Progress 10/01/06 to 09/30/09
Outputs
OUTPUTS: Prevalence and pathogenetic mechanisms of chlamydial infection: The working hypothesis of this investigation was that the main effect of chlamydial infection on milk would be due to interstitial colonization of the mammary gland. However, the rare detection of chlamydiae in milk samples strongly indicates that chlamydial colonization of the bovine mammary gland under conditions of an endemic C. pecorum infection of a herd is rare (<1% of udder quarter samples). The corollary of this finding is that the influence of chlamydial infection on the mammary gland primarily results from systemic effects of mucosal infections rather than from local effects of direct udder colonization by Chlamydia spp. Association of C. pecorum infection with milk production: Chlamydial PCR detection significantly associated with higher daily milk yield (36.24 vs 29.41 lb, P=0.004), higher milk fat (2.81 vs 2.66%, P=0.034), and lower milk protein (2.90% vs. 3.20%, P<0.001). Chlamydial detection in milk associated with higher milk SCC (5.31 vs. 5.09 log10 SCC, P<0.0001) and lower milk protein (2.73 vs. 3.05%, P<0.0001). Thus, detectable chlamydial infection was more frequently observed in higher producing cows, and detection of C. pecorum in this endemic herd infection suggested lower resistance rather than lower production of these cows. However, chlamydial infection of the mammary gland had a significant added effect on the inflammatory status of the mammary gland. Principal component and cluster analyses: Cases were separated in clusters of significantly different cases. Milk production in cluster 1 with significantly higher anti-C. pecorum serum IgM than cluster 2 is characterized by significantly higher yield and fat and lower SCC than cluster 2. Thus, a low immune response to chlamydial infection, as indicated by serum anti-C. pecorum IgM, but not the infection itself, associates with highly significant milk production losses. Since these losses are associated with mucosal, but largely not mammary gland chlamydial infections, they are likely precipitated by circulating inflammatory mediators and their influence on the bovine liver, the central metabolic organ in high-performance dairy cows. To test this hypothesis, additional serum parameters were analyzed. Serum cholesterol in dairy cows is an indicator of liver production of cholesterol transport proteins and is elevated in healthy and high milk yield- and fat-producing dairy cows as compared to low-producing cows under inflammatory stress, and is considered a "negative acute phase protein". Serum globulin is reduced in high-producing cows and is considered a surrogate of acute phase proteins with extended serum half-life. In fact, high producing cows in cluster 1 had highly significantly elevated serum cholesterol and highly significantly reduced serum globulin levels as compared to low-producing cluster 2 cows. These results strongly suggest that clinically asymptomatic chlamydial infection in cows with low anti-chlamydial immunity results in significant inflammatory stress on the liver and highly significantly reduces milk quality and production efficiency by up to 20%. PARTICIPANTS: The lead Graduate student on this project, Sudhir Ahluwalia, applied in June 2009 for funding by the Alabama EPSCoR Graduate Research Scholars Program and, based on the progress in this project, received 6 months of funding at $25,000/year until his PhD graduation in December 2009. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of this study shift the paradigm for the mechanisms of asymptomatic chlamydial dairy herd disease from the local effect of an isolated mammary gland infection to a systemic effect mediated largely by metabolic stress on the liver by circulating inflammatory mediators released from mucosal infection sites. The fact that it is the immune response to the chlamydial infection that determines the outcome, rather than the omnipresent infection itself, suggests a promising potential for immunoprophylactic and immunotherapeutic intervention to ameliorate the profound production losses incurred by endemic chlamydial infection of dairy cows. The project will provide value to the citizens of Alabama by meeting needs of Alabama agriculture through i) improving understanding and treatment of dairy herd health; ii) enabling better maintenance of milk quality and production; iii) contributing knowledge to vaccine-mediated prevention of bovine mastitis, thus reducing the prophylactic and therapeutic use of antibiotics.
Publications
- Ahluwalia, S., H. Maxwell, D. M. Carpenter, Y. Li, E. Chowdhury, and B. Kaltenboeck. 2009. Characterization of mastitis caused by natural Chlamydia spp. infection of dairy cows. Oral presentation. Proceedings of the 90th Annual Meeting of the Conference of Research Workers in Animal Disease. Chicago, Illinois, December 2009: Abstract 11.
- Ahluwalia, S. K., E. Heinen, J. Kronewetter, R. Schneider, M. Mueller, N. Schmeer, and B. Kaltenboeck. 2010. Post-abortion effects of Chlamydia abortus infection on the ovine mammary gland. Submitted for publication in Infection and Immunity.
- Ahluwalia, S. K., E. Heinen, R. Schneider, M. M. Wittenbrink, N. Schmeer, and B. Kaltenboeck. 2008. OmpA and antigenic diversity of bovine Chlamydophila pecorum strains. Proceedings of the 89th Annual Meeting of the Conference of Research Workers in Animal Disease. Chicago, Illinois, December 2008: Abstract 6, p. 130.
