Soil-transmitted nematodes Strongyloides spp. parasitize a range of vertebrates, including endangered great apes and humans. Since 2018, the Mountain Gorilla Veterinary Project has observed an increase in gastrointestinal parasitic infections, including suspected strongyloidiasis in mountain gorillas in Bwindi Impenetrable National Park (BINP), Uganda. To investigate the prevalence of Strongyloides and potential cross-host transmission, we conducted noninvasive fecal sampling of mountain gorillas, olive baboons, domestic dogs and pigs. Samples were examined by coproscopy and real-time PCR followed by sequencing of the HVR-IV 18S rRNA and cox1 genes. Strongyloides prevalence was higher in mountain gorillas ranging near the park edge compared to forest interiors inside the park and among infant and juvenile mountain gorillas compared to other age groups. Strongyloides fuelleborni, a known zoonotic species, was identified in both baboons and mountain gorillas, suggesting sharing this particular parasite between the two primate species with potential implications for humans surrounding the park. In domestic animals, S. ransomi was detected in pigs, a zoonotic lineage S. stercoralis haplotype was detected in one dog, and another haplotype closely related to S. ransomi and S. venezuelensis was identified in another dog. The presence of zoonotic S. fuelleborni and S. stercoralis in Bwindi ecosystem poses a threat to mountain gorilla as well as human public health. Our study underscores the need for continued monitoring and community sensitization about the risk of cross-species transmission of these parasites.

The invasive nematode Angiostrongylus cantonensis (rat lungworm) can cause eosinophilic meningitis in humans. Once restricted to Southeast Asia, A. cantonensis nematodes are now widespread across the tropics and have been reported in Europe. Tenerife, in the Canary Islands, and the Mediterranean region are emerging hotspots. We surveyed gastropods, rats, and lizards across Tenerife and detected the parasite in all host groups at 2.4%- 41.6% prevalence. Using species distribution models, we identified precipitation seasonality as the main driver of habitat suitability; tree cover and climatic variability primarily shaped prevalence patterns. Modeling showed suitable habitats in northeastern Tenerife and several western Canary Islands but limited overlap with areas of dense human population. Multivariate environmental similarity surface analysis comparison with another A. cantonensis hotspot, Hawaii, USA, revealed similar environments across the archipelago, except for the novel northeastern Tenerife area. Although no human infections have been reported, continued vigilance is warranted because A. cantonensis nematodes are established in Tenerife.

Emerging infectious diseases are one of the biggest challenges in a globalized world. To date, resources have been allocated to prevent and control the spread of zoonotic and livestock pathogens. We argue that, in line with the One Health approach, equitable efforts, financial resources, attention, and coordination are required for wildlife-only pathogens to halt biodiversity loss. Deploying the amphibian fungus Batrachochytrium salamandrivorans as a model, we demonstrate the unbalanced efforts among countries in Europe regarding surveillance, disease response, prevention, public outreach, and research. We compare investments with B. salamandrivorans l-free countries such as the United States, concluding that structural resources are urgently needed to curb the effects of this fungus within Europe and beyond. We encourage dialogue among authorities, researchers, and stakeholders and propose a coordinated European Union-level program of €6-10 million over 5-7 years to implement B. salamandrivorans action plans and define structural funding requirements for future wildlife disease mitigation.

Strongyloides stercoralis nematode infection occurs in ≈600 million persons worldwide and is listed by the World Health Organization as a neglected tropical disease. Understanding zoonotic potential is critical, especially in areas where humans, domestic animals, and wildlife interact. We explored cross-species sharing of Strongyloides roundworms by analyzing fecal samples from humans, dogs, and nonhuman primates in the Dzanga-Sangha Protected Areas, Central African Republic. We detected positive samples by quantitative PCR and assessed genetic diversity through amplification of the 18S rRNA HVR-IV region and cox1, followed by high-throughput sequencing. Strongyloides prevalence was high in humans, dogs, and gorillas. S. stercoralis haplotype A roundworm dominated in humans but appeared in dogs and apes, whereas S. fuelleborni roundworm was present in all hosts. Shared species and haplotypes indicated zoonotic transmission. Our findings highlight the need for molecular surveillance and emphasize the role of dogs and nonhuman primates as reservoirs, complicating efforts to control infections in human populations.

