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Antibody response patterns in goats experimentally infected with Mycoplasma agalactiae
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Small R u m i n a n t Research ELSEVIER
Small Ruminant Research 14 (1994) 79-81
Antibody response patterns in goats experimentally infected with
Mycoplasma agalactiae S.A. Hasso a'*, A.H. A l - O m r a n b ~'Department~["Veterina~ Medicine and Therapeutics, College of Veterinary Medicine, Universityof Baghdad, P.O. Box 28061, Dawoodi, AI-Ameria, lraq bDepartment ~)fMicrobiology, College of Veterinary Medicine. Universi~ ~f Baghdad, AI-Ameria, lraq Accepted 25 August 1993
Abstract
The enzyme linked immuno-sorbent assay (EL1SA) was used to evaluate humoral immunoresponse to Mycoplasma agalactiae. Four groups of goats were inoculated via different routes with the microorganism. Serum samples were collected at monthly intervals for five successive months. Peak antibody levels were detected at months 3-4 in the subcutaneously and orally inoculated groups, while a higher peak since the first month was detected in the intramammary inoculated group. The intravenously inoculated group revealed very slight increases in antibody levels. The best route of inoculation for high and protective antibody levels was the intramammary route. Keywords: Mycoplasma agalactiae; Humoral antibody; Goat; Immunoresponse; Inoculation route
1. Introduction
Levels and durations of antibody production alter vaccination are well known to be affected by the kind of vaccine, and route of administration in general. Similar events usually occur after introduction of the living microorganism naturally or experimentally by different routes. Sensitivity of the technique is important in clarifying the antibody response. ELISA, due to its greater sensitivity than CFT (Schearen and Nicolet, 1982; Lambert, 1985; Lambert and Cabasse, personal communication), was chosen to measure the humoral immune response in goats experimentally infected by different routes with Mycoplasma agalactiae, the causative agent of contagious agalactia (a chronic disease of sheep and goats manifested clin*Corresponding author. 0921-4488/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1092 1 -448 8 ( 93 ) E 0 0 9 7 - C
ically by mastitis, arthritis and keratoconjunctivitis) (Lambert, 1987).
2. Materials and methods
Isolation of M. agalactiae, cultural media and inocula preparation (9 × 109 CFU/ml) were as described by Hasso et al. (1993). Isolates were identified by the growth inhibition test (disc method) as described by Clyde (1983) against M. agalactiae antisera.
Inoculation of experimental animals Twenty-eight locally bred, black goats (16 nonpregnant, nine pregnant and three males) free from M. agalactiae were divided into four inoculation groups (intravenous, intramammary, subcutaneous and oral). Four goats from each group with three females and one
80
S.A. Hasso, A.H. Al-Omran / Small Ruminant Research 14 (1994) 79-81
male goat (except for the intramammary group no male), were inoculated with M. agalactiae (5 ml intravenously, 2 ml intramammary, 5 ml subcutaneously, and 10 ml inocula+25 ml sterile milk orally). Two goats were not inoculated but remained with the inoculated ones in order to contract the infection (contact control), and one goat was separated and received broth only (group control). There was no contact between goats of the different inoculated groups. Serum samples were collected at monthly intervals from the day of inoculation until sacrifice of all goats 5 months later.
ELISA technique The cell lysate method was used as described by Cassel and Brown (1983). Serum samples were serially diluted over antigen-coated microtitre plates (Linbrow). Optical densities at mid dilution 1 : 160 were used to plot the graphs. Briefly, the cell lysate was prepared by diluting cell suspensions in 0.05 M carbonate/bicarbonate buffer (pH 10.0). After 10-15 min incubation at 37°C a 2% w/v boric acid was added to stop lysis, later protein concentration was determined according to the method of Bradford (1967). Antigen concentration of 10/zg/ml in carbonate buffer was used to coat a 96-well microtitre plate. Rabbit anti-goat IgGHRP was prepared by conjugation of a commercial preparation of rabbit anti-IgG (Sigma Chemical Company) to horseradish peroxidase (HRP type VI Sigma Chemical Company) following the method of Goding (1986).
