Outcomes of patients in Chagas disease of the central nervous system: a systematic review.

Chagas disease is a parasitic infection caused by the protozoan Trypanosoma cruzi. One of the complications of the disease is the infection of the central nervous system (CNS), as it can result from either the acute phase or by reactivation during the chronic phase, exhibiting high mortality in immunocompromised patients. This systematic review aimed to determine clinical and paraclinical characteristics of patients with Chagas disease in the CNS. Articles were searched from PubMed, Scopus and LILACS until January 2023. From 2325 articles, 59 case reports and 13 case series of patients with Chagas in the CNS were retrieved from which 138 patients were identified. In this population, 77% of the patients were male, with a median age of 35 years old, from which most of them came from Argentina and Brazil. Most of the individuals were immunocompromised from which 89% were HIV-positive, and 54 patients had an average of 48 cells per mm3 CD4+ T cells. Motor deficits and seizures were the most common manifestation of CNS compromise. Furthermore, 90 patients had a documented CNS lesion by imaging from which 89% were supratentorial and 86% were in the anterior/middle cranial fossa. The overall mortality was of 74%. Among patients who were empirically treated with anti-toxoplasma drugs, 70% died. This review shows how Chagas disease in the CNS is a devastating complication requiring prompt diagnosis and treatment to improve patients' outcomes.

False positive serology of prepandemic chagasic samples with SARS-CoV-2 antigen.

OBJECTIVE: To determine whether prepandemic sera from patients with Chagas disease recognise SARS-CoV-2 antigens. MATERIALS AND METHODS: Forty sera from patients with Chagas disease were tested for the presence of IgG cross-reactivity against the nucleocapsid protein (NP) and spike (S) SARS-CoV-2 proteins by ELISA. Positive samples were tested again using a different ELISA and CLIA, both against NP. RESULTS: None of the sera from patients with Chagas disease, previously confirmed as positive for the presence of anti-Trypanosoma cruzi antibodies reacted against the SARS-CoV-2 S protein, and six samples tested positive for the NP antigen (15%). The six positive samples were re-tested, five remained positive by ELISA and all were negative by CLIA. CONCLUSION: According to our data, false-positive results might be a concern in the detection of SARS-CoV-2 antibodies in patients with Chagas disease.

Diminished mitogen-induced T cell proliferation by Trypanosoma cruzi antigens associated with antigen-presenting cell modulation and CD3 signaling.

Chronic infection by Trypanosoma cruzi decreases T cell proliferation and it is most likely accompanied by changes in signals required for activation. We assessed the effect of T. cruzi antigens on mitogen-induced proliferation of T cells from uninfected individuals and the association with the expression of molecules involved in antigen presentation, T cell costimulation and activation, and cytokine production. T. cruzi antigen exposure reduced mitogen-induced proliferation of CD4+ and CD8+ T cells in PBMC cultures, but only reduced mitogen-induced proliferation in the CD4+ T cells from sorted cell cultures cocultured with antigen-pulsed CD3- cells. CD40/CD80 and CD86 expression were reduced in antigen-pulsed DCs and monocytes, respectively. TNF-α, IL-10 and CCL17 levels were increased in cultures with antigen-pulsed CD3- cells, while CD3ζ chain expression was reduced in T cells from cultures with antigen. Our findings suggest that T. cruzi could alter T cell proliferation indirectly by downregulating costimulatory molecules and inducing the secretion of IL-10 and directly by decreasing TCR signaling.

Antiparasitic Treatment Induces an Improved CD8+ T Cell Response in Chronic Chagasic Patients.

Chagas disease is a chronic infection caused by Trypanosoma cruzi, an intracellular protozoan parasite. Chronic chagasic patients (CCPs) have dysfunctional CD8+ T cells that are characterized by impaired cytokine production, high coexpression of inhibitory receptors, and advanced cellular differentiation. Most patients diagnosed in the chronic phase of Chagas disease already exhibit heart involvement, and there is no vaccination that protects against the disease. Antiparasitic treatment is controversial as to its indication for this stage of the disease. There is a lack of biological markers to evaluate the effectiveness of antiparasitic treatment, and little is known about the effect of the treatment on CD8+ T cells. Thus, the aim of the current study was to analyze the early effects of antiparasitic treatment on CD8+ T cells from CCPs with asymptomatic clinical forms of disease. To evaluate the CD8+ T cell subsets, expression of inhibitory receptors, and functionality of T cells in CCPs, PBMCs were isolated. The results showed that treatment of CCPs with the asymptomatic form of the disease induces an increase in the frequency of CD8+ central memory T cells and terminal effector T cells, a decrease in the coexpression of inhibitory receptors, an improved Ag-specific CD8+ T cell response exhibited by the individual production of IFN-γ or IL-2, and a multifunctional CD8+ T cell profile of up to four functions (IFN-γ+IL-2+Perforin+Granzyme B+). These findings suggest that, in CCPs, antiparasitic treatment improved the quality of Ag-specific CD8+ T cell responses associated with a decrease in inhibitory receptor coexpression, which could serve as biomarkers for monitoring the effectiveness of antiparasitic treatment.

