Infection and molecular identification of ascaridoid nematodes from the important marine food fish Japanese threadfin bream Nemipterus japonicus (Bloch) (Perciformes: Nemipteridae) in China.

The Japanese threadfin bream N. japonicus (Bloch) (Perciformes: Nemipteridae) is an important marine food fish in Asia. However, our present knowledge of the occurrence of its nematode parasites is still limited. In the present study, the species composition and infection rate of ascaridoid nematodes in N. japonicus from the South China Sea, were studied for the first time. Five ascaridoid species, namely Anisakis typica (L3), Hysterothylacium amoyense (L3), Hysterothylacium sp. IV-A (L3), adult of H. thalassini and Raphidascaris lophii (L3), were identified using integrative taxonomy. Hysterothylacium amoyense was the most prevalent species (prevalence 47.2%, mean intensity 14.9 ± 17.1). Hysterothylacium thalassini and R. lophii were reported in the Japanese threadfin bream for the first time. Two different genotypes of A. typica (overall prevalence of 3.4%; mean intensity 1.7 ± 0.9) were found in the South China Sea for the first time. The unique restriction polymorphism patterns of three species of Hysterothylacium are provided for rapid diagnosis. Our present results indicate that RFLP analysis of ITS region, using the restriction enzymes HhaI and RsaI, represents a simple and practical method for large-scale surveys of Hysterothylacium for seafood industry.

Baylisascaris procyonis infection in raccoons: A review of demographic and environmental factors influencing parasite carriage.

Baylisascaris procyonis, the roundworm of raccoons (Procyon lotor), is an emerging helminthic zoonosis in North America. Since the larval form is capable of causing neurological disease in more than 150 species of birds and mammals including humans, understanding factors that influence carriage of the parasite by raccoons is important for mitigating risk. This review examines the current literature to identify major demographic and environmental risk factors associated with B. procyonis carriage in wild raccoons. Raccoon age and season of sample collection were most commonly identified as risk factors, with increased prevalence found in juvenile animals and when sample collection occurred in the fall. Human urbanization and agricultural land use were also observed as potential risk factors; however, there are inconsistencies in the direction of influence these risk factors have on the prevalence of infection. Further investigation into the role of environmental risk factors is required to better understand how human activities influence parasite carriage in raccoons. Additionally, future research using multivariable statistical models guided by epidemiological principles to control for confounding variables and identify interaction effects will help clarify the effect of these demographic and environmental factors. Developing a better understanding of the primary risk factors for parasite carriage in raccoons will help identify areas of higher risk for environmental contamination and will aid in the development and refinement of education and management programs to reduce the risk of human exposure.

Heterakis gallinarum, the Cecal Nematode of Gallinaceous Birds: A Critical Review.

Heterakis gallinarum is a heavily prevalent poultry parasite that thrives in the ceca of various species of gallinaceous birds. It is a small roundworm, measuring between 4 and 15 mm long, in the family Heterakidae. Heterakis gallinarum has a direct life cycle not requiring an intermediate host to complete development, and it is generally believed that poultry raised at high density on litter are at greatest risk for accumulating large numbers of the nematode. This species typically only causes mild pathology that does not significantly affect bird performance. However, H. gallinarum is recognized as an economically important parasite by the poultry industry because its ovum serves as the vector for the protozoal parasite Histomonas meleagridis, the cause of histomonosis in poultry. Diagnosis of the nematode typically relies on fecal egg counts, which are prone to false negative diagnoses. Molecular tools are available for studying the nematode and diagnosing infected flocks. Treating and preventing H. gallinarum infection is made difficult due to the low efficacy of anthelmintics for eradicating H. gallinarum from infected birds and of disinfectants for destroying H. gallinarum ova on contaminated farms.

