This article was downloaded by: [Erciyes University] On: 25 December 2014, At: 02:20 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
Immunity in foot-rot infection a
b
K.M. Moriarty M.Sc. Ph.D. , B.S. Cooper B.V.Sc Ph.D. & B.R. Ingram N.Z.C.S.
a
a
Department of Veterinary Pathology and Public Health , Massey University , Palmerston North b
Glaxo Laboratories , Palmerston North Published online: 02 Dec 2011.
To cite this article: K.M. Moriarty M.Sc. Ph.D. , B.S. Cooper B.V.Sc Ph.D. & B.R. Ingram N.Z.C.S. (1976) Immunity in foot-rot infection, New Zealand Veterinary Journal, 24:1-2, 9-10, DOI: 10.1080/00480169.1967.34267 To link to this article: http://dx.doi.org/10.1080/00480169.1967.34267
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
1976
NEW ZEALAND VETERINARY JOURNAL
9
IMMUNITY IN FOOT-ROT INFECTION K. M. MORIARTY, B. S. COOPER and B. R. INGRAM*
Downloaded by [Erciyes University] at 02:20 25 December 2014
INTRODUCTION
LIMITED preliminary observations (Coop" er, 1972) suggested that cell-mediated im~ munity (CMi) might be involved in the response of sheep to Fusiformis nodosus infection. To explore this possibility, lymphocyte:s from experimentally infected animals showing seveTe fOOlt-rOlt; lesions were tested fo;r their ability to undergo transformation in vitro in response to F. nodosus antigens. MATERIALS ANP METHODS
EXPERIMENTAL INFECTIONS The method of Egerton et al. (1969) was followe:d. Sheep with no history of previous foot-rot infections were housed for 7 days on wet straw. Following this maceration process, F. nodosus organisms grown on solid medium were applied to the interdigital skin and held in position by wet cotton wool and foot bandages for 5 days. LYMPHOCYTE CULTURES Six weeks after infection, the animals were bled and lymphocytes were harvested by filtration of buffy-coat preparations through cotton wool columns. Following washing in 0.01 M phosphate buffered saline (PBS), pH 7.2. the cells were cultured at a concentration of 1 X lCJ6/ml in 2 ml cultures in an atmosphere of 5 % CO 2 in air. The culture medium: was Eagle's minimal essential ntedium supplemented with heat-inactivated foe,tal calf sennn and antibiotics. The antigenic material used to attempt lymphocyte stimulation was prepared from F. nodosus organisms of the sam~ stock as was used for infection of the *K. M. Moriarty, M.Sc., P,h.D., Department of Veterinary Pathology and Public Health, Massey University, Palmerston North. B. S. Cooper, B.V.Sc., Ph.D .. Glaxo Labo'ratories, Palmerston North. B. R. Ingram, N.Z.C.S., Department of Veterinary Path· ology and Public Health, Massey University, Palmersion North.
sheep. The cells were disintegrated in a French press and the- resulting material dialyserd against PBS beforre being Millipor~filtered (0.45 m[J.). Pilot studies showed that concentrations of this material greater than 1 :25 inhibite,d lymphocyte activity. Consequently, two further dilutions of the antigen were used: Fnod1, a 1 :25 dilution of the starting material, and Fnod2, a 1 : 125 dilution. The following cultures were. established in triplicate: Lymphocytes alone - i.e., unstimulated cells. Lymphocytes stimulated non-specifically with the mitogen phytohaemagglutinint (PHA, 50 [J.g/ml). Lymphocytes exposed to Fnodl. Lymphocytes e;xposed to' Fnod2. Lymphocytes stimulated with both PHA and Fnodl. Lymphocytes stimulated with both PHA and Fnod2. The last two cultures were established with cells from sheep No.1 only. The cultures were maintained at 37" C for 3 days before being pulsed with tritiated thymidine:!: (2 (J..C/ml). The cells were harvested and their uptake of the isotope measured as described previously (Moriarty, 1973). RESULTS
The activities, in counts/min, were averaged for each group of triplicate cultures. The results (Table 1) are presented as the ratios of the activities of eitheir the PHA, the antigen, or the PHA and antigen stimulated cultures to the activities of the unstimulated cultures. The addition of PHA to the cultures stimulated cell activity, indicating that culture conditions were adequate for' continued cell viability. In contrast to this tWellcome Research Laborato1J'. Beckenham, Kent, England. :j:Radiochemical Centre, Amersham, England.