Reduced skeletal muscle fiber size following caloric restriction is associated with calpain-mediated proteolysis and attenuation of IGF-1 signaling.

Caloric restriction decreases skeletal muscle mass in mammals, principally due to a reduction in fiber size. The effect of suboptimal nutrient intake on skeletal muscle metabolic properties in neonatal calves was examined. The longissimus muscle (LM) was collected after a control (CON) or caloric restricted (CR) diet was cosnumed for 8 wk and muscle fiber size, gene expression, and metabolic signal transduction activity were measured. Results revealed that CR animals had smaller (P < 0.05) LM fiber cross-sectional area than CON, as expected. Western blot analysis detected equivalent amounts of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) but reduced (P < 0.05) amounts of the splice-variant, PGC1α-4 in CR LM. Expression of IGF-1, a PGC1α-4 target gene, was 40% less (P < 0.05) in CR than CON. Downstream mediators of autocrine IGF-1 signaling also are attenuated in CR by comparison with CON. The amount of phosphorylated AKT1 was less (P < 0.05) in CR than CON. The ratio of p4EBP1T37/46 to total 4EBP1, a downstream mediator of AKT1, did not differ between CON and CR. By contrast, protein lysates from CR LM contained less (P < 0.05) total glycogen synthase kinase-3ß (GSK3ß) and phosphorylated GSK3ß than CON LM, suggesting blunted protein synthesis. Smaller CR LM fiber size associates with increased (P < 0.05) calpain 1 (CAPN1) activity coupled with lower (P < 0.05) expression of calpastatin, the endogenous inhibitor of CAPN1. Atrogin-1 and MuRF expression and autophagy components were unaffected by CR. Thus CR suppresses the hypertrophic PGC1α-4/IGF-1/AKT1 pathway while promoting activation of the calpain system.

Inhibitory Receptor Expression on CD8+ T Cells Is Linked to Functional Responses against Trypanosoma cruzi Antigens in Chronic Chagasic Patients.

In mammals, chronic diseases resulting from infectious agents have been associated with functional T cell response deficiency, a high frequency of terminally differentiated T cells, the presence of monofunctional Ag-specific T cells, and increased expression of inhibitory receptors. Similar to other chronic diseases, the progressive loss of certain functional activities during Trypanosoma cruzi infection might result in the inability to control replication of this parasite. To examine this hypothesis, we evaluated the differentiation and cell effector function of CD8(+) T cells and characterized the expression of inhibitory receptors and the presence of the parasite in the bloodstream of chagasic patients. The results showed that patients at an advanced severe disease stage had a higher frequency of terminally differentiated CD8(+) T cells than patients at an early stage of the disease. A monofunctional CD8(+) T cell response was observed in patients at an advanced stage, whereas the coexpression of markers that perform three and four functions in response to parasite Ags was observed in patients at a less severe disease stage. The frequency of CD8(+) T cells producing granzyme B and perforin and those expressing inhibitory receptors was higher in symptomatic patients than in asymptomatic patients. Taken together, these findings suggest that during the course of Chagas disease, CD8(+) T cells undergo a gradual loss of function characterized by impaired cytokine production, the presence of advanced differentiation, and increased inhibitory receptor coexpression.

Altering the motility of Trypanosoma cruzi with rabbit polyclonal anti-peptide antibodies reduces infection to susceptible mammalian cells.