Estudio Recapitulativo- Heterakis gallinarum, el nematodo cecal de las aves gallináceas: una revisión crítica. Heterakis gallinarum es un parásito avícola muy prevalente que se desarrolla en el ciego de varias especies de aves gallináceas. Es un pequeño gusano redondo, que mide entre 4 y 15 mm de largo, de la familia Heterakidae. Heterakis gallinarum tiene un ciclo de vida directo que no requiere de un huésped intermedio para completar el desarrollo y en general se cree que las aves criadas en alta densidad en cama tienen un mayor riesgo de acumular grandes cantidades de nematodos. Esta especie generalmente solo causa una patología leve que no afecta significativamente el rendimiento de las aves. Sin embargo, H. gallinarum es reconocido como un parásito económicamente importante para la industria avícola debido a que sus huevos sirven como vector para el parásito protozoario Histomonas meleagridis, que es la causa de la histomoniasis en las aves. El diagnóstico del nematodo generalmente se basa en el recuento de huevos fecales, que puede establecer diagnósticos falsos negativos. Las herramientas moleculares están disponibles para estudiar el nematodo y diagnosticar las parvadas infectadas. El tratamiento y la prevención de la infección por H. gallinarum se dificultan debido a la baja eficacia de los antihelmínticos para erradicar H. gallinarum de las aves infectadas y de los desinfectantes para destruir los huevos de H. gallinarum en granjas infestadas.

Allopatric speciation of Meteterakis (Heterakoidea: Heterakidae), a highly dispersible parasitic nematode, in the East Asian islands.

To clarify how the species diversity of highly dispersible parasites has developed, molecular phylogenetic analyses of Meteterakis spp., multi-host endoparasitic nematodes of reptiles and amphibians from the East Asian islands, were conducted. The results demonstrated the existence of two major clades, the J- and A-groups, with exclusive geographic ranges that are discordant with the host faunal province. However, diversification within the J-group was concordant with the host biogeography and suggested co-divergence of this group with vicariance of the host fauna. In contrast, the phylogenetic pattern within the A-group was discordant with host biogeography and implied diversification by repeated colonization. In addition, the mosaic distribution pattern of a J-group and an A-group species in the Japanese Archipelago, along with comparison of population genetic parameters and the genetic distance from their closest relatives, suggested the initial occurrence of a J-group lineage followed by exclusion in the western part of this region caused by invasion of an A-group lineage. Thus, the present study suggested that the species diversity of highly dispersible parasites including Meteterakis is formed not only by co-divergence with host faunal vicariance but also by peripatric speciation and exclusive interactions between species.

Genetic polymorphism of Baylisascaris procyonis in host infrapopulations and component populations in the Central USA.

Baylisascaris procyonis is a nematode of significant concern to public and domestic animal health as well as wildlife management. The population genetics of B. procyonis is poorly understood. To gain insights into patterns of genetic diversity within (infrapopulation level) and among (component population level) raccoon (Procyon lotor) hosts, and specifically to assess the relative importance of indirect and direct transmission of the parasite for explaining observed population structure, we collected 69 B. procyonis from 17 wild raccoons inhabiting five counties in Missouri and Arkansas, USA. Informative regions of mitochondrial (CO1, CO2) and nuclear (28S, ITS2) genes were amplified and the distribution and genetic variability of these genes were assessed within and across raccoons. Concatenation of the CO1 and CO2 mtDNA sequences resulted in 5 unique haplotypes, with haplotype diversity 0.456 ±â€¯0.068. The most common haplotype occurred in 94% of raccoons and 72.5% of B. procyonis. Sequences for 28S rDNA revealed four unique nuclear genotypes, the most common found in 100% of raccoons and 82.6% of B. procyonis. ITS2 genotypes were assessed using fragment analysis, and there was a 1:1 correspondence between 28S and ITS-2 genotypes. Infrapopulation variation in haplotypes and genotypes was high and virtually all hosts infected with multiple sequenced nematodes also harbored multiple haplotypes and genotypes. There was a positive relationship between the size of the analyzed infrapopulation (i.e., the number of nematodes analyzed) and the number of haplotypes identified in an individual. Collectively this work emphasizes the importance of indirect transmission in the lifecycle to this parasite.

