All experiments were conducted according to the National Standards for Laboratory Animals Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018), and were approved by the Animal Care and Use Committee of the Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences (Jinan, China). Mammary tissue samples were collected from eight healthy and eight mastitic Chinese Holstein cows (650–750 kg) at 2.8 to 3.0 years of age during their first lactation at a commercial slaughter plant in Jinan, China. The duration of the experiment was 3 months, and no animal succumbed during that time. Prior to the collection of mammary tissues, the animal health and behavior were monitored daily. The mastitic cows were selected according to their clinical symptoms, and the mastitis infections were caused only by S. aureus. The healthy cows were not affected by pathogen infection and had no clinical symptoms (heat, pain, redness, swelling of the udder and milk clotting). The mammary tissues were sampled at the time of slaughter and frozen immediately in liquid nitrogen.

An E.Z.N.A^® Mollusc RNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) was used to extract the total RNA from the mammary samples, and cDNA was synthesized using a PrimeScript™ Reverse Transcription (RT) reagent kit with gDNA Eraser (Perfect Real Time; Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The primer sequences of the CCL5 gene (GenBank accession no. NM_175827.2) and KB-SNP1 were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) and Primer-BLAST software (www.ncbi.nlm.nih.gov/tools/primer-blast/; Table I), and synthesized by Beijing TsingKe Biological Technology Co., Ltd. (Beijing, China). The polymerase chain reaction (PCR) was performed as follows: 94°C for 4 min, 94°C for 30 sec, 68°C for 30 sec, 72°C for 35 sec (35 cycles), and 72°C for 10 min. DNA bands were separated by 1% agarose gel electrophoresis and eluted using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd., Beijing, China). The method of identifying the splice variants was performed as described previously (18). The PCR products were subcloned into the pEASY-T3 cloning vector (TransGen Biotech Co., Ltd., Beijing, China) following purification, and the mixture was transformed into E. coli DH5α competent cells (Takara Biotechnology Co., Ltd.). The positive clones were selected and sequenced by BGI LifeTech Co., Ltd. (Beijing, China) and DNAMAN v5.2.2 software (Lynnon LLC, San Ramon, CA, USA) was used to identify the possible splice variants through multiple sequence alignments with the CCL5-reference mRNA.

An E.Z.N.A^® Mollusc RNA kit was used to extract the total RNA from the mammary samples, and cDNA was synthesized as aforementioned. SYBR-Green PCR Master Mix (Tiangen Biotech Co., Ltd.) was used for RT-quantitative (q)PCR in a BioRad CFX96 Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. The primer sequences for CCL5 and β-actin (GenBank accession no. NM_173979.3; www.ncbi.nlm.nih.gov/genbank) were designed using Primer Premier 5.0 and Primer-BLAST software, and the primer sequences of CCL5-reference and CCL5-AS (Table II) were designed based on the results for the splice variants of the CCL5 gene. The primer sequences were synthesized by Beijing TsingKe Biological Technology Co., Ltd. The relative expression values for the RT-qPCR assay were calculated with β-actin as the endogenous control, and the template that did not undergo the reverse transcription reaction served as the negative control. qPCR was performed as follows: 95°C for 30 sec, 95°C for 15 sec, 60°C for 30 sec, 72°C for 35 sec (40 cycles).

Exonic splice enhancer (ESE)finder 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi) was used to predict the alterations in the binding site of the splicing factor, and the alternative splice site prediction was achieved with ASSP (http://wangcomputing.com/assp/index.html).

The 598 bp genomic fragment of the CCL5 gene from bovine genomic DNA, including intron 1, intron 2 and exon 2, was amplified to evaluate the in vitro splicing with mini-genes, and the primer sequences of KB-SNP1 are presented in Table I. The segment with the wild-type, AA, or the mutant type, TA, of the CCL5 gene was cloned into the pSPL3 vector (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following digestion with EcoRI and XhoI. The clones were transformed into Trans5a cells (TransGen Biotech Co., Ltd.) which were plated on agar containing 100 mg/ml ampicillin, and incubated at 37°C overnight. The positive colonies were cultured in lysogeny broth at 37°C overnight, and an Endo-free Plasmid Mini kit II (Omega Bio-Tek, Inc.) was used to isolate the plasmids according to the manufacturer's protocols. The presence of the correct sequences was verified through sequencing constructs by Beijing TsingKe Biological Technology Co., Ltd.

Bovine mammary epithelial cells (MAC-T) and 293T cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing penicillin/streptomycin (100 IU/ml; 0.1 mg/ml) with 10% fetal bovine serum in an atmosphere of 5% CO₂ in air at 37°C. The cells were transferred to six-well culture plates, and transfected with 4 µg of wild-type or mutant mini-gene constructs using Lipofectamine^® 2000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 h in Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.), according to manufacturer's protocol, when the cells had grown to 80–85% confluence. A subset of cells were transfected with pSPL3 lacking the CCL5 insert, and other cells were treated with only transfection reagent as the control.

