Biomedical subjects

W R Waters

Publications and source records attributed to W R Waters.

At least 19 recordsLinked to original sources

PubMed2007

High growth rate fails to enhance adaptive immune responses of neonatal calves and is associated with reduced lymphocyte viability.

The objective of the study was to evaluate the effects of 3 targeted growth rates on adaptive (i.e., antigen-specific) immune responses of preruminant, milk replacer-fed calves. Calves (9.1 +/- 2.4 d of age) were assigned randomly to one of 3 dietary treatments to achieve 3 targeted daily rates of gain [no growth (maintenance) = 0.0 kg/d, low growth = 0.55 kg/d, or high growth = 1.2 kg/d] over an 8-wk period. The NRC Nutrient Requirements of Dairy Cattle calf model computer program was used to estimate the milk replacer intakes needed to achieve target growth rates. All calves were fed a 30% crude protein, 20% fat, all-milk protein milk replacer reconstituted to 14% dry matter. Diets were formulated to ensure that protein would not be limiting. All calves were vaccinated 3 wk after initiation of dietary treatments with Mycobacterium bovis, strain bacillus Calmette-Guerin and ovalbumin. Growth rates for no-growth (0.11 kg/d), low-growth (0.58 kg/d), and high-growth (1.16 kg/d) calves differed throughout the experimental period. Blood glucose concentrations in high-growth calves increased with time and were higher than in low- and no-growth calves. Mononuclear and polymorphonuclear leukocyte percentages in peripheral blood were unaffected by growth rate but did change with advancing age. Percentages of CD4(+) T cells increased with age in no-growth and low-growth calves, a characteristic of maturation, but failed to increase in high-growth calves. Growth rate did not affect the percentages of CD45RO(+) (memory) CD4(+) and CD8(+) T cells, antigen (i.e., ovalbumin)-specific serum IgG concentrations, or antigen (i.e., purified protein derivative)-induced IFN-gamma and nitric oxide secretion by mononuclear cell cultures. Antigen-elicited cutaneous delayed-type hypersensitivity responses of no-growth calves exceeded responses of low-growth, but not high-growth, calves. In resting- and antigen-stimulated cell cultures, viabilities of CD4(+), CD8(+), and gammadeltaTCR(+) T cells from high-growth calves were lower than those of the same T cell subsets from no-growth and low-growth calves. Alternatively, resting cultures of mononuclear leukocytes from high-growth calves produced more nitric oxide than those from no-growth and low-growth calves. In conclusion, adaptive immune responses were affected minimally by growth rate. The results suggest that protein-energy malnutrition in the absence of weight loss is not detrimental to antigen-specific responses of neonatal vaccinated calves and that a high growth rate does not enhance these responses. The negative effect of a high growth rate on the viability of circulating T cell populations may influence infectious disease resistance of the calf.

Adjuvants, Immunologic↗

PubMed2006

Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle.

The Bovigam assay is approved for use within the United States as a complementary tuberculosis test. Prior to whole blood culture and the ensuing ELISA to detect interferon-(IFN)-gamma, samples are subjected to various holding time/temperature combinations due, in part, to practical constraints associated with shipment of samples to approved laboratories. To evaluate these effects, 5-month-old Holstein calves (n = 7) received 10(3) cfu Mycobacterium bovis by aerosol. Heparinized blood was collected 2 months after challenge and held at 4 or 22 degrees C for 0, 8 or 24 h prior to culture with mycobacterial antigens or pokeweed mitogen (PWM). Responses of samples held for 8 or 24 h were comparable and lower than responses of cultures prepared immediately after collection, regardless of holding temperature. Differences in responses of samples held at 4 degrees C versus 22 degrees C were also minimal. A subset of samples was held for 2 h at 37 degrees C at the beginning of the holding period. This subset of samples had diminished responses to all stimulants and increased holding times (i.e., 24 h versus 8 h) negatively impacted the response. Pre-processing conditions, particularly delays in set-up and initial high sample temperatures, reduces IFN-gamma responses of cells from infected cattle increasing the risk of false negatives in this assay of regulatory importance.

