Release of Angiostrongylus cantonensis larvae from live intermediate hosts under stress.

The metastrongyloid nematode Angiostrongylus cantonensis causes eosinophilic meningitis in a variety of homeothermic hosts including humans. Third-stage infectious larvae develop in gastropods as intermediate hosts. Humans are usually infected by intentional or incidental ingestion of an infected mollusk or paratenic host (poikilothermic vertebrates and invertebrates). The infection may also hypothetically occur through ingestion of food or water contaminated by third-stage larvae spontaneously released from gastropods. Larvae are thought to be released in greater numbers from the intermediate host exposed to stress. This study aimed to compare larval release from stressed with unstressed gastropods. Experimentally infected Limax maximus and Lissachatina fulica were exposed to a stress stimulus (shaking on an orbital shaker). The mucus was collected before and after the stress and examined microscopically and by qPCR for the presence of A. cantonensis larvae and their DNA. In the case of L. maximus, no larvae were detected microscopically in the mucus, but qPCR analysis confirmed the presence of A. cantonensis DNA in all experimental replicates, without clear differences between stressed and non-stressed individuals. In contrast, individual larvae of A. cantonensis were found in mucus from Li. fulica after stress exposure, which also reflects an increased number of DNA-positive mucus samples after stress. Stress stimuli of intensity similar to the transport or handling of mollusks can stimulate the release of larvae from highly infected intermediate hosts. However, these larvae are released in small numbers. The exact number of larvae required to trigger neuroangiostrongyliasis is unknown. Therefore, caution is essential when interacting with potential intermediate hosts in regions where A. cantonensis is endemic.

First insight into strongylid nematode diversity and anthelmintic treatment effectiveness in beef cattle in the Czech Republic explored by HTS metagenomics.

Parasitic diseases and mitigation of their effects play an important role in the health management of grazing livestock worldwide, with gastrointestinal strongylid nematodes being of prominent importance. These helminths typically occur in complex communities, often composed of species from numerous strongylid genera. Detecting the full diversity of strongylid species in non-invasively collected faecal samples is nearly impossible using conventional methods. In contrast, high-throughput amplicon sequencing (HTS) can effectively identify co-occurring species. During the four-year project, we collected and analysed faecal samples from beef cattle on >120 farms throughout the Czech Republic. Strongylids were the predominant nematodes, detected in 56% of the samples, but at a low level of infection. The apparent limitations in identifying strongylid taxa prompted this pilot study on a representative group of samples testing positive for strongylids using ITS-2 metabarcoding. The most widespread genera parasitizing Czech cattle were Ostertagia (O. ostertagi) and Oesophagostomum spp., followed by Trichostrongylus and Cooperia, while Bunostomum, Nematodirus and Chabertia were present only in a minority. As comparative material, 21 samples of cattle from the Danube Delta in Romania were used, which, in contrast, were dominated by Haemonchus placei. Finally, the effect of ivermectin treatment was tested at two Czech farms. After treatment with the anthelmintic, there was a shift in the strongylid communities, with a dominance of Cooperia and Ostertagia.

Rat lungworm survives winter: experimental overwintering of Angiostrongylus cantonensis larvae in European slugs.

The rat lungworm Angiostrongylus cantonensis is a metastrongyloid nematode that causes neurological disorders in its accidental hosts, including humans. This invasive pathogen is native to Southeast Asia and adjacent regions and is gradually expanding its distribution to tropical and subtropical areas with new foci discovered near temperate regions. The parasite has a complex life cycle with a range of gastropods serving as intermediate hosts. A broad spectrum of poikilotherm vertebrates and invertebrates can serve as paratenic hosts. Since it has already been demonstrated that other, non-zoonotic metastrongyloids can survive in their intermediate hosts during the winter, the aim of our study was to evaluate the survival of A. cantonensis third-stage larvae in experimentally infected slugs (Limax maximus) kept at 4.5­7°C for 60 days. Third-stage larvae of A. cantonensis survived the period of low temperature and remained capable of infecting definitive hosts (laboratory rats) afterwards, even though their numbers dropped significantly. These results suggest that further spread to higher latitudes or altitudes is possible in areas with sufficient abundance of definitive hosts, since low winter temperatures are not necessarily an obstacle to the spread of the parasite.

Range of metastrongylids (superfamily Metastrongyloidea) of public health and veterinary concern present in livers of the endemic lizard Gallotia galloti of Tenerife, Canary Islands, Spain.

BACKGROUND: Endemic lizards of the genus Gallotia are of high ecological value to the terrestrial ecosystem of the archipelago of the Canary Islands, being potent seed spreaders as well as an important component of the diet of other vertebrates. The endemic lizard Gallotia galloti in Tenerife has recently been reported to be a paratenic host of Angiostrongylus cantonensis, an invasive metastrongylid with zoonotic potential that is associated with rats as definitive hosts. However, microscopic examination of G. galloti tissue samples also revealed the presence of other metastrongylid larvae inside granulomas on the liver of this reptile. The aim of this study was to investigate the presence of helminths other than A. cantonensis in tissues of G. galloti from Tenerife. METHODS: A multiplex-nested PCR targeting the internal transcribed spacer 1 was designed that enabled the species-specific detection of A. cantonensis, Angiostrongylus vasorum, Aelurostrongylus abstrusus, Crenosoma striatum and Crenosoma vulpis. Liver samples from 39 G. galloti were analysed. RESULTS: Five metastrongylids were detected: A. cantonensis (15.4% of samples analysed), A. vasorum (5.1%), Ae. abstrusus (30.8%), C. striatum (30.8%) and undetermined metastrongylid sequences (12.8%). Co-infection was highly prevalent among the lizards which tested positive. CONCLUSIONS: The study provides a new specific tool for the simultaneous detection of a range of metastrongylids of veterinary importance as well as new data on the circulation of metastrongylids in an ecosystem dominated by lizards.

The winner takes it all: dominance of Calicophoron daubneyi (Digenea: Paramphistomidae) among flukes in Central European beef cattle.

In Europe, paramphistomosis caused by Paramphistomum spp. was historically regarded as being of minor importance. However, Calicophoron daubneyi has recently been recognized as an emerging pathogen in Europe due to its increasing prevalence and negative impact on livestock production. In search for paramphistomid flukes, 5573 beef cattle fecal samples from 115 farms across the whole Czech Republic were examined from March 2019 to June 2021. The eggs of paramphistomid flukes were identified in 29.9% of samples. Internal transcribed spacer 2 sequences from 90 adult flukes and 125 fecal samples collected across Czech Republic confirmed C. daubneyi infection in the Czech beef cattle. Ninety mitochondrial DNA sequences obtained from adult C. daubneyi specimens revealed 13 individual haplotypes, two of them recorded for the first time. Although C. daubneyi is a new parasite in beef cattle herds in the Czechia, it clearly dominates the parasitological findings in the country's beef cattle. The common occurrence of C. daubneyi in most of the beef cattle herds indicates environmental conditions suitable also for the life cycle of Fasciola hepatica and risk of its emergence.