Dactylomyza gibsoni n. g., n. sp. (Digenea: Opecoelidae) from Schuettea woodwardi (Waite) (Monodactylidae) from off Western Australia

An opecoelid digenean, Dactylomyza gibsoni n. g., n. sp. is described and figured from Schuettea woodwardi (Waite), a monodactylid from off the coast of Western Australia. The new genus conforms to the concept of the opecoelid subfamily Opecoelinae. The resemblance of the new genus to three other opecoelid genera, Pseudopecoeloides Yamaguti, 1940, Opecoeloides Odhner, 1928 and Poracanthium Dollfus, 1948, is discussed. Dactylomyza n. g. is distinguished from these morphologically similar worms on the basis of its median genital pore, ventral sucker appendages, uroproct and the absence of an accessory sucker. Pseudopecoeloides equesi Manter, 1947 is transferred to the new genus as Dactylomyza equesi (Manter, 1947) n. comb.     

Length‐weight relationship and seasonal effects of the Summer Monsoon on condition factor of Terapon jarbua (Forsskål, 1775) from the wider Gulf of Aden including Socotra Island

The present study investigates the length‐weight relationship and condition factor of populations of the Indo‐Pacific fish Terapon jarbua ( Forsskål, 1775 ) collected in the wider Gulf of Aden, notably from Socotra Island and the Hadramout coast of Yemen. This region displays a monsoon climate, with wide seasonal variations affecting estuarine habitats. A total of 620 specimens collected in estuaries and at sea were measured and weighed during field surveys carried out in 2007 and 2008 during pre‐ and post‐ South‐Western monsoon periods. The length‐weight relationship of the studied populations of Terapon jarbua was determined as W = 0.0288 × SL^2.99, with r² = 0.96 and consistent with existing data on the species from other regions. Significant seasonal differences were found in Fulton’s condition factor of Terapon jarbua.     

Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay

Among the previously established enzyme-linked immunosorbent assays (ELISAs), an ELISA using the full length of a recombinant thrombospondin-related adhesive protein of Babesia gibsoni (rBgTRAPf) is considered as the most sensitive diagnostic method for the detection of an antibody to B. gibsoni in dogs. However, the expression of rBgTRAPf in high concentration is poor and, thus, limits its usefulness as a diagnostic antigen. To improve its expression level, we have truncated BgTRAPf into two fragments having either an N- or a C-terminus (BgTRAPn or BgTRAPc, respectively). The expression of BgTRAPc protein in Escherichia coli yielded adequate recombinant protein. The specificity and sensitivity of ELISAs with the truncated proteins were determined using dog sera experimentally infected with B. gibsoni and specific pathogen-free (SPF) dog sera. A total of 254 field dog sera were examined by the ELISA with rBgTRAPn, rBgTRAPc, and rBgTRAPf as well as by an indirect fluorescent antibody test (IFAT). The specificity of rBgTRAPc was the highest (97.15%), and its kappa value was more (0.8003) than rBgTRAPn (0.7083). With a sufficient level of expression as well as higher specificity and reliable sensitivity, rBgTRAPc appears to be a potential candidate antigen for the serodiagnosis of B. gibsoni infection in dogs.     

Use of a doxycycline-enrofloxacin-metronidazole combination with/without diminazene diaceturate to treat naturally occurring canine babesiosis caused by <Emphasis Type="Italic">Babesia gibsoni</Emphasis>

Canine babesiosis is an important worldwide, tick-borne disease caused by hemoprotozoan parasites of the genus Babesia. Babesia gibsoni is the predominant species that causes canine babesiosis in Taipei, Taiwan. It is a small pleomorphic intraerythrocytic parasite that can cause erythrocyte destruction and hemolytic anemia. Efficacy of oral administration of a doxycycline-enrofloxacin-metronidazole combination with and without injections of diminazene diaceturate in the management of naturally occurring canine babesiosis caused by B. gibsoni was evaluated retrospectively. The overall efficacy of this combination of doxycycline-enrofloxacin-metronidazole in conjunction with and without administration of diminazene diaceturate was 85.7% and 83.3%, respectively; with a mean recovery time of 24.2 and 23.5 days, respectively. Concomitant use of intramuscular diminazene diaceturate may not improve the efficacy of a doxycycline-enrofloxacin-metronidazole combination in management of canine babesiosis caused by B. gibsoni.     

