Citokine expression characterizing a Th1 and Th2 response in spontaneously aborted bovine fetuses at mid- and late-gestation

Abstract

Bovine abortion is one of the most important problems of livestock production and health, causing severe economic loss. The worldwide diagnosis of bovine abortion is difficult, with a definitive etiological diagnosis having been reported in no more than 50% of cases. In Argentina, certain infectious agents are present with greater relative frequency as causal agents of abortion, with neosporosis, brucellosis and infection by the Bovine Viral Diarrhea Virus being among the most frequently diagnosed diseases and behaving as obligate intracellular agents. However, a significant number of fetuses remain without an etiological diagnosis, although they present inflammatory lesions compatible with an infectious agent. Additionally, scarce information has been published on the fetal immune response in spontaneous abortions due to infectious agents. The objective of this work was to evaluate the mRNA expression of cytokines in bovine fetuses spontaneously aborted by intracellular infectious agents and by possible infectious etiology, received at INTA Balcarce (2022-2023). Fetal age was estimated according to morphometric characteristics. Fetuses between 4-6 months (n=6) and ≥7 months of gestation (n=7) were selected in which N. caninum, B. abortus and BVD were detected by PCR or isolation and compatible lesions were observed, without the presence of any other infectious agent. Bovine fetuses with inflammatory lesions and no detection of infectious agents ≥7 months of gestation were also analyzed (n = 5). Finally, bovine fetuses with no inflammatory lesions and absence of infectious agents [between 4-6 months (n=2); ≥7 months of gestation (n=7); control] were also tested. Fetal spleen was collected and total RNA was extracted using Trizol, followed by DNase I digestion and cDNA synthesis using MMLV reverse transcriptase. The relative mRNA levels of cytokines characterizing a Th1 (IL-8, IL-12, IFN-γ, TNF-α) and Th2 (IL-4) response were determined by RT-qPCR. Results were expressed as the mean value of the change in mRNA levels with respect to expression in control fetuses. Mid-gestation fetuses aborted by intracellular agents showed significant upregulation of IFN-γ and IL-8 Th1 cytokines compared to control fetuses. In contrast, fetuses ≥7 months gestation showed significant downregulation of IFN-γ and IL-8 Th1 cytokines compared to control fetuses. Fetuses ≥7 months gestation by probable infectious etiology (PIE), showed only downregulation of IFN-γ compared to control fetuses. Finally, fetuses ≥7 months gestation aborted by intracellular agents showed significant upregulation of IL-4 Th2 and downregulation of IL-8 Th1 cytokines compared to fetuses of PIE. These results could be associated with an acute infection during mid-gestation with intracellular agents, with a Th1-type response. However, after this stage of the disease, fetuses aborted by intracellular agents in the last third of gestation would seem to mount a Th2 response. Additionally, cytokine expression between fetuses aborted by intracellular agents and by PIE showed significant differences, suggesting the involvement of other agents in the latter group. This study highlights the differences in the type of immune response observed between fetuses aborted by different agents at different gestational stages. Greater efforts should be made to characterize the causes of abortion.