Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection

PLOS ONE, Dec 2019

Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNA-Seq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies.

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Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection

January Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection Rohini Chopra-Dewasthaly 0 1 Melanie Korb 1 Rene Brunthaler 1 Reinhard Ertl 1 0 , Vienna , Austria 1 Editor: Glenn F. Browning, The University of Melbourne , AUSTRALIA Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNASeq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies. - Data Availability Statement: All sequencing reads are available in the European Nucleotide Archive (www.ebi.ac.uk/arrayexpress/) under the accession number E-MTAB-5216. Funding: This work was supported by the Austrian Science Fund (FWF) (P23595-B20 to RCD, Joachim Spergser and Renate Rosengarten) and a supplementary funding by the University of Veterinary Medicine Vienna (PP0201127201).The funders had no role in study design, data collection Introduction Mycoplasmas are one of the smallest and simplest microbes that cause difficult-to-eradicate chronic infections by complex unknown pathogenicity factors [ 1, 2 ]. Mycoplasma mastitis is one such worldwide problem. Amongst different causative species Mycoplasma bovis is the and analysis, decision to publish, or preparation of the manuscript. most important species in cattle, whereas its very close phylogenetic relative Mycoplasma agalactiae, inflicts similar severe mastitis in small ruminants by complex immuno-inflammatory pathophysiological processes that are yet to be fully comprehended [3±5]. M. agalactiae is the main etiological agent of contagious agalactia (CA) syndrome in sheep and goats and causes major economic losses, especially due to its persistence and shedding in milk for many years even after antibiotic treatment of the initial infection [6±9]. Its socio-clinical significance can be stressed by the fact that unlike the economically important M. bovis infections, CA is enlisted as a notifiable disease by the World Organization for Animal Health [10]. Despite its importance, the complex host-pathogen interactions of M. agalactiae enabling survival in the mammary gland are not completely understood. We had recently identified potential pathogenicity factors by negative selection of transposon mutants that were unable to survive in the experimentally infected udders [ 11, 12 ]. Although the exact role of most of the identified factors remains to be elucidated, pyruvate dehydrogenase was shown to be involved in cell invasiveness [13], a strategy that M. agalactiae likely employs to invade mammary cells to avoid antibiotics and host defenses, and to spread systemically to new host niches [ 14, 15 ]. Another study has described neutrophil extracellular trap (NET) formation in vivo in the mammary gland and milk of sheep naturally infected with M. agalactiae [16]. Although mycoplasma liposoluble proteins are demonstrated to induce NET release, the exact role of the recently identified phase variable β-(1!6)-Glucan is yet to be elucidated in its disease progression [ 17 ]. With respect to the involved host factors, except for a couple of immune response reports, that too in goats, where under natural conditions four other mycoplasma species are known to cause clinically indistinguishable syndrome [ 4, 18 ], the dynamics of M. agalactiae induced host responses in the mastitic sheep mammary gland are almost completely unknown. One study has described proteomic changes occurring in milk fat globules (MFG) du (...truncated)


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Rohini Chopra-Dewasthaly, Melanie Korb, René Brunthaler, Reinhard Ertl. Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection, PLOS ONE, 2017, Volume 12, Issue 1, DOI: 10.1371/journal.pone.0170015