Development of a nucleic acid lateral flow immunoassay for simultaneous detection of Listeria spp. and Listeriamonocytogenes in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food sample

Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns

Nucleic Acid Lateral Flow Immunoassay for the Detection of Pathogenic Bacteria from Food

Nucleic acid lateral flow immunoassay (NALFIA) is a method combining molecular biological principle of detection with immunochemical principle of visualisation. Following isolation of DNA from the sample, a duplex PCR with two primer sets, of which one was labelled with biotin and the other with digoxigenin or fluorescein, respectively, was performed. The PCR solution and carbon particles conjugated with avidin are directly added to the nitrocellulose membrane with two test lines of immobilised antibodies specific for digoxigenin and fluorescein. The appearance of a black line indicates the presence of specific amplicon. We would like to present the NALFIA for the simultaneous detection of L. monocytogenes in particular and the genus Listeria in general, in food. Bacteria from the genus Listeria frequently contaminate a large variety of foods. Occurrence of Listeria strains in food may indicate errors in good hygienic and manufacturing practice, only L. monocytogenes is a significant human and animal pathogen responsible for the serious illness listeriosis. Conventional microbiological methods for L. monocytogenes detection are laborious and take several days to achieve a confirmed identification

THE DYNAMICS OF ANTIGEN SPECIFIC PROLIFERATIVE RESPONSES OF LYMPHOCYTES AT EARLY STAGES OF BOVINE PARATUBERCULOSIS INFECTION

The present study was aimed to quantify the dynamics of early antigen specific proliferative responses of lymphocytes to (protein) antigens associated with experimentalMycobacterium avium subsp. paratuberculosis (Mp) infection cattle. The data were collected from20 experimentally infected calves, and 10 uninfected control animals, during the first 2 years oftheir lives. Several purified protein derivative antigens (Ppdp, Ppda, and Ppdb), tworecombinant Mp heatshock proteins (Hsp65 and Hsp70) and whole bacteria (sonicated Mpstrain 316F) were used to measure lymphocyte proliferation in a lymphocyte proliferationassay. Data were analyzed using a linear mixed effect (LME) model. The results showedsignificant group and timed effects for all antigens tested. At several time points, the responsesin the infected group were found significantly higher as compared to control group. The Ppdantigens induced similar lymphocyte proliferation patterns, as compared to whole bacteriaantigen and Hsp70. These results indicated that the antigen specific proliferative responses oflymphocytes differs for different antigens, probably related to differences in their availabilityduring different stages of infection. The application of LME model is a useful tool for analyzingthe quantitative longitudinal datasets. Keywords: dynamics, Mp, antigen, LM

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein–Friesian heifers

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne’s disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein–Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13567-022-01087-0

Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays

Carbon nanoparticles (CNPs) labeled with reporter molecules can serve as signaling labels in rapid diagnostic assays as an alternative to gold, colored latex, silica, quantum dots, or up-converting phosphor nanoparticles. Detailed here is the preparation of biomolecule-labeled CNPs and examples of their use as a versatile label. CNPs can be loaded with a range of biomolecules, such as DNA, antibodies, and proteins (e.g., neutravidin or a fusion protein of neutravidin with an enzyme), and the resulting conjugates can be used to detect analytes of high or low molecular mass

Defining Fatty Acid Changes Linked to Rumen Development, Weaning and Growth in Holstein-Friesian Heifers

After birth, as effectively monogastric animals, calves undergo substantial physiological changes to become ruminants by 3 months of age and reach sexual maturity at approximately 15 months of age. Herein, we assess longitudinal metabolomic changes in Holstein-Friesian (HF) heifers from birth until sexual maturity during this developmental process. Sera from 20 healthy, HF heifers were sampled biweekly from 2 weeks of age until 13 months of age and then monthly until 19 months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares discriminant analysis (PLS-DA) and unsupervised hierarchical clustering analysis (HCA) of the derived metabolomes indicated changes detectable in heifers’ sera over time. Time series analyses identified 30 metabolites that could be related to rumen development and weaning at ~3 months of age. Further time series analysis identified 40 metabolites that could be correlated with growth. These findings highlight the role of acetic acid and 3-phenylpropionate (3-PP) in rumen development and growth, suggest that weaning induces elevated levels of fatty acyls in response to a post-weaning stress-induced innate immune response and demonstrate the utilization of fatty acyls in growth. The identified metabolites offer serum metabolites which could inform the nutrition and healthy development of heifers

Metabolomic Changes in Naturally MAP-Infected Holstein–Friesian Heifers Indicate Immunologically Related Biochemical Reprogramming

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes weight loss, diarrhoea, and reduced milk yields in clinically infected cattle. Asymptomatic, subclinically infected cattle shed MAP bacteria but are frequently not detected by diagnostic tests. Herein, we compare the metabolite profiles of sera from subclinically infected Holstein-Friesian heifers and antibody binding to selected MAP antigens. The study used biobanked serum samples from 10 naturally MAP-infected and 10 control heifers, sampled monthly from ~1 to 19 months of age. Sera were assessed using flow infusion electrospray-high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least-squares discriminant analyses (PLS-DA) and hierarchical cluster analysis (HCA) of the data discriminated between naturally MAP-infected and control heifers. In total, 33 metabolites that differentially accumulated in naturally MAP-infected heifers compared to controls were identified. Five were significantly elevated within MAP-infected heifers throughout the study, i.e., leukotriene B4, bicyclo prostaglandin E2 (bicyclo PGE2), itaconic acid, 2-hydroxyglutaric acid and N6-acetyl-L-lysine. These findings highlight the potential of metabolomics in the identification of novel MAP diagnostic markers and particular biochemical pathways, which may provide insights into the bovine immune response to MAP

Влияние фенилгидразина и алкилсульфатов на осмотическую чувствительность эритроцитов млекопитающих

Показано, что чувствительность эритроцитов млекопитающих к гипертоническому стрессу при 37 °С после обработки фенилгидразином зависит от видовой принадлежности клеток. Установлено, что модификация фенилгидразином эритроцитов млекопитающих приводит к снижению антигемолитической активности алкилкилсульфатов в условиях гипертонического стресса.Показано, що чутливість еритроцитів ссавців до гіпертонічного стресу при 37 °С після обробки фенілгідразином залежить від видової приналежності клітин. Встановлено, що модифікація фенілгідразином еритроцитів ссавців призводить до зниження антигемолітичної активності алкілсульфатів в умовах гіпертонічного стресу.It is shown that the sensitivity of mammalian erythrocytes to hypertonic stress at 37 °C after the treatment with phenylhydrazine depends on the species of a cell. We have established that the modification with phenylhydrazine of mammalian erythrocytes leads to a decrease of the alkyl sulfates antihemolytic activity under hypertonic stress