Veterinary Microbiology, 31 ( 1992 ) 243-249 Elsevier Science Publishers B.V., Amsterdam
243
ELISA tests for antibodies in experimental bovine tuberculosis J. Hanna a, S.D. Neill a and J.J.
O'Brien b
"Dept. of Agriculture for Northern Ireland, Veterinary Research Laboratories, Stoney Road, Belfast BT4 3SD, UK bVeterinary Division, Dept. of Agriculture for Northern Ireland, Dundonald House, Belfast BT4 3SB, UK (Accepted 16 October 1991 )
ABSTRACT Hanna, J., Neill, S.D. and O'Brien, J.J., 1992. ELISA tests for antibodies in experimental bovine tuberculosis. Vet. Microbiol., 31: 243-249. It was shown that 104 cfu of a field isolate of Mycobacterium bovis caused illness in five experimentally infected calves; one of these died. One of three contact calves also became clinically infected. Considerable variation in the humoral response of the affected animals was demonstrated by EL1SAs using purified protein derivative (PPD) and phosphatide antigens. The inoculation of antigens used in the comparative tuberculin skin test significantly enhanced the level of PPD antibodies in the affected animals whereas that of the apparently non-infected contact animals remained unchanged.
INTRODUCTION
Cattle affected with bovine tuberculosis are usually identified by using an intradermal tuberculin test. Although widely used, such a test may suffer from lack of specificity due to antigens cross-reacting with mycobacteria other than Mycobacterium bovis (Chaparas and Maloney, 1978 ) or with other microorganisms (Thorns and Morris, 1986 ). Although it is generally considered that the immune response to mycobacterial infection is primarily cell mediated, many attempts have been made to develop serodiagnostic tests, including complement fixation (Ellis et al., 1964), haemagglutination (Takahashi and Ono, 1961 ) ELISA (Reggiardo et al., 1980; Thoen et al., 1984). ELISA has also been used to study the onset and development of the humoral response of calves experimentally infected with M. bovis (Hanna et al., 1989 ). The purpose of this study was to investigate further the humoral responses in calves experimentally infected with a field isolate of M. bovis and also the responses of three calves introduced to the experimentally infected animals. 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
244
J. HANNA ET AL.
Experimental animals Eight calves 4-7 months old were obtained from tuberculosis-free farms with no history ofM. bovis infection for the previous seven years. The experiment was carried out in a high security isolation house held under negative pressure with expelled air filtered through absolute filters. The calves were fed normal rations.
Experimental infection As described by (Neill et al. (1988) (expt 2) five of the calves were inoculated intranasally with 104 cfu of a field strain of M. boris which had been isolated from bovine nasal secretions. The inoculum was delivered into the posterior nasal cavity on two consecutive days.
Contact calves One calf (A467) was placed in contact with the five experimentally infected animals at the time of inoculation. Two other calves ( 1074 and 1211 ) were introduced to the group 84 days after inoculation.
Specimen collection Specimens of blood, faeces and nasal mucus were collected from each calf at least twice before inoculation, on days 3, 5, 7, 12 and 14 after inoculation and thereafter at weekly intervals. All calves were examined clinically at times of specimen collection. ELISA were carried out on sera using purified protein derivative PPD and phosphatide antigens as previously described (Hanna et al., 1989 ). Faeces and nasal mucus were cultured for M. bovis as previously described (Neill et al., 1988).
Skin testing All calves were tuberculin tested using the comparative intradermal test (avian and bovine PPD-antigens, Central Veterinary Laboratory, Weybridge ). Measurement of skin thickness was taken at the time of initial testing and 72 hours later. Calf A462 was not skin tested as this animal died prior to the date selected for testing. A465 and 2149 were tested at 48 days post infection (pi) calves 1245 and 2148 were tested at 192 and 267 days pi; calf A467 was tested at 164 and 267 days after introduction. Calves 1074 and 1211 were tested at 80 and 183 days after introduction. RESULTS
Clinical manifestations Details of clinical manifestation of the experimentally infected calves are presented elsewhere (Neill et al. (1988) (expt 2) Of the contact calves A467 showed marked deterioration in clinical condition accompanied with audible
245
ELISA TESTS FOR ANTIBODIES IN EXPERIMENTAL BOVINE TUBERCULOSIS 160
140
120
o'~ ",d" L,d v
100
lad ._1
80
._1 laJ
40
20
o
t
4
-20
20
60
1 O0
140
180
220
260
300
DAYS AFTER INOCULATION 160
140
120
0"~ laJ Lul :::) .--I .,¢ (/I .--I
100
8O
60
40
20
ol -20
~
~ 20
t 60
100
140
180
t 220
260
300
DAYS AFTER INOCULATION
Fig. 1. ELISA values of innoculated calves as assessed by ELISA employing (a) phosphatide antigen (b) PPD antigen. © calf2149; • calf2148; • calf A465; • calf A462; A calf 1245. T Denotes the dates on which calves 2149 and A465 were tuberculin tested. T Denotes the dates on which calves 2148 and 1245 were tuberculin tested.