10
l'mw
TABLE 1: RATIOS OF THE ACTIVITIES, OF STIMULATED LYMPHOCYTE CULTURES TO THOSE OF UNSTIMULATED, CULTURES
Cultures Unstimulated +PHA + Fnodl + Fnod2 + PHA + Fnod1 + PHA + Fnod2
Downloaded by [Erciyes University] at 02:20 25 December 2014
PHA Fnodl Fnod2 NT
1
2
1.0
1.0
8.5
1.1 1.1
7.8 8.1
13.9 1.0 1.1
NT NT
Sheep No.
3
1.0
S.1
1.1
0.9 NT NT
VOL. 24
ZEALAND VETERINARY JOURNAL
4
5
1.0
1.0
Mean
1.0
IS. 1 10.4 0.6 1.1 1.0 0.9 1.1 1.0 NT NT NT NT 9.3
phytohaemagglutinin, SOp,g/ml F. nodosus extract, 1:25 dilution F. nodosus extract, 1: 125 dilution Not tested
non-specific stimulation by PHA, neither concentration of the F. nodosus antigen, when used alone, increased lymphocyte activity by any appreciable extent. That this lack of, stimulation was not due to a toxic effect of Fnodl or Fnod2 was shown by the observation that neither of these antigen concentrations suppressed cell activity when used in conjunction with PHA. DISCUSSION
These findings suggest that CMI, as judged by lymphocyte transformation, does not occur in sheep infected with F. nodosus. Alternative explanations can be made for these findings. First, neither Fnodl nor Fnod2 may have contained sufficient amountso,f any determinants needed to further stimulate lymphocytes from infected animals. Fractionation of these antigens to separate stimulatory from inhibit Dry components and their subsequent assay would be a formidable task. Secondly, It is possible that the 6week interval between infection and the collection of lymphocytes was insufficient to allow complete expression of CMI capacity. Thirdly, while lymphocyte transfDrmation induced by non-specific
mitogens provides a convenient index of total eMI capacity (Bloom, 1971 ) the technique, in SDme situations, may not be sufficiently sensitive to de.tect an increase in the activity of a limited number of cells in a lymphocyte population. The relative roles of antibody mediated. immunity and eMI in resistance to footrot infections has not be.en evaluated. While large amounts of passively administered antibody protect sheep against infection with F. nodosus (Egerton and Merritt, 1973), natural infections, which elicit only low levels of antibody, dO' not confer immunity. Protection can, however, be achieved by vaccinatiDn. Whether vaccination achieves its result by inducing sufficiently high le.vels of antibody or by pro!01oting some degree of eMI is not known, SUMMARY
LymphDcytes from sheep infe:cted with foot-rot did not undergo transformation when cultured in the presence of Fusiformis nodosus antigens. The rok Q1f cellmediated immunity in relation to footrot Df she.ep remains uncertain. REFERENCES Bloom, B. R. (1971): In Advances in Immunology (F. J. Dixon and r. H. Humphrey, Eds.), 13: 101-208. Academic Press, New York. Cooper, B. S. (1972): Ph.D. thesis, Department of Veterinary Pathology and Public Health, Massey University. Egerton, J. R.; Merritt, G. C. (1973): Serology of foot-rot: antibodies against Fusiformis nodosus in normal, affected, vaccinated and pass.ively iinmunised sheep. Aust. vet. f" 49: 139-4S. Egerton, r. R.; Roberts, D. S.; Parsonson, I. M. (1969): The aetiology and pathogenesis of O'yine foot-rot. I. A 'histological study ,of the bacterial invasion. ,. comp, Path., 79: 207-16. Moriarty, K. M, (1973): A possible deficiency' of cd I-mediated immunity in the opossum. Trichosurus vulpecula, in relation to tuberculosis. N.Z. vet. f., 21: 167-9.
