Worm Fragments Research Articles

Description and circadian rhythms of Chandlerella sinensis Li, 1933 (Nematoda; Onchocercidae), with remarks of microfilariae effects on the host health.

During investigation of common linnet (Linaria cannabina) blood using the buffy coat method one bird with microfilariae in the blood was found. The morphometric description of adult worms corresponded to the Chandlerella sinensis. This species was found for the first time in common linnets. DNA sequences of cox1 and 28S gene fragments of adult worm recovered during necropsy was identical to that from the microfilariae in the bird blood. Phylogenetic analysis of the cox1 gene fragment clustered this parasite with Chandlerella quiscali. Histological examination revealed the presence of microfilariae in the lumen of small capillaries and other blood vessels in different organs, but no inflammations were notice. The greatest number of microfilariae was in the lungs. Even if there was no inflammation, but vessels associated with the lungs were markedly distended with blood, parabronchial walls were thickened and, in some cases, almost completely obstructing the lumen. The large number of microfilariae in lungs indicates possible disturbance of gas exchange in the lungs adversely affected the ability of the bird to exercise and made breathing difficult at rest. The investigation of circadian rhythm of the microfilariae showed that C. sinensis microfilariae in blood of common linnet were more numerous at night and morning and less numerous at midday. The survival rate of mosquitoes infected with C. sinensis microfilariae was significantly lower than that of uninfected mosquitoes.

Genetic features of the marine polychaete Sirsoe methanicola from metagenomic data

The methane ice worm Sirsoe methanicola is the only marine polychaete species observed to colonize the methane hydrates of the Gulf of Mexico. Methane hydrates are ephemeral features of deep-sea cold seeps, and finding worm-colonized hydrates is rare; thus, little is known about these organisms. Recent metagenomic analysis predicted prokaryotic taxa and pathways from S. methanicola gut contents and worm fragments. Here, we increase the genetic information known about S. methanicola by assembling its nuclear rRNA genes (18S rRNA and 28S rRNA), mitochondrial genome (mitogenome), and other protein-coding genes from metagenomic data. Assembled 18S rRNA and 28S rRNA gene sequences of S. methanicola were near-identical to previously reported S. methanicola sequences. The 17,403-bp mitogenome of S. methanicola is the first mitogenome sequence of the family Hesionidae, consisting of 39.03% G+C content, 13 protein-coding genes, 24 tRNAs (including two split trnM genes), and 2 rRNA genes. Protein-coding genes in the S. methanicola metagenomes assigned to the phylum Annelida were involved in cell adhesion, signaling, ubiquitin system, metabolism, transport, and other processes. From the metagenomes, we also found 42 homologs of the cytochrome P450 (CYP) superfamily putatively involved in polycyclic aromatic hydrocarbon (PAH) metabolism. Our results encourage further studies into the genetic adaptations of S. methanicola to its methane hydrate habitat, especially in the context of deep-sea ecology and nutrient cycling.

Dominance of Sulfurospirillum in Metagenomes Associated with the Methane Ice Worm (Sirsoe methanicola).

Sirsoe methanicola, commonly known as the methane ice worm, is the only macrofaunal species known to inhabit the Gulf of Mexico methane hydrates. Little is known about this elusive marine polychaete that can colonize rich carbon and energy reserves. Metagenomic analysis of gut contents and worm fragments predicted diverse metabolic capabilities with the ability to utilize a range of nitrogen, sulfur, and organic carbon compounds through microbial taxa affiliated with Campylobacterales, Desulfobacterales, Enterobacterales, SAR324, Alphaproteobacteria, and Mycoplasmatales. Entomoplasmatales and Chitinivibrionales were additionally identified from extracted full-length 16S rRNA sequences, and read analysis identified 196 bacterial families. Overall, the microbial community appeared dominated by uncultured Sulfurospirillum, a taxon previously considered free-living rather than host-associated. Metagenome-assembled genomes (MAGs) classified as uncultured Sulfurospirillum predicted thiosulfate disproportionation and the reduction of tetrathionate, sulfate, sulfide/polysulfide, and nitrate. Microbial amino acid and vitamin B12 biosynthesis genes were identified in multiple MAGs, suggesting nutritional value to the host. Reads assigned to aerobic or anaerobic methanotrophic taxa were rare. IMPORTANCE Methane hydrates represent vast reserves of natural gas with roles in global carbon cycling and climate change. This study provided the first analysis of metagenomes associated with Sirsoe methanicola, the only polychaete species known to colonize methane hydrates. Previously unrecognized participation of Sulfurospirillum in a gut microbiome is provided, and the role of sulfur compound redox reactions within this community is highlighted. The comparative biology of S. methanicola is of general interest given research into the adverse effects of sulfide production in human gut microbiomes. In addition, taxonomic assignments are provided for nearly 200 bacterial families, expanding our knowledge of microbiomes in the deep sea.

