Gene expression profile in zebu dairy cows (Bos taurus indicus) with mastitis caused by Streptococcus agalactiae

Abstract

a b s t r a c tMastitis is an inflammatory response in the mammary gland caused by an influx of somatic cells, composed mainly of neutrophils, macrophages and lymphocytes. The speed and efficacy of the host's immune response to the invasive pathogen affects the establishment, persistence and severity of the infection. To characterize the gene expression and response mechanism to infection by Streptococcus agalactiae (S. agalactiae) in zebu dairy cows of the Gyr breed, we carried out a transcriptome study of the cells present in the milk from 17 animals. Milk samples were collected before inoculation (hour 0) and 4, 9 and 24 h after inoculation of the bacteria into one of the quarters and at 0 and 24 h from one of the quarters not inoculated. The transcriptome analysis was done by the microarray and real-time PCR techniques. The microarray technique revealed the existence of 32 differentially expressed genes between inoculation and 4 h afterward. The validation of these results by real-time PCR was done for eight genes. Besides these eight genes, the expression of six others was evaluated by real-time PCR even though they did not present a significant difference by the microarray technique. Of the 14 genes analyzed by real-time PCR, all showed a significant difference in expression for at least one of the comparisons between times. This analysis indicated an increase in the expression of all the genes that presented a significant difference in relation to hour 0, with most of them presenting maximum expression 24 h after inoculation of the pathogen. Comparison of the gene expression between the inoculated and non-inoculated quarters showed greater expression in nine genes in the inoculated quarters. Analyses of gene networks revealed three modules with distinct characteristics 24 h after inoculation and showed that some mechanisms are altered in Gyr dairy cows after infection of the mammary gland by S. agalactiae. In this study it was possible to verify changes in the expression of at least 14 genes related to the immune response of zebuine animals against intramammary infection caused by S. agalactiae. These genes can play important roles in fighting intramammary infection and maintaining the tissue during infection.

Isolated perfused udder model for transcriptome analysis in response toStreptococcus agalactiae

“…These results are in accordance with findings of Fonseca et al . (2015) who showed that CCL2 was up-regulated in milk samples after inoculation with the same strain in the udders. Similarly to our results, only a few studies demonstrated that the chemokine CCL5 (also known as RANTES) is strongly expressed in the infected udder as well as in inflammation stimulated MEC (Günther et al ., 2009, 2011).…”

“…In contrast, the transcriptional profile of bovine mammary tissue after challenge with S. agalactiae is poorly characterized. Only a few studies have investigated the changes in gene expression in milk samples after IMI with S. agalactiae (Fonseca et al ., 2011, 2015).…”

“…This observation confirmed results from Fonseca et al . (2015) who observed increased expression of both TLR2 and TLR4 in milk samples after experimental in vivo infection with S. agalactiae. These findings indicate that TLR2 , TLR4 and CD14 receptors are regulated jointly at the beginning of the innate immune response in mammary alveolar tissue.…”

“…The left fore and rear quarters (IQ-inoculated quarters) were inoculated with the same strain of S. agalactiae used by Fonseca et al . (2015). To prepare the inoculum, the culture was resuspended in 1× PBS (2.7 m m KCl; 2 m m K 3 PO 4 ; 137 m m NaCl; 10 m m NaH 2 PO 4 ) to a final concentration of 100 CFU/ml determined by spectrophotometer.…”

“…These studies demonstrate the importance of dissecting the immune response of the mammary gland to different etiological agents, using transcriptomic profiling to understand the patho-physiology of mastitis and to develop appropriate control strategies. Nevertheless, despite the great significance of S. agalactiae as a causative agent, only a few studies using microarray technique have analyzed the changes in expression of cytokines and chemokines from milk samples during mastitis infection by this pathogen (Fonseca et al ., 2011, 2015). Furthermore, the majority of studies used infection models that measured the response relatively late after experimental inoculation (Bannernam et al ., 2004 a , 2004 b ; Petzl et al ., 2008) or during acute naturally acquired clinical mastitis (Fonseca et al ., 2011) when significant necrotic damage may have been evident.…”