Semen quality and pathological lesions in rams naturally infected with Brucella ovis during an outbreak in Saskatchewan

Abstract

This report documents clinical signs, semen parameters, and reproductive organ lesions in rams during a natural Brucella ovis outbreak. Eleven rams from 1 flock underwent 2 breeding soundness evaluations and necropsies. Brucella ovis was isolated from all semen ejaculates and confirmed via whole-genome sequencing. Despite above-threshold scrotal circumferences, all rams were categorized as unsatisfactory due to poor semen quality and leukocytes in semen. Semen motility values in 9 of 11 infected rams and morphologically normal sperm in all infected rams were below the recommended threshold values. Necropsies revealed gross lesions in 7 of 11 rams, with histopathology revealing inflammatory lesions in the epididymides, ductus deferens, ampullae, and seminal vesicles in all rams and in the testes in 9 of 11 rams. Inflammatory infiltrates were primarily lymphoplasmacytic and neutrophilic, with fibrosis, vasculitis, mineralization, and spermatic granulomas also observed.

Key clinical message:

Gross and histopathological lesions as well as semen characteristics were described in rams naturally infected with B. ovis. Most were subclinical cases. Veterinary practitioners can utilize changes in semen quality and the presence of leukocytes as potential indicators of B. ovis infection in rams, which may prompt further confirmatory testing.

Brucella ovis is a Gram-negative coccobacillus that, in rams, predominantly causes a sexually transmitted disease leading primarily to ovine infectious epididymitis, orchitis, and infertility. Although the primary effect is on rams, B. ovis infection can also potentially result in abortions in ewes and perinatal mortality in neonatal lambs (1,2). Inoculation studies in rams have shown an incubation period of 3 to 17 wk. Typically, rams can be infected for 2 to 4 y and may become chronic carriers, shedding bacteria intermittently in semen, urine, and genital secretions (1,2). In ewes, the infection is cleared quickly; however, ewes serve as a vector, transmitting disease from infected to noninfected rams in multi-sire breeding situations (1–3). The disease also can be transmitted among rams due to homosexual behavior (4). All Brucella species exhibit a shared mechanism of infection involving the invasion of host immune cells and a tropism for genital tissues (4,5). Natural B. ovis infection has also been reported in farmed red deer, and experimental infection can be induced in goats, bighorn sheep, cows, and other deer species (1,2). Unlike other species within this genus, B. ovis is not known to be zoonotic. This feature is attributed to specific genetic modifications and a rough phenotype that render B. ovis avirulent for humans (6,7). In contrast, B. canis, which also shares the rough phenotype of B. ovis, is a zoonotic pathogen (8).

Contagious epididymitis in rams due to B. ovis infection, was reported first in New Zealand in 1953 (7–9) and subsequently from other locations, including North America, during the latter half of 1950s (1,7,9,10). However, limited information is available on semen characteristics and necropsy findings in naturally infected rams across Canada, including Saskatchewan. In a single report on sheep presented for necropsies during 1976 and 1977 at the Western College of Veterinary Medicine in Saskatoon, 1 of 8 rams seropositive for B. ovis, originating from a flock affected by late-term abortions, showed orchitis and epididymitis but was culture-negative for B. ovis (11). Within Canada, B. ovis infection is currently listed in the annually notifiable diseases category by the Canadian Food Inspection Agency. Provincial categorization for regulation of this disease varies (12).

Over the past 7 decades, B. ovis has been studied intensively with regards to disease epidemiology, pathogenesis, and control. Typically, B. ovis infection is suspected in rams that exhibit testicular and epididymal lesions during clinical examinations or breeding soundness evaluations (BSE) (1,2), though other bacteria may also be involved (13–16). The effects of B. ovis on ram semen parameters and reproductive tissues have been extensively studied using inoculation experiments (17). However, only a limited number of studies have investigated the pathological findings and semen characteristics of rams naturally infected with B. ovis (18–20). These studies relied on either archived reproductive tissues or B. ovis-seropositive rams with unknown bacterial shedding status in semen. A longitudinal study on naturally infected rams reported fluctuations in seroconversion, bacterial shedding in semen, and pathological lesions, but did not provide information on semen quality or BSE outcomes (21). Our report addresses these gaps by providing comprehensive information on clinical observations, semen quality, and associated pathological changes in the reproductive organs of rams naturally infected with B. ovis and actively shedding the bacteria in semen. Furthermore, this case series presents an outbreak of B. ovis infection in a Saskatchewan sheep flock for which all bacterial isolates were confirmed via whole-genome sequencing.

