis with severe necrotic changes despite of slight inflammation was also noted. Interstitial edema and focal perivascular inflammation were observed, and interstitial edema was severe especially in the pampiniform plexus (Fig. 3b). In the testis, seminiferous tubules consisted only of Sertoli cells arranged in a single layer, and no germ cells or spermatozoa were present (Fig. 3f). Moreover, Leydig cells had focally increased in number. Orchitis and epididymitis were not observed.

Immunohistochemistry (IHC) using primary antibodies for CD3 (1:100; mouse monoclonal antibody; Dako, Glostrup, Denmark) and CD20 (1:400; rabbit polyclonal antibody; Thermo Fisher Scientific, Fremont, CA, USA) was conducted to identify the subtypes of lymphocytes infiltrating the testicular arterial walls. Sections were pretreated with citrate buffer, pH 6.0, at 98°C for 15 min, and endogenous peroxidase was blocked with 3% H₂O₂. After incubation with primary antibodies at 4°C overnight, sections were incubated with MAX-PO polymer reagent (Nichirei Bioscience, Tokyo, Japan) at room temperature for 30 min. Each bound primary antibody was visualized using 3–3′-diaminobenzidine (Nichirei Bioscience), and sections were counterstained with hematoxylin.

To investigate whether immune-mediated mechanisms, such as the production of autoantibodies to the testicular arteries, were involved in the testicular arteritis of the present case, we performed indirect immunofluorescence (IF) examinations on sections of normal testicular arteries from a control case using serum from the present case. Sections of normal testicular arteries were prepared from an approximately 4-year-old male heavy draft horse, which had no lesions in the reproductive organs and died of peritonitis due to perforation of the small colon. In the indirect IF protocol, after heat-induced antigen retrieval, sections were incubated with diluted serum from the present case (1:10, 1:50, and 1:100) at room temperature for 2 hr. The sections were then incubated with anti-horse IgA (alpha chain, 1:200, goat polyclonal antibody; Bethyl Laboratories, Montgomery, TX, USA), anti-horse IgG (heavy and light chain, 1:200, rabbit polyclonal antibody; Bethyl Laboratories), or anti-horse IgM (µ chain, 1:200, goat polyclonal antibody; Bethyl Laboratories) antibodies at 4°C overnight, followed by reaction with goat anti-rabbit IgG (H+L) Alexa Fluor^TM 594 (1:400, Thermo Fisher Scientific) or donkey anti-goat IgG (H+L) Alexa Fluor^TM 488 (1:400, Thermo Fisher Scientific) at 37°C for 30 min. For counterstaining of nuclei, 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) was applied. As a negative control, the serum of the present case was replaced with that of a healthy 7-year-old male heavy draft horse.

Virological examinations were also conducted to investigate whether EAV, equine infectious anemia virus (EIAV), equine herpes virus (EHV), and equine adenovirus, which are known to cause vasculitis in horses, were associated with arteritis in the present case. The involvement of EAV was examined by an enzyme-linked immunosorbent assay (ELISA) provided by Nisseiken (Tokyo, Japan). The involvement of EIAV was assessed using an agar gel immunodiffusion test with an equine infectious anemia virus antibody test kit (VMRD, Pullman, WA, USA). For assessment of the involvement of EHV-1, EHV-4, and equine adenovirus-1 by quantitative PCR (qPCR), DNA was extracted from a formalin-fixed paraffin-embedded block of the pampiniform plexus using a commercial kit (NucleoSpin tissue, Macherey-Nagel, Düren, Germany). The primer pair used in the present study was based on those in previous studies [1,2,3]. Beta-2-microglobulin was used as the endogenous control for qPCR examinations.

The results of IHC for CD3 and CD20 revealed that CD3-positive T lymphocytes were more predominant than CD20-positive B lymphocytes in the foci of arteritis (Fig. 3g, 3h). In indirect IF examinations of anti-IgA, IgG, and IgM antibodies using sections of normal testicular arteries, there were no apparent positive reactions in sections incubated with the serum of the present case compared with those incubated with the serum of the control case. The results of IF examinations suggested that there was no evidence of IgA-, IgG-, or IgM autoantibodies to the testicular arteries being produced in the present case. Also, examinations for viral pathogens (EAV, EIAV, EHV-1, EHV-4, and equine adenovirus 1) revealed no associations for these pathogens with arteritis in the present case.

