Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the River Danube using mitochondrial DNA barcodes.

BACKGROUND: Metacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data. METHODS: Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Stúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the 'barcode' region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene. RESULTS: Only lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and 'D. mergi Lineage 2', and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of 'D. mergi Lineage 2'. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and 'D. mergi Lineage 2' (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene. CONCLUSIONS: This first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.

Microhabitat Selection and Eyefluke Infection Levels in the Western Mosquitofish (Gambusia affinis).

Several trematode species infect the eyes of fish as second intermediate hosts. In most cases the definitive host is a piscivorous bird. Studies of a few species have shown an increase in transmission due to decreased visual acuity of the fish host. However, this may vary depending on trematode microhabitat choice within the eye. Some trematode species are found in the lens, some are found in the vitreous humor, and others have been reported from the retina. Here we report 3 genera of eyeflukes in 3 locations of the eye in the intermediate fish host, Gambusia affinis . Clinostomum metacercariae were found attached to the outer sclera within the eye orbit, and Diplostomum metacercariae were found in the lens. Posthodiplostomum metacercariae were confirmed by histology to reside between the choroid and pigmented retina. Posthodiplostomum metacercariae were found in both eyes of all 20 fish examined and in high intensities (up to 27 metacercariae per eye). High trematode intensities between the choroid and pigmented retina found in this study may disrupt vision in this fish host. Our study is the first to document the microhabitat of all 3 trematode metacercariae within the eye of G. affinis .

Infectious agents in congenital cataract in a tertiary care referral center in North India.

Congenital cataract has the potential for inhibiting early visual development. Intrauterine infections with Rubella virus, Herpes simplex virus (HSV) and Toxoplasma gondii plays an important role in the development of congenital cataract. The study included 120 children under the age of 6 years presenting with congenital cataract and diagnosed using serology and polymerase chain reaction (PCR). The IgM positivity for rubella, HSV, T. gondii was found to be 5.8%, 1.6% and 8.3% respectively. The overall PCR positivity was found to be 40(33.3%), 25 (20.8%) and 39 (32.5%) for rubella, HSV and T. gondii with mean copy number of 1599 copies/µL; 1716 copies/µL and 1503 copies/µL respectively. Infective etiology significantly contributes to the causation of congenital cataract particularly for rubella virus which is a potentially eradicable disease. This study provides an epidemiological data for rubella, HSV and T. gondii in children with congenital cataract and highlights the need to introduce rubella vaccine in the National Immunization Programme of India.

Community structure of Diplostomum spp. (Digenea: Diplostomidae) in eyes of fish: main determinants and potential interspecific interactions.

Parasite communities in freshwater fish vary annually and seasonally and can be influenced by the length, age, sex and phylogeny of hosts, and by associations among parasite species. We assessed the influence of these factors in species of Diplostomum in the eyes of 828 fish in 20 different species collected in a single lake in early summer or autumn over a 5year interval. Strong negative associations were found between five pairs of Diplostomum spp. and associations were strongest among abundant species. Most interspecific associations occurred among species inhabiting the lens, suggesting competitive interactions. However, the strongest association occurred between two Diplostomum spp. that inhabit different tissues (i.e., the vitreous humour and lens), indicating processes other than direct competition. Seasonal variation was small compared with inter-annual variation. Infection intensities were high in 2006 and decreased dramatically in 2010 and 2011. Infracommunity composition and structure showed no clear correspondence to the ecology or phylogeny of host species. Host length and age, but not sex, had significant effects on the structure of Diplostomum infracommunities in lenses. However, a significant amount of variance in lens infracommunities could not be explained, indicating the potential importance of other factors such as resistance or exposure in determining infracommunity structure.

Penetration and migration success of Diplostomum spp. cercariae in Arctic charr.

We exposed 2 groups of young-of-the-year Arctic charr (Salvelinus alpinus) singly to 54 +/- 2 (mean +/- SE) Diplostomum spp. cercariae that had emerged from 4 randomly sampled snail hosts (Lymnaea stagnalis). The rearing tanks of the fish received Diplostomum spp. cercariae via the incoming water; therefore, 18 of the 36 fish had parasites in their eyes before the experimental exposure. Of the Diplostomum spp. cercariae presented to the fish, 19% penetrated and 46% of those that had penetrated the fish migrated successfully to the lens of the fish eye. The migration success of Diplostomum spp. from the site of penetration to the fish eye lens was lower in the previously parasitized (37.0 +/- 8.7% [mean +/- SE] adjusted with the number of penetrated cercariae) than in the unparasitized fish (55.3 +/- 8.8%) and differed between the individual snail host from which the cercariae had emerged. In addition, the migration success of Diplostomum spp. decreased with an increase in the number of the cercariae that penetrated the fish. At the individual snail host level, there seemed to be a trade-off between penetration and migration ability of the cercariae. The results indicate that success of Diplostomum spp. in penetration and especially in migration to the fish eye is affected by both the molluscan first intermediate host and the piscine second intermediate host.