CASE DESCRIPTIONS

Eleven rams, aged 1 to 5 y and representing various breeds (Dorset, Dorper, Texel, Southdown, Romanov, Cheviot, Ile de France, and Canadian Arcott), were part of a broader study on BSE criteria. These rams were used for breeding in a multi-sire group during the 2021 to 2022 natural breeding season. In June 2022, a routine BSE — comprising a physical examination, reproductive assessment including scrotal circumference measurement, and detailed semen analysis with gross and microscopic evaluation — revealed unsatisfactory semen parameters in all but 1 ram. Two rams exhibited unilaterally enlarged epididymides and 1 ram exhibited unilaterally shrunken testes. Subsequently, semen ejaculates from all 11 rams were submitted to Prairie Diagnostic Services (Saskatoon, Saskatchewan) (PDS) for bacteriological culture on Skirrow agar. Brucella ovis was isolated from all 11 rams and confirmed using whole-genome sequencing. Following recommendations, the producer decided to cull and donate all the rams with no treatment administered.

In August 2022, a follow-up BSE was done that included a general clinical assessment focusing on body condition scores (5-point scale in which 1 is very thin and 5 is very fat), teeth, eye condition, leg conformation, hooves, and gait. The reproductive examination involved a visual assessment of scrotal skin for lesions and palpation of scrotal contents to evaluate size and consistency, with particular attention to the testes and parts of the epididymis. Testicular mobility within the scrotum was assessed by gently moving testes dorsally and ventrally within the scrotal sac. Scrotal circumference was measured for each ram using a scrotal tape (ReliaBull; Western Drug Distribution Centre, Edmonton, Alberta). In addition, the presence or absence of penile protrusion during electroejaculation and the number of stimulations required for semen ejaculation were recorded.

Semen analysis was conducted following the collection of semen ejaculates via electroejaculation. A 2-minute transrectal massage was applied to the accessory sex glands using the well-lubricated probe of a commercially available handheld electroejaculator (Lane Manufacturing, Lebanon, Oregon, USA). Electroejaculation was carried out, with the 1st stimulation lasting 6 s, followed by up to 8 additional stimulations each lasting 8 s, with 2 to 4 s between stimulations. Most rams (10/11) ejaculated within 3 stimulations, and the semen in each case was collected into a plastic cone attached to a calibrated plastic test tube at the tapered end. Collected semen ejaculates were evaluated both grossly and microscopically. Gross evaluation included assessment of semen volume, color, and density; microscopic evaluation involved assessing motility and sperm morphology (eosin-nigrosin stain), as well as the presence (> 2 in high-power field, 100×) or absence of leukocytes in the stained semen smears. Leukocytes in semen were also confirmed by Feulgen staining. In addition, a portion of each semen ejaculate was submitted to PDS for bacteriological isolation and whole-genome sequencing of isolates positive for B. ovis. The resulting nucleotide sequences for each B. ovis isolate were uploaded to public databases for molecular typing and microbial genome diversity (PubMLST, www.pubmlst.org) to identify any matches in the existing Brucella sequence-typing database. Furthermore, the semen ejaculates were processed for culturing by PDS, per their routine methods, to isolate and identify both aerobic and anaerobic bacteria.

A 5-year old healthy ram from a different flock enrolled in the larger study, confirmed negative for B. ovis in semen through bacteriological culture and meeting satisfactory BSE criteria, was selected as a negative control. Gross and histopathological evaluations were done on all rams (11 infected rams plus the control) by a Board-certified pathologist (SS). Tissue samples were collected from the testes (parenchyma and rete testes), epididymides (head, body, and tail), ductus deferens, prostate gland, bulbourethral gland, ampullae, seminal vesicles, prepuce, and penis of every ram for histopathological evaluation. Histopathological study was conducted at PDS using hematoxylin and eosin-stained tissue sections and following routine protocols.

