2. Diagnostics 1 and 2

2.1

Time: Wednesday 15th May 11:00-12:30
Venue: Meeting room "Nørrebros Runddel"
Moderator: Karl Pedersen

 

O2.1-1 - On-farm diagnostics and integration in udder health consulting

11:00-11:30 hrs

Presenter

Volker Krömker, University of Applied Sciences and Arts, Germany

Abstract

The antibiotic therapy of mastitis is a core element of good udder health work. The extent to which antibiotic therapy can increase bacteriological cure rates and thus clinical cure and recurrence frequency depends on various criteria. An essential feature is the causative bacterial agent. A fast identification - i.e. usually within 24 hours - is required for consideration in therapeutic decision making. With the help of modern on-farm diagnostics, this can be achieved in a user-friendly way and become a central element of modern mastitis therapy. For most dairy farms, this means a reduction in the locally applied antibiotic doses while maintaining bacteriological cure rates.

 

O2.1-2 - On-farm culture - diagnostic validation and apparent pathogen prevalences in Denmark

11:30-11:45 hrs

Authors

Astrup LB., Center for Diagnostics, Danish Technical University. Kgs. Lyngby. Denmark
Pedersen K., National Veterinary Institute. Uppsala. Sweden
Farre M., SEGES. Aarhus. Denmark

Abstract

Little is known on the diagnostic accuracy in veterinary practice and general prevalence of mastitis pathogens in Denmark. Therefore, a pilot project was designed to examine the mastitis pathogens in the diagnostic samples used in veterinary practices.

In total, 14 veterinary clinics provided all of their diagnostic mastitis cultures during the last 4 month of 2016. The cultures were from clinical mastitis and were cultured on Selma Selma® agar plates. They were cultured according to diagnostics protocol at the clinic. After diagnosis was completed, the clinics send the plates to DTU for validation. In total, 492 agar plates were obtained.

At DTU the plates were first screened as quality-control before enrolled. All contaminated plates (? 2 types of bacteria on the plate) were discharged. This left 266 agar plates to be included in the study (158 plates were omitted as contaminated and 68 were omitted as growth negative).

From the 266 plates bacterial colonies were sub-cultured on calves blood agar and incubated for 37 °C for 24 h. Then the sub-cultures was examined with MALSI-TOF (Bruker daltonics).

The MALDI-TOF analysis of the sub-cultures showed a considerable variation within bacterial genuses and specieses as compared to the diagnoses reported from the clinics. Also, the study revealed a high prevalence of both contaminated samples and incorrect veterinary diagnostics.

The study demonstrates that many bacteria prevails in acute mastitis in Danish dairy cows. Also the study disclose a considerable challenge in obtaining pure cultures in mastitis diagnostics.

 

O2.1-3 - Evaluation of three cultural diagnostic tests for the fast identification of mastitis pathogens

11:45-12:00 hrs

Authors

Braker, N., Christian Albrechts-University, Kiel Germany
Haeussermann, A., Christian Albrechts-University, Kiel Germany
Hartung, E., Christian Albrechts-University, Kiel Germany
Knappstein, K., Max Rubner-Institute, Kiel Germany

Abstract

Different test systems are available for fast identification of bacterial mastitis pathogens in order to enable target-oriented application of antibiotics in cases of clinical mastitis. The objective of the study was to evaluate the following commercially available test systems: mastDecide, QuIdee GmbH, Homberg, DE; VetoRapid, Vetoquinol GmbH, Ismaning, DE; a combination of Aerobic Count-PetrifilmTM and Coliform Count-PetrifilmTM, 3M Deutschland GmbH, Neuss, DE. Tests were applied simultaneously according to manufacturers´ instructions. The standard cultural method according to the guidelines of the German Veterinary Association served as reference.

253 samples from clinical and subclinical mastitis were collected on 19 farms and sent via over night express to the laboratory of the Max Rubner-Institute. 34 samples (14.1 %) classified as negative by the reference method were correctly identified by mastDecide, VetoRapid and PetrifilmTM in 88.2 %, 70.6 %and 50.0 %, respectively. Sensitivity for gram-positive and gram-negative mastitis pathogens was between 70.7 and 85.4 % for VetoRapid and PetrifilmTM, but only 58.8 % (gram-positive) and 48.8 % (gram-negative) for mastDecide. Specificity of all three tests was high for gram-negative bacteria (77.4 to 90.3 %). Specificity of VetoRapid (68.0 %) and PetrifilmTM (65.3 %) for gram-positive pathogens was low compared to mastDecide (89.3 %).

VetoRapid allowed a preliminary identification of Streptococcus agalactiae or Staphylococcus aureus. In addition, this test was able to detect atypical mastitis pathogens after 48 hours of incubation.

Contamination of samples which occurred in 35 samples (14.5 %) could not be detected by mastDecide and only with difficulty by PetrifilmTM. Thus a high quality of sampling is necessary to interpret tests results correctly.

According to the results and the easy handling procedure the VetoRapid seems most suitable of for usage in mastitis diagnosis by veterinary practitioners.

 

O2.1-4 - Sociological aspects of introducing a mastitis rapid test - compliance, concerns and expectations

12:00-12:15 hrs

Authors

Anne Schmenger, Hochschule Hannover, University of Applied Sciences and Arts
Dr. Stefanie Leimbach, Hochschule Hannover, University of Applied Sciences and Arts
Prof. Dr. med. vet. habil. Volker Krömker , Hochschule Hannover, University of Applied Sciences and Arts

Abstract

More and more dairy farmers want to use fewer antibiotics in their animal health management. This can be achieved through the use of cultural bacteriological on-farm tests. On the other hand, farmers and veterinarians have concerns about the use of on-farm culture systems and post-introduction therapy outcomes.

From November 2015 to February 2018, five farms were scientifically supported in the implementation of a selective mastitis therapy concept with the novel mastDecide® rapid test (Quidee GmbH, Homberg, Germany). Therapy results and treatment expenditure were recorded and compared with previous conventional therapy concepts. The compliance of the implementation with the specifications was investigated.