Description and genetic characterization of a new Contracaecum larval type (Nematoda: Anisakidae) from Australia.

Nematode parasites belonging to the genus Contracaecum are economically important parasites with zoonotic significance. Adult Contracaecum spp. are found in the stomach of marine mammals or piscivorous birds, and larval stages infect a wide range of invertebrates and fish species. Human infection with Contracaecum larvae has been reported in Australia and other countries after the consumption of infected fish. Although the genus Contracaecum comprises numerous species, thus far only four Contracaecum larval types have been specifically identified, therefore their life cycle and biology are not yet fully understood. In this study, a new Contracaecum larva (type IV), found in the intestinal tissue of carp caught from Coonancoocabil Lagoon, New South Wales, Australia, is described and characterized genetically. It was identified as Contracaecum bancrofti, a unique Australian species reported previously from the Australian pelican in Northern Territory, New South Wales, Victoria and South Australia. This study highlights the role of migratory birds and introduced fish species, such as carp, in distributing zoonotic pathogens not only across the continent but also from marine to freshwater systems. Coonancoocabil Lagoon is located in the Murrumbidgee Valley National Park, a highly managed conservation area with native fish in wetlands located in close proximity to several fish farms and hatcheries. Infection of a highly resilient invasive fish species, such as carp, with a zoonotic parasite of low host specificity, such as Contracaecum larvae, should be alarming for aquaculture and environmental authorities.

Raccoon roundworm (Baylisascaris procyonis) as an occupational hazard: 1. Knowledge of B. procyonis and attitudes towards it and other zoonoses among wildlife rehabilitators.

Wildlife rehabilitators are at risk of zoonotic diseases because they often have prolonged contact with many species of wildlife and their bodily fluids. Raccoon roundworm (Baylisascaris procyonis) is a common zoonotic parasite of raccoons that has the potential to cause severe or fatal neurologic disease in a broad variety of hosts if the eggs within raccoon faeces are ingested. We administered an online survey to wildlife rehabilitators to assess their knowledge regarding aspects of transmission, biology and disease caused by B. procyonis, and also to evaluate attitudes towards wildlife diseases and B. procyonis as an occupational hazard. Knowledge was assessed using multiple choice and true-false questions; attitudes were measured using Likert-type items. A total of 659 complete or near-complete responses (missing fewer than three knowledge or attitudes items and/or non-response to some demographic fields) were collected. The median knowledge score was 7/14 questions correct (range: 0-14 correct). Generally, individuals with higher levels of education and rehabilitation experience, veterinary professionals and those who are members of professional wildlife rehabilitation groups scored above the median significantly more often (p < .01). Significantly more rehabilitators who were located in the south-east and those with part-time or infrequent commitments scored below the median overall knowledge score. There was general agreement that B. procyonis is a health risk of rehabilitators and that measures should be taken to control transmission to people and animals. Some factors explaining differences in attitudes include setting of rehabilitation (home versus animal care facility), veterinary profession, region, membership in a wildlife rehabilitation group and rehabilitation of raccoons. Findings emphasize the importance of awareness and mentorship to inform rehabilitators on the potential risks of B. procyonis and other potential zoonoses within captive wildlife settings, and the important role of professional wildlife rehabilitator groups in disseminating educational materials.

Introduced Rats and an Endemic Roundworm: Does Rattus rattus Contribute to Baylisascaris procyonis Transmission in California?

