DIFFERENTIAL SOMATIC CELL COUNT AND OTHER ADVANCEMENTS IN

DIFFERENTIAL SOMATIC CELL COUNT AND OTHER ADVANCEMENTS IN DAIRY HERD MANAGEMENT DR. DANIEL SCHWARZ, FOSS, DENMARK VETRNY JENIKOV, 10 APRIL 2018

THE FUTURE OF GLOBAL FOOD RESOURCES +60% Increasing demand for food of consistent and safe quality +9 billion World population will continue to grow in size +4. 9 billion 2018 The global middle class will more than double – from today’s 2 billion 2030 2

OUR MISSION THE INDUSTRY’S BEST DEDICATED ANALYTICAL SOLUTIONS We contribute to the sustainable use of our planet’s agricultural resources and thus to the nutrition and health of the people of the world. We provide the Industry’s best Dedicated Analytical Solutions, which add value to our customers by improving quality and optimising food and agricultural production. 3

INDUSTRY LEADING SOLUTIONS FOR INDUSTRY LEADING CUSTOMERS RAW MILK TESTING DAIRY GRAIN, MILLING & OILS WINE MEAT FEED & FORAGE OTHER INDUSTRIES LABORATORIES 4

MILK VALUE CHAIN Dairy Raw Milk Testing g in t s te t en m e ov g pr iry rd e h e tt n e im in st ym a P Da Dairy farm Receiving points At-line/In-line production Quality Control Laboratories Finished products RAW MATERIAL PROCESSING FINISHED GOODS Payment, segregation and quality control of raw material Improved predictability and control of manufacturing processes Safe products and compliance with regulatory requirements 5

RAW MILK TESTING • Solutions in operation in 80+ countries • 3, 000+ Milko. Scan™ sold (7 generations) • 3, 000+ Fossomatic™ sold (7 generations) • 1, 200+ Bacto. Scan™ sold (3 generations) • Solutions in compliance with international and national standards (e. g. IDF/ISO, EU, NCIMS/FDA)

MILK TESTING – 60 YEARS OF INNOVATION 1950 s 1960 s 1970 s 1980 s 1990 s 2000 s 2016

THE NEW COMBIFOSS 7 DC

7 TH GENERATION COMBIFOSS – EVOLUTION OF PARAMETERS Differential SCC Urea Citric Acid Freezing Point Depression Free Fatty Acids Casein p. H Fatty acid profile Ketosis Adulteration Solids SCC Fat Protein Lactose CF 215 1970 s CF 360 1980 s CF 4000 1980 s CF 5000 1990 s CF 6000 2000 s CF FT+ 2010 s CF 7 DC 2016

7 TH GENERATION COMBIFOSS – PARAMETERS 19 parameters in 6 sec. DONE • • • Fat Crude protein True protein Lactose Citric acid Urea Solids non fat Total solids Casein p. H-value • • • Freezing point depression Free fatty acids Fatty acid profile 1 Fatty acid profile 2 Adulteration Acetone BHB Somatic cell count Differential somatic cell count

APPLICATION OF DATA FROM RAW MILK TESTING

MASTITIS

FACTS ABOUT MASTITIS • Inflammation of the mammary gland, mainly caused by pathogenic bacteria • Multifactorial disease (environment, keeping, feeding) • Costs of mastitis: 50 -950 €/cow (Huijps et al. , 2008; Heikkilä et al. , 2012) • Mastitis diagnosis: somatic cell counts (SCC) and bacteriology

DEVELOPMENT OF UDDER HEALTH SCC 600, 000 cells/ml 200, 000 cells/ml 1971 e. g. Sampimon et al. , 2005 2002 Time

THE VALUE OF WORKING WITH SCC 30 Proportion of cows on farm (%) Farm 25 20 15 SCC, bulk tank, in 1, 000 cells/ml Number of cows Cost of mastitis (€), per herd per cow 750 226 95, 000 420 210 246 40, 000 162 farm with mastitis problem 10 normal farm 5 0 <50 50 -100 101 -200 201 -500 501 -1000 SCC range (x 1, 000 cells/ml) >1000

ECONOMIC LOSSES DUE TO MASTITIS l l i b 2 € 3 y l l a u n n a n o i

DAIRY HERD IMPROVEMENT TESTING – THE FUTURE • “We need to provide dairy farmers with more information for improved decision making through DHI testing. ” • Mastitis: Differential Somatic Cell Count (DSCC) as a new, additional indicator 30 Proportion of herd (%) 25 20 15 farm with mastitis problem normal farm 10 5 0 <50 50 -100 101 -200 201 -500 501 -1000 SCC range (x 1, 000 cells/ml) >1000