In more than half of the cases the farmers deviated from the specifications of the therapy concept. Based on the available information and education, farmers still overestimated the effect of antibiotics in many cases and hoped to positively influence the cure rate. In some cases, however, where the on-farm test recommended an antibiotic, farmers left their animals untreated. This suggests that at least some farmers are more likely to trust their gut feeling and easily return to old habits. However, they saved both local and systemic antibiotics while the cure rates remained the same. Farmers reported using between 15 minutes and half an hour per mastitis case for documentation and clean sampling. The performance and evaluation of mastDecide® was assessed as simple.

In order to change the treatment, the long-term change of established behavioural patterns must be achieved. Communicating results and experiences can help veterinarians and farmers introduce a selective mastitis treatment approach and estimate the effort required to implement it.

 

O2.1-5 - Differential somatic cell count - a tool to improve diagnosis of cows with intramammary infections

12:15-12:30 hrs

Authors

Nyman A.-K., Växa Sverige, Department of Animal Health and Development, Stockholm, Sweden
Landin, H, Växa Sverige, Department of Animal Health and Development, Stockholm, Sweden
Lärk, Henrik, Eurofins Milk Testing Sweden AB, Jönköping, Sweden
Westerber, F, Eurofins Milk Testing Sweden AB, Jönköping, Sweden

Abstract

Somatic cell count (SCC) is the mastitis monitoring tool with the highest test performance in identifying cows with subclinical mastitis. However, the accuracy of SCC in correct identifying cows with or without intramammary infection (IMI) is barely 80%. The differential somatic cell count (DSCC), measuring the proportion of neutrophils, macrophages and lymphocytes of the SCC, could improve the interpretation of the SCC and hence, improve the diagnosis of cows with IMI. A new analysis instrument has been developed making it possible to measure DSCC routinely in test-milk samples and the aim of this project was to evaluate if this DSCC could improve the diagnostic performance of identifying cows with IMI.

In this cross-sectional study 20 randomly selected dairy herds with an annual BMSCC of >200 000/ml and a herd size of 100-200 cows were recruited. Monthly test-milk samples were collected during five consecutive month and analysed according to Fossomatic™ 7 and FossomaticTM 7 DC. Approximately 10% of the milk samples from each herd were randomly selected for analysis of presence of bacterial DNA using Thermo Scientific PathoProof Mastitis Complete-16 kit. IMI was defined to be present when the was a very high (+++) or high (++) level of DNA in the milk sample. The distributions of SCC, DSCC and DNA findings were summarized using descriptive statistics and associations between them were investigated using appropriate statistical analyses for normal, ordinal and binomial distributed data.

In the so far 1 338 analysed milk samples SCC varied between 1 000 - 15 057 000/ml, DSCC varied between 0 - 94.3% (i.e. the proportion of neutrophils), and 33% of the samples came from udders considered IMI positive. The preliminary analyses show a significant association between SCC - DSCC combinations and ct-levels. The statistical analyses are ongoing investigating cut-offs for SCC and DSCC with highest accuracy in diagnosing IMI.

 

2.2

Time: Thursday 16th May 09:00-10:30
Venue: Meeting room "Nørrebros Runddel"
Moderator: Lærke Astrup

 

O2.2-1 - Assessment of Subclinical Mastitis Diagnostic Accuracy by Differential Cell Count

09:00-09:15 hrs

Authors

Lucio Zanini, Dairy farmer ass. Lombardia ARAL
Diego Vairani, Dairy farmer ass. Lombardia ARAL
Micaela Cipolla, University of Milan Dept.Vet.Med.
Nicoletta Rizzi, Dairy farmer ass. Lombardia ARAL
Paolo Marconi, Dairy farmer ass. Lombardia ARAL
Francesca Dell'Orco, University of Milan Dept.Vet.Med.

Abstract

Recently availability of high-throughput milk analysers performing DSCC on milk, allowed designing studies aiming to identify subclinical mastitis in individual milk samples applying DSCC. The presentation reports the result of Italian studies performed under field conditions.

The first study considered 4386 milk test samples from four dairy herds with different size, management and milking management. DSCC data were analysed by ROC procedure. This procedure allows identifying the threshold giving the highest accuracy and the highest combined value for sensitivity and specificity, among all the possible thresholds. Among the different ways used to classify milk samples, the analysis applied to days in milk (3 classes) showed to have the best perfomances and the specific thresholds were identified.

A second study aimed to assess the frequency of low SCC (50.000 cells/ml.

The third study aimed to evaluate the use of DSCC on quarter milk samples. The study involved more than 1,000 samples and DSCC showed to add useful information on conventional analysis based on bacteriological assay and SCC. Bacteriologically positive samples have a significant different DSCC pattern, even when SCC was below 100,000 cell/ml, helping in identify false-negative results, particularly when contagious pathogens are involved.

Overall, these results show as DSCC will help the improvement of mastitis diagnosis and will help dairy farmers to increase the levels of herd management and efficiency.

 

O2.2-2 - PCR detection of staphylococcus aureus and streptococcus agalactiae in pool milk samples

09:15-09:30 hrs

Authors

Schlez K., Hessian State Laboratory, Gießen, Germany
Hechinger S., Hessian State Laboratory, Gießen, Germany
Herrmann D. C., INDICAL BIOSCIENCE GmbH, Leipzig, Germany
Labitzke M., INDICAL BIOSCIENCE GmbH, Leipzig, Germany
Gaunitz C., INDICAL BIOSCIENCE GmbH, Leipzig, Germany
Schroeder C., INDICAL BIOSCIENCE GmbH, Leipzig, Germany affiliation
Zschöck M., Hessian State Laboratory, Gießen, Germany

Abstract

Subclinical mastitis is one of the most common diseases in dairy farming. In this study, we evaluated the bactotype Mastitis HP3 multiplex real-time (q)PCR for the detection of Staphylococcus (S.) aureus, Streptococcus (Sc.) agalactiae and Mycoplasma bovis as a screening method for continuous monitoring of contagious pathogens in dairy herds.

