Parasitic egg recognition using convolution and attention network.

Intestinal parasitic infections (IPIs) caused by protozoan and helminth parasites are among the most common infections in humans in low-and-middle-income countries. IPIs affect not only the health status of a country, but also the economic sector. Over the last decade, pattern recognition and image processing techniques have been developed to automatically identify parasitic eggs in microscopic images. Existing identification techniques are still suffering from diagnosis errors and low sensitivity. Therefore, more accurate and faster solution is still required to recognize parasitic eggs and classify them into several categories. A novel Chula-ParasiteEgg dataset including 11,000 microscopic images proposed in ICIP2022 was utilized to train various methods such as convolutional neural network (CNN) based models and convolution and attention (CoAtNet) based models. The experiments conducted show high recognition performance of the proposed CoAtNet that was tuned with microscopic images of parasitic eggs. The CoAtNet produced an average accuracy of 93%, and an average F1 score of 93%. The finding opens door to integrate the proposed solution in automated parasitological diagnosis.

GEOGRAPHICAL VARIATION OF ENTEROPARASITISM IN THE SOUTHERN SEA LION OTARIA FLAVESCENS (SHAW, 1800) OFF THE COAST OF CHILE AS EVIDENCED WITH COPROLOGICAL TESTS: IMPLICATONS FOR ZOONOTIC RISKS.

The scarce information on the helminth fauna in otariids from the Southeastern Pacific comes mainly from stranded individuals or killed for that purpose. In this study, we compared the abundance and composition of enteroparasitic assemblages of Otaria flavescens using coprological techniques. Three sampling localities from north to south spanning 2,200 km off the Chilean coast were considered (Iquique, Viña del Mar, and Talcahuano). In all, 60 fecal samples were collected, and eggs belonging to 5 taxa were found in 91.6% of the samples. They were the anisakid nematodes Contracaecum and Pseudoterranova, the cestode Adenocephalus (syn. Diphyllobothrium), the trematode Ogmogaster, and the acanthocephalan Corynosoma. Samples from southern Chile (Talcahuano) showed the highest prevalence. Adenocephalus eggs had the highest prevalence and abundance in Iquique and Talcahuano, whereas Ogmogaster was the less prevalent and abundant in all sampling localities. Corynosoma eggs had similar prevalence and abundance among sampling localities, and Pseudoterranova eggs were absent in Iquique and with median prevalence values in Viña del Mar and Talcahuano. Thus, the composition of parasite egg assemblages was different between sampling localities. These differences between sampling localities may help to explain differential records of some zoonotic parasitoses such as pseudoterranovosis and diphyllobothriosis in Peru and Chile, where consumption of raw or marinated fish (ceviche) is common. For example, the lower diversity of parasite egg assemblages in the northern Chilean coast may be due to the absence or lower abundance of first intermediate/paratenic hosts of Pseudoterranova.

Two Helminthic Cases of Human Mummy Remains from Joseon-Period Graves in Korea.

Our previous research on coprolite specimens from the mummies of Joseon Dynasty (1392-1910 CE) has revealed various species of parasite eggs. Herein, we added 2 new helminthic cases of human remains from Joseon-period graves in the Republic of Korea (Korea). The organic materials precipitated on the hip bones of 2 half-mummied cases (Goryeong and Gwangmyeong cases) were collected, rehydrated, and examined by a microscope. In the sample from Goryeong-gun (gun=County), ova of Trichuris trichiura, Clonorchis sinensis, and Metagonimus spp. were detected, and eggs of T. trichiura and A. lumbricoides were found from the sample of Gwangmyeong-si (si=City). By adding this outcome to the existing data pool, we confirm our previous estimates of Joseon-period parasite infection rates. The overall rates of A. lumbricoides, T. trichiura, and C. sinensis decreased dramatically from Joseon to the modern period. In Goryeong mummy specimen, we also found Metagonimus spp. eggs that has rarely been detected in archaeological samples so far.