Breeding soundness evaluation findings

The BSE outcomes for each B. ovis-infected and the noninfected ram in August are provided in Table 1. Physical examinations of all 11 B. ovis infected rams were unremarkable, with body condition scores ranging from 2 to 3.5 on a 5-point scale. All rams had satisfactory scrotal circumferences (mean ± SD: 36 ± 2.5 cm) for their respective age groups (≥ 30 cm for rams under 14 mo and ≥ 33 cm for rams over 14 mo), and penile protrusion was observed during ejaculation in all rams. The only notable findings were unilateral epididymal tail enlargement in 2 rams and 1 ram with a small and firm right testis. Semen volume (1.2 ± 0.5 mL), density, and color were unremarkable. However, both gross motility (poor in 9 rams, good and fair in 1 ram each) and individual progressive motility (24 ± 17%, excluding 2 infected rams) were below the recommended levels for rams. Eosin-nigrosin-stained semen smears of all infected rams revealed a low percentage of normal spermatozoa (41 ± 12%), with high proportions of detached heads (40 ± 15%) and midpiece defects (14.6 ± 14%) (Figure 1). In addition, leukocytes were present in the semen smears of all rams, ranging from a few to abundant. The B. ovis-negative healthy ram was 5 y old with an unremarkable physical examination and was characterized as a satisfactory potential breeder (Table 1).

TABLE 1.

Summary of breeding soundness evaluation parameters for Brucella ovis-infected rams and noninfected (control) ram.

Ram	Breed	SC (cm)	Gross motility	Individual motility (%)	Live (%)	Normal (%)	Head (%)	MP (%)	PP (%)	Detached head (normal, %)	Detached head (abnormal, %)	Proximal droplets (%)	Scrotal palpation findings
1	Texel	37.5	P	45	50	44	3	4	—	48	1	—	Enlarged epididymal tail (right)
2	Southdown	35.5	P	20	50	41	—	1	—	56	2	—	Enlarged epididymal tail (left)
3	Romanov	39	P	10	75	31	—	28	—	23	—	18	—
4	Texel	34	P	10	50	14	1	42	—	44	—	—	—
5	Texel	32	P	40	70	48	—	8	—	36	4	4	Small and firm testis (right)
6	Texel	33	F	70	80	67	—	11	—	21	—	1	—
7	Cheviot	40	P	10	45	54	1	32	—	11	2	—	—
8	Ile de France	37.5	P	50	50	45	3	5	—	41	5	2	—
9	Arcott	36	G	60	85	57	—	6	—	34	3	—	—
10	Dorset	37.5	P	10	40	28	1	12	—	58	1	—	—
11	Dorset	34.5	P	20	50	38	2	8	1	49	2	—	—
Control	Arcott	38.5	G	90	90	92	—	4	—	2	—	2	—

Head — Head defects; MP — Midpiece defects; PP — Principal piece defects; SC — Scrotal circumference.

For gross motility: F — Fair; G — Good; P — Poor.

Semen smears stained with eosin-nigrosin were used for sperm morphology and live/dead counts.

FIGURE 1.

Characteristic sperm morphology in Brucella ovis-infected rams. A — Morphologically normal sperm (arrowhead), detached sperm heads (asterisks), and distal midpiece reflexes (arrows). B — Leukocytes (isolated or clumps) alongside dead sperm. C — Neutrophils (arrows). Eosin-nigrosin staining (A, B) and Feulgen staining (C); 100× magnification.