The introduced black rat, Rattus rattus, occurs throughout the native range of the raccoon roundworm, Baylisascaris procyonis, and might incorporate into its life cycle if rats consume parasite eggs, acquire viable infections, and are eaten by raccoons. Although rats forage at raccoon latrines, their role in B. procyonis transmission remains unknown. Here I tested the potential for rats to amplify B. procyonis transmission in California by surveying wild rodents for B. procyonis and conducting scavenger trials with the use of motion-activated cameras. Rattus rattus were infected with B. procyonis at intensities more than 100 times greater than that of co-occurring native Reithrodontomys megalotis and Peromyscus maniculatus. Rodent carcasses were scavenged by opossums, skunks, and raccoons, suggesting that these rodents, particularly R. rattus, contribute to B. procyonis transmission in this coastal California ecosystem.

Validation of a commercial kit aimed to the detection of pathogenic anisakid nematodes in fish products.

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.

Great sandeel (Hyperoplus lanceolatus) as a putative transmitter of parasite Contracaecum osculatum (Nematoda: Anisakidae).

In the Baltic Sea, the great sandeel (Hyperoplus lanceolatus), a common fish species that inhabits sandy substrates, is an important element in the diet of marine mammals, sea birds, and piscivorous fish. It can also act as a transmitter of parasites to its predators, but parasitological studies on this species are limited. We sampled great sandeels in April 2016 in two areas of the southern Baltic Sea (SB-south of Bornholm and GG-the Gulf of Gdansk) and found parasitic anisakid nematodes in both locations. The only one species of nematode parasites has been detected: liver worm (Contracaecum osculatum) with average prevalence of infection 8.96%. This is the first study to report on infection of H. lanceolatus with anisakid nematodes in the southern Baltic Sea and our results suggest that the great sandeel may play a role in the transmission of liver worm in the food web of this marine environment.

Baylisascaris procyonis Roundworm Seroprevalence among Wildlife Rehabilitators, United States and Canada, 2012-2015.

Baylisascaris procyonis roundworms can cause potentially fatal neural larva migrans in many species, including humans. However, the clinical spectrum of baylisascariasis is not completely understood. We tested 347 asymptomatic adult wildlife rehabilitators for B. procyonis antibodies; 24 were positive, suggesting that subclinical baylisascariasis is occurring among this population.

Raccoon Roundworm Infection Associated with Central Nervous System Disease and Ocular Disease - Six States, 2013-2015.

Baylisascaris procyonis, predominantly found in raccoons, is a ubiquitous roundworm found throughout North America. Although raccoons are typically asymptomatic when infected with the parasite, the larval form of Baylisascaris procyonis can result in fatal human disease or severe neurologic outcomes if not treated rapidly. In the United States, Baylisascaris procyonis is more commonly enzootic in raccoons in the midwestern and northeastern regions and along the West Coast (1). However, since 2002, infections have been documented in other states (Florida and Georgia) and regions (2). Baylisascariasis is not a nationally notifiable disease in the United States, and little is known about how commonly it occurs or the range of clinical disease in humans. Case reports of seven human baylisascariasis cases in the United States diagnosed by Baylisascaris procyonis immunoblot testing at CDC are described, including review of clinical history and laboratory data. Although all seven patients survived, approximately half were left with severe neurologic deficits. Prevention through close monitoring of children at play, frequent handwashing, and clearing of raccoon latrines (communal sites where raccoons defecate) are critical interventions in curbing Baylisascaris infections. Early treatment of suspected cases is critical to prevent permanent sequelae.

Permeability and Viability of Baylisascaris procyonis Eggs in Southern Texas Soils.