An efficient method for collecting the full-length adults, fragments, and eggs of Trichobilharzia spp. from the liver of definitive hosts.

Precise identification of avian schistosomes in the genus Trichobilharzia at the species level is difficult and requires both traditional morphological and molecular techniques. To obtain satisfactory results by traditional methods, the characteristics of the intact adults or large fragments of male and females are necessary. The present study aimed to introduce a more efficient method for collecting eggs and both fragments and intact worms for morphological identification of visceral Trichobilharzia spp. Thirty-eight domestic ducks (twenty-eight fresh and ten frozen) were studied. For fresh samples, warm saline (40-45°C) was injected into the portal vein or liver tissue, followed by slicing of the liver to small pieces in a large Petri dish. All materials were then transferred into the laboratory sieves arranged from the largest to the smallest mesh size and while crushed with the hand, washed, and filtered using a trigger water sprayer. The collected materials were studied under a stereomicroscope for parasite eggs, fragments, and full-length worms. Out of 28 freshly killed ducks, 19 (67.9%) and of 10 frozen ducks 6 (60%) were positive for visceral Trichobilharzia spp. The full-length worms and large fragments of male worms were mostly recovered with the mesh no. 150 (diameter of 106μm) and small fragments, especially of females, and eggs with the mesh no. 270 (diameter of 53μm). In addition to large numbers of fragments, 15 full-length adults were obtained from fresh and 2 from frozen ducks. The number of collected full-length adults was related to the worm burden. Since morphological description of different species of the genus Trichobilharzia is primarily based on the availability of adult worms, the application of methods that provide a higher number of intact males and females will result in better characterization of the species and deposition of appropriate voucher specimens. These results show the present method as a suitable tool for the collection of quality adults of visceral Trichobilharzia spp. in ducks.

Feline heartworm (Dirofilaria immitis) infection: first case report of serological diagnosis in Brazil, confirmed by molecular assay

The clinical importance of heartworm infection in cats has indeed increased in recent years. Dirofilaria immitis infection has been reported worldwide in cats and continues to be regularly diagnosed in endemic areas. The diagnosis can be overlooked easily, especially in Brazil, where there is not a specific feline immunodiagnostic test, forcing the veterinarians to use a test made for the canine host. In 2015, a 10-year-old female neutered cat was diagnosed with D. immitis using an antigen serological test, based on imunocromatography and designed for dogs. The modified Knott test was negative. As the disease progressed, the cat showed clinical signals of respiratory distress, such as dyspnoea and polypnea in addition to prostration and emaciation, and died a few weeks after the diagnosis. During necropsy, one adult nematode was found in the pulmonary artery. D. immitis infection was confirmed by molecular amplification, performed in the worm fragment. This is the first report of serological diagnosis of feline dirofilariasis in Brazil. A chemoprophylaxis routine in cats should be done, as is done in dogs from endemic areas.

Https://ijmrr.medresearch.in/index.php/ijmrr/article/view/973

Background: Bancroftian Filariasisisatropical and sub tropical disease caused by Wuchereriabancrofti and transmitted by the Culexmosquitoes. The diagnosis of it is conventionally made by demonstrating microfilaria eintheperipheral blood smear. Microfilariae and adult filarial worm have been incidentally detected in fine needle aspirate of swelling at various sites in amicrofilaremic patients. Currently 1.4 billion people worldwide are at risk of being infected. In India 600 million people are at risk of filariasis.Cytology has important role in the diagnosis of occult Filariasis. Objectives: To discuss the role of FNAC in diagnosing occult Filariasis. Material and Methods: This is a retrospective study carried out in the Department of Pathology at SRMS IMS, Bareilly, Uttar Pradesh. Aspiration was made by 22-23 gauze needle, smears were stained by Papanicolaou and May-GrünwaldGiemsa stain. Result:16 cases of cytological specimensincidentallyshowed microfilaria without any clinical suspicion which included breast lumps (three cases), thyroid swelling (three cases), lymph nodes (two cases), subcutaneous swelling (two cases), ascitic fluid (one case), pleural fluid (one case), lung mass (one case), scrotal swelling (one case), cervical smear (one case) and space occupying lesion in liver (one case). Fragments of adult worm were reported in four cases. Only one patient with scrotal swelling had microfilaremia. Conclusion:FNAC can play an important role in diagnosing occult Filariasis in clinically unsuspected cases.

