J.R. Campos

Endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis

Transient endometritis after breeding is necessary for clearance of bacteria and spermatozoa; however, in a subpopulation of mares, the inflammation fails to resolve in a timely fashion. The objective of this study was to describe the uterine inflammatory response in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) during the first 24 h after induction of uterine inflammation.Twelve mares were classified as susceptible (nZ6) or resistant (nZ6) to PBIE. Mares were inseminated over five estrous cycles and endometrial biopsies were collected at one time point per cycle before (0) and 2, 6, 12, and 24 h after insemination. qPCR analysis for IL1B, IL6, IL8, IFNG, TNF (TNFA), IL10, and IL1RN was performed, and endometrial inflammatory cells were counted for each sample. Relative quantification values reported fold changes in mRNA expression from 0 h values. A general pattern of expression post insemination was observed in both groups of mares. Cytokine mRNA increased at 2 h, peaked between 2 and 12 h, and then decreased.Differences were detected between groups of mares 6 h after challenge; resistant mares had higher mRNA expression of IL6, IL1RN,and IL10 than susceptible mares. Susceptible mares had an increased number of polymorphonuclear neutrophils in the endometrium 2 and 12 h after breeding when compared with resistant mares. These findings describe an inherent difference in the initial immune response to insemination and may help explain the transient nature of inflammation in resistant mares, whereas susceptible mares develop a persistent inflammation.

Susceptibility to persistent breeding-induced endometritis in the mare: Relationship to endometrial biopsy score and age, and variations between seasons

The objectives were to: (1) investigate the associations of age and endometrial biopsy score with uterine fluid retention after insemination; and (2) determine if a strict classification of susceptibility to persistent breeding-induced endometritis (PBIE) based on biopsy score, endometrial cytology, and fluid retention after inseminations, is consistent over subsequent breeding seasons. In Experiment 1, 57 mares were inseminated with 10(9) freeze-killed sperm during estrus and evaluated for uterine fluid retention 48 h and 96 h after insemination. Comparisons were made between fluid retention and biopsy score or age. In Experiment 2, a subset of 14 mares was classified for susceptibility to persistent breeding-induced endometritis in two subsequent breeding seasons. Biopsy score and age were associated with fluid retention (P < 0.001). In addition, age was related to biopsy score (P < 0.001). Of the mares examined for susceptibility, 36% (5 of 14) changed status during subsequent seasons. Three mares changed to a more severe classification (intermediate to susceptible, or resistant to intermediate), whereas two mares changed to a less severe classification (susceptible to intermediate).

Assessment of correlation between in vitro CD3+ T cell susceptibility to EAV infection and clinical outcome following experimental infection.

In a recent study, we demonstrated that the virulent Bucyrus strain (VBS) of EAV could infect in vitro a small population of CD3(+) T lymphocytes from some but not all horses. Furthermore, we have shown that a common haplotype is associated with this in vitro CD3(+) T cell susceptibility/resistance phenotype to EAV infection. In this study, we investigated whether the differences in the susceptibility or resistance of CD3(+) T cells in vitro correlate with the outcome and severity of clinical signs in vivo. Thus, horses were divided into two groups based on their CD3(+) T cell susceptible or resistant phenotype. Following experimental inoculation with the recombinant VBS of EAV, horses were assessed for presence and severity of clinical signs, duration and magnitude of virus shedding, as well as production of proinflammatory and immunomodulatory cytokines in peripheral blood mononuclear cells using real-time quantitative RT-PCR. The data showed that there was a significant difference between the two groups of horses in terms of cytokine mRNA expression and evidence of increased clinical signs in horses possessing the in vitro CD3(+) T cell resistant phenotype. This is the first study to provide direct evidence for a correlation between variation in host genotype and phenotypic differences in terms of the extent of viral replication, presence and severity of clinical signs and cytokine gene expression caused by infection with virulent EAV.

Semen quality of stallions challenged with the Kentucky 84 strain of equine arteritis virus

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P ≤ 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline.

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract

ABSTRACT Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation. IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallions reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.