Case Report: Dendritic Cells and Macrophages Capture Sperm in Chronically Inflamed Human Epididymis.

Chronic inflammation of the male genital tract is thought to be a primary etiological factor of male infertility. The abundance and activation of macrophages and dendritic cells in patients with chronic inflammation of genital tract were closely associated with oligozoospermia and asthenospermia. Chronic epididymitis appears to be more important than seminal vesiculitis or prostatitis due to the direct interaction between spermatozoa and epididymal inflammatory cells. In this study, we present a case report of a 41-year-old male with oligoasthenospermia and chronic epididymitis. Hematoxylin-eosin staining and immunofluorescence analyses showed that antigen presenting cells including macrophages and dendritic cells were found capturing spermatozoa in the lumen of cauda epididymis. To our knowledge, this is the first case report that directly observed dendritic cells capturing spermatozoa in the lumen of an inflamed epididymis. This finding directly explains chronic epididymitis as the possible cause of oligospermia in patients.

Differential Immune Response to Infection and Acute Inflammation Along the Epididymis.

The epididymis is a tubular structure connecting the vas deferens to the testis. This organ consists of three main regions-caput, corpus, and cauda-that face opposing immunological tasks. A means of combating invading pathogens is required in the distally located cauda, where there is a risk of ascending bacterial infections originating from the urethra. Meanwhile, immune tolerance is necessary at the caput, where spermatozoa with immunogenic neo-antigens originate from the testis. Consistently, when challenged with live bacteria or inflammatory stimuli, the cauda elicits a much stronger immune response and inflammatory-inflicted damage than the caput. At the cellular level, a role for diverse and strategically positioned mononuclear phagocytes is emerging. At the mechanistic level, differential expression of immunoprotective and immunomodulatory mediators has been detected between the three main regions of the epididymis. In this review, we summarize the current state of knowledge about region-specific immunological characteristics and unveil possible underlying mechanisms on cellular and molecular levels. Improved understanding of the different immunological microenvironments is the basis for an improved therapy and counseling of patients with epididymal infections.

The Immune Characteristics of the Epididymis and the Immune Pathway of the Epididymitis Caused by Different Pathogens.

The epididymis is an important male accessory sex organ where sperm motility and fertilization ability develop. When spermatozoa carrying foreign antigens enter the epididymis, the epididymis shows "immune privilege" to tolerate them. It is well-known that a tolerogenic environment exists in the caput epididymis, while pro-inflammatory circumstances prefer the cauda epididymis. This meticulously regulated immune environment not only protects spermatozoa from autoimmunity but also defends spermatozoa against pathogenic damage. Epididymitis is one of the common causes of male infertility. Up to 40% of patients suffer from permanent oligospermia or azoospermia. This is related to the immune characteristics of the epididymis itself. Moreover, epididymitis induced by different pathogenic microbial infections has different characteristics. This article elaborates on the distribution and immune response characteristics of epididymis immune cells, the role of epididymis epithelial cells (EECs), and the epididymis defense against different pathogenic infections (such as uropathogenic Escherichia coli, Chlamydia trachomatis, and viruses to provide therapeutic approaches for epididymitis and its subsequent fertility problems.

Region-specific immune responses to autoimmune epididymitis in the murine reproductive tract.

Epididymitis is a common pathology of the male reproductive tract, potentially leading to infertility. Studies on bacterial epididymitis indicate that the cauda epididymis is more susceptible to inflammatory damage than the caput. These regional differences in immunoregulation are further investigated using an experimental autoimmune epididymo-orchitis model. Adult mice were immunized against testicular antigens and tissues were collected at 30 and 50 days following the first immunization. Epididymitis developed progressively; 70% of the mice developed disease at 30 days after the initial immunization and 93% at 50 days. Epididymitis was characterized by epithelial damage, immune cell infiltrates and fibrosis in the cauda, with minimal changes in the corpus, while the caput was unaffected. The incidence of epididymitis was greater than that of orchitis but similar to vasitis. The severity of epididymitis was positively correlated with the orchitis severity. Expression of key genes implicated in epididymal immunoregulation, inflammation and fibrosis, such as Ido1, Tnf, Tgfb1, Ccl2, Il1b, Il10, Cx3cl1 and Col1a1, was unchanged in the caput but increased in proportion to damage severity in the cauda at 50 days. Activin receptor mRNA expression in the cauda was negatively correlated with disease severity. These data suggest that the cauda is highly susceptible to inflammatory damage following an autoimmune challenge but the caput is minimally affected. This may be because the cauda is required to combat ascending infections through a robust inflammatory response, while the caput provides a more tolerogenic environment in order to protect the auto-antigenic sperm released from the testis.

