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Species of the genus Dipetalonema are parasitic nematodes of the family Onchocercidae (Nematoda; Filarioidea) which infect the peritoneal cavity of Neotropical primates. Of these, six species have been taxonomically described, two of these have been reported infecting the black-faced spider monkey (Ateles chamek): Dipetalonema gracile and Dipetalonema yatesi. Description of Dipetalonema species have been based on morphological characteristics, and their phylogenetic relationships remain unresolved. A few molecular studies have been carried out in Dipetalonema spp. infecting Neotropical primates. Seven filarioid nematodes (6 females and one male) recovered from one A. chamek in the Peruvian Amazon rainforest were morphologically identified as D. yatesi and molecularly characterized. A multi-locus genetic analysis of nuclear ribosomal region (18S) and mitochondrial (cox1, 12S, and nad5) gene sequences supported D. yatesi as a distinct lineage and yielded a highly resolved phylogenetic lineage tree for this filarioid genus of Neotropical primates. Our results highlighted that Dipetalonema species are divided in two well-supported clades, one containing D. yatesi and D. caudispina, and the second containing D. robini, D. gracile, and D. graciliformis. Due to sequence ambiguities from GenBank entries, relationships among isolates of D. gracile and D. graciliformis cannot be fully resolved, which requires further investigation. However, this suggests that these could represent a species complex. Our study confirms that D. yatesi is a valid species and constitutes the first molecular phylogenetic analysis of this parasite in black-faced spider monkeys., Graphical abstract Image 1, Highlights • Dipetalonema yatesi from the black-faced spider monkey (Ateles chamek) in Peru. • Characterization of D. yatesi nuclear ribosomal and mitochondrial gene sequences. • Phylogenetic inference of relationships among Dipetalonema species. • Dipetalonema yatesi and D. caudispina form a well-supported clade. • Relationships among D. gracile and D. graciliformis isolates are not fully resolved.

Molecular characterization studies on Setaria equina are limited. The present study aimed to characterize S. equina at the cytochrome c oxidase gene and to examine its phylogenetic relationships with other filarid species. Sequence analysis showed 100% nucleotide homology with an S. equina sequence from Italy (AJ544873). However, both sequences exhibited 7 nucleotide substitutions from a S. equina donkey isolate from Egypt (MK541847). Overall, S. equina formed a monophyletic sister group to Setaria tundra. All Setaria spp. examined formed a separate group on the phylogenetic tree that was related to corresponding Onchocerca spp. and Dirofilaria spp. clades. Human filarid worms-Brugia spp. and Wuchereria spp. grouped in a separate clade alongside Theilezia spp. Dipetalonema spp.-formed a separate group at the top of the tree.

Six Dipetalonema species have been reported from Neotropical monkeys, Dipetalonema gracile, Dipetalonema graciliformis and Dipetalonema caudispina being the dominant species found in French Guiana primates. Adult filarioids isolated from the abdominal cavity of tamarins (Saguinus midas) in French Guiana were morphologically and molecularly identified as D. graciliformis. Phylogenetic analysis based on DNA and amino acid sequences of the cox1 gene as well as the concatenated sequences of the cox1 and the 18S genes indicated that D. graciliformis belongs to the clade 4 (ONC4) of Onchocercidae. Blast analysis of the 18S rDNA revealed that D. graciliformis in the studied tamarins is conspecific with the filarioid circulating in howler monkeys (Alouatta macconnelli) in French Guiana, previously referred to as unidentified Onchocercidae species.

Although little studied, infections with nematodes of the Onchocercidae Leiper, 1911, predominated by the genera Dipetalonema Diesing, 1861 and Mansonella Faust, 1929, are frequent in wild primates and human populations in the Neotropical forest areas. This study reports natural infections with Dipetalonema freitasi Bain, Diagne & Muller, 1987 and D. gracile (Rudolphi, 1809) in two free-living species of pitheciid primates, extending the known geographical distribution of these species to the forest of the Peruvian Amazon. Adult worms were recovered from the thoracic and abdominal cavities of two species of monkeys, Pithecia monachus monachus (E. Geoffroy Saint-Hilaire) and Cacajao calvus ucayalii (Thomas) (Primates: Pitheciidae), collected along the Yavari-Mirin River basin and analysed via light and scanning electron microscopy. Both host species represent new host records for D. freitasi and D. gracile. Morphometric data are also presented for the sampled filarial worms in addition to morphological details obtained through light and electron microscopy examination of D. freitasi specimens.

Cameline filarosis is an important parasitic disease having an economic impact on the camel industry around the world. However, there has been no study on filarosis in Bactrian camels of Mongolia. Therefore, the aim of the present study was to detect and identify microfilariae of Deraiophoronema evansi (D. evansi) in Bactrian camels from three provinces, located in southern and southwestern Mongolia. Blood samples were obtained from 400 healthy two-humped camels of different ages and both sexes. All blood samples were analysed using a variety of diagnostic techniques. Microfilariae were detected in 30 Bactrian camels (7.5%) by the Knott technique, while 13 Bactrian camels (3.3%) tested positive in a direct smear test. D. evansi was detected in 18 Bactrian camels (4.5%) by PCR assay. Prevalence was shown to be high among Bactrian camels in the age group up to 5 years, while the lowest positive results were obtained for Bactrian camels in the 5–10-year age group and the over 10-year age group. To confirm the morphological identification, D. evansi–COI gene sequences were subjected to phylogenetic analyses. The D. evansi–COI gene sequences from Mongolian two-humped camels were identical to sequences from Iranian one-humped camels and were clustered together with these sequences in the phylogeny. This is the first report of molecular detection and identification of microfilariae of D. evansi in Bactrian camels of Mongolia.

