Metabolomics (Non-Targeted) of Induced Type 2 Diabetic Sprague Dawley Rats Comorbid with a Tissue-Dwelling Nematode Parasite.

Type 2 diabetes is a non-communicable metabolic syndrome that is characterized by the dysfunction of pancreatic ß-cells and insulin resistance. Both animal and human studies have been conducted, demonstrating that helminth infections are associated with a decreased prevalence of type 2 diabetes mellitus (T2DM). However, there is a paucity of information on the impact that helminths have on the metabolome of the host and how the infection ameliorates T2DM or its progression. Therefore, this study aimed at using a non-targeted metabolomics approach to systematically identify differentiating metabolites from serum samples of T2DM-induced Sprague Dawley (SD) rats infected with a tissue-dwelling nematode, Trichinella zimbabwensis, and determine the metabolic pathways impacted during comorbidity. Forty-five male SD rats with a body weight between 160 g and 180 g were used, and these were randomly selected into control (non-diabetic and not infected with T. zimbabwensis) (n = 15) and T2DM rats infected with T. zimbabwensis (TzDM) (n = 30). The results showed metabolic separation between the two groups, where d-mannitol, d-fructose, and glucose were upregulated in the TzDM group, when compared to the control group. L-tyrosine, glycine, diglycerol, L-lysine, and L-hydroxyproline were downregulated in the TzDM group when compared to the control group. Metabolic pathways which were highly impacted in the TzDM group include biotin metabolism, carnitine synthesis, and lactose degradation. We conclude from our study that infecting T2DM rats with a tissue-dwelling nematode, T. zimbabwensis, causes a shift in the metabolome, causing changes in different metabolic pathways. Additionally, the infection showed the potential to regulate or improve diabetes complications by causing a decrease in the amino acid concentration that results in metabolic syndrome.

Metabolomics of Type 2 Diabetes Mellitus in Sprague Dawley Rats-In Search of Potential Metabolic Biomarkers.

Type 2 diabetes mellitus (T2DM) is an expanding global health concern, closely associated with the epidemic of obesity. Individuals with diabetes are at high risk for microvascular and macrovascular complications, which include retinopathy, neuropathy, and cardiovascular comorbidities. Despite the availability of diagnostic tools for T2DM, approximately 30-60% of people with T2DM in developed countries are never diagnosed or detected. Therefore, there is a strong need for a simpler and more reliable technique for the early detection of T2DM. This study aimed to use a non-targeted metabolomic approach to systematically identify novel biomarkers from the serum samples of T2DM-induced Sprague Dawley (SD) rats using a comprehensive two-dimensional gas chromatography coupled with a time-of-flight mass spectrometry (GCxGC-TOF/MS). Fifty-four male Sprague Dawley rats weighing between 160-180 g were randomly assigned into two experimental groups, namely the type 2 diabetes mellitus group (T2DM) (n = 36) and the non-diabetic control group (n = 18). Results from this study showed that the metabolite signature of the diabetic rats was different from that of the non-diabetic control group. The most significantly upregulated metabolic pathway was aminoacyl-t-RNA biosynthesis. Metabolite changes observed between the diabetic and non-diabetic control group was attributed to the increase in amino acids, such as glycine, L-asparagine, and L-serine. Aromatic amino acids, including L-tyrosine, were associated with the risk of future hyperglycemia and overt diabetes. The identified potential biomarkers depicted a good predictive value of more than 0.8. It was concluded from the results that amino acids that were associated with impaired insulin secretion were prospectively related to an increase in glucose levels. Moreover, amino acids that were associated with impaired insulin secretion were prospectively related to an increase in glucose levels.

The impact of Escherichia coli contamination products present in recombinant African horse sickness virus serotype 4 proteins on the innate and humoral immune responses.

Transcriptome analysis was used to characterise the in vitro primary and secondary immune responses induced in horse peripheral blood mononuclear cells (PBMC) stimulated for 24 h with the individual recombinant proteins of a virulent AHSV serotype 4 (AHSV4) field isolate (rAHSV4 proteins) that were previously expressed in Escherichia coli (E. coli). The results showed that the E. coli contamination products greatly affected the innate and humoral immune response transcripts. Hence, the impact of E. coli contamination products present in the individual rAHSV4 proteins on the translational immune response was determined. The combined amplification effects of synergistic pattern recognition receptors (PRRs), TNF-α and IL-1ß signalling induced potent pro-inflammatory responses that were too overwhelming for the anti-inflammatory cytokines and regulators to control. In addition to inducing robust B cell and antibody-mediated responses, lipopolysaccharide (LPS) activation of the innate-like B cells and subsequent polyreactive (natural) antibody responses could potentially contribute to endotoxin tolerance.

Apoptosis versus survival of African horse sickness virus serotype 4-infected horse peripheral blood mononuclear cells.