246
J. HANNA ET AL. 160
140
120
03 i.i v ILl =:) .--I
O3 .--I hi
100
80
60
40
20
z~ 40
80
.t 120
~
160
t 200
24-0
280
320
280
320
DAYS AFTER INTRODUCTION 160
140
120
.~ hi
100
I,I :,,--I ~ .< >,
80
V) ,,--I I,I
60
40
20
.t 0
4-0
80
120
160
~
t 200
24-0
DAYS AFTER INTRODUCTION
Fig. 2. ELISA values of contact calves as assessed by ELISA employing (a) phosphatide antigen (b) PPD antigen. • calf 121 l; • calf 1073; a calf A467. Denotes the dates on which calves 1072 and 1211 were tuberculin tested. Denotes the dates on which calf A467 was tuberculin tested.
ELISA TESTS FOR ANTIBODIES IN EXPERIMENTAL BOVINE TUBERCULOSIS
247
exhalation from 45 days to 155 days post introduction. However this animal recovered and was in good condition at slaughter. Calves 1074 and 1211 introduced to the group 84 days after inoculation remained in good condition throughout the experiment. ELISA Figure 1 shows the humoral responses occurring in experimentally infected calves during the course of this study. Antibody levels demonstrated by an ELISA using a phosphatide antigen are presented in Fig. l a. The humoral response of calf 2149 which was severely affected c o m m e n c e d at 25 d pi. In calves 1245 and 2148 which were less severely affected the onset of the response was much later at 78 and 90 d pi respectively. Antibody levels of PPD presented in Fig. lb show that prior to intradermal testing only calf 1245 had developed a humoral response to this antigen. Antibody levels of calves 1245 and 2148 were enhanced following intradermal testing. The humoral response of the contact calves are presented in Fig. 2. The results of the ELISA using a phosphatide antigen (Fig. 2a) shows that only calf 467 developed a significant increase in antibody which occurred following the second intradermal test. The antibody levels to P P D are presented in Fig. 2b. In calf 467, a significant increase in antibody levels occurred after intradermal testing. Skin test With the exception of contact calves 1074 and 1211 all other calves reacted positively to skin tests carried out. Post-mortem and bacteriology Each animal was subjected to a detailed post-mortem and bacteriological examination. The results of the experimentally infected calves are reported elsewhere (Neill et al. 1988) (Expt 2). Of the three contact calves only in A467 was a lesion observed. M. bovis was isolated from this lesion which was located in the retropharangeal lymph node. DISCUSSION
In this experiment as in a previous study (Hanna et al., 1989 ), infection was readily established in all of the animals by intranasal inoculation. Those animals reacted to the intradermal tuberculin test. Infection was confirmed by necropsy and cultural findings (Neill et al. ( 1988 ). It was shown in a previous study (Hanna et al., 1989) that 106 cfu of a field isolate o f M . bovis could rapidly produce severe disease in cattle and eventually cause death. In this present study it has been shown that whilst the reduced inoculum did delay the onset of disease, three out of the five calves developed a peracute