Animals↗

PubMed2006

Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries.

Bighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (INOS) inhibitor, N(G)-monomethyl-L-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.

Antigens, CD↗

PubMed2006

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii.

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

Animals↗

PubMed2006

Early antibody responses to experimental Mycobacterium bovis infection of cattle.

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.

Animals↗

PubMed2006

A highly sensitive and subspecies-specific surface antigen enzyme- linked immunosorbent assay for diagnosis of Johne's disease.

Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 microg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.

Animals↗

PubMed2006

Immune responses of elk to initial and booster vaccinations with Brucella abortus strain RB51 or 19.

Previous studies have suggested that currently available brucellosis vaccines induce poor or no protection in elk (Cervus elaphus nelsoni). In this study, we characterized the immunologic responses of elk after initial or booster vaccination with Brucella abortus strains RB51 (SRB51) and 19 (S19). Elk were vaccinated with saline or 10(10) CFU of SRB51 or S19 (n=seven animals/treatment) and booster vaccinated with a similar dosage of the autologous vaccine at 65 weeks. Compared to nonvaccinates, elk vaccinated with SRB51 or S19 had greater (P<0.05) antibody responses to SRB51 or S19 after initial vaccination and after booster vaccination. Compared to nonvaccinated elk, greater (P<0.05) proliferative responses to autologous antigen after initial vaccination occurred at only a few sample times in SRB51 (6, 14, and 22 weeks) and S19 (22 weeks) treatment groups. In general, proliferative responses of vaccinates to nonautologous antigens did not differ (P>0.05) from the responses of nonvaccinated elk. Gamma interferon production in response to autologous or nonautologous Brucella antigens did not differ (P>0.05) between controls and vaccinates after booster vaccination. Flow cytometric techniques suggested that proliferation occurred more frequently in immunoglobulin M-positive cells, with differences between vaccination and control treatments in CD4+ and CD8+ subset proliferation detected only at 22 weeks after initial vaccination. After booster vaccination, one technique ([3H]thymidine incorporation) suggested that proliferative responses to SRB51 antigen, but not S19 antigen, were greater (P<0.05) in vaccinates compared to the responses of nonvaccinates. However, in general, flow cytometric and other techniques failed to detect significant anamnestic responses to autologous or nonautologous Brucella antigens in S19 or SRB51 vaccinates after booster vaccination. Although some cellular immune responses were detected after initial or booster vaccination of elk with SRB51 or S19, our data suggest that responses tend to be transient and much less robust than previously reported in SRB51-vaccinated cattle (Bos taurus) or bison (Bison bison). These data may explain why the vaccination of elk with S19 and SRB51 induces poor protection against brucellosis.

Animals↗

PubMed2006

Diagnostic implications of antigen-induced gamma interferon production by blood leukocytes from Mycobacterium bovis-infected reindeer (Rangifer tarandus).

The only approved method of tuberculosis (TB) surveillance of reindeer within the United States is tuberculin skin testing; however, skin testing has an apparent lack of specificity, since numerous reindeer are classified as reactors, yet Mycobacterium bovis is not isolated from tissues upon necropsy. The objective of this study was to evaluate the ability of an in vitro assay (the Cervigam assay) to detect gamma interferon (IFN-gamma) produced by blood leukocytes in response to mycobacterial antigens from M. bovis-infected reindeer. Thirteen male reindeer approximately 9 months of age were inoculated with 10(5) CFU M. bovis in their tonsillar crypts. Stimulation of whole-blood cultures with a mitogen resulted in significant production of IFN-gamma compared to that by nonstimulated samples. Responses by infected reindeer to M. bovis purified protein derivative (PPD) were as much as 3.5-fold higher than those by noninfected reindeer (n = 4). Despite differences in responses to PPD by the two groups, reindeer within the noninfected group had responses of >0.1 change in optical density (DeltaOD) (a level generally considered positive) to PPD. Mean responses by infected reindeer to a rESAT-6-CFP-10 fusion protein (Mycobacterium tuberculosis complex specific) were as much as 20-fold higher than respective responses by noninfected reindeer at all time points. Additionally, responses by 3/4 noninfected reindeer were <0.1 DeltaOD (considered negative) at each time point. To further evaluate the specificity of the assay, samples were collected from reindeer in a TB-free herd. All reindeer had responses to mitogen; however, only 1 of 38 had a response to PPD, and none of the reindeer responded to rESAT-6-CFP-10. Together, these findings indicate that IFN-gamma-based tests may prove useful for TB surveillance of reindeer.