Next-generation sequencing of DNA from resting eggs: signatures of eutrophication in a lake's sediment.

Hatching resting stages of ecologically important organisms such as Daphnia from lake sediments, referred to as resurrection ecology, is a powerful approach to assess changes in alleles and traits over time. However, the utility of the approach is constrained by a few obstacles, including low and/or biased hatching among genotypes. Here, we eliminated such bottlenecks by investigating DNA sequences isolated directly (i.e. without hatching) from resting eggs found in the sediments of Lake Constance spanning pre-, peri-, and post-eutrophication. While we expected genome-wide changes, we specifically expected changes in alleles related to pathways involved in mitigating effects of cyanobacterial toxins. We used pairwise FST-analyses to identify transcripts that showed strongest divergence among the four different populations and a clustering analysis to identify correlations between allele frequency shifts and changes in abiotic and biotic lake parameters. In a cluster that correlated with the increased abundance of cyanobacteria in Lake Constance we find genes that have been reported earlier to be differentially expressed in response to the cyanobacterial toxin microcystin and to microcystin-free cyanobacteria. We further reveal the enrichment of gene ontology terms that have been shown to be involved in microcystin-related responses in other organisms but not yet in Daphnia and as such are candidate loci for adaptation of natural Daphnia populations to increased cyanobacterial abundances. In conclusion this approach of investigating DNA extracted from Daphnia resting stages allowed to determine frequency changes of loci in a natural population over time.

Investigation of possible alternate animal reservoir hosts of Opisthorchis viverrini.

Chronic opisthorchiasis caused by Opisthorchis viverrini (O. viverrini) adversely affects human health and is associated with a fatal bile duct cancer (cholangiocarcinoma). Although cats and dogs are known animal reservoir hosts of opisthorchiasis, there is limited information about whether other fish-eating animals are fluke reservoirs. Wetlands along Chi River of Thailand have abundant intermediate host snails and fish for O. viverrini and diverse other animal species. This led to our investigation into whether other fish-eating animals can also become infected and be alternate reservoir hosts for human opisthorchiasis. Our preliminary study focused on the O. viverrini infection status of crab-eating or long-tailed macaques roaming in Kosumpi National Forest Park in Chi River Basin, Kosumpisai District of Mahasarakam Province, and rodents, small residential mammals and fish-eating birds living in Lawa wetland complex of Khon Kaen Province. Fecal samples of each animal were collected and modified formalin ether concentration technique was applied to identify infections. Additionally, participatory epidemiology was used to retrieve information from local communities on opisthorchiasis transmission in these animals. No O. viverrini infection was found in any fecal samples suggesting that monkeys, rodents, small residential mammals and birds in these two wetlands probably do not serve as alternate reservoir hosts of O. viverrini.

Unscrambling the oocyte and the egg: clarifying terminology of the female gamete in mammals.

Most reproductive biologists who study female gametes will agree with the 16th century anatomist William Harvey's doctrine: 'Ex Ovo Omnia'. This phrase, which literally translates to 'everything from the egg', recognizes the centrality of the egg in animal development. Eggs are most impressive cells, capable of supporting development of an entirely new organism following fertilization or parthenogenetic activation. Not so uniformly embraced in the field of reproductive biology is the nomenclature used to refer to the female germ cell. What is an oocyte? What is an egg? Are these terms the same, different, interchangeable? Here we provide functional definitions of the oocyte and egg, and how they can be used in the context of mammalian gamete biology and beyond.

Helminth Egg Automatic Detector (HEAD): Improvements in development for digital identification and quantification of helminth eggs and their application online.

