Novel High-Throughput Multiplex qPCRs for the Detection of Canine Vector-Borne Pathogens in the Asia-Pacific.
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SUBMITTER: Huggins L
PROVIDER: S-EPMC8161336 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundIn Southeast Asia, the canine vector-borne pathogens Babesia spp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, haemotropic mycoplasmas and Dirofilaria immitis cause significant morbidity and mortality in dogs. Moreover, dogs have also been implicated as natural reservoirs for Rickettsia felis, the agent of flea-borne spotted fever, increasingly implicated as a cause of undifferentiated fever in humans in Southeast Asia. The objective of this study was to determine the prevalence and diversity of canine vector-borne pathogens in 101 semi-domesticated dogs from rural Cambodia using molecular diagnostic techniques.ResultsThe most common canine vector-borne pathogens found infecting dogs in this study were Babesia vogeli (32.7 %) followed by Ehrlichia canis (21.8 %), Dirofilaria immitis (15.8 %), Hepatozoon canis (10.9 %), Mycoplasma haemocanis (9.9 %) and "Candidatus Mycoplasma haematoparvum" (2.9 %). A high level of co-infection with CVBD agents (23.8 %) was present, most commonly B. vogeli and E. canis. Naturally occurring R. felis infection was also detected in 10.9 % of dogs in support of their role as a natural mammalian reservoir for flea-borne spotted fever in humans.ConclusionsThis study reports for the first time, the prevalence and diversity of CVBD pathogens in dogs in Cambodia. In total, five species of CVBD pathogens were found infecting semi-domesticated dogs and many were co-infected with two or more pathogens. This study supports the role of dogs as natural mammalian reservoirs for R. felis, the agent of flea-borne spotted fever in humans.
| S-EPMC4862146 | biostudies-literature
Project description:BackgroundVector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP .ResultsWe tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample.ConclusionsBloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.
| S-EPMC8325813 | biostudies-literature
Project description:Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens (TBPs), a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of TBPs, and which can also be customized to monitor emerging diseases.
| S-EPMC4114295 | biostudies-literature
Project description:Canine vector-borne pathogens (CVBP) have a worldwide distribution and show a high prevalence in tropical countries such as Sri Lanka. Some CVBP are zoonotic, with dogs identified as reservoir hosts for human subcutaneous dirofilariasis and potentially for spotted fever rickettsioses and re-emergent brugian filariasis in Sri Lanka, making these pathogens emerging public health issues in the country. Veterinarians are crucial in monitoring, preventing, and controlling these pathogens in dogs. Therefore, it is imperative to understand veterinarians' knowledge, attitude, and practices (KAP) regarding CVBP to mitigate their impact. A survey was designed and administered electronically to veterinarians residing and practising in Sri Lanka. Responses were evaluated using descriptive, univariable, and multivariable analyses to investigate associations between demographic factors, knowledge, attitude, and practices related to CVBP. Out of the 170 participating veterinarians, nearly 70% had moderate or high knowledge. However, the awareness of zoonotic pathogens, Brugia spp. (16%) and Rickettsia conorii (18%), was low, and a considerable number of veterinarians were unaware of the zoonotic nature of Dirofilaria repens. Based on multivariable analysis adjusting for experience and self-rated knowledge, new graduates had higher odds of knowledge compared to experienced veterinarians (OR 5.7, 95% CI 1.7-23, p = 0.028). Questions assessing the attitude towards CVBP indicated that most participating veterinarians comprehend and agree with their importance. Nearly all participants agreed that ectoparasite control is the best option to prevent CVBP infections (91%, 153/167) and that for effective treatment of CVBP, a definitive diagnosis is required (81%, 135/167). However, veterinarians recommended suboptimal treatments for some CVBP, like Babesia gibsoni. Better practices were associated with being a companion animal practitioner (OR 2.4, 95% CI 1.1-5.7, p = 0.032) and having a low to moderate canine caseload (OR 3.6, 95% CI 1.3-10.4, p = 0.038). Limited knowledge of zoonotic CVBP among veterinarians, along with suboptimal treatment, might contribute to dogs acting as reservoirs and high prevalence of these pathogens in Sri Lanka. Therefore, continued veterinary education is recommended to improve knowledge and practices, which in turn will help to improve the diagnosis, treatment, and control of these infections in Sri Lanka to ensure the well-being of dogs and humans.