Heartworm-associated respiratory disease (HARD) induced by immature adult Dirofilaria immitis in cats

BackgroundA controlled, blind research study was conducted to define the initial inflammatory response and lung damage associated with the death of immature adult Dirofilaria immitis in cats as compared with cats developing adult heartworm infections and cats on preventive medication.MethodsThree groups of cats were utilized, 10 per group. All cats were infected with 100 third-stage (L3) larvae by subcutaneous injection. Group A cats were treated topically with selamectin (Revolution®; Zoetis) per label directions at 28 days post infection (PI) and once monthly for 8 months. Group B cats were treated orally with ivermectin (Ivomec®; Merial) at 150 μg/kg at 70 days PI, then every 2 weeks for 5 months. Group C cats were untreated PI. At baseline (Day 0) and on Days 70, 110, 168, and 240 PI, peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected on all cats. Upon completion of the study (Day 245), cats were euthanized and necropsies were conducted.ResultsResults were analyzed statistically between groups by ANOVA and by paired sample T testing for changes within the group over time. The selamectin-treated cats (Group A) did not develop radiographically evident changes throughout the study and were free of adult heartworms or worm fragments at necropsy. The heartworm life cycle was abbreviated with oral doses of ivermectin (Group B), shown by the absence of adult heartworms or worm fragments at necropsy. The early stage of immature adult worm in Group B cats, however, did induce severe pulmonary airway, interstitial, and arterial lung lesions, revealing that the abbreviated infection is a significant cause of respiratory pathology in cats. Cats in Groups B and C could not be differentiated based on radiographic changes, serologic antibody titers, complete blood count, or bronchoalveolar lavage cytology at any time point throughout the study. Eighty percent of cats in Group A and 100% of cats in Groups B and C became heartworm antibody positive at some time point post infection.ConclusionsThe clinical implications of this study are that cats that become infected with immature adult heartworms may not develop fully mature heartworms and are only transiently heartworm antibody positive, but do develop Heartworm-Associated Respiratory Disease (HARD).

An unusual cause of obstructive jaundice in a 2-year-old child

Background and Objectives:Pancreaticobiliary ascariasis is common problem in tropical countries. The roundworm in the bile duct can cause biliary colic, obstructive jaundice, or pancreatitis. Live Ascaris worms are usually diagnosed on ultrasonography (USG) or endoscopic ultrasonographic (EUS) showing characteristic features of linear mobile echogenic structure with central anechoic lumen. However, the worm can die inside the common bile duct (CBD) and create a foreign body acting as a nidus for stone or sludge formation. Obstructive jaundice due to dead Ascaris is a rare but important cause in the developing World. As the worm shrivels up after death, accurate identification requires a high index of suspicion. The features of dead worm on EUS include hyperechoic structure without any acoustic shadow.Methods:We present a case of a 2-year-old Indian female child referred with biliary colic and jaundice for the last 3 weeks. Abdominal USG revealed multiple ill-defined, oval, hyperechoic shadows near the lower end of dilated CBD. MRCP showed multiple intraluminal curvilinear, hypointense areas in lower CBD.Results:Linear EUS from the duodenal bulb revealed dilated CBD with multiple hyperechoic structures without acoustic shadowing. It showed 2–6 mm sized curvilinear, disc-shaped, short-segment echogenic structures with central anechoic core parallel and equidistant from each other, suggestive of recently broken down parallel fragments of round worms. The central anechoic core represents the digestive tract of Ascaris. After multiple balloon sweeps on ERCP, creamy white structures and yellow-colored material were removed suggestive of recently fragmented Ascaris.Conclusions:Biliary ascariasis should be considered in any child presenting with obstructive jaundice in endemic regions.