Baylisascaris procyonis is a nematode whose definitive host is the raccoon ( Procyon lotor ). Adult parasites are not particularly pathogenic to raccoons; however, larvae in intermediate hosts can cause visceral, ocular, and neural larva migrans. Humans serve as dead-end hosts, and pathological responses are similar to those found in infected intermediate hosts. Infected raccoons expose intermediate hosts through their feces, which can contain millions of B. procyonis eggs. Our objective was to determine how the quantity and viability of B. procyonis eggs in soil changed over time within different soil texture, moisture, and sun exposure. To examine egg survival and movement through soil we placed 100 B. procyonis eggs on 100 squares in 48 boxes representing a full factorial treatment of soil texture, moisture, and sun level. We monitored egg percolation and survival for 2 yr, removing (at 0, 1, 3, 6, 12, 18, and 24 mo) 5 squares from each box and counting the number of viable eggs at each depth in the soil column. Dry soils were mainly impermeable; even after 2 yr, >60% of B. procyonis eggs remained on the surface of all soil textures. Wet soils were more permeable than dry soils, but even in wet sandy soils where the greatest egg movement occurred, it required 1 yr before 60% of eggs transitioned from the soil surface to the next soil depth. For all soil textures, moistures, sun exposures, and depths, >92% of B. procyonis eggs remained viable after 2 yr in the southern Texas environment. Therefore, high exposure risk exists for potential hosts because B. procyonis eggs remain viable on or near the soil surface for at least 2 yr.

Patterns of latrine use by raccoons (Procyon lotor) and implication for Baylisascaris procyonis transmission.

Mammals often use latrine sites for defecation, yet little is known about patterns of latrine use in many common species such as raccoons (Procyon lotor). Because raccoon latrines are important foci for the transmission of raccoon roundworm (Baylisascaris procyonis), documenting metrics of raccoon latrine use may have public health implications. Although some studies have provided evidence that multiple raccoons visit single latrine sites, exact latrine visitation patterns of raccoons have never been documented. We monitored raccoon latrine usage using proximity-logging collars placed at 15 latrine sites. We found that latrine sites were visited by multiple raccoons (range 1-7), and raccoons visited as many as six latrines during a 2-wk period. No sex differences were found in the number of latrines visited or time spent during visits. We posit that the use of multiple latrine sites by raccoons may lead to the pattern that rates of B. procyonis infection at latrines are greater than infection rates found in individual raccoon fecal samples. This in turn could lead to greater transmission of B. procyonis to paratenic hosts. Our results support the conclusion that raccoon latrines can be major foci for the infection and spread of B. procyonis.

Baylisascaris procyonis infection in white-footed mice: predicting patterns of infection from landscape habitat attributes.

There is a growing body of evidence that habitat fragmentation resulting from anthropogenic land use can alter the transmission dynamics of infectious disease. Baylisascaris procyonis , a parasitic roundworm with the ability to cause fatal central nervous system disease in many mammals, including humans, is a zoonotic threat, and research suggests that parasite recruitment rates by intermediate hosts are highly variable among forest patches in fragmented landscapes. During 2008, we sampled 353 white-footed mice ( Peromyscus leucopus ) from 22 forest patches distributed throughout a fragmented agricultural ecosystem to determine the influence of landscape-level habitat attributes on infection rates of B. procyonis in mice. We characterized each mouse in terms of infection status and intensity of infection, and calculated (on a patch-wide basis) prevalence, mean abundance of B. procyonis , and mean intensity of infection. We used an information-theoretic approach to develop a suite of candidate models characterizing the influence of landscape attributes on each of our measured characteristics of B. procyonis infection in white-footed mice, based on previous knowledge of raccoon ( Procyon lotor ) ecology and B. procyonis distribution in agricultural ecosystems. We observed evidence of B. procyonis infection in mice across all 22 habitat patches sampled. However, parasite recruitment rates and intensity were highly variable among patches, and the results of our analyses suggest that spatial variability in B. procyonis infections was primarily driven by emergent properties of fragmented ecosystems. In particular, prevalence, abundance, and intensity of B. procyonis infections in mice were negatively associated with the size and connectivity of forest patches. These results support previous studies indicating that habitat fragmentation can alter the transmission dynamics of infectious disease, and suggest that factors below the scale of landscape, i.e., fine-scale habitat structure or demographic and behavioral attributes of intermediate and/or definitive hosts, also may be important for predicting patterns of B. procyonis infection in intermediate hosts.