| S-EPMC11309419 | biostudies-literature
Project description:BackgroundVector-borne diseases (VBDs) have been increasingly reported in dogs and cats worldwide. However, no data are currently available regarding canine and feline VBDs in Qatar and limited information is available from other Persian Gulf countries.MethodsBlood samples from 98 client-owned animals (i.e. 64 dogs and 34 cats) living in Doha (Qatar) were collected and the presence of genomic DNA of Anaplasma spp., Babesia spp., Dirofilaria spp., Ehrlichia spp., Hepatozoon spp., Mycoplasma spp. and Rickettsia spp. was assessed by polymerase chain reaction (PCR), real time-PCR (rt-PCR) and sequence analysis.ResultsOf the 64 dogs, 12 (18.8%) were infected with at least one pathogen (i.e. 7.8% with Mycoplasma spp., 4.7% with Babesia vogeli, 3.1% with Ehrlichia canis, and 1.6% with Anaplasma platys, Babesia gibsoni and Hepatozoon canis, each). One of the 12 dogs was co-infected with B. vogeli and E. canis. Of the 34 cats, seven (20.6%) animals were infected with at least one pathogen (i.e. 5.9% were positive for Mycoplasma spp., and 2.9% for Babesia felis, B. vogeli, E. canis, "Candidatus Mycoplasma haemominutum" and Mycoplasma haemofelis, each). No dogs or cats were positive for Dirofilaria spp. or Rickettsia spp.ConclusionsAlthough the sample sizes of dogs and cats herein analysed was moderately small, data from this study report the occurrence of A. platys, B. vogeli, B. gibsoni, E. canis, H. canis and Mycoplasma spp. in domestic dogs and of B. felis, B. vogeli, "Candidatus M. haemominutum", E. canis and M. haemofelis in domestic cats from Qatar. Further investigations along with prophylactic measures are strongly recommended in order to reduce the risk of dogs and cats acquiring VBDs in Qatar.
| S-EPMC5479033 | biostudies-literature
Project description:The aim of the study was to establish the prevalence of Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi in dogs in eastern Poland and to determine the factors associated with exposure (seroposity) or infection (PCR). Anti-A. phagocytophilum, anti-B. burgdorferi and anti-E. canis antibodies were determined in 400 dogs, using the SNAP 4Dx ® test (IDEXX Laboratories). In addition, PCRs were performed for the detection of E. canis, A. phagocytophilum and B. burgdorferi DNA. In reference to the risk factor analysis, a regression logistic model was determined for each aetiological agent. The overall seroprevalence was highest for B. burgdorferi (11.0 %), followed by A. phagocytophilum (8.0 %) and E. canis (1.5 %). Eleven healthy dogs were found to be infected with A. phagocytophilum, as determined by PCR, while the remainder were seronegative. For B. burgdorferi, the DNA of the spirochetes was detected in the blood of 20 dogs, while the presence of anti-B. burgdorferi IgG was detected in the sera of ten of these. For E. canis, none of the dogs tested positive by PCR. Tick control was included as a protective factor for A. phagocytophilum and B. burgdorferi, while the origin (rural) was included as a risk factor for B. burgdorferi and A. phagocytophilum infection. In addition, breed (pure) was a risk factor for B. burgdorferi infection, and sex (female) was a risk factor for E. canis.
| S-EPMC4759218 | biostudies-literature
Project description:In French Guiana, canine heartworm disease is well known, but the diversity of filarial parasites of dogs remains largely unknown. A total of 98 canine blood samples from Cayenne and Kourou were assessed by a blood wet mount preparation, heartworm antigen test and molecular exploration of filarioid and Wolbachia DNAs, followed by a multiplex species-specific qPCR's identification and a subsequent sequencing analysis. Thereafter, a phylogeny based on maximum likelihood was carried out to facilitate specific identification. Five dogs were microfilaremic. Heartworm antigens were detected in 15 (15.3%) dogs. Of these, six (6.1%) were considered as occult infections as neither microfilariae nor Dirofilaria immitis DNA were detected. The 11 (11.2%) D. immitis isolates corresponded to a low virulent strain. Six of the D. immitis isolates were positive for Wolbachia endosymbionts of D. immitis belonging to the clade C DNA. Acanthocheilonema reconditum DNA was detected in 3 (3.1%) samples. Of these latter, one was found co-infected with the Brugia sp. genotype and the DNA of the clade D of the Wolbachia endosymbiont of Brugia species. This latter was also detected in two filarioid DNA-free samples. Finally, two samples were positive for Cercopithifilaria bainae genotype, which is distinct from those identified in Europe. The present study highlights the urgent need to implement chemoprophylaxis associated with anti-Wolbachia drugs to control these potential zoonoses.
