Detection Of Canine Distemper Virus Research Articles

Molecular Detection of Canine Distemper Virus Among Dogs Showing Neurologic and Non-neurologic Forms of Disease

Background: Canine distemper (CD) is the dogs’ most contagious and lethal viral disease. Despite the widespread use of vaccines to control CD, the prevalence of the CD virus (CDV) has increased at an alarming rate in recent years. Objectives: To identify the genotypes responsible for the neurological and non-neurological clinical forms of CD and to investigate the presence of the virus in the neurological and non-neurological forms of the disease. Methods: In this descriptive-analytical study, the samples were collected from 70 CD suspected unvaccinated dogs with clinical signs of distemper. All cases were tested with rapid tests and separated into 3 groups based on clinical signs and symptoms. Cerebral spinal fluid (CSF), respiratory secretion, and fecal samples of allall 70 cases were examined for reverse transcription polymerase chain reaction (RT-PCR). After sequencing the hemagglutinin gene (H gene), phylogenetic analysis of the gene isolated from CDVs was carried out using MEGA software, version 7. Results: The RT-PCR results showed that the respiratory secretion sample in the non-neurological CDV group (85%) and the neurological CDV group (80%) had the highest level of virus contamination. However, in the non-neurological CDV group, the CSF sample (40%) had a high level of infection. In neurotic groups, cases older than 12 months showed the highest percentage of distemper contamination, and in the non-neurologic CDV group, those between 3 and 6 months were more involved. Sequencing and phylogenetic analysis of the H gene revealed the CDV as a member of the endemic Arctic-like genetic lineage. Conclusion: The genotypic examination of the hemagglutinin gene of the distemper virus reveals that the recent isolates of neurologic and non-neurologic clinical forms of CDV in Iran are similar. In positive rapid test cases, the PCR test of respiratory secretions for virus detection ranks with the highest sensitivity. In neurologic cases with negative rapid test results, PCR of CSF had the highest sensitivity, so it may serve as a diagnostic criterion.

Recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) designed for rapid detection of canine distemper virus.

In the present study, recombinase polymerase amplification (RPA) was combined with the colloidal gold lateral flow dipstick (LFD) method to establish a new, stable, and efficient assay for the detection of canine distemper virus (CDV). We designed a set of specific primers labeled with biotin and a specific probe labeled with dSpacer and C3 spacer, according to the conserved region in the N-terminal gene sequence of CDV. The reaction conditions and systems were then optimized, and the sensitivity and specificity were analyzed for potential clinical application. The results showed that the RPA-LFD assay for CDV detection was successfully established. We also found that the temperature in a closed fist (35°C) is optimal for the RPA reaction. The optimal ratio of primer to probe was 2:1. The minimum detection limit of the RPA-LFD assay was 1 × 101 the median tissue culture infective dose (TCID50)/mL. Using this assay with samples from experimentally infected dogs, CDV was detected in nasal secretions, eye secretions, and blood on the fourth day post infection. In summary, this novel RPA-LFD assay for CDV detection is simple to use, and preliminary findings indicate its high specificity and sensitivity.

Novel high-coverage primers for detection of canine morbillivirus by end-point and real-time RT-PCR assays

Canine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100 % (194/194) and 99 % (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15 % (29/194) and 9 % (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.001 TCID50/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32 %) positive samples selected for comparison purposes. In addition, our assays did not amplify other canine viruses associated with respiratory and neurological diseases: canine adenovirus 2, canine parainfluenza virus 2, canine herpesvirus 1 and rabies virus. Overall, we describe a high-coverage primer set for CDV detection, which represents an attractive tool for laboratory diagnosis of canine distemper.

Optimization and Comparison of Real-time Reverse Transcription PCR with Conventional PCR for Detection of Canine Distemper Virus

Optimization and Comparison of Real-time Reverse Transcription PCR with Conventional PCR for Detection of Canine Distemper Virus

Trace detection of canine distemper virus based on Michelson-interferometer sensing probe.

A single-mode-fiber (SMF)-multimode-fiber (MMF)-tri-core-fiber (TCF) Michelson probe structure is proposed for trace detection of canine distemper virus (CDV). One end of the TCF is cut flat and fused with the multimode fiber, and the other end is coated with a silver film to enhance the reflection, and an optic-fiber sensing probe with SMF-MMF-TCF structure is obtained. The (PDDA/PSS)3 multilayer film is modified on the surface of the fiber by layer-by-layer self-assembly method as a polyelectrolyte binder to immobilize CDV antibodies to form a (PDDA/PSS)3 /CDV antibody composite membrane for specific detection of CDV antigens. The response-recovery test of the sensor is performed to verify its repeatability. The detection limit, the sensitivity, and the linear fitting degree for CDV antigen are 0.1236 pg/mL, 1.1776 dB/(pg/mL), and 0.9899, respectively. At the same time, the stability, selectivity, and clinical samples of the sensors were also verified.