Background Dirofilaria immitis is responsible for heartworm disease in dogs in endemic areas worldwide. Screening for this infection is done by blood tests. Antigen testing is the most sensitive method to detect an infection with adult (female) worms. Microscopic examination of a blood smear or Knott’s test can be used to detect circulating microfilariae, the infective larvae. To increase the sensitivity of the antigen test by decreasing the false negative test results, heating of the blood sample has been recommended in recent guidelines. Heating is believed to remove blocking immune-complexes. Circulating microfilariae are not specific findings for heartworm infection, as other nematodes (among others, Acanthocheilonema dracunculoides) can also result in microfilaremia. Although the type of microfilariae cannot be determined by microscopy alone, real-time PCR can reliably identify the infecting nematode species. Correct identification of the parasite is of major importance, as an infection with D. immitis requires antiparasitic therapy, whereas A. dracunculoides is thought to be a clinically irrelevant coincidental finding. The present case report describes a microfilaremic dog where the initial antigen test for D. immitis turned positive after heat treatment, whereas real-time PCR revealed that the microfilariae were A. dracunculoides (syn. Dipetalonema dracunculoides). Results A circa 5-year old, asymptomatic Spanish mastiff dog was referred for heartworm therapy because microfilariae were found via a screening blood test. The dog was recently imported to the Netherlands from Spain, where it had been a stray dog. Antigen tests on a plasma sample for D. immitis were performed with three different test kits, which all turned out to be negative. However, heat treatment of two of these samples were carried out and both of them led to a positive antigen test result. Real-time PCR showed that the circulating microfilariae belonged to A. dracunculoides species. Three administrations of moxidectin spot-on at monthly intervals resulted in a negative antigen and a negative Knott’s tests one month after the last treatment. Conclusions We conclude that heat treatment of initially negative blood samples for D. immitis could lead to false positive antigen test results if the dog is infected with A. dracunculoides.

At least two species of filarial worms, Dirofilaria immitis and Acanthocheilonema (Dipetalonema) odendhali, infect otariid pinnipeds, including the California sea lion (Zalophus californianus). To date, evidence of infection in sea lions has come from dead or captive animals, and little is known about filariasis in free-living populations. We sampled 45 California sea lion adults and 197 pups captured at 12 rookeries from different ecological regions within the Gulf of California and detected and quantified D. immitis and A. odendhali microfilariae in blood smears. We investigated differences in prevalence and parasite load (intensity of infection) among ecological regions. Microfilariae were detected in the blood of 35 of the 45 (77.78%) adult females and in 1 of the 197 (0.51%) pups examined. The average burden of A. odendhali per microlitre of blood was nearly twice that of D. immitis. Prevalence and intensity of infection differed significantly among regions, being highest for colonies within the northern and northcentral regions and lowest in the southern region. Dirofilaria immitis and A. odendhali infections displayed a similar spatial pattern of prevalence. Colony density inversely predicted the prevalence of microfilariae. Based on the clinical parameters typically associated with filarial infections in carnivores and physical examinations, none of the sea lions appeared to have evidence of disease. This is a first approximation to investigate the prevalence of microfilaria infections in free-ranging California sea lions and to explore their relevance to population health.

Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.

Dipetalonema gracile (Rudolphi, 1809) (Filarioidea: Onchocercidae) is one of six species of cavities filarial parasites of Neotropical non-human primates. The present study recorded the occurrence of D. gracile, provides morphological and morphometric data and extends the geographical distribution. Adult filariae were obtained from the thoracic and abdominal cavities of 38 specimens of woolly monkey, which were used for local human consumption, in the northeastern Peruvian Amazon. Male and female filarids were processed and analysed using light and scanning electron microscopy. Details of the cephalic papillae, post-cloacal bands and papillae, vulva, phasmid position and lateral appendages are showed by scanning electron microscopy and is recorded the occurrencce of Lagothrix poeppigii monkey as a new host of this filaria in the Yavari-Mirin river basin, Peruvian Amazon. Resumo Dipetalonema gracile (Rudolphi, 1809) (Filarioidea: Onchocercidae), é uma das seis espécies de filarias parasitas de primatas não humanos neotropicais. O presente trabalho registra a ocorrência de D. gracile , proporciona dados morfológicos e morfométricos e amplia sua distribuição geográfica. Filárias adultas foram obtidas da cavidade torácica e abdominal de 38 espécimes de macaco barrigudo, que foram utilizados para consumo humano local. Essa espécie de primatas não humanos neotropicais tem distribuição ao nordeste da Amazônia peruana. Filarídeos machos e fêmeas foram processados e analisados pela microscopia de luz e microscopia eletrônica de varredura. Detalhes das papilas cefálicas, bandas e papilas pós-cloacais, vulva, posição dos fasmídeos e apêndices laterais são mostradas pela microscopia eletrônica de varredura e registra-se a ocorrência do macaco Lagothrix poeppigii como novo hospedeiro desta filaria na bacia do rio Yavari-Mirin na Amazônia peruana.