DIFFERENTIAL SCC (DSCC) The concept and method

CELLS IN MILK 1. Lymphocytes 2. Polymorphonuclear neutrophils (PMN) 3. Macrophages Microscope spot, milk slide Sordillo and Nickerson, 1988; Nickerson, 1989; Paape et al. , 2002; Oviedo-Boyso et al. , 2007

SCC MASTITIS CASCADE Macrophages PMN Macrophages Time Lymphocytes play minor role only

THE FOSS DSCC METHOD – FOSSOMATIC 7 DC Analysis of SCC Analysis of DSCC (cells/ml) FL 2 macrophages DSCC (%) PMN + lymphocytes FL 1 Damm et al. , 2017; Schwarz, 2017 Patented technology

EVOLUTION OF DSCC Differentiation of cells… …valuable …scientific method …not feasible in connection with DHI programmes 2014 and before New Fossomatic 7 DC with DSCC capability (600 samples/hour) 2017 Focus on practical application of DSCC 2018

DEVELOPMENT OF DSCC AND SCC DURING CONTROLLED MASTITIS

STUDY PLAN • 8 healthy cows recruited Infection with either S. aureus or E. coli Sampling scheme: Days post infection Time -3 14 -2 1 -1 0 2 0. 5 3 1 1. 5 2 2. 5 4 • Sample types: quarter foremilk, cow-composite • Parameters: SCC, DSCC, bacteriological examination Wall et al. , 2018 3 5 4 5 6

S. AUREUS – SCC AND DSCC RESULTS 110 7 to s ge a h p o n a, b r c a io 80 m t c e m f o n r i f a r s e t n f o 70 ) a i t a a l C u C p o S 60 p D a l l f e o a f c ase o e e r g 50 c n n i a ch 90 5. 5 5 a 4. 5 4 3. 5 r a e Cl 1 Wall et al. , 2018 N M P , . e (i. b DSCC (%) SCC (LOG) 6 3 100 b 6. 5 b a S. aureus control 40 2 3 Time 4 5 1 2 3 Time 4 5 a, b: indicate significant differences (P < 0. 05)

E. COLI – SCC AND DSCC RESULTS 7 6. 5 5 4 3. 5 3 b 100 b to s ge a h p o r c ion a m ect 80 a m o r inf r f s e t a n f a o a 70 ti C) a a l u SC a p o p D 60 l l f e o f c ase o e e r g c 50 n n i a , h. c e. r i ( a e l 40 C 90 N M P b DSCC (%) SCC (log) 6 110 b 1 Wall et al. , 2018 2 3 Time 4 5 1 2 3 Time a E. coli control 4 5 a, b: indicate significant differences (P < 0. 05)

S. AUREUS INFECTION – INDIVIDUAL COW RESULTS 180 100 90 140 100 80 60 hen w y l nt 60 40 0 70 ven e C SC D y l n o s t f i sh e d i ev Days post infection Wall et al. , 2018 C C S de o m ly e t ra r c n i S. aureus control 50 40 30 -3 -2 -1 0 0. 5 1 1. 5 2 2. 5 3 4 5 6 7 14 DSCC (%) 120 20 s ea 80 -3 -2 -1 0 0. 5 1 1. 5 2 2. 5 3 4 5 6 7 14 SCC (x 1, 000 cells/m. L) 160 Days post infection

LEARNINGS • Determination of the stage of mastitis using combination of SCC and DSCC: • Early stage: High SCC (>200, 000 cells/ml), high DSCC values (>86%) • Late stage: High SCC (>200, 000 cells/ml), low DSCC values (<86%) • Clearer reflection of an individual quarter in a cowcomposite sample not only SCC increase but also the shift of cell populations (DSCC) reflected Wall et al. , 2018

DSCC AS ADDITIONAL INDICATOR FOR UDDER HEALTH/MASTITIS MONITORING

EXAMPLE FOR DSCC DATASET 100 2 normal 90 2 80 2 likely to be infected normal likely to be infected 70 1 active. . DSCC (%) Column 1 60 1 inactive inflammatory response 50 1 40 1 30 1 20 0 10 0 Schwarz, 2018 50 100 150 200 250 300 200 300 SCC (x (x 1, 000 cells/ml) 350 400 450 500