Trace detection of canine distemper virus based on surface-functionalized optic-fiber Mach–Zehnder interferometer and the Vernier effect

A surface-functionalized cascaded optic-fiber Mach–Zehnder interferometer (MZI) is proposed and fabricated for trace detection of canine distemper virus (CDV). The Vernier effect is formed based on single-mode fiber (SMF)-dislocation single-mode fiber-SMF. The two MZIs serve as the sensing arm and reference arm, respectively. The CDV antibody is modified on the sensing arm to detect CDV antigen, and the surface structure and morphology are analyzed by the atomic force microscope. The sensitivity of the cascaded MZI sensor is about 12 times than that of a single MZI. In the range of 0.1–104 pg ml−1 CDV, the detection limit of the cascaded MZI sensor is 0.1687 pg ml−1, and the response time is 25 min. The sensor also has good temperature, time stability and excellent selectivity. The biosensor has the advantages of low cost, easy preparation, high sensitivity and good selectivity, and has potential application for trace CDV detection.

A recent distemper virus outbreak in the growing canine populations of Galapagos Islands: a persistent threat for the endangered Galapagos Sea Lion.

Canine Distemper Virus (CDV) is a highly contagious virus that can cross mammalian species barriers and has widespread impacts on both domestic animals and wildlife populations. This study describes a recent outbreak of CDV in the Galapagos Islands in 2019. A total number of 125 dogs with clinical signs compatible with CDV were included in this study. Nasal swabs were taken and analyzed by RT-qPCR for the detection of CDV, resulting in a positivity rate of 74.4% (IC95%, 66-81%). Among the CDV positive dogs, 82.2% presented with respiratory signs, 48.8% neurological signs, and 28.9% gastrointestinal signs. CDV has been previously reported in the domestic canine population of the Galapagos Islands in 2001 and 2004. The current study shows how CDV is still a threat for the endemic and endangered Galapagos sea lion, despite recent policies for dog population control and CDV vaccination.

Rapid molecular detection and isolation of canine distemper virus in naturally infected dogs

The canine distemper virus (CDV), which infects dogs and a broad range of animal species, remains a serious concern in Türkiye and across the world. The current study shows that CDV can be detected and isolated rapidly and specifically in naturally infected dogs. Whole blood, nasal swab, ocular swab, rectal swab, and urine samples from 50 stray dogs were used in the study (n = 250). The presence of the CDV genome was confirmed in 105 (42%) samples using one-Step real-time RT-PCR. In total, 39 dogs were diagnosed with CDV infection based on the detection of cytopathic effects in MDCK, which was verified by the fluorescent antibody technique. A total of 12 one-Step real-time RT-PCR negative samples, consisting of 4 rectal swabs and 8 urine samples, were found to be positive by virus isolation. Blood, nasal swab, ocular swab (P<0.01, r = 1), rectal swab (P<0.01, r = 0.844), and urine samples (P<0.01, r = 0.697) all showed positive correlations in the tests for viral genome detection and virus isolation. CPE levels of high 37 (31.62%), medium 26 (22.23%) and low 54 (46.15%) were detected in a total of 117 (46.8%) samples with viral growth in cell culture. The highest CPE levels detected by FAT were for rectal swab and urine samples. In conclusion, the one-step real-time RT-PCR method on rectal swab samples proved to be a very sensitive method for the rapid and reliable CDV detection. Besides, non-modified MDCK can be used to isolate CDV from naturally infected dogs.

Novel optical fiber Vernier immunosensor based on cascading Sagnac loops embedded with excessively tilted fiber grating for specific detection of canine distemper virus.