Vector competence ofRhipicephalus sanguineus andDermacentor variabilis for American isolates ofBabesia gibsoni

To determine the identity of the tick vector of enzooticBabesia gibsoni in California, two common ixodid ticks were allowed to engorge uponB. gibsoni infected dogs. Sporozoites were observed in the salivary glands of prefed nymphalRhipicephalus sanguineus ticks that fed as larvae onB. gibsoni-infected dogs. A higher proportion (31%) of nymphal ticks that prefed on an uninfected dog for 48 hours contained sporozoites in their salivary glands than did ticks which had fed for 24 hours (13%). Sporozoites were not observed in the salivary glands of prefedR. sanguineus nymphs which were derived from the eggs of adult females that fed on an infected dog, in adults that were fed as nymph on an infected dog, or in the nymphal and adult uninfected controls.Dermacentor variabilis ticks appeared not to become infected. Although attempts to transmitB. gibsoni to susceptible, splenectomized dogs were unsuccessful,R. sanguineus would appear to be the most likely tick vector to maintain this piroplasm in California. This study was supported by grants from the Companion Animal Disease Laboratory, School of Veterinary Medicine, University of California, Davis.     

In vitro screening of novel anti-Babesia gibsoni drugs from natural products

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoni in vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC₅₀) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.     

Dactylostomum cribbi n. sp., a new opecoeline digenean from the Australian freshwater fish Gadopsis marmoratus Richardson

An opecoelid digenean, Dactylostomum cribbi n. sp., is described from the intestine of the freshwater fish Gadopsis marmoratus from Victoria, Australia, and its pathology commented upon. Its systematics is discussed in relation to related opecoelid genera, such as Coitocaecum Nicoll, 1915 (syn. Ozakia Wisniewski, 1933) and Nicolla Wisniewski, 1934, and the species of Dactylostomum Woolcock, 1935 occurring in Australasian waters. D. cribbi is smaller and less elongate than most species of Dactylosto- and Coitocaecum. The zoogeography of this and related forms is commented upon.     

Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis

A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity.     

Field observations on the lepidophagous teleost Terapon jarbua (Forsk»l)

Synopsis Foraging behaviour of the lepidophagous Indo-Pacific teleost Terapon jarbua in the Bulolo and Mtumbane estuaries of southern Africa is described. These observations suggest that scale removal from large fish and the complete ingestion of fish fry represents a modified form of predation. Prey reaction to T. jarbua shoals is also discussed.     

The life cycle of Haplorchis pumilio (Trematoda: Heterophyidae) from the Indian region

The life cycle of the heterophyid fluke, Haplorchis pumilio is elucidated for the first time from the Indian region. Various stages in the life cycle were established based on observations made on natural infections found in snails and fish in a freshwater stream at Visakhapatnam, India and experimental infections carried out in the laboratory. The thiarid snail, Thiara tuberculata served as the first intermediate host and a wide range of freshwater fish as second intermediate hosts. Natural infections with adult flukes were found in the piscivorous birds Ardeola grayii and Bubulcus ibis. Adults were raised experimentally in day-old chicks. Distinguishing features of the cercaria of H. pumilio are: a large body size (200-224 x 92-96 micro m), body-tail ratio of 1:2.1 and densely distributed pigment granules in the parenchyma imparting a brownish tinge to the body. Natural infections with metacercariae were found in the freshwater fish Channa punctatus, C. orientalis, Puntius sophore, Gambusia affinis and fingerlings of Cyprinus carpio and Liza macrolepis. Additionally, experimental infections were established in Therapon jarbua, Esomus danricus and Oreochromis mossambica. Metacercariae were embedded in the caudal muscles of fish and heavy infections induced mortality. Metacercariae were infective at about 15 days of age.     

Purification of Onchocerca gibsoni microfilariae

Forsyth K. P., Copeman D. B. and Mitchell G. F. 1982. Purification of Onchocerca gibsoni microfilariae. International Journal for Parasitology12: 53–57. Viable microfilariae from the bovine parasite, Onchocerca gibsoni, were purified to a level suitable for immunochemical analysis and without appreciable loss in numbers by low speed centrifugation on Ficoll-paque. Microfilariae were obtained by incubation of intact O. gibsoni nodules or from worm fragments dissected from nodules. After purification, microfilariae were determined as viable by motility, ability to establish in mice and to incorporate ³⁵S-methionine into large numbers of proteins in vitro as determined by two-dimensional gel electrophoresis and fluorography.     

