The SNAP 4DX Plus (IDEXX Laboratories Inc., Westbrook, Maine, USA), an ELISA-based rapid assay, was performed to test the presence of Dirofilaria immitis antigen, and antibodies to Anaplasma phagocytophilum/A. platys, Borrelia burgdorferi, Ehrlichia canis/Ehrlichia ewingii, following the manufacturer’s recommendations. Briefly, three drops of canine serum were mixed with two drops of the conjugate in a fresh tube, and added to the sample well of the SNAP device. As soon as the sample-conjugate mixture reaches the activation circle, the device is activated. Results were manually read 8 min after activating the device. Color development in sample spots indicates a positive reaction. The sensitivity and specificity of the SNAP 4DX Plus test in dogs is 99 and 99.3% for heartworm disease, 90.3 and 94.3% for anaplasmosis, 94.1 and 96.2% for Lyme disease, and 97.1 and 95.3% for ehrlichiosis (IDEXX Laboratories, Inc.).
Snap 4dx plus
Manufactured by IDEXX
Sourced in United States
The SNAP 4DX Plus is a rapid in-clinic test that detects the presence of antibodies to Anaplasma, Ehrlichia, and Borrelia burgdorferi (the causative agent of Lyme disease), as well as the presence of Dirofilaria immitis (heartworm) antigen, in canine and feline patients.
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11 protocols using snap 4dx plus
1
Rapid Canine Infectious Disease Screening
2
Canine Vector-Borne Disease Diagnostic
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Anticoagulated blood was used on the same day to detect antibodies to Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, A. platys, and Borrelia burgdorferi, and antigen of Dirofilaria immitis using a cage-side immunochromatographic test (Snap 4Dx Plus, IDEXX laboratories, Westbrook, ME, USA), according to the manufacturer’s instructions. The packed cell volume (PCV) was measured using the EDTA-stored blood using a portable centrifuge (ZIPocrit, Shanghai LW Scientific Co. Ltd., Shanghai, China). The blood smears were evaluated using the technique described by Allison et al. [6 (link)]; forty medium power (X400) fields were evaluated for each smear.
3
Comprehensive Screening of Canine Vector-Borne Diseases
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Faecal samples were processed with Baermann-Wetzel technique [29 ] for the detection of broncho-pulmonary larvae and by a modified McMaster technique with a cut-off of 15 eggs per gram (epg) [30 –33 ].
Blood was collected from a peripheral vein (jugular or cephalic) using a standard technique and stored in 2.5 ml anticoagulant (K3 EDTA) tube and in 2.5 ml cloth activator tube. A complete cells blood count (CBC), including red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT), white blood cells (WBC) and platelets (PLT), was performed on all blood samples using an automated haematology analyser (Benesphera **H32 VET, Avantor Performance Materials Inc., Center Valley, PA, USA).
The presence of circulating microfilariae was assessed using a modified Knott’s test [34 (link)] on Days -7 and 168. Observed microfilariae were identified to species level using morphometric criteria [34 (link)].
Sera samples collected on Days -7 and 168 were tested by rapid ELISA assays for L. infantum using the Canine Leishmania Antibody Test Kit produced by IDEXX® (IDEXX laboratories, Westbrook, ME, USA), and for Anaplasma spp., Borrelia spp., Ehrlichia spp., and D. immitis using SNAP® 4Dx Plus (IDEXX laboratories, Westbrook, ME, USA) following the producer's recommendations.
Blood was collected from a peripheral vein (jugular or cephalic) using a standard technique and stored in 2.5 ml anticoagulant (K3 EDTA) tube and in 2.5 ml cloth activator tube. A complete cells blood count (CBC), including red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT), white blood cells (WBC) and platelets (PLT), was performed on all blood samples using an automated haematology analyser (Benesphera **H32 VET, Avantor Performance Materials Inc., Center Valley, PA, USA).
The presence of circulating microfilariae was assessed using a modified Knott’s test [34 (link)] on Days -7 and 168. Observed microfilariae were identified to species level using morphometric criteria [34 (link)].
Sera samples collected on Days -7 and 168 were tested by rapid ELISA assays for L. infantum using the Canine Leishmania Antibody Test Kit produced by IDEXX® (IDEXX laboratories, Westbrook, ME, USA), and for Anaplasma spp., Borrelia spp., Ehrlichia spp., and D. immitis using SNAP® 4Dx Plus (IDEXX laboratories, Westbrook, ME, USA) following the producer's recommendations.
4
Canine Leishmaniasis Diagnostic Criteria
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Dogs were prospectively recruited at the internal medicine service of the Hospital Veterinario Valencia Sur (Spain) when they fulfilled the following inclusion criteria: 1) presence of generalized lymphadenomegaly or increased sized of popliteal and/or prescapular lymph nodes, 2) confirmed diagnosis of CanL, 3) negative for other vector-borne pathogens using the SNAP®4Dx®Plus (IDEXX Laboratories Inc., Westbrook, ME, USA) including antigen detection for Dirofilaria immitis (heartworm). and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia. canis, Ehrlichia ewingii and Borrelia burgdoferi. and 4) no development of lymphoma at 6-month follow up. Follow-up was done by the responsible clinician according to the published guidelines for practical management of Canl,[1 (link)] including clinical history and complete physical examination, routine laboratory tests (complete blood count, biochemical profile, serum electrophoresis and complete urinalysis when needed and serology). All samples were collected as part of routine diagnostic procedures and before any other diagnostic or therapeutic procedure was carried out. Animals were included in the study only after the obtention of the signed informed consent by the owner. This study was conducted according to European legislation (86/609/EU).