Phylogenetic Placement of a Schistosome from an Unusual Marine Snail Host, the False Limpet (Siphonaria lessoni) and Gulls (Larus dominicanus) from Argentina with a Brief Review of Marine Schistosomes from Snails

In the blood fluke family Schistosomatidae, marine snails are well known as intermediate hosts. Eight families of marine snails have thus far been reported to host schistosomes across the world, most of which have been implicated in human cercarial dermatitis (HCD) outbreaks. As part of our larger effort to define the species diversity and biology of schistosomes in Argentina, in particular their role in causing HCD, we searched in the marine pulmonate snail (Siphonaria lessoni) for a schistosome species described previously from S. lessoni from southern Argentina. Additionally, gulls (Larus dominicanus) collected from a different project locality (inland) were examined, because they are known to spend time in the intertidal regions. Schistosome sporocysts were found in S. lessoni, and a small worm fragment was retrieved from a gull. Molecular phylogenies for 28S, ITS1-5.8S-ITS2, and cox1 genes revealed that the specimens from the gull and S. lessoni grouped closely together, suggesting they are conspecifics. Also, ITS1-5.8S-ITS2 sequences suggested one of the schistosomes from S. lessoni and a schistosome from a South African penguin were also conspecifics. Further study is needed to verify if these specimens comprise a distinct marine clade within the larger avian schistosome clade that is comprised mostly of species using freshwater snail hosts. Thus far, it appears this group of marine schistosomes may be more likely found in the southern hemisphere. It is unclear if the observed distribution pattern of schistosomes in Siphonaria is a result of sampling bias and/or indicative of a specific bird-snail-schistosome association. It is clear they are sharply differentiated from the basal marine clade of avian schistosomes that includes Austrobilharzia.

ANALISIS DEL CONTENIDO ESTOMACAL DEL CAMARON TITI Xiphopenaeus kroyeri (HELLER) (CRUSTACEA: NATANTIA: PENAEIDAE)

Juveniles and adults of the seabob Xiphopenaeus kroyeri (Heller) were collected at monthly invervals between September 1988 and June 1988. The food items were determined in order of descending importance as: detritus and fragments of bivalve shells, polychaete worms, foraminiferans, and crustaceans. There was a difference in the feeding habits between the rainy seasson (September to December) and the dry season (January to June) with items like rotifers, sponges and radiolarians appearing only during the dry season. Analysis of the frequency of occurrence of different food items showed that groups like rotifers and bryozoans appear more frequently in juveniles. Measurements of protein and carbohydrate levels in the stomach contents revealed the highest peaks of proteins for adults in September (92.03 ug/mg dry weight of stomach contents) and for juveniles in December (81.27 ug/mg), and of carbohydrates for adults in October (45.81 ug/mg) and for juveniles in December (35.25 ug/mg). Quantitative differences in proteins and carbohydrates between the two size groups were not statistically significant.

Biliary ascariasis in the etiology of recurrent pyogenic cholangitis in an endemic area

Aims: The role of biliary ascariasis in etiology of recurrent pyogenic cholangitis in endemic areas has not been well studied. Methods: We studied 30 patients of recurrent pyogenic cholangitis with brown pigment stones and 30 patients with cholesterol gallstones. Stones from 22 patients (11 brown pigment stones and 11 gallstones) were examined by infrared spectrophotometry to substantiate the classification of stones done on visual inspection. Biliary calculi were considered to be associated with Ascaris lumbricoides in those with: previous documented evidence of biliary ascariasis, bile specimens containing ova of Ascaris lumbricoides on light microscopy and worms, worm fragments or their ova forming the nidus of stones on histological examination. Results: Bile cultures grew microorganisms in 24 (80%) patients with recurrent pyogenic cholangitis while only one patient with gallstones grew Escherichia coli from bile. None of the patients in either group had past or persisting evidence of clonorchiasis. Five patients had previous documentation of biliary ascariasis. In three patients bile contained ova of Ascaris lumbricoides and stones in 22 patients contained worm, worm fragments or their ova on histological examination. Overall 24 (80%) patients with recurrent pyogenic cholangitis had past or persistent evidence of biliary ascariasis while only one patient with gallstones had worm fragments on histopathology of stones (p ＜ 0.001). Conclusion: We conclude that in endemic areas bacterial infection of the bile ducts in recurrent pyogenic cholangitis occurs with biliary invasion by Ascaris lumbricoides and dead worms, their fragments or ova form nidus of brown pigment stones formation in such patients.