Dipetalonema caudispina(Molin, 1858) andD. gracile(Rudolphi, 1809) (Filarioidea: Onchocercidae) are two of six known species of filarial nematodes that parasitize Neotropical non-human primates. Adult filariae were collected from the thoracic and abdominal cavities of 38 of 44 specimens ofSapajus macrocephalus(Spix, 1823) and nine of ten specimens ofCebus albifrons(Humboldt, 1812) (Primates: Cebidae), distributed in the Yavarí-Mirín river basin and used locally for human consumption. Co-occurrence ofD. caudispinaandD. gracileis reported for the first time, with a prevalence of 18.5% (10 of 54 hosts examined). Our finding ofD. caudispinaandD. gracilein cebids from the Peruvian Amazon constitutes a new geographical record for both filariae, two new host records forD. caudispina,and the first report ofD. gracileinS. macrocephalus. In addition, we provide morphometric data forD. caudispina, complementing the original description, as well as scanning electron microscopy details on the structure of the area rugosa and number of caudal papillae in males.

Following chronic retinol (vitamin A) deprivation leading to exhaustion of liver vitamin A reserves below 50 I.U. per liver hamsters were fed diets either deficient in (“Rd”: 250 I.U.A/kg in experiment I, 1000 I.U.A/kg in experiment II) or enriched with retinol (“Rw”: 10000 I.U.A/kg in experiment I and II). After 4 weeks some of the animals (36 in experiment I, 30 in II) were infected with 150 3rd-stage larvae of D. viteae, while clean animals were kept as controls. The retinol status, the immune response (indirect fluorescent antibody test: IFAT) and parasitological parameters were examined up to 8 (experiment I) and 12 weeks (experiment II) post infection (p.i.). Rd hamsters had levelling off of weight gain or weight loss, severely deficient retinol levels in serum and liver, and high mortality. Weight gain was less in infected than in uninfected hamsters, and the capacity of infected Rw animals to restore liver retinol was significantly lower than that of uninfected Rw animals. IFAT titres were similar in Rd and in Rw animals, but microfilaraemia was significantly enhanced at 8 and 10·5 weeks p.i. in Rd hamsters. While the number of worms recovered from Rd and Rw hamsters was similar, there was a significant increase in the ratio of female to male worms in Rd hamsters. Rd hamsters in experiment I produced 3·3 times the worm mass per 100 g body-weight than Rw hamsters. Also, the average mass per female worm was significantly higher in Rd than in Rw hamsters, and this parameter was negatively correlated with the liver retinol concentration in experiment I (r=−0·89). Retinol deficiency has a marked effect on growth and fertility of D. viteae in hamsters.

Parasite-host relationships are influenced by several factors intrinsic to hosts, such as social standing, group membership, sex, and age. However, in wild populations, temporal variation in parasite distributions and concomitant infections can alter these patterns. We used microscropy and molecular methods to screen for naturally occurring haemoparasitic infections in two Neotropical primate host populations, the saddleback (Leontocebus weddelli) and emperor (Saguinus imperator) tamarin, in the lowland tropical rainforests of southeastern Peru. Repeat sampling was conducted from known individuals over a three-year period to test for parasite-host and parasite-parasite associations. Three parasites were detected in L. weddelli including Trypanosoma minasense, Mansonella mariae, and Dipetalonema spp., while S. imperator only hosted the latter two. Temporal variation in prevalence was observed in T. minasense and Dipetalonema spp., confirming the necessity of a multi-year study to evaluate parasite-host relationships in this system. Although callitrichids display a distinct reproductive dominance hierarchy, characterized by single breeding females that typically mate polyandrously and can suppress the reproduction of subdominant females, logistic models did not identify sex or breeding status as determining factors in the presence of these parasites. However, age class had a positive effect on infection with M. mariae and T. minasense, and adults demonstrated higher parasite species richness than juveniles or sub-adults across both species. Body weight had a positive effect on the presence of Dipetalonema spp. The inclusion of co-infection variables in statistical models of parasite presence/absence data improved model fit for two of three parasites. This study verifies the importance and need for broad spectrum and long-term screening of parasite assemblages of natural host populations., Graphical abstract Image 1, Highlights • Co-infection data improves statistical models of parasite prevalence. • Longitudinal sampling is key to unraveling parasite-host relationships in nature. • Host specificity of blood parasites among sympatric Callitrichine primates. • Sex roles in cooperatively breeding primates may not impact parasitism.

ES-62 is an immunomodulatory phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae. Previously, the use of knockout mice has revealed the effects of ES-62 on macrophages and dendritic cells to be dependent on TLR4. However, it is possible that ES-62 may interact with additional proteins on the surfaces of target cells and hence that cells may vary with respect to receptor usage. In this study, we identified by molecular weight, proteins that interact with ES-62 and found differences amongst the immune system cells studied. Thus, whereas lymphocytes appear to have two major interacting proteins of ∼135 and ∼82 kDa, U937 monocytes only contain an ES-62-binding protein of the latter molecular weight. Binding to the proteins on B cells and U937 cells was blocked by PC, suggesting a critical role for this ES-62 moiety in facilitating interaction. Finally, ES-62 binding is followed by internalization in both macrophages and B cells but only in the former was absence of TLR4 found to block internalization. These findings are consistent with differences in receptor usage by ES-62 amongst different cell-types.