Differential tissue-specific damage caused by bacterial epididymo-orchitis in the mouse.

Ascending bacterial urinary tract infections can cause epididymo-orchitis. In the cauda epididymidis, this frequently leads to persistent tissue damage. Less coherent data is available concerning the functional consequences of epididymo-orchitis on testis and caput epididymidis. This in vivo study addresses the functional and spatial differences in responsiveness of murine epididymis and testis to infection with uropathogenic Escherichia coli (UPEC). Whole transcriptome analysis (WTA) was performed on testis, caput, corpus and cauda epididymidis of adult C57BL/6 J wildtype mice. Following UPEC-induced epididymo-orchitis in these mice, epididymal and testicular tissue damage was evaluated histologically and semi-quantitatively at 10 days and 31 days post-inoculation. Expression of inflammatory markers and candidate antimicrobial genes were analysed by RT-qPCR. WTA revealed distinct differences in gene signatures between caput and cauda epididymidis, particularly amonst immunity-related genes. Cellular and molecular signs of testicular inflammation and disruption of spermatogenesis were noticed at day 10, but recovery was observed by day 31. In contrast to the cauda, the caput epididymidis did not reveal any signs of gross morphological damage or presence of pro-inflammatory processes despite confirmed infection. In contrast to beta-defensins, known UPEC-associated antimicrobial peptides (AMP), like Lcn2, Camp and Lypd8, were inherently highly expressed or upregulated in the caput following infection, potentially allowing an early luminal protection from UPEC. At the time points investigated, the caput epididymidis was protected from any obvious infection/inflammation-derived tissue damage. Studies addressing earlier time-points will conclude whether in the caput epididymidis a pro-inflammatory response is indeed not essential for effective protection from UPEC.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord.

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.

Damaged male germ cells induce epididymitis in mice.

Epididymitis can be caused by infectious and noninfectious etiological factors. While microbial infections are responsible for infectious epididymitis, the etiological factors contributing to noninfectious epididymitis remain to be defined. The present study demonstrated that damaged male germ cells (DMGCs) induce epididymitis in mice. Intraperitoneal injection of the alkylating agent busulfan damaged murine male germ cells. Epididymitis was observed in mice 4 weeks after the injection of busulfan and was characterized by massive macrophage infiltration. Epididymitis was coincident with an accumulation of DMGCs in the epididymis. In contrast, busulfan injection into mice lacking male germ cells did not induce epididymitis. DMGCs induced innate immune responses in epididymal epithelial cells (EECs), thereby upregulating the pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), as well as the chemokines such as monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-5 (MCP-5), and chemokine ligand-10 (CXCL10). These results suggest that male germ cell damage may induce noninfectious epididymitis through the induction of innate immune responses in EECs. These findings provide novel insights into the mechanisms underlying noninfectious epididymitis, which might aid in the diagnosis and treatment of the disease.

Melatonin protects against lipopolysaccharide-induced epididymitis in sheep epididymal epithelial cells in vitro.

