AN IN-LINE TEST FOR THE COLORIMETRIC DETERMINATION OF SOMATIC CELL COUNT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 0206499
• Grant No.: 2006-33610-16756
• Proposal No.: 2006-00118
• Project Start Date: May 15, 2006
• Project End Date: Jan 14, 2007
• Grant Year: 2006
• Program Code: [8.3]- (N/A)

Recipient Organization
PORTASCIENCE INC.
337 TOM BROWN ROAD
MOORESTOWN,NJ 08057

Performing Department
(N/A)

Non Technical Summary
Many attempts had been made to bring faster testing to the cow-side. The introduction of automatic milking systems (AMS) has the potential to enhance milk production and milk quality. In addition, the size of dairy farms has been increasing, with the increasing need for better cow management. Many of these larger farms are moving to AMS. For this reason, there has been increasing interest in the development of a new type of in-line sensor. In-line SCC sensors are designed to take samples directly from the milking lines and measure signals that may reflect the health of an animal. The proposed research is directed at demonstrating the feasibility of using a simple colorimetric in-line quantitative test to measure somatic cells in milk samples. Since over 90% of somatic cells are white blood cells (WBC) or leukocytes, the proposed method, that utilizes the enzymatic properties of WBC, will directly determine the somatic cell count, yielding quantitative cow side results for the milk of individual cows. The proposed analytical system (Porta-IL) will use an inexpensive photometer and liquid reagents for the detection of sub-clinical mastitis. The proposed test will produce accurate quantitative SCC measurements in approximately 90 seconds.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3499	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3499 - Dairy cattle, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

dairy cattle

milk

milk quality

mastitis

equipment

robotic systems

subclinical mastitis

somatic cell count

colorimetric assay

automatic milking systems

cowside testing

inline testing

Goals / Objectives
The goal of the Phase I program is to demonstrate that it is feasible to design and develop an accurate and inexpensive in-line SCC measurement system. The technical objectives are as follows: *-Develop a stable reagent buffer system that will detect 50,000 cells/mL in 90 seconds. *-Build a working prototype that delivers accurate volumes of milk, buffer, dye substrate, mixes and measures color intensity of the solution. *-The prototype should deliver precise and accurate SCC counts relative to reference method.

Project Methods
Task 1: Demonstrate a 90 second assay time standard curve The objective is to further develop a liquid reagent system by: 1) optimizing the buffer solution to reduce the assay time to 90 seconds. Buffer solution is added to change the pH of the matrix and surfactant to aid in mixing and dissolution of the dye substrate. We will investigate potential accelerators such as alcohols, surfactants, and additional buffer systems to increase speed speed to 90 seconds. Phase I experiments will use a manual mixing protocol with a LED detection system. We will assay samples ranging from <10,000 through 4,000,000 cells/mL (n=50). 2) Reagent stability. The dye substrate is moisture sensitive and light sensitive. Both accelerated and real time stability studies will be run to ensure the final liquid reagent will be room temperature stable. Task 2: Build a flow cell based in-line prototype Construct in-line prototype instrument. 1. Fluidic controls, Designed with 1 peristaltic pump and 6 valves, controlling sample and reagent measurements, mixing, and washing steps. Six valves simplify the initial sequencing design and will eventually be cut to 3. The instrument will also contain reagent, buffer, and waste bottles. 2. Optical detection, A bubble detector (inexpensive) will be used to measure exact sample and reagent volumes. The different fluid segments will be separated by air columns of (bubbles). The leading or trailing edge of a bubble will allow accurate fluid volume measurement by using a LED based detector. An optical flow cell with a path length of 0.3 mm, a LED light source, and a silicon detector will measure the optical intensity of the reaction mixture color. The SCC will be shown on a digital display. Fluid movements and signal processing will be controlled by off-the-shelf electronic control boards. 3. Temperature control, A temperature controlled heating element will be designed into the flow cell and used to maintain a constant 37C assay temperature. Because the assay system is enzymatic based, a constant reaction temperature will ensure assay accuracy. The proposed assay protocol follows: 1. Milk is pumped into the system by opening the first valve. 2. Reagent and buffer are introduced into the flow mixture by opening and closing the second and third valves. 3. The mixture is pumped back and forth through the flow line to achieve complete mixing. 4. The mixture is then pumped into the optical flow cell. 5. The optical intensity of the mixture is measured after 90 seconds. 6. The flow cell is flushed using buffer solution and is ready for the next sample. Task 3: Prototype evaluation A standard curve will be integrated into the system software for precision and accuracy testing. Milk samples and FOSS analysis will be provided by Dr. Ynte Schukken, director of the Quality Milk Promotion Services at Cornell University. Prototype Precision: We will test three samples (n=10 each) with SCC levels at approximately 100,000, 500,000 and 4,000,000. Accuracy: 50 samples to be provided by Milk Quality of Cornell, SCC levels to range from <100,000 through 4,000,000.

