Rapid detection of Mucorales in human blood and urine samples by functionalized Heusler magnetic nanoparticle assisted customized loop-mediated isothermal amplification.

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01 ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.

Mucormycosis is a challenging disease to diagnose early. This study introduces a sensitive and rapid diagnostic approach using Loop-mediated isothermal amplification technology. Testing blood and urine samples from 30 patients revealed promising sensitivity and repeatability, indicating its potential for non-invasive diagnosis.

Determining the Prevalence of Avian Chlamydiosis in Wild Amazona Species From Brazil Using Molecular Testing and Clinical Signs.

Avian chlamydiosis is a disease that occurs in birds, especially parrots, and is caused by the Gram-negative bacterium Chlamydia psittaci. Wild Animal Screening Centers in Brazil receive, maintain, treat, and place (preferably to nature) wild animals recovered from illegal trafficking. We performed molecular testing for avian chlamydiosis in parrots from the genus Amazona that were presented to these centers. Cloacal swab samples were collected from 59 parrots (Amazona species) and transported in aqueous or culture medium. The samples were subsequently submitted for DNA extraction by the boiling method, polymerase chain reaction (PCR) amplification using CPF/CPR primers, and agarose gel electrophoresis. Conjunctivitis, nasal discharge, and poor body condition were the clinical signs associated with a differential disease diagnosis of avian chlamydiosis. Transport medium did not have an effect on the test results. The prevalence of C psittaci in the samples was 37% (22/59, 95% confidence interval: 25-49). There was a significant (P = 0.009) association between the PCR test results and clinical signs. Follow-up testing was conducted on a subgroup of 14 individuals that initially tested negative on PCR; 50% (7/14) of these birds were found to be positive within 24 days of the first test. The results of this study confirm the feasibility of using the CPF/CFP primer-based PCR to detect C psittaci in Amazona species, describe a less costly method of transporting biological material for DNA extraction, and evaluate the temporal aspect for obtaining positive results through molecular testing for C psittaci in Amazona species.

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Development of a TaqMan loop-mediated isothermal amplification assay for the rapid detection of pigeon paramyxovirus type 1.

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies µL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati.

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15­120 min), and different concentrations of malachite green dye (0.004­0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10­100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.

Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.

BACKGROUND: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for "naked eye" end-point detection when testing 'real-world' clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. RESULTS: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91-100%. CONCLUSIONS: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing 'real-world' samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

Application of the FTA elute card coupled with visual colorimetric loop-mediated isothermal amplification for the rapid diagnosis of Streptococcus agalactiae in farmed tilapia (Oreochromis niloticus).

A method combining the FTA Elute card and visual colorimetric loop-mediated isothermal amplification (FTA-e/LAMP) was tested to diagnose Streptococcus agalactiae infections in vitro and in vivo. FTA-e/LAMP consists of two main steps: first, the FTA card is used to extract DNA and then a colorimetric loop-mediated isothermal amplification (LAMP) reaction is carried out on the extracted DNA. In vitro sensitivity was 1.9 x 102  CFU/mL, and regarding specificity, all nine S. agalactiae strains tested positive. All Streptococcus spp. tested negative, except for S. dysgalactiae, thereby indicating the need for another set of primers to distinguish this species from S. agalactiae. To diagnose S. agalactiae infections using FTA-e/LAMP in vivo, two experimental trials on juvenile Oreochromis niloticus infected with bovine or piscine strains were carried out. Sensitivity in symptomatic fish was 100%, and 50.7% of fish without signs were positive. All negative control fish tested negative (n = 28). No bacteria were detected after 16 days post-infection (dpi). Accuracy during the first week (1-7 dpi) was 89% and decreased to 44% thereafter (10-22 dpi). FTA-e/LAMP results suggest that this method is a promising tool for early and fast diagnosis of S. agalactiae on tilapia farms.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with colorimetric gold nanoparticle (AuNP) probe assay for visual detection of tilapia lake virus (TiLV) in Nile and red hybrid tilapia.