Effect of pre-cardiac and adult stages of Dirofilaria immitis in pulmonary disease of cats: CBC, bronchial lavage cytology, serology, radiographs, CT images, bronchial reactivity, and histopathology

A controlled, blind study was conducted to define the initial inflammatory response and lung damage associated with the death of precardiac stages of Dirofilaria immitis in cats as compared to adult heartworm infections and normal cats. Three groups of six cats each were used: UU: uninfected untreated controls; PreS I: infected with 100 D. immitis L3 by subcutaneous injection and treated topically with selamectin 32 and 2 days pre-infection and once monthly for 8 months); IU: infected with 100 D. immitis L3 and left untreated. Peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected from all cats on Days 0, 70, 110, 168, and 240. CT images were acquired on Days 0, 110, and 240. Cats were euthanized, and necropsies were conducted on Day 240 to determine the presence of heartworms. Bronchial rings were collected for in vitro reactivity. Lung, heart, brain, kidney, and liver tissues were collected for histopathology. Results were compared for changes within each group. Pearson and Spearman correlations were performed for association between histologic, radiographic, serologic, hematologic and bronchoalveolar lavage (BAL) results. Infected cats treated with selamectin did not develop radiographically evident changes throughout the study, were heartworm antibody negative, and were free of adult heartworms and worm fragments at necropsy. Histologic lung scores and CT analysis were not significantly different between PreS I cats and UU controls. Subtle alveolar myofibrosis was noted in isolated areas of several PreS I cats and an eosinophilic BAL cytology was noted on Days 75 and 120. Bronchial ring reactivity was blunted in IU cats but was normal in PreS I and UU cats. The IU cats became antibody positive, and five cats developed adult heartworms. All cats with heartworms were antigen positive at one time point; but one cat was antibody positive, antigen negative, with viable adult females at necropsy. The CT revealed early involvement of all pulmonary arteries and a random pattern of parenchymal disease with severe lesions immediately adjacent to normal areas. Analysis of CT 3D reconstruction and Hounsfield units demonstrated lung disease consistent with restrictive pulmonary fibrosis with an interstitial infiltrate, absence of air trapping, and decrease in total lung volume in Group IU as compared to Groups UU and PreS I. The clinical implications of this study are that cats pretreated with selamectin 1 month before D. immitis L3 infection did not become serologically positive and did not develop pulmonary arterial hypertrophy and myofibrosis.

The detection of different Dirofilaria species using direct PCR technique

A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 μl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.

Glycyrrhetinic acid and its analogs: A new class of antifilarial agents

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5μM; IC50: 1.20μM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a–7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50μM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC50 values were found to be 8.8 and 2.2μM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100mg/kg, s.c. ×5days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.

Effectiveness of synthetic trioxolane OZ78 against Schistosoma japonicum in mice and rabbits

Antischistosomal activities of a synthetic peroxide OZ78 (an ozonide carboxylic acid) against Schistosoma japonicum have been studied in mice and rabbits. Among 132 mice used, 30 of them were infected with 80-100 S. japonicum cercariae for collection of juvenile and adult schistosomes applied in in vitro tests. The remaining 102 mice were infected with 40 schistosome cercariae used for experimental treatment. Other 13 rabbits infected each with 200 schistosome cercariae were treated orally with OZ78 42 days post-infection. Most treated mice and rabbits were sacrificed 4 weeks post-treatment to collect residual schistosomes for evaluation of the drug efficacy. OZ78 and its sodium salt (OZ78-Na salt) 10-60 μg/mL alone exhibited no in vitro effect against day 14, day 21 schistosomula, and day 35 adult schistosomes. But OZ78 and OZ78-Na salt 10 and 20 μg/mL together with hemin 80 μg/mL showed decrease in worm motor activity and severe damage to the worm tegument and intestine, and all worms died within 3 days post-incubation. After infected mice were treated orally with OZ78 at a single dose of 400 mg/kg for 1 day, 34.9% of the worms shifted to the liver. Three and 7 days post-treatment, 100% of the worms were recovered from the liver. Fourteen days post-treatment, 92.3% of the worms still remained in the liver and 7.7% of the worms returned back to the mesenteric veins. Male and female worms shifted to the liver revealed in apparent shrinkage, degeneration of worm body, depigmentation in gut, and disappearance of ova in the uterus of some female worms. Meanwhile, dead worm and dead worm fragments were found in the liver tissues. In mice infected with various stages of schistosomes and treated orally with single OZ78 400 mg/kg, moderate or potential effect of the drug against day 0 (3-h-old worm), day 7, day 14, and day 21 juvenile worms and day 28, day 35 as well as day 42 adult worms were observed, the differences of total or female worm burdens between each treated group and control group were statistically significant (P < 0.01 or P < 0.05). Among the various stages of juveniles, day 7 worms were more susceptible to OZ78 with worm reduction of 83.8%, while the effect of OZ78 against day 28 to day 42 adult worms were similar. Finally, rabbits infected with adult schistosomes and treated with OZ78 at a single dose of 45 mg/kg or a daily dose of 35 mg/kg for three consecutive days resulted in significantly lower total and female worm burdens in comparison with that of control (P < 0.05) with total and female worm reductions of 84.1% and 84.7% as well as 74.3% and 77.4%, respectively. The results demonstrate that OZ78 possesses effect against both juvenile and adult S. japonicum in mouse model, and also shows effect against adult schistosomes in rabbits.

