• View in gallery

    Mean serum S:P ratios for IgG1 antibodies against Mycoplasma bovis in control (n = 40 [gray bars]) and vaccinated (38 [black bars]) cows at 60 and 39 days prior to expected parturition date. Day 0 represents actual date of parturition. aSignificant (P < 0.05) difference, compared with the control group. bSignificant (P < 0.05) difference, compared with prior measurement for the control group. cSignificant (P < 0.05) difference, compared with prior measurement for the vaccinated group. dSignificant (P < 0.05) difference, compared with value at −60 days prior to expected parturition for the control group. eSignificant (P < 0.05) difference, compared with value at −60 days prior to expected parturition for the vaccinated group.

  • View in gallery

    Mean colostrum S:P ratios for IgG1 antibodies against Mycoplasma bovis between control (n = 28 [gray bars]) and vaccinated (26 [black bars]) cows and mean milk S:P ratios between control (n = 32 [gray bars]) and vaccinated (27 [black bars]) cows. See Figure 1 for key.

  • 1.

    Hale HH, Helmboldt CF, Plastridge WN, et al. Bovine mastitis caused by a Mycoplasma species. Cornell Vet 1962;52:582591.

  • 2.

    Garrity GM, Boone DR, Castenholz RW, eds. Bergey's manual of systematic bacteriology. Vol 1. 2nd ed. New York: Springer-Verlag Inc, 2001.

  • 3.

    Zar JH. Biostatistical analysis. 4th ed. Upper Saddle River, NJ: Prentice-Hall Inc, 1999.

  • 4.

    Kutner MH, Nachtsheim C, Neter J. Applied linear regression models. 4th ed. Boston, NY: McGraw-Hill Book Co/Irwin, 2004.

  • 5.

    Ishikawa H. Observation of lymphocyte function in perinatal cows and neonatal calves. Nippon Juigaku Zasshi 1987;49:469475.

  • 6.

    Kashiwazaki Y, Maede Y, Namioka S. Transformation of bovine peripheral blood lymphocytes in the perinatal period. Nippon Juigaku Zasshi 1985;47:337339.

    • Search Google Scholar
    • Export Citation
  • 7.

    Kehrli ME Jr, Nonnecke BJ, Roth JA. Alterations in bovine lymphocyte function during the periparturient period. Am J Vet Res 1989;50:215220.

    • Search Google Scholar
    • Export Citation
  • 8.

    Mallard BA, Dekkers JC, Ireland MJ, et al. Alteration in immune responsiveness during the peripartum period and its ramification on dairy cow and calf health. J Dairy Sci 1998;81:585595.

    • Search Google Scholar
    • Export Citation
  • 9.

    Detilleux JC, Kehrli ME Jr, Stabel JR, et al. Study of immunological dysfunction in periparturient Holstein cattle selected for high and average milk production. Vet Immunol Immunopathol 1995;44:251267.

    • Search Google Scholar
    • Export Citation
  • 10.

    Concha C. Cell types and their immunological functions in bovine mammary tissues and secretions—a review of the literature. Nord Vet Med 1986;38:257272.

    • Search Google Scholar
    • Export Citation
  • 11.

    Watson DL. Immunological functions of the mammary gland and its secretion—comparative review. Aust J Biol Sci 1980;33:403422.

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Determination of serologic and colostral response in late-gestation cows vaccinated with a Mycoplasma bovis bacterin

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  • 1 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 2 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 3 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 4 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 67432.
  • | 5 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 6 Department of Animal Science, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211.

Abstract

Objective—To determine whether vaccinating cows during late gestation against Mycoplasma bovis will result in adequate concentrations of M bovis–specific IgG1 in serum, colostrum, and milk.

Animals—78 dairy cows.

Procedures—Serum samples were obtained 60 and 39 days prior to expected parturition in vaccinated and control cows from a single herd. Serum and colostrum samples were also obtained at parturition. Milk samples were obtained 7 to 14 days after parturition. Samples were analyzed for anti–M bovis IgG1 concentrations.

Results—Prior to vaccination, control and vaccinated cows had similar anti–M bovis IgG1 concentrations. After initial vaccination and subsequent booster and at parturition, there was a significant difference between the 2 groups, with vaccinated cows having higher IgG concentrations. Colostrum from vaccinated cows had higher anti–M bovis IgG1 concentrations, compared with control cows; however, IgG1 concentrations in milk did not differ between the 2 groups.

