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      Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows

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          Abstract

          In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells ( Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species ( E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

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          Defining postpartum uterine disease in cattle.

          Uterine function is often compromised in cattle by bacterial contamination of the uterine lumen after parturition, and pathogenic bacteria often persist, causing uterine disease, a key cause of infertility in cattle. However, the definition or characterization of uterine disease frequently lacks precision or varies among research groups. The aim of the present paper was to provide clear clinical definitions of uterine disease that researchers could adopt. Puerperal metritis should be defined as an animal with an abnormally enlarged uterus and a fetid watery red-brown uterine discharge, associated with signs of systemic illness (decreased milk yield, dullness or other signs of toxemia) and fever > 39.5 degrees C, within 21 days after parturition. Animals that are not systemically ill, but have an abnormally enlarged uterus and a purulent uterine discharge detectable in the vagina, within 21 days post partum, may be classified as having clinical metritis. Clinical endometritis is characterised by the presence of purulent (> 50% pus) uterine discharge detectable in the vagina 21 days or more after parturition, or mucuopurulent (approximately 50% pus, 50% mucus) discharge detectable in the vagina after 26 days post partum. In the absence of clinical endometritis, a cow with subclinical endometritis is defined by > 18% neutrophils in uterine cytology samples collected 21-33 days post partum, or > 10% neutrophils at 34-47 days. Pyometra is defined as the accumulation of purulent material within the uterine lumen in the presence of a persistent corpus luteum and a closed cervix. In conclusion, we have suggested definitions for common postpartum uterine diseases, which can be readily adopted by researchers and veterinarians.
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            Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle.

            Bacteria contaminate the uterus of most dairy cattle after parturition and endometritis causes infertility. An endometritis score can be ascribed based on the vaginal mucus character and odour but it is not clear if the clinical score reflects the number of uterine bacteria or the inflammatory response. The present study tested the hypothesis that clinical evaluation of endometritis reflects the number of bacteria present in the uterus, and the acute phase protein response. Swabs (n = 328) were collected from the uterine lumen of dairy cattle, 21 and 28 days postpartum, vaginal mucus was scored for character and odour, and blood samples collected for acute phase protein measurement. Bacteria were identified following aerobic and anaerobic culture, and the bacterial growth density was scored semi-quantitatively. When bacteria were categorised by their expected pathogenic potential in the uterus, purulent or fetid odour vaginal mucus was associated with the growth density of pathogenic bacteria but not opportunist contaminants. When bacteria were analysed independently, Arcanobacterium pyogenes, Proteus and Fusobacterium necrophorum growth densities were associated with mucopurulent or purulent vaginal mucus. The bacterial growth densities for A. pyogenes, Escherichia coli, non-hemolytic Streptococci, and Mannheimia haemolytica were associated with a fetid mucus odour. Peripheral plasma concentrations of alpha(1)-acid glycoprotein were higher if there was a fetid compared with a normal vaginal mucus odour (1.50 +/- 0.09 mg/mL versus 1.05 +/- 0.02 mg/mL, P < 0.001), but did not differ significantly between vaginal mucus character scores. The evaluation of the character and odour of vaginal mucus reflects the number of bacteria in the uterus, and the acute phase protein response.
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              X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli.

              Type 1 pili-adhesive fibers expressed in most members of the Enterobacteriaceae family-mediate binding to mannose receptors on host cells through the FimH adhesin. Pilus biogenesis proceeds by way of the chaperone/usher pathway. The x-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli at 2.5 angstrom resolution reveals the basis for carbohydrate recognition and for pilus assembly. The carboxyl-terminal pilin domain of FimH has an immunoglobulin-like fold, except that the seventh strand is missing, leaving part of the hydrophobic core exposed. A donor strand complementation mechanism in which the chaperone donates a strand to complete the pilin domain explains the basis for both chaperone function and pilus biogenesis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                17 March 2014
                : 9
                : 3
                : e91734
                Affiliations
                [1]Department of Population Medicine & Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America
                Universidad Nacional de La Plata, Argentina
                Author notes

                Competing Interests: Rodrigo Carvalho Bicalho, Robert Owen Gilbert, Marcela Luccas de Souza Bicalho, and Vinicius Silva Machado have a pending patent entitled “Vaccine for the prevention of uterine diseases” (Application number 61/731,333) on the vaccine technology described in this manuscript. This study was funded by Merck Animal Health. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

                Conceived and designed the experiments: RCB VSM ROG SHC. Performed the experiments: VSM MLdSB EBdSM RR BLR SL TS AK CF EKG. Analyzed the data: RCB VSM. Contributed reagents/materials/analysis tools: RCB. Wrote the paper: VSM RCB GO.

                Article
                PONE-D-13-39721
                10.1371/journal.pone.0091734
                3956715
                24638139
                766a9603-b2a4-459f-aa2f-7ab0d4570e21
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 27 September 2013
                : 14 February 2014
                Page count
                Pages: 11
                Funding
                This study was funded by a grant from Merck Animal Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Agriculture
                Animal Management
                Animal Welfare
                Immunology
                Vaccination and Immunization
                Vaccine Development
                Clinical Immunology
                Immunity
                Veterinary Science
                Animal Types
                Large Animals
                Veterinary Diseases
                Veterinary Virology
                Veterinary Medicine
                Livestock Care
                Veterinary Epidemiology
                Veterinary Microbiology
                Veterinary Pathology
                Medicine and Health Sciences

                Uncategorized
                Uncategorized

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