We used pool milk samples collected in the framework of the national brucellosis, leucosis and BHV1 surveillance programs and bacteriologically negative milk spiked with S. aureus or Sc. agalactiae. Pools consisted of up to 50 individual milk samples.

Sensitivity testing, using spiked milk samples, revealed comparable results between this qPCR and traditional bacterial culture. Sc. agalactiae could be detected in artificially created, spiked pool milk samples by qPCR when at least two weakly positive individual samples were added (end concentration 1x10^3 CFU/ml in the pool). In contrast, only one S. aureus containing individual milk sample is necessary to obtain a positive test result in spiked pool milk (end concentration 1x10^3 CFU/ml in the pool) by culture or the bactotype Mastitis qPCR.

When investigating pool milk samples, collected randomly from 127 Hessian dairy farms, 21 (11%) were S. aureus positive in the qPCR. In three of these 21 dairy holdings, culture results of quarter foremilk samples were available which confirmed the positive qPCR findings.

By means of our results, the bactotype Mastitis HP3 multiplex qPCR is suitable for the detection of contagious mastitis pathogens in pool milk samples as a screening test.

The screening is recommended to be carried out at regular time intervals. Positive qPCR results of any given pool sample should then be followed up by cultural examination or qPCR testing of the quarter milk samples to confirm and identify the positive udder quarters.

 

O2.2-3 - Development of a novel milk ELISA test for the detection of S. aureus infections

09:30-09.45 hrs

Authors

Anton Pernthaner , Koru Diagnostics Ltd
Rebecca White , LIC
Shona Pryor, LIC
Patricia Rubio Reyes, Koru Diagnostics Ltd
Roland Schaap , Koru Diagnostics Ltd
Madeleine Palmer , LIC
Aaron Yang , Massey University
Varsha Boby, LIC
Mike Seawright , Koru Diagnostics Ltd
Rosemary Thresher , LIC

Abstract

Background: The detection of Staphylococcus aureus in milk samples has traditionally been conducted by bacterial culture, and more recently, by PCR analysis. These methods rely on the sample containing live bacteria or bacterial DNA for successful identification, however S. aureus is shed intermittently and can avoid detection. Repeated testing within several weeks is therefore required to establish the true infection status. Neither of these traditional tests are easily adapted for high throughput screening. Tests that detect specific biomarkers of S. aureus infection, regardless of whether the pathogen is being shed, would substantially enhance the diagnosis of the infection.

Methods: We developed an ELISA test that detects antibodies specific to S. aureus in milk. The test was validated for samples that were collected from individual cows during routine herd testing. Over 680 samples from cows with a somatic cell count greater than 250,000/ml were collected from 12 farms during the first half of the lactation. These samples were then tested by ELISA and real-time PCR . 250 cows were confirmed as infected with S. aureus. The herd test ELISA and PCR data were then used to establish sensitivity and specificity for each test using a Bayesian latent class model for analysis.

Results: The new S. aureus ELISA test had significantly higher sensitivity (0.92; 95% PI 0.87-0.96) than the PCR test (0.73; 95% PI 0.67-0.79). Both tests had high levels of specificity (ELISA 0.91; 95% PI 0.82-0.98; PCR 0.92; 95% PI 0.87-0.96). Conclusion: The new ELISA can be used to confidently screen cows with elevated somatic cell counts to identify S. aureus infections in, enabling farmers and veterinarians to make informed herd management and animal treatment decisions. The test can easily be integrated and automated in routine testing platforms for herd test milk samples.

 

O2.2-4 - Sensitivity and Specificity of PCR and Bacterial Culture of S. aureus based on Bayesian LCA

09:45-10:00 hrs

Authors

Nils Toft, Division for Diagnostics and Scientific Advice, National Veterinary Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
Tariq Halasa, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark
Søren S. Nielsen, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark
Silke Hechinger, Landesbetrieb Hessisches Landeslabor (LHL), Schubertstraße 60, Haus 13 35392, Gießen, Germany
Carsten Kirkeby, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark

Abstract

Bacterial culture (BC) and qPCR are among the most used tests for intra-mammary infections (IMI) in dairy cattle. However, their sensitivity (Se) and specificity (Sp) are unknown because the true infection status of the udder is unknown. Samples are either taken on quarter or cow level, which must be assumed to impact the sensitivity (Se) and specificity (Sp). In this study we use latent class analysis (LCA) to estimate the Se and Sp of two tests for Staph. aureus on the same population on both quarter and cow level. To our knowledge, LCA has not yet been applied to qPCR and BC for the detection of Staph. aureus in both composite and aseptically collected quarter milk samples. Thus we are able to evaluate the two tests and how they perform on both quarter and cow level. We used Bayesian LCA on a dataset from one sampling day in a Danish dairy herd to estimate the Se and Sp for detecting Staph. aureus at cow and quarter level. We used Ct cut-offs of 32 and 37 cycles for the qPCR. Using a cut-off of 37 cycles, we estimated Se and Sp for BC to be 62.2% and 90.5% at cow level, and 90.7% and 93.5% at quarter level, respectively. The estimated Se and Sp for qPCR at cow level were 82.7% and 84.1%, respectively, and 80.0% and 96.8% at quarter level. Sp was therefore estimated to be higher for both BC and qPCR at quarter level. Se was also highest for BC at quarter level, but the opposite was true for qPCR. The same pattern was found using a Ct cut-off at 32 cycles, but with different estimates. The results show that qPCR with a Ct cut-off at 37 had a higher Se than BC for composite DHI samples and it is therefore more suitable as a routine screening test for Staph. aureus. However, BC on quarter samples gave the highest Se and Sp and should therefore be used for confirmatory testing.

 

O2.2-5 - General discussion

P4 - Pathogens associated with chronic mastitis in dairy cows and their susceptibility pattern to antimicrobials

Presenter

H. Markiewicz, Laboratory Examination of Milk, Institute of Experimental Biology, Kazimierz Wielki University in Bydgoszcz
W. Krumrych, Department of Immunobiology, Institute of Experimental Biology, Kazimierz Wielki University in Bydgoszcz
R. Golda, Department of Immunobiology, Institute of Experimental Biology, Kazimierz Wielki University in Bydgoszcz

Abstract

The desirability of treating chronic mastitis is disputable. The low effectiveness of treatment is the result of, among others, the lack of targeted treatment.

