scholarly journals Cattle Husbandry in Ethiopia Is a Predominant Factor Affecting the Pathology of Bovine Tuberculosis and Gamma Interferon Responses to Mycobacterial Antigens

2006 ◽  
Vol 13 (9) ◽  
pp. 1030-1036 ◽  
Author(s):  
Gobena Ameni ◽  
Abraham Aseffa ◽  
Howard Engers ◽  
Douglas Young ◽  
Glyn Hewinson ◽  
...  
Keyword(s):  
Bovine Tuberculosis ◽  
Control Program ◽  
Economic Problem ◽  
Gamma Interferon ◽  
Dominant Factor ◽  
Zebu Cattle ◽  
Holstein Cattle ◽  
Ifn Γ ◽  
Mycobacterial Antigens ◽  
Cattle Husbandry

ABSTRACT Bovine tuberculosis is a major economic problem and a potential public health risk. Improved diagnostics like the gamma interferon (IFN-γ) test with ESAT6 and/or CFP10 could contribute to the control program. We assessed IFN-γ responses in zebu (Ethiopian Arsi breed) and Holstein cattle kept indoors or in a pasture to tuberculin purified protein derivative (PPD) and an ESAT6-CFP10 protein cocktail. Furthermore, the intensity and distribution of pathology of bovine tuberculosis were compared between the two breeds. Our data demonstrated significantly (all P < 0.02) higher IFN-γ responses to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail in Holstein than in zebu cattle, while lesion severities in infected animals and tuberculin skin test responses did not differ significantly (P > 0.05) between the two breeds. Holstein cattle that were kept indoors produced significantly (all P < 0.01) higher IFN-γ levels in response to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail than did Holstein cattle kept in a pasture. Moreover, lesion severity was significantly higher in Holstein cattle kept indoors (P = 0.001) than in those kept in the pasture. Lesions were localized predominantly in the digestive tract in cattle kept in a pasture, while they were localized in the respiratory tract in cattle kept indoors. In conclusion, in Holstein cattle, husbandry was a dominant factor influencing the severity of tuberculosis lesions and IFN-γ responses to mycobacterial antigens compared to breed. A difference in the cellular immune response between zebu and Holstein cattle was observed, while tuberculosis lesion severities were identical in the two breeds, when both were kept in a pasture.

scholarly journals Recognition of Mycobacterial Antigens Delivered by Genetically Detoxified Bordetella pertussis Adenylate Cyclase by T Cells from Cattle with Bovine Tuberculosis

2004 ◽  
Vol 72 (11) ◽  
pp. 6255-6261 ◽  
Author(s):  
H. Martin Vordermeier ◽  
Marcela Simsova ◽  
Katalin A. Wilkinson ◽  
Robert J. Wilkinson ◽  
R. Glyn Hewinson ◽  
...  
Keyword(s):  
T Cells ◽  
Adenylate Cyclase ◽  
Fusion Protein ◽  
Bordetella Pertussis ◽  
Bovine Tuberculosis ◽  
Economic Problem ◽  
T Cell Recognition ◽  
Ifn Γ ◽  
Mycobacterial Antigens ◽  
Immunodiagnostic Tests

ABSTRACT The exponential increase in the incidence of tuberculosis in cattle over the last two decades in the British national herd constitutes a significant economic problem. Therefore, research efforts are under way to develop cattle tuberculosis vaccines and specific diagnostic reagents to allow the distinction of vaccinated from infected animals. Mycobacterial antigens like ESAT-6 and CFP10 allow this distinction. This study investigates whether fusion protein of ESAT-6 or CFP10 with genetically detoxified Bordetella pertussis adenylate cyclase (CyaA) are recognized by Mycobacterium bovis-infected cattle more effectively than conventional recombinant proteins are, thus enhancing sensitivity or reducing the amount of antigens required. By measuring the frequencies of gamma interferon (IFN-γ)-producing cells, we were able to show that the presentation of CFP10 as a CyaA fusion protein enhanced the molecular efficiency of its recognition 20-fold, while the recognition of ESAT-6 was not improved by CyaA delivery. Furthermore, in the whole-blood IFN-γ test currently used in the field, the delivery of CFP10 and ESAT-6 by fusion to CyaA increased the amount of IFN-γ produced and hence the proportion of infected animals responding to CFP10. The improved T-cell recognition of CyaA336/CFP10 was found to be dependent upon interaction with CD11b. In addition, presentation of CyaA336/CFP10 to CD4+ T cells was chloroquine sensitive, and CFP10 delivery by CyaA resulted in its accelerated presentation to T cells. In conclusion, the use of CyaA fusion proteins with ESAT-6 and CFP10 has the potential to improve the sensitivity of immunodiagnostic tests detecting bovine tuberculosis in cattle.

