Abstract Book of EAVLD2012 - eavld congress 2012

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S1 - P - 22 MILK AMYLOID A AND ITS USEFULNESS IN THE LABORATORY DIAGNOSIS OF MASTITIS Gabriel Kováč 1 , Csilla Tóthová 1 , Oskar Nagy 1 , Martin Kováč 2 1 University of Veterinary Medicine and Pharmacy, Clinic for Ruminants, Košice, Slovak Republic 2 Regional Veterinary and Food Administration, Košice, Slovak Republic Dairy cows, mastitis, milk amyloid A, haptoglobin, serum amyloid A Introduction Mastitis is recognized as one of the most important diseases affecting dairy cattle. Raised levels of major acute phase proteins in cattle, haptoglobin and serum amyloid A, have previously been shown in milk from cows with clinical mastitis as a result of the leakage of these proteins from the blood to the milk (Eckersall et al., 2001). Further investigations showed an extrahepatic synthesis of specific isoform of amyloid A directly in mammary epithelial cells, the so called milk amyloid A (MAA) (McDonald et al., 2001). Therefore, this work was aimed at the evaluation of the influence of clinical and sub-clinical mastitis in dairy cows on the concentrations of MAA in milk samples, and selected acute phase proteins in blood serum. Materials & methods Into the evaluation we included 41 dairy cows of a low-land black spotted breed and its crossbreeds with various clinical findings on the mammary gland. These cows were in the 3rd – 4th lactation, but not in the period shortly after parturition. Clinical examination of the mammary gland was performed by visual inspection and palpation. Clinical mastitis was diagnosed by the presence of observable signs of inflammation in the infected quarter such as swelling, heat, pain or redness, and by the presence of clots and flakes in the milk, or by its abnormal color or consistency. To detect sub-clinical mastitis the Californian Mastitis Test (CMT) was performed. According to the results of the clinical examination of the udder and to the results of CMT the animals were divided into 4 groups: Group I – cows without clinical changes on the mammary gland and with negative CMT (n = 7), Group II – cows without clinical changes on the mammary gland and with weakly positive CMT (n = 12), Group III – cows without clinical changes on the mammary gland and with strongly positive CMT (n = 13), Group IV – cows with clinical changes on the mammary gland and changes in milk appearance (n = 9). Milk samples were collected into plastic tubes by hand-stripping. Blood samples were collected by direct puncture of v. jugularis. Milk samples were used to assess the concentrations of milk amyloid A (M-SAA, ng/ml), and haptoglobin (Hp, mg/ml) and serum amyloid A (SAA, μg/ml) were assessed in blood samples. Haptoglobin was assessed using commercial colorimetric kits (Tridelta Development, Ireland) in microplates. SAA and M-SAA were analysed by commercial ELISA kits (Tridelta Development, Ireland). The optical densities were read on automatic microplate reader Opsys MR (Dynex Technologies, USA) at an optical density of 630 nm for Hp, and 450 nm using 630 as reference for SAA and M-SAA. The analysis of the significance of differences in measured values between cows with various clinical findings on the mammary gland was performed by Kruskal-Wallis nonparametric ANOVA test and Dunn's Multiple Comparisons Test. Results M-SAA concentrations in milk samples differed significantly between the groups (P < 0.001), with concentrations in samples from cows with clinical mastitis (group IV) being significantly higher than in samples from groups I and II (P < 0.001 and P < 0.05, respectively, Table 1). The concentrations of M-SAA in milk samples increased with increasing CMT score. The serum concentrations of Hp showed also tendency of gradual significant increase with increasing CMT score and clinical changes on the mammary gland (P < 0.05, Table 1). The highest mean Hp concentration we found in cows with clinically manifested signs of mastitis. Similarly, SAA concentrations differed significantly between the evaluated groups of cows (P < 0.05), with the highest mean concentration in animals with clinical signs of mastitis. However, the differences in the obtained results of Hp and SAA concentrations between the evaluated groups of cows were less significant (P < 0.05) compared with MAA concentrations. The mean SAA concentrations found in cows from group I and group II were roughly uniform. Table 1: Concentrations of evaluated variables in dairy