Helminths are parasitic worms that constitute a major public health problem. Conventional analytical techniques to evaluate helminth eggs in environmental samples rely on different steps, namely sedimentation, filtration, centrifugation, and flotation, to separate the eggs from a variety of particles and concentrate them in a pellet for direct observation under an optical microscope. To improve this process, a new approach was implemented in which various image processing algorithms were developed and implemented by a Helminth Egg Automatic Detector (HEAD). This allowed identification and quantification of pathogenic helminth eggs of global medical importance and it was found to be useful for relatively clean wastewater samples. After the initial version, two improvements were developed: first, a texture verification process that reduced the number of false positive results; and second, the establishment of the optimal thresholds (morphology and texture) for each helminth egg species. This second implementation, which was found to improve on the results of the former, was developed with the objective of using free software as a platform for the system. This does not require the purchase of a license, unlike the previous version that required a Mathworks® license to run. After an internal statistical verification of the system was carried out, trials in internationally recognized microbiology laboratories were performed with the aim of reinforcing software training and developing a web-based system able to receive images and perform the analysis throughout a web service. Once completed, these improvements represented a useful and cheap tool that could be used by environmental monitoring facilities and laboratories throughout the world; this tool is capable of identifying and quantifying different species of helminth eggs in otherwise difficult environmental samples: wastewater, soil, biosolids, excreta, and sludge, with a sensitivity and specificity for the TensorFlow (TF) model in the web service values of 96.82% and 97.96% respectively. Additionally, in the case of Ascaris, it may even differentiate between fertile and non-fertile eggs.

A new polychelidan lobster preserved with its eggs in a 165 Ma nodule.

Crustacean eggs are rare in the fossil record. Here we report the exquisite preservation of a fossil polychelidan embedded within an unbroken nodule from the Middle Jurassic La Voulte-sur-Rhône Lagerstätte (France) and found with hundreds of eggs attached to the pleon. This specimen belongs to a new species, Palaeopolycheles nantosueltae sp. nov. and offers unique clues to discuss the evolution of brooding behaviour in polychelidan lobsters. In contrast to their development, which now relies on a long-lived planktic larval stage that probably did not exist in the early evolutionary steps of the group, the brood size of polychelidan lobsters seems to have remained unchanged and comparatively small since the Jurassic. This finding is at odds with reproductive strategies in other lobster groups, in which a long-lived planktic larval stage is associated with a large brood size.

The invasive Asian tiger mosquito Aedes albopictus in Romania: towards a country-wide colonization?

Currently, five invasive Aedes mosquito species are of concern in Europe according to the European Centre for Disease Prevention and Control: Aedes aegypti, Ae. albopictus, Ae. atropalpus, Ae. japonicus, and Ae. koreicus. Among these, only Ae. albopictus was reported to occur in Romania, in Bucharest. The aim of this study was to update the knowledge on the distribution of this invasive mosquito species in Romania, by investigating new potential locations. Monitoring of Ae. albopictus was carried out between May 2017 and October 2018. Three types of traps (CDC-Gravid Traps, CDC miniature Light Traps, ovitraps) were placed in 53 localities in 13 counties at sites suitable for container-breeding mosquitoes. Collected adult mosquitoes were counted and identified according to morphological criteria. Larvae were found present in domestic containers and rain catch basins. Aedes albopictus adults and eggs were collected in 10 localities in eight counties across Romania. Our study confirms nine new localities and seven counties where Ae. albopictus became established in Romania, highlighting the need for surveillance to further assess the species' distribution and abundance, as well as the pathogen transmission risk related to that vector species.

A potential global surveillance tool for effective, low-cost sampling of invasive Aedes mosquito eggs from tyres using adhesive tape.