- Kaltenboeck, B., E. Heinen, R. Schneider, M. M. Wittenbrink, and N. Schmeer. 2009. OmpA and antigenic diversity of bovine Chlamydophila pecorum strains. Veterinary Microbiology 135:175-180.
- Ahluwalia, S., H. Maxwell, D. M. Carpenter, Y. Li, E. Chowdhury, and B. Kaltenboeck. 2009. Characterization of mastitis caused by natural Chlamydia spp. infection of dairy cows. Poster presentation at the 4th Biennial Meeting of the Chlamydia Basic Research Society, Little Rock, Arkansas, March 2009.
- Ahluwalia, S., D. M. Carpenter, H. Maxwell, and B. Kaltenboeck. Endemic Chlamydia pecorum infections reduce milk production in dairy cows via inflammatory liver injury. 2010. Submitted for publication in the Proceedings of the 12th International Symposium on Human Chlamydial Infection, Fuschl, Austria, June 2010.
- Ahluwalia, S., D. M. Carpenter, H. Maxwell, and B. Kaltenboeck. 2010. Endemic Chlamydia pecorum infections reduce milk production in dairy cows via inflammatory liver injury. Manuscript in preparation.
- Ahluwalia, S. K., J. Kronewetter, R. Schneider, M. Mueller, E. Heinen, N. Schmeer, and B. Kaltenboeck. 2008. Increased resistance against staphylococcal/streptococcal mastitis after chlamydial abortion in sheep. Presentation at the 4th Workshop of the National German Veterinary Reference Laboratory for Psittacosis at the Federal Institute for Animal Health - Friedrich-Loeffler Institute, Jena, Germany, September 2008: Abstract 13.
- Ahluwalia, S. K., E. Heinen, R. Schneider, M. M. Wittenbrink, N. Schmeer, and B. Kaltenboeck. 2008. OmpA and antigenic diversity of bovine Chlamydia pecorum strains. Proceedings of the Phi Zeta Research Emphasis Forum, College of Veterinary Medicine, Auburn University. Auburn, AL, November 2008: O4.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Sampling for research objective 1 has been performed from August 2007 to April 2008. A total of 23 dairy cows were sampled every other week for 20 weeks. Five of these cows were from dairy herd of the Auburn University Large Animal Clinic, and 18 from the EV Smith Dairy Unit of the Alabama Agricultural Experiment Station in Shorter, AL. Data about milk kg, protein, fat, milk bacterial culture, and somatic cells per quarter have been collected and tabulated for all 10 sampling time points. Total nucleic acids from 1,150 samples (40 milk samples and 10 vaginal cytobrush samples per cow x 23 cows) have been extracted and analyzed by single Chlamydia 23S rDNA real-time PCR. Ten (43.5%) of the cows were positive for Chlamydia spp. in any of the samples, with 6 cows positive only in vaginal cytobrush specimens, 2 positive only in milk samples, and 2 cows in both types of specimens. These data allow already major conclusions: 1) as anticipated, chlamydial infection is endemic in the Auburn and EV Smith dairy herds, and 2) even at endemic infections the Chlamydia detection rate in milk, despite extensive sampling, is too low for epidemiological analysis of the impact of chlamydial infection on milk production (quality and quantity) in dairy herds. Previous experiences in sheep and cows had shown that the intracellular chlamydiae can be much more frequently detected in mammary gland tissue biopsies than in the corresponding milk. Next we proceeded to a preliminary analysis of the data by repeated measure ANOVA of the milk somatic cell counts across the course of the complete sampling period. The results of this analysis confirm previous data that had indicated that any detection of chlamydiae in dairy cows associated with increased milk somatic cell counts. Interestingly, however, confirming data from a sheep abortion study, cows that had shown episodes of mastitis caused by extracelluar bacteria (e.g., Streptococcus agalactiae, Staphylococcus aureus) and were Chlamydia-positive had lower milk somatic cell counts than cows that were Chlamydia-negative. These data suggest that low-level inflammatory responses to chronic chlamydial infections reduce the severity of mastitis caused by extracellular bacterial pathogens. PARTICIPANTS: The lead Graduate student on this project, Sudhir Ahluwalia, applied in December 2007 for funding by the Alabama EPSCoR Graduate Research Scholars Program and, based on the progress in this project, received one-year funding at $25,000/year. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Overall, our preliminary data allow already major conclusions that will impact the continuation of this study and further analyses of the interaction between chlamydial infection and bovine mastitis: 1) Detection of chlamydiae in the bovine mammary gland will have to rely on tissue biopsies. Objective 2 of this study addresses precisely this question and will be initiated in September 2008. 2) Detection of chlamydiae on mucosal epithelium (vagina) correlates highly significantly with milk somatic cell counts. Therefore, in epidemiological studies, non-invasive sampling at mucosal sites (vagina, conjunctiva) will be sufficient to establish ongoing chlamydial infection. This will remove a major impediment to the study of bovine chlamydial mastitis.