The aim of this study was to identify pathogens and assess the in vitro antimicrobial susceptibility of bacteria isolated from milk.

272 milk samples come from mastitic cows which were at least twice empirically treated ineffectively. After the treatment, there were observed only clinical recover and, during two-three months, a relapse. Samples originated from different farms mainly located in central part of Poland. Milk samples were sent to Laboratory Examination of Milk in Bydgoszcz in 2018 for a bacteriological identification and tested for susceptibility to the antibiotic used. The results were interpreted as sensible, intermediate and resistant according to CLSI guidelines.

The bacteriological examinations were performed according to generally accepted principles.

All Str. spp. belong to CAMP-negative streptococci. 89% strains of Str. spp. were aesculin splitting on Edward's media. Str. spp. were identified in 23.5% of samples and in 3.3% together with E. coli. Staph. aureus as a single pathogen was identified in 7% of samples and together with Str. spp. in 3.7%.

CNS were diagnosed in 15.4% of samples and together with Str. spp. also in 15.4%.

E.coli was diagnosed in 5.9% of samples.

Str. spp. were isolated together from 45.9% of milk samples. The mixed infection was 22.4%.

Yeast-like fungi were isolated in 6.6% of samples.

80% of isolates Str. spp. were sensitive to amoxycillin, 79.7% to bacitracin, 71.4% to pirlimycin, 65.7 to cefalexin, 53% to tylosin but only 7.5% to penicillin.

87% of isolates CNS were sensitive to cefalexin, 88.6% to bacitracin, 77% to pirlimycin, 84% to amoxycillin/clavulanic acid and 28.6% to penicillin.

Conclusion

The main etiological chronic mastitis agents are environmental streptococci and coagulase - negative staphylococci (CNS). The knowledge of the pathogen and its sensitivity may be helpful when making a decision to cull chronic mastitis dairy cows.

 

Table 1. Microorganisms isolated from chronic mastitis in dairy cows

Microorganisms n %
Str. species 64 23,5
CNS 42 15,4
CNS+Str. spp. 42 15,4
Staph. aureus 19 7
Staph. aureus+Str. spp. 10 3,7
E.coli 16 5,9
E.coli+Str. spp. 9 3,3
Gram-negative bacilli other than E.coli 4 1,5
Gram-positive bacilli 3 1,1
Trueperella pyogenes 8 2,9
Corynebacterium spp. 10 3,7
Yeast-like fungi 16 6,6
Prototheca spp. 2 0,7
Negative 11 4
Contaminated 16 5,9
Total 272 100

 

POSTERS

P5 - European project MOLOKO: a 21th century on-farm multiplex plasmonic sensor for udder health

Authors

Dr. Stefano Toffanin, CNR-ISMN Istituto per lo studio dei Materiali Nanostrutturati
Dr. Jeroen Peters, RIKILT - Wageningen University & Research
Dr. Mark Whatton, QuadraChem Laboratories Ltd (QCL)
Dr. Tarja Nevanen, VTT Technical Research Centre Of Finland Ltd
Dr. Stefania Leonardi, MILKLINE S.R.L
Dr. Chiara Frazzoli, ISS Istituto Superiore di Sanità
Prof. Franco Marabelli, PLASMORE S.R.L

Abstract

MOLOKO is a European project comprising 12 partners from across Europe, intended to optimise production throughout the milk supply chain.

The MOLOKO biosensor under development will allow to increase and facilitate the food safety and food quality of milk and dairy products implementing multiple diagnostic tests in one single measurement. The innovative surface plasmon resonance optical biosensor, combined with miniaturized optoelectronic devices with plasmonic material, will enable on-site, point of care and automated quantitative analysis of potential milk contaminants and indicators of milk quality to be completed within 5 minute.

In order to achieve this ambitious result, MOLOKO partners have decided to both research potential antibiotics in milk but also to create a diagnostic tool useful for the reduction of antibiotics prescriptions.

One of MOLOKO diagnostic target molecules is lactoferrin, that is synthesized by neutrophilic polymorphonuclear leukocytes and glandular epithelial cells and secreted in milk in response to the presence of the major mastitis pathogens (E. coli, S. aureus, St. agalactiae and St. uberis) due to its immunological role in competing with bacteria for iron, and thus preventing them from its availability for living processes.

Lactoferrin is a model high molecular weight compound (HMW) that can be used in the on-farm version of the MOLOKO sensor as an indicator for udder health.

The resulting MOLOKO diagnostic sensor will be useful in the daily cow-by-cow evaluation, giving the farmer an early alert of the sanitary status of the animal. In addition, it has the potential to act as a predictive marker of future infections in the individual quarters of dairy heifers as well as a marker for the evaluation of the immunity of the mammary gland (innate or vaccine induced).

This work has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 780839 (MOLOKO Project) www.moloko-project.eu

 

P6 - MALDI-TOF is a rapid and reliable tool for identification of bacteria from bovine mastitis

Authors

Nonnemann B., Technical University of Denmark, DK-2800, Lyngby, Denmark
Lyhs U., University of Helsinki, FI-00014 Helsinki, Finland
Svennesen L., University of Copenhagen, DK-1870 Frederiksberg C, Denmark
Kristensen K.A., Technical University of Denmark, DK-2800 Lyngby, Denmark
Klaas I., DeLaval International AB, 147 21 Tumba, Sweden
Pedersen K., National Veterinary Institute, Ulls väg 2B, 751 89 Uppsala, Sweden

Abstract

MALDI-TOF MS has gained acceptance in the human and veterinary clinical microbiological laboratory. It is recognized as a rapid and reliable technology for identification of clinical bacteria and fungi. However, there are only few reports on the application of MALDI TOF on mastitis bacteria. The present study was undertaken to evaluate MALDI-TOF on isolates from bovine mastitis and improve its performance by amending spectra to the database.