A novel optical fiber Vernier effect (VE) biosensor based on cascading Sagnac loops embedded with excessively tilted fiber grating (ExTFG) is proposed for the label free and specific detection of canine distemper virus (CDV). The VE was realized by cascading two different Sagnac loops with similar free spectrum range (FSR), one of which was integrated with panda-type polarization maintaining fiber (PMF) as the reference loop, and the other was embedded with ExTFG as the sensing loop. Owning to the amplified function of the VE, the refractive index (RI) sensitivity of the proposed sensing structure reached -1914.89 nm/RIU, which is approximately 12 times higher than that of the single ExTFG based RI sensor. Furthermore, the ExTFG in sensing loop was modified by graphene oxide (GO) and bio-functionalized by the CDV monoclonal antibodies (anti-CDV MAbs) for the specific detection of the CDV. Experimental results show that the proposed optical fiber Vernier sensor could detect the CDV in buffer solution with concentration as low as 1 pg/mL, and the sensitivity was about -1.18 nm/[log(mg/ml)] in the concentration range of 1 pg/mL ~ 50 ng/mL. The excellent specific and clinical properties of the biosensor were verified by immunoassays for fetal bovine serum, Toxoplasma gondii, rabies virus and CDV serum in sequence. Due to the sensitivity amplification function of VE, dense comb spectrum of the Sagnac loop and the stable interference spectra maintained by the polarized light, the proposed biosensor possesses the combined advantages of high sensitivity, high Q-factor and high stability, which may have potential applications in biosensing fields.

Canine Distemper Virus in Autochtonous and Imported Dogs, Southern Italy (2014-2021).

Simple SummaryIn the period 2014–2021, the circulation of CDV in dogs of Southern Italy was investigated. In this time span a reduction in the circulation of CDV was observed, with a higher frequency of detection of the pathogen in imported dogs (18.4%) compared to stray (7.4%) and household (3.9%) animals. These results underline the effectiveness of the prophylaxis strategy on autochthonous dogs as well as the importance of continuous surveillance of CDV, especially in imported dogs.This study aims to investigate the presence of canine distemper virus (CDV) infection in 949 autochthonous or illegally imported dogs from Southern Italy, over a period of eight years (2014–2021). CDV RNA was detected in 6.8% (65/949) of the animals tested, with no detection of CDV in dogs sampled in 2020–2021. The frequency of CDV detection was higher in imported dogs (19/103, 18.3%) with respect to stray (27/365, 7.4%) and household dogs (19/481, 3.9%). On sequence and phylogenetic analyses of selected strains, the analyzed viruses belonged to the Arctic clade, which has already been reported in Italy and in Europe. The results of our study may suggest a reduction of CDV circulation in Southern Italy, while at the same time highlighting the need for strict controls on dog importation, in order to prevent the introduction of viruses from endemic countries.

An Integrated, Tentative Remote-Sensing Approach Based on NDVI Entropy to Model Canine Distemper Virus in Wildlife and to Prompt Science-Based Management Policies.

Simple SummaryCanine distemper virus (CDV) is a pathogen that affects wildlife with particular regard to Canidae family such as red foxes, wolves, etc. In this study, we focus on CDV outbreaks in the Aosta Valley territory, an alpine region in the NW of Italy which was affected by important waves of this disease during the years 2015–2020 (hereinafter called τ). Ground data are collected on the entire territory at a municipality level. The detection of the canine distemper virus is performed by means of real-time PCR. By adopting satellite remote-sensing data, we notice that CDV trends are strongly related to anomalies in the NDVI entropy changes through (τ). A tentative local model is developed concerning on-the-ground data, helping veterinarians, foresters, and wildlife ecologists enforce management health policies in a One Health perspective.Changes in land use and land cover as well as feedback on the climate deeply affect the landscape worldwide. This phenomenon has also enlarged the human–wildlife interface and amplified the risk of potential new zoonoses. The expansion of the human settlement is supposed to affect the spread and distribution of wildlife diseases such as canine distemper virus (CDV), by shaping the distribution, density, and movements of wildlife. Nevertheless, there is very little evidence in the scientific literature on how remote sensing and GIS tools may help the veterinary sector to better monitor the spread of CDV in wildlife and to enforce ecological studies and new management policies in the near future. Thus, we perform a study in Northwestern Italy (Aosta Valley Autonomous Region), focusing on the relative epidemic waves of CDV that cause a virulent disease infecting different animal species with high host mortality. CDV has been detected in several mammalian from Canidae, Mustelidae, Procyonidae, Ursidae, and Viverridae families. In this study, the prevalence is determined at 60% in red fox (Vulpes vulpes, n = 296), 14% in wolf (Canis lupus, n = 157), 47% in badger (Meles meles, n = 103), and 51% in beech marten (Martes foina, n = 51). The detection of CDV is performed by means of real-time PCR. All the analyses are done using the TaqMan approach, targeting the chromosomal gene for phosphoprotein, gene P, that is involved in the transcription and replication of the virus. By adopting Earth Observation Data, we notice that CDV trends are strongly related to an altitude gradient and NDVI entropy changes through the years. A tentative model is developed concerning the ground data collected in the Aosta Valley region. According to our preliminary study, entropy computed from remote-sensing data can represent a valuable tool to monitor CDV spread as a proxy data predictor of the intensity of fragmentation of a given landscape and therefore also to monitor CDV. In conclusion, the evaluation from space of the landscape variations regarding the wildlife ecological corridors due to anthropic or natural disturbances may assist veterinarians and wildlife ecologists to enforce management health policies in a One Health perspective by pointing out the time and spatial conditions of interaction between wildlife. Surveillance and disease control actions are supposed to be carried out to strengthen the usage of geospatial analysis tools and techniques. These tools and techniques can deeply assist in better understanding and monitoring diseases affecting wildlife thanks to an integrated management approach.

