Characterization of buffalo interleukin 8 (IL-8) and its expression in endometritis

Abstract

River buffalo (Bubalus bubalis bubalis) with a population over 135 million heads is an important livestock. Interleukin 8 (IL-8) is a member of the chemokine family and is an important chemoattractant for neutrophils associated with a wide variety of inflammatory diseases such as endometritis. Tissue samples from the mammary gland, uterus and ovary were obtained from river buffalo (Mediterranean type) with and without endometritis. Bacteriological examination showed the presence of both gram positive and negative in all buffalo with endometritis. RNA extraction and complementary DNA (cDNA) synthesis were conducted from all tissues. Specific primer for IL8 full coding regions was designed using known cDNA sequences of Bubalus bubalis, Genbank accession number AY952930.1. IL-8 gene expression was investigated in buffalo tissues. Expression of IL-8 in buffalo with endometritis was found to increase significantly over buffalo without endometritis only in the uterus (P = 0.0159). PCR products from uterus tissues (target organs) of buffalo with and without endometritis, were purified and sequenced. No polymorphic sites were detected in the investigated samples. IL-8 cDNA nucleotide sequences of buffalo with and without endometritis were 100% identical (accession number JX413057). Buffalo IL8 cDNAs were compared with corresponding sequences of member of subfamily Bovinae (buffalo and cattle) and subfamily Caprinae (sheep and goat). IL-8 species specific differences were identified.

Keywords: Endometritis; Gene expression; Interleukin 8; Polymorphic sites; Water buffalo.

Figure 1

Mean normalized expression in IL-8 mRNA gene expression in uterus of Egyptian buffalo with (w) and without (w/o) endometritis using quantitative real time PCR. Constitutive production of IL8 full spliced transcript (306 bp) detected by RT-PCR, RPLPO is the housekeeping gene used as an internal control. Data are presented as the average ± SD from three experiments.

Figure 2

Egyptian buffalo IL-8 cDNA nucleotide sequence.

Figure 3

Buffalo IL-8 transcript showing (underlined) the conserved ELRCXC; GPH motifs; the four highly conserved cysteine residues.

Figure 4

Alignment of IL-8 complete coding region nucleotide sequence between Egyptian buffalo, Bubalus bubalis (B.b), Bos taurus (B.t), Bos indicus (B.i), Ovis aries (O.a), Ovis canadensis (O.c) and Capra hircus (C.h).

Figure 5

Alignment of IL-8 transcripts of buffalo (Bubalus sp.), cattle (Bos sp.), sheep subspecies (O. aries and O. canadensis) and Goat (Capra sp.).