A total of 530 bacterial cultures were tested. Of these, 30 were reference strains, while 413 were from milk samples or cultures from cows with clinical or subclinical mastitis, submitted to the laboratory by veterinary practitioners. The remaining 87 were recovered from milk samples from cows with subclinical mastitis.

MALDI-TOF was performed on an Autoflex Speed (Bruker Daltonics). Score values ?2.0 were considered identified correctly to species level, while score values between 1.7 and 2.0 were considered identified only to genus level. Isolates with scores values below 2.0 were further subjected to 16S rDNA sequencing for identification. Once identified, a reference mass spectrum for each new entry was created and amended to the existing database.

MALDI-TOF performed satisfactorily on the reference cultures, but spectra from three cultures, Streptococcus canis, Streptococcus uberis and Micrococcus luteus, were added to the database to improve accuracy. All 87 isolated from subclinical cases were identified correctly. The 413 isolates from clinical and subclinical cases belonged to 24 different genera and 63 different species. The Gram positive isolates were dominated by staphylococci, in particular Staph. aureus, and streptococci, in particular S. uberis and S. dysgalactiae. The Gram negative isolates were dominated by Enterobacteriales, in particular E. coli followed by Klebsiella.

In conclusion, MALDI-TOF is rapid and cost effective for identification of mastitis bacteria belonging to a plethora of genera and species.

 

P7 - Haptoglobin and ?-lactalbumin, biomarkers for selective dry cow therapy?

Authors

Emily L. O'Reilly, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland
Lorenzo Viora , School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland
Nicola Brady, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland
Theo Pepler, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland
Ruth Zadoks, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland
David Eckersall, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland

Abstract

Selective dry cow therapy (SDCT) is a treatment strategy designed to reduce antimicrobial usage, with selection largely based on SCC or clinical mastitis history, which are used as proxies for current infection status. There have been limited attempts to evaluate whether measuring inflammatory biomarkers in milk results in more accurate selection of infected cows for DCT. Haptoglobin (Hp) and ?-lactolbumin (LA), positive and negative biomarkers for inflammation respectively, were assessed for their diagnostic accuracy in identifying quarters sub-clinically infected with major mastitis pathogens (MMP). SCC (based on milk recording), bacteriology (culture) and Hp and LA concentrations were determined in 304 quarter milk samples collected aseptically at dry-off from 103 cows. In quarters infected with a MMP (n=17), Hp and SCC were significantly higher (p < 0.0001 for both) than in those that were not (n=323) but LA did not differ significantly (p=0.1082). From a generalised linear regression (GLR) model applied to the data we found no significant interaction between Hp and LA in identifying quarters with MMP. A treatment decision rule based on both Hp and LA was found to have greater in-sample sensitivity than SCC >= 200,000 (1.00 vs 0.94). The biomarker-based decision rule has reduced specificity compared to SCC (0.43 vs 0.61), and a worse misclassification rate (0.54 vs 0.37, McNemar test p-value < 0.0001). Fitting a boosted regression tree (BRT) model to the data revealed that there was a significantly smaller misclassification rate (0.04) using both Hp and LA than the common treatment decision rule of SCC >= 200,000 (p < 0.0001). However, the sensitivity of the BRT model was poorer (0.59, compared to 0.94 for SCC). Further investigations, focused on a range of biomarkers in a larger data set would further elucidate whether additional inflammatory biomarkers, used independently or in conjunction with other data such as SCC, could refine decision making at dry-off.

 

P9 - Quality-assessment of E. coli diagnostics in Danish veterinary practice

Authors

Astrup LB., Center for Diagnostics, Danish Technical University. Kgs. Lyngby. Denmark
Chehabi CN., Center for Diagnostics, Danish Technical University. Kgs. Lyngby. Denmark
Farre M., SEGES. Aarhus. Denmark

Abstract

The environmental pathogen Escherichia coli a frequently isolated pathogens in clinical bovine mastitis in dairy herds with low-somatic cell count. Therefore, we set up the present study to estimate the accuracy of E. coli-diagnostics performed in veterinary practices in Denmark.

Milk samples were obtained from 5 Danish veterinary clinics. The clinics dispatched all milk samples from clinical mastitis diagnosed as E. coli, and all milk samples from clinical mastitis that caused diagnostic difficulties at the veterinary clinic. The samples were collected from late May until first of October 2018. After diagnosis, the samples were frozen and send to DTU for diagnostic validation. In total, 62 presumed E. coli milk samples and 256 milk samples of diagnostic difficulties were included in the study. At DTU the samples were analyzed with Maldi-tof (Bruker Daltonics).

The results showed a diagnostic accuracy of 90 % on presumed E. coli, and a rate of false negative E. coli of 8 % on samples causing diagnostic difficulties only. For both the false positives and most of the false negative veterinary diagnose E. coli was diagnosed as various Gram-positive bacteria.

The E. coli isolates were whole genome sequenced (66 isolates in total). MLST analysis identified 28 different types. Each type was identified in 1 to 18 samples. The analysis also showed that some types were farm-specific whereas others occurred in multiple farms. Also, some farms had only one type whereas others had up to 5 different types.

Altogether, the present study indicates that flawed diagnoses might undermine efficient prevention and control of E. coli mastitis. Accordingly, mastitis control programs need to investigate and correct the diagnostic accuracy of veterinary clinics.

 

P9 - Patterns of antibiotic resistance in major pathogens from clinical mastitis in Denmark 2018

Authors

Mille Agerbo, Technical University of Denmark, Kgs. Lyngby, Denmark
Michael Farre, SEGES, Aarhus, Denmark
Bettina Nonnemann, Technical University of Denmark, Kgs. Lyngby, Denmark
Lærke Boye Astrup, Technical University of Denmark, Kgs. Lyngby, Denmark

Abstract

Antibiotic resistance is an international threat to human and animal welfare. In Denmark, the level of antibiotic resistance in mastitis pathogens is monitored on a yearly basis. The yearly monitoring provides data on the actual situation as well as the development in resistance-dynamics. This study reports the preliminary results on the 2018 monitoring of resistance in major pathogens from clinical Danish mastitis.