lntramammary
Intravenous
1.4 1.2 E 1.0
Z
C
1.8 0.6 ,4 0.4 d
0.2 0
I
l
I
I
I
1
2
3
z,
5
Subcutaneous
Oral
1.2 1.0 O.B 0.6
c5 d 0.4 0.2 0
I
5
I
I
I
12325
I
Months Post-lnoculation
Fig. 1. Antibody titre in goats. Experimentally inoculated goats (O); contact control goats (O) and group control goats (&).
levels were detected in the remaining two groups of inoculation. The group control goats of all routes of inoculations showed no increase in the levels of antibodies throughout the experiment except for the group control goat of the subcutaneously inoculated group which manifested an unsteady increase in its antibody level reaching a peak at month 5 (Fig. IC).
3. Results 4. Discussion
Each route of inoculation had its characteristic pattern of response and antibody production. The intramammary inoculated goats produced highest levels in the shortest time (within the first month), remained at peak level for 4 months, then declined (Fig. 1B). The subcutaneously inoculated goats reached their peak level at month 3 then declined (Fig. 1C). The orally inoculated goats reached their peak level at month 4 then declined (Fig. 1D), while a very slight antibody response was detected in the intravenously inoculated goats (Fig. 1A). The contact control goats of the intramammary and subcutaneously inoculated groups manifested slight rises in antibody levels, while no increase in antibody
Contagious agalactia of sheep and goats has more than one mode of transmission. The natural portals of entry are the mouth and through the teat meatus (Gillespie and Timoney, 1981; Lambert, 1987). Infection by a single portal of entry does not produce all clinical manifestations (mastitis, arthritis and keratoconjunctivitis) in the same animal, as reported by Cottew and Leach (1969), and detected in this experiment (absence of keratoconjunctivitis, only mild conjunctivitis), in spite of four different routes of inoculation being used. However, differences in the severity of clinical signs were detected between the groups along with differences in their immune response.
S.A. Hasso, A.H. Al-Omran / Small Ruminant Research 14 (1994) 79-81
The intramammary inoculated goats manifested a dramatic increase in antibody level (highest) and were somewhat protected against severe clinical signs, while clinical signs of the subcutaneously inoculated goats (lover level of antibodies) were severe and the antibody level was not protective against severe clinical signs. Goats inoculated orally manifested mild clinical signs although their antibody levels were lower than the subcutaneously inoculated goats, while the intravenously inoculated goats also manifested mild clinical signs along with a very slight increase in antibody level when compared with other groups. Such low grades of antibody formation by the intravenous inoculation method were observed by Toshkov et al. (1974), and were suspected to be due to percipitins and mycoplasma growth inhibitors, while the low level of antibodies in the orally inoculated goats could be due to inhibitors present in the gastro-intestinal tract. Such inhibitors, local or systemic, are suspected when considering the amount of inocula, i.e., 2 ml (1 m l = 9 × 109 C F U ) intramammary produced highest and fastest levels of antibody, while 5 ml intravenously and 10 ml orally produced very slight and low increases in antibody levels, respectively. Four blood isolates ofM. agalactiae (one from intravenously and subcutaneously inoculated groups, and two from the orally inoculated group) were recovered throughout the length of the experiment (including febrile periods ), none were from the intramammary inoculated goats. Such findings solidifies the possibility that antibodies at a certain level in the intramammary inoculated goats were protective against mycoplasmemia and severe clinical signs. Persistence of antibodies in high levels for a long duration can be more protective (intramammary inoculated goats) than a peak with a short duration (subcutaneously inoculated goats). Peak antibody levels of the remaining contact control goats were not protective, some goats (intramammary and subcutaneous inoculation groups) contracted infection, manifested clinical signs, one from the subcutaneously inoculated group died during a febrile period, M. agalactiae were recovered from some goats before and after sacrifice. The group control goats were normal throughout the experiment, except for the unexplained increase of anti-
81
body levels of the group control goat belonging to the subcutaneous inoculation group.
5. Conclusion
The production mechanism of high and fast antibody levels by the intramammary route of inoculation (within 1 month) needs to be considered in immunization against M. agalactiae and its possible application to other microorganisms and routes of inoculation.