Animals↗

PubMed2006

Effects of different tuberculin skin-testing regimens on gamma interferon and antibody responses in cattle experimentally infected with Mycobacterium bovis.

Although tuberculin skin testing has been a hallmark of bovine tuberculosis eradication campaigns, it lacks sensitivity, can be confounded by exposure to nontuberculous mycobacteria, and cannot be repeated for 60 days due to desensitization. To overcome these difficulties, an effective whole-blood cellular immunoassay for bovine gamma interferon (IFN-gamma) has been developed. The IFN-gamma test is commonly used in conjunction with tuberculin skin testing as a confirmatory test following a positive response to the caudal fold test (CFT). The present study was conducted to determine the effect of different tuberculin skin-testing regimens on IFN-gamma and antibody production by using calves that were experimentally infected with Mycobacterium bovis. Holstein calves were CFT tested 60 days after inoculation and the comparative cervical test (CCT) was conducted 7 (7-day CCT) or 55 (55-day CCT) days after the CFT. In both the 7-day CCT and 55-day CCT groups, IFN-gamma responses increased 3 days after the CFT; this was immediately followed by a decrease to pre-skin test levels 7 days after the CFT. In both groups, the application of the CCT at 7 or 55 days after the CFT resulted in no significant increase in IFN-gamma production. The administration of the CFT and the CCT to M. bovis-inoculated cattle boosted antibody responses to M. bovis PPD, rMPB83, ESAT-6, and the fusion protein Acr1-MPB83. The boosting effect was more pronounced in the 55-day CCT group. Increases in either IFN-gamma or antibody production were not seen in noninoculated cattle. Measurement of both IFN-gamma and antibody responses after skin testing may be useful in identifying M. bovis-infected cattle; however, the timing of collection of such samples may influence interpretation.

Animals↗

PubMed2006

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus).

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.

Anaplasma↗

PubMed2005

New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry.

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.

Animals↗

PubMed2005

Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis.

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.

Animals↗

PubMed2005

Development of an adult-like cell-mediated immune response in calves after early vaccination with Mycobacterium bovis bacillus Calmette-Guérin.

Effects of neonatal vaccination on antigen-specific cellular and humoral immune responses of dairy calves have not been well described. The purpose of this study was to characterize the ontogeny of the adaptive immune response in calves sensitized to the attenuated strain of Mycobacterium bovis, bacillus Calmette-Guerín. Holstein bull calves were nonvaccinated (n = 6, vaccination controls) or vaccinated subcutaneously (n = 6) with bacillus Calmette-Guerín at 1 and 7 wk of age. Composition and functional capacities of blood mononuclear cell populations from calves were evaluated at 1 (prevaccination), 3, 6, 7, 8, 9, and 12 wk of age. Young adults (nulliparous heifers, n = 4) vaccinated in an identical manner were sampled concurrently to evaluate effects of animal maturity on the development of the adaptive immune response. Responses of nonvaccinated calves to recall antigen (Mycobacterium bovis purified protein derivative) ex vivo and in vivo (i.e., cutaneous delayed-type hypersensitivity) were minimal or nonexistent. Responses of cells from vaccinated calves and young adults to recall antigen, however, were evident as early as wk 2 after primary vaccination. Antigen-induced T cell subset proliferation, and secretion of interferon-gamma, nitric oxide, and tumor necrosis factor-alpha by cells from vaccinated calves were comparable to or greater than responses of vaccinated adults during the 11-wk study. Eleven weeks after primary vaccination, cutaneous responses of vaccinated calves and young adults to intradermal administration of antigen were pronounced and comparable, demonstrating the capacity of the bovine neonate to develop a vigorous cell-mediated immune response in vivo. Antibody responses (i.e., antibody concentrations in sera and in supernatants from antigen-stimulated cultures of blood mononuclear cells) of vaccinated calves, in contrast, were markedly lower than parallel responses of vaccinated adults. In conclusion, these results suggest that the bovine neonate can mount a vigorous, adult-like cell-mediated immune response when vaccinated at an early age.