Detection of canine distemper virus in stray and pet dogs in Mosul city, Iraq

The current work was carried out during the period from September, 2020 to March, 2021 in Mosul city, Iraq, with the objectives to detection of canine distemper virus (CDV) for the first time in Mosul using microscopic examination of blood smears, rapid serum antigen test and sandwich ELISA test as well as determine the agreement between the different diagnostic methods. A total of 92 blood samples were collected from suspected dogs with CDV (69 stray dogs and 23 Pet dogs). A primarily detection of CDV in blood smear by the presence of inclusion bodies in erythrocytes and leukocytes, followed by detection of CDV antigen in serum using rapid test and sandwich ELISA test. Results indicate that the infection rates of CDV in dogs were 32.6%, 13%, and 19.5% using microscopic examination, rapid test and sandwich ELISA test respectively. A statistically significant higher infection rate was reported in stray dogs compared to pet dogs based on all diagnostic tests used in this study. Based on Kappa values 0.413, 0.675, 0.745, there were moderate agreement between microscopic examination and rapid test, and substantial agreement between microscopic examination and sandwich ELISA test, also between rapid test and sandwich ELISA test respectively. In conclusions, CDV is widespread in dogs in Mosul city, Iraq and all tests used in this study are efficient for detection CDV based on compatibility between them.

Detection of canine distemper virus in cerebrospinal fluid, whole blood and mucosal specimens of dogs with distemper using RT-PCR and immunochromatographic assays.

BackgroundAccurate diagnosis of canine distemper (CD), a highly contagious and acute viral disease, cannot be made solely based on clinical signs and haematological findings, but serological and molecular methods compatible with clinical signs are also required. The type of sample and method of tissue sampling are also very important. Sometimes in chronic cases, the canine distemper virus (CDV) may not be detected in blood and conjunctival specimens but can be detected in cerebrospinal fluid (CSF).ObjectivesThe aim of this study was to evaluate and compare the suitability of CSF samples with whole blood and conjunctival samples in the detection of CDV.MethodsThe CDV was detected in CSF, whole blood and mucosal specimens in 20 dogs with obvious neurological with or without systemic signs congruous with CD by RT‐PCR and rapid immunochromatographic (IC) antigen test kit assays.ResultsRapid kit results were positive for mucosal swabs in 10 cases (50%) and for CSF in 17 cases (85%); RT‐PCR results from whole blood were positive in 11 cases (55%) and from CSF in 16 cases (80%).ConclusionsOur results revealed that dogs with neurological signs showing simultaneous or recent systemic symptoms, whole blood, CSF and mucosal swabs are suitable for the diagnosis of CDV by RT‐PCR and rapid IC antigen test kits, but dogs with neurological symptoms that are systematically asymptomatic or have had systemic signs for a long time, whole blood and mucosal swabs are not good samples while CSF is a good one.