Conclusions and Clinical Relevance—Vaccination of late-gestation cows resulted in increased concentrations of anti–M bovis IgG1 in colostrum. However, ingestion of colostrum by calves may not guarantee protection against M bovis infection.

Abstract

Objective—To determine whether vaccinating cows during late gestation against Mycoplasma bovis will result in adequate concentrations of M bovis–specific IgG1 in serum, colostrum, and milk.

Animals—78 dairy cows.

Procedures—Serum samples were obtained 60 and 39 days prior to expected parturition in vaccinated and control cows from a single herd. Serum and colostrum samples were also obtained at parturition. Milk samples were obtained 7 to 14 days after parturition. Samples were analyzed for anti–M bovis IgG1 concentrations.

Results—Prior to vaccination, control and vaccinated cows had similar anti–M bovis IgG1 concentrations. After initial vaccination and subsequent booster and at parturition, there was a significant difference between the 2 groups, with vaccinated cows having higher IgG concentrations. Colostrum from vaccinated cows had higher anti–M bovis IgG1 concentrations, compared with control cows; however, IgG1 concentrations in milk did not differ between the 2 groups.

Conclusions and Clinical Relevance—Vaccination of late-gestation cows resulted in increased concentrations of anti–M bovis IgG1 in colostrum. However, ingestion of colostrum by calves may not guarantee protection against M bovis infection.

Mycoplasma bovis, a bovine bacterial pathogen, has been implicated as a cause of contagious mastitis, respiratory tract disease, arthritis, otitis media and interna, and a host of other disease processes in cattle. It was first identified as a contagious bovine mastitis pathogen in 1961,1 and much effort has been made toward establishing proper diagnostic, treatment, prevention, and control measures.

Mycoplasma bovis–associated diseases are costly to beef and dairy production systems worldwide. Cattle producers incur costs to treat infected animals as well as replace those culled from the production facility. Dairy farmers may incur additional costs in the form of decreased premiums or penalties in bulk tank sales.

Like other Mycoplasma spp, M bovis lacks a typical bacterial cell wall.2 Traditional bacteriocidal antimicrobials, such as ceftiofur and penicillin G procaine, which inhibit bacterial cell wall synthesis, have little effect against M bovis. Therefore, when faced with treating an M bovis – associated respiratory tract disease in their cattle, producers may perceive that a treatment failure has occurred if cattle are treated with such antimicrobials without results of bacteriologic culture. Vaccinating dams against respiratory tract pathogens, such as infectious bovine rhinotracheitis virus, parainfluenza 3 virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, Mannhaemia hemolytica, and Pasteurella multocida, prior to calving has resulted in increases in specific IgG concentrations against these pathogens, which are then passively transferred to calves via colostrum.

The purpose of the controlled trial reported here was to determine whether vaccinating cows during late gestation against M bovis would result in adequate concentrations of M bovis–specific IgG1 in serum, colostrum, and milk.

Materials and Methods

Cattle and study design—Seventy-eight Holstein cows with confirmed expected parturition dates were used. The study population originated from and was housed at the University of Missouri-Columbia Foremost Dairy. Cows had ceased lactation 0 to 6 days prior to enrollment. Upon initial enrollment, a coin flip was used to allocate the initial cow into its respective study group with binary allocation into the 2 study groups that followed. Cows assigned to the vaccinated group were given 2 mL of M bovis bacterina SC in the neck area on the right side. Simultaneously, a 10-mL blood sample was collected aseptically via the coccygeal vein. Cows assigned to the control group received no vaccination. A blood sample was collected from these cows in the same fashion. Three weeks later, a booster vaccination of the same product was given to the vaccinated group and a second blood sample was obtained in the same fashion. A blood sample was obtained from the control cows at the same time and in the same fashion. Cows were monitored for impending parturition by farm management personnel. At the time of calving, a third blood sample was obtained. All blood samples were centrifuged, and the serum sample was decanted into a 5-mL serum collection tube. Cow number, sample number, and collection date were used to identify samples. Samples were frozen at −10°C until serologic analysis was performed. At calving, a 10-mL composite sample of colostrum was obtained from each cow in an aseptic fashion and collected into a 10-mL tube. Colostrum samples were frozen at −10°C until serologic analysis was performed. A composite milk sample was collected in a similar way at 7 to 13 days of lactation.

