Establishment and application of multiplex PCR for rapid detection of three mink diarrhea-associated viruses.

In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and enteritis virus (MEV). Three compatible sets of primers specific for each virus were designed respectively based on their conserved sequences. After optimization of the crucial factors such as primer concentration and annealing temperature in single and multiple amplification, three specific fragments were simultaneously amplified with the highest sensitivity and specificity in one PCR reaction. The fragments amplified were 259 bp (MCV)，455 bp (MBoV) and 671 bp (MEV). The sensibility of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There were no cross-reactions with some relevant pathogens like mink coronavirus, canine distemper virus, and aleutian mink disease virus. In our study we analyzed viral DNA in mink fecal samples by multiplex PCR assay from China, which revealed the occurrence of MCV, MBoV, and MEV as 3.1 %, 5.7 %, and 9.8 %, respectively. The testing results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100 %. These results indicated that the method could provide technical support for rapid detection of the three diarrhea-associated viruses, and epidemiological investigation of mink viral diarrhea.

Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay.

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/µL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar's test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

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.

Field application of a combined serological-molecular testing protocol for diagnosing genital leptospirosis in naturally infected cows with gestational losses.

BACKGROUND: Bovine genital leptospirosis (BGL) causes chronic reproductive disease in cattle. This study aimed to apply a combined serological-molecular testing protocol under field conditions for diagnosing BGL in cows with gestational losses. METHODS: Three beef herds with reproductive failures were studied, and 60 cows with gestational losses (20 from each herd) were randomly selected for laboratory diagnosis of BGL. In addition, 40 cows with normal pregnancy were included as a control. Blood samples were collected from all 100 cows for microscopic agglutination testing, and cervicovaginal mucus (CVM) samples were collected from 28 cows with gestational losses and 20 control cows for lipL32-PCR. RESULTS: All herds had high Leptospira seroreactivity (>65%), mainly against serogroup Sejroe. Ten of the 28 CVM samples from cows with gestational losses were PCR-positive, while all samples from the control group were negative (p < 0.05). LIMITATIONS: Unfortunately, the positive samples did not amplify in secY-PCR for nucleotide sequencing, which would allow the identification of leptospiral strains. CONCLUSION: Serology was sufficient to indicate leptospirosis at the herd level, but the definitive diagnosis of BGL was only possible using CVM PCR. Although seroreactivity against serogroup Sejroe has been associated with gestational losses, this is the first study to conduct CVM PCR as a confirmatory test for BGL diagnosis in extensive beef herds under field conditions.

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.

Dogs naturally infected by Rangelia vitalii, Babesia canis vogeli, and Ehrlichia canis in São Paulo, Brazil / Cães naturalmente infectados por Rangelia vitalii, Babesia canis vogeli, e Ehrlichia canis em São Paulo, Brasil

Several agents can cause hemoparasitic diseases in dogs, and blood-sucking arthropods transmit these diseases. These agents can cause several clinical manifestations and, in some cases, can kill the host. Because these agents are essential in animal health, this study aims to detect the frequency of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, and Rangelia vitalii by real-time PCR and Babesia vogeli in dogs in the southern region of the city of São Paulo, São Paulo. Of the 98 dog samples, 18 (18.4%) tested positive with real-time polymerase chain reaction for at least one studied agent. Of these 18 samples, 17 tested positive for a single agent (11.2% for B. canis vogeli, 1.02% for R. vitalii, and 5.1% for E. canis), and one showed co-infection with B. canis vogeli and R. vitalii. The results demonstrate the presence of hemoparasites in the studied animals, which can influence the quality and life expectancy of these animals. The Rangeliadetection warns small animal clinicians to include it as a differential diagnosis for hemoparasitosis.(AU)

As hemoparasitoses em cães podem ser causadas por diversos agentes, sendo essas doenças transmitidas por artrópodes hematófagos. Esses agentes podem causar diversas manifestações clínicas e, em alguns casos, podem matar o hospedeiro. Este estudo teve como objetivo detectar por PCR em tempo real a frequência de Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii e Babesia canis vogeli em amostras de cães da zona sul da cidade de São Paulo, Brasil. Das 98 amostras de cães, 18 (18,4%) testaram positivo com reação em cadeia da polimerase em tempo real para pelo menos um agente estudado. Destas 18 amostras, 17 testaram positivo para um único agente (11,2% para B. canis vogeli, 1,02% para R. vitalii e 5,1% para E. canis), e uma apresentou coinfecção com B. canis vogeli e R. vitalii. Os resultados demonstram a presença de hemoparasitas nos animais estudados, o que pode influenciar a qualidade e a expectativa de vida desses animais. Além disso, é o primeiro relato da detecção de R. vitalli na zona sul de São Paulo e serve de alerta para os clínicos de pequenos animais incluírem esse agente como diagnóstico diferencial para as hemoparasitoses.(AU)

An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats.

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.

RNA in situ hybridisation as a molecular diagnostic technique targeting IBA-1 and CD204 in canine histiocytic sarcoma.