(Received far publication March 24, 1975)
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
Immunity in foot-rot infection a
b
K.M. Moriarty M.Sc. Ph.D. , B.S. Cooper B.V.Sc Ph.D. & B.R. Ingram N.Z.C.S.
a
a
Department of Veterinary Pathology and Public Health , Massey University , Palmerston North b
Glaxo Laboratories , Palmerston North Published online: 02 Dec 2011.
To cite this article: K.M. Moriarty M.Sc. Ph.D. , B.S. Cooper B.V.Sc Ph.D. & B.R. Ingram N.Z.C.S. (1976) Immunity in foot-rot infection, New Zealand Veterinary Journal, 24:1-2, 9-10, DOI: 10.1080/00480169.1967.34267 To link to this article: http://dx.doi.org/10.1080/00480169.1967.34267
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
1976
NEW ZEALAND VETERINARY JOURNAL
9
IMMUNITY IN FOOT-ROT INFECTION K. M. MORIARTY, B. S. COOPER and B. R. INGRAM*
Downloaded by [Erciyes University] at 02:20 25 December 2014
INTRODUCTION
LIMITED preliminary observations (Coop" er, 1972) suggested that cell-mediated im~ munity (CMi) might be involved in the response of sheep to Fusiformis nodosus infection. To explore this possibility, lymphocyte:s from experimentally infected animals showing seveTe fOOlt-rOlt; lesions were tested fo;r their ability to undergo transformation in vitro in response to F. nodosus antigens. MATERIALS ANP METHODS
EXPERIMENTAL INFECTIONS The method of Egerton et al. (1969) was followe:d. Sheep with no history of previous foot-rot infections were housed for 7 days on wet straw. Following this maceration process, F. nodosus organisms grown on solid medium were applied to the interdigital skin and held in position by wet cotton wool and foot bandages for 5 days. LYMPHOCYTE CULTURES Six weeks after infection, the animals were bled and lymphocytes were harvested by filtration of buffy-coat preparations through cotton wool columns. Following washing in 0.01 M phosphate buffered saline (PBS), pH 7.2. the cells were cultured at a concentration of 1 X lCJ6/ml in 2 ml cultures in an atmosphere of 5 % CO 2 in air. The culture medium: was Eagle's minimal essential ntedium supplemented with heat-inactivated foe,tal calf sennn and antibiotics. The antigenic material used to attempt lymphocyte stimulation was prepared from F. nodosus organisms of the sam~ stock as was used for infection of the *K. M. Moriarty, M.Sc., P,h.D., Department of Veterinary Pathology and Public Health, Massey University, Palmerston North. B. S. Cooper, B.V.Sc., Ph.D .. Glaxo Labo'ratories, Palmerston North. B. R. Ingram, N.Z.C.S., Department of Veterinary Path· ology and Public Health, Massey University, Palmersion North.
sheep. The cells were disintegrated in a French press and the- resulting material dialyserd against PBS beforre being Millipor~filtered (0.45 m[J.). Pilot studies showed that concentrations of this material greater than 1 :25 inhibite,d lymphocyte activity. Consequently, two further dilutions of the antigen were used: Fnod1, a 1 :25 dilution of the starting material, and Fnod2, a 1 : 125 dilution. The following cultures were. established in triplicate: Lymphocytes alone - i.e., unstimulated cells. Lymphocytes stimulated non-specifically with the mitogen phytohaemagglutinint (PHA, 50 [J.g/ml). Lymphocytes exposed to Fnodl. Lymphocytes e;xposed to' Fnod2. Lymphocytes stimulated with both PHA and Fnodl. Lymphocytes stimulated with both PHA and Fnod2. The last two cultures were established with cells from sheep No.1 only. The cultures were maintained at 37" C for 3 days before being pulsed with tritiated thymidine:!: (2 (J..C/ml). The cells were harvested and their uptake of the isotope measured as described previously (Moriarty, 1973). RESULTS
The activities, in counts/min, were averaged for each group of triplicate cultures. The results (Table 1) are presented as the ratios of the activities of eitheir the PHA, the antigen, or the PHA and antigen stimulated cultures to the activities of the unstimulated cultures. The addition of PHA to the cultures stimulated cell activity, indicating that culture conditions were adequate for' continued cell viability. In contrast to this tWellcome Research Laborato1J'. Beckenham, Kent, England. :j:Radiochemical Centre, Amersham, England.