Serologic, colostrum, and milk analysis—Analysis of serum, colostrum, and milk samples was performed by American Animal Health Laboratory in Grand Prairie, Tex. Serum samples were analyzed for M bovis –specific IgG1 concentration. Colostrum and milk samples were analyzed for M bovis –specific IgG1 concentrations. The technical staff was unaware of the study group from which each sample originated.

ELISA—An ELISA was developed and used for analysis of submitted samples. Mycoplasma bovis –coated plates prepared by American Animal Health Laboratory were used. Plates were blocked with gelatin. Bovine serum samples were diluted 1:100 with PBS-Tween 20 solution. One hundred microliters of sample was used. Serum samples collected from M bovis –infected cattle were used as positive controls at 2X serial dilution starting at 1:100. Fetal bovine serum at a 1:100 dilution was used as a negative control. Samples were incubated at 37°C for 1 to 2 hours. Samples were washed 3 times with dH20 or PBS-Tween 20 solution. One hundred microliters of anti–bovine IgG1 for serum, milk, and colostrum analysis was added with horseradish peroxidase conjugate. Samples were again incubated for 1 to 2 hours at 37°C. Samples were washed 6 times with dH20 or PBS-Tween 20 solution. One hundred microliters of 3,3′,5,5′-tetramethylbenzidine substrate was added and left to develop for 10 minutes. One hundred microliters of 0.5M H2SO4 was added to stop the reaction. Optical density was read with a microplate reader with dual wavelength of 450 and 630 nm. To minimize ELISA plate-to-plate variability, S:P ratios were calculated for each sample by use of the following formula:

article image

where OD450 is optical density measured at a wavelength of 450 nm.

Statistical analysis—Anti–M bovis IgG1 antibody responses are indicated as mean ± SEM S:P ratios. Variance ratio tests were performed for each comparison between groups. Significant differences in variance were adjusted by use of the Welch approximation. Mean serum, colostrum, and milk IgG1 antibody response were compared between the 2 groups by use of the Student t test. Mean serum IgG1 antibody response in groups were compared by use of paired t tests.3

Linear regression models that predicted maternal anti–M bovis IgG1 S:P ratios at parturition as a function of maternal serum anti–M bovis IgG1 S:P ratios 39 days prior to parturition were constructed. Linear regression models that predicted colostral anti–M bovis IgG1 S:P ratios as a function of maternal serum anti–M bovis IgG1 S:P ratios 39 days prior to parturition and at actual date of parturition were also constructed.4 Variables with associated values of P ≤ 0.05 were included. The regression coefficient, P value, and correlation coefficient were reported for each significant regression model.

Differences were considered significant at P < 0.05. All statistical analyses were performed by use of specialized software.b

Results

Serologic results—The 2 groups consisted of 40 control cows and 38 vaccinated cows. Mean S:P ratio values between the control and vaccinated groups were similar upon enrollment in the study and at concurrent primary vaccination (0.2813 ± 0.012 and 0.2514 ± 0.021, respectively). Mean S:P ratio values were significantly different between the control and vaccinated groups at the time of second vaccination 3 weeks following enrollment (0.3228 ± 0.019 and 0.5535 ± 0.031, respectively). Mean S:P ratio values were significantly different between the control and vaccinated groups at the time of parturition as well (0.2321 ± 0.023 and 0.3090 ± 0.030, respectively; Figure 1). Mean ± SD nonlactating periods for the cows in the control and vaccinated groups were 53.51 ± 20 days and 55.00 ± 20.54 days, respectively (P = 0.75).

There was a significant difference in mean S:P ratio values between values measured at 39 versus 60 days prior to expected parturition and between parturition and 39 days prior to expected parturition in the control and vaccinated groups. There was also a significant difference between values measured at 60 days prior to expected parturition date and actual date of parturition in the control and vaccinated groups.

Figure 1—
Figure 1—

Mean serum S:P ratios for IgG1 antibodies against Mycoplasma bovis in control (n = 40 [gray bars]) and vaccinated (38 [black bars]) cows at 60 and 39 days prior to expected parturition date. Day 0 represents actual date of parturition. aSignificant (P < 0.05) difference, compared with the control group. bSignificant (P < 0.05) difference, compared with prior measurement for the control group. cSignificant (P < 0.05) difference, compared with prior measurement for the vaccinated group. dSignificant (P < 0.05) difference, compared with value at −60 days prior to expected parturition for the control group. eSignificant (P < 0.05) difference, compared with value at −60 days prior to expected parturition for the vaccinated group.

Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.912

Colostrum—Colostrum samples from 28 control cows and 26 vaccinated cows were available for analysis. Colostrum samples from 24 of the cows available from the serology study were not available for analysis. These samples were not collected at the time of parturition or appropriate aliquots were not obtained from collected colostrum before the samples were frozen. Mean colostrum IgG1 S:P ratio values were lower in the control group (Figure 2) . Mean S:P ratio values between the control and vaccinated groups were significantly (P = 0.011) different (0.3546 ± 0.050 and 0.5661 ± 0.087, respectively).

Figure 2—
Figure 2—

Mean colostrum S:P ratios for IgG1 antibodies against Mycoplasma bovis between control (n = 28 [gray bars]) and vaccinated (26 [black bars]) cows and mean milk S:P ratios between control (n = 32 [gray bars]) and vaccinated (27 [black bars]) cows. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.912

Milk—Milk samples from 32 control cows and 27 vaccinated cows were available for analysis. Milk samples from 19 of the cows available from the serology study were not available for analysis. These samples were not collected at the appropriate time according to the study protocol of 7 to 13 days of lactation or these cows had been culled soon after parturition. Mean milk IgG1 S:P ratio values were lower in the control group (Figure 2). However, mean S:P ratio values were not significantly (P = 0.322) different between the control and vaccinated groups (0.5585 ± 0.047 and 0.6511 ± 0.079, respectively). A significant difference in mean S:P ratio was found between colostrum and milk of cows in the control group (P = 0.013) but not in cows in the vaccinated group (P = 0.437).

Regression modeling—Predicting maternal anti–M bovis IgG1 S:P ratios at parturition as a function of maternal serum anti–M bovis IgG1 S:P ratios 39 days prior to parturition yielded a significant linear model, as did predicting colostral anti–M bovis IgG1 S:P ratios as a function of maternal serum anti–M bovis IgG1 S:P ratios 39 days prior to expected parturition (Table 1). Predicting colostral anti–M bovis IgG1 S:P ratios as a function of maternal serum anti–M bovis IgG1 S:P ratios at actual date of parturition did not yield a significant linear model.

Table 1—

Results of linear regression models predicting maternal anti—Mycoplasma bovis IgG1 S:P ratios at parturition as a function of maternal serum anti—M bovis IgG1 S:P ratios 39 days prior to parturition, predicting colostral anti—M bovis IgG1 S:P ratios as a function of maternal serum anti—M bovis IgG1 S:P ratios 39 days prior to parturition, and predicting colostral anti—M bovis IgG1 S:P ratios as a function of maternal anti—M bovis IgG1 S:P ratios at parturition. Y-intercepts, regression coefficients, and correlation coefficients are not reported for nonsignificant linear models.

ComparisonY-intercept (anti—M bovis IgG1 S:P ratio)Regression coefficient (anti—M bovis IgG1 S:P ratio)r2P value
Maternal serum 39 days prior to and at actual parturition0.07350.45970.273< 0.001
Maternal serum 39 days prior to expected parturition and colostrums0.09460.86900.1310.01
Maternal serum at actual parturition and colostrumsNSNSNA0.401

NS = Not significant. NA = Not applicable.

Discussion

In the present study, on the basis of herd records, cases of Mycoplasma spp mastitis were not recorded and results of random bulk tank bacteriologic cultures for Mycoplasma spp were negative. To the authors' knowledge, cows enrolled in this controlled trial had never received a prior M bovis bacterin. As expected, all cows entering this trial had statistically similar anti–M bovis S:P ratios. At the time of booster vaccination, there was a substantially larger mean serum S:P ratio in the vaccinated group versus the control group, and this continued though parturition. There was a noticeable decrease in mean serum S:P ratios at time of calving. If this variable is used to gauge the overall immune status of the periparturient dams, these findings are consistent with other studies.5–7 These alterations of immune status in dairy cows begin approximately 3 weeks prior to calving and continue until 3 weeks after calving.8 Serum IgG1 concentrations typically decrease at time of parturition.7,9 Although the present study only evaluated mean concentrations of anti–M bovis IgG1, there was a difference between mean IgG1 concentrations at calving versus enrollment 60 days prior to parturition in both groups, with vaccinated cows having higher concentrations at calving.

Predicting the concentration of anti–M bovis IgG1 in the serum of cows at parturition can be accomplished by knowledge of serum concentrations approximately 5 to 6 days before parturition. High serum concentration before parturition is associated with high serum concentration at parturition, but the correlation is moderately weak. The same correlation cannot be made between serum IgG1 concentration at parturition and colostrum concentration of IgG1, which appeared to have no linear relationship.