Eleven veterinary clinics provided their milk samples to the study chronologically for two consecutive weeks in October 2018. The samples were collected with no selection other than that contaminated samples were omitted. Contaminated samples were defined as presence of ?3 types of bacteria in a milk sample. The samples were send frozen to the lab at DTU along with the veterinary diagnosis. At DTU the samples were thawed, plated on blood agar and incubated for 24 h at 37 °C. After incubation the bacterial cultures were examined for quality and diagnosed with Maldi-tof. After diagnosis the samples were analyzed with MIC-panels. In total 185 milk samples were included in the MIC-analysis. The samples composed 30 of each of the pathogens S. aureus, S. dysgalaciae, S. uberis, E. coli and Klebsiella spp., and 5 S. agalactiae, respectively. MIC panels of 13 S. uberis and 22 S. aureus are currently completed. The preliminary results on S. uberis compares well to former Danish results. All S. uberis are resistant to sulphamethoxazole and the majority is resistant to streptomycin as well. Also, some S. uberis were in the intermediary interval for penicillin which mimics international reportings on S. uberis resistance. Likewise, the S. aureus isolates from 2018 resembles former Danish results in that the highest levels of resistance are found to spectinomycin and sulphametoxazole. However, the exact comparison between the resistance patterns and levels in mastitis pathogens in 2018 compared with former years still await.

 

P10 - Fast and convenient nucleic acid extraction method for mastitis diagnostic

Authors

Anne Quijada , Thermo Fisher Scientific, Lissieu, France
Emeline Ripoche , Thermo Fisher Scientific, Lissieu, France
Quoc Hoang, Thermo Fisher Scientific, Austin, TX, USA
Denisse Meza , Thermo Fisher Scientific, Austin, TX, USA
Robert Tebbs, Thermo Fisher Scientific, Austin, TX, USA

Abstract

Identifying pathogens in mastitic milk traditionally requires culturing, which is labor-intensive and timeconsuming. The advent of molecular testing provides a convenient alternative. In combination with VetMAX™ MastiType multiplex qPCR kits, the Animal Health group at Applied Biosystems™ Thermo Fisher Scientific now offers a new easy-to-use and fast nucleic acid extraction method formastitis diagnostic.

MagMAX™ CORE Nucleic Acid Purification Kit is a universal magnetic bead-based separation system designed for rapid purification of high-quality DNA and RNA for downstream molecular analysis. The new MagMAX™ CORE Mastitis and Panbacteria Module is offered here in combination with the universal kit to aid in the processing of all bacteria, especially those important for the detection of mastitis in cattle and from fresh, frozen, or preserved milk samples. The protocol offers benefits such as minimal hands-on time and easy-to-use (no centrifugation and no aspiration steps), fast (less than 1 hour for 96 samples) and convenient with both automated (using the KingFisher™ Purification System) and manual purification methods.

Nucleic acid from milk samples coming from North-America and Europe were extracted using this new extraction method and our current available method (PathoProof™). The extracted DNA were tested with three different 4-plexed VetMAX™ MastiType multiplex qPCR kits allowing a total detection of 19 species.

MagMAX CORE extraction method has been validated on a broad range of mastitic milk field samples containing each of the target organisms, including gram-positive and gram-negative bacteria. It provides equivalent or better DNA recovery compared to the leading competitor with minimal extraction and reagent preparation time, to get faster diagnostic results.

MagMAX CORE extraction method is part of a complete mastitis solution designed to provide rapid and accurate results in order to ensure the efficacy of surveillance and control programs.

 

P11 - Bacterial findings at clinical mastitis in Swedish dairy cows

Authors

Helle Ericsson Unnerstad, National Veterinary Institute, Uppsala, Sweden
Johan Waldner, District Veterinary Organization, Jönköping, Sweden
Karin Persson Waller, National Veterinary Institute, Uppsala, Sweden

Abstract

Clinical mastitis is one of the most common diagnoses that give rise to antibiotic treatment in Swedish dairy cows. Knowledge of causative bacterial agents is important for optimal handling of clinical mastitis both on individual and herd level.

From October 2013 to December 2018 milk samples from dairy cows with clinical mastitis were taken by field veterinarians in the District Veterinary Organization. Inclusion criteria were lactating cow with visible pathological changes of the milk indicating inflammation. Participating veterinarians were situated in most parts of Sweden and were told to collect milk from the 3 first cases each month that fulfilled the criteria. Samples were sent to the National Veterinary Institute and were cultured on bovine blood agar according to accredited routines. Species identification was done by MALDI-TOF MS. All isolated staphylococci were tested for beta lactamase production by the clover leaf method.

During the study period, 733 cows were sampled. Cultivation resulted in 827 bacteriological diagnoses. The dominating pathogen isolated was Staphylococcus aureus with 28% of the diagnoses. Of the S. aureus-isolates, 3% were beta lactamase producing. Streptococcus dysgalactiae and Escherichia coli constituted 15% each of the bacteriological diagnoses, Streptococcus uberis 11%, Trueperella pyogenes 8%, Klebsiella spp. and coagulase negative staphylococci 3% each, Streptococcus agalactiae 1% and various other bacteria 6%. Samples with no growth or contamination constituted 5% each of the diagnoses. The bacterial panorama, with S. aureus as dominating bacterial diagnosis, has not shifted significantly compared to a similar investigation in 2002-2003.

 

P12 - On-farm testing on a Stick

Authors

Tomas Ussing, FluimediX
Marie Skovmøller, FluimediX

Abstract

The need for diagnostic tools in connection with the day-to-day -decision making in the modern milk production facility, has been eminent for years, however - easily accessible tools have been absent.