Tilapia is one of the major aquaculture species with a global economic significance. Despite a high scale of production worldwide, mortality in many tilapia cultures has recently become a problem concerned with not only intensive farming but also the prevalence of infectious pathogens. Tilapia lake virus (TiLV) has emerged as a serious single-stranded RNA disease agent that thus far has continued to cause a number of incidences across the continents. Conventional PCR-based molecular detection techniques, despite having high sensitivity for TiLV, are not best suited for the onsite identification of infected fish mainly due to their requirement of laboratory resources and extended assay turnaround time. To address this practical limitation, we have developed a novel colorimetric assay based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and gold nanoparticle (AuNP)-labelled oligonucleotide reporter probe targeting the viral genomic segment 9 that enables the assay to be completed within an hour. This technique has been shown to be compatible with a rapid nucleic extraction method that does not demand centrifugation steps or any benchtop laboratory equipment. When validated with field-acquired tilapia samples, our RT-LAMP-AuNP assay exhibited a near-perfect agreement with the semi-nested RT-PCR assay recommended by OIE with Cohen's κ coefficient of .869, yet requiring significantly less time to perform.

Rapid visual detection of Aeromonas salmonicida by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

To make crucial prevention, reduce fish losses and minimize the economic damage of diseases on the fish farm owners, a rapid detection of fish pathogens is mandatory. In this study, a loop-mediated isothermal amplification assay combined with hydroxynaphthol blue dye (LAMP-HNB) was developed and used for the rapid detection of Aeromonas salmonicida that caused significant economic losses in fish farming. Firstly, a pair of outer and inner primers specific for conserved fragment of vapA gene in A. salmonicida were designed and synthesized. Secondly, by optimizing the reaction conditions including reaction temperature, time, Mg2+ concentration, dNTP concentration and primer ratio, a LAMP-HNB assay was successfully established for the detection of A. salmoncida. Thirdly, the assay showed good specificity with no false-positive and false-negative results, and good sensitivity with the detection limit of 3.077 × 10-6  ng/µl, which was 102 times more sensitive than the conventional PCR. Finally, the LAMP-HNB assay was validated by the fish samples inoculated with different concentrations of A. salmoncida. This is the first development of rapid visual detection of A. salmonicida based on LAMP-HNB assay, which has great application prospect and market for diagnostic testing, health certification and active surveillance programmers.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

A modified loop-mediated isothermal amplification method for detecting avian infectious laryngotracheitis virus.

This study was aimed to establish a highly specific and sensitive loop-mediated isothermal amplification (LAMP) method for diagnosing avian infectious laryngotracheitis (AILT). DNA was extracted from isolated infectious laryngotracheitis virus (ILTV) strains and control samples, followed by PCR using three sets of six specific primers. The detection efficiency of the LAMP assay was evaluated by the turbidity and calcein methods. The sensitivity of LAMP was then assessed using a concentration gradient followed by a specificity analysis. Furthermore, the detection efficiency of LAMP and PCR was compared. Finally, a clinical test was performed to evaluate the value of the LAMP assay. The optimal temperature for the LAMP reaction was 66 °C. Meanwhile, the primers selected for the LAMP assay were highly specific for the target virus. The sensitivity of the turbidity and calcein methods for LAMP was consistent. The minimum detection concentration of LAMP was 0.06 pg/µL, which was 100-fold higher than that of PCR. Furthermore, the results from clinical samples showed that the LAMP method could identify AILT from many samples. The newly designed LAMP assay was an effective method for AILT detection at an optimal temperature of 66 °C with a minimum detection concentration of 0.06 pg/µL.

An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases.

Present-day diagnostic tools and technologies for canine diseases and other vector-borne parasitic diseases hardly meet the requirements of an efficient and rapid diagnostic tool, which can be suitable for use at the point-of-care in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been always a method of choice in the development and validation of quick, precise, and sensitive diagnostic assays for pathogen detection and to reorganize point-of-care (POC) molecular diagnostics. In this study, we have demonstrated an efficient detection system for parasitic vector-borne pathogens like Ehrlichia canis and Hepatozoon canis by linking the LAMP assay to a smartphone via a simple, inexpensive, and a portable "LAMP box," All the components of the LAMP box were connected to each other wirelessly. This LAMP box was made up of an isothermal heating pad mounted below an aluminum base which served as a platform for the reaction tubes and LAMP assay. The entire setup could be connected to a smartphone via an inbuilt Wi-Fi that allowed the user to establish the connection to control the LAMP box. A 5 V USB power source was used as a power supply. The sensitivity of the LAMP assay was estimated to be up to 10-6 dilution limit using the amplified, purified, and quantified specific DNA templates. It can also serve as an efficient diagnostic platform for many other veterinary infectious or parasitic diseases of zoonotic origin majorly towards field-based diagnostics.

Development of a loop-mediated isothermal amplification assay based on RoTat1.2 gene for detection of Trypanosoma evansi in domesticated animals.