10
l'mw
TABLE 1: RATIOS OF THE ACTIVITIES, OF STIMULATED LYMPHOCYTE CULTURES TO THOSE OF UNSTIMULATED, CULTURES
Cultures Unstimulated +PHA + Fnodl + Fnod2 + PHA + Fnod1 + PHA + Fnod2
Downloaded by [Erciyes University] at 02:20 25 December 2014
PHA Fnodl Fnod2 NT
1
2
1.0
1.0
8.5
1.1 1.1
7.8 8.1
13.9 1.0 1.1
NT NT
Sheep No.
3
1.0
S.1
1.1
0.9 NT NT
VOL. 24
ZEALAND VETERINARY JOURNAL
4
5
1.0
1.0
Mean
1.0
IS. 1 10.4 0.6 1.1 1.0 0.9 1.1 1.0 NT NT NT NT 9.3
phytohaemagglutinin, SOp,g/ml F. nodosus extract, 1:25 dilution F. nodosus extract, 1: 125 dilution Not tested
non-specific stimulation by PHA, neither concentration of the F. nodosus antigen, when used alone, increased lymphocyte activity by any appreciable extent. That this lack of, stimulation was not due to a toxic effect of Fnodl or Fnod2 was shown by the observation that neither of these antigen concentrations suppressed cell activity when used in conjunction with PHA. DISCUSSION
These findings suggest that CMI, as judged by lymphocyte transformation, does not occur in sheep infected with F. nodosus. Alternative explanations can be made for these findings. First, neither Fnodl nor Fnod2 may have contained sufficient amountso,f any determinants needed to further stimulate lymphocytes from infected animals. Fractionation of these antigens to separate stimulatory from inhibit Dry components and their subsequent assay would be a formidable task. Secondly, It is possible that the 6week interval between infection and the collection of lymphocytes was insufficient to allow complete expression of CMI capacity. Thirdly, while lymphocyte transfDrmation induced by non-specific
mitogens provides a convenient index of total eMI capacity (Bloom, 1971 ) the technique, in SDme situations, may not be sufficiently sensitive to de.tect an increase in the activity of a limited number of cells in a lymphocyte population. The relative roles of antibody mediated. immunity and eMI in resistance to footrot infections has not be.en evaluated. While large amounts of passively administered antibody protect sheep against infection with F. nodosus (Egerton and Merritt, 1973), natural infections, which elicit only low levels of antibody, dO' not confer immunity. Protection can, however, be achieved by vaccinatiDn. Whether vaccination achieves its result by inducing sufficiently high le.vels of antibody or by pro!01oting some degree of eMI is not known, SUMMARY
LymphDcytes from sheep infe:cted with foot-rot did not undergo transformation when cultured in the presence of Fusiformis nodosus antigens. The rok Q1f cellmediated immunity in relation to footrot Df she.ep remains uncertain. REFERENCES Bloom, B. R. (1971): In Advances in Immunology (F. J. Dixon and r. H. Humphrey, Eds.), 13: 101-208. Academic Press, New York. Cooper, B. S. (1972): Ph.D. thesis, Department of Veterinary Pathology and Public Health, Massey University. Egerton, J. R.; Merritt, G. C. (1973): Serology of foot-rot: antibodies against Fusiformis nodosus in normal, affected, vaccinated and pass.ively iinmunised sheep. Aust. vet. f" 49: 139-4S. Egerton, r. R.; Roberts, D. S.; Parsonson, I. M. (1969): The aetiology and pathogenesis of O'yine foot-rot. I. A 'histological study ,of the bacterial invasion. ,. comp, Path., 79: 207-16. Moriarty, K. M, (1973): A possible deficiency' of cd I-mediated immunity in the opossum. Trichosurus vulpecula, in relation to tuberculosis. N.Z. vet. f., 21: 167-9.
(Received far publication March 24, 1975)