BACKGROUND: The international movement of used tyres is a major factor responsible for global introductions of Aedes invasive mosquitoes (AIMs) (Diptera: Culicidae) that are major disease vectors (e.g. dengue, Zika, chikungunya and yellow fever). Surveillance methods are restricted by expense, availability and efficiency to detect all life stages. Currently, no tested method exists to screen imported used tyres for eggs in diapause, the life stage most at risk from accidental introduction. Here we test the efficiency of adhesive tape as an affordable and readily available material to screen tyres for eggs, testing its effect on hatch rate, larval development, DNA amplification and structural damage on the egg surface. RESULTS: We demonstrated that the properties of adhesive tape can influence pick up of dormant eggs attached to dry surfaces. Tapes with high levels of adhesion, such as duct tape, removed eggs with high levels of efficiency (97% ± 3.14). Egg numbers collected from cleaned used tyres were found to explain larval hatch rate success well, particularly in subsequent larval to adult emergence experiments. The strength of this relationship decreased when we tested dirty tyres. Damage to the exochorion was observed following scanning electron microscopy (SEM), possibly resulting in the high variance in the observed model. We found that five days was the optimal time for eggs to remain on all tested tapes for maximum return on hatch rate success. Tape type did not inhibit amplification of DNA of eggs from three, five or ten days of exposure. Using this DNA, genotyping of AIMs was possible using species-specific markers. CONCLUSIONS: We demonstrated for the first time that adhesive tapes are effective at removing AIM eggs from tyres. We propose that this method could be a standardised tool for surveillance to provide public health authorities and researchers with an additional method to screen tyre cargo. We provide a screening protocol for this purpose. This method has a global applicability and in turn can lead to increased predictability of introductions and improve screening methods at high risk entry points.

The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage.

The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is the most widely distributed tick species globally. Throughout the world there are at least two divergent lineages on dogs that are traditionally grouped into what was known as R. sanguineus. The species R. sanguineus was recently redescribed using a neotype reported from countries with a temperate climate. The second lineage distributed in countries with primarily tropical climates is currently designated R. sanguineus s.l. tropical lineage. Here, we present a comprehensive genetic evaluation of Australian brown dog ticks from across the continent that complements the morphological study of R. sanguineus sensu Roberts (1965). A total of 294 ticks were collected from dogs around Australia - including New South Wales, Queensland, the Northern Territory and Western Australia - for morphological identification. All ticks were morphologically identified as R. sanguineus sensu Roberts (1965). DNA was isolated from a single leg from morphologically characterised individuals from New South Wales (n = 14), Queensland (n = 18), Northern Territory (n = 7) and Western Australia (n = 13), together with ticks from Fiji (n = 1) and the Seychelles (n = 1) for comparison with Australian ticks. The study revealed three cox1 haplotypes clustered only with R. sanguineus s.l. tropical lineage'. An updated distribution of R. sanguineus sensu Roberts (1965) is compared to the 1965 distribution. In the Australian context, R. sanguineus s.l. has appeared in north-western New South Wales but remains absent from coastal New South Wales. Despite both temperate and tropical climates being present in Australia, only R. sanguineus s.l. tropical lineage was found. The evidence does not support the presence of the strictly defined brown dog tick, R. sanguineus by Nava et al. (2018) in Australia, because the examined ticks are genetically and morphologically distinct. We recommend using the term brown dog tick, R. sanguineus sensu Roberts (1965) for specimens from Australia.

A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis.

BACKGROUND: Zoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp. METHODS: We compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites. RESULTS: The protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile. CONCLUSION: The new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

Gastrointestinal Parasites of Vervet Monkeys (Chlorocebus pygerythrus) in a High Latitude, Semi-Arid Region of South Africa.