Melatonin has protective effects against inflammation but its role in epididymitis is unknown. We addressed this in the present study using lipopolysaccharide (LPS)-stimulated sheep epididymal epithelial cells as an in vitro inflammation model. We found that interleukin (IL)-1ß, IL-6, tumor necrosis factor α, and cyclooxygenase (COX)-2 mRNA levels; COX-2 and Toll-like receptor (TLR)-4 protein levels; and nuclear factor (NF)-κB p65 phosphorylation were increased by LPS treatment. These effects were reversed in a dose-dependent manner by melatonin (10-11-10-7 M). Quantitative reverse transcription PCR and immunofluorescence analyses showed that the melatonin receptors MT1 and MT2 were expressed in sheep epididymal epithelial cells. The inhibitory effect of melatonin on inflammation was abrogated by the MT1 and MT2 receptor antagonist luzindole and the MT2 ligand 4-phenyl-2-propanamide tetraldehyde. Thus, melatonin exerted anti-inflammatory effect in epididymal epithelial cells by inhibiting TLR4/NF-κB signaling, suggesting its potential as an effective drug for the treatment of epididymitis in sheep.

Meningitis and epididymitis caused by Toscana virus infection imported to Switzerland diagnosed by metagenomic sequencing: a case report.

BACKGROUND: We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland. CASE PRESENTATION: The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days. CONCLUSIONS: This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.

ASSOCIATION OF MEAN PLATELET VOLUME AND THE MONOCYTE/LYMPHOCYTE RATIO WITH BRUCELLA-CAUSED EPIDIDYMO-ORCHITIS.

We evaluated the association between the mean platelet volume (MPV) and monocyte/lymphocyte ratio (MLR) with brucella-caused epididymo-orchitis to determine if they could be used to differentiate between brucella and non-brucella epididymo-orchitis. The charts of 88 patients with non-brucella and 14 patients with brucella epididymo-orchitis were retrospectively reviewed. Brucellosis was diagnosed by isolating Brucella spp from a blood culture or from a serum agglutination titer ≥ 1:160 along with accompanying clinical findings. The patients with brucella epididymo-orchitis were significantly more likely to have a lower MPV and a higher MLR than those with non-brucella epididymo-orchitis. Using a MPV cut-off level of less than 9.25 fl to differentiate brucella from non-brucella epididymo-orchitis gives a sensitivity of 78.6%, a specifity of 78.4%, a positive predictive value of 36.7% and a negative predictive value of 95.8%. Using a MLR cut-off level of greater than 0.265 to differentiate brucella from non-brucella epididymo-orchitis gives a sensitivity of 71.4%, a specifity of 65.9%, a positive predictive value of 25% and a negative predictive value of 93.5.%. MPV and MLR values may assist in differentiating between brucella and non-brucella epididymo-orchitis.

Characterisation of dendritic cell subsets in chronically inflamed human epididymis.

Infection and inflammation of the genital tract are thought to be a primary aetiological factor of male infertility. Chronic epididymitis appears to be more important than prostatitis or seminal vesiculitis due to the direct interaction between sperm cells and epididymal epithelium. Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells that play a crucial role in the regulation of the immune response and immunological tolerance. The aim of this study was to investigate the expression and characteristic of different DC subsets in chronic inflammation of human epididymis and controls. Our study demonstrated that normal human epididymis contained only immature CD1a(+) DCs, CD11c(+) myeloid DCs (mDCs) and CD209(+) DCs whereas CD123(+) plasmacytoid DCs and CD83(+) mature DCs were virtually absent. The number of both CD11c(+) IL-23(+) mDCs and CD123(+) pDCs were significantly elevated in inflamed epididymis; meanwhile the mDC populations of CD1a(+), CD209(+) immature DCs and CD83(+) mature DCs also increased in inflamed group. Moreover, Th17 (CD4(+) IL-17(+)) cells were predominantly distributed under chronic inflammation of human epididymis. Taken together these results suggest that epididymal DCs might play a pivotal role in the development of chronic epididymitis and induce an increased recruitment of Th17 cells under inflammatory conditions.

CLINICAL AND ANAMNESTIC CHARACTERISTICS OF DEVELOPMENT OF ANTISPERM IMMUNITY IN INFERTILE MEN.