5
Multiplex Serological Assay for Canine Pathogens
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A quantitative enzyme-linked immunosorbent assay (LEISCAN® enzyme-linked immunosorbent assay [ELISA]) was performed for Leishmania infantum antibody detection (IDEXX Barcelona, Spain), which has a cut-off of 0.55, with values > 0.55 considered positive. A commercial qualitative assay kit (SNAP 4DX Plus IDEXX, Hoofddorp, Netherlands) was employed for the detection of E. canis/ewingii, B. burgdorferi, and A. phagocytophilum/platys antibodies, and D. immitis antigen.
6
Heartworm Antigen Detection in Dogs
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Following submission of owner’s consent for their animals to participate in the study, 20 dogs testing positive for adult D. immitis antigens (SNAP® 4DX Plus®, IDEXX Laboratories Inc., Westbrook, ME, USA) were eligible to participate in the study. Dogs had to be 1–10 years-old, be clinically healthy, have a platelet count > 150 × 103/µl, and be free of left ventricular function disorders and severe concomitant diseases. There were no size or sex restrictions, but some breeds (collies, shelties and Australian shepherds), females at any stage of pregnancy, and dogs that had been on doxycycline or MLs in the past 6 months were not eligible for enrollment.
7
SNAP 4DX Plus Leishmania Diagnosis
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Twenty-one plasma samples that were considered Leishmania-positive or undetermined in DPP, ELISA, or both methods were tested on the SNAP 4DX Plus (Idexx Laboratories) according to the manufacturer’s instructions. SNAP 4DX Plus detects Dirofilaria immitis antigens and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, and Erlichia canis in serum, plasma or whole blood canine samples.
8
Canine Vector-Borne Disease Screening
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Peripheral blood (0.5–1.0 ml) was collected from a vessel on dog’s arm in EDTA tubes. According to the instructions by the manufacturer, whole blood samples from dogs were tested with the ELISA kit SNAP® 4Dx® Plus from IDEXX® (Westbrook, Maine, USA). The SNAP tests were performed on site immediately after blood sampling. This assay screens for the simultaneous qualitative detection of a circulating carbohydrate of D. immitis adult female antigen, and antibodies, both IgG and IgM, against proteins from Ehrlichia spp., Anaplasma spp. and B. burgdorferi. Reported sensitivity and specificity of in-clinic ELISA for detection of antibodies are 96.2 and 100% for Ehrlichia spp., 99.1 and 100% for Anaplasma spp., and 98.8 and 100% for B. burgdorferi. Reported sensitivity and specificity for detection of heartworm antigens are 99.2 and 100%, respectively (Chandrashekar et al., 2010 (link)). The tests were read visually.
9
Canine Heartworm Screening Protocol
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The data was limited to that of blood samples obtained from dogs over 12 months of age to avoid bias due to the long prepatent period of the infection and that collected by attending veterinarians of private clinics or hospitals located in one of the five municipalities of Baixada Fluminense (Metropolitan Rio de Janeiro). The data included: (i) D. immitis antigen detection test results (lateral flow immunochromatographic assay – Alere™ Dirofilariasis Ag Test Kit; BioNote, Inc., Republic of Korea, or enzyme immunoassay – SNAP® 4Dx® Plus; IDEXX Laboratories, Westbrook, MN, United States); (ii) results of modified Knott’s test to detect microfilariae (15 (link)); and (iii) unexpected findings obtained during blood smear for CBC or hemoparasite investigation. When an infection was detected in a dog using one technique, results from other methods were excluded to avoid duplication. When antigen detection test result was available, it was considered first. Knott’s test results were considered when the antigen test result was unavailable, and blood smear results were considered only when none of the other were available. In these cases, the presence of microfilariae was recorded.
10
Canine Leishmaniasis Co-Infections Assessment
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On day 0 (D0), each dog was photographed, clinically examined, assessed for ectoparasites, and collared with Scalibor (4% Delthametrin) (MSD, Animal Health, Brazil). All data, including sex, age, weight, and clinical signs were recorded in individual files. The clinical status of each animal was determined by a score based on systemic, dermatological, and ocular signs (S1 Table ). In addition, animals were screened using a qualitative ELISA test (SNAP 4DX Plus, Idexx Laboratory, Westbrook, Maine, USA) according to the manufacturer’s instructions. This test was able to detect Dirofilaria immitis antigens and antibodies against Anaplasma spp., Ehrlichia spp., and Borrelia burgdorferi.
It was noteworthy that most dogs had co-infections (revealed by the SNAP analyses) thus making it difficult to report with total accuracy their exclusively Leishmania-related symptoms: in the Miltefosine group, 8/10 of the dogs were infected by Ehrlichia canis, and 1/10 infected by D. immitis; in the IN immunotherapy group, 9/10 of the dogs were infected by E. canis, 2/10 infected by D. immitis and 1/10 infected by Anaplasma platys; in the combined treatment group, 8/10 were infected by E. canis, and 4/10 infected by D. immitis (Table 1 ).
It was noteworthy that most dogs had co-infections (revealed by the SNAP analyses) thus making it difficult to report with total accuracy their exclusively Leishmania-related symptoms: in the Miltefosine group, 8/10 of the dogs were infected by Ehrlichia canis, and 1/10 infected by D. immitis; in the IN immunotherapy group, 9/10 of the dogs were infected by E. canis, 2/10 infected by D. immitis and 1/10 infected by Anaplasma platys; in the combined treatment group, 8/10 were infected by E. canis, and 4/10 infected by D. immitis (
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