Angiostrongylus cantonensis is an invasive parasitic nematode and zoonotic pathogen responsible for eosinophilic meningitis. Originally native to Southeast Asia, it is now globally distributed across tropical and subtropical regions and is approaching Europe, with Tenerife as a key hotspot. This study investigates the distribution and prevalence of A. cantonensis in Tenerife across three host groups (rats, gastropods, lizards). Based on prevalence data, we modelled its potential distribution using species distribution models (SDMs) and compared climatic conditions with Hawaii, a region with frequent human cases. Field surveys confirmed A. cantonensis in endemic and introduced gastropods (25.6%; 179/698), rats (21.5%; 14/79), and lizards (24.0%; 31/129), with local prevalence ranging from 2.4% to 41.6%. MaxEnt and Boosted Regression Tree models identified precipitation seasonality as the main driver of distribution, while prevalence was influenced primarily by tree cover density and climatic variability. Northeastern Tenerife, La Gomera, La Palma, and El Hierro showed the highest habitat suitability. However, overlap with densely populated areas was limited, possibly explaining the absence of reported human cases. The MESS analysis, based on climatic data from Hawaii, indicated moderate to high environmental similarity across most of the Canary Islands, except in northeastern Tenerife, where conditions were outside the range observed in Hawaii. A. cantonensis is firmly established in Tenerife, but human cases remain absent, likely due to limited human exposure, cultural practices, and geographic separation of parasite hotspots from urban zones. Our findings highlight the importance of integrating ecological and epidemiological data in zoonotic risk assessments. ### Competing Interest Statement The authors have declared no competing interest.

Angiostrongylus cantonensis is a mollusk-borne parasitic nematode originating in Southeast Asia. Commonly known as the rat lungworm, it uses rats as definitive hosts, though other mammals, including humans, can be infected and typically suffer from neurological disorders. This study focuses on the parasite's presence in its gastropod intermediate hosts in several urban and rural areas in Aceh province, Sumatra, Indonesia. Samples of Achatina (Lissachatina) fulica (161) and Pomacea sp. (90) were collected in eight localities in Banda Aceh. Additionally, 531 edible freshwater snails belonging to the genus Sulcospira sp. from three different localities in Aceh province were obtained in wet markets. All samples were examined by LAMP and qPCR for the A. cantonensis DNA. No samples of Sulcospira sp. and Pomacea sp. tested positive. 13.4% of L. fulica tested positive, with the highest prevalence in urban areas of Banda Aceh. The ITS1 sequences obtained from positive samples using conventional PCR confirmed 100% identity with A. cantonensis. The present study confirms, for the first time, the presence of the zoonotic parasite A. cantonensis in Banda Aceh, Sumatra, Indonesia. Notably, the handling and consumption of snails sold at wet markets do not appear to increase the risk of eosinophilic meningitis in this region. However, the relatively high prevalence of A. cantonensis in urban land snails underscores the need for continued vigilance and public health awareness.

The rat lungworm Angiostrongylus cantonensis is a zoonotic metastrongyloid nematode currently considered an emerging pathogen. Originating in Southeast Asia, this nematode has spread to tropical and subtropical parts of the world via its invasive rodent and gastropod hosts.On the island of Tenerife in the Canary archipelago, the A. cantonensis invasion was recognized more than a decade ago. The endemic lizard Gallotia galloti has been identified as a paratenic host of this nematode in the Canary Island ecosystem. Because this lizard species is the most abundant reptile in Tenerife, we tested its suitability as a possible sentinel for A. cantonensis presence. Lizards were captured alive in nine localities, spanning an environmental gradient across the island. Tail muscle tissue was obtained by provoked caudal autotomy and tested for the nematode infection by a species-specific qPCR. Infection intensities were assessed by detecting A. cantonensis DNA quantities based on a calibrated standard curve. Of the 129 samples tested, 31 were positive. The prevalence varied among localities, with the highest (63.6%) recorded in a humid laurel forest. Even though the prevalence in Valle San Lorenzo was the lowest, this is the first record of A. cantonensis from the arid south of Tenerife. Variation in prevalence at different localities was significantly and positively correlated with increasing vegetation cover and negatively correlated with seasonal variability of precipitation, as determined by Spearman correlation coefficients. Fisher's exact test was used to determine the variation in the prevalence of A. cantonensis among adult males, females, and juveniles and showed no significant difference. Also, there was no significant difference in infection intensity between males and females (as determined by GEE-g). We demonstrated that provoking caudal autotomy can be an effective non-lethal method of A. cantonensis mapping in island ecosystems with abundant lizard species, particularly those with a sharp climatic and vegetation gradient, from xeric to humid conditions.