The aim of the research was to establish connection between the roles of infertility duration, patient age, various extragenital and genital pathologies in ASA pathogenesis. The work is based on retrospective analysis of 496 outpatient cards, which covered detailed anamnestic data and results of past complex clinico-laboratory tests. Criteria for inclusion in the study was infertility during marriage for at least 18 month, high concentration of ASA-s in booth blood and sperm. It was found that there is no statistically significant connection between the ASA and past illnesses, profession, family history, accompanying autoimmune diseases and extragenital pathologies. Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis compared to other STDs, cause statistically significant increase of ASAs concentration in blood serum, as well as in ejaculate (p<0,001). Testicle associated pathologies plays an important role in development of antisperm immunity, especially orchitis and epididymitis, during which statistically insignificant increase of levels of ASA has been detected in blood, as well as in semen (p<0,001). Thus, we can conclude that, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, orchitis and epididymitis causes malfunction of blood-testicular barrier, which resulted in inflammatory and toxic damage of spermatogenic epithelium, which in turn plays significant role in the development of autoimmune infertility.

Primary cell cultures for understanding rat epididymal inflammation.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development.

Exercise training protects against an acute inflammatory insult in mouse epididymal adipose tissue.

Exercise training reduces systemic and adipose tissue inflammation. However, these beneficial effects seem to be largely tied to reductions in adipose tissue mass. The purpose of the present study was to determine if exercise training confers a protective effect against an acute inflammatory challenge. We hypothesized that the induction of inflammatory markers, such as interleukin 6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), and TNF-α by the beta-3 adrenergic agonist CL 316,243 would be reduced in adipose tissue from trained mice and this would be associated with reductions in transient receptor potential cation channel 4 (TRPV4), a protein recently shown to regulate the expression of proinflammatory cytokines. Exercise training (4 wk of treadmill running, 1 h/day, 5 days/wk) increased markers of skeletal muscle mitochondrial content and the induction of PPAR-gamma coactivator 1 alpha in epididymal adipose tissue. The mRNA expression of IL-6, SOCS3, and TNFα were not different in subcutaneous and epididymal adipose tissue from sedentary and trained mice; however, the CL 316,243-mediated induction of these genes was attenuated ∼50% in epididymal adipose tissue from trained mice as were increases in plasma IL-6. The effects of training were not explained by reductions in lipolytic responsiveness, but were associated with decreases in TRPV4 protein content. These results highlight a previously unappreciated anti-inflammatory effect of exercise training on adipose tissue immunometabolism and underscores the value of assessing adipose tissue inflammation in the presence of an inflammatory insult.

Uropathogenic Escherichia coli modulates innate immunity to suppress Th1-mediated inflammatory responses during infectious epididymitis.

Infectious epididymitis in men, a frequent entity in urological outpatient settings, is commonly caused by bacteria originating from the anal region ascending the genitourinary tract. One of the most prevalent pathogens associated with epididymitis is Escherichia coli. In our previous study, we showed that semen quality is compromised in men following epididymitis associated with specific E. coli pathovars. Thus, our aim was to investigate possible differences in immune responses elicited during epididymitis following infection with the uropathogenic E. coli (UPEC) strain CFT073 and the nonpathogenic enteric E. coli (NPEC) strain 470. Employing an in vivo experimental epididymitis model, C57BL/6 mice were infected with UPEC CFT073, NPEC 470, or phosphate-buffered saline (PBS) as a sham control for up to 7 days. After infection with NPEC 470, the expression of proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in the epididymis was significantly increased. Conversely, UPEC CFT073-challenged mice displayed inflammatory gene expression at levels comparable to sham PBS-treated animals. Moreover, by day 7 only NPEC-infected animals showed activation of adaptive immunity evident by a substantial influx of CD3+ and F4/80+ cells in the epididymal interstitium. This correlated with enhanced production of Th1-associated cytokines IL-2 and gamma interferon (IFN-γ). Furthermore, splenocytes isolated from UPEC-infected mice exhibited diminished T-cell responses with significantly reduced secretion of IL-2 and IFN-γ in contrast to NPEC-infected animals. Overall, these findings provide new insights into understanding pathogen-specific modulation of host immunity during acute phases of epididymitis, which may influence severity of disease and clinical outcomes.