cows Variable I II III IV P < M- x 325.7 A,B 1433.1 a 3910.4 A 6073.8 B,a SAA ±sd 173.8 949.2 2145.8 4414.0 0.001 Hp x 0.046 0.122 0.299 0.329 ±sd 0.053 0.263 0.314 0.339 0.05 SAA x 29.7 27.6 a 48.2 71.5 a ±sd 27.6 28.0 42.5 31.5 0.05 The same indexes in lines mean significance of differences in values between the groups: a – P < 0.05; A, B – P < 0.01 Discussion & conclusions The results of our study indicate that inflammatory diseases of the mammary gland lead to an increase in the concentrations of M-SAA. Compared to Hp and SAA, M-SAA is synthesized directly in the mammary epithelia of the udder in response to infection (Jacobsen et al. 2005). Therefore, M-SAA is believed to be a more sensitive indicator of mastitis; it accumulates in milk only during mammary inflammation. Petersen et al. (2005) reported that M-SAA concentrations, similarly to our results, were higher in quarters with mastitis compared to healthy quarters. Moreover, mastitis may activate systemic inflammatory reactions, including the induction of the synthesis of acute phase proteins by the liver. The results of our study showed that the concentrations of Hp and SAA were higher in serum from cows with clinical mastitis, and increased with increasing CMT score. These results are in line with previous studies (Eckersall et al. 2001). It appears that localized severe inflammation of the udder is sufficiently intense to induce a measurable systemic acute phase response. The concentrations of measured acute phase proteins had a tendency to be higher in the serum from the cows with local signs of mastitis and also in cows without clinical changes on the mammary gland, but with positive CMT. However, the finding that the differences in Hp and SAA concentrations observed between the groups of cows were less significant than the differences in M-SAA concentrations means that the measuring of serum concentrations of some acute phase proteins would be less useful to the evaluation of the severity of mastitis than the measuring of the concentrations of M-SAA directly in milk samples. Generated data suggest the usefulness of M-SAA for diagnosis of bovine sub-clinical mastitis, as well as in the determination of the severity of mastitis, suggesting that its assessment as a part of the laboratory diagnosis would be a valuable supplementation to the proper clinical diagnosis and determination of other blood laboratory parameters. Acknowledgements This work was supported by VEGA Scientific Grant No 1/0592/12 from the Ministry of Education, and by Slovak Research and Development Agency under contract No. APVV-0475-10. References 1. Eckersall, PD, Young, FJ, McComb, C, Hogarth, CJ, Safi, S, Weber, A, McDonald, T, Nolan, AM, Fitzpatrick, JL (2011). Acute phase proteins in serum and milk from dairy cows with clinical mastitis. Vet Rec, 148, 35-41 2. Jacobsen, S, Niewold, TA, Kornalijnslijper, E, Toussaint, MJM, Gruys, E (2005). Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk. Vet Immunol Immunopathol, 104, 21-31 3. McDonald, TL, Larson, MA, Mack, DR, Weber, A (2001). Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A3 (M-SAA3) into colostrums. Vet Immunol Immunopathol, 83, 203-211 4. Petersen, HH, Gardner, IA, Rossitto, P, Larsen, HD, Heegaard, PMH (2005). Accuracy of milk amyloid A (MAA) concentration and somatic cell count for diagnosing bovine mastitis. Proceedings of the 5 th International Colloquim on Animal Acute Phase Protein, Dublin, Ireland, March 14-15, 2005, 43-44
S1 - P - 23 RELIABLE AND EARLY DETECTION OF SALMONELLA IN PIGS Tilman Kühn 1 , Patrik Buholzer 2 1 Synlab.vet Leipzig, Leipzig, Germany 2 Prionics AG, Schlieren, Switzerland Introduction Salmonellosis is one of the most important zoonotic diseases and can cause serious clinical symptoms in humans. Pigs have been recognized as an important source of Salmonella infections. The existence of this disease presents great risks for human health. Salmonella infections of animals intended for the food industry play an important role in public health as these animals are considered to be the major source of human Salmonella infections. The PrioCHECK Salmonella Ab porcine 2.0 was developed for large-scale screening of pigs and for application in Salmonella control programs. Here we show that the test clearly discriminates positive and negative samples through its large dynamic range. As the test also allows earlier detection of a Salmonella infection in pigs, than competitor tests, it is the diagnostic tool of choice for