Gross and histopathological findings

During necropsy, various gross lesions were identified in the reproductive organs of infected rams, but not in those of the noninfected ram. Overall, 7 infected rams exhibited gross lesions in at least 1 reproductive organ or tissue (Figure 2). Specifically, the lesions included reddish fluid in the vaginal cavity (Figure 3 A); thickening of the tunica vaginalis; multiple firm adhesions (Figure 3 A) of the vaginal tunic to the parietal tunic covering the testicle or tail of the epididymis; scattered bilateral red foci of hemorrhages in the testicular parenchyma (Figure 3 B); and gritty, white, focal mineralization in the testicular parenchyma beneath the tunica albuginea (Figure 3 C). Two rams that exhibited unilateral epididymitis had pale-yellow foci of purulent/caseous exudate in the tail of the epididymis (Figure 3 D). In addition, 1 ram had a small and firmer testicle with an epididymal cyst in the caput, and another ram exhibited an enlarged unilateral prescapular lymph node containing thick, yellow pus.

FIGURE 2.

Proportions of Brucella ovis-infected rams (n = 11) exhibiting gross and histopathological lesions during necropsy examination.

FIGURE 3.

Gross lesions in Brucella ovis-infected rams. Adhesions between vaginal and parietal tunics covering the tail of the epididymis (arrow in A), multiple red foci in testicular parenchyma (arrows in B), gritty white foci in testicular parenchyma (arrows in C), and multiple yellowish foci of caseous exudate in the body of the epididymis (arrows in D).

Histopathological changes were observed primarily in the testes, epididymis, ductus deferens, seminal vesicles, ampulla, and prepuce of all infected rams (Figure 4), and not in those of the noninfected ram. Among the 11 infected rams with histopathological lesions, 6 exhibited variable amounts of focal tubular degeneration, mineralization, and fibrosis of the testicular parenchyma, involving the interstitium and seminiferous tubules (Figure 4). In addition, these rams showed fibrotic changes and granulomatous inflammation characterized by macrophages, multinucleated giant cells, scattered lymphocytes, and plasma cells in the interstitium. In 1 ram, interstitial edema was noted in specific areas of testes, with edema and vasodilatation in the rete testes. Another ram exhibited proliferation of thin-walled, blood-filled vascular structures in the interstitium in certain sections. Rete lesions were identified in 6 rams and were characterized by scattered infiltrates of lymphocytes and plasma cells, with some neutrophils and macrophages in the interstitium.

FIGURE 4.

Histopathological changes in testes and epididymis of a noninfected ram (A to D) versus Brucella ovis-infected rams (E to L). Multilayered seminiferous tubules with mature spermatozoa toward lumen in testicular parenchyma of a noninfected ram (A, 10×). Tubular degeneration (arrows, E) and mineralization (arrows, I) in the seminiferous tubules of a B. ovis-infected ram (10×). Sections of head (B), body (C), and tail (D) of the epididymis of the noninfected ram (10×). Tubular degeneration (arrow) and lymphoplasmacytic infiltrates (asterisk) in the section of epididymal head (F, 10×). Head of the epididymis (J, 40×) with lymphoplasmacytic infiltration (arrow). Body of the epididymis (G, 10×) with tubular degeneration (arrow), lymphocytes and plasmacytes in the interstitium, and peritubular fibrosis (asterisk). Spermatic granuloma (arrow) adjacent to the body of the epididymis (K, 4×). Section of epididymal tail of an infected ram (H, 10×) with epithelial hyperplasia (thick arrow), and intraepithelial cysts with neutrophils and cellular debris (thin arrow). Tail of the epididymis (L, 10×) of another infected ram with epithelial hyperplasia and severe intraepithelial lumina formation (asterisks). Note the neutrophilic accumulation (#) in the lumen and engulfment of neutrophils and necrotic debris into the epithelium (arrow). Hematoxylin and eosin staining.

Histopathological lesions were observed throughout all parts of the epididymides in infected rams (Figure 4). In 8 of the 11 infected rams, the head of the epididymides exhibited scattered infiltrates of lymphocytes and plasma cells (Figure 4), interstitial edema, and thickened fibrotic tunics. One ram displayed fibrosis and infiltrates of lymphocytes and plasma cells around ductules, suggesting ductular atrophy in the head of the epididymides. The body of the epididymides in 9 of 11 infected rams showed lymphoplasmacytic and neutrophilic infiltrates, interstitial fibrosis (Figure 4), edema, congestion, thickened fibrotic tunics with neovascularization, spermatic granuloma formation (Figure 4), foci of mineralization, tubular atrophy, multifocal vasculitis, epithelial hyperplasia, and the formation of intraepithelial lumina. Inflammatory changes in the tail of the epididymides were present in all 11 infected rams (Figure 4) and included lymphoplasmacytic and neutrophilic infiltrates with epithelial hyperplasia; intraepithelial lumina formation; infiltration of lymphocytes, plasma cells, and neutrophils in the interstitium; increased interstitial fibrosis; transmigration of neutrophils and lymphocytes through the epithelium; and neutrophilic clusters with vasculitis. One ram displayed unilateral spermatic granuloma and tunica-vaginitis in the histologic section.