Given a changing climate and large-scale human migration, understanding infectious diseases in wildlife and the factors that drive the spread of these diseases is becoming increasingly important. Owing to the close phylogenetic relationship between nonhuman primates and humans, primate parasites are of particular interest due to the potential for zoonotic disease transmission and for the study of social transmission within gregarious social groups. There is a wide range of social and environmental factors that influence the prevalence and transmission of pathogens, and identifying these, and their effects, is crucial to understanding the population-level consequences of climate change for animals that live in obligate social groups. Here we investigated gastrointestinal parasite species richness and used fecal egg counts to estimate worm intensities in 3 vervet monkey troops (Chlorocebus pygerythrus) in a high latitude, semi-arid region of South Africa. This region is characterized by unpredictable rainfall and temperature extremes in summer and winter. We identified the gastrointestinal parasites in the population and explored potential demographic predictors, namely sex and troop membership, of parasite species richness and estimated intensity. Additionally, we assessed whether there was short-term intra-individual, inter-sample consistency in egg counts. Six species of gastrointestinal helminths were identified from 3 study troops, with egg counts ranging from 0 eggs/g to 1,100 eggs/g. Neither age nor sex predicted species richness or estimated intensity. This population had the highest prevalence of parasites with an insect vector compared with all other vervet populations studied, and distinctively high prevalences of Trichostrongylus sp. (71%) and Ternidens sp. (27%). Additionally, we found intra-individual egg count consistency in the short term (mean: 32 days).

Two new species of ascaridoid nematodes in Brazilian Crocodylomorpha from the Upper Cretaceous.

Two new ascaridoid species, Bauruascaris cretacicus n. gen. et n. sp., and Bauruascaris adamantinensis n. gen. et n. sp., are described based on the fossils of eggs preserved in 80-70 million year old phosphatized coprolites of Crocodyliformes, chronologically assigned to the Upper Cretaceous (Campanian/Maastrichtian age), collected from sedimentary rocks of the Bauru Group, Adamantina Formation in the municipality of Santo Anastácio, in the southwestern region of the state of São Paulo, Brazil, South America. This paper describes the oldest ascaridoid species ever recorded in Crocodylomorpha. Hence, this article contributes to the body of knowledge about the evolutionary history of this nematode group. It also offers a clue about the composition of the parasite fauna of these reptiles from the Late Cretaceous, which is still unknown despite numerous studies about various aspects of their biology and the pioneering paleoparasitological analysis of animal coprolites by South American researchers.

Egg cases of the graytail skate Bathyraja griseocauda and the cuphead skate Bathyraja scaphiops from the south-west Atlantic Ocean.

Egg cases of Bathyraja griseocauda were larger (140-142 mm in length) than those of Bathyraja scaphiops (88-90 mm in length) and their surface was relatively smooth, without denticles, prickles or any ornamentation. Egg cases of B. scaphiops had a relative coarse surface, covered with prickles of similar size. An identification key for the all described egg cases from Bathyraja occurring in the south-west Atlantic Ocean is provided.

Coprodiagnosis of Echinococcus granulosus infection in dogs from Ankara, Turkey.

The present study were undertaken to compare two isolation techniques (centrifugal flotation and sedimentation) for recovering taeniid eggs from faecal samples, to identify E. granulosus DNA from taeniid eggs by PCR, and to determine the prevalence of E. granulosus in dogs in villages. Faecal samples were collected from 100 dogs in Ankara province. Taenia spp. eggs were found in 27% of dogs faeces. Echinococcus granulosus-specific PCR was obtained in 14 (51.85%) of the taeniid eggs-positive samples. As well as finding Taenia eggs in dogs' faeces, we also found eggs of some helminthic parasites; such as Dipylidium caninum, Toxocara canis, Toxascaris leonina, Trichuris sp., Capillaria sp., Filaroides sp., Dioctophyme renale, Linguatula serrata, hookworm, Dicrocoelium sp., Fasciola sp. and Ascaridia galli. Significantly, more dogs excreting taeniid eggs were diagnosed with the sedimentation method (n=27) as compared to the flotation method (n=10).

Quantifying the risk of zoonotic geohelminth infections for rural household inhabitants in Central Poland.