Quantitative retrotransposon anchored PCR confirms transduction efficiency of transgenes in adult Schistosoma mansoni

A quantitative retrotransposon anchored PCR (qRAP) that utilizes endogenous retrotransposons as a chromosomal anchor was developed to investigate integration of transgenes in Schistosoma mansoni. The qRAP technique, which builds on earlier techniques, (i) Alu-PCR which has been used to quantify lentiviral (HIV-1) proviral insertions in human chromosomes and (ii) a non-quantitative retrotransposon anchored PCR known to detect the presence of transgenes in the S. mansoni genome, was tested here in a model comparison of retrovirus-transduced adult schistosomes in which one group included intact worms, the other included fragments of adult worms. At the outset, after transducing intact and viable fragments of schistosomes with reporter RNAs, we observed more reporter activity in fragments of worms than in intact worms. We considered this simply reflects the increased surface area in fragments compared to intact worms exposed to the exogenous reporter genes. Subsequently, intact worms and worm fragments were transduced with pseudotyped virions. Transgene integration events in genomic DNA extracted from the virion-exposed worms and worm fragments were quantified by the qRAP, which revealed that fragmenting adult schistosomes resulted in increased density of proviral integrations. The qRAP findings confirmed the likely value of this qRAP technique for quantification of transgenes integrated in schistosome chromosomes. Last, considering the absence of schistosome cell or tissue lines, primary culture of fragmented worms offers an opportunity to optimize transgenesis, and other functional genomic approaches.

Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis

A controlled laboratory study was conducted to evaluate the efficacy of four commercial products administered as a single treatment for the prevention of heartworm disease caused by Dirofilaria immitis in dogs.Forty-four commercially sourced Beagle dogs, 6–7 months of age, were received at the test site (Auburn University, Department of Pathobiology) on Study Day (SD) −72 to begin acclimation. On SD −30, each dog was inoculated subcutaneously with 100 infective, third-stage D. immitis larvae (MP3 strain, TRS Laboratories, Inc., Athens, GA). On SD −1, 40 dogs weighing 18.2–25.3 lbs were ranked by decreasing body weight and randomized to five groups of eight dogs each. On SD 0, the dogs assigned to Group 1 were treated orally with ivermectin/pyrantel pamoate chewable tablets, Group 2 dogs were treated orally with milbemycin oxime flavored tablets, Group 3 dogs were treated with selamectin topical solution, and Group 4 dogs were treated with imidacloprid/moxidectin topical solution. Group 5 dogs remained nontreated. Dosages for dogs in Groups 1–4 were based on the individual body weight of each dog and current labeled dose banding for each commercial product. All dogs were fasted overnight prior to treatment. Food was returned four hours after treatment. Animals were observed for abnormal clinical signs involving eyes, feces, respiration, behavioral attitude, locomotion/musculature, or skin conditions at prescribed intervals immediately after treatment and at twice daily intervals thereafter. On SD 90, whole blood was collected and tested for adult heartworm antigen. On SDs 119/120, the dogs were euthanized and subjected to necropsy examination for recovery of adult D. immitis and/or worm fragments.At necropsy, all 8 dogs in the nontreated group were infected with adult D. immitis (34–70 worms/dog, geometric mean (GM)=51.6 worms/dog). One or more adult D. immitis and/or worm fragments were recovered from 7 of 8 of the dogs each in Groups 1–3 (87.5% were heartworm positive). The respective GM worm burdens/dog for Groups 1–3 was 2.3, 2.4, and 2.3 which resulted in 95.6, 95.4 and 95.5% efficacy, respectively. No worms were recovered from any of the 8 dogs in Group 4 resulting in 100% efficacy.