Trypanosoma cruzi's trypomastigotes are highly active and their incessant motility seems to be important for mammalian host cell infection. The kinetoplastid membrane protein-11 (KMP-11) is a protein expressed in all parasite stages, which induces a cellular and humoral immune response in the infected host, and is hypothesized to participate in the parasite's motility. An N-terminal peptide from KMP-11, termed K1 or TcTLE, induced polyclonal antibodies that inhibit parasitic invasion of Vero cells. The goal of this study was to evaluate the motility and infectivity of T. cruzi when exposed to polyclonal anti-TcTLE antibodies. Rabbits were immunized with TcTLE peptide along with FIS peptide as an immunomodulator. ELISA assay results showed that post-immunization sera contained high titers of polyclonal anti-TcTLE antibodies, which were also reactive against the native KMP-11 protein and live parasites as detected by immunofluorescence and flow cytometry assays. Trypomastigotes of T. cruzi were incubated with pre- or post-immunization sera, and infectivity to human astrocytes was assessed by Giemsa staining/light microscope and flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled parasites. T. cruzi infection in astrocytes decreased approximately by 30% upon incubation with post-immunization sera compared with pre-immunization sera. Furthermore, trypomastigotes were recorded by video microscopy and the parasite's flagellar speed was calculated by tracking the flagella. Trypomastigotes exposed to post-immunization sera had qualitative alterations in motility and significantly slower flagella (45.5 µm/s), compared with those exposed to pre-immunization sera (69.2 µm/s). In summary, polyclonal anti-TcTLE serum significantly reduced the parasite's flagellar speed and cell infectivity. These findings support that KMP-11 could be important for parasite motility, and that by targeting its N-terminal peptide infectivity can be reduced.

Effects of nicotinamide riboside in ovo feeding on high-yield broiler performance, meat quality, and myopathy incidence.

Introduction: The objective of this study was to examine the effects of in ovo nicotinamide riboside (NR) feeding on high-yield broiler growth and meat quality. Methods: Fertilized Cobb 700 by-product eggs (N = 3,240) were randomly assigned to one of four in ovo treatments and injected with 0 (0NR), 250 (250NR), 500 (500NR), or 1,000 (1,000NR) mM NR at incubation-day 10. Chicks were hatched, vent sexed, and randomly placed 18 per pen in one of 32 floor pens. On day 48, birds were processed and deboned. Results: There were dose effects for all part weights (p < 0.05). Pectoralis major weight of 250, 500, and 1,000NR carcasses were heavier than 0NR (p < 0.03) but did not differ from remaining NR doses (p > 0.26). Pectoralis minor weight of 250NR carcasses was greater (p < 0.01) than 0NR and did not differ from other NR tenders (p > 0.21). Pectoralis minor weight of 500 and 1,000NR carcasses was greater than 0NR (p < 0.09), but did not differ (P = 0.82) from each other. There were no dose effects for all Pectoralis major and minor myopathy scores and incidence except incidence of tenders scoring "0" and "1" for woody-like tender. Percentage of NR1,000 tenders scoring 0 and 1 for woody-like tender were less than and greater than all other treatments, respectively (p < 0.05). There were no differences among remaining NR doses and NR0 tenders (p > 0.10). There were dose effects for muscle fiber number (P = 0.03). There tended to be more muscle fibers within 250 and 1,000NR muscles compared to 0NR (p < 0.09). Pectoralis major muscle from 500NR did not differ in muscle fiber number compared to 250 and 1,000NR (p > 0.18), but had more (p < 0.01) fibers than 0NR muscle. There tended to be more fibers in 250 and 1,000NR muscles compared to 0NR muscle (p < 0.09). Discussion: Nicotinamide riboside in ovo feeding caused birds to produce heavier parts; however, myopathy scores and incidence were minimally affected which may have been due greater muscle fiber number.

National Beef Quality Audit-2022: Transportation, mobility, live cattle, and hide assessments to determine producer-related defects that affect animal welfare and the value of market cows and bulls at processing facilities.