Publications
- Ahluwalia, J. Kronewetter, R. Schneider, M. Mueller, E. Heinen, N. Schmeer, and B. Kaltenboeck. Differential immune response against Chlamydophila abortus associates with differential severity of staphylococcal and streptococcal mastitis in postpartum and post-abortion sheep. Poster presentation at the 3rd Biennial Meeting of the Chlamydia Basic Research Society, Louisville, Kentucky, March 2007.
- Biesenkamp-Uhe, C., Y. Li, H.-R. Hehnen, K. Sachse, and B. Kaltenboeck. 2007. Therapeutic Chlamydophila vaccine against bovine mastitis. Infection and Immunity 75:870-877.
- Kaltenboeck, B. The intriguing question of clinically silent Chlamydia infections in farm animals. Oral presentation. Proceedings of the 24th Symposium of the Veterinary Comparative Respiratory Society, Jena, Germany. October 2006: 75-78.
- Reinhold, P. and B. Kaltenboeck. Impact of clinically inapparent infections with chlamydiae on animal health in cattle. Oral presentation. Proceedings of the 13th International Conference on Production Diseases in Farm Animals, Leipzig, Germany, July 2007: 514-525.
- Li, Y., Borovkov, A., A. Loskutov, S. Ahluwalia, C. Wang, D. Gao, K. F. Sykes, and B. Kaltenboeck. Identification of Chlamydophila pecorum vaccine candidate genes by expression library immunization. Oral presentation. Proceedings of the 88th Annual Meeting of the Conference of Research Workers in Animal Disease. Chicago, Illinois, December 2007: Abstract 17.
- Ahluwalia, J. Kronewetter, R. Schneider, M. Mueller, E. Heinen, N. Schmeer, and B. Kaltenboeck. Differential immune response against Chlamydophila abortus associates with differential severity of staphylococcal and streptococcal mastitis in postpartum and post-abortion sheep. Oral presentation. Proceedings of the 88th Annual Meeting of the Conference of Research Workers in Animal Disease. Chicago, Illinois, December 2007: Abstract 18.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Research on objective 1 (the main objective) has been initiated in January 2007. From January through May 2007 all methods for this objective were established and extensively tested by pilot sampling of cows. During this work it became apparent, that the quantification of milk yield of individual quarter by means of electronic measurement in the Lactocorder was not sufficiently accurate. For this reason, this approach was abandoned. As a better replacement, a quarter milk meter was developed with the help of Mr. Mark Williams, a dairy specialist at the Dairy Herd Improvement Association located in the Agricultural Center at Louisiana State University in Baton Rouge, LA. This milk meter was constructed from four Tru-Test WB Ezi Test Milk Meters that are mounted on one bracket. Collected via a quarter milking claw, milk from each individual udder quarter is sampled by one each of the four parallel milk meters, subsequently combined into the collection vacuum hose and bulk milk
tank. This assembly allows statistically accurate quantification and sampling of the milk of each udder quarter and represents a major improvement for the research project. Due to breeding synchronization, the vast majority of cows at the dairy herd of the Auburn University Large Animal Clinic calve from August to December. For this reason, continuous sampling started in August 2007. Since the number of available cows was potentially not sufficient for the project, additional cows are sampled at the EV Smith Dairy Unit of the Alabama Agricultural Experiment Station in Shorter, AL. These animals are on a breeding schedule similar to the AU-LAC dairy herd. Currently 12 cows are sampled every other week, and the enrollment of 12-18 more cows is anticipated by October 2007. Therefore, completion of sampling is anticipated for March 2008, followed soon by the completion of continuously performed analyses of specimens. Therefore, the project proceeds at the anticipated time table.
PARTICIPANTS: Bernhard Kaltenboeck, Professor, Project Director, Department of Pathobiology, Auburn University, directs project. Sudhir Ahluwalia, Graduate Research Assistant, Department of Pathobiology, Auburn University, performs sampling of cows and sample processing. Herris Maxwell, Associate Clinical Professor, Department of Clinical Sciences, Auburn University, oversees clinical sampling. Calvin MacCarthy, Manager, EV Smith Dairy Research Station, Alabama Agricultural Extension Service, Shorter, AL, oversees samling at EV Smith Research Station.
TARGET AUDIENCES: Dairy research community worldwide, Cattle Veterinary Practitioners, Cattlemen, Dairyowners
PROJECT MODIFICATIONS: No major modificiation of goals and procedures of the projects were necessary.
Impacts
The project will provide value to the citizens of Alabama by meeting needs of Alabama agriculture through i) improving understanding and treatment of dairy herd health; ii) enabling better maintenance of milk quality and production; iii) contributing knowledge to vaccine-mediated prevention of bovine mastitis, thus reducing the prophylactic and therapeutic use of antibiotics.
Publications
- Presentation at the Department of Pathobiology on April 26, 2007, as part of the departmental seminar series by Sudhir Ahluwalia, the lead graduate student on the project: Title: Ruminant Mastitis Associated with Chlamydophila spp. Infection: Prevalence, Mechanism and Treatment. This presentation served to introduce the graduate research project of Dr. Ahluwalia and to fulfill in part the annual requirement for original scientific presentations by Biomedical Sciences Graduate students in the College of Veterinary Medicine.
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