Despite the economic importance of camels, the parasites that affect them have not received adequate attention so far and molecular studies are scarce compared to other livestock. In this study, we characterized peripheral blood microfilariae in 200 healthy one-humped camels (Camelus dromedarius) from south-east Iran by microscopy and molecular tools to receive a more detailed insight into prevalence and species that affect them. Moreover, adult specimens of the filarial nematode Dipetalonema evansi were collected from the carcass of an infected animal. Microscopic examination was performed on Giemsa-stained blood smears, and blood was also spotted on Whatman FTA(®) cards for DNA analysis. Genomic DNA was extracted, and PCR was carried out for the detection of filaroid helminths, followed by sequence analysis of positive samples. Four samples were positive for microfilariae by microscopy, while 16 animals (8 %) were positive by PCR. Sequence analysis revealed D. evansi in all cases. Phylogenetic analysis of a cytochrome C oxidase subunit I (COI) sequence of filaroid nematodes showed that most species in a single genus cluster in the same clade; however, D. evansi and D. gracile are not monophyletic and branch rather at the base of the tree. Further studies on the life cycle of D. evansi, specifically the identification of intermediate host(s), have become feasible with the provision of the first specific COI sequences in this study.

Canine arthropod-borne infections are of major interest in small animal practice and have been widely investigated in Central and Western Europe. However, only limited epidemiological data are available from South-Eastern European countries, although diseases including babesiosis or dirofilariosis are widely recognised as important canine infections in these countries. A steadily increasing number of dogs imported from South-Eastern Europe into Germany require particular attention by small animal practitioners. In this study, a total of 216 dogs [29 local Romanian pet dogs presented at Salvavet Veterinary Clinic in Bucharest, Romania, and 187 imported stray dogs from Romania (n = 109) and Hungary (n = 78) into Germany] were screened by molecular biological, serological and haematological methods for canine arthropod-borne infections. Eleven different parasitic and bacterial vector-borne pathogens-Babesia canis canis, Babesia canis vogeli, Babesia gibsoni, Babesia felis-like, Hepatozoon canis, Leishmania spp., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum, Anaplasma phagocytophilum and Mycoplasma haemocanis-were detected. Fifty-six percent of the dogs were positive by direct methods. B. canis canis was the most prevalent pathogen in dogs imported to Germany (42.8%) and dogs submitted for clinical consultation in Bucharest (44.8%). Our data strongly suggest the introduction of an adjusted screening panel in dogs from South-East Europe in view of increasing importation of dogs into Germany.

Tropomyosins of invertebrates are pan-allergens responsible for wide spread allergic reactions against seafood and arthropods. As invertebrate tropomyosins are highly conserved, helminth tropomyosins are likely to show properties similar to these medically important allergens. Studies with a monoclonal antibody, NR1, raised against tropomyosin of the rodent filarial nematode Acanthocheilonema viteae revealed a B cell epitope common to helminths and marine mollusks, which does not occur in vertebrate tropomyosin. This antibody detected tropomyosin of A. viteae, other filariids, nematodes, trematodes and a cestode, and recognized as well tropomyosin of oyster, squid and octopus, but not of arthropods and vertebrates. Immunohistological analyses of A. viteae, Onchocerca volvulus and other nematodes using NR1 showed that tropomyosin is located in the fibrillar part of the body wall muscles and the uterus, and is also conspicuous in muscles of the pharynx, the vagina and other organs of the nematodes. The abundance of a pan-allergen like tropomyosin in parasitic worms and the counterintuitive, but well documented protection against allergic reactivity by some chronic helminth infections is discussed.

The heartworm Acanthocheilonema spirocauda (Leidy, Proc Acad Nat Sci Philadelphia 10:110-112, 1858) Anderson, 1992 is described from material collected from harbour seals in Scandinavia and compared with types and other specimens described by Anderson (Can J Zool 37:481-493, 1959) from harbour seals in eastern USA. Most morphometric characters of the material from USA fall within the ranges established for the Scandinavian one. Some intraspecific variability in the organisation of papillae on the male tail was detected among the Scandinavian specimens. Differences between the specimens from Scandinavia and Eastern USA are also found in the organisation of papillae on the tail of males and females. An excretory pore was not discernible, but a clearly hemizonid-like structure is described. For the first time, scanning electron micrographs present external morphological structures of the species.

Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae , a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.

In an attempt to study the occurrence of concomitant immunity in filarial infections, jirds (Meriones unguiculatus) were experimentally infected with Acanthocheilonema viteae, and patent animals were superinfected with a defined dose of A. viteae stage 3 larvae (L3). Infected animals harbored significantly less worms deriving from the superinfection than the control group (P < 0.05, 56.2%, and 63.4% protection), as shown by analysis of female worms 6 wk after superinfection on the basis of their developmental status and their length. This protection was not due to contact with L3 antigens because a significant reduction of worm burdens deriving of a superinfection was also observed after subcutaneous implantation of a single female worm (P < 0.05, 40.2% and 64.9% protection). The induced protective responses target L3 and restrict their migration because an established infection resulted in a reduction of L3 recovery (95.6% and 94.3%, P < 0.001) from tissues of jirds at day 5 after superinfection. Other data show that L3 from a superinfection are trapped within eosinophil-rich granulomas, which is likely to create unfavorable conditions for the worms and to lead to later death. Taken together, established A. viteae-infections partially protect hosts against homologous superinfection by an immune-mediated mechanism and, thus, regulate the population density of the parasites within the host by concomitant immunity.