In 1 ram, the ductus deferens section was not obtained (missing data). The ductus deferens (Figure 5) of the other 10 infected rams and ampullae (Figure 5) of all infected rams exhibited lymphoplasmacytic and neutrophilic infiltrates within the adventitia, along with infiltration of neutrophils, lymphocytes, and plasma cells in the lamina propria; epithelial hyperplasia; transepithelial migration of neutrophils and lymphocytes; and neutrophil clusters within the epithelium. All infected rams exhibited bilateral seminal adenitis characterized by lymphoplasmacytic and neutrophilic infiltrates (Figure 5). Some glands contained eosinophilic material mixed with sloughed cells, cellular debris, and nonviable neutrophils. In 1 ram, congestion, interstitial edema, and hemorrhage were also observed in the vesicular glands. Among the remaining 10 rams, only 1 exhibited prostate lesions, with a few scattered lymphocytes and plasma cells in the interstitium (Figure 5). The prostatic urethra of every infected ram showed multifocal infiltration of lymphocytes and plasma cells in the lamina propria. Three rams had some scattered lymphocytes and plasma cells in the interstitia of the bulbourethral glands (Figure 5).

FIGURE 5.

Histopathological changes in accessory sex glands of a noninfected ram (A to E) versus Brucella ovis-infected rams (F to J). Ductus deferens (F) and ampulla (G) of infected rams showing lymphocytes, plasma cells, neutrophils (asterisks), epithelial hyperplasia (arrows), and inflammatory cell transmigration into the epithelium. Seminal vesicle (H) with epithelial hyperplasia (arrows), interstitial infiltration of lymphocytes and plasma cells (asterisks), and acini with cellular debris (#). Prostate gland section (I) shows basal cell hyperplasia (asterisk) and lymphocytes, neutrophils, and plasma cells in the interstitium. Bulbourethral gland (J) showing scattered infiltrations of lymphocytes and plasma cells (asterisk) in the glandular epithelium and interstitium. Hematoxylin and eosin staining; 10× magnification (A to J).

In every infected ram, preputial sections exhibited infiltration of lymphocytes, plasma cells, eosinophils, and neutrophils in the dermis and around blood vessels, hair follicles, and sebaceous glands. Focal folliculitis and scattered lymphoid aggregates were observed in the lamina propria-submucosa of the inner preputial mucosa. Penile section of only 1 infected ram showed infiltrates of lymphocytes and plasma cells in the lamina propria of the penile urethra, with inflammatory cells multifocally transmigrating to the urothelium. None of the reproductive tract (Figure 4) or accessary gland (Figure 5) tissues from the noninfected ram exhibited the inflammatory lesions described herein.

DISCUSSION

This report is unique in that it provides detailed descriptions of both gross and histopathological lesions in reproductive tissues, along with BSE parameters, in rams diagnosed with B. ovis infection and active bacterial shedding in the semen. All B. ovis isolates matched 21 loci selected for multilocus genotyping and were identified as B. ovis sequence type 13. This sequence type represents 23 isolates from 3 continents listed in public databases for molecular typing and microbial genome diversity.