The objective of this study was to evaluate the risk of soil transmitted zoonotic helminth infections for families with young children, inhabitants of villages in the Mazowieckie Province of Central Poland. Epidemiological survey was conducted at 33 randomly selected households with 2-3 children present. Examination of soil samples from yards surrounding the houses for the presence of geohelminth eggs was conducted, the households were inspected, and family members interviewed using a designed questionnaire. Among 55 localities examined, i.e. 33 backyards, 10 vegetable gardens and 12 sandpits, contamination was found in 2 backyards (6.1%) and 1 sandpit (8.3%) at 3 households (9.1%). Of the total 550 examined soil samples, 4 (0.7%) were found to contain Trichuris and Toxocara eggs, with an average density of 1.5 and 2.0 eggs per sample. The study showed a low level of soil contamination, which was the result of inhabitants care about the sanitation of their domiciles. However, the results of the questionnaire survey demonstrated the need to warn rural residents about the risk factors for zoonotic helmints infections. In particular, parents should be advised how to minimize the threat of parasitic diseases for children in the rural environment. The presented study showed that promotional campaigns implemented in recent years on the prevention of parasitic zoonoses have had little effect to increase the awareness of the rural community. The present results confirmed that Toxocara is the most common zoonotic agent among geohelminths in the rural environment.

A Multiplex PCR Assay for Differentiating Coconut Rhinoceros Beetle (Coleoptera: Scarabaeidae) From Oriental Flower Beetle (Coleoptera: Scarabaeidae) in Early Life Stages and Excrement.

The coconut rhinoceros beetle, Oryctes rhinoceros (L.), is a major pest of coconut and other palm trees. An incipient coconut rhinoceros beetle population was recently discovered on the island of Oahu, Hawaii and is currently the target of a large, mutiagency eradication program. Confounding this program is the widespread presence of another scarab beetle on Oahu, the oriental flower beetle, Protaetia orientalis (Gory and Percheron 1833). Eggs, early life stages, and fecal excrement of coconut rhinoceros beetle and oriental flower beetle are morphologically indistinguishable, thereby creating uncertainty when such specimens are discovered in the field. Here, we report the development of a multiplex PCR assay targeting cytochrome oxidase I of coconut rhinoceros beetle and oriental flower beetle that can rapidly detect and distinguish between these insects. This assay also features an internal positive control to ensure DNA of sufficient quantity and quality is used in the assay, increasing its reliability and reducing the chances of false negative results.

A SIMPLE AND EFFECTIVE MULTIPLEX PCR TECHNIQUE FOR DETECTING HUMAN PATHOGENIC TAENIA EGGS IN HOUSEFLIES.

Taenia solium, T. saginata, and T. asiatica are cestode pathogens causing taeniasis in humans. Houseflies can transfer Taenia eggs to food. However, houseflies are thought to carry only small numbers of Taenia eggs, sometimes fewer than 10. Although several PCR-based methods have been developed to detect Taenia DNA, these require more than 10 eggs for adequate detection. We developed a multiplex PCR method with high specificity for the discrimination among the eggs of the three Taenia species, T. solium, T. saginata, and T. asiatica, using 18S ribosomal DNA (rDNA) as a genetic marker. This technique was found to be highly sensitive, capable of identifying the Taenia species from only one egg. This multiplex PCR technique using 18S rDNA specific primers should be suitable to diagnose Taenia eggs.

Morphological Differentiation of the Eggs of North American Container-Inhabiting Aedes Mosquitoes.

Aedes aegypti , Ae. albopictus, Ae. japonicus japonicus, and Ae. triseriatus eggs are described with the aid of color images and images from scanning electron microscopy. All eggs were broadly cigar shaped with Ae. triseriatus and Ae. j japonicus eggs being dull or matte black, while Ae. albopictus and Ae. aegypti eggs were shiny jet black. Aedes triseriatus eggs were lighter in color and had a rougher appearance when compared to those of Ae. j. japonicus. Aedes albopictus and Ae. aegypti eggs can be differentiated by the distinct presence of micropylar collars in Ae. aegypti. Our results reduce the need to rear these mosquitoes, reducing labor and time while providing an accurate identification of the vector mosquitoes.