Molecular identification and larval morphological description of Contracaecum pelagicum (Nematoda: Anisakidae) from the anchovy Engraulis anchoita (Engraulidae) and fish-eating birds from the Argentine North Patagonian Sea.

Anisakids use invertebrates as paratenic and/or intermediate hosts as a basic feature of larval transmission. The third-stage larva usually develops in invertebrates which are prey items of finfish paratenic hosts. Contracaecum larvae molt twice inside the egg and hatch as free third-stage larvae ensheathed in the second-stage larval cuticle. Copepods act as paratenic or obligatory hosts, usually ingesting these free L3 larvae, and fish act as intermediate/paratenic or metaparatenic hosts preying on infected copepods. Fish-eating birds acquire L3 larvae by ingesting infected fish where they develop into the fourth-stage larvae and adults. Objectives of this work were to establish the specific correspondence between Contracaecum pelagicum L3 larvae parasitizing the anchovy Engraulis anchoita, and the adults parasitizing the Magellanic penguin Spheniscus magellanicus and the Imperial shag Phalacrocorax atriceps through the use of molecular markers; and, to evaluate the anisakid L3 larval recruitment and infection caused by ingestion of anchovy by S. magellanicus. Sixteen specimens of Contracaecum L3 larvae were analyzed from E. anchoita from Bahía Engaño, Chubut, eight adult nematodes from S. magellanicus and six adult specimens from P. atriceps both from the Valdés Peninsula, Chubut. All nematodes were sequenced for three genes: mitochondrial cytochrome oxidase 2 (mtDNA cox2), mitochondrial ribosomal RNA (rrnS), and the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA region. Phylogenetic analyses were performed by using Maximum Parsimony (MP) analysis by PAUP. In addition, studies under SEM and LM were carried out on L3 larvae. All L3 individuals from E. anchoita, adults from S. magellanicus, and P. atriceps clustered in the same clade, well supported in the MP tree inferred from the mtDNA cox2, and rrnS gene sequences analyses. Further, the sequence alignments of L3 larvae and adults of C. pelagicum here obtained at the ITS-1 and ITS-2 regions of the rDNA matched the sequences of C. pelagicum previously deposited by us in GenBank. Nematode recruitment (Ro) was equal to 33.07 (7.20-91.14) L3 larvae for C. pelagicum in each penguin's meal of anchovy. The MP tree topologies obtained from mtDNA cox2 and rrnS genes demonstrated that specimens of Contracaecum L3 larvae from E. anchoita and C. pelagicum from S. magellanicus as well as from P. atriceps constitute a unique clade, well-distinct and supported from all the others formed by the Contracaecum spp. sequenced so far for these genes. Molecular markers are considered to be an effective tool to elucidate larval transmission. The Contracaecum L3 larval recruitment value showed that many worms fail to establish in the bird digestive tract, probably because they are below a critical size. Further work is needed to elucidate other factors (e.g., physiological, immunological) that control nematode populations in the penguin digestive tract.

Effects of urbanization on prevalence of Baylisascaris procyonis in intermediate host populations.

Baylisascaris procyonis is an intestinal parasite of raccoons (Procyon lotor) that can also infect humans and a wide range of wildlife species. Prevalence of B. procyonis in raccoon populations appears to decrease as the landscape urbanizes, but less is known about prevalence in the small-mammal intermediate hosts of the parasite. We measured prevalence of B. procyonis in populations of mice (Peromyscus spp.) in forest preserves along a gradient of urbanization in Illinois. Prevalence in the mouse intermediate host exhibited a trend opposite raccoons: prevalence increased as surrounding human population density increased. This counterintuitive result may be due to higher overall environmental loads of B. procyonis in urban areas due to higher raccoon densities. Our results emphasize the need to understand fully the transmission dynamics of B. procyonis in its definitive and intermediate hosts in order to design and implement effective strategies to mitigate zoonotic risks to humans.