Molecular Detection of Canine Distemper Virus in Dogs in Baghdad Province, Iraq

Canine distemper (CD) is an infectious disease that affects dogs and is extremely contagious and lethal, with a high mortality and morbidity rates. It infects a broad variety of animals, including primates, cetaceans, and carnivores causing a multi-systemic pathological condition. This study aimed to detect canine distemper virus (CDV) in blood samples of dogs clinically suspected with distemper at the Baghdad Veterinary Hospital, Baghdad, Iraq. CDV nucleoprotein gene (N) was detected in the whole blood of 46 dogs using reverse transcription-PCR (RT-PCR). The partially amplified (591 bp) fragment of the N gene was detected in 12 of 46 (26%) blood samples of dogs examined. Based on the partial sequencing data of the N gene, three local isolates might be similar to the NCBI-BLAST reference CDV virus isolates FJ977579.1 China, AF378705.1 USA, and AF305419.1 UK, while other strains EU072200.1 Hungary, AF164967.1 Switzerland, KU578257.1 Germany, and AB474397.1 Japan were found to be rather distinct. The isolates displayed a higher level of similarity with the Snyder Hill CDV strain and Onderstepoort CDV strain. There was less homology with the CDV strain A75/17 of Switzerland and 007Lm CDV strain of Japan. In conclusion, this study confirmed that CDV infection is present in domestic dogs in Iraq. This may indicate a risk of the disease spreading to parts of the country that may be disease-free.

A multiplex nanoparticle-assisted polymerase chain reaction assay for detecting three canine epidemic viruses using a dual priming oligonucleotide system

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 μL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

Development of recombinase polymerase amplification assays for rapid and visual detection of canine distemper virus infecting giant panda

BackgroundCanine distemper virus (CDV) is an enveloped negative-strand RNA virus that exhibits a high mutation rate and continuously expands the range of hosts. Notably, CDV has infected giant panda with spill over from viral reservoirs in canines. Giant pandas (Ailuropoda melanoleuca), especially captive pandas, are known to be susceptible to natural infection with CDV. The high fatality rate of CDV poses a serious threat to the safety of the giant panda population. However, vaccines or drugs for canine distemper in giant pandas have not been developed to date. Therefore, a rapid test that can achieve accurate onsite detection of CDV is important to enable the timely implementation of control measures. In this study, we established a nucleic acid visualization assay for targeting the CDV N gene by using combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF).ResultsThe RT-RPA-VF assay does not require sophisticated equipment, and it was determined to provide rapid detection at 35 °C for 30 min, while the limit of detection was 5 × 101 copies/μl RNA transcripts and 100.5 TCID50 ml− 1 viruses. The results showed that the assay was high specific to CDV and had no cross-reactivity with other viruses infecting the giant panda. Compared with RT-qPCR, RT-RPA-VF assay had a sensitivity of 100% and a specificity of 100% in 29 clinical samples. The coincidence rate between RT-RPA-VF and RT-qPCR was 100% (kappa = 1), indicating that the RT-RPA-VF assay possessed good diagnostic performance on clinical samples.ConclusionsThe RT-RPA-VF provides a novel alternative for the simple, sensitive, and specific identification of CDV and showed great potential for point of care diagnostics for captive and wild giant panda.

Molecular and Immunologic Detection of Canine Distemper Virus (CDV) and Investigation of Co-infection with Candida albicans in Dogs

Background: Canine distemper (CDV) is a highly transmissible serious disease of carnivores. Distemper virus has immunosuppression effects which, in turn, causes opportunistic infections. Objectives: The present study was performed to detect CDV by the genomic and immunological methods and investigate its co-infection with Candida albicans. Methods: In this study, from spring of 2018 to winter of 2019, blood, eye, respiratory and digestive system samples were collected from 50 CDV suspected dogs and 50 seemingly healthy dogs. Rapid distemper immunochromatography kit was applied for the primary detection of CDV. RT-PCR and PCR tests were performed using special primers for molecular investigation. Results: Using immunochromatography, twenty-nine cases and one case had positive results for CDV among dogs suspected of having the disease and seemingly healthy dogs, respectively. After RT-PCR and PCR assays, 37 samples were CDV-positive, and four were C. albicans-positive in the first group. While three CDV and one C. albicans-positive samples were found in the second group. In total, the frequency of co-infection was 4%. Conclusions: In the present study, there was an association between distemper and C. albicans using statistical tests. Conducting such studies in an appropriate sample population gives more accurate results.

A Canine Distemper Virus Retrospective Study Conducted from 2011 to 2019 in Central Italy (Latium and Tuscany Regions).

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the “Europe wildlife” lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies.

Development of a double monoclonal antibody\u2013based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus

Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores.Key points• Three CDV mAbs that recognized different epitopes and bound to virion were generated.• The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed.• The sandwich ELISA is an ideal method for detecting CDV infections in the field.

Specific Detection of Canine Distemper Virus Using Highly Conserved L Gene Sequences

Specific Detection of Canine Distemper Virus Using Highly Conserved L Gene Sequences