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1. For long term storage specimens should be kept below 20 C Whole blood collected by venipuncture should be stored at 2 8 C if the test is to be run within 2 days of collection Do not freeze whole blood specimens Bring specimens to room temperature prior to testing Frozen specimens must be completely thawed and mixed well prior to testing Soecimens should not be frozen and thawed repeatedly If soecimens are to be shipped they should be packed in compliance with local transportation regulations for etiologic agents KRUUSE labsolutions Test Procedure for Serum and Plasma 1 Take a blood sample to prepare the serum or plasma sample Open the pouch remove the pipette and pick up the sample material with the pipette Place one drop of serum or plasma with the pipette into the sample well Wait a few seconds until the sample fluid is completely absorbed Negative Result The test is negative when only the control line C is visible in the reaction field No Ehrlichia canis antibodies were detected in the sample material 2 Open the buffer and add 2 drops of the buffer into the sample well If the liquid is not running well up the strip after 1 5 minute add an additional drop of buffer into the sample well Invalid Result 3 Read the results within 5 10 minutes of starting the test If no control line is visible after the test was performed the test is invalid In this case the test might not have been correctly
2. Only for veterinary and professional use For single use only Use the test cassette within 10 minutes ofopening Please use an appropriate amount of sample Do not apply sample solution to the reaction field Avoid cross reactions by using a new sample tube for each sample Do not touch the reaction field Use only the original buffer provided in the kit Sample material could be infectious Be careful with waste disposal Do not use cassettes after the expiry date Do not use the test if the packaging is damaged Consider the test results as invalid after indicated time Reagents Materials Instruments I Contents 5 test cassettes with pipette and drying bag 5 sample tubes 1 bottle with 2 5 ml reagent buffer 1 instruction manual Il Additional Necessary Equipment Timer Sample Preparation KRUUSE Ehrlichia Quick is developed for examinations of whole blood serum and plasma ATTENTION Samples and other materials should be handled as infectious materials mace recommendations for blood samples Separate serum or plasma from blood as soon as possible to avoid haemolysis Heparin or EDTA blood can be used for the plasma extraction Use only clear non haemolyzed specimens Use of whole blood my decrease the sensitivity of the test result Testing should be performed immediately after sample collection Do not leave the specimens at room temperature for prolonged periods Serum and plasma specimens may be stored at 2 8 C for up to 3 days
3. the sample material BA If a faint test line appears the test result is still positive The red colour 4 C Symbols Used in the test region will vary depending on the concentration of Ehrlichia canis antibodies present in the sample material Lot number Expiry date KRUUSE laDSO UTIONS
4. KRUUSE Ehrlichia Quick Rapid test for detection of Ehrlichia antibodies in whole blood serum or plasma Instruction Manual KRUUSE Ehrlichia Quick is used for the rapid assay detection of specific antibodies against Ehrlichia canis in whole blood serum or plasma of dogs Ehrlichia canis Canine monocytic ehrlichiosis CME is a vector borne disease transmitted by ticks The pathogen Ehrlichia canis E canis is a Rikettsiaceae gram negative obligate intracellular bacteria and is transmitted by the brown dog tick Rhipicephalus sanguineus R sanguineus The disease occurs in dogs especially in southern Europe After an incubation period of 8 to 20 days the course of the disease is divided into three phases In the first acute phase the clinical symptoms are not pathognomonic and rather mild with nonspecific clinical signs such as fatigue fever swollen lymph nodes anorexia and dyspnoea The pathogen infects lymphocytes and monocytes The second subclinical phase may last from several months to years and is characterized by pathogen persistence with an increased antibody production These dogs appear clinically healthy The third phase is chronic CME characterized by complex clinical symptoms resulting from different organ manifestations of the pathogen and persistent antibody production The consequences can be immune complex mediated glomerulopathies arthropathies solenomegaly or neurological symptoms Nonspecific symptoms such as fe