The cells were collected at 24 h post-transfection, and the total RNA was extracted, reverse transcribed into cDNA, and PCR was performed as described above. The pSPL3 vector specific primers were SD6 (5′-TCTGAGTCACCTGGACAACC-3′) and SA2 (5′-ATCTCAGTGGTATTTGTGAGC-3′). The PCR products were separated on 2% agarose gels, and the DNA bands were extracted with a Gel Extraction kit (Omega Bio-Tek, Inc.) and analyzed by direct sequencing by Beijing TsingKe Biological Technology Co., Ltd.

Relative quantification of CCL5-reference and CCL5-AS mRNA expression was performed using the 2^−ΔΔCq analysis method for relative real-time PCR (19). The data for the relative expression levels of CCL5-reference and CCL5-AS mRNA were analyzed using a completely randomized design with eight animals per group, via a mixed-model analysis of variance using Proc Mixed in SAS (version 9.1; SAS Institute, Cary, NC, USA). The comparisons among the relative expression levels of the different groups were performed using the Duncan method, controlling the experiment wise type ± error equal to 0.05. Data are presented as least squares means. P<0.05 was considered to indicate a statistically significant difference.

The cDNA from mammary tissues was used to amplify the full-length CCL5 transcript with specific primers for CCL5 cDNA (Table I). As a result, two PCR amplification fragments were detected in the mastitis mammary gland tissues, while only one fragment was detected in the healthy tissues (Fig. 1A). A total of two PCR bands of different sizes were recovered and subsequently sequenced. The common PCR fragment was the expected CCL5 product (566 bp), and the shorter PCR fragment was a novel splice variant with a size of 454 bp. By comparing with the reference genomic sequence of the bovine CCL5 gene (GenBank accession no. NC_007317.6) and the reference mRNA sequence (GenBank accession no. NM_175827.2), it was observed that the shorter splice variant, designated CCL5 transcript variant CCL5-AS, lacked the entirety of exon 2 with a 112 bp deletion (Fig. 1B and C). Furthermore, the putative protein of CCL5-AS was missing the key Chemokine_CC domain when compared with the CCL5-reference protein (Fig. 1D), indicating the loss of the normal function of CCL5 in the immune and inflammatory processes.

Through the RT-qPCR, the relative expression levels of CCL5-reference and CCL5-AS were compared in the mammary gland tissues of cows. The expression of CCL5-reference mRNA in the healthy tissue was significantly higher compared with that in the mastitic tissue. Moreover, it was demonstrated that the CCL5-reference was the most abundant transcript in the healthy and mastitic tissues, which is consistent with the RT-PCR results. The novel transcript CCL5-AS was expressed in the mastitic mammary tissues with a low abundance, whereas it was undetectable in the normal tissues (Fig. 2). These results supported the hypothesis that CCL5-AS was specific to mastitic tissue.

To analyze the molecular basis of the aberrant CCL5-AS splice variant, a pair of primers (KB-SNP1; Table I) was designed to amplify a 598 bp fragment harboring exon 2 of CCL5. A total of two novel SNPs, one SNP (g.1647C>T) in exon 2 (a synonymous mutation) and the other SNP (g.1804G>A) in the intron 2 region, were identified in the Holstein cow population by sequencing the PCR amplification products (Fig. 3A). To investigate whether the SNPs affected the alternative splicing of exon 2, ESEfinder 3.0 software was used to predict alterations in the splice sites and factors in the ESE motif. The prediction demonstrated that the introduction of allele T, relative to allele C, in the locus g.1647C>T resulted in the deletion of two binding sites of auxiliary splicing proteins, serine/arginine-rich splicing factor 1 (SRSF1) and SRSF1 (IgM-BRCA1); however, the introduction of allele A, relative to allele G, in the locus g.1804G>A led to the deletion of two IgM-BRCA1 binding sites, and increased the SRSF5 (SRp40) binding site (Fig. 3B). A total of two SNPs (g.1647C>T and g.1804G>A) located in the ESE motif region were in complete linkage disequilibrium, constituting two haplotypes (C-G and T-A) of SNPs (g.1647C>T and g.1804G>A), which may putatively induce the production of a novel splice variant of the bovine CCL5 gene.

To determine whether the two SNPs (g.1647C>T and g.1804G>A) of CCL5 led to the abrogation of the predicted SR protein motifs and caused AS, the 598 bp genomic fragment spanning 268 bp of intron 1, 112 bp of exon 2 and 218 bp of intron 2 was amplified, which harbored the two haplotypes (C-G and T-A), and the two fragments were cloned into the pSPL3 vector (Fig. 4A). The CCL5 splicing mini-gene reporter assay demonstrated that transfecting the two plasmids, with the wild haplotype (C-G) and mutant haplotype (TA), respectively, into the 293T cells produced the same RT-PCR amplification product with a size of 375 bp, while the negative pSPL3-control generated a product of 263 bp (Fig. 4B). The results suggested that the haplotypes of the two SNPs (g.1647C>T and g.1804G>A) were not responsible for the production of CCL5-AS.