References Bradford, M.M., 1967. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72: 248-254. Cassell, G.H. and Brown, M.B., 1983. Enzyme-linked immunosorbent ~ssay (ELISA) for detection of anti-mycoplasmal antibody. In: Razin, S. and Tully, J.G. (Editors). Methods in Mycoplasmology. Vol. 1. Academic Press, New York, pp. 457-469. Clyde, W.A., 1983. Growth inhibition tests. In: Razin, S. and Tully, J.G. (Editors). Methods in Mycoplasmology, Vol. 1. Academic Press, New York, pp. 405--410. Cottew, G.S. and Leach, R.H., 1969. Mycoplasmas of the cattle, sheep and goats. In: Hayrick, L. (Editor). The Mycoplasmatales and L-phase of Bacteria. Meredith Corporation, New York, pp. 527-570. Gilliespie, J.H. and Timoney, J.F., 1981. The mycoplasmas. Part I11. In: Hagan and Bruner's: Infectious Diseases of Domestic Animals. 7th edn. Cornell University Press, Ithaca, NY, pp. 289. Goding, J.W., 1986. Monoclonal antibodies: Principles and Practice. second edition. Academic Press, Harcourt Brace Jovanovich Publisher, London, pp. 82-85. Hasso, S.A., AI-Aubaidi, J.M. and AI-Darraji, A.M., 1993. Contagious Agalactia in goats: It's severity as related to the route of infection and pregnancy. Small Rumin. Res., 10: 263-275. Lambert, M., 1985. Use of computerized ELISA for the serological diagnosis of contagious agalactiain small ruminants. Revue Med. Vet., 136: 303-306. Lambert, M., 1987. Contagious agalactia of sheep and goats. Rev. Sci. Tech. Oft. Int. Epiz., 6:699-711. Schearen, W. and Nicolet, J., 1982. Micro-ELISA for detecting contagious agalactia in goats. Schweizer Archiv. Tierheilkunde, 124: 163-177. Toshkov, A.S., Mihailova, L., Shirova, L. and Shabanov, M., 1974. Infection and immunity in mycoplasmoses. 1. Some peculiarities in the course of experimental agalactiae in sheep and goats. Bull. Inst. Microbiol., 25: 3945.
.
.
.
L
Small R u m i n a n t Research ELSEVIER
Small Ruminant Research 14 (1994) 79-81
Antibody response patterns in goats experimentally infected with
Mycoplasma agalactiae S.A. Hasso a'*, A.H. A l - O m r a n b ~'Department~["Veterina~ Medicine and Therapeutics, College of Veterinary Medicine, Universityof Baghdad, P.O. Box 28061, Dawoodi, AI-Ameria, lraq bDepartment ~)fMicrobiology, College of Veterinary Medicine. Universi~ ~f Baghdad, AI-Ameria, lraq Accepted 25 August 1993
Abstract
The enzyme linked immuno-sorbent assay (EL1SA) was used to evaluate humoral immunoresponse to Mycoplasma agalactiae. Four groups of goats were inoculated via different routes with the microorganism. Serum samples were collected at monthly intervals for five successive months. Peak antibody levels were detected at months 3-4 in the subcutaneously and orally inoculated groups, while a higher peak since the first month was detected in the intramammary inoculated group. The intravenously inoculated group revealed very slight increases in antibody levels. The best route of inoculation for high and protective antibody levels was the intramammary route. Keywords: Mycoplasma agalactiae; Humoral antibody; Goat; Immunoresponse; Inoculation route
1. Introduction
Levels and durations of antibody production alter vaccination are well known to be affected by the kind of vaccine, and route of administration in general. Similar events usually occur after introduction of the living microorganism naturally or experimentally by different routes. Sensitivity of the technique is important in clarifying the antibody response. ELISA, due to its greater sensitivity than CFT (Schearen and Nicolet, 1982; Lambert, 1985; Lambert and Cabasse, personal communication), was chosen to measure the humoral immune response in goats experimentally infected by different routes with Mycoplasma agalactiae, the causative agent of contagious agalactia (a chronic disease of sheep and goats manifested clin*Corresponding author. 0921-4488/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1092 1 -448 8 ( 93 ) E 0 0 9 7 - C
ically by mastitis, arthritis and keratoconjunctivitis) (Lambert, 1987).