| S-EPMC7285362 | biostudies-literature
Project description:The prevalence of tick-borne infections has been steadily increasing in both number and geographic distribution in the United States and abroad. This increase, in conjunction with the continued recognition of novel pathogens transmitted by ticks, has made accurate diagnosis of these infections challenging. Mainstay serologic tests are insensitive during the acute phase of infection and are often cross-reactive with similar pathogenic and nonpathogenic organisms. Further, they are unable to reliably differentiate active versus past infection which can lead to misdiagnosis and incorrect understanding of the epidemiology and incidence of specific tick-borne pathogens. We evaluated a novel multiplexed high-definition PCR (HDPCR) Tickborne Panel (TBP) assay (ChromaCode, Carlsbad, CA) for the detection of nine tick-borne pathogens or groups associated with human illness. The HDPCR technology enables multiplex identification of multiple targets in a single fluorometric channel based on fluorescent signal modulation using a limiting probe design. A collection of 530 whole-blood specimens collected from patients being evaluated for tick-borne infections, in addition to a panel of 93 simulated specimens, were used to challenge the HDPCR TBP. The results were compared to a clinically validated traditional multiplexed PCR test with additional sequence analysis and clinical history collected to aid in resolving discrepancies. Among clinical specimens the TBP demonstrated 100% sensitivity for the identification of Anaplasma phagocytophilum, Borrelia miyamotoi, Borrelia mayonii, and Rickettsia rickettsii The sensitivity for identification of B. burgdorferi was 44.4% compared to a composite gold standard. Among simulated specimens containing single or multiple targets present at 103 to 105 copies/PCR, the sensitivity of TBP was 100% for all targets, with a combined specificity of 99.5%. Of note, an increased rate of false-positive results was observed among simulated specimens that contained multiple targets. Based on these data, we find the HDPCR TBP to be a useful adjunct for the diagnosis of tick-borne infections in patients with suspected tick-borne illness.
| S-EPMC6812998 | biostudies-literature
Project description:BackgroundVector-borne pathogens are the subject of several investigations due to the zoonotic concern of some of them. However, limited data are available about the simultaneous presence of these pathogens in cats and their ectoparasites. The aim of the present study was to define the species of ectoparasites found on cats as well as to investigate vector-borne pathogens in cats and their ectoparasites in southern Italy.MethodsBlood from 42 cats and fleas or flea pools (n = 28) and ticks (n = 73) collected from them were investigated by quantitative PCR for the detection of vector-borne pathogens. Feline serum samples were tested by IFAT to detect IgG antibodies against Leishmania infantum, Bartonella henselae, Rickettsia conorii, Rickettsia felis, Rickettsia typhi, Babesia microti, Ehrlichia canis and Anaplasma phagocytophilum antigens.ResultsOnly one flea species (Ctenocephalides felis) and four tick species belonging to the genera Rhipicephalus and Ixodes were identified on cats from southern Italy. Molecular evidence of Bartonella spp., Rickettsia spp., hemoplasmas, Babesia vogeli and L. infantum was found in ectoparasites (fleas and/or ticks) while DNA from Hepatozoon felis and Ehrlichia/Anaplasma spp. was not detected. Likewise, DNAs from Bartonella, hemoplasma and Leishmania were the only pathogens amplified from feline blood samples. Cats had also antibodies against all the investigated pathogens with the exception of Rickettsia typhi. Agreement between serological and molecular results in individual cats and their ectoparasites was not found. The only exception was for Bartonella with a fair to moderate agreement between individual cats and their ectoparasites. Bartonella clarridgeiae was the species most frequently found in cats and their fleas followed by B. henselae.ConclusionsIn conclusion, cats harboring ticks and fleas are frequently exposed to vector-borne pathogens. Furthermore, ticks and fleas harbored by cats frequently carry pathogens of zoonotic concern therefore appropriate feline ectoparasiticide preventative treatments should be used in cats.
| S-EPMC4862052 | biostudies-literature
Project description:A major challenge in many clinical diagnostic applications is the measurement of low-abundance proteins and other biomolecules in biological fluids. Digital technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional protein detection methods. However, current digital ELISA technologies still possess insufficient sensitivities for many rare protein biomarkers and require specialized instrumentation or time-consuming workflows that have limited their widespread implementation. To address these challenges, we have developed a more sensitive and streamlined digital ELISA platform, Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), which attains low attomolar limits of detection, with an order of magnitude enhancement in sensitivity over these other methods. MOSAIC uses a rapid, automatable flow cytometric readout that vastly increases throughput and is easily integrated into existing laboratory infrastructure. As MOSAIC provides high sampling efficiencies for rare target molecules, assay bead number can readily be tuned to enhance signal-to-background with high measurement precision. Furthermore, the solution-based signal readout of MOSAIC expands the number of analytes that can simultaneously be measured for higher-order multiplexing with femtomolar sensitivities or below, compared with microwell- or droplet-based digital methods. As a proof of principle, we apply MOSAIC toward improving the detectability of low-abundance cytokines in saliva and ultrasensitive multiplexed measurements of eight protein analytes in plasma and saliva. The attomolar sensitivity, high throughput, and broad multiplexing abilities of MOSAIC provide highly accessible and versatile ultrasensitive capabilities that can potentially accelerate protein biomarker discovery and diagnostic testing for diverse disease applications.
| S-EPMC9499451 | biostudies-literature