An Alexandrium Spp. Cyst Record from Sequim Bay,Washington State,USA, and its Relation to Past Climate Variability1

Since the 1970s, Puget Sound, Washington State, USA, has experienced an increase in detections of paralytic shellfish toxins (PSTs) in shellfish due to blooms of the harmful dinoflagellate Alexandrium. Natural patterns of climate variability, such as the Pacific Decadal Oscillation (PDO), and changes in local environmental factors, such as sea surface temperature (SST) and air temperature, have been linked to the observed increase in PSTs. However, the lack of observations of PSTs in shellfish prior to the 1950s has inhibited statistical assessments of longer‐term trends in climate and environmental conditions on Alexandrium blooms. After a bloom, Alexandrium cells can enter a dormant cyst stage, which settles on the seafloor and then becomes entrained into the sedimentary record. In this study, we created a record of Alexandrium spp. cysts from a sediment core obtained from Sequim Bay, Puget Sound. Cyst abundances ranged from 0 to 400 cysts · cm^?3 and were detected down‐core to a depth of 100 cm, indicating that Alexandrium has been present in Sequim Bay since at least the late 1800s. The cyst record allowed us to statistically examine relationships with available environmental parameters over the past century. Local air temperature and sea surface temperature were positively and significantly correlated with cyst abundances from the late 1800s to 2005; no significant relationship was found between PDO and cyst abundances. This finding suggests that local environmental variations more strongly influence Alexandrium population dynamics in Puget Sound when compared to large‐scale changes.     

Heterosigma akashiwo in central California waters

Heterosigma akashiwo (Hada) gives rise to red tides along the Atlantic and Pacific coasts and is known to produce brevetoxins. This investigation establishes baseline information showing the presence of H. akashiwo along the central California coast based on water samples collected from the Santa Cruz pier in Monterey Bay (on the open coast) and the Berkeley pier in San Francisco Bay. Light and electron microscopy as well as two species-specific DNA probe methods based on cell homogenates preparations were employed to detect H. akashiwo during the 2001–2002 field study. The DNA probe methods consisted of a sandwich hybridization assay (SHA), which targets ribosomal RNA (rRNA), and an end-point polymerase chain reaction (PCR) assay, which targets internal transcribed spacer (ITS) sequences of rRNA genes. The SHA was used to provide semi-quantitative data showing the intermittent presence of the species during a 13-month period in Monterey Bay. Samples that showed a variety of responses in the SHA (negative as well as the highest) were then subjected to the PCR assay in an attempt to confirm species identification using an independent DNA probe method that employs cell homogenates; samples included those from Monterey Bay and one from a red tide event in San Francisco Bay. SHA and PCR assays agreed on the presence or absence of H. akashiwo. Gene products from two field samples positive for H. akashiwo by PCR were cloned and sequenced and found to be identical to those of that species in GenBank. When the same samples were viewed by light microscopy, however, H. akashiwo cells were only seen in the sample with the highest abundance of that species, as evidenced by SHA. It was extremely difficult to recognize naturally occurring H. akashiwo using light microscopy in field samples that had been preserved with Lugol's iodine, including samples that gave positive results by cell homogenate methods. Results of this study indicate that H. akashiwo is present along the open California coast and could easily be missed in routine phytoplankton surveys. Despite its presence, H. akashiwo does not appear to routinely bloom with sufficient densities to cause harmful outbreaks of the frequency and severity documented in some other coastal environments. Molecular identification techniques may be the preferred approach over light microscopy when there is a need to rapidly screen many samples for fragile, harmful species and those that are otherwise problematic to identify based on their gross morphology alone.     