Mastomys coucha and jirds infected with Acanthocheilonema viteae, a filarial species free of endosymbiontic bacteria of the genus Wolbachia, suffer lethal side effects after effective microfilaricidal therapy with diethylcarbamazine and levamisole, whereas, M. coucha infected with the Wolbachia-infested species Brugia malayi or Litomosoides carinii tolerate corresponding treatment. Mortality in A. viteae infected, treated animals varied with microfilariae density in the blood. It was up to 100% in highly microfilaraemic M. coucha and jirds, but low or absent in animals with low microfilariae counts. Deaths occurred in most cases 5-24 h after treatment. Characteristic symptoms in animals, which died subsequently were a rapid drop in body temperature by 4-7 degrees C, an increase in hematokrit values by up to 10% and a moderate blood acidosis. Lethal effects in A. viteae infections did not depend on a particular status of hypersensitivity of the animals since desensitization procedures, which protected infected M. coucha against an otherwise lethal intravenous challenge with A. viteae homogenate did not protect against adverse reactions to a subsequent microfilaricidal treatment. The animals were protected from treatment induced death by injection of N-LMMA. Thus the final morbific agent seems NO. The data show that adverse effects after effective microfilaricidal therapy may be caused by microfilariae derived components different from Wolbachia-released LPS.

The development of a compound with activity against filarial nematodes (a ‘macrofilaricide’) has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.

Octopamine acts as an important neurotransmitter and neuromodulator in arthropods, mollusks, and nematodes. In mammals, however, no definite function for this amine has yet been described. By virtue of this difference in the neurophysiological requirement of the mammalian host and nematodes, octopamine offers good opportunity for exploring this area deeply with a view to identify a unique target for filarial chemotherapy. Results of the present study indicated that Acanthocheilonema viteae, the rodent filarial parasite, utilized tyrosine as a precursor for producing octopamine and some other biogenic amines. Octopamine exhibited specific saturable binding with the membrane prepared from the anterior portion of the filariid. This amine induced concentration dependent increase in the membrane potential which possibly caused tonic paralysis of the filariid. The rate of micro filarial release by the female worms also declined in the presence of this amine. The study thus provided preliminary evidences for the presence of an octopamine neurotransmitter system and also about some of the roles it plays in A. viteae.

The present study determined the prevalence and geographical distribution of Dirofilaria immitis and other filariae, from dogs in littoral areas of Parana state, in Brazil. This survey spanned eight months, between 1998 and 1999, and was also designed to compare the efficacy of different tests for diagnosis of heartworm infection in that area. Blood samples were collected from 256 native-owned dogs distributed along the Parana coastal area. Five diagnostic procedures were used: direct smear examination, the Knott’s modified test, filtration assay, and two heartworm antigen detection kits. A follow-up imaging exam was performed to support the heartworm diagnosis. The imaging diagnosis included radiographic and ultrasonographic exams of six dogs that had positive results for the heartworm antigen detection kits, but showed different microfilarial burdens. The presence and severity of radiographic and ultrasonographic signs were compared with the results obtained in microfilariae detection and antigen tests. Diagnostic parasitology results indicated that 31.25% of the dogs were microfilaremic. Three different microfilariae were recovered: D. immitis, Dipetalonema reconditum, and the third (mf3) was not identified. D. reconditum was the species with the highest prevalence: 22.6%. In general, D. immitis prevalence was 5.47% (28.57% occult infections), but it varied along the coast and the range was from 0 to 20%. No correlation could be established between the overall scores for microfilarial counts (small or large numbers) and the severity of radiographic results or the likelihood of detecting filariae in the pulmonary artery using echocardiography. The finding of a different type of microfilaria (mf) suggested the existence of a third species in Parana state, whose prevalence was 4.68%. These results show that to obtain a reliable diagnosis of heartworm infection, antigen detection kits are indicated. Knott’s test or filtration should be performed to confirm microfilaremia and not for diagnosis of heartworm infection. Imaging tests support parasitology exams and add more about severity of infection. The northern areas, specially Guaraquecaba and Ilha das Pecas, presented the highest number of heartworm-infected dogs.

Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites,Acanthocheilonema viteaeandBrugia pahangi. ForB. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages ofA. viteaediffered greatly from each other with a pattern of increased mRNA production with maturation. As a ruleA. viteaehad higher levels of ES-62 mRNA thanB. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, asA. viteaeonly secreted ~3 times as much ES-62 asB. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Both Dirofilaria immiti and Dipetalonema reconditum may be found in blood of infected dogs but it is not easy to distinguish D. immitis from D. reconditum in morphology. We cloned and sequenced the contiguous internal transcribed spacer (ITS) region, ITS1-5.8S-ITS2, of these two different parasites and published on GenBank as AF217800 for D. immiti and AF217801 for D. reconditum in this study. We designed two pairs of specific primers derived from ITS2 being used for polymerase chain reaction (PCR). The amplicons of ITS2 from D. immiti and D. reconditum are 302 and 348bp, respectively. Moreover, the limitation for amplifying ITS2 gene using this PCR demonstrated that 1 x 10(-2) microfilaria of each species of parasite smashed or even with mixed samples could be detected and the PCR products were predicted as the same as that described above. Thus, D. immiti and D. reconditum could be differentially diagnosed by this specific PCR. Seventeen clinical cases were evaluated and all of them were correctly identified. In this study, ITS1-5.8S-ITS2 of D. immiti or D. reconditum were the first time sequenced and analyzed. No significant similarity of ITS1 and ITS2 between D. immiti and D. reconditum could be observed.

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis , Acanthocheilonema viteae and Brugia malayi . The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5×100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by ≥95%) determined 3 and 7 days after treatment were 3.125–6.25 and 6.25–12.5 mg/kg, respectively. Using the spot on formulation, doses of 6.25 mg/kg ( L. sigmodontis ), 12.5 mg/kg ( A. viteae ) and 25 mg/kg ( B. malayi ) were required to cause reductions of microfilaraemia levels by ≥95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100 mg/kg independent of the route of administration). Repeated oral treatment (100 mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi . However, single doses of 6.25 and 25 mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi , respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.