The National Beef Quality Audit (NBQA)-2022 serves as a benchmark of the current market cow and bull sectors of the U.S. beef industry and allows comparison to previous audits as a method of monitoring industry progress. From September 2021 through May 2022, livestock trailers (n = 125), live animals (n = 5,430), and post-slaughter hide-on animals (n = 6,674) were surveyed at 20 commercial beef processing facilities across the U.S. Cattle were transported in a variety of trailer types for an average distance of 490.6 km and a mean transport time of 6.3 h. During transit, cattle averaged 2.3 m2 of trailer space per animal indicating sufficient space was provided according to industry guidelines. Of all trailers surveyed, 55.3% transported cattle from an auction barn to a processing facility. When surveyed, 63.6% of all truck drivers reported to be Beef Quality Assurance certified. The majority (77.0%) of cattle were sound when evaluated for mobility. Mean body condition scores (9-point scale) for beef cows and bulls were 3.8 and 4.4, respectively, whereas mean body condition scores (5-point scale) for dairy cows and bulls were 2.3 and 2.6, respectively. Of the cattle surveyed, 45.1% had no visible live animal defects, and 37.9% had only a single defect. Of defects present in cows, 64.6% were attributed to an udder problem. Full udders were observed in 47.5% of all cows. Nearly all cattle were free of visible abscesses and knots (97.9% and 98.2%, respectively). No horns were observed in 89.4% of all cattle surveyed. Beef cattle were predominantly black-hided (68.9% and 67.4% of cows and bulls, respectively). Holstein was the predominant dairy animal observed and accounted for 85.7% of the cows and 98.0% of the bulls. Only 3.1% of all animals had no form of identification. Findings from the NBQA-2022 show improvements within the industry and identify areas that require continued education and research to improve market cow and bull welfare and beef quality.

Sulfocerebrosides upregulate liposome uptake in human astrocytes without inducing a proinflammatory response.

Astrocytes are involved in the pathogenesis of demyelinating diseases, where they actively regulate the secretion of proinflammatory factors, and trigger the recruitment of immune cells in the central nervous system (CNS). Antigen presentation of myelin-derived proteins has been shown to trigger astrocyte response, suggesting that astrocytes can directly sense demyelination. However, the direct response of astrocytes to lipid-debris generated during demyelination has not been investigated. The lipid composition of the myelin sheath is distinct, presenting significant amounts of cerebrosides, sulfocerebrosides (SCB), and ceramides. Studies have shown that microglia are activated in the presence of myelin-derived lipids, pointing to the possibility of lipid-induced astrocyte activation. In this study, a human astrocyte cell line was exposed to liposomes enriched in each myelin lipid component. Although liposome uptake was observed for all compositions, astrocytes had augmented uptake for liposomes containing sulfocerebroside (SCB). This enhanced uptake did not modify their expression of human leukocyte antigen (HLA) molecules or secretion of chemokines. This was in contrast to changes observed in astrocyte cells stimulated with IFNγ. Contrary to human monocytes, astrocytes did not internalize beads in the size-range of liposomes, indicating that liposome uptake is lipid specific. Epifluorescence microscopy corroborated that liposome uptake takes place through endocytosis. Soluble SCB were found to partially block uptake of liposomes containing this same lipid. Endocytosis was not decreased when cells were treated with cytochalasin D, but it was decreased by cold temperature incubation. The specific uptake of SCB in the absence of a proinflammatory response indicates that astrocytes may participate in the trafficking and regulation of sulfocerebroside metabolism and homeostasis in the CNS.

Influence of age and vaccination interval on canine parvovirus, distemper virus, and adenovirus serum antibody titers.

Canine core vaccine titer screenings are becoming increasingly popular in veterinary practice as a tool to guide vaccination decisions, despite a lack of supportive, peer-reviewed evidence-based literature. Additionally, it has been suggested that the canine core vaccine duration of host protective immunity can persist past the currently recommended vaccination interval. Thus, this study evaluated serum antibody titers against three core antigens in dogs with known vaccination histories and lifestyles, analyzing the effect of life stage, exposure risk, and time since last vaccination (TSLV). Clinically healthy dogs (n = 188) presenting to the primary care services of three colleges of veterinary medicine were selected to represent a variety of ages, breeds, and vaccination history. Serum antibody titers for canine parvovirus (CPV), canine distemper virus (CDV), and canine adenovirus-2 (CAV2) were measured via virus neutralization and hemagglutination inhibition. CAV2 and CPV titers decreased, while CDV titers had a decreasing trend with increasing time since last vaccination or vaccination interval. When assessing circulating antibody levels historially associated with protective immunity across various vaccination intervals, 62% (95%CI 36-82%; 8/13) of dogs had positive titers for CDV 5 years post last vaccination, while 92% (95%CI 67-99%; 12/13) of dogs were positive for CAV2 and CPV. Both advanced age and life stage were associated with lower titers and thus, identify a canine population cohort likely at higher disease risk. The results of this study revealed that patient duration of core vaccine-mediated immunity changes with a number of variables, with animal aging and time since vaccination influencing host humoral immunity. This provides further support for the performance of canine core antibody titers to assess whether a vaccine booster and/or specific type of booster is warranted.

