82 research outputs found
Evaluating contribution of the cellular and humoral immune responses to the control of shedding of \u3cem\u3eMycobacterium avium\u3c/em\u3e spp. \u3cem\u3eparatuberculosis\u3c/em\u3e in cattle
Mycobacterium avium spp. paratuberculosis (MAP) causes a persistent infection and chronic inflammation of the gut in ruminants leading to bacterial shedding in feces in many infected animals. Although there are often strong MAP-specific immune responses in infected animals, immunological correlates of protection against progression to disease remain poorly defined. Analysis of cross-sectional data has suggested that the cellular immune response observed early in infection is effective at containing bacterial growth and shedding, in contrast to humoral immune responses. In this study, 20 MAP-infected calves were followed for nearly 5 years during which MAP shedding, antigen-specific cellular (LPT) and humoral (ELISA) immune responses were measured. We found that MAP-specific cellular immune response developed slowly, with the peak of the immune response occurring one year post infection. MAP-specific humoral immunity expanded only in a subset of animals. Only in a subset of animals there was a statistically significant negative correlation between the amount of MAP shedding and magnitude of the MAP-specific cellular immune response. Direct fitting of simple mechanistic mathematical models to the shedding data suggested that MAP-specific immune responses contributed significantly to the kinetics of MAP shedding in most animals. However, whereas the MAP-specific cellular immune response was predicted to suppress shedding in some animals, in other animals it was predicted to increase shedding. In contrast, MAP-specific humoral response was always predicted to increase shedding. Our results illustrate the use of mathematical methods to understand relationships between mycobacteria and immunity in vivo but also highlight problems with establishing cause-effect links from observational data
Vaccination induced antibodies to recombinant avian influenza a virus M2 protein or synthetic M2e peptide do not bind to the M2 protein on the virus or virus infected cells.
BACKGROUND: Influenza viruses are characterized by their highly variable surface proteins HA and NA. The third surface protein M2 is a nearly invariant protein in all Influenza A strains. Despite extensive studies in other animal models, this study is the first to describe the use of recombinant M2 protein and a peptide coding for the extracellular part of the M2 protein (M2e) to vaccinate poultry. METHODS: Four groups of layer chickens received a prime-boost vaccination with recombinant M2 protein, M2e, a tetrameric construct from M2e peptide bound to streptavidin and a control tetrameric construct formulated with Stimune adjuvant. RESULTS: We determined the M2-specific antibody (Ab) responses in the serum before vaccination, three weeks after vaccination and two weeks after booster, at days 21, 42 and 56 of age. The group vaccinated with the M2 protein in combination with Stimune adjuvant showed a significant Ab response to the complete M2 protein as compared to the other groups. In addition an increased Ab response to M2e peptide was found in the group vaccinated with the M2e tetrameric construct. None of the vaccinated animals showed seroconversion to AI in a commercial ELISA. Finally no Ab’s were found that bound to M2 expressed on in vitro AI infected MDCK cells. CONCLUSION: Although Ab’s are formed against the M2 protein and to Streptavidin bound M2e peptide in a tetrameric conformation these Ab’s do not recognize of M2 on the virus or on infected cells
A longitudinal study of factors influencing the result of a mycobacterium avium ssp. paratuberculosis antibody ELISA in milk of dairy cows
The influence of milk yield and milk composition
on the diagnosis of Mycobacterium avium ssp. paratuberculosis
(MAP) by milk ELISA in the context of
the total IgG secretion patterns in milk throughout
lactation and serum concentrations were investigated.