Colostrogenesis begins about 15 days prior to parturition. This event is marked by active and selective transport of immunoglobulin, generally of the IgG1 subclass, into the mammary gland.10,11 Colostral quality and subsequent calf health can be influenced by vaccination status of the dam, exposure of the dam to infectious diseases, production of protective antibodies, and antibody transport into the mammary gland.8 In the present study, vaccinated cows produced colostrum higher in anti–M bovis IgG1 concentration than did their nonvaccinated herdmates. Again, the purpose of this study was not to determine the effective concentration of colostrum IgG1 needed to protect calves against M bovis. Calves have not been challenged with M bovis exposure to determine if any protection is provided via colostrum of vaccinated dams.

Analysis of milk, however, revealed no significant differences between the 2 study groups. With the purpose of vaccinating cows in late gestation for the sole benefit of promoting immunity in neonatal calves, milk anti–M bovis IgG1 is of less benefit than colostral anti–M bovis IgG1. Calf feeding practices in modern dairy production systems usually involve feeding a balanced milk replacer, rather than dump milk from lactating cows. However, milk anti–M bovis IgG1 concentration is greater than colostrum anti–M bovis IgG1 concentration. Higher IgG1 concentrations are expected in colostrum than in milk. Further investigation into the colostrum and milk ELISA used in this study is needed to further validate their practical usage as rapid tests for determining whether adequate or protective concentrations of anti–M bovis IgG1 are present in dams.

ABBREVIATIONS

dH20

Distilled, deionized water

S:P

Sample to positive

a.

Pulmo-Guard MpB, Boehringer Ingelhiem Vetmedica Inc, St Joseph, Mo.

b.

Intercooled Stata for Windows, version 9.0, Stata Corp, College Station, Tex.

References

  • 1.

    Hale HH, Helmboldt CF, Plastridge WN, et al. Bovine mastitis caused by a Mycoplasma species. Cornell Vet 1962;52:582591.

  • 2.

    Garrity GM, Boone DR, Castenholz RW, eds. Bergey's manual of systematic bacteriology. Vol 1. 2nd ed. New York: Springer-Verlag Inc, 2001.

  • 3.

    Zar JH. Biostatistical analysis. 4th ed. Upper Saddle River, NJ: Prentice-Hall Inc, 1999.

  • 4.

    Kutner MH, Nachtsheim C, Neter J. Applied linear regression models. 4th ed. Boston, NY: McGraw-Hill Book Co/Irwin, 2004.

  • 5.

    Ishikawa H. Observation of lymphocyte function in perinatal cows and neonatal calves. Nippon Juigaku Zasshi 1987;49:469475.

  • 6.

    Kashiwazaki Y, Maede Y, Namioka S. Transformation of bovine peripheral blood lymphocytes in the perinatal period. Nippon Juigaku Zasshi 1985;47:337339.

    • Search Google Scholar
    • Export Citation
  • 7.

    Kehrli ME Jr, Nonnecke BJ, Roth JA. Alterations in bovine lymphocyte function during the periparturient period. Am J Vet Res 1989;50:215220.

    • Search Google Scholar
    • Export Citation
  • 8.

    Mallard BA, Dekkers JC, Ireland MJ, et al. Alteration in immune responsiveness during the peripartum period and its ramification on dairy cow and calf health. J Dairy Sci 1998;81:585595.

    • Search Google Scholar
    • Export Citation
  • 9.

    Detilleux JC, Kehrli ME Jr, Stabel JR, et al. Study of immunological dysfunction in periparturient Holstein cattle selected for high and average milk production. Vet Immunol Immunopathol 1995;44:251267.

    • Search Google Scholar
    • Export Citation
  • 10.

    Concha C. Cell types and their immunological functions in bovine mammary tissues and secretions—a review of the literature. Nord Vet Med 1986;38:257272.

    • Search Google Scholar
    • Export Citation
  • 11.

    Watson DL. Immunological functions of the mammary gland and its secretion—comparative review. Aust J Biol Sci 1980;33:403422.

Contributor Notes

Dr. Calloway's present address is Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.

Supported by Boehringer Ingelhiem Vetmedica Inc, St Joseph, Mo.

The authors thank Julie Holle, Kathy Smith, and Dr. Craig Jones for technical assistance.

Address correspondence to Dr. Calloway.