2. Materials and methods
Isolation of M. agalactiae, cultural media and inocula preparation (9 × 109 CFU/ml) were as described by Hasso et al. (1993). Isolates were identified by the growth inhibition test (disc method) as described by Clyde (1983) against M. agalactiae antisera.
Inoculation of experimental animals Twenty-eight locally bred, black goats (16 nonpregnant, nine pregnant and three males) free from M. agalactiae were divided into four inoculation groups (intravenous, intramammary, subcutaneous and oral). Four goats from each group with three females and one
80
S.A. Hasso, A.H. Al-Omran / Small Ruminant Research 14 (1994) 79-81
male goat (except for the intramammary group no male), were inoculated with M. agalactiae (5 ml intravenously, 2 ml intramammary, 5 ml subcutaneously, and 10 ml inocula+25 ml sterile milk orally). Two goats were not inoculated but remained with the inoculated ones in order to contract the infection (contact control), and one goat was separated and received broth only (group control). There was no contact between goats of the different inoculated groups. Serum samples were collected at monthly intervals from the day of inoculation until sacrifice of all goats 5 months later.
ELISA technique The cell lysate method was used as described by Cassel and Brown (1983). Serum samples were serially diluted over antigen-coated microtitre plates (Linbrow). Optical densities at mid dilution 1 : 160 were used to plot the graphs. Briefly, the cell lysate was prepared by diluting cell suspensions in 0.05 M carbonate/bicarbonate buffer (pH 10.0). After 10-15 min incubation at 37°C a 2% w/v boric acid was added to stop lysis, later protein concentration was determined according to the method of Bradford (1967). Antigen concentration of 10/zg/ml in carbonate buffer was used to coat a 96-well microtitre plate. Rabbit anti-goat IgGHRP was prepared by conjugation of a commercial preparation of rabbit anti-IgG (Sigma Chemical Company) to horseradish peroxidase (HRP type VI Sigma Chemical Company) following the method of Goding (1986).
lntramammary
Intravenous
1.4 1.2 E 1.0
Z
C
1.8 0.6 ,4 0.4 d
0.2 0
I
l
I
I
I
1
2
3
z,
5
Subcutaneous
Oral
1.2 1.0 O.B 0.6
c5 d 0.4 0.2 0
I
5
I
I
I
12325
I
Months Post-lnoculation
Fig. 1. Antibody titre in goats. Experimentally inoculated goats (O); contact control goats (O) and group control goats (&).
levels were detected in the remaining two groups of inoculation. The group control goats of all routes of inoculations showed no increase in the levels of antibodies throughout the experiment except for the group control goat of the subcutaneously inoculated group which manifested an unsteady increase in its antibody level reaching a peak at month 5 (Fig. IC).
3. Results 4. Discussion
Each route of inoculation had its characteristic pattern of response and antibody production. The intramammary inoculated goats produced highest levels in the shortest time (within the first month), remained at peak level for 4 months, then declined (Fig. 1B). The subcutaneously inoculated goats reached their peak level at month 3 then declined (Fig. 1C). The orally inoculated goats reached their peak level at month 4 then declined (Fig. 1D), while a very slight antibody response was detected in the intravenously inoculated goats (Fig. 1A). The contact control goats of the intramammary and subcutaneously inoculated groups manifested slight rises in antibody levels, while no increase in antibody
Contagious agalactia of sheep and goats has more than one mode of transmission. The natural portals of entry are the mouth and through the teat meatus (Gillespie and Timoney, 1981; Lambert, 1987). Infection by a single portal of entry does not produce all clinical manifestations (mastitis, arthritis and keratoconjunctivitis) in the same animal, as reported by Cottew and Leach (1969), and detected in this experiment (absence of keratoconjunctivitis, only mild conjunctivitis), in spite of four different routes of inoculation being used. However, differences in the severity of clinical signs were detected between the groups along with differences in their immune response.