Testicular inflammation as a new manifestation of IgG4-associated disease.

IgG4-related disease has properties of a systemic disorder but simultaneously is associated with a growing list of organ-specific manifestations including autoimmune pancreatitis, IgG4-associated cholangitis, IgG4-related kidney disease, and IgG4-associated prostatitis. In this study, we present, to the best of our knowledge, the first case of a patient with multiorgan IgG4-related disease who lost his testes because of IgG4-related testicular inflammation. We postulate that IgG4-related disease in the urogenital tract is not restricted to IgG4-related kidney disease and prostatitis, but that this rare disorder may also affect the testis.

[Diagnosis and management of adverse events occuring during BCG therapy for non-muscle invasive bladder cancer (NMIBC): review of the Cancer Committee of the French Association of Urology]. / Diagnostic et prise en charge des événements indésirables survenant au décours des instillations endovésicales de BCG pour le traitement des tumeurs de vessie n'infiltrant pas le muscle (TVNIM) : revue du comité de cancérologie de l'Association française d'urologie.

INTRODUCTION: Intravesical BCG immuno-therapy with maintenance therapy is considered as the standard treatment for non-muscle invasive bladder cancer with high risk of recurrence and progression. In practice, adverse events (AEs) of BCG therapy could restrict its prescription by urologists. The aim of this article was to present a review of these AEs and of their management. MATERIALS AND METHODS: A bibliographic research in French and English using Medline(®) and Embase(®) with the keywords "BCG", "bladder", "complication", "toxicity", "adverse reaction", "prevention" and "treatment" was performed. RESULTS: The main mechanism of AEs of BCG are infectious (cystitis, fever), immuno-allergic (granulomatous prostatitis, epididymo-orchitis, and granulomatous reactions) and auto-immune (arthralgies, rash). Management of AEs is based on their pathophysiological mechanisms. Classifications of BCG therapy AEs based on clinical features allow to adapt their treatments. CONCLUSION: The combination of antibiotics directed against BCG, steroid or non-steroidal anti-inflammatory medication and symptomatic treatment is currently the triad on which is set up the appropriate treatment of severe AEs. Reductions of BCG doses and ofloxacin medication after instillation decrease the frequency and severity of minor and moderate AEs. Severe or more than 7 days long infectious AEs, immuno-allergic AEs or auto-immune during more than 7 days impose cessation of BCG immuno-therapy.

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats.

OBJECTIVE: Male rats transgenic for HLA-B27 and human ß(2) -microglobulin (hß(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hß(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS: Male B27/hß(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. RESULTS: In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION: Autoimmune EO develops spontaneously in HLA-B27/hß(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.

Vaccination programs for reproductive disorders of small ruminants.

Vaccines are available for the control of contagious epididymitis and abortion in small ruminants, although many of them have significant limitations either in efficacy or safety to both the animals vaccinated and to the people handling the vaccine or animals. Shelf-life of vaccines should be extended and improved, so that the vaccine remains effective with longer term storage and ideally without refrigeration, so that use in under-developed rural areas is not restricted (e.g., Brucella melitensis, Toxoplasma gondii). The vaccines should not be dangerous for veterinarians or producers to handle (again as examples, B. melitensis, T. gondii). The vaccines should prevent shedding of the organism, in order to prevent spread of the disease causal agent through the sale of vaccinated but shedding animals (e.g., inactivated killed Chlamydophila abortus vaccines), as well as to prevent possible exposure to people handling those vaccinated animals. Production of vaccines using zoonotic disease agents is problematic and sometime dangerous, which increases regulatory restrictions and reduces availability of those vaccines (e.g., C. abortus, Coxiella burnetii). Development of subunit recombinant DNA vaccines may offer a method to increase access to these important vaccines, as long as they are also effective, prevent shedding and remain cost effective. It is important that these vaccines are brought to international commercial production. As many of these disease agents are zoonotic and prevalent world-wide, improvement in vaccine efficacy and safety is of extreme importance.