Salmonella control programs. Materials & methods Two separate experiments were performed. In the first experiment serum samples of ten pigs (4 negative and 6 Salmonella positive samples) were tested with both the PrioCHECK Salmonella Ab porcine 2.0 and a competitor test. Additionally, the Salmonella status was determined in ten meat juice samples with both tests. In the second experiment, 40 pigs were experimentally inoculated with live Salmonella vaccine. Serum samples were taken at the day of inoculation, 3, 6 and 9 weeks post inoculation and were tested with the PrioCHECK Salmonella Ab porcine 2.0 and two competitor tests both validated for use in Germany. The results of the three tests were compared. Results In the first experiment, both tests identified all positive and negative samples correctly, but the positive samples gave a much higher values with the PrioCHECK Salmonella Ab porcine 2.0 and therefore results were more clear-cut than those obtained with the competitor test. The average percentage positivity (PP) of positive samples of the PrioCHECK Salmonella Ab porcine 2.0 exceeded that of the competitor test by over 70 %. However, the PP values of negative samples were equal to, or lower than those of the competitor test. This means that the test does not generally generate high values, but is specific for positive samples. The PrioCHECK Salmonella Ab porcine 2.0 also earlier identifies animals experimentally infected with Salmonella. In serum samples of experimentally infected pigs, the PrioCHECK Salmonella Ab porcine 2.0 identified 25 samples as positive at 3 weeks post inoculation. 17 out of these 25 samples were missed by at least one competitor test (13 samples) at three weeks post inoculation, or only detected by the other tests at a later time post inoculation (4 samples). Discussion & conclusions The data show that the results obtained with PrioCHECK Salmonella Ab porcine 2.0 showed a large dynamic range and therefore allow reliable discrimination between positive and negative samples. This dynamic range means that the cut-off of 40% PP -- implemented in most countries -- could easily be lowered to 20% without loss of specificity. The application of the PrioCHECK Salmonella Ab porcine 2.0 reliably helps control Salmonella infections even early after infection. References 1. B. Nielsen, D. Baggesen, F. Bager, J. Haugegaard, P. Lind Veterinary Microbiology 47 (1995) 205-218 2. H.M.F. van der Heijden. First International Ring Trial of ELISA’s for Salmonella- antibody. Berl. Münch. Tierärztl Wschr. (2001) 389-392. 3. PrioCHECK® Salmonella Ab porcine 2.0, Prionics AG http://www.prionics.com/diseases-solutions/salmonellosis/ Food safety, diagnostic test, Salmonellosis
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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worked with many diagnostic compani
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Products and services for scientist
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16:4518.15 20:0023:00 [S1.]Oralpres
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Day4 Wednesday,4July2012 4 th sessi
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Oral presentations “General Sessi
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antigens or attenuated vaccines can
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conditions, e.g. Staphylococcus aur
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S1 - O - 1 ORAL FLUIDS - SAMPLE MAT
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S1 - O - 03 INTRODUCTION RATE OF A
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S1 - O -05 VALIDATION OF A COMPETIT
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S1 - O - 07 SUBTYPING OF SWINE INFL
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S1 - O - 13 THE FIRST YEAR OF OBLIG
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S1 - O - 15 SEROLOGICAL ANALYSIS AN
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S1 - O - 17 RING TEST EVALUATION FO
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S4 - P - 01 MOLECULAR CHARACTERIZAT
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S4 - P - 11 EFFECTIVE TESTING STRAT
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List of Abstracts (in order of numb
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S1-P-03 BALLAGI ANDREA S1-P-04 BENI
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S3-P-06 KELLER SELINA S3-P-07 KÖNI
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