Underlying genetic architecture of resistance to mastitis in dairy cattle: A systematic review and gene prioritization analysis of genome-wide association studies.

Mastitis, the most frequent disease in dairy cattle. Resistance to mastitis is a complex, polygenic trait controlled by several genes, each with small effects. Genome-wide association studies have been widely used to identify genomic variants associated with complex traits, including resistance to mastitis, to elucidate the underlying genetic architecture of the trait. However, no systematic review and gene prioritization analysis have been conducted to date on GWAS results for resistance to mastitis in dairy cattle. Hence, the objective was to perform a systematic review and gene prioritization analysis of GWAS studies to identify potential functional candidate genes associated with resistance to mastitis-related traits in dairy cattle. Four electronic databases were searched from inception to December 2020, supplemented with multiple sources of gray literature, to identify eligible articles. Annotation for genes and quantitative trait loci (QTL), and QTL enrichment analysis were conducted using GALLO. Gene prioritization analysis was performed by a guilty-by-association approach using GUILDify and ToppGene. From 52 articles included within this systematic review, 30 articles were used for further functional analyses. Gene and QTL annotation resulted in 9,125 and 43,646 unique genes and QTL, respectively, from 39 studies. In general, overlapping of genes across studies was very low (mean ± SD = 0.02% ± 0.07%). Most annotated genes were associated with somatic cell count-related traits and the Holstein breed. Within all annotated genes, 74 genes were shared among Holstein, Jersey, and Ayrshire breeds. Approximately 7.5% of annotated QTL were related to QTL class "health." Within the health QTL class, 2.6 and 2.2% of QTL were associated with clinical mastitis and somatic cell count-related traits. Enrichment analysis of QTL demonstrated that many enriched QTL were associated with somatic cell score located in Bos taurus autosomes 5, 6, 16, and 20. The prioritization analysis resulted in 427 significant genes after multiple test correction (false discovery rate of 5%) from 26 studies. Most prioritized genes were located in Bos taurus autosomes 19 and 7, and most top-ranked genes were from the cytokine superfamily (e.g., chemokines, interleukins, transforming growth factors, and tumor necrosis factor genes). Although most prioritized genes (397) were associated with somatic cell count-related traits, only 54 genes were associated with clinical mastitis-related traits. Twenty-four genes (ABCC9, ACHE, ADCYAP1, ARC, BCL2L1, CDKN1A, EPO, GABBR2, GDNF, GNRHR, IKBKE, JAG1, KCNJ8, KCNQ1, LIFR, MC3R, MYOZ3, NFKB1, OSMR, PPP3CA, PRLR, SHARPIN, SLC1A3, and TNFRSF25) were reported for both somatic cell count and clinical mastitis-related traits. Prioritized genes were mainly associated with immune response, regulation of secretion, locomotion, cell proliferation, and development. In conclusion, this study provided a fine-mapping of previously identified genomic regions associated with resistance to mastitis and identified key functional candidate genes for resistance to mastitis, which can be used to develop enhanced genomic strategies to combat mastitis by increasing mastitis resistance through genetic selection.

Establishment of a Three-Dimensional In Vitro Model of Equine Papillomavirus Type 2 Infection.

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis.

Prevalence of Equus caballus Papillomavirus Type-2 Infection and Seropositivity in Asymptomatic Western Canadian Horses.

Equus caballus papillomavirus type 2 (EcPV-2) has been recognized as a potential cause of a subset of genital squamous cell carcinomas (SCCs) in horses. In the current study, we measured EcPV-2 seropositivity in 50 healthy horses from Western Canada, and these were compared to a herd of horses with known EcPV-2 exposure. Second, the presence of EcPV-2 DNA was measured using EcPV-2-specific PCR (polymerase chain reaction), performed on a variety of tissues collected at necropsy from 70 horses that lacked any history, gross, or histologic evidence of neoplasia or papillomavirus-associated disease. EcPV-2-specific RNA in situ hybridization (R-ISH) was performed on PCR-positive samples to identify the specific tissues infected. The prevalence of asymptomatic infection with EcPV-2 in Western Canadian horses was 20/70 (29%). Exposure to EcPV-2 as measured by seropositivity was 18/50 (36%). EcPV-2 positivity by anatomic location, as measured by R-ISH, was as follows: penis 10/29 (35%), vulva 5/34 (15%), eyelid 8/68 (12%), oral mucosa 7/65 (11%), skin from muzzle 7/68 (10%), and retropharyngeal lymph node 2/64 (3%). The youngest horses with EcPV-2 infection, based on PCR, were fetuses, suggesting for the first time that vertical transmission of EcPV-2 occurs in horses. The current study observed an increased prevalence of EcPV-2 as compared to previous studies. We suggest that this difference is due to our use of biopsies in place of superficial swabs. We propose that EcPV-2 infection in asymptomatic horses is more common than previously reported and that the virus' role in equine genital SCCs may be more complex than originally thought.

Prevalence and Prognostic Impact of Equus caballus Papillomavirus Type 2 Infection in Equine Squamous Cell Carcinomas in Western Canadian Horses.