Role of fine needle aspiration cytology in diagnosis of filarial infestation

Filariasis is a tropical disease transmitted by the Culex mosquitoes. The diagnosis of it is conventionally made by demonstrating microfilariae in the peripheral blood smear. However; microfilariae and adult filarial worm have been incidentally detected in fine needle aspirates of various lesions in clinically unsuspected cases. The cases of filariasis diagnosed by fine needle aspiration cytology (FNAC) were retrieved from the archives of the Cytopathology laboratory between the periods of January 1998 to February 2009. Both Papanicolaou- and May-Grunwald-Giemsa-stained smears were available in all the cases. A total of 26 diagnosed cases of filariasis were found, of which 19 were related to funiculo-epididymitis, four presented with breast lump, and three cases with lymphadenopathy. Smears revealed fragments of adult worm in 12 cases including 10 gravid female worm containing eggs and microfilariae and two male adult worm; whereas in remaining 14 cases only microfilariae or eggs were seen. Unfertilized eggs were seen in three cases and fertilized eggs were seen in five cases. Tissue response in the form of eosinophils in 16 cases, acute inflammatory exudate in five cases, macrophages in 22 cases, epitheloid cell granulomas in five cases, giant cells in four cases, lymphocytes in 10 cases, and plasma cells in three cases were seen. Adherence of polymorphonuclear leukocytes, eosinophils, and epithelioid cells were observed in four cases. On conclusion, filaria may affect the epididymis, spermatic cord, breast, and lymph node, and the accurate diagnosis can be easily and conveniently achieved by FNAC without any requirement of biopsy.

Heartworm (Dirofilaria immitis) infection in a leopard (Panthera pardus pardus) housed in a zoological park in north-eastern Italy

Canine heartworm (cHW) disease is now recognised as potential cause of serious disease in cats and other felids, especially in endemic areas. In March 2009, a 23-years-old male African leopard (Panthera pardus pardus) housed in a zoological park located in the Province of Padova (Veneto Region), a cHW endemic area of the north-eastern Italy, died and was immediately necropsied. A cloth completely occluding the pyloric lumen was considered the presumptive cause of death. During necropsy, six nematodes (4 males and 2 females) were found within the right ventricle of the heart and the pulmonary artery. Diagnosis of HW (Dirofilaria immitis) infection was carried out by morphological features of adult worms and microfilariae, and then confirmed by detection of circulating HW antigens using a commercial SNAP kit (IDEXX Laboratories inc., USA). D. immitis infection was also confirmed by PCR amplification of the 5S ribosomal spacer region, performed on worm fragments and microfilaraemic blood samples obtained from the right ventricle of the heart. A glomerulonephritis of immuno-mediated origin and most likely associated with the HW infection is also reported. HW chemoprophylaxis and annual serological testing on wild felids housed outdoors in endemic cHW disease areas are recommended. This is the first diagnosis of D. immitis infection in an exotic felid in Italy.

Analysis of cell proliferation and migration during regeneration in Lumbriculus variegatus (Clitellata: Lumbriculidae)

Abstract Regeneration of anterior and posterior structures following fragmentation is a well-known characteristic of Lumbriculus variegatus, a freshwater oligochaete. Elucidation of cellular activities occurring during regeneration in oligochaetes has relied on classic histological methods. In this project, molecular and chemical approaches were used to analyze the role of cell proliferation and cell migration in L. variegatus regeneration. Mitosis was blocked through incubation of worm fragments in 2.5 mM colchicine or 25 μg/ml vinblastine sulfate. Worm fragments incubated in these drugs no longer regenerated heads or tails. Because colchicine and vinblastine may affect cellular processes other than mitosis, more direct evidence of cell proliferation was obtained by soaking regenerating worms in 5 mM bromodeoxyuridine (BrdU). At various times following BrdU treatment, worms were fixed and macerates of regenerating tissues were prepared. The dispersed cells were stained with antibodies against BrdU follow...