Expanding on our previous work, this study used transcriptome analysis of RNA sequences to investigate the various factors that contributed to either inducing apoptosis that resulted in cell death or promoting the survival of African horse sickness virus serotype 4 (AHSV4)-infected horse peripheral blood mononuclear cells (PBMC) after 24 h. Apoptosis is a host defense mechanism that prevents virus replication, accumulation and spread of progeny viruses. AHSV4-infected PBMC were killed via the intrinsic and the perforin/granzyme pathways of apoptosis during the attenuated AHSV4 (attAHSV4) in vivo primary and secondary immune responses. Trained innate immunity played an important role in circumventing viral interference that resulted in the elimination of AHSV4-infected PBMC through the intrinsic and the extrinsic pathways of apoptosis during the virulent AHSV4 (virAHSV4) in vitro secondary immune response. Oxidative stress in conjunction with IRE1α pro-apoptotic signaling played a major role in the induction of the intrinsic pathway of apoptosis and cytotoxic lymphocytes induced the perforin/granzyme or extrinsic pathways of apoptosis. In contrast, AHSV4-infected PBMC survived during the virAHSV4 in vitro primary immune response, which allows unrestrained viral replication. The virAHSV4 interference with the innate immune response resulted in impaired NK cell responses and delayed immune responses, which together with the antioxidant defense system promoted AHSV4-infected PBMC survival.

A Pilot Study on the Microbiome of Amblyomma hebraeum Tick Stages Infected and Non-Infected with Rickettsia africae.

Variation in tick microbiota may affect pathogen acquisition and transmission but for many vector species, including Amblyomma hebraeum, components and determinants of the microbiome are unidentified. This pilot study aimed to determine baseline microbial community within A. hebraeum nymphs infected- and non-infected with Rickettsia africae from the environment, and within adult ticks infected- and non-infected with R. africae collected from cattle sampled from two locations in the Eastern Cape province of South Africa. Adult A. hebraeum ticks (N = 13) and A. hebraeum nymph (N = 15) preliminary screened for R. africae were randomly selected and subjected to Illumina sequencing targeting the v3-v4 hypervariable regions of the 16S rRNA gene. No significant difference in microbial community composition, as well as rarefied OTU richness and diversity were detected between adults and nymphs. Nymphs showed a higher richness of bacterial taxa indicating blood-feeding could have resulted in loss of microbial diversity during the moulting stage from nymph to adult. Core OTUs that were in at least 50% of nymphs and adults negative and positive for Rickettsia at 1% minimum relative abundance were Rickettsia, Coxiella and Ruminococcaceae UCG-005 with a single genus Arsenophonus occurring only in nymphs negative for Rickettsia. Ehrlichia spp. was present in only four nymphal ticks positive for Rickettsia. Interestingly, Rickettsia aeschlimannii was found in one nymph and one adult, indicating the first ever detection of the species in A. hebraeum. Furthermore, A. hebraeum harboured a Coxiella-like endosymbiont, which should be investigated further as Coxiella may affect the viability and transmission of other organisms.

Virulent African horse sickness virus serotype 4 interferes with the innate immune response in horse peripheral blood mononuclear cells in vitro.

African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a double stranded RNA (dsRNA) virus of the genus Orbivirus, family Reoviridae. For the development of new generation AHS vaccines or antiviral treatments, it is crucial to understand the host immune response against the virus and the immune evasion strategies the virus employs. To achieve this, the current study used transcriptome analysis of RNA sequences to characterize and compare the innate immune responses activated during the attenuated AHSV serotype 4 (attAHSV4) (in vivo) and the virulent AHSV4 (virAHSV4) (in vitro) primary and secondary immune responses in horse peripheral blood mononuclear cells (PBMC) after 24 h. The pro-inflammatory cytokine and chemokine responses were negatively regulated by anti-inflammatory cytokines, whereas the parallel type I and type III IFN responses were maintained downstream of nucleic acid sensing pattern recognition receptor (PRR) signalling pathways during the attAHSV4 primary and secondary immune responses. It appeared that after translation, virAHSV4 proteins were able to interfere with the C-terminal IRF association domain (IAD)-type 1 (IAD1) containing IRFs, which inhibited the expression of type I and type III IFNs downstream of PRR signalling during the virAHSV4 primary and secondary immune responses. Viral interference resulted in an impaired innate immune response that was not able to eliminate virAHSV4-infected PBMC and gave rise to prolonged expression of pro-inflammatory cytokines and chemokines during the virAHSV4 induced primary immune response. Indicating that virAHSV4 interference with the innate immune response may give rise to an excessive inflammatory response that causes immunopathology, which could be a major contributing factor to the pathogenesis of AHS in a naïve horse. Viral interference was overcome by the fast kinetics and increased effector responses of innate immune cells due to trained innate immunity and memory T cells and B cells during the virAHSV4 secondary immune response.