In our case series, only 3 B. ovis-infected rams exhibited clinical signs (epididymal enlargement or shrunken testes) during physical examination. Despite adhesions between the vaginal and parietal tunics in some infected rams, the testes remained mobile within their respective scrota. Previous reports suggested that 30 to 50% of rams with B. ovis infection develop visible or palpable lesions in the testes and/or epididymides (1). Thus, subclinical infection could easily have been missed during routine BSE or physical examinations. However, the rams were actively shedding the bacteria in their semen. These findings align with recent research indicating that B. ovis infection often lacks distinct, pathognomonic clinical symptoms and is frequently subclinical (3,18). Consequently, the disease may circulate within a flock without being detected by producers and can even go unnoticed during general physical examinations by veterinarians. We did not conduct ultrasound examination of the scrotal contents or accessory sex glands of the rams described herein. Based on the gross lesions observed in B. ovis-infected rams, clinicians may consider using ultrasound to assess subclinical cases. However, ultrasound may lack specificity, as inflammatory or necrotic lesions of these tissues could result from transient conditions or age-related changes.

We observed that semen volume, density, and color were unremarkable, but individual motility was poor and sperm morphology was abnormal. This, along with the presence of leukocytes in the semen, indicates that a microscopic evaluation is more informative than gross evaluation of semen. However, other infections in the reproductive tract, such as Actinobacillus seminis, Histophilus ovis, Haemophilus spp., Corynebacterium pseudotuberculosis ovis, Chlamydophila abortus, and Brucella melitensis, can also lead to poor sperm motility and morphological abnormalities (9,17,18). In the current case series, semen from B. ovis-infected rams was also culture-positive for other bacteria in 10/11 cases, with 7/11 semen ejaculates containing 1 additional bacterium and 3/11 yielding 2 additional bacteria. The seminal bacteria identified included Corynebacterium cystitidis (4/11), Acinetobacter sp. (3/11), Staphylococcus sp. (3/11), Trueperella pyogenes (2/11), Pseudomonas sp. (1/11), and Streptococcus ovis (1/11). It remains unclear whether the presence of these other bacteria was incidental or whether any associations contributing to reproductive diseases exist between B. ovis and the other cultured bacteria. We observed high proportions of loose heads, distal midpiece reflexes, and leukocytes in the stained smear during BSE of B. ovis-infected rams. Given the contagious nature of B. ovis and its association with male infertility, observations of poor semen quality, characteristic sperm morphology, and the presence of leukocytes in semen during routine BSE of rams should raise suspicion and warrant testing for B. ovis infection.

Both gross and histopathological examinations revealed a combination of acute and chronic inflammatory changes in all parts of the epididymides, ampulla, seminal vesicles, and prostatic urethra. These inflammatory lesions were consistent with previous reports on rams naturally infected with B. ovis (4,17,18,21). The rams in the current case series were exposed to an older ram in a multi-sire breeding group that had been diagnosed with clinical contagious epididymitis and pus in the semen 8 mo before necropsy. The inflammatory lesions in the epididymis and accessory sex glands could explain the presence of leukocytes, sperm defects, and poor motility, potentially due to a combination of defective maturation during epididymal transit and abnormal glandular secretions. Despite the erythritol content being 3× higher in the seminal vesicles than in the testes of rams (22), the exact reason for the preference of B. ovis for the epididymides and accessory glands remains unclear, especially since the bacteria cannot catabolize the sugars commonly used by other Brucella species. It is likely that B. ovis relies on alternative sources, such as sugars and flavoproteins, for energy metabolism to support its survival and pathogenicity (23,24). Although the origin of the B. ovis infection could not be confirmed, the outbreak — primarily subclinical — in a single flock underscores the need for a province-wide strategy to screen Saskatchewan sheep flocks for potential B. ovis infection.

In conclusion, our findings underscore the presence of subclinical B. ovis infections even during outbreaks, despite lesions being widespread throughout the reproductive tissue. This observation emphasizes the importance of detecting such cases during BSE. Collectively, the poor semen quality parameters and the presence of leukocytes in semen identified in this case series could serve as useful indicators for identifying potentially infected rams and warranting follow-up testing to confirm the prevalence.

Funding Statement

The authors extend their gratitude to the Agriculture Development Fund (#20200237), the Saskatchewan Sheep Development Board, the ADF Experiential Discovery and Learning through Research in Food Animal Veterinary Medicine Grant (WCVM), and the Western College of Veterinary Medicine at the University of Saskatchewan, for funding.