The early containment of trypanosomosis depends on early, sensitive, and accurate diagnosis in endemic areas with low-intensity infections. The study was planned to develop a simple read out loop-mediated isothermal amplification (LAMP) assay targeting a partial RoTat1.2 VSG gene of Trypanosoma evansi with naked eye visualization of LAMP products by adding SYBR® Green I dye. The visual results were further confirmed with those of agarose gel electrophoresis, restriction enzyme digestion of LAMP products with AluI, and sequencing of the PCR products using LAMP outer primers. The LAMP primers did not show cross reactivity and non-specific reactions with regional common hemoparasitic DNA revealing high specificity of the assay. The threshold sensitivity level of the LAMP assay was determined to be 0.003 fg compared to 0.03 fg RoTat1.2 amplified DNA fragments of T. evansi by PCR assay. Moreover, assessment of 500 blood samples collected from unhealthy domestic animals in field suspected for various hemoparasitic infections was carried out for the presence of T. evansi by microscopy, RoTat1.2 VSG PCR, and LAMP assay. LAMP could detect T. evansi in 36 samples, while PCR and microscopy could detect 33 and 12 samples, respectively. All the samples positive by microscopy and PCR were also confirmed positive by the LAMP assay. The current LAMP assay has appealing point of care characteristics to visually monitor the results, lessen the need of post DNA amplification procedure, and enable this method to be applied as a rapid and sensitive molecular diagnostic tool in under resourced laboratories and field setup.

Comparison of PCR-based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy.

Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order to adopt this diagnostic method, the Italian National Reference Centre for Equine Diseases (NRC-ED) conducted a preliminary comparison between an end-point PCR, nested PCR, real-time PCR, and commercial real-time PCR, for the detection of T. equi and B. caballi, respectively. One hundred and three field samples, collected during spring-summer 2013 in Latium and Tuscany regions, were employed for the study, and results discordant between detection assays were confirmed by sequencing. The reference assay was defined as that showing the highest sensitivity, and the relative sensitivity (rSe) and specificity (rSp) of the other methods were estimated referring to this assay. Agreement between methods was estimated by calculating the concordance between each pair of methods. Although no statistical differences were detected among PCR-based methods, the non-commercial real-time PCR assays seemed to be the most suitable for detection of T. equi and B. caballi, respectively. An important advantage of direct PCR detection of the pathogen, in comparison to indirect detection using serological methods, is that it allows specific treatment against the causative pathogen species responsible of the infection as well as for the definition of the infectious status of an animal for international movement.

Perspectives and challenges in validating new diagnostic technologies.

This paper focuses on several new diagnostic technologies, which are set to dominate the testing landscape in the near future and have applications in animal health diagnostics, namely: next-generation sequencing, assays to detect biomarkers, and point-of-care tests. An example of real-time loop-mediated isothermal amplification validation is also provided. Validating these new technologies presents several challenges, which are addressed in this paper.

Les auteurs s'intéressent à plusieurs nouvelles technologies de diagnostic appelées à occuper, dans un futur proche, une place de choix dans le paysage du dépistage et dont il existe déjà des applications en santé animale, à savoir : le séquençage de nouvelle génération, la détection de biomarqueurs et les tests utilisables sur le lieu des soins. Ils décrivent par ailleurs l'exemple de la validation d'une amplification isotherme à médiation par boucle en temps réel. La validation de ces nouvelles technologies présente un certain nombre de difficultés, que les auteurs examinent en détail.

Los autores se centran en varias tecnologías de nuevo cuño que están llamadas a dominar el panorama de las pruebas de diagnóstico en un futuro próximo y que tienen aplicaciones de diagnóstico en sanidad animal, a saber: la secuenciación de próxima generación, los ensayos de detección de marcadores biológicos y las pruebas practicadas en el lugar de consulta. También ofrecen un ejemplo de validación de una técnica de amplificación isotérmica mediada por bucles en tiempo real. La validación de estas nuevas tecnologías presenta varias dificultades, que los autores examinan en estas líneas.

Molecular Detection of Feline Leukemia Virus in Oral, Conjunctival, and Rectal Mucosae Provides Results Comparable to Detection in Blood.

