Browsing by Author "Heiser A"

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    A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.
    (Frontiers Media S.A., 2023-05-22) Ogbuigwe P; Roberts JM; Knox MA; Heiser A; Pita A; Haack NA; Garcia-Ramirez JC; Velathanthiri N; Biggs PJ; French NP; Hayman DTS; Xu R
    Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.
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    Growth performance, antibody response, and mammary gland development in New Zealand dairy replacement bovine heifers fed low or high amounts of unpasteurized whole milk
    (Oxford University Press on behalf of the American Society of Animal Science, 2022-10-28) Khan MA; Heiser A; Maclean PH; Leath SR; Lowe KA; Molenaar AJ
    This study evaluated the influence of feeding low and high preweaning allowances of unpasteurized whole milk (MA) on intake, selected blood metabolites, antibody response, mammary gland growth, and growth of New Zealand (NZ) dairy heifers to 7 mo of age. At 10 ± 2 d of age (study day 0), group-housed (six·pen-1) heifer calves (Holstein-Friesian × Jersey) were allocated to low (4 L whole milk·calf-1·d-1; n = 7 pens) or high (8 L whole milk·calf-1·d-1; n = 7 pens) MA for the next 63 d. Calves were gradually weaned between days 63 ± 2 and 73 ± 2. Calves in each pen had ad-libitum access to clean water, pelleted calf starter, and chopped grass hay from day 1 to 91 ± 2 d. At 92 ± 2 d, all calves were transferred to pasture, grazed in a mob, and their growth and selected blood metabolites were measured until day 209. All animals were weighed weekly during the indoor period (to day 91) and then at days 105, 112, 128, 162, 184, and 209. Skeletal growth measurements and blood samples to analyze selected metabolites were collected at the start of the experiment, weaning, and then postweaning on day 91, and day 201. Specific antibodies against Leptospira and Clostridia were quantified in weeks 7, 13, and 27. Mammary glands were scanned using ultrasonography at the start of the experiment, weaning, and day 201. Feeding high vs. low amounts of MA increased the preweaning growth in heifer calves (P = 0.02) without negatively affecting postweaning average daily gain (ADG) (P = 0.74). Compared with heifers fed with low MA, high MA fed heifers had a greater increase in antibodies against Leptospira and Clostridia by 13 wk of age (P = 0.0007 and P = 0.06, respectively). By 27 wk of age, the antibody response was the same in heifers offered low or high MA. There was no effect of MA on the total size of the mammary gland, measured by ultrasonography, at weaning and 7 mo of age. However, the greater MA was associated with more mammary parenchyma (P = 0.01) and less mammary fat pad (P = 0.03) in back glands at 7 mo of age compared with heifers fed lower MA. In conclusion, feeding a high vs. a low amount of unpasteurized whole milk increased the preweaning growth of New Zealand replacement heifers without negatively affecting their ADG during postweaning under grazing conditions. Feeding more (8 vs. 4 L·d-1) unpasteurized whole milk positively affected antibody responses early in life and mammary gland composition by 7 mo of age in dairy heifers reared for pasture-based dairy systems.
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    Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.
    (Springer Nature Limited, 2022-06-21) Khanum S; Carbone V; Gupta SK; Yeung J; Shu D; Wilson T; Parlane NA; Altermann E; Estein SM; Janssen PH; Wedlock DN; Heiser A
    In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.