DSCC VALIDATION ACTIVITIES Project Objective Status Denmark Longitudinal study to develop general guidelines for application of DSCC in practise In progress Germany Implementation of DSCC in routine DHI analysis – development of application guidelines In progress University Value of DSCC for selective dry cow therapy In progress DHI laboratory, North America DSCC for enhanced analysis of udder health in fresh lactating cows In progress DHI laboratory, North America Investigation of correlation between DSCC and udder health status in a longitudinal study - selective dry cow therapy In progress DHI laboratory, Europe DSCC as parameter for improved microbiological testing Kick-off: Q 2 2018

LITERATURE

KETOSIS

KETOSIS – THE PROBLEM • Negative energy balance • Incidence: 25 to 60% • Prevalence: 5 to 30% • Costs per case: € 260 34 Mc Art et al. , 2013, 2015; Mahrt et al. , 2015

KETOSIS – DEFINITION & TESTING • Ketone bodies elevated in blood • No visible symptoms – need for measurement of ketone bodies in blood, milk, or urine (Andersson, 1988) • Cow-side tests labour-intensive (Iwersen et al. , 2009) • Availability of DHI samples and FTIR technology

FOSS’S KETOSIS SCREENING TOOL

FTIR – BHB PREDICTION MODEL Indirect calibration developed: Fat Absorbance Fingerprint Protein area c t e ti a l e rr o C e s o BHB D s i m he : y r t ( 2 8. 0 ow t n o Wavenumber l. a t o R e 2 7 00 )

WORKING WITH FOSS’S KETOSIS SCREENING TOOL

REAL LIFE EXAMPLE – KETOSIS MANAGEMENT CÉTOLAB Herd Results April 2013 BHB values in cows with less than 90 days in milk Type II** Type I* Days in milk *Type I (Fresh cow; Production > Dry matter intake, NEB) **Type II (Starts before calving; “fat cow syndrome”; insulin resistance) Advisor’s suggestion: “Focus first on dry cow (faroff) rations as they obviously bring too much energy. “

Test day milk yield (kg) MILK BHB AND MILK YIELD 41 39 a b a a 37 35 3 kg/day c b parity 1 parity 2 33 31 a parity 3 a 29 b 27 25 <0. 15 -0. 19 Milk BHB (mmol/l) Santschi et al. 2016 ≥ 0. 20 a-c = P <0. 05 n = 498, 310

Test day SCC (k cells/ml) MILK BHB AND MASTITIS c 500 b 400 300 200 184 k cells/ml a c c b a a parity 1 parity 3 b a-c = P <0. 05 n = 498, 310 100 <0. 15 Santschi et al. 2016 parity 2 0. 15 -0. 19 Milk BHB (mmol/l) ≥ 0. 20

DEVELOPMENT OF KETOSIS PREVALENCE OVER TIME +9% +10% -8% -1% -0. 6% -9% +0. 6% -1% Prevalence of ketosis (low, medium, high risk) in Canada (Valacta), France (CLASEL) and Belgium (region Flanders) and the Netherlands (Qlip) in 2012 and 2014, respectively. Data for Belgium and the Netherlands are expressed as ketosis yes (high risk) or no (low risk). Schwarz et al. 2015

FATTY ACID ANAYLIS

FATTY ACID CALIBRATIONS • Chain length • Short Chain Fatty Acids (SCFA): C 4: 0, C 6: 0, C 8: 0, C 10: 0 • Medium Chain Fatty Acids (MCFA): C 12: 0, C 14: 0, C 16: 0 • Long Chain Fatty Acids (LCFA): C 18: 0, C 18: 1, C 18: 2 • Degree of unsaturation • Saturated Fatty Acids (SFA) • Mono Unsaturated Fatty Acids (MUFA) • Poly Unsaturated Fatty Acids (PUFA) • Major fatty acids • • C 14: 0 C 16: 0 C 18: 1

REAL LIFE EXAMPLES • Visiolait – optimisation of feeding: • Energy and protein efficiency • Rumen activity • Health and fertility Used successfully in France and Germany • National milk laboratories, UK: • Fatty acid profiling as basis for production of value-added dairy products

A MESSAGE TO TAKE HOME Raw milk samples hold a wealth of information – milk quality and dairy herd management + SCC and DSCC as a new tool to improve mastitis management € Other value-added services: Ketosis screening and fatty acid analysis successfully used in many countries around the world + € First indications: e. g. , differentiation of early vs. late stage of mastitis Various practical applications in development das@foss. dk @Schwarz. D 123 @FOSSAnalytical www. linkedin. com/in/daniel-schwarz 84 www. linkedin. com/company/6750/