Abstract Background Borrelia miyamotoi and Borrelia burgdorferi sensu lato (s.l.) are important zoonotic agents transmitted by Ixodes ricinus ticks, which are widely distributed across Central Europe. Understanding the spatial distribution of these pathogens’ prevalence will help identify areas with increased infection risk and facilitate the implementation of effective preventive measures. Methods We analysed 12,955 I. ricinus ticks collected from 142 towns in the Czech Republic between 2016 and 2018. The ticks were pooled into 2591 groups of five and tested using duplex quantitative polymerase chain reaction (qPCR) for the presence of B. burgdorferi s.l. and B. miyamotoi. For each location, we estimated the overall prevalence of both agents using the EpiTools Epidemiological Calculator for pooled samples and calculated the minimum infection rate (MIR). To assess the potential risk of infection, we combined data on the abundance of nymphs and females with pathogen prevalence at each sampled site. Using a geographic information system (GIS), we mapped the MIR and infection risk of both Borrelia species across all 142 sampled locations and employed a geostatistical method (ordinary kriging) to predict MIR values and infection risk as continuous surfaces across the entire country. Results We detected B. miyamotoi in 110 localities and B. burgdorferi s.l. in all 142 localities. The estimated prevalence of B. miyamotoi and B. burgdorferi s.l. in the collected ticks was 2.1% (95% confidence interval [CI] 1.8–2.3) and 27.1% (95% CI 26.0–28.3), respectively. For B. miyamotoi, we identified previously unknown, geographically distinct hotspots of MIR up to 8.3%, with MIR slightly higher in females (2.3%) than in males (1.9%) and nymphs (1.8%), though the difference was not statistically significant. In contrast, B. burgdorferi s.l. exhibited ubiquitous presence, with consistently high prevalence nationwide, showing similar MIRs in females (16.2%) and males (16.1%), and slightly lower in nymphs (15.6%). The highest infection risk for B. miyamotoi was 12.4 infected vectors per hour in southeastern Moravia, while the highest risk for B. burgdorferi s.l. reached 78.6 infected vectors per hour in the Bohemian-Moravian Highlands. Conclusions Borrelia miyamotoi is widespread, forming distinct high-prevalence areas in certain regions. Borrelia burgdorferi s.l. demonstrates consistently high prevalence across most of the country, except for a few localized areas such as southwestern Czechia. Both pathogens exhibit natural nidality, forming regions with elevated prevalence and infection risk. Long-term time-series data are needed to confirm the spatio-temporal stability of these hotspots. Graphical Abstract

Cysts and trophozoites of vestibuliferid ciliates and larvae of Strongyloides were found in fecal samples from captive orangutans Pongo pygmaeus and P. abelii from Czech and Slovak zoological gardens. As comparative material, ciliates from semi-captive mandrills Mandrillus sphinx from Gabon were included in the study. Phylogenetic analysis of the detected vestibuliferid ciliates using ITS1-5.8s-rRNA-ITS2 and partial 18S ribosomal deoxyribonucleic acid (rDNA) revealed that the ciliates from orangutans are conspecific with Balantioides coli lineage A, while the ciliates from mandrills clustered with Buxtonella-like ciliates from other primates. Morphological examination of the cysts and trophozoites using light microscopy did not reveal differences robust enough to identify the genera of the ciliates. Phylogenetic analysis of detected L1 larvae of Strongyloides using partial cox1 revealed Strongyloides stercoralis clustering within the cox1 lineage A infecting dogs, humans, and other primates. The sequences of 18S rDNA support these results. As both B. coli and S. stercoralis are zoonotic parasites and the conditions in captive and semi-captive settings may facilitate transmission to humans, prophylactic measures should reflect the findings.