Equus caballus papillomavirus type-2 (EcPV-2) has been proposed as a causal factor in equine genital squamous cell carcinoma (SCC). This study had 2 objectives: first, calculate the frequency of papillomavirus (PV) and EcPV-2 infection in papillomas, carcinomas in situ (CIS), and SCCs in Western Canadian horses; and second, determine if EcPV-2 status of equine SCCs is associated with overall survival (OS). EcPV-2 status of 115 archived tissue samples, spanning 6 years, was determined using broad spectrum (MY09/11) and EcPV-2-specific polymerase chain reaction (PCR) assays, EcPV-2-E6/E7 chromogenic RNA in situ hybridization (R-ISH), and amplicon sequencing. A retrospective survey gathered data on history, outcome, breeding, treatment, and rationales of referring veterinarians when managing PV-associated diseases. Histologic grade and completeness of surgical margins of SCCs were also considered. EcPV-2 DNA was identified in 10/58 (17%) SCC, 8/27 (30%) papillomas, 0/5 CIS, and 0/11 lesions identified as "other." Overall, 18/101 (18%) of these lesions were positive for EcPV-2. EcPV-2 was identified in 10/35 (29%) SCCs arising from genital tissues but in 0/22 SCCs from other locations. There was no association between breeding history and EcPV-2 status of genital SCCs. EcPV-2 status of genital SCCs was not associated with OS (P = .76). The strongest negative predictors of OS were a lack of treatment (P < .01) and recurrence post-treatment (P < .01). Weaker predictors of OS included older age at time of diagnosis (P = .02). Completeness of margins at surgical excision, concurrent disease, treatment type, anatomic location of the SCC (anogenital vs other), and histologic grade of the SCC did not influence OS (P > .1).

Paving the way for more precise diagnosis of EcPV2-associated equine penile lesions.

BACKGROUND: There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is causally associated with the development of equine genital squamous cell carcinomas (SCCs). Early stages of disease present clinically as plaques or wart-like lesions which can gradually progress to tumoural lesions. Histologically these lesions are inconsistently described as benign hyperplasia, papilloma, penile intraepithelial neoplasia (PIN), carcinoma in situ (CIS) or SCC. Guidelines for histological classification of early SCC precursor lesions are not precisely defined, leading to potential misdiagnosis. The aim of this study was to identify histologic criteria and diagnostic markers allowing for a more accurate diagnosis of EcPV2-associated equine penile lesions. RESULTS: A total of 61 archived equine penile lesions were histologically re-assessed and classified as benign hyperplasia, papilloma, CIS or SCC. From these, 19 representative lesions and adjacent normal skin were comparatively analysed for the presence of EcPV2 DNA and transcripts using PCR and RNA in situ hybridisation (RISH). All lesional samples were positive by EcPV2 PCR and RISH, while adjacent normal skin was negative. RISH analysis yielded signal distribution patterns that allowed distinction of early (hyperplasia, papilloma) from late stage lesions (CIS, SCC). Subsequently, the 19 lesions were further assessed for expression of p53, Ki67, MCM7 and MMP1 by immunohistochemistry (IHC). All four proteins were expressed in both normal and lesional tissue. However, p53 expression was up-regulated in basal keratinocyte layers of papillomas, CIS and SCCs, as well as in upper keratinocyte layers of CIS and SCCs. MCM7 expression was only up-regulated in upper proliferating keratinocyte layers of papillomas, CIS and SCCs. CONCLUSION: This study proposes combining a refined histological protocol for analysis of equine penile lesions with PCR- and/or RISH based EcPV2-screening and p53/MCM7 IHC to more accurately determine the type of lesion. This may help to guide the choice of optimum treatment strategy, especially at early stages of disease.

Interferon epsilon is constitutively expressed in equine endometrium and up-regulated during the luteal phase.

Interferon epsilon (IFNE) is type I interferon which stands out through its unusual expression profile and differing regulation compared to classic type I interferons such as interferon alpha and interferon beta. Unlike other type I interferons, the expression of IFNE is not stimulated through exposure to viral agents. Expression of IFNE is most abundant in mouse and human endometrium where it is constitutively expressed in luminal and glandular epithelial cells and expression levels are up-regulated with estrogen exposure. The aim of the current study was to determine whether a cycle or pregnancy dependent expression pattern of IFNE is existent in equine endometrium and to localize IFNE expression within the endometrium. Additionally, endometrial explant culture and culture of mixed epithelial/stromal cells populations was used to determine the effects estrogen and seminal plasma on IFNE transcript abundance. Samples collected during diestrus and pregnancy expressed significantly higher levels of IFNE than samples obtained from anestrous or estrous mares (P < 0.001). Exposure of mixed endometrial epithelial/stromal cell populations and endometrial explants to 10% seminal plasma and estradiol 17-beta did not affect IFNE expression levels (P > 0.05). Upon in situ hybridization, staining was exclusively present in luminal and glandular epithelial cells, with stromal displaying absent staining intensity. Both diestrous and pregnant samples were characterized by markedly stronger staining of glandular epithelial cells than anestrous and estrous samples. The progesterone-dependent increase in IFNE abundance during the estrous cycle likely implies that IFNE is part of the innate immune system in endometrium that gives protection against uterine infections during progesterone-dominated phase of the estrous cycle.