Progress 05/15/06 to 01/14/07

Outputs
The objective of the Phase I study was to develop a fast, in-line, colorimetric, quantitative system to determine somatic cell counts (SCC) in the foremilk of cows. The following text summarizes the specific aims and their outcomes. Specific Aim Ia--Reduce the assay time to 90 seconds or less.--Three chemical approaches were investigated to decrease assay time; (1) increasing hydrolytic activity, (2), generating a homogeneous sample, and (3) reducing steric hindrance. Status--Aim Ia successfully completed. Specific Aim Ib--Preserve the stability of dye-substrate solution and buffer solution for 1 year at 22-25C.--To determine the stability of the dye-substrate, spectral analysis of freshly prepared dye-substrate was compared to spectra from solutions kept at 2-8C, 22-25C and 35-39C.--After 4 months at accelerated temperature (35-39C), the dye-substrate solution maintained over 98 percent purity, predictive of 1 year stability at lower temperatures.--Status--Aim Ib was successfully completed. Specific Aim II--Build a working system that can sip milk samples, add buffer/dye solutions, mix, and generate a read out at a fixed timed interval.--Three problems were encountered with initial prototype; (1) measurement of milk, buffer, and dye-substrate reagent volumes using bubble detector was not accurate, (2) back and forth rocking of the reagent mixture did not produce adequate mixing, and (3) the optical detection system, using transmittance mode, yielded a low signal to noise ratio due to the opacity of the milk. The prototype was redesigned to address these problems as follows; (1) the bubble detector was replaced with volume dispensing by counting pump motor steps, (2) the mixing cell was vented to introduce air used for mixing, and (3) the transmittance mode was changed to a reflectance mode measurements.--Status--Aim II was successfully completed. Specific Aim III--Using the finished prototype, generate a dose response for milk samples with SCC levels of 50,000 cells/mL through 3,000,000 cells/mL with acceptance criteria of r equal to 0.9 and precision not to exceed a CV of 15 percent.--74 fresh milk samples were analyzed by a Fossomatic flow cytometer, by DCC analysis, and by the prototype unit. The prototype collected reflectance values as Analog-to-Digital Units that were converted to SCC values. The SCC values ranged from 0 to 2,656,000 cells/mL. The correlation of the prototype system versus both the FOSS and DCC systems was 0.94. Based on the FOSS system and at 200,000 cells/mL, sensitivity was 95.1 percent and specificity 90.1 percent. Precision testing used 3 samples from the 74 collected. Each sample was assayed 10 times with prototype. Sample 1 mean was 550,400 with a std dev of 52,900 and a CV of 9.6 percent. Sample 2 mean was 470,800 with a std dev of 56,200 and a CV of 11.9 percent. Sample 3 mean was 380,400 with a std dev of 57,500 and a CV of 7.2 percent.--Status--Aim III was successfully completed. Final Status--PortaScience successfully completed all of its Phase I specific aims and will be submitting an application for Phase II funding. Patents and Inventions Patent 11/512,498 applied for on 08/30/2006.

Impacts
The proposed instrument system, for determining somatic cell counts, can provide a fast quantitative, inexpensive means of isolating sick cows and poor quality milk so the milk can be diverged prior to reaching the bulk tank. The system will allow dairymen to identify cows in the early stages of infection, resulting in higher quality milk to the public and increased revenue to the dairyman.

Publications

• No publications reported this period