Bay 44-4400 was used as a spot on formulation and administered in single doses of 25 and 100 mg/kg to Acanthocheilonema viteae, Brugia malayi, and Litomosoides sigmodontis infected Mastomys coucha on various dates during prepatency, aiming to affect third stage larvae, fourth stage larvae or preadult worms. Microfilaraemia levels were controlled in comparison to untreated controls until necropsies were performed 100 days p.i. (A. viteae, L. sigmodontis) and 150 days p.i. (B. malayi) to determine the numbers of surviving worms and the condition of intrauterine developing stages. A significant proportion (86-100%) of larval and preadult stages of A. viteae were killed by Bay 44-4400 at a dose of 100 mg/kg. A dose of 25 mg/kg had only insignificant effects on the developing parasites, however, it strongly reduced microfilaraemia levels caused by surviving worms in the early phase of patency. Larval and preadult B. malayi and L. sigmodontis were not killed by Bay 44-4400 to a significant degree. Microfilaraemia developing by surviving parasites was generally and significantly reduced throughout the observation period when treatment was performed to affect the preadult parasites. In the other cases variable results were obtained. Intrauterine early embryonic stages were found to be pathologically altered in worms which had been treated at a preadult stage.

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.

Four new species of Molinema (Filarioidea: Onchocercidae), parasites of echimyid rodents in South America, are described: M. algardneri n. sp. from Proechimys amphichoricus, M. barbarae n. sp. from P. cayennensis, both in Venezuela (Rio Negro and Cerro La Neblina, respectively), M. peruviensis n. sp. from P. steerei in Peru (lower Urubamba), and M. nattereri n. sp. (= Filaria diacantha Molin, 1858 pro parte) from Echimys ? didelphoides (= Loncheres rufa) in Brazil (Matto Grosso). They differ from each other and from the previously described species in the following characters: flat or concave head, cephalic ratio (distance between cephalic papillae in median view/lateral view), size and shape of the buccal capsule, length of the oesophagus, cuticular ornamentation of the female body, length of the ovijector, thick or slim female tail, area rugosa, long or short filament in the left spicule, heel in the right spicule, respective position of postcloacal papilla pairs 5 and 6, shape of the caudal lappets, terminal point present or absent, and the microfilariae. Molinema, which belongs to the worldwide Dipetalonema line, is distributed in two of the ten families of the Caviomorpha (South American Hystrichognathi have extended their distribution in South America since the early Oligocene). Nine species are parasitic in the Echimyidae (suborder Caviida); they have a short oesophagus and a complete or reduced set of precloacal papillae (four or three pairs). Two species are parasitic in the Erethizontidae (Erethizontida) and might be more primitive in having a well-developed oesophagus. However, the type-species M. diacantha, of which a female specimen was studied, has a large buccal capsule and has evolved concave head, while M. arbuta has a reduced buccal capsule and primitive flat head. This last species is a parasite of a Nearctic porcupine and probably represents a small line of Molinema which migrated to the north with its hosts when communications were established between the two American continents 3 mya (Pleistocene). M. sprenti, the unique parasite of the Castoridae in North America, is considered to be derived from this group by capture.

Six synthetic 2H-1-benzopyran-2-one (cournarin) derivatives (CDRI compounds # 1, 2, 3, 4, 5 and 6) were evaluated for filaricidal activity against Litomosoides carinii and Acanthocheilonema viteae infections in cotton rats (Sigmodon hispidus) and Mastomys coucha respectively. Significant effects on macrofilariae (>80% death/sterilisation) were detected with compounds #2, 3 and 6 against L. carinii and/or A. viteae. Thus detection of filaricidal activity in benzopyrones, which are so far known for anti-inflammatory activity, provides a new lead for development of better filaricidal agents for combating filariasis.

Ov20 is a structurally novel 20-kDa retinol binding protein secreted by Onchocerca volvulus . Immunological and biological investigation of this protein has been hampered by the inability to maintain O. volvulus in a laboratory setting. In an effort to find a system more amenable to laboratory investigation, we have cloned, sequenced, and expressed cDNA encoding homologues of Ov20 from two closely related filarial species, Brugia malayi (Bm20) and Acanthocheilonema viteae (Av20). Sequence comparisons have highlighted differences in glycosylation of the homologues. We present here an analysis of mouse immune responses to Ov20, Bm20, and Av20. The results suggest a strong genetic restriction in response to native Bm20 that is overcome when recombinant, nonnative material is used. Reactivity of human filarial sera to the three recombinant proteins confirmed previous specificity studies with Ov20 but highlighted important differences in the reactivity patterns of the O. volvulus and B. malayi homologues that may be due to differences in glycosylation patterns. Ov20 is a dominant antigen in infected individuals, while Bm20 is not. The availability of the B. malayi homologue enabled us to use defined murine reagents and inbred strains for genetic analysis of responsiveness in a way that is not possible for Ov20. However, the close sequence similarity between Ov20 and Av20 suggests that the A. viteae model may be more suited to the investigation of the biological functions of Ov20.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES–62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae . Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway – phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

Evaluation of antifilarial activity of new potential agents in vivo is extremely time consuming and uneconomic. In the present study effort has been made to develop an in vitro screening method using Acanthocheilonema viteae, a subcutaneously dwelling rodent filariid with anaerobic metabolic characteristics like human filariids, W. Bancrofti/Brugia malayi as test parasite. Motility test and tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) based colorimetric assay were used as parameters in in vitro assay. Results showed that 92.3% of compounds (in vivo active) could be picked up in the in vitro assay when both adults and microfilarae (mf) were used simultaneously. Mf and adult stages separately detected, respectively, 84.6 and 69.2% of in vivo active compounds. The adults and mf separately and both the life stages together exhibited, respectively, 80.0, 50.0 and 80.0% false positive results in the in vitro test with in vivo inactive compounds. It is felt that mf stage when used in in vitro test using motility and MTT assays as parameters would be useful in primary screening of new potential filaricides.