248
J. H A N N A ET AL.
infection and died or were euthanised approximately 48 d after inoculation. Despite the onset of clinical illness in the inoculated calves ( 1245, 2148 ) and the contact calf (A467) they recovered and were in good body condition at the time of slaughter. Infection was detected by tuberculin testing, and confirmed at necropsy by histological examination and bacteriological culture. These findings demonstrate, as observed on many occasions in the field, that infected animals can appear to be in good clinical conditions. The onset of the response to the phosphatide antigen in calves 1245 and 2148 at 47 and 90 d pi respectively was much later than the 14 d reported in the previous study (Hanna et al., 1989). Therefore, there appeared to be a relationship between the onset of the humoral response and the level of the inoculating dose. The failure to demonstrate an antibody response in the tuberculous contact calf A467 prior to skin testing, is at variance with the suggestion ofTakahashi et al. ( 1961 ) that the phosphatide antibody levels reflect the extent and activity of the disease. Up to the time of the first intradermal test only calf 1245 had produced a humoral response detected by the ELISA to P P D antigen. The onset of this response was approximately 80 d after that of the appearance of significant levels of antibodies demonstrated by the ELISA to phosphatide antigen. This finding supports the suggestion in our previous study (Hanna et al. 1989) that antibodies to the various components of M. boris may appear at different stages during the course of the disease. Following the first application of the skin test there were moderate and transient increases in antibodies in the three clinically affected animals (two inoculated, and one contact ). No demonstrable response was detected in the other two contact calves in the group. After the second skin test the humoral responses of the three clinically affected animals were significantly enhanced. The preliminary analysis of a current study into the use of the ELISA in the field has suggested the enhancement effect described here may be quite specific and useful in the diagnosis of tuberculous animals hitherto deemed to be inconclusive by the skin test. (Hanna and Neill unpublished data). Although this p h e n o m e n o n has been previously reported (Thoen et al. 1983 and Rittaco et al. 1987), it should be seriously considered in assessing the efficacy of any serological test using PPD antigen to detect tuberculous animals. It would be essential to establish if any of the animals under scrutiny have previously been skin tested. Failure to do so might falsely enhance the sensitivity of the test employed. ACKNOWLEDGEMENTS The authors acknowledge the excellent technical assistance of Mr. E. Walton, Mr. S. McBride and Mrs. D. Monteith.
EL1SA TESTS FOR ANTIBODIES IN EXPERIMENTAL BOVINE TUBERCULOSIS
249
REFERENCES Chaparas, S.D. and Maloney, C.J., 1978. An analysis of cross reactions among mycobacteria by in vivo and in vitro assays of cellular hypersensitivity. Am. Rev. Respir. Dis., 117: 897-902. Ellis, E.M., Wright, H.S. and Richards, W.D., 1965. The evaluation of an antigen for the complement fixation test in tuberculosis of cattle. Am. Rev. Respir. Dis., 93: 749-753. Hanna, J., NeiU, S.D. and O'Brien, J.J., 1989. Use of PPD and phosphatide antigens to detect the serological response in experimental bovine tuberculosis. Res. Vet. Sci., 47: 43-47. Neill, S.D., Hanna, J., O'Brien, J.J. and McCracken, R.M., 1988. Excretion of Mycobacterium bovis by experimentally infected cattle. Vet. Rec., 123: 340-343. Reggiardo, Z. and Middlebrook, G., 1974. Serologically active glyeolipid families from Mycobacterium boris. Am. J. Epidemiol., 100: 469-476. Rittacco, V., De Kantor, I.N., Barrera, L., Nader, A., Bernardelli, A., Torrea, G., Errico, F. and Fleiss, E., 1987. Assessment of the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for the detection of mycobacterial antibodies in bovine tuberculosis. J. Vet. Med., B34:119-125. Takahashi, Y. and Ono, K., 1961. Study on the passive haemagglutination reaction by the phosphatide of M. tuberculosis. Am. Rev. Respir. Dis., 83, 381-385. Takahashi, Y., Ondera, T. and Yamamoto, K., 1961. The behaviour of three different kinds of antibodies in tuberculosis: antiprotein, antipolysacharide and antiphosphatide. J. Exp. Med., 114: 555-567. Thoen, C.O., Hall, M.R., Peterburg, T.A. and Harrington, R. Jr., 1983. Detection of mycobacterial antibodies in sera of cattle experimentally exposed to Mycobacterium bovis by use of a modified enzyme-linked immunosorbent assay. AM. Assoc. Vet. Diagnost., 26th Ann. Proc., 25-38. Thorns, C.J. and Morris, J.A., 1986. Shared epitopes between mycobacteria and other microorganisms. Res. Vet. Sci., 41: 275-276.