Aging↗

PubMed2005

Effects of age and nutrition on expression of CD25, CD44, and L-selectin (CD62L) on T-cells from neonatal calves.

Effects of the plane of nutrition and age on the proliferation and activation of lymphocyte subsets from milk replacer-fed calves were investigated in vitro. Holstein calves were fed a standard (0.45 kg/d of a 20% crude protein, 20% fat milk replacer, n = 4) or intensified (1.14 kg/d of a 28% crude protein, 20% fat milk replacer, n = 4) diet from 1 to 8 wk of age. Average daily weight gain of intensified-diet (0.66 kg/d) calves was greater than that of standard-diet (0.27 kg/d) calves. Relative to the pokeweed mitogen-induced responses of CD4(+) cells from steers (5 to 6 mo of age), CD4(+) cells from 1-wk-old calves showed decreased proliferative activity, delayed increase in CD25 expression, and no demonstrable increase in CD44 expression or decrease in CD62L expression. Calf CD8(+) and gammadeltaT-cell receptor(+) cells, unlike T-cells from the older animals, did not demonstrate decreased expression of CD62L after stimulation with mitogen. The increased expression of CD44 by mitogen-stimulated gammadeltaT-cell receptor(+) cells from older animals was not seen in gammadeltaT-cell receptor(+) cells from 1-wk-old calves. At wk 8 of age, mitogen-induced proliferation and expression of activation antigens by T-cells from standard-fed calves were similar to responses of T-cells from steers indicating rapid maturation of T-cell function during the neonatal period. Feeding calves an intensified milk replacer was associated with decreased proliferation of mitogen-stimulated CD4(+), CD8(+), and gammadeltaT-cell receptor(+) cells; decreased CD25 expression by mitogen-stimulated CD4(+) and CD8(+) cells; and decreased CD44 expression by mitogen-stimulated CD8(+) cells. These results indicate that the functional capacity of the calf's T-cell population becomes more adult-like during the first weeks of life and suggest that nutrition modulates T-cell function during this period of immune maturation.

Aging↗

PubMed2004

Mycobacterium bovis infection of vitamin D-deficient NOS2-/- mice.

Vitamin D deficiency is associated with an increased risk for tuberculosis infection. Studies using in vitro systems indicate that 1,25-dihydroxyvitamin D(3) [i.e. 1,25(OH)(2)D(3)], the most active form of the vitamin, enhances mycobacterial killing by increasing nitric oxide (NO) production. To evaluate concurrently the role of 1,25(OH)(2)D(3) and NO on the host response to tuberculosis infection, mice deficient in NO synthase 2 (NOS2(-/-)) and/or vitamin D were aerosol-challenged with Mycobacterium bovis and subsequently evaluated for mycobacterial colonization and lesion formation. Infected NOS2(-/-) mice developed severe necrotizing pyogranulomatous inflammation of the lungs with heavy M. bovis colonization and systemic dissemination of the bacillus. Colonization and lung lesion area of NOS2(-/-) mice exceeded that of NOS2(+/+) mice. Additionally, disease progression was more rapid in NOS2(-/-) mice than in NOS2(+/+) mice. Lung colonization and lesion area of vitamin D deficient mice exceeded that of vitamin D replete mice, regardless of NOS2 phenotype. However, effects of vitamin D on colonization, but not lesion area, were more pronounced in NOS2(+/+) mice than in NOS2(-/-) mice. These findings are consistent with the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing through a NO-dependent mechanism. As responses of NOS2(-/-) mice were affected by 1,25(OH)(2)D(3) deficiency, albeit to a lesser extent than were those of NOS2(+/+) mice, NO-independent actions of 1,25(OH)(2)D(3) also likely exist.