Summary objective To determine mitogenic and antigen-specific cellular immune responses of two species of rodents, viz. Merionesunguiculatus and Mastomyscoucha to assess the usefulness of the A. viteae/Mastomys model for cellular immune studies in experimental filariasis. methods Lymphocyte blast transformation test (LTT) using spleen cells of normal and A. viteae infected animals. results The proliferative response of gerbils was much higher than that of Mastomys to both ConA and filarial antigens. Cells of both species of rodents did not respond to microfilarial (mf) antigen, however, their mitogenic response differed during infection. Some degree of nonspecific suppression was observed in gerbils during prepatent and patent stages of infection, while Mastomys revealed highest proliferation during patent microfilaraemia. Mastomys cells did not respond to adult or mf antigen,while adult-specific proliferation was detected in the case of gerbils. conclusion The A. viteae/gerbil model shows more similarity to human filarial infection regarding cellular immune response. Markedly low responsiveness of a high percentage of Mastomys and wide variations in the cellular response to nonspecific mitogen limit the usefulness of Mastomyscoucha in immunological studies, especially cellular immunity.

cDNA, synthesised from total RNA from Acanthocheilonema viteae , was amplified by PCR with a primer derived from the spliced leader 1 sequence of nematodes and oligo-dT. Due to the great number of side products observed in the reaction, a biotinylated oligo-dT primer was used for cDNA-synthesis and the first cycles of PCR. After binding of the PCR products to streptavidin/paramagnetic particles, the (+) strands of the cDNAs were recovered and reamplified. Analysis of the PCR products obtained revealed the presence of full-length cDNAs of at least 1.7 kbp in size in amplified total cDNA from microfilariae, postinfective L3, and adult worms. The total cDNA, from only 20 ex vivo recovered postinfective L3, was efficiently amplified.

A purified 41-kDa protein of the rodent filaria Acanthocheilonema viteae was shown to protect jirds against a challenge infection. Subcutaneous immunization with the protein reduced the number of adult worms by up to 65% and the number of circulating microfilariae declined by up to 93% in these animals. The protein is located in the muscle tissues of adult worms and was identified as tropomyosin by N-terminal sequencing of the purified protein.

The major excretory-secretory product of the rodent filarial nematode Acanthocheilonema viteae is a 62 kDa glycoprotein (ES-62), which has phosphorylcholine, attached to the N-linked carbohydrates. In this paper, we describe structural studies of N-glycans released from ES-62 by peptide N-glycosidase F. Three major classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine resides. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine. The implications of these results with respect to the probable glycosylation pathways operating in A. viteae are discussed.

Three species of rodents were immunized with 50 irradiated (35 krad) stage-3 larvae (L3) of the filaria Acanthocheilonema viteae and challenged with an infection of normal L3. The immunization induced a significant reduction of the worm burden developing from the challenge infection in all host species, the jird (Meriones unguiculatus), the multimammate rat (Mastomys coucha) and the golden hamster (Mesocricetus auratus). The induced resistance was highest in jirds (92.5 +/- 9.7) followed by golden hamsters (59.4 +/- 26.6) and multimammate rats (55.1 +/- 40.4). The time course of antibody response against antigens of L3, adult worms and microfilariae, as studied by ELISA, showed quantitative and qualitative differences between the species. The antibody response against L3 antigens in immunoblots was similar in all species. Only one of the golden hamsters developed an antibody response against the surface of vector derived L3, while sera of jirds and multimammate rats did not react with L3 surface.

Background Considering the increasing importance of small animals travel medicine and the spread of filariae with zoonotic potential to non-endemic European areas, routine filarial diagnosis in dogs is becoming important. Dirofilaria immitis, D. repens, Acanthocheilonema dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae (mf) which can be differentiated to species level by the acid phosphatase activity patterns or by PCR. Available data on the size of the mf vary considerably in the literature. The aim of this study was to validate morphometric criteria for filarial identification in blood samples of dogs after concentration of mf with the modified Knott’s technique. Methods Morphometric analysis of 10 mf from samples identified to species level by acid phosphatase activity and partially confirmed by PCR were performed with specimens from 377 dogs. Results The mean length and width of D. immitis mf from 60 dogs were 301.77±6.29 μm and 6.30±0.26 μm, of D. repens mf from 171 dogs 369.44±10.76 μm 8.87±0.58 μm, of A. dracunculoides mf from 133 dogs 259.43±6.69 μm and 5.09±0.47 μm and of A. reconditum mf from 13 dogs 264.83±5.47 μm and 4.63±0.52 μm. For a subset of 30 samples, morphometric analysis was repeated with identical results in two laboratories. Furthermore, the size of mf concentrated and fixed by the Knott’s technique was shown to be stable over 105 days. Conclusions The Knott’s test enables to clearly distinguish between D. immitis, D. repens and Acanthocheilonema spp. However, due to the overlapping size ranges of A. dracunculoides and A. reconditum, biochemical or molecular methods are required to distinguish these two species.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

A total of 65 compounds, most of which were from chemical classes having members known to be active against one or more parasitic organisms, were evaluated against Brugia pahangi and Acanthocheilonema viteae for macrofilaricidal activity in male Mongolian jirds (Meriones unguiculatus). Sixteen of the 65 compounds tested suppressed the number of parasites. Of these 16, three were suppressive for B. pahangi, 10 for A. viteae, and three for both parasites. The antibiotic nigericin and the antihistaminic isothipendyl were found to be most active.