BACKGROUND: Canine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA-1, ionised calcium-binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features. OBJECTIVES: This study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single-molecule RNA visualisation in formalin-fixed paraffin-embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis for IBA-1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA-ISH for IBA-1 and CD204 was performed on the DH82 cell line to validate the RNA-ISH probes. RNA-ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA-1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls. RESULTS: RNA-ISH and IHC showed moderate to strong expression for IBA-1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA-ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration. CONCLUSION: RNA-ISH for CD204 and IBA-1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.

LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY FOR DETECTION OF ASIAN FISH TAPEWORM, SCHYZOCOTYLE ACHEILOGNATHI (YAMAGUTI, 1934) [SYN. BOTHRIOCEPHALUS ACHEILOGNATHI].

The Asian fish tapeworm (Schyzocotyle acheilognathi syn. Bothriocephalus acheilognathi) (AFT) is an invasive parasite that can infect many species of fish, although most hosts are primarily members of Cyprinidae. Pathogenicity has most often been reported in aquaculture settings in fry and fingerling stages of carp (Cyprinus spp.). More recently, it has been shown to cause growth retardation in the endangered bonytail chub (Gila elegans) and found to be widespread in populations of endangered humpback chub (Gila cypha) in the Colorado River, Grand Canyon, Arizona. AFT spreads most often through the transport of infected fish, particularly baitfish. Despite its harmful potential, there is no efficient or accurate ante mortem test to detect AFT in water or fish samples before transport. Herein, we report on the development of a sensitive and specific loop-mediated isothermal amplification (LAMP) assay to detect the parasite in under 30 min from laboratory prepared samples. Six LAMP primers were designed to amplify a variable region of the 18S ribosomal RNA gene in AFT with the detection and quantification of DNA on a real-time fluorometer. The limit of detection was 1 × 101 copies/µl of DNA extracted from as few as 2 AFT eggs. Future application of our assay would be a low-cost test to rapidly and accurately detect AFT DNA from environmental samples on-site so that preventive actions can be taken to halt the spread of the AFT through the movement of infected fish.

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.

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.

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.

Broad detection and quick differentiation of bovine viral diarrhea viruses 1 and 2 by a reverse transcription loop-mediated isothermal amplification test.

For broad detection of pestivirus A (bovine viral diarrhea virus 1: BVDV1) and pestivirus B (BVDV2) by a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test, the P25 primer set was designed using nucleotide sequences of 5'-UTR region of 1454 BVDVs. The base coverage of each primer against diverse BVDVs were more than 99% in each base position. The one step LAMP test with the P25 primer set could detect both BVDV1 (TK) and BVDV2 (KZ), but did not amplify 5 other bovine viruses. Detection limit of the LAMP test was 103 copies of synthesized DNAs, and 10-3 and 10-4 dilutions of viral RNAs of TK and KZ strains, respectively, whereas that with current Aebischer's primer set was 10-2 dilution and negative of these RNAs, respectively. All of the 63 viral RNA samples of persistently infected (PI) cattle, consisting of the 1a (12), 1b (31), 1c (11), and 2a (9) subgenotypes, were broadly detected with the P25, while only 65% of them were positive with Aebischer's primer set. The validation study showed that the RT-LAMP test with the P25 had 100% sensitivity and 100% specificity against that with updated Vilcek's PCR primers. Also, by using the P26 primer set which contained 3 species-specific primers, all 63 RNA samples were clearly distinguished from BVDV1 or BVDV2 by the typing RT-LAMP test. These results indicate that the one step RT-LAMP test using P25 or P26 primer sets would be useful for broad detection and rapid differentiation of BVDV1 and BVDV2.

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.

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 Anaplasma phagocytophilum in hair and spleen of cats revealed a possible underestimation of feline vector-borne pathogens.

Feline Vector-Borne Diseases show increased global prevalence and some Anaplasma and Ehrlichia species may pose a risk to human health. The diagnosis of Anaplasma and Ehrlichia species infection in cats is achieved by the combined use of different methods as cytologic examination evidencing intracytoplasmic morulae, serologic tests and molecular assays. The peripheral whole blood is considered the sample of choice for Anaplasma and Ehrlichia species DNA detection in cats, but false negative results are reported leading to underestimation of infection prevalence. In order to have a more accurate assessment of the spread of feline vector-borne pathogens, the presence of Anaplasma spp. and Ehrlichia spp. DNA in 37 owner and shelter cats subjected to necropsy were prospectively investigated by testing in end-point PCR spleen, bone marrow, blood clot and hair samples. The bacteria identified were genetically characterised. Three shelter cats tested positive for A. phagocytophilum DNA in spleen (one cat) or in hair samples (two cats). None of the cats tested positive in bone marrow and blood samples. From the results obtained, it can be assumed that the use of spleen or hair samples could allow a more reliable detection of A. phagocytophilum DNA in cats with blood tested negative. In the phylogeny constructed with a fragment of the heat shock (groEL) gene nucleotide sequences, all the identified A. phagocytophilum clustered with bacteria infecting a wide range of hosts, including humans, showing a potential zoonotic role.

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.

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 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.