Project description:The gastrointestinal nematode Ostertagia ostertagi is one of major causal agents that contribute to production inefficiency in cattle industry in the temperate region of the world. One of pathophysiological factors that lead to reduced weight gain and milk yield is altered gastrointestinal functions, resulting from considerable tissue damage in the abomasal mucosa during infections. Protective immunity to Ostertagia ostertagi infections in cattle develops very slowly. Resistance to reinfection becomes manifest only after a prolonged period of exposure. Mechanisms underlying the development of protective immunity remain largely unexplored. Immune animals, with significantly reduced worm burdens, were developed after multiple drug-attenuated experimental infections and were compared to the primary infected group and their respective uninfected controls. In this study, transcriptomic analysis identified 3 signaling pathways, the complement system, leukocyte extravasation and acute phase responses, significantly impacted during both primary and repeat infections. The markedly increased mRNA levels of complement components C3, factor B (CFB), and factor I (CFI) in the abomasal mucosa of the infected cattle were confirmed using quantitative PCR. Western blot analysis established the presence of elevated levels of activated C3 proteins in the mucosa. One of the iniators of local complement activation could be related with secretory IgA and IgM because infections significantly upregulated expression of J chain (IGJ) as well as polymeric Ig receptor (PIGR) and an IgM-specific receptor (FAIM3), suggesting sustained increase in both synthesis and transepithelial transport of IgA and IgM during the infection. The elevated levels of pro-inflammatory cytokines, such as IL-4 and IL-1Î², during the infection may be involved in gene regulation of complement components. Our data suggested enhanced tissue repair and mucin secretion in immune animals may also contribute to protective immunity. Our results presented the first piece of evidence that local complement activation may be involved in the development of long term protective immunity and provided a novel mechanistic insight into resistance against Ostertagia ostertagi in cattle. There were four treatment groups: naive control (never infected), primary infection, drug-attenuated control, and drug-attenuated 5th reinfection. Each group had 4 biolgical replicates. A total of 16 arrays were used for this experiment. The 2 major contrast were 1). The primary infection vs naive control; and 2). The drug-attenuated 5th reinfection vs the drug-attenuated control.

Project description:The gastrointestinal nematode Ostertagia ostertagi is one of major causal agents that contribute to production inefficiency in cattle industry in the temperate region of the world. One of pathophysiological factors that lead to reduced weight gain and milk yield is altered gastrointestinal functions, resulting from considerable tissue damage in the abomasal mucosa during infections. Protective immunity to Ostertagia ostertagi infections in cattle develops very slowly. Resistance to reinfection becomes manifest only after a prolonged period of exposure. Mechanisms underlying the development of protective immunity remain largely unexplored. Immune animals, with significantly reduced worm burdens, were developed after multiple drug-attenuated experimental infections and were compared to the primary infected group and their respective uninfected controls. In this study, transcriptomic analysis identified 3 signaling pathways, the complement system, leukocyte extravasation and acute phase responses, significantly impacted during both primary and repeat infections. The markedly increased mRNA levels of complement components C3, factor B (CFB), and factor I (CFI) in the abomasal mucosa of the infected cattle were confirmed using quantitative PCR. Western blot analysis established the presence of elevated levels of activated C3 proteins in the mucosa. One of the iniators of local complement activation could be related with secretory IgA and IgM because infections significantly upregulated expression of J chain (IGJ) as well as polymeric Ig receptor (PIGR) and an IgM-specific receptor (FAIM3), suggesting sustained increase in both synthesis and transepithelial transport of IgA and IgM during the infection. The elevated levels of pro-inflammatory cytokines, such as IL-4 and IL-1β, during the infection may be involved in gene regulation of complement components. Our data suggested enhanced tissue repair and mucin secretion in immune animals may also contribute to protective immunity. Our results presented the first piece of evidence that local complement activation may be involved in the development of long term protective immunity and provided a novel mechanistic insight into resistance against Ostertagia ostertagi in cattle.

Project description:Introduction Chlamydia trachomatis (C. trachomatis) is a Gram-negative bacterium and a common human pathogen. The World Health Organization (WHO) estimates that over 130 million people are infected with C. trachomatis globally each year and with increasing incidence. C. trachomatis causes long-lasting and recurrent infections that over time induce severe tissue damage in the female genital tract that can lead to ectopic pregnancy and infertility. Thus, the human immune system fails to control and eradicate C. trachomatis during primary infection and fails to develop protective immunity against secondary infections. In vivo infection models, using complement knock out mice, suggest that the complement system is critically involved in both anti-chlamydial immunity and infection-induced pathology. To increase our understanding of complement-mediated immunity against C. trachomatis we analyzed global complement deposition on serum-incubated C. trachomatis by mass spectrometry. Methods Purified C. trachomatis was incubated in seronegative normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) for 30 min, thoroughly washed, and processed for mass spectrometry. All samples were lysed, reduced and alkylated and digested with trypsin. Some samples were chemically modified to acetylate free amino groups (N-terminal and lysine amino groups) before trypsin digestion. Peptides were analyzed on a UltimateTM 3500 RSLCnano coupled to a Q Exactive HF-X mass spectrometer. Raw data files were searched against the Uniprot human reference proteome using MaxQuant. Results We demonstrate that C. trachomatis elicits potent complement activation demonstrated by deposition of both early and late complement factors together with several complement regulators. We further demonstrate proteolytically processing of complement C3b to “inactive” C3 cleavage fragments. Conclusion We demonstrate the deposition of several novel complement-associated proteins and -cleavage fragments on the surface of C. trachomatis.

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