S.A. Hasso, A.H. Al-Omran / Small Ruminant Research 14 (1994) 79-81
The intramammary inoculated goats manifested a dramatic increase in antibody level (highest) and were somewhat protected against severe clinical signs, while clinical signs of the subcutaneously inoculated goats (lover level of antibodies) were severe and the antibody level was not protective against severe clinical signs. Goats inoculated orally manifested mild clinical signs although their antibody levels were lower than the subcutaneously inoculated goats, while the intravenously inoculated goats also manifested mild clinical signs along with a very slight increase in antibody level when compared with other groups. Such low grades of antibody formation by the intravenous inoculation method were observed by Toshkov et al. (1974), and were suspected to be due to percipitins and mycoplasma growth inhibitors, while the low level of antibodies in the orally inoculated goats could be due to inhibitors present in the gastro-intestinal tract. Such inhibitors, local or systemic, are suspected when considering the amount of inocula, i.e., 2 ml (1 m l = 9 × 109 C F U ) intramammary produced highest and fastest levels of antibody, while 5 ml intravenously and 10 ml orally produced very slight and low increases in antibody levels, respectively. Four blood isolates ofM. agalactiae (one from intravenously and subcutaneously inoculated groups, and two from the orally inoculated group) were recovered throughout the length of the experiment (including febrile periods ), none were from the intramammary inoculated goats. Such findings solidifies the possibility that antibodies at a certain level in the intramammary inoculated goats were protective against mycoplasmemia and severe clinical signs. Persistence of antibodies in high levels for a long duration can be more protective (intramammary inoculated goats) than a peak with a short duration (subcutaneously inoculated goats). Peak antibody levels of the remaining contact control goats were not protective, some goats (intramammary and subcutaneous inoculation groups) contracted infection, manifested clinical signs, one from the subcutaneously inoculated group died during a febrile period, M. agalactiae were recovered from some goats before and after sacrifice. The group control goats were normal throughout the experiment, except for the unexplained increase of anti-
81
body levels of the group control goat belonging to the subcutaneous inoculation group.
5. Conclusion
The production mechanism of high and fast antibody levels by the intramammary route of inoculation (within 1 month) needs to be considered in immunization against M. agalactiae and its possible application to other microorganisms and routes of inoculation.
References Bradford, M.M., 1967. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72: 248-254. Cassell, G.H. and Brown, M.B., 1983. Enzyme-linked immunosorbent ~ssay (ELISA) for detection of anti-mycoplasmal antibody. In: Razin, S. and Tully, J.G. (Editors). Methods in Mycoplasmology. Vol. 1. Academic Press, New York, pp. 457-469. Clyde, W.A., 1983. Growth inhibition tests. In: Razin, S. and Tully, J.G. (Editors). Methods in Mycoplasmology, Vol. 1. Academic Press, New York, pp. 405--410. Cottew, G.S. and Leach, R.H., 1969. Mycoplasmas of the cattle, sheep and goats. In: Hayrick, L. (Editor). The Mycoplasmatales and L-phase of Bacteria. Meredith Corporation, New York, pp. 527-570. Gilliespie, J.H. and Timoney, J.F., 1981. The mycoplasmas. Part I11. In: Hagan and Bruner's: Infectious Diseases of Domestic Animals. 7th edn. Cornell University Press, Ithaca, NY, pp. 289. Goding, J.W., 1986. Monoclonal antibodies: Principles and Practice. second edition. Academic Press, Harcourt Brace Jovanovich Publisher, London, pp. 82-85. Hasso, S.A., AI-Aubaidi, J.M. and AI-Darraji, A.M., 1993. Contagious Agalactia in goats: It's severity as related to the route of infection and pregnancy. Small Rumin. Res., 10: 263-275. Lambert, M., 1985. Use of computerized ELISA for the serological diagnosis of contagious agalactiain small ruminants. Revue Med. Vet., 136: 303-306. Lambert, M., 1987. Contagious agalactia of sheep and goats. Rev. Sci. Tech. Oft. Int. Epiz., 6:699-711. Schearen, W. and Nicolet, J., 1982. Micro-ELISA for detecting contagious agalactia in goats. Schweizer Archiv. Tierheilkunde, 124: 163-177. Toshkov, A.S., Mihailova, L., Shirova, L. and Shabanov, M., 1974. Infection and immunity in mycoplasmoses. 1. Some peculiarities in the course of experimental agalactiae in sheep and goats. Bull. Inst. Microbiol., 25: 3945.