Feline leukemia virus (FeLV) infection causes immunosuppression, degeneration of the hematopoietic system, and fatal neoplasms. FeLV transmission occurs mainly by close social contact of infected and susceptible cats. Developing procedures for the diagnosis of feline retroviruses is crucial to reduce negative impacts on cat health and increase the number of animals tested. Blood collection requires physical or chemical restraint and is usually a stressful procedure for cats. Our objective was to evaluate the use of samples obtained from oral, conjunctival, and rectal mucosae for the molecular diagnosis of FeLV. Whole blood and oral, conjunctival, and rectal swabs were collected from a total of 145 cats. All samples were subjected to the amplification of a fragment of the gag gene of proviral DNA. Compared to blood samples used in this study as a reference, the accuracies for each PCR were 91.72, 91.23, and 85.50% for samples obtained by oral, conjunctival, and rectal swabs, respectively. The diagnostic sensitivity and specificity were 86.11 and 97.26% for the oral swabs, 90 and 92.59% for the conjunctival swabs, and 74.24 and 95.77% for the rectal swabs, respectively. The kappa values for oral, conjunctival, and rectal swabs were 0.834, 0.824, and 0.705, respectively. The diagnosis of these samples showed the presence of proviral DNA of FeLV in oral and conjunctival mucosae. In conclusion, mucosal samples for the molecular diagnosis of FeLV are an excellent alternative to venipuncture and can be safely used. It is faster, less laborious, less expensive, and well received by the animal.

Development of a recombinase polymerase amplification fluorescence assay to detect feline coronavirus.

Feline coronavirus (FCoV) is classified into two pathotypes: the avirulent feline enteric coronavirus (FECV), and the virulent feline infectious peritonitis virus (FIPV). Rapid pathogen detection, which is efficient and convenient, is the best approach for early confirmatory diagnosis. In this study, we first developed and evaluated a rapid recombinase polymerase amplification (RPA) detection method for FCoV that can detect FCoV within 15 min at 39 °C. The detection limit of that assay was 233 copies/µL DNA molecules per reaction. The specificity was high: it did not cross-react with canine distemper virus (CDV), canine coronavirus (CCoV), canine adenovirus (CAV), feline calicivirus (FCV), feline herpesvirus (FHV), or feline parvovirus (FPV). This assay was evaluated using 42 clinical samples (30 diarrhea samples and 12 ascites samples). The coincidence rate between FCoV-RPA and RT-qPCR for detection in clinical samples was 95.2%. In summary, FCoV-RPA analysis provides an efficient, rapid, and sensitive detection method for FCoV.

Bioassay-based detection of Toxoplasma gondii in free-range chickens from Khorramabad, Iran: comparison of direct microscopy and semi-nested PCR.

This study aimed to investigate the occurrence of anti-Toxoplasma gondii antibodies in free-range chickens from Khorramabad, western Iran, and also to compare the performance of direct microscopy and semi-nested PCR in mice bioassayed with tissues from seropositive chickens. We investigated 97 serum samples from free-range chickens, using the modified agglutination test (MAT). Tissues from all seropositive chickens (MAT ≥ 1:10) were bioassayed in mice. All inoculated mice were examined by direct microscopy and a semi-nested PCR targeting the 529 bp repeat element (RE) of the parasite. Anti-T. gondii antibodies were detected in 21.6% of chicken sera. Eighteen of 21 (85.7%) seropositive chickens were positive in mouse bioassay using molecular DNA detection. However, biological forms of the parasite were isolated only from 11 (52.3%) seropositive chickens. Compared with semi-nested PCR, the sensitivity of direct microscopy was 62.1%. It can be concluded that although direct microscopy is a rapid and specific method for the detection of T. gondii, it does not detect the parasite in all experimentally infected mice. The low sensitivity of direct microscopy highlights the need for molecular techniques, such as RE-based semi-nested PCR, to increase the sensitivity of the mouse bioassay.

Potential of cell-free DNA as a screening marker for parasite infections in dog.

Parasitic infections are common in stray dogs and accurate knowledge of parasite communities in dogs would provide insight into the epidemiology of parasitic diseases. In this study, we used Illumina sequencing technology to evaluate cell-free DNA (cfDNA) as a marker for screening of parasitic infections in dogs. Plasma samples from 14 stray dogs captured in Bangladesh were used in the experiments. An average of 2.3 million reads was obtained for each sample. BLASTn analysis identified 150 reads with high similarity with parasites from 19 different genera. In particular, we detected sequences of Babesia spp. in five dogs; consistent with this, a previous study using conventional PCR showed that four of these dogs were positive for B. gibsoni. Several reads with similarity to Leishmania and filarial nematodes were also identified. These findings indicate that cfDNA in blood can be a potential screening marker for identifying parasite diversity in dogs.