On-farm analysis using agar-plates has been ported from the veterinary's laboratory and into the barn. The limited success of the otherwise rugged and proven technology, stems from the fact that processes, routines and standards that may be considered "first page in the book" for vets and trained laboratory technicians, may pose a hindering barrier for the average farmer and his staff. Today's dairy farmer is experiencing stress from all aspects of their business, so the efforts associated with adopting new tools and procedures should be minimal, otherwise they are not adopted into the daily routine. We have devised a range of "stick tests", that - in terms of ease of use - are akin to pregnancy tests from a super market, that we wish to present. In line with the before mentioned level of stress, it is our belief, that the test(s) should provide simple answers, and that the availability of multiple tests for different situations and - not least - for different farmers, is the way to achieving widespread usage of on-farm -analysis. Our first test is thus a Gram-positive/negative -test that will provide two simple answers:

1. Are a given set of mastitis symptoms caused by bacteria?
2. And - if so - is it a Gram-negative or a Gram-positive bacteria?

Provided with the answer to these questions the farmer will enable to make a decision on whether or not to treat a given quarter, as a (mild) Gram-negative bacterial infection is often cleared by the animal without antibiotic treatment and because penicillins have no effect on Gram-negative bacteria.

In most cases, this is all the answer you want, and you want it without being dependent of making arrangements with and sending milk samples of to external laboratories.

The answer will be provided within 8 to 16 hours.

 

P13 - Identification of Streptococcus uberis Using Phenotipic Methods and Mass Spectrometry

Authors

Larissa Martins, School of Veterinary Research and Animal Sciences/ University of São Paulo
Renata de Freitas Leite, School of Veterinary Research and Animal Sciences/ University of São Paulo
Gustavo Freu, School of Veterinary Research and Animal Sciences/ University of São Paulo
Tiago Tomazi, School of Veterinary Research and Animal Sciences/ University of São Paulo
Marcos Veiga dos Santos, School of Veterinary Research and Animal Sciences/ University of São Paulo

Abstract

Streptococcus uberis causing bovine intramammary infections may be prone to misidentification using phenotypic methods. The method of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used for identification of mastitis causing pathogens. The aim of this study was to evaluate the agreement between microbiological culture and MALDI-TOF MS to identify mastitis-causing Strep. uberis. A total of 147 isolates previously identified as Strep. uberis by microbiological culture were selected for this study. These isolates were obtained from clinical mastitis and were cryopreserved. Thus, Strep. uberis isolates were recultured and a colony was submitted to MALDI-TOF MS. MALDI-TOF MS scores ?2 indicate species-level identification, scores ?1.7 but

 

P14 - Phylogenique identification study of staphylococcus aureus involved in mastitis cases in Oran, Algeria

Authors

Benhamed Dadija, laboratory of applied, university of Oran, Algéria. university of sciences and technology of Oran m-b.
Mezouar Ismahene, university of sciences and technology of Oran m-b
Baaj Salim, university of sciences and technology of Oran m-b
Gautier Philippe, microbiologie-risques infectieux, université de Rennes
Kihal Mebrouk, laboratory of applied, university of Oran, Algéria
Donnio Pierre Yves, microbiologie-risques infectieux, université de Rennes

Abstract

Introduction:

S. aureus mastitis are considered one of the major diseases in dairy cattle. This study determines phylogenetic profile of S. aureus isolated from Bovines mastitis in Oran.

Materiels et methods:

MALDI-TOF,spa, MLST have identified.The molecular profile toxin was demonstrated byPCR. Antibiotic resistance of S. aureus and confirmed by amplification of the mecA gene.

Results:

Results of spa typing, variety (T267, T021 and t007).MLST; reveals different(ST39, ST2598 and ST97).Toxin research shows that only some strains proved carriers of different virulence genes; pvlgene.Other strains were positive for tst gene (TSST-1), 100% of the S aureus isolates identified were SASM.this work have determined the phylogenetic profile, toxic and sensitivity profile to méticiline strains.

Strains found produisantes as PVLare sensitive to methicillin, don't belong to ST30 commonly found in humans in Algeria, relation to the type of stem ST97 are of bovine origin.

Conclusion:

the results obtained in this work have determined the phylogenetic profile, toxic and sensitivity profile of the méticiline strains studied. Stem found produisantes as the Panton-Valentine leukocidin are sensitive to méticilline. Cette study is the first molecular characterization study of animal strains of S. aureus isolated in the region of Oran.

 

P15 - Investigation of the added value of differential somatic cell count (DSCC) for detection of mastitis

Authors

Carsten Kirkeby, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark
Nils Toft, Division for Diagnostics and Scientific Advice, National Veterinary Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
Daniel Schwarz, Foss Analytical A/S, Foss Allé 1, 3400 Hillerød, Denmark
Michael Farre, SEGES Livestock Innovation, 8200 Aarhus, Denmark
Tariq Halasa, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark

Abstract

Intra-mammary infections (IMI) are a major problem in Danish dairy herds and worldwide. The somatic cell count (SCC) is routinely used as an indicator for IMI in milk from dairy cows. SCC represents the number of white blood cells in the milk. We here investigate the potential of a new available parameter, differential somatic cell count (DSCC), for detection of IMI using regularly collected milk recording samples. DSCC represents the proportion of polymorphonuclear neutrophils (PMNs) and lymphocytes in relation to macrophages, and could therefore provide more detailed information about the immune status within the udder. We here evaluate the DSCC as an additional IMI indicator in two Danish dairy herds. All milking cows in two commercial dairy herds were sampled monthly during a period of one year. Herds 1 and 2 included 180 and 360 milking cows, respectively. We measured SCC, DSCC and detected pathogens using bacterial culture and PCR in all samples. The data were combined with records on all cow-related data. We constructed a dataset including all infections in general in each herd as well as sub-datasets, where detected pathogens were grouped into three groups: Major, Minor and Other pathogens. In a first analysis, using regression modelling, we found that DIM, parity and infection with a pathogen had significant impact on the DSCC, both when using BC and PCR for pathogen detection. In a second analysis, using BC only, we found that, DSCC did contribute significantly to detect infections in general in both herds, even when SCC was already known. Furthermore, DSCC contributed significantly to detect infections with Minor pathogens in Herd 2. When using PCR in the second analysis, DSCC contributed significantly to detect Other pathogens in Herd 1. These results showed overall that DSCC had added value for detection of IMI when the SCC is already known.