Animals↗

PubMed2004

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Amino Acid Sequence↗

PubMed2004

Antigen recognition by serum antibodies in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis.

White-tailed deer (Odocoileus virginianus) have emerged as reservoirs of bovine tuberculosis in northern America. For tuberculosis surveillance of deer, antibody-based assays are particularly attractive because deer are handled only once and immediate processing of the sample is not required. Sera collected sequentially from 25 Mycobacterium bovis-infected and 7 noninfected deer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for immunoglobulin specific to M. bovis antigens. Various routes of experimental M. bovis infection, such as intratonsillar inoculation (n = 11), aerosol (n = 6), and exposure to infected deer (in contact, n = 8), were studied. Upon infection, specific bands of reactivity at approximately 24 to 26 kDa, approximately 33 kDa, approximately 42 kDa, and approximately 75 kDa to M. bovis whole-cell sonicate were detected by immunoblot. Lipoarabinomannan-specific immunoglobulin was detected as early as 36 days postchallenge, and responses were detected for 94% of intratonsillarly and "in-contact"-infected deer. In MAPIA, sera were tested with 12 native and recombinant antigens coated on nitrocellulose. All in-contact-infected (8 of 8) and 10 of 11 intratonsillarly infected deer produced antibody reactive with one or more of the recombinant/native antigens. Responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Additionally, three of six deer receiving a very low dose of M. bovis via aerosol exposure produced antibody specific to one or more recombinant proteins. M. bovis was isolated from one of three nonresponding aerosol-challenged deer. Of the 12 antigens tested, the most immunodominant protein was MPB83; however, a highly sensitive serodiagnostic test will likely require use of multiple antigens.

Animals↗

PubMed2004

Immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination.

The objective was to evaluate cellular immune response of captive white-tailed deer (Odocoileus virginianus) to live Mycobacterium bovis bacille Calmette Guerin (BCG) vaccination and to determine diagnostic implications of these responses. In vitro proliferative and interferon-gamma (IFN-gamma) responses to M. bovis purified protein derivative (PPD) were detected beginning 9 days postvaccination. Responses to Mycobacterium avium PPD, however, generally exceeded responses to M. bovis PPD. Interferon-gamma responses to M. avium PPD were not detected prior to vaccination nor in nonvaccinated deer, suggesting that vaccination with BCG boosted prior quiescent M. avium-sensitized cells. Both CD4+ and gammadelta T cells from vaccinated deer proliferated in response to M. bovis PPD stimulation. Intradermal administration of M. bovis PPD resulted in increases in skin thickness of vaccinated deer beginning 24 hr postinjection. Such early reactions were characterized by edema and minimal mononuclear cell infiltration, whereas later reactions (i.e., 72 hr postinjection) were more typical of delayed type hypersensitivity. Upon in vitro activation with pokeweed mitogen, CD44 expression increased and CD62L expression decreased on lymphocytes from deer regardless of vaccination status. Likewise, M. bovis PPD stimulation of lymphocytes from vaccinated deer resulted in increases in CD44 expression and decreases in CD62L expression. These findings demonstrate the potential of BCG vaccination to elicit strong cell-mediated immune responses and appropriate alterations in CD44 and CD62L expression with in vitro stimulation of white-tailed deer lymphocytes. In relation to M. bovis diagnosis, vaccination of white-tailed deer with BCG can induce skin test responses that classify the animal as a tuberculosis reactor. In contrast, BCG vaccination will likely not interfere with tuberculosis testing by the IFN-gamma assay.

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