Epidemic of unilateral panuveitis in children from Brazilian Amazonia: clinical and etiological aspects in seven patients.

OBJECTIVES: To describe clinical presentation and results of diagnostic and therapeutic procedures in seven children from an epidemic of panuveitis in the Brazilian Amazonia, as well as environmental analysis and etiological aspects involved. METHODS: Patients underwent full pediatric and ophthalmic examinations, B-scan, ultrasound biomicroscopy, and serological tests. Ocular samples were thoroughly analyzed, including two enucleation specimens. Environmental investigation encompassed water, soil, and river fauna. RESULTS: All patients had bathed in the waters of a regional river, the Araguaia. Six of them presented with intermediate uveitis, with snowbanking. Five had cataract and four showed inferior endothelial opacity, with localized anterior synechiae. One showed total leukoma, with flat anterior chamber. Only two had active uveitis, one of them with anterior chamber nodule. Serology revealed high prevalence of anti-Toxocara canis immunoglobulin G (IgG) antibodies. In three cases, vitreous and lens samples disclosed spicules of freshwater sponges Drulia uruguayensis and D. ctenosclera, also detected in the waters of the river. CONCLUSION: Freshwater sponge spicules could be potential new etiological agents of ocular pathology, but further studies are needed, considering the heterogeneity of the ocular lesions and results of serological and environmental studies.

[A study on the helminth fauna linked to seasonal changes and size of the fish host, Leuciscus cephalus L., from Lake Dam Orenler, Afyonkarahisar]. / Mevsimsel degisime ve boy büyüklügüne bagli olarak Leuciscus cephalus L.'un (Orenler Baraj Gölü, Afyonkarahisar) helmint faunasi uzerine bir arastirma.

In this study, the presence of helminths on 103 Leuciscus cephalus from the Orenler Dam Lake, Turkey was investigated from July 2007 to May 2008. Five parasite species were found in the host fish: Of these species Dactylogyrus vistuale was found in the gills (42.7%, 10.6+/-14.8 parasite/fish), Diplostomum sp. in the lens of eyes (12.6%, 4.1+/-4.0), Bothriocephalus acheilognathi in the intestine (23.3%, 4.8+/-10.4), Ligula intestinalis in the body cavity (12.6%, 2.1+/-1.2), and Pomphorhynchus laevis in the intestine (32.4%,.4.6+/-5.5). Infection data for parasite species were determined using seasonal data and size distribution of the host fish.

Infectivity of Diplostomum spp. in Arctic charr: aspects of exposure duration and cercariae morphology.

The diplostomid flukes, Diplostomum spp., infect fish and cause cataract opacities in the eye lens. The effect of exposure dose on abundance of Diplostomum spp. eye flukes in fish is known, but the effect of the duration of cercariae exposure has not been studied. However, under natural conditions, the temporal window for a successful cercaria attachment on fish is very short and, consequently, differences in infectivity of eye fluke cercariae, in the short-exposure durations of a few seconds, are probably biologically the most meaningful. We investigated infectivity of Diplostomum spp. cercariae originating from snail hosts in 3 lakes (3 Lymnaea stagnalis populations and 1 Radix balthica population) in 6 exposure times, ranging from 5 sec to 15 min, in young-of-the-year Arctic charr Salvelinus alpinus. In addition, we compared the infectivity to the cross-morphology of the cercariae. In the long-exposure duration, i.e., > or = 5 min, infectivity of Diplostomum spp. did not vary between the snail host species (L. stagnalis and R. balthica) of the same lake or across the L. stagnalis populations of 3 different lakes. However, in the short-exposure duration, i.e., < or = 60 sec, Diplostomum spp. cercariae shed from L. stagnalis had higher infectivity than did cercariae from R. balthica of the same lake. This indicates that that there is an interaction between length of cercariae exposure and origin of Diplostomum spp., and that the duration of exposure may influence the results when fish are experimentally infected. Within a lake, cercariae shed from L. stagnalis were also smaller than cercariae shed from R. balthica.

[The occurrence of helminth parasites in Vimba (Vimba vimba L. 1758) of Golbasi (Bursa) Dam Lake, Turkey]. / Gölbasi Baraj Gölü (Bursa)'ndeki Egrez Baliklari (Vimba vimba L. 1758)'nda Görülen Helmint Parazitler.