243
ELISA tests for antibodies in experimental bovine tuberculosis J. Hanna a, S.D. Neill a and J.J.
O'Brien b
"Dept. of Agriculture for Northern Ireland, Veterinary Research Laboratories, Stoney Road, Belfast BT4 3SD, UK bVeterinary Division, Dept. of Agriculture for Northern Ireland, Dundonald House, Belfast BT4 3SB, UK (Accepted 16 October 1991 )
ABSTRACT Hanna, J., Neill, S.D. and O'Brien, J.J., 1992. ELISA tests for antibodies in experimental bovine tuberculosis. Vet. Microbiol., 31: 243-249. It was shown that 104 cfu of a field isolate of Mycobacterium bovis caused illness in five experimentally infected calves; one of these died. One of three contact calves also became clinically infected. Considerable variation in the humoral response of the affected animals was demonstrated by EL1SAs using purified protein derivative (PPD) and phosphatide antigens. The inoculation of antigens used in the comparative tuberculin skin test significantly enhanced the level of PPD antibodies in the affected animals whereas that of the apparently non-infected contact animals remained unchanged.
INTRODUCTION
Cattle affected with bovine tuberculosis are usually identified by using an intradermal tuberculin test. Although widely used, such a test may suffer from lack of specificity due to antigens cross-reacting with mycobacteria other than Mycobacterium bovis (Chaparas and Maloney, 1978 ) or with other microorganisms (Thorns and Morris, 1986 ). Although it is generally considered that the immune response to mycobacterial infection is primarily cell mediated, many attempts have been made to develop serodiagnostic tests, including complement fixation (Ellis et al., 1964), haemagglutination (Takahashi and Ono, 1961 ) ELISA (Reggiardo et al., 1980; Thoen et al., 1984). ELISA has also been used to study the onset and development of the humoral response of calves experimentally infected with M. bovis (Hanna et al., 1989 ). The purpose of this study was to investigate further the humoral responses in calves experimentally infected with a field isolate of M. bovis and also the responses of three calves introduced to the experimentally infected animals. 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
244
J. HANNA ET AL.
Experimental animals Eight calves 4-7 months old were obtained from tuberculosis-free farms with no history ofM. bovis infection for the previous seven years. The experiment was carried out in a high security isolation house held under negative pressure with expelled air filtered through absolute filters. The calves were fed normal rations.
Experimental infection As described by (Neill et al. (1988) (expt 2) five of the calves were inoculated intranasally with 104 cfu of a field strain of M. boris which had been isolated from bovine nasal secretions. The inoculum was delivered into the posterior nasal cavity on two consecutive days.
Contact calves One calf (A467) was placed in contact with the five experimentally infected animals at the time of inoculation. Two other calves ( 1074 and 1211 ) were introduced to the group 84 days after inoculation.
Specimen collection Specimens of blood, faeces and nasal mucus were collected from each calf at least twice before inoculation, on days 3, 5, 7, 12 and 14 after inoculation and thereafter at weekly intervals. All calves were examined clinically at times of specimen collection. ELISA were carried out on sera using purified protein derivative PPD and phosphatide antigens as previously described (Hanna et al., 1989 ). Faeces and nasal mucus were cultured for M. bovis as previously described (Neill et al., 1988).
Skin testing All calves were tuberculin tested using the comparative intradermal test (avian and bovine PPD-antigens, Central Veterinary Laboratory, Weybridge ). Measurement of skin thickness was taken at the time of initial testing and 72 hours later. Calf A462 was not skin tested as this animal died prior to the date selected for testing. A465 and 2149 were tested at 48 days post infection (pi) calves 1245 and 2148 were tested at 192 and 267 days pi; calf A467 was tested at 164 and 267 days after introduction. Calves 1074 and 1211 were tested at 80 and 183 days after introduction. RESULTS
Clinical manifestations Details of clinical manifestation of the experimentally infected calves are presented elsewhere (Neill et al. (1988) (expt 2) Of the contact calves A467 showed marked deterioration in clinical condition accompanied with audible
245
ELISA TESTS FOR ANTIBODIES IN EXPERIMENTAL BOVINE TUBERCULOSIS 160
140
120
o'~ ",d" L,d v
100
lad ._1
80
._1 laJ
40
20
o
t
4
-20
20
60
1 O0
140
180
220
260
300
DAYS AFTER INOCULATION 160
140
120
0"~ laJ Lul :::) .--I .,¢ (/I .--I
100
8O
60
40
20
ol -20
~
~ 20
t 60
100
140
180
t 220
260
300
DAYS AFTER INOCULATION
Fig. 1. ELISA values of innoculated calves as assessed by ELISA employing (a) phosphatide antigen (b) PPD antigen. © calf2149; • calf2148; • calf A465; • calf A462; A calf 1245. T Denotes the dates on which calves 2149 and A465 were tuberculin tested. T Denotes the dates on which calves 2148 and 1245 were tuberculin tested.