Background Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve the accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae , the only other neurotropic Angiostrongylus species, which is native to Australia. Methodology/Principal Findings Cerebrospinal fluid (CSF) from dogs (2020-2022) with a presumptive diagnosis of A. cantonensis infection were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR. A newly designed LAMP assay targeting AcanR3390 in a diagnostic laboratory setting was directly compared to the reference ultrasensitive qPCR for determination of presence of A. cantonensis DNA to aid the diagnosis of CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa=0.81, 95%CI 0.69-0.92; n =93) and rapid single-step lysis of archived CSF samples (Kappa=0.77, 95%CI 0.59-0.94; n =52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox 1. Conclusions/Significance The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in CSF of dogs and performs comparably to laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for effective diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension for humans. Authors summary A potentially fatal disease, neural angiostrongyliasis, is caused by the rat lungworm ( Angiostrongylus cantonensis ). The parasite migrates into the spinal cord and brain of accidental hosts, such as humans and dogs, after ingestion of infective larvae. Recently, an ultrasensitive molecular assay which can detect tiny fragments of the parasite’s DNA was developed and has been used for confirmatory diagnosis. Although this assay outperforms previously developed assays, it requires clean DNA with specialised equipment in a laboratory setting. There is an urgent need for an alternative diagnostic method which is sensitive and portable, for deployment in the field and in the hospitals in remote areas or in low-income countries. The authors developed a fast and portable loop-mediated isothermal amplification (LAMP) assay that compares favourably to the ultra-sensitive PCR assay when tested using cerebrospinal fluid from dogs on the Australian east coast with presumptive neural angiostrongyliasis. Considering a ‘One Health’ approach to diagnostics, this assay enables portable emergency diagnostics equally suitable to humans, dogs and wildlife. The newly developed assay will also enable water supplies to be screened, as well as crustaceans and molluscs used as potential food sources, for presence of the parasite.

Ophidiomyces ophidiicola (Oo) is a snake fungal pathogen that causes ophidiomycosis. The disease manifests as dermatomycosis and/or systemic mycosis, and can be fatal. It occurs in free-ranging snakes in Asia, Europe and the USA and has also been demonstrated in captive snakes. We tested for the presence of Oo in free-ranging snake populations in the Czech and Slovak Republics (n = 420) between 2019 and 2022, focusing mainly on grass snakes (Natrix natrix) and dice snakes (Natrix tessellata), as well as various captive exotic species (n = 207). After collecting skin swabs, we tested for Oo using the qPCR method. We confirmed fragmented occurrence of Oo in the Czech Republic (total prevalence 15%) and recorded Oo in the Slovak Republic for the first time (total prevalence 33.9%). The highest prevalence was observed in N. tessellata (20.2%), which appears to be the most susceptible species. The pathogen was not detected in captive snakes.

Co-existence of bats with a wide range of infectious agents relates to their co-evolutionary history and specific physiology. Here, we examined blood samples collected during hibernation and the post-hibernation period to assess the influence of trypanosomes and babesias on the health status of 50 Noctule bats (Nyctalus noctula) using nested PCR. The impact of blood parasites on health was assessed by analysis of haematology and blood chemistry parameters in 21 bats. Prevalence of trypanosomes (Trypanosoma dionisii and T. vespertilionis) and babesia (Babesia vesperuginis) was 44% and 8%, respectively. Analysis of blood parameters indicated impact of babesia on acid–base balance. Blood chemistry parameters showed a significant decrease in total dissolved carbon dioxide and bicarbonate, increased anion gap, and no change in blood pH, suggesting compensated metabolic acidosis. Adverse effects of babesia were only apparent in hibernating bats. Our results suggest differences in the pathogenicity of trypanosomes and babesia in bats. While trypanosomes in general had no significant impact on the health status, we observed alterations in the blood acid–base balance in Babesia-infected bats during hibernation. Despite being infected, Babesia-positive bats survived hibernation without showing any clinical signs.

We used quantitative PCR to detect Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) in 135 samples of Pelophylax esculentus complex water frogs from 41 Latvian populations. We detected Bd in 18 populations of water frogs. None of the samples was positive for Bsal.

Chromogranin A has been reported to be processed in vivo by an as yet undefined proteinase(s) suggesting that it is a precursor of biologically active peptides such as pancreastatin. In this study, plasma kallikrein was used as a model proteinase to identify the cleavage sites exposed in bovine parathyroid chromogranin A. Purified bovine parathyroid chromogranin A was digested with human plasma kallikrein. The proteolytic fragments produced were isolated by HPLC and chemically characterized by amino acid composition and sequence analysis. The combined results indicate that the enzyme has preference for specific single Arg residues, cutting C-terminal to this amino acid, although certain pairs of basic sites were also cleaved. The characterized fragments were released in a selective manner from the whole molecule with rapid production of the fragments covering positions 1–247 and 352–358.