This paper reports the development of the canid filarial worm, Dipetalonema dracunculoides, in the brown dog tick, Rhipicephalus sanguineus by determining whether development is similar within larval, nymphal and adult stages of infected ticks. This study demonstrates that only infected nymphal ticks can support the complete development of the filarial worm. Infected larval ticks are not suitable intermediate hosts, nor are infected adults. Development depends on some stage-specific property of the vector, and the maturation to the infective stage is stimulated during the course of the nymphal-adult moult.

SUMMARYAfter i.v. injection of 305 × 103 microfilariae (mf) per animal (50 g) into naive jirds, 50·8% of them could be recovered at autopsy 15 min later. Of these, 65·8% were calculated to be in the peripheral circulating blood (PCB) and were completely intact; 18·6% were recovered by perfusion of the lungs and 13·6% from the liver. In both organs about half the mf were associated with adherent lymphocytes and neutrophils but a few were partly disintegrated. Only 2·6% were recovered from the kidneys and the spleen. In long-term injection experiments using the same inoculum size the autopsy was done 15 min and 1, 3 and 6 weeks post-injection (p.i.) of mf into naive jirds. Throughout the experimental period the density of mf remained more or less constant in the PCB, but 3 weeks p.i. the density in the lungs increased up to 14 times to that in the PCB, whereas in the liver it decreased at the same time to a density similar to that in the PCB. In patent animals with adult worms delivering mf these were distributed as follows: 34·7% were calculated to be in the PCB; 24·4% were obtained by perfusion from the lungs and 22·0% from the liver; the rest were found in the kidneys (16·6%) and spleen (2·3%). In the lungs and the liver about 5/6 were associated with adherent cells, partly disintegrated or as fragments. In view of the fact that very few mf become disintegrated immediately after i.v. injection and also from their extremely long sojourn in the PCB, a low turnover rate of mf is presumed. The limited fecundity of the female adults combined with the 20 weeks median circulation time of injected mf in the PCB supports this view.

The effects of K+ on muscle contractility were explored in the filarial nematode Acanthocheilonema viteae (Dipetalonema viteae). The parasite was slit open longitudinally and mounted in a smooth muscle chamber that was filled with aerated (95% N2-5% CO2) physiological solution at 37 degrees C. KCl at concentrations ranging from 20 to 100 mM induced a rapid isotonic contraction of the filarial muscle. The maximal response from KCl was similar to the maximal response to acetylcholine chloride (ACh). When KCl was applied for several minutes, tolerance frequently occurred. Contractions were also induced by K2SO4 but not by NaCl, Na2SO4 or sucrose. Nifedipine was more than 10 times as potent in reducing the KCl-induced contraction as in reducing that caused by ACh. The KCl-induced contraction was abolished in a Ca-free physiological medium containing ethyleneglycol-bis-(beta-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA, 10(-4) M). Low [Ca2+]/[Mg2+] solutions blocked the spontaneous activity, the KCl-induced contractions, and the ACh-induced contractions. KCl also induced contractions in denervated muscle strips, supporting the hypothesis that K+ acts directly on the muscle cells. These results indicate that K+ can depolarize the muscle membrane and induce a muscle contraction that is dependent on extracellular calcium ions.

Background Hippobosca longipennis (the 'dog louse fly') is a blood sucking ectoparasite found on wild carnivores such as cheetahs and lions and domesticated and feral dogs in Africa, the Middle East and Asia, including China. Known as an intermediate host for Acanthocheilonema dracunculoides and a transport host for Cheyletiella yasguri, it has also been suggested that H. longipennis may be a vector for other pathogens, including Acanthocheilonema sp.? nov., which was recently reported to infect up to 48% of dogs in northern India where this species of fly is known to commonly infest dogs. To test this hypothesis, hippoboscid flies feeding on dogs in Ladakh in northern India were collected and subjected to microscopic dissection. Results A total of 12 infective larvae were found in 10 out of 65 flies dissected; 9 from the head, 2 from the thorax and 1 from the abdomen. The larvae averaged 2, 900 (± 60) μm in length and 34 (± 5) μm in width and possessed morphological features characteristic of the family Onchocercidae. Genetic analysis and comparison of the 18S, ITS-2, 12S and cox-1 genes confirmed the identity of the larvae as the Acanthocheilonema sp.? nov. reported in dogs in Ladakh. Conclusion This study provides evidence for a potential intermediate host-parasite relationship between H. longipennis and the canine Acanthocheilonema sp.? nov. in northern India.

A dog naturally infected with Dipetalonema dracunculoides and having a microfilaremia of 6050 microfilariae per mm3 of blood was used as source of infection. Experimentally cultivated nymphs of Rhipicephalus sanguineus were fed on the donor dog. Once engorged, ninety-three nymphs were removed and kept at 30°C and RH 90% until they moulted to the adult stage. To study the development of microfilariae in the vector, ten ticks were dissected at Day 37 post-infection. Adult infected ticks were fed on two uninfected dogs. The observed pre-patent periods were 69 and 76 days. Trans-stadial transmission of Dipetalonema dracunculoides by Rhipicephalus sanguineus was demonstrated.