246
J. HANNA ET AL. 160
140
120
03 i.i v ILl =:) .--I
O3 .--I hi
100
80
60
40
20
z~ 40
80
.t 120
~
160
t 200
24-0
280
320
280
320
DAYS AFTER INTRODUCTION 160
140
120
.~ hi
100
I,I :,,--I ~ .< >,
80
V) ,,--I I,I
60
40
20
.t 0
4-0
80
120
160
~
t 200
24-0
DAYS AFTER INTRODUCTION
Fig. 2. ELISA values of contact calves as assessed by ELISA employing (a) phosphatide antigen (b) PPD antigen. • calf 121 l; • calf 1073; a calf A467. Denotes the dates on which calves 1072 and 1211 were tuberculin tested. Denotes the dates on which calf A467 was tuberculin tested.
ELISA TESTS FOR ANTIBODIES IN EXPERIMENTAL BOVINE TUBERCULOSIS
247
exhalation from 45 days to 155 days post introduction. However this animal recovered and was in good condition at slaughter. Calves 1074 and 1211 introduced to the group 84 days after inoculation remained in good condition throughout the experiment. ELISA Figure 1 shows the humoral responses occurring in experimentally infected calves during the course of this study. Antibody levels demonstrated by an ELISA using a phosphatide antigen are presented in Fig. l a. The humoral response of calf 2149 which was severely affected c o m m e n c e d at 25 d pi. In calves 1245 and 2148 which were less severely affected the onset of the response was much later at 78 and 90 d pi respectively. Antibody levels of PPD presented in Fig. lb show that prior to intradermal testing only calf 1245 had developed a humoral response to this antigen. Antibody levels of calves 1245 and 2148 were enhanced following intradermal testing. The humoral response of the contact calves are presented in Fig. 2. The results of the ELISA using a phosphatide antigen (Fig. 2a) shows that only calf 467 developed a significant increase in antibody which occurred following the second intradermal test. The antibody levels to P P D are presented in Fig. 2b. In calf 467, a significant increase in antibody levels occurred after intradermal testing. Skin test With the exception of contact calves 1074 and 1211 all other calves reacted positively to skin tests carried out. Post-mortem and bacteriology Each animal was subjected to a detailed post-mortem and bacteriological examination. The results of the experimentally infected calves are reported elsewhere (Neill et al. 1988) (Expt 2). Of the three contact calves only in A467 was a lesion observed. M. bovis was isolated from this lesion which was located in the retropharangeal lymph node. DISCUSSION
In this experiment as in a previous study (Hanna et al., 1989 ), infection was readily established in all of the animals by intranasal inoculation. Those animals reacted to the intradermal tuberculin test. Infection was confirmed by necropsy and cultural findings (Neill et al. ( 1988 ). It was shown in a previous study (Hanna et al., 1989) that 106 cfu of a field isolate o f M . bovis could rapidly produce severe disease in cattle and eventually cause death. In this present study it has been shown that whilst the reduced inoculum did delay the onset of disease, three out of the five calves developed a peracute