Environmental DNA (eDNA) is becoming an indispensable tool in biodiversity monitoring, including the monitoring of invasive species and pathogens. Aquatic chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) are major threats to amphibians. However, the use of eDNA for detecting these pathogens has not yet become widespread, due to technological and economic obstacles. Using the enhanced eDNA approach (a simple and cheap sampling protocol) and the universally accepted qPCR assay, we confirmed the presence of Bsal and Bd in previously identified sites in Spain, including four sites that were new for Bsal. The new approach was successfully tested in laboratory conditions using manufactured gene fragments (gBlocks) of the targeted DNA sequence. A comparison of storage methods showed that samples kept in ethanol had the best DNA yield. Our results showed that the number of DNA copies in the Internal Transcribed Spacer region was 120 copies per Bsal cell. Eradication of emerging diseases requires quick and cost-effective solutions. We therefore performed cost-efficiency analyses of standard animal swabbing, a previous eDNA approach, and our own approach. The procedure presented here was evaluated as the most cost-efficient. Our findings will help to disseminate information about efforts to prevent the spread of chytrid fungi.

AbstractDiagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false‐positive or false‐negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above‐mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non‐diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin‐embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Small mammals are important reservoirs of multiple pathogens transmittable to humans. Rodent populations are highly dynamic, passing through multiannual cycles with densities changing in several orders of magnitude. Such variable pools of hosts shape the intensity of pathogen spread among the animals and risks of spillover to humans. We describe such dynamic system within a sample set of 13 small mammal species and six potentially zoonotic pathogens (bacteria Anaplasma, Rickettsia, Leptospira, Borrelia, Bartonella, and protist Babesia) present in surroundings of a small village in south-eastern part of the Czech Republic. This article presents results of a six-year-long study at the site. The observed prevalence of the selected pathogens varied greatly among years and host species. The dominant rodents (Apodemus sp., Apodemus agrarius and Myodes glareolus) harboured all tested pathogens and multi-infections were not rare – we found up to four pathogens in some individuals. We observed surge in Borrelia burgdorferi s.l. prevalence following the overall rodent population increase in 2014 and 2019, with A. agrarius and Microtus arvalis being the most infected species. Rickettsia sp. prevalence reached 24 % in the shrew Crocidura suaveolens, thus this potential neglected reservoir host deserves further attention.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is implicated in global declines of amphibian populations and has been documented in African specimens originally collected as far back as the 1930s. Numerous recent surveys focusing on regional pathogen prevalence have greatly increased the number of known occurrences of Bd in African species, but few studies have focused on continental distribution patterns. We analyzed all known positive occurrences of Bd in African amphibians to date, including newly reported data from Cameroon, the Democratic Republic of the Congo, Gabon, Namibia, and the Republic of the Congo. Records from both Namibia and the Republic of the Congo reported herein represent first positive occurrences for these countries. With this most comprehensive sampling of the African continent to date we identified patterns of Bd-positive occurrences associated with (1) location (i.e., biogeographic region, country), (2) taxonomy, (3) life history, and (4) threat of extinction. We used fine-grained (30 arc seconds) environmental niche models (ENMs) to predict the continental distribution of Bd and identify hotspots for the pathogen, including areas not previously modeled to have high suitability for the fungus, and areas of high amphibian biodiversity from which Bd has not yet been documented. Our ENMs predicted that the environmentally suitable range of Bd encompasses vast areas of high amphibian biodiversity, including the Congo Basin and the Albertine Rift. Although our ENMs indicated that West Africa is environmentally suitable for Bd, the fungus has not been reported west of the Dahomey Gap. Likewise, the ENMs also identified regions across the Congo Basin and coastal Angola that are environmentally suitable for the pathogen but from which Bd has not yet been reported, underscoring a need for Bd surveys in these regions. Although amphibian declines in Africa have not been directly attributed to chytridiomycosis, Bd has been detected in over one fifth of the most-threatened African amphibians. Given the presence of the hypervirulent Bd global panzootic lineage (BdGPL) in Africa, we believe that the threat of Bd as a novel pathogen may be underestimated and that focused research is urgently needed to identify which species are susceptible to Bd-driven declines.

Background. Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. Methods. In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. Results. The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. Conclusion. These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Anaplasma phagocytophilum and Rickettsia spp. are vector-borne zoonotic bacteria, which are clinically important especially in immunocompromised patients. There are large gaps in the current knowledge of their geographic distribution and prevalence in both their vectors and hosts. Our aim was to develop reliable and easy detection method for both these pathogens. We made a new hydrolysis probe based duplex Real-Time PCR assay based on previous studies. We optimized the assays and tested them to provide reliable recommended procedures with a sensitivity to a minimum of 10 target DNA copies per sample. The assays were designed to be specific for A. phagocytophilum and in the same reaction detect multiple species of rickettsiae. We designed gBlock quantification standards that provide the option to identify differences in pathogen load among different samples in subsequent studies.