5. carried out the test may have passed the expiry date or the test was exposed for too long to ambient air outside the sealed pouch If this occurs a new test must be conducted ATTENTION The results must be read within 10 minutes of starting the test The test and the result are considered as invalid after that period Remarks The test results should always be judged in connection with the Test Procedure for Whole Blood anamnestic response and clinical context All test results obtained with whole blood 1 Take a blood sample Open the pouch remove the pipette and pick up the samples which do not correlate with the clinical picture should be repeated with a sample material with the pipette and place 1 drop of the whole blood into the plasma or serum sample All doubtful test results should be repeated after approx sample tube 4 weeks Open the buffer and add 3 drops of the buffer into the sample tube Storage 2 Close the sample tube and shake it gently Open the sample tube again and KRUUSE Ehrlichia Quick must be stored at room temperature 2 C to 30 C pick up the sample material with the pipette Slowly add 2 drops of the sample material with the pipette into the sample well add the first drop to the sample Disposal well wait a few seconds until the drop is absorbed then add the second drop p Asafe disposal is recommended Sample material and test cassettes should be collected in a sealable plastic bag If th
6. e liquid is not running well up the strip after 1 5 minute add an additional drop of buffer into the sample well Test Performance Characteristics PE N Ehrlichia canis IFAT 3 Read the results within 5 10 minutes of starting the test antibodies Positive Negative KRUUSE Ehrlichia Positive 48 1 Quick Negative r A a Sensitivity 94 12 2 Specificity 95 24 o ES amp References Selbitz HJ Truyen U 2011 16 3 4 Gattung Ehrlichia in Mayr A M Rolle Hrsg Medizinische Mikrobiologie Infektions und Seuchenlehre Enke Verlag Stuttgart 9 Auflage Bauer C Brahm R Daugschies A Kietzmann M Kohn B Moritz A Schnieder T Wendland B 2011 Empfehlung zur Bekampfung von durch Vektoren Ubertragenen Krankheiten bei Hunden und Katzen Kleintierpraxis 56 7 373 385 Naucke T J Lorentz S Menn B Mencke N 2011 Pravalenz von durch Ektoparasiten bertragenen Infektionskrankheiten CVBDs bei Import und Reisebegleitenden Hunden in Deutschland und Pr vention von CVBDs Tierdztliche Umschau 66 311 317 Test Evaluation The results of the test can be read 5 10 minutes after starting the test Note A detection of antibodies in the sample material is not possible until 3 4 weeks after infection Positive Result The test is positive when the control line C and the test line T are visible in the reaction field Antibodies against Ehrlichia canis are ee eeeheiimaceaiale detected in
7. n and finally a wicking pad which takes up the fluid applied to the test cassette poem Vicks Y gray yyy 4 Sample Pad Conjugat Pad Reaktionsmatrix Adsorbent Pad Upon application onto the test strip Ehrlichia canis antibodies in the sample material react with the gold labelled antigens in the conjugate pad The fluid then migrates up the strip by capillary action In the test line region the Ehrlichia canis antibody gold marked antigen complexes are captured by the immobilised capture reagents so that a red line is generated The presence of a red line in the test line region indicates a positive result If the sample does not contain Ehrlichia canis antibodies no line will form in the test line region Additionally a red line must form in the control line region independently of the presence of Ehrlichia canis antibodies in the specimen The control line serves as an internal control and indicates that sufficient sample fluid has been applied to the test strip and that proper wicking of the membrane has occurred www kruuse com The Test Cassette The test strip is located behind the plastic cover The sample well is on the right side The reaction field is located in the middle of the test cassette The test and the control region are located in the reaction field The labelling above the reaction field shows the test and control region Sample well for blood sample Reaction field test and control region CAUTION
8. ver anorexia and lethargy also continue A serological test for antibodies against Ehrlichia canis should always be interpreted in conjunction with the current clinical symptoms The detection of antibodies is possible after the seventh day of infection and indicates a past contact with the pathogen However the seroconversion can sometimes take up to four weeks But even dogs with an infection stopped at an early stage may have low antibody titers for several months and are serologically negative Here regular examination is advised With the KRUUSE Ehrlichia Quick veterinarians have a useful tool for the accurate and rapid detection of an infection with Ehrlichia canis at an early stage so correct treatment and prevention can be implemented Test Principle KRUUSE Ehrlichia Quick rapid assay is a sandwich immunoassay which was developed for professional use by veterinarians The assay uses specific antibodies which capture Ehrlichia canis antibodies from the sample and thus visualise the agent KRUUSE Ehrlichia Quick is a highly sensitive immunoassay and comes in a handy test cassette in which a membrane is included Explanation of the Testing Process KRUUSE Ehrlichia Quick test consists of a sample pad a conjugate pad which contains Ehrlichia canis specific goldlabeled antigens a membrane pre coated with Ehrlichia canis specific capture antigens immobilized in the test line region and control antibodies immobilized in the control line regio

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