In this study, the occurrence of helminth parasites in Vimba of Golbasi Dam Lake was investigated monthly from May 2003 to April 2004. During the study, a total of 62 Vimba specimens were examined for helminth parasites. A total of 4 species of helminth parasites were found on 43 fish examined as follows: (Dactylogyrus sphyrna (Monogenea), Diplostomum sp. (Digenea), Caryophyllaeus laticeps (Cestoda), Contracaecum sp. (Nematoda). D. sphyrna seen on gills of host fish was the dominant parasite species found in Vimba. A total of 308 parasites were recorded on 30 of the 62 fish examined (48.39%). The second dominant species was Diplostomum sp. of which 233 were found in the eye lens of 29 of the 62 hosts examined (46.77%). C. laticeps was the third dominant parasite in this study and a total of 37 parasites were recorded in 8 of 62 fish (12.90%). As to Contracaecum sp., only 4 specimens were found in 3 fish (4.84%).

Toxoplasma gondii detection by nested polymerase chain reaction in lens aspirate and peripheral blood leukocyte in congenital cataract patients: the first report from a tertiary eye hospital in India.

PURPOSE: To detect T. gondii DNA and specific antibodies in lens aspirates (LA) and peripheral blood leucocytes (PBL) of congenital cataract patients. METHODS: ELISA for T. gondii antibodies on sera nPCR for T. gondii DNA (B1 gene) on LA and PBL were performed for 52 patients. RESULTS: T. gondii DNA was detected in 29 (55.8%) of the 52 patients (LA-14, PBL-13, LA and PBL-2, and specific IgM in 2 sera). nPCR in PBL was more sensitive than ELISA (p<0.005). CONCLUSION: nPCR is a sensitive technique to detect T. gondii from LA and PBL in congenital cataract patients.

[Features of pathogenesis of Diplostomum infection in riverine Abbottina rivularis (Cypriniformes: Cyprinidae) from Primor'e]. / Ob osobennostiakh patogeneza pri diplostomozakh u rechnoi Abbottiny Abbottina rivularis (Cypriniformes: Cyprinidae) iz Primor'ia.

Autopsies of Abbottina rivularis from southern Primorye (drainage-basin of Artyomovka River, Razdolnaya River and Khanka Lake) and southern Sakhalin Island (Maloye Chibisanskoye Lake) revealed high indices of diplostomum-infected lenses of these fishes. The metacercariae have been identified as Diplostomum parviventosum Dubois, 1932, D. huromense (La Rue, 1927), D. helveticum (Dubois, 1929), D. mergi Dubois, 1932, Diplostomum sp. The most lenses of parasitized eyes possessed dorsally situated sperical protrusions of the lens capsule ("cyst"). Earlier, this phenomenon was found by Larson (1965) in naturally infected bullheads (Ictalurus). The metacercarial infection level and its dynamics, age-composition of metacercariae in both lenses and "cysts", and "cysts" production are discussed.

Studies on the infectivity of Diplostomum spathaceum in rainbow trout (Oncorhynchus mykiss).

The infectivity of Diplostomum spathaceum (Digenea: Trematoda) cercariae to rainbow trout and the efficacy of the diplostomule migration to the lens following different routes of administration was examined. The optimum age of infectivity for cercariae was between 0-5 h after liberation from the snail and for intraperitoneally injected diplostomules, 5 h post-transformation in vitro through fish skin. After exposure of the entire fish body or head to cercariae, metacercariae first appeared in the lens at 5 h and their numbers gradually increased until 22 h. Following exposure of the tail region of rainbow trout to cercariae, metacercariae first appeared in the lens at 14 h. Significantly more metacercariae established in the lens of fish following exposure of the fish head compared with the tail region; 40% of penetrating cercariae reached the lens of fish following exposure of the head or entire body, 20% of cercariae or diplostomules injected either intraperitoneally, intramuscularly or intracardially reached the lens while only 5% of cercariae established as metacercariae following exposure of the tail region.

Metacercariae of Diplostomum spathaceum in the eyes of fishes from Yellowstone Lake, Wyoming.

Fish were collected from Yellowstone Lake, Wyoming (USA). Metacercariae of Diplostomum spathaceum was found in the lens of 11 of 12 longnose suckers (Catostomus catostomus). The mean number of metacercariae per sucker was 59 and the average age of the fish was 11.6 yr. There was no correlation between age and intensity of parasites (r = 0.24). Of 10 cutthroat trout (Salmo clarki) examined, there were metacercariae present in six. The metacercariae were found outside of the lens tissue in the trout; they occurred in the vitreous humor and the retina. These may be a different species from those found in the suckers.