The immunomodulatory potential of phage therapy to treat acne: a review on bacterial lysis and immunomodulation.

Background: Characterized by an inflammatory pathogenesis, acne is the most common skin disorder worldwide. Altered sebum production, abnormal proliferation of keratinocytes, and microbiota dysbiosis represented by disbalance in Cutibacterium acnes population structure, have a synergic effect on inflammation of acne-compromised skin. Although the role of C. acnes as a single factor in acne development is still under debate, it is known that skin and skin-resident immune cells recognize this bacterium and produce inflammatory markers as a result. Control of the inflammatory response is frequently the target for acne treatment, using diverse chemical or physical agents including antibiotics. However, some of these treatments have side effects that compromise patient adherence and drug safety and in the case of antibiotics, it has been reported C. acnes resistance to these molecules. Phage therapy is an alternative to treat antibiotic-resistant bacterial strains and have been recently proposed as an immunomodulatory therapy. Here, we explore this perspective about phage therapy for acne, considering the potential immunomodulatory role of phages. Methodology: Literature review was performed using four different databases (Europe PubMed Central-ePMC, Google Scholar, PubMed, and ScienceDirect). Articles were ordered and selected according to their year of publication, number of citations, and quartile of the publishing journal. Results: The use of lytic bacteriophages to control bacterial infections has proven its promising results, and anti-inflammatory effects have been found for some bacteriophages and phage therapy. These effects can be related to bacterial elimination or direct interaction with immune cells that result in the regulation of pro-inflammatory cytokines. Studies on C. acnes bacteriophages have investigated their lytic activity, genomic structure, and stability on different matrices. However, studies exploring the potential of immunomodulation of these bacteriophages are still scarce. Conclusions: C. acnes bacteriophages, as well as other phages, may have direct immunomodulatory effects that are yet to be fully elucidated. To our knowledge, to the date that this review was written, there are only two studies that investigate anti-inflammatory properties for C. acnes bacteriophages. In those studies, it has been evidenced reduction of pro-inflammatory response to C. acnes inoculation in mice after bacteriophage application. Nevertheless, these studies were conducted in mice, and the interaction with the immune response was not described. Phage therapy to treat acne can be a suitable therapeutic alternative to C. acnes control, which in turn can aid to restore the skin's balance of microbiota. By controlling C. acnes colonization, C. acnes bacteriophages can reduce inflammatory reactions triggered by this bacterium.

Effects of in ovo injection of nicotinamide riboside on high-yield broiler myogenesis.

The objective of this study was to determine the effects of in ovo injection of high-yield broiler embryos with nicotinamide riboside (NR) on pectoralis major muscle (PMM) development, growth, and gene expression. Fertilized Cobb 700 broiler eggs were randomly assigned to one of four treatments within a 2 × 2 factorial design. Factor 1 consisted of NR dose (DOS) with eggs receiving 0 or 2.5 mM NR. Factor 2 consisted of injection location (LOC), with treatments injected into either the yolk sac or albumen. At day 10 of incubation, 100 µL of the assigned NR dose was injected into the yolk sac of the developing embryo and chicks were euthanized within 24 h of hatching. Chick PMM and individual fiber morphometrics, and expression of genes associated with cell cycle progression were analyzed. There were DOS × LOC interactions for hatched chick PM weight and length (P < 0.04). When NR was injected into the albumen, PMM weight decreased (P < 0.05); when NR was injected into the yolk, PMM weight increased (P < 0.05). Pectoralis major length was not affected (P > 0.05) when NR was injected into the albumen but was increased (P < 0.05) when NR was injected into the yolk. There was a DOS × LOC interaction (P = 0.04) for muscle fiber density and tended to be a DOS × LOC interaction (P = 0.07) for muscle fiber CSA. Pectoralis major muscle fiber density was not affected when NR was injected into the albumen (P > 0.05), but density increased when NR was injected into the yolk (P < 0.05). There were DOS × LOC interactions for hatched chick COXII, cyclin D, and SIRT1 expression (P ≤ 0.04), which may indicate NR improves skeletal muscle development and growth by enhancing myoblast proliferation during embryonic development.