scholarly journals Assessment of the specificity of a gamma-interferon test performed with specific antigens to detect bovine tuberculosis, after non-negative results to intradermal tuberculin testing

2019 ◽  
Vol 6 (1) ◽  
pp. e000335 ◽  
Author(s):  
Anne Praud ◽  
Clémence Bourély ◽  
Maria-Laura Boschiroli ◽  
Barbara Dufour
Keyword(s):  
Bovine Tuberculosis ◽  
Gamma Interferon ◽  
Skin Tests ◽  
Negative Results ◽  
In Series ◽  
Specific Antigens ◽  
Ifn Γ ◽  
Tuberculin Testing ◽  
Cattle Herds ◽  
Higher Sensitivity

In cattle herds in France, cervical skin tests (STs) using simple intradermal tuberculin (SIT) are performed to detect bovine tuberculosis (bTB). When positive results are found on ST screening, the herd is considered to be ‘under suspicion’ and confined, raising economic issues. The suspicion can be lifted by carrying out a single intradermal cervical comparative test (SICCT) at least six weeks later.The authors conducted an experimental study in France between 2013 and 2015 to assess the accuracy of the gamma-interferon test (IFN-γ), used in series after a non-negative result to ST screening, and to study the possibility of replacing the SICCT performed six weeks later by an IFN performed within a few days. Data were collected concerning 40 infected and 1825 bTB-free animals from herds with non-negative results to ST screening. This study showed that the IFN-γ test based on specific antigens and performed within a few days of a non-negative result to the ST has higher sensitivity than the SICCT performed six weeks later and equal specificity. The IFN test is more convenient to perform; however, it is more expensive. The IFN-γ test based on MIX antigens may be a useful alternative to the SICCT, to shorten the confinement period of suspect herds without underdetecting bTB.

scholarly journals Distinct Response Kinetics of Gamma Interferon and Interleukin-4 in Bovine Tuberculosis

2000 ◽  
Vol 68 (9) ◽  
pp. 5393-5400 ◽  
Author(s):  
S. G. Rhodes ◽  
N. Palmer ◽  
S. P. Graham ◽  
A. E. Bianco ◽  
R. G. Hewinson ◽  
...  
Keyword(s):  
Bovine Tuberculosis ◽  
Gamma Interferon ◽  
Interleukin 4 ◽  
Early Increase ◽  
Onchocerca Ochengi ◽  
Ifn Γ ◽  
Bovine Tuberculin ◽  
Kinetics Of ◽  
Experimental Challenge

ABSTRACT This study shows that gamma interferon (IFN-γ) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected with Mycobacterium bovis. The different kinetics of the IFN-γ and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN-γ was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose of M. bovis which did not result in the development of lesions, positive DTH skin test, or substantial IFN-γ responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors showed increased IFN-γ and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of theM. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematode Onchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.

scholarly journals Investigation of the Role of CD8+ T Cells in Bovine Tuberculosis In Vivo

2003 ◽  
Vol 71 (8) ◽  
pp. 4297-4303 ◽  
Author(s):  
B. Villarreal-Ramos ◽  
M. McAulay ◽  
V. Chance ◽  
M. Martin ◽  
J. Morgan ◽  
...  
Keyword(s):  
Lymph Node ◽  
Bovine Tuberculosis ◽  
The Other ◽  
Cd8 Cells ◽  
Cd8 Cell ◽  
Ifn Γ ◽  
Mycobacterial Antigens

ABSTRACT Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8+ T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulent M. bovis strain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-γ) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-γ and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response to M. bovis in cattle by contributing to the IFN-γ response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.

scholarly journals Evaluation of Gamma Interferon (IFN-γ)-Induced Protein 10 Responses for Detection of Cattle Infected with Mycobacterium bovis: Comparisons to IFN-γ Responses

2012 ◽  
Vol 19 (3) ◽  
pp. 346-351 ◽  
Author(s):  
W. R. Waters ◽  
T. C. Thacker ◽  
B. J. Nonnecke ◽  
M. V. Palmer ◽  
I. Schiller ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Similar Pattern ◽  
Gamma Interferon ◽  
Content Type ◽  
Purified Protein Derivative ◽  
Testing Protocol ◽  
Archived Samples ◽  
Ifn Γ ◽  
Highly Correlated ◽  
Mycobacterial Antigens