A 2-yr trial was performed in which 1,410 dairy cows
were sampled monthly and MAP milk ELISA status
and milk yield and composition were determined. Data
were analyzed by mixed model analysis. Milk yield
was found to significantly influence ELISA results
expressed as sample-to-positive (S/P) ratios. For each
5-kg increase in milk, the S/P ratio has to be multiplied
by 0.89; therefore, high milk yield can change the MAP
milk ELISA outcome of a cow in early infection from
positive to negative. Parity influenced ELISA outcome
significantly, indicating that cows with a parity >1 are
more likely to be identified by milk testing. Also, herd
was an important predictor, showing that herd prevalence
influences the milk ELISA strongly. Other factors
influencing the S/P ratios were protein concentration,
somatic cell count, and days in milk. The IgG concentration
and mass excreted per day were determined
longitudinally in a subset of 41 cows of which samples
and data of a complete lactation were available. Again,
the IgG concentration in milk was mainly influenced by milk yield. The total IgG mass secreted per day in milk
was found to be relatively constant, with a mean of
8.70 ± 5.38 g despite an increasing IgG concentration
in serum at the same time. The variation of IgG concentration
in milk can be mainly attributed to dilution
through changes in milk yield. This supports the assumption
that concentrations of MAP-specific antibodies
are influenced by changes in milk yield similarly. In
conclusion, we confirmed that antibody concentrations, and therefore MAP ELISA outcome, were influenced by
milk yield, herd, and parity. To enhance performance,
milk ELISA tests should either be performed in early or
late lactation, when milk yield is low. From a management
perspective, sampling should be done during early
lactation before cows are bred again. Based on the slow
progressive infection dynamics, only first-parity cows
should be preferentially tested at the end of their first
lactation to avoid false-negative results.The study was partly funded by Stichting Mesdagzuivelfonds
(NLTO, Leeuwarden, The Netherlands).http://www.journalofdairyscience.orgam201
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
Intestinal infection following aerosol challenge of calves with Mycobacterium avium subspecies paratuberculosis
A challenge experiment was performed to investigate whether administration of Mycobacterium avium subsp. paratuberculosis (MAP) via the respiratory route leads to MAP infection in calves. Eighteen calves from test negative dams were randomly allocated to four groups. Six calves were challenged with MAP nasally and six calves were challenged by transtracheal injection; three orally challenged calves served as positive controls, and three non challenged calves as negative controls. The challenge was performed as a nine-fold trickle dose, 107 CFU in total. Blood and faecal samples were collected frequently. Calves were euthanized three months post-challenge and extensively sampled. Blood samples were tested for the presence of antibodies and interferon gamma producing cells by ELISA. Faecal and tissue samples were cultured in a liquid culture system and the presence of MAP was confirmed by IS900 realtime PCR. Fourteen out of fifteen calves had no MAP antibody response. The negative controls remained negative; all positive controls became infected. Two nasally challenged calves showed a Purified Protein Derivative Avian (PPDA) specific interferon gamma response. In all nasally challenged calves, MAP positive intestinal samples were detected. In three calves of the nasal group MAP positive retropharyngeal lymph nodes or tonsils were detected. In all calves of the transtracheal group MAP positive intestinal tissues were detected as well and three had a MAP positive tracheobronchial lymph node. These findings indicate that inhalation of MAP aerosols can result in infection. These experimental results may be relevant for transmission under field conditions since viable MAP has been detected in dust on commercial dairy farms
Subpopulations of bovine WC1+ γδ T cells rather than CD4+CD25highFoxp3+ T cells act as immune regulatory cells ex vivo
Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4+CD25high T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4+CD25high T cells and γδ T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4+CD25high T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4+CD25low T cells, WC1+, WC1.1+ and WC1.2+ γδ T cells, NK cells, CD8+ T cells and CD14+ monocytes. Only the WC1.1+ and WC1.2+ γδ T cells and CD14+ monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4+CD25highFoxp3+ and CD4+CD25low T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the γδ T cell population, more precisely in the WC1.1+ and the WC1.2+ subpopulation, major populations present in blood of cattle in contrast to non-ruminant species
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
Immune response of cattle immunized with a conjugate of the glycolipid glucose monomycolate and protein
Strong anti glycolipid IgG responses can occur in humans and animals, but contrary to anti protein responses and anti
glycoprotein responses, the exact mechanism of induction is unknown. We have previously shown that experimental
immunization with the glycolipid glucose monomycolate (GMM) causes the development of specific T cell responses, but
not of anti GMM antibodies. However, cattle naturally infected with Mycobacterium avium ssp. paratuberculosis produce high
levels of anti GMM IgG. In the present study, we tested whether vaccination with GMM conjugated to a protein mimics
natural infection in its capacity to induce the production of antibodies against GMM. Cattle were immunized (n = 5 per group)
with GMM conjugated to a protein, or GMM and protein non-conjugated and administered at contralateral locations, or
carrier only. Although immunization with the GMM-protein conjugate vaccine and the non-conjugated vaccine induced
protein specific antibody responses, GMM specific antibodies were not detected in either of the groups. In conclusion, the
generation of isotype-switched anti lipid antibodies appears to require more than providing peptide epitopes for T helper cells
to support glycolipid specific B cells in antibody production.Supplementary data associated with this article can be found as a separated attachment.Van Rhijn is supported by a NWO Meervoud subsidy (#836.08.001), and T.K.A. Nguyen is supported by the Ministry of Education and Training of Vietnam according to Decision 322/QD-TTg for Overseas Training Projects.http://www.sciencedirect.com/science/journal/0165242
Isotype specific antibody responses to Mycobacterium Avium subspecies Paratuberculosis antigens are associated with the use of biological therapy in Inflammatory Bowel Disease
Background: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn’s disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility.Methods: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS].Results: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44–5.01; and 2.60, 1.46–4.64, respectively]. No associations were seen for risk of surgery [p-values > 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance.Conclusions: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found
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