 

P16 - Differential Somatic Cell Count as a new & inexpensive supplementary tool for mastitis screening

Authors

Daniel Schwarz, FOSS, Hilleroed, Denmark
Debora E. Santschi, Valacta, Ste-Anne-de-Bellevue, Canada
Anne-Marie Christen, Valacta, Ste-Anne-de-Bellevue, Canada
Simon Dufour, Faculty of Veterinary Medicine, University of Montreal, Canada
Daniel M. Lefebvre, Valacta, Ste-Anne-de-Bellevue, Canada

Abstract

Mastitis, in particular in its subclinical form, is the most costly disease in milk production causing substantial financial losses to the dairy industry. Somatic cell count (SCC) is broadly used as an indicator for mastitis or intramammary infections (IMI) and thus the basis for mastitis management programmes, e.g. through monthly milk recording/dairy herd improvement (DHI) testing. While SCC represents the total number of cells in milk, the new Differential Somatic Cell Count (DSCC) parameter represents the combined proportion of polymorphonuclear neutrophils and lymphocytes as a percentage. The objective of our study was to investigate the diagnostic abilities of DSCC as a new supplementary parameter for mastitis screening. Metered DHI samples of, in total, 969 dairy cows from 11 herds were collected once per month for a period of 4 months. The IMI status was determined using sterile hand-stripped samples and the Maldi-ToF method. The pathogens detected were categorized as no, minor, major or other pathogens. The results of our study showed that the DSCC parameter is significantly affected by the IMI status and the cow's parity but not by days in milk or test day milk weight, while SCC was affected by all of these four factors. DSCC results were significantly higher in cows with IMI by major pathogens compared to cows with no IMI or IMI by minor or other pathogens. SCC results were also significantly higher in cows with IMI by major pathogens as compared to all other groups. While the diagnostic abilities of DSCC and SCC alone each were at the same level, working with the combination of DSCC and SCC generally led to an increase in sensitivity but a decrease in specificity. In conclusion, the results of our study showed that the combination of DSCC and SCC opens up the possibility to further improve mastitis screening based on milk recording/DHI samples and thus an actual tool for dairy farmers will be developed in a next step.

 

P17 - Mastitis diagnosis utilizing a turnkey solution from Thermo Fisher Scientific's Animal Health

Authors

Anne Quijada , Thermo Fisher Scientific, Lissieu, France
Emeline Ripoche , Thermo Fisher Scientific, Lissieu, France
Quoc Hoang , Thermo Fisher Scientific, Austin, TX, USA
Denisse Meza, Thermo Fisher Scientific, Austin, TX, USA
Robert Tebbs , Thermo Fisher Scientific, Austin, TX, USA

Abstract

INTRODUCTION

Identifying pathogens in mastitic milk traditionally required culturing, which is labor-intensive and timeconsuming. The advent of qPCR testing provides a faster and much more convenient alternative. The Animal Health group at Applied Biosystems™ (AB) Thermo Fisher Scientific now offers a complete mastitis solution.

MATERIALS AND METHODS

The solution includes a new module to be use with the MagMAX™ CORE Nucleic Acid Purification Kit for DNA isolation protocol. This kit offers benefits such as ambient-temperature shipping and storage, inclusion of all reagents in one box, and verified automated processing on the Thermo Fisher™ King Fisher™magnetic particle processor. The extracted DNA can then be tested with three different 4-plexed VetMAX™ MastiType PCR kits, on either the 7500 or QuantStudio 5 (QS5) series of AB Real-Time PCR Systems. Data analysis for each of these uses a new cloud-based software with a user-friendly graphical interface.

RESULTS

The MagMAX™ CORE Nucleic Acid Purification Kit has been validated on a broad range of mastitic milk field samples containing each of the target organisms. Extracts have been validated on the three different 4-plexed VetMAX™ MastiType PCR kits (Micro4, Myco8 and Multi panels) allowing a total detection of 19 species. This entire workflow takes about 3 hours and provides equivalent results on the 7500 and QS5 Real-Time PCR Systems.

CONCLUSION

Detection of mastitic pathogens infections needs diagnostic tools that are easy to handle and can provide rapid and accurate results in order to ensure the efficacy of surveillance and control programs.

The Thermo Fisher Scientific mastitis solution is designed to meet these expectations.

These 3 kits are for Veterinary Use Only.

 

P18 - High-resolution Immunophenotyping in bovine blood and milk

Authors

Sabine Farschtschi, Department of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich
Prof. Dr. Michael Pfaffl, Department of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich

Abstract

The determination of a differential cell count (DCC) in milk is a promising method to identify inflamed mammary glands, even in an early or subclinical stage. Moreover, the DCC could even have the potential to give information about the health condition of a cow.

By means of flow cytometry we established a high-resolution DCC to detect 13 populations and subpopulations of the major immune cells parallel in blood and milk using fluorochrome conjugated antibodies that bind to the cell-specific surface molecules (cluster of differentiation, CD). Furthermore, we determine the amount of epithelial cells with a fluorochrome conjugated pan-cytokeratin antibody and we measure the viability of all detected cells using a live/dead stain.

In this study we have three different experimental setups. Within our long-term immunophenotyping project we monitor initially eight cows throughout their entire lactation (ca. 305 days). In the early lactation phase (ca. 100 days) blood and milk samples are taken twice a week, during the remaining lactation we take both samples once a week. In another experiment, three groups of five healthy cows each were immunized with regular vaccines as a controlled immune stimulus and sampled before and after the vaccinations. Furthermore, we closely sample every diseased cow at our research station. In addition to our analysis, we send blood samples of all cows to a veterinary laboratory to determine further circulating biomarkers, e.g. haptoglobin, calcium and several metabolic parameters.

Integrating all these blood and milk derived biomarkers, we can study the influence of (subclinical) inflammations, calcium deficiency or metabolic disorders on the immune status. The reference values we obtain can serve as a health indicator of a single animal or can be used as an early warning system in dairy farming.