ABSTRACTGamma interferon (IFN-γ)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker ofMycobacterium tuberculosisinfection of humans. The aim of the current study was to compare IP-10 and IFN-γ responses uponMycobacterium bovisinfection in cattle by using archived samples from two aerosol inoculation studies. In the first study (104CFUM. bovisby aerosol,n= 7),M. bovispurified protein derivative (PPDb)-specific IP-10 and IFN-γ gene expression was detected as early as 29 days after challenge. PPDb-specific IP-10 and IFN-γ mRNA responses followed a similar pattern of expression over the course of this study and were highly correlated (r= 0.87). In the second study (105CFUM. bovisby aerosol,n= 5), IP-10 and IFN-γ (protein) responses to mycobacterial antigens were compared following challenge. IFN-γ responses to mycobacterial antigens were detected at 29 days after challenge and were sustained during the remainder of the study. IFN-γ responses to mycobacterial antigens exceeded corresponding responses in nonstimulated cultures. IP-10 responses to mycobacterial antigens exceeded preinfection responses at 7, 29, and 63 days after challenge. In contrast to IFN-γ responses, IP-10 responses to mycobacterial antigens generally did not exceed the respective responses in nonstimulated cultures. IP-10 responses to medium alone and to mycobacterial antigens followed a similar pattern of response. Correlations between IP-10 and IFN-γ (protein) responses were modest (r≈ 0.50 to 0.65). Taken together, these findings do not support the use of IP-10 protein as a biomarker for bovine tuberculosis using the current testing protocol and reagents; however, mRNA-based assays may be considered for further analysis.

scholarly journals Clinical and Diagnostic Developments of a Gamma Interferon Release Assay for Use in Bovine Tuberculosis Control Programs

2013 ◽  
Vol 20 (12) ◽  
pp. 1827-1835 ◽  
Author(s):  
K. E. Bass ◽  
B. J. Nonnecke ◽  
M. V. Palmer ◽  
T. C. Thacker ◽  
R. Hardegger ◽  
...  
Keyword(s):  
Bovine Tuberculosis ◽  
Second Generation ◽  
Gamma Interferon ◽  
Tuberculosis Control ◽  
Content Type ◽  
Control Programs ◽  
Release Assay ◽  
Ifn Γ ◽  
First And Second Generation ◽  
And Control

ABSTRACTCurrently, the Bovigam assay is used as an official supplemental test within bovine tuberculosis control programs. The objectives of the present study were to evaluate twoMycobacterium bovis-specific peptide cocktails and purified protein derivatives (PPDs) from two sources, liquid and lyophilized antigen preparations. PPDs and peptide cocktails were also used for comparison of a second-generation gamma interferon (IFN-γ) release assay kit with the currently licensed first-generation kit (Bovigam; Prionics AG). Three strains ofM. boviswere used for experimental challenge:M. bovis95-1315,M. bovisRavenel, andM. bovis10-7428. Additionally, samples from a tuberculosis-affected herd (i.e., naturally infected) were evaluated. Robust responses to both peptide cocktails, HP (PC-HP) and ESAT-6/CFP10 (PC-EC), and the PPDs were elicited as early as 3 weeks after challenge. Only minor differences in responses to Commonwealth Serum Laboratories (CSL) and Lelystad PPDs were detected with samples from experimentally infected animals. For instance, responses to LelystadM. avium-derived PPD (PPDa) exceeded the respective responses to the CSL PPDa inM. bovisRavenel-infected and control animals. However, a 1:4 dilution of stimulated plasma demonstrated greater separation of PPDb from PPDa responses (i.e., PPDb minus PPDa) with the use of Lelystad PPDs, suggesting that Lelystad PPDs provide greater diagnostic sensitivity than CSL PPDs. The responses to lyophilized and liquid antigen preparations did not differ. Responses detected with first- and second-generation IFN-γ release assay kits (Bovigam) did not differ throughout the study. In conclusion, antigens may be stored in a lyophilized state without loss in potency, PC-HP and PC-EC are dependable biomarkers for aiding in the detection of bovine tuberculosis, and second-generation Bovigam kits are comparable to currently used kits.

scholarly journals Gamma Interferon Responses Induced by a Panel of Recombinant and Purified Mycobacterial Antigens in Healthy, Non-Mycobacterium bovis BCG-Vaccinated Malawian Young Adults