Inflammation of the testicular arteries in horses commonly occurs as a consequence of the migration of strongyle larvae and EAV infection [4, 9, 11]. A previous study focusing on equine testicular lesions due to invasion of the larvae of nematodes revealed that testicular vasculitis was characterized by the infiltration of lymphocytes, hemosiderin-laden macrophages, mast cells, and eosinophils [6, 10]. Moreover, parasitic migration tracts, which are characterized by hemorrhage and the infiltration of lymphocytes, eosinophils, and mast cells in the acute stage and granulomatous inflammation in the chronic stage, were frequently observed in the affected testis and epididymis. Peri-orchitis, epididymitis, sperm granuloma, and testicular degeneration were also reported in some parasitic cases [4, 6, 10]. In the present case, the bilateral testes had degenerated; however, parasitic migration tracts, orchitis, and epididymitis were not detected. The inflammatory cells observed in the arteritis were mainly T lymphocytes and macrophages. Therefore, the arteritis of the present case did not appear to be due to migrating larvae.

Vasculitis due to EAV infection is characterized by the infiltration of lymphocytes with fibrinoid necrosis in the tunica media of small arteries [9]. Since arteritis occurs systemically, edema is frequently observed in the scrotum, periorbital region, and subcutaneous tissue of the limbs and abdomen of affected horses. In addition to edema, EAV-infected horses present various clinical signs, such as fever, ocular and nasal discharges with rhinitis and conjunctivitis, and skin rash [9, 11]. In Japan, equine viral arteritis due to EAV infection is designated as a notifiable infectious disease by the Act on Domestic Animal Infectious Diseases Control, and its occurrence has not been reported to date [7]. Based on the absence of systemic clinical signs, the epidemiological context, and the results of ELISA tests, the arteritis in the present case was not considered to be associated with EAV infection.

In a previous case report of a stallion with unilateral swelling of the scrotum, pampiniform phlebitis induced enlargement of the pampiniform plexus, which was similar to the gross lesion in the present case. Since the nature of phlebitis was pyogenic, the authors suspected that bacterial infection was the cause of the vasculitis [12]. On the other hand, in the present case, lesions were detected bilaterally, and the nature of the arteritis was non-suppurative with fibrinoid necrosis. Bacterial growth was not observed in aerobic or anaerobic cultures of the fluid that accumulated in the scrotum. Therefore, bacterial infection was unlikely to be the cause of the arteritis in the present case.

The results of histopathological, indirect IF, and viral examinations failed to reveal a direct association of pathogens or an autoimmune abnormality with the arteritis in the present case. The use of some drugs, such as itraconazole, ivermectin, and trimethoprim-sulfadiazine, may cause vasculitis in animals [9]. However, the present case had no recent history of administration of any drugs. Food hypersensitivity and poisoning by toxic plants may cause vasculitis in animals [9]. Since there were no other horses with scrotal enlargement at the farm at which the present case was kept, poisoning did not appear to be the cause of arteritis. There was also no evidence to suggest that food hypersensitivity was the cause of the arteritis, and we were unable to elucidate the precise pathogenesis of the present case.

In the present case, apparent abnormalities were not detected clinically before or after castration, except for scrotal swelling, and the prognosis after castration has been favorable. Therefore, arteritis may be confined to testicular arteries in the reproductive organs. The arteritis in the present case had similar histological features to those observed in equine viral arteritis, such as lymphocyte infiltration and fibrinoid necrosis of the vascular walls [9]. Although the cause is currently unknown, veterinarians should be aware that lesions similar to equine viral arteritis may be encountered, even in countries in which equine viral arteritis has not yet occurred. In addition to viral examinations, the presence or absence of systemic clinical signs or lesions was considered to be important in differentiating the current condition from equine viral arteritis-related one.

Histologically, testicular degeneration, such as the disappearance of germinal cells, was observed in the present case. The causes of testicular degeneration range from aging to toxicosis, and changes in blood flow are one of the main causes [4]. In consideration of the age and clinical course of the present case, in which no abnormalities other than in the testes were observed, the testicular degeneration appeared to be due to inflammation of the testicular arteries.

In conclusion, the underlying cause of inflammation of the testicular arteries in horses has not been investigated in detail, except for examinations for strongyle larvae and EAV infection. Although we examined testicular arteritis in a horse without evidence of larvae migration or EAV infection pathologically, the precise pathogenesis remains unclear. Spermatogenesis was not observed in the testes of our case, and this suggests that inflammation of the testicular arteries may have a negative impact on equine reproduction. Therefore, it is important to elucidate the precise pathogenesis of inflammation of the testicular arteries, as in the present case, and further studies with more cases are required.