Broiler chicken weight gain is a result of genetics and nutrition, with increased muscle mass attributed to accelerated embryonic myogenesis and posthatch muscle growth. During the avian incubation period, in ovo injection may be used as a strategy to deliver exogenous supplements into growing embryos for improving skeletal muscle development and growth. Nicotinamide riboside (NR), a vitamin B3 analog, is a human performance supplement used to stimulate mitochondria biogenesis and elevate tissue NAD+ levels. Research showed injecting NR into the chick embryonic yolk sac increased breast muscle weight and muscle satellite cell numbers and proliferation rate. Therefore, our objective was to determine the effects of in ovo injection of high-yield broilers with NR on broiler breast muscle development and growth. Our study showed in ovo injection of NR into the yolk sac increased hatched chick breast muscle morphometrics, which coincided with an increase in muscle fiber density and tended to decrease fiber cross-sectional area. Increased Sirtuin1 and cyclin D mRNA expression of hatched chicks from eggs injected with 2.5 mM NR into yolk sac indicate a potential NR regulated Sirtuin1/cyclin D molecular mechanism mediating chicken muscle early development.

Trypanosoma cruzi-specific CD8+ T cells and other immunological hallmarks in chronic Chagas cardiomyopathy: Two decades of research.

Trypanosoma cruzi, the causal agent of Chagas disease, has coexisted with humans for thousands of years. Therefore, the parasite has developed several mechanisms of antigenic variability that has allowed it to live inside the cells and evade the host immune response. Since T. cruzi displays an intracellular cycle-stage, our research team focused on providing insights into the CD8+ T cells immune response in chronic Chagas cardiomyopathy. We began our work in the 2000s studying parasite antigens that induce natural immune responses such as the KMP11 protein and TcTLE, its N-terminal derived peptide. Different approaches allowed us to reveal TcTLE peptide as a promiscuous CD8+ T cell epitope, able of inducing multifunctional cellular immune responses and eliciting a humoral response capable of decreasing parasite movement and infective capacity. Next, we demonstrated that as the disease progresses, total CD8+ T cells display a dysfunctional state characterized by a prolonged hyper-activation state along with an increase of inhibitory receptors (2B4, CD160, PD-1, TIM-3, CTLA-4) expression, an increase of specific terminal effector T cells (TTE), a decrease of proliferative capacity, a decrease of stem cell memory (TSCM) frequency, and a decrease of CD28 and CD3ζ expression. Thus, parasite-specific CD8+ T cells undergo clonal exhaustion, distinguished by an increase in late-differentiated cells, a mono-functional response, and enhanced expression of inhibitory receptors. Finally, it was found that anti-parasitic treatment induces an improved CD8+ T cell response in asymptomatic individuals, and a mouse animal model led us to establish a correlation between the quality of the CD8+ T cell responses and the outcome of chronic infection. In the future, using OMICs strategies, the identification of the specific cellular signals involved in disease progression will provide an invaluable resource for discovering new biomarkers of progression or new vaccine and immunotherapy strategies. Also, the inclusion of the TcTLE peptide in the rational design of epitope-based vaccines, the development of immunotherapy strategies using TSCM or the blocking of inhibitory receptors, and the use of the CD8+ T cell response quality to follow treatments, immunotherapies or vaccines, all are alternatives than could be explored in the fight against Chagas disease.

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application.

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human ß-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

The effects of in ovo nicotinamide riboside dose on broiler myogenesis.

The objective of this study was to determine the effects of in ovo injection of nicotinamide riboside (NR) on broiler embryonic myogenesis. Fertilized Cobb 500 broiler eggs (N = 240) were sorted by weight and within each strata, randomly assigned to 1 of 4 NR dose treatments (0 mmol, 250 mmol, 500 mmol, or 1 mol; final concentration in yolk of 0, 2.5, 5.0, or 10.0 mmol) of NR. At day 10 of incubation, 100 µL of the assigned NR dose was injected into the yolk sac of the developing embryo, and chicks were euthanized within 24 h of hatching. Pectoralis major muscle (PMM) and individual fiber morphometrics were collected. Chicks injected with NR had greater PMM weight and length (P < 0.01), but did not differ from each other (P > 0.14). Chicks from eggs injected with NR had greater PMM weight and width than control chicks (P < 0.01), but did not differ from each other (P = 0.86). Chicks from eggs injected with 500 mmol NR had greater PMM depth than control and 1M chicks (P < 0.04), which did not differ (P = 0.24) from each other. Chicks from eggs injected with 250 mmol NR did not differ in PMM length compared with all other treatments (P > 0.06). There was no treatment effect (P = 0.20) for PMM fiber cross-sectional area; however, there was a treatment effect (P < 0.01) for muscle fiber density. Chicks from eggs injected with 1 mol NR had greater fiber density than all other treatments (P < 0.01). Chicks injected with 250 and 500 mmol NR had greater fiber density than control chicks (P < 0.01), but did not differ (P < 0.06) from each other. Injecting developing embryos at day 10 of incubation increased hatched chick PMM morphometrics, which were partly because of the NR catalyzed increase in muscle fiber density.