[Adaptibility of Diplostomum cercariae in carp and the effect of a prior infestation]. / Prizhivaemost' u karpa tserkarii diplostom i vliianie na nee predshestvuiushchego zarazheniia.

Experiments on the infection of carp fry with cercariae of Diplostomum have shown that cercariae of Diplostomum spathaceum can penetrate crystalline lens of larvae of these fishes in three days after their hatching and that the adaptability of cercariae of D. paracaudum to carp is much higher than that of cercariae of D. spathaceum. After the first infection with cercariae a relative postinfectious immunity against infection with cercariae of this or close species of this genus arises in carp fry.

[Enzyme activity of metacercariae in the genus Diplostomum (Trematoda, Diplostomidae)]. / O fermentativnoi aktivnosti metatserkaii roda Diplostomum (Trematoda, Diplosotmidae).

Studies were conducted of the content of ferments with which metacercariae of trematodes of the genus Diplostomum affect the tissues of the host's crystalline lens. The following ferments were found in exudates of metacercariae: pepsin, cathepsin, hyaluronidase and lipase. Their activity depends to a great extent on temperature. Considerable changes in the biochemical content of crystalline lens infected with the parasites were found. The character and force of pathogenic effect of diplostomatids on fishes depend on the factors associated with activation and inhibition of ferments of metacercariae, temperature and the presence of ions-inhibitors in particular.

Observations on the occurrence of Diplostomum spathaceum and Schistocephalus sp. in ninespine sticklebacks (Pungitius pungitius) from the Belcher Islands, Northwest Territories, Canada.

All 175 ninespine sticklebacks, Pungitius pungitius (Linné), collected from the Belcher Islands were parasitized by Diplostomum spathaceum (Rudolphi) and 43% by Schistocephalus sp. D. spathaceum metacercariae were mostly confined to a dorso-ventral band encircling the lens of the eye, and were concentrated in the anterio-dorsal sector of this band. The central area of the lens was thus relatively free of diplostomula, probably minimizing interference with the vision of the fish. The frequency distribution of D. spathaceum abundance in P. pungitius was closely approximated by a negative binomial, while that for Schistocephalus was best fitted by a Poisson. None of the fish condition factors examined appeared related to the intensity of the parasitic infections.

An experimental approach to the study of intraocular Toxocara canis.

An experimental study of nematode endophthalmitis due to T canis and review of the literature has been presented. Six owl monkeys were infected either by nasogastric tube using embryonated T canis eggs or by carotid or intravitreal injection of second stage larvae. The clinical manifestations, especially ocular, were observed and various diagnostic tests performed. Only minimal or no intraocular changes were seen after systemic infection but significant abnormalities such as retinal hemorrhages and venous dilation were noted after intravitreal infection. Motile larvae were observed in the lenses of three eyes and in the vitreous of five eyes and, probably a sixth, after intravitreal injection. The intensity and timing of the intraocular reaction seemed to correlate with the infecting dose and apparent disappearance of larvae from the eye. Pathologic confirmation of larvae in the lens was obtained in one eye. A marked inflammatory reaction occurred in eyes receiving intraocular infection but none was seen in eyes with only systemic infectin. Various laboratory and serologic studies were performed, including the ELISA test, which were used to evaluate systemic as well as intraocular responses to infection with T canis. The two monkeys infected by nasogastric tube gave a positive ELISA response in the serum but intraocular fluids gave a negative response in all monkeys including those infected syst:mically and/or intraocularly. Problems in the understanding of clinical aspects of the disease, laboratory diagnosis and treatment are discussed. The need for future experimental studies is emphasized.

Life history and habitat analysis of the eye fluke Diplostomum spathaceum (trematoda: diplostomatidae) in Utah.

Diplostomum spathaceum which is widespread throughout Utah in desert, mountain valley, and high alpine lakes, causes a disease known as diplostomatosis or eye fluke disease. Snails (756) were examined and two species, Lymnaea stagnalis and L. palustris were positive for D. spathaceum. Examination of 838 fish, which included 19 species, revealed 10 species (Salmo trutta, Catostomus discobulus, Salmo clarki, Micropterus salmoides, Catostomus platyrhynchus, Salmo gairdneri, Gila atraria, Catostomus ardens, Salvelinus fontinalis, and Richardsonius balteatus) positive for metacercariae of D. spathaceum. The only avian hosts positive for adult Diplostomum were Larvus californicus and L. delawarensis.