Host and Parasite Interactions During Single and Concurrent Infections with Eimeria nieschulzi and E. separata in the Rat1

SYNOPSIS. Some effects of 2 species of Eimeria in the rat were studied in single and concurrent infections. Groups included singly infected E. nieschulzi and E. separata controls (15 rats each) and 4 groups of 5 rats each infected with E. separata (shorter life cycle) at different days of an established E. nieschulzi infection. Experiments were conducted using inoculations of approximately 3,000, 5,000, and 7,000 sporulated oocysts of each species. Hosts showed consistently poor weight gain on day 3 postinoculation (PI) in E. separata infections and usually showed weight loss on days 7 and/or 8 PI in E. nieschulzi infections. Spleen weights indicated that the hosts responded immunologically to both parasites. Small intestine weights increased during E. nieschulzi infections as did cecum-colon weights during E. separata infections. In severe infections both species seemed to cause pathologic change in tissues at a distance from the site of endogenous development. Fecal collections were made at 24-hr intervals from the time of 1st inoculation till the end of patency. Patency usually began on day 7 PI for E. nieschulzi and on day 4 PI for E. separata with oocyst discharge peaking on days 8 and 5, respectively. In 2 instances, a biphasic pattern of oocyst discharge was found for E. separata during concurrent infections. The occurrence of interspecific interactions was indicated by an increased discharge of oocysts of E. separata, including those with the ability to sporulate, in doubly-infected animals as compared with those in singly-infected controls. It was suggested that the increased oocyst discharges may have resulted from additional production of merozoites during schizogony. Concurrent infections weakened the host more than single infections and this seemingly allowed more multiplication of the parasites.     

Babesia gibsoni: Detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis

In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis.     

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein gene of Babesia gibsoni isolates in dogs in South India

Babesia gibsoni, the causative agent of canine piroplasmosis, is a tick-borne intraerythrocytic protozoan parasite predominantly reported in Asian countries. The present study aimed at genotypic characterization of B. gibsoni isolates prevalent in dogs in Kerala, a southern state of India. Blood samples were collected from 272 dogs in Kerala and B. gibsoni infection was detected by microscopy and polymerase chain reaction (PCR). Molecular confirmation of B. gibsoni parasites was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR detected a higher percentage of dogs (40.44%) positive for B. gibsoni infection than microscopy where 15.81% dogs were detected positive for infection. Genetic characterization of B. gibsoni isolates (n = 11) prevalent in dogs in the state of Kerala was carried out by PCR amplification and sequencing of the 855 bp thrombospondin-related adhesive protein (TRAP) gene fragment. Phylogenetic analysis of the B. gibsoni TRAP (BgTRAP) gene revealed that B. gibsoni isolates from Kerala formed a distinct cluster with the isolates from north India and Bangladesh, away from other East Asian isolates. Nucleotide analysis of the tandem repeats of BgTRAP gene showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh but not by the isolates of East Asian countries. The results of the present study further confirmed that B. gibsoni parasites in a distinct genetic clade are endemic in dogs in India and Bangladesh. However, elaborate studies are required for better understanding of the genetic diversity of B. gibsoni.     

Observations on the morphology and life-cycle of Procerovum varium (Onji & Nishio, 1916) (Trematoda: Heterophyidae)

The life-cycle of Procerovum varium (Digenea: Haplorchiinae) was studied experimentally and the morphology of stages in the life-cycle has been described and illustrated. Infections with adult flukes were found in the pond heron Ardeola grayii and heavy infections with metacercariae were found, attached to the liver of the fish Oryzias melastigma (Oryziatidae) occurring in a freshwater stream situated in Visakhapatnam, India. The cercariae developing in the snail Thiara tuberculatapossessed typical haplorchiine features and were characterised by the presence of numerous cystogenous glands. Early stages of metacercarial development occurred free in the muscles of the fish intermediate host. The larvae reached the liver at 5 days post-infection, encystment commenced 2 days later and 15-day-old metacercariae were found to be infective to chicks, ducks and mice that served as suitable experimental hosts. The adult flukes obtained from natural and experimental infections showed many intraspecific variations, especially in the size and shape of the expulsor which depend on the quantity of sperm it contains. The validity of various species described in the genus and differentiated on the basis of differences in the size of the expulsor has been examined. It is concluded that only three species of the genus, namely P. varium, P. cheni and P. calderoni, are valid. "P. sisoni" of Chen (1949) is confirmed as a synonym of P. varium. P. varium is reported for the first time from India.