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth.

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during embryonic and fetal development. This area continues to be an area of focus because muscle fiber number is established during this time and determines the basis for all future growth. In poultry, numerous studies demonstrated in ovo feeding of growth factors, vitamins, or other nutrients improved chick embryonic muscle and intestinal development. Improving in ovo muscle development could benefit the poultry industry by possibly influencing meat yield, growth rate, or myopathy conditions. During the past five years, the Gonzalez Laboratory at the University of Georgia developed a nicotinamide riboside in ovo feeding methodology for broiler-chicken embryos, which altered muscle development. When injected into a developing embryo's yolk sac, nicotinamide riboside increased pectoralis major muscle weight and muscle fiber density at hatch. This protocol will demonstrate a methodology to accurately and reproducibly conduct in ovo feeding studies utilizing commercial standard- and high-yielding broiler embryos. These data and methods will allow other research groups to perform in ovo feeding studies with much success and reproducibility.

Measurement of the three-axis vibration, temperature, and relative humidity profiles of commercial transport trailers for pigs.

The objective of this study was to collect and interpret three-axis acceleration, temperature, and relative humidity data from six locations within commercial transport trailers shipping market-weight pigs. Transport was observed in Kansas (n = 15) and North Carolina (n = 20). Prior to loading, three-axis accelerometers were affixed to six locations on the trailers: top fore (TF), top center (TC), top aft (TA), bottom fore (BF), bottom center (BC), and bottom aft (BA) compartments. Data were post-processed to calculate root-mean-square (RMS) accelerations and vibration dose values (VDV) in the vertical direction and the horizontal plane. These values were compared with exposure action values (EAV) and exposure limit values (ELV), vibration levels deemed uncomfortable and potentially dangerous to humans. Additionally, RMS and VDV were compared among the trailer compartments. The vertical RMS accelerations for all compartments exceeded the EAV for loads measured in Kansas, and for the majority of the compartments measured in North Carolina. Many compartments, specifically the BA compartment from all trips, exceeded the vertical ELV. Regardless of where the data were collected, fewer compartments exceeded the EAV in the horizontal orientation. Only BA compartments exceeded the ELV in the horizontal orientation. There were Area × Level interactions for vertical and horizontal RMS and VDV (P < 0.01). The BF compartment had a greater vertical RMS value than the TF, TC, and BC (P < 0.02) compartments, but did not differ (P = 0.06) from the TA compartment. The vertical RMS of the TA compartment did not differ from the TF, TC, and BC compartments (P > 0.13). The BF compartment had a greater (P = 0.02) vertical VDV value than the TC location, but did not differ from the other locations (P > 0.16). All other locations did not differ in vertical VDV (P > 0.12). The BF compartment had greater horizontal RMS than the TC and TA compartments (P < 0.01), but did not differ from TF and BC compartments (P > 0.12). All other compartments did not differ in horizontal RMS (P > 0.34). All compartments, aside from the BA compartment, did not differ in horizontal VDV (P > 0.19). Vibration analyses indicated the BA compartment had the greatest vertical and horizontal vibrations and a large percentage of the compartments exceed the EAV and ELV, which indicated pigs may have experienced uncomfortable trips that could cause discomfort or fatigue.

Effects of extended postmortem aging and intramuscular location on protein degradation, muscle fiber morphometrics, and tenderness of beef longissimus lumborum and semitendinosus steaks.