248
J. H A N N A ET AL.
infection and died or were euthanised approximately 48 d after inoculation. Despite the onset of clinical illness in the inoculated calves ( 1245, 2148 ) and the contact calf (A467) they recovered and were in good body condition at the time of slaughter. Infection was detected by tuberculin testing, and confirmed at necropsy by histological examination and bacteriological culture. These findings demonstrate, as observed on many occasions in the field, that infected animals can appear to be in good clinical conditions. The onset of the response to the phosphatide antigen in calves 1245 and 2148 at 47 and 90 d pi respectively was much later than the 14 d reported in the previous study (Hanna et al., 1989). Therefore, there appeared to be a relationship between the onset of the humoral response and the level of the inoculating dose. The failure to demonstrate an antibody response in the tuberculous contact calf A467 prior to skin testing, is at variance with the suggestion ofTakahashi et al. ( 1961 ) that the phosphatide antibody levels reflect the extent and activity of the disease. Up to the time of the first intradermal test only calf 1245 had produced a humoral response detected by the ELISA to P P D antigen. The onset of this response was approximately 80 d after that of the appearance of significant levels of antibodies demonstrated by the ELISA to phosphatide antigen. This finding supports the suggestion in our previous study (Hanna et al. 1989) that antibodies to the various components of M. boris may appear at different stages during the course of the disease. Following the first application of the skin test there were moderate and transient increases in antibodies in the three clinically affected animals (two inoculated, and one contact ). No demonstrable response was detected in the other two contact calves in the group. After the second skin test the humoral responses of the three clinically affected animals were significantly enhanced. The preliminary analysis of a current study into the use of the ELISA in the field has suggested the enhancement effect described here may be quite specific and useful in the diagnosis of tuberculous animals hitherto deemed to be inconclusive by the skin test. (Hanna and Neill unpublished data). Although this p h e n o m e n o n has been previously reported (Thoen et al. 1983 and Rittaco et al. 1987), it should be seriously considered in assessing the efficacy of any serological test using PPD antigen to detect tuberculous animals. It would be essential to establish if any of the animals under scrutiny have previously been skin tested. Failure to do so might falsely enhance the sensitivity of the test employed. ACKNOWLEDGEMENTS The authors acknowledge the excellent technical assistance of Mr. E. Walton, Mr. S. McBride and Mrs. D. Monteith.
EL1SA TESTS FOR ANTIBODIES IN EXPERIMENTAL BOVINE TUBERCULOSIS
249
REFERENCES Chaparas, S.D. and Maloney, C.J., 1978. An analysis of cross reactions among mycobacteria by in vivo and in vitro assays of cellular hypersensitivity. Am. Rev. Respir. Dis., 117: 897-902. Ellis, E.M., Wright, H.S. and Richards, W.D., 1965. The evaluation of an antigen for the complement fixation test in tuberculosis of cattle. Am. Rev. Respir. Dis., 93: 749-753. Hanna, J., NeiU, S.D. and O'Brien, J.J., 1989. Use of PPD and phosphatide antigens to detect the serological response in experimental bovine tuberculosis. Res. Vet. Sci., 47: 43-47. Neill, S.D., Hanna, J., O'Brien, J.J. and McCracken, R.M., 1988. Excretion of Mycobacterium bovis by experimentally infected cattle. Vet. Rec., 123: 340-343. Reggiardo, Z. and Middlebrook, G., 1974. Serologically active glyeolipid families from Mycobacterium boris. Am. J. Epidemiol., 100: 469-476. Rittacco, V., De Kantor, I.N., Barrera, L., Nader, A., Bernardelli, A., Torrea, G., Errico, F. and Fleiss, E., 1987. Assessment of the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for the detection of mycobacterial antibodies in bovine tuberculosis. J. Vet. Med., B34:119-125. Takahashi, Y. and Ono, K., 1961. Study on the passive haemagglutination reaction by the phosphatide of M. tuberculosis. Am. Rev. Respir. Dis., 83, 381-385. Takahashi, Y., Ondera, T. and Yamamoto, K., 1961. The behaviour of three different kinds of antibodies in tuberculosis: antiprotein, antipolysacharide and antiphosphatide. J. Exp. Med., 114: 555-567. Thoen, C.O., Hall, M.R., Peterburg, T.A. and Harrington, R. Jr., 1983. Detection of mycobacterial antibodies in sera of cattle experimentally exposed to Mycobacterium bovis by use of a modified enzyme-linked immunosorbent assay. AM. Assoc. Vet. Diagnost., 26th Ann. Proc., 25-38. Thorns, C.J. and Morris, J.A., 1986. Shared epitopes between mycobacteria and other microorganisms. Res. Vet. Sci., 41: 275-276.