2003 ◽  
Vol 10 (4) ◽  
pp. 602-611 ◽  
Author(s):  
Gillian F. Black ◽  
Rosemary E. Weir ◽  
Steven D. Chaguluka ◽  
David Warndorff ◽  
Amelia C. Crampin ◽  
...  
Keyword(s):  
Young Adults ◽  
Extrapulmonary Tuberculosis ◽  
Gamma Interferon ◽  
African Population ◽  
Mantoux Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Population Exposure ◽  
Environmental Mycobacteria ◽  
Ifn Γ ◽  
Mycobacterial Antigens

ABSTRACT We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-γ) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.

scholarly journals Identification of Vaccine Candidate Peptides in the NcSRS2 Surface Protein of Neospora caninum by Using CD4+ Cytotoxic T Lymphocytes and Gamma Interferon-Secreting T Lymphocytes of Infected Holstein Cattle

2005 ◽  
Vol 73 (3) ◽  
pp. 1321-1329 ◽  
Author(s):  
Lauren M. Staska ◽  
Christopher J. Davies ◽  
Wendy C. Brown ◽  
Travis C. McGuire ◽  
Carlos E. Suarez ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
T Lymphocyte ◽  
Peripheral Blood ◽  
Vaccine Development ◽  
Neospora Caninum ◽  
Gamma Interferon ◽  
Target Cells ◽  
Holstein Cattle ◽  
Ifn Γ

ABSTRACT Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-γ) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-γ secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-γ enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-γ ELISPOT and in vitro by measuring T-lymphocyte IFN-γ production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-γ-secreting T lymphocytes in cattle with varied MHC genotypes.

scholarly journals Mycobacterium bovis BCG Producing Interleukin-18 Increases Antigen-Specific Gamma Interferon Production in Mice

2002 ◽  
Vol 70 (12) ◽  
pp. 6549-6557 ◽  
Author(s):  
Franck Biet ◽  
Laurent Kremer ◽  
Isabelle Wolowczuk ◽  
Myriam Delacre ◽  
Camille Locht
Keyword(s):  
Critical Role ◽  
Gamma Interferon ◽  
Cell Mediated Immunity ◽  
Mycobacterial Antigen ◽  
Interleukin 18 ◽  
Ifn Γ ◽  
Macrophage Colony ◽  
Humoral Responses ◽  
Mycobacterial Antigens ◽  
Recombinant Bcg

ABSTRACT Interleukin-18 (IL-18) and IL-12 play a critical role in the expression of cell-mediated immunity involved in host defense against intracellular pathogens. Both cytokines are produced by macrophages and act in synergy to induce gamma interferon (IFN-γ) production by T, B, and natural killer cells. In the present study, we analyzed both cellular and humoral responses upon infection with IL-18-secreting BCG of BALB/c and C3H/HeJ mice, two strains known to differ in their ability to support the growth of BCG. The cDNA encoding mature IL-18 was fused in frame with the alpha-antigen signal peptide-coding sequence, cloned downstream of the mycobacterial hsp60 promoter and expressed in BCG. IL-18 produced by the recombinant BCG strain was functional, as judged by NF-κB-mediated luciferase induction in a tissue culture assay. When susceptible mice were infected with IL-18-producing BCG, their splenocytes were found to produce higher amounts of Th1 cytokines after stimulation with mycobacterial antigens than the splenocytes of mice infected with the nonrecombinant BCG. This was most prominent for IFN-γ, although the mycobacterial antigen-specific secretion of granulocyte-macrophage colony-stimulating factor and IL-10 was also augmented after infection with the recombinant BCG compared to infection with nonrecombinant BCG. In contrast, the immunoglobulin G levels in serum against mycobacterial antigens were lower when the mice were infected with IL-18-producing BCG compared to infection with nonrecombinant BCG. The IL-18 effect was delayed in BALB/c compared to C3H/HeJ mice. These results indicate that the production of IL-18 by recombinant BCG may enhance the immunomodulatory properties of BCG further toward a Th1 profile. This may be particularly useful for immunotherapeutic or prophylactic interventions in which a Th1 response is most desirable.

scholarly journals Interpretation of the Gamma Interferon Test for Diagnosis of Subclinical Paratuberculosis in Cattle

2002 ◽  
Vol 9 (2) ◽  
pp. 453-460 ◽  
Author(s):  
G. Jungersen ◽  
A. Huda ◽  
J. J. Hansen ◽  
P. Lind
Keyword(s):  
Bovine Tuberculosis ◽  
Preventive Measures ◽  
Clinical Signs ◽  
Cross Reactivity ◽  
Gamma Interferon ◽  
Test Results ◽  
Young Stock ◽  
Purified Protein Derivative ◽  
Interpretation Criteria ◽  
Ifn Γ

ABSTRACT A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-γ) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-γ responses were significantly reduced (P < 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-γ test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.

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