The objective of this study was to determine effects of extended aging and intramuscular location on Warner-Bratzler shear force (WBSF), muscle fiber cross-sectional area (CSA), and protein degradation of semitendinosus (ST) and longissimus lumborum (LL) steaks. Left ST and LL were removed from 40 carcasses at 6 d postmortem. The ST was fabricated into five locations (LOC), with LOC 1 being most proximal and LOC 5 being most distal. The posterior LL was fabricated into 3 LOC, with LOC 1 being most anterior. Vacuum sealed ST steaks were aged 7, 14, 28, 56, or 112 d postmortem, while LL steaks were aged 7, 28, or 112 d postmortem at 2 ± 1 °C. A steak from each LOC was assigned to WBSF or laboratory analyses. There were no Day of Aging (DOA) × LOC interactions for all dependent variables (P > 0.06). There were DOA effects for ST and LL WBSF values and degraded 38-kDa desmin (DES; P < 0.01). Day-7 ST-steak WBSF value was greater than all other days (P < 0.01) and day-14 steaks had greater WBSF value than remaining days (P < 0.05). Day-28 ST-steak WBSF values were greater than day 56 and 112 (P < 0.01), which did not differ (P = 0.53). In the LL, day-7 steaks had greater WBSF values than the other two timepoints (P < 0.01) and day-28 steaks had greater (P < 0.01) WBSF values than day-112 steaks. Degraded ST 38-kDa DES content was less on day 7 and 14 compared to all other days (P < 0.03), but did not differ (P = 0.79) from each other. Days 28 and 56 38-kDa DES content was less than day 112 (P < 0.01), but did not differ (P = 0.34) from each other. Degraded LL 38-kDa DES content was less on day 7 than day 28 and 112 (P < 0.02), which did not differ (P = 0.67). There were LOC effects for only ST WBSF and muscle fiber CSA (P < 0.05). Semitendinosus steak LOC 1 and 2 had greater WBSF values than all other locations (P < 0.01), but did not differ (P = 0.32) from each other. Semitendinosus steak LOC 3 and 5 had greater WBSF values than LOC 4 (P < 0.01), but did not differ (P = 0.85) from each other. The CSA of all ST fiber types were largest in LOC 1 compared to all other fiber types (P < 0.01). The CSA of all LOC 2 and 3 fiber types was greater than LOC 4 and 5 (P < 0.01), but were not different from each other (P > 0.81), and LOC 4 had greater CSA than LOC 5 (P < 0.01). Steak aging WBSF value improvements seemed proteolysis catalyzed, while the ST intramuscular tenderness gradient was more likely due to muscle fiber CSA.

In ovo feeding of nicotinamide riboside affects broiler pectoralis major muscle development.

The objective of this study was to examine the effect of nicotinamide riboside (NR) on pectoralis major muscle (PM) development and growth. Fertilized Cobb 500 broiler eggs (N = 156; average weight of 70.3 g) were ordered by weight, and within each four egg strata, eggs were randomly assigned to treatments within a 2 × 2 factorial arrangement. Factor 1 consisted of NR treatment with eggs receiving 0 or 250 mM NR. Factor 2 consisted of injection location, with treatments injected into either the yolk sac or albumen. Eggs were incubated at a temperature of 37 °C and a relative humidity of 40 ± 2% for the first 18 d of incubation and humidity was increased to 60 ± 2 °C for the final 3 d. On day 10 of incubation, eggs were injected in their designated location with 100 µL of 0.9% sterile saline containing the assigned NR dose. Chicks were hatched, euthanized, and morphometric measurements of the body and left PM were collected. The left PM was also analyzed for muscle fiber cross-sectional area (CSA) and density. There were no treatment × location or main effects for all body morphometric measurements (P > 0.07), except chest width of chicks from eggs injected in the yolk were wider (P = 0.01) than chicks from eggs injected in the albumen. There were only treatment × location interactions for PM weight and length (P < 0.01). When NR was injected into the albumen, PM weight did not differ (P = 0.09); however, when NR was injected into the yolk sac, PM weight increased (P < 0.01). When NR was injected into both locations, PM length increased (P < 0.01), but increased to a greater extent when NR was injected into the yolk sac. There were treatment main effects for PM width and depth (P < 0.01), with NR injected chicks having PM with greater width and depth. There were no treatment × location or main effects for PM fiber CSA (P > 0.06). There was a treatment × location interaction (P < 0.01) for fiber density. When NR was injected into the albumen, fiber density did not differ (P = 0.09); however, when NR was injected into the yolk sac, fiber density increased (P < 0.01). Injecting NR into the yolk sac of the developing embryo at day 10 of incubation increased PM development which was due to an increase in muscle density.