Transgenesis in Strongyloides and related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing.

Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.

Imaging trematode and nematode parasites.

Recent advances in molecular genetics and in imaging mean that it is now increasingly feasible to image biological processes within helminth parasites and to visualize interactions between worms and their hosts. Moreover, other innovative imaging approaches that are not dependent on transgenic parasites have been applied to, and or developed for, the study of helminth parasites and have provided novel and important insights into the biology of these important pathogens.

Transgenesis and neuronal ablation in parasitic nematodes: revolutionary new tools to dissect host-parasite interactions.

Ease of experimental gene transfer into viral and prokaryotic pathogens has made transgenesis a powerful tool for investigating the interactions of these pathogens with the host immune system. Recent advances have made this approach feasible for more complex protozoan parasites. By contrast, the lack of a system for heritable transgenesis in parasitic nematodes has hampered progress toward understanding the development of nematode-specific cellular responses. Recently, however, significant strides towards such a system have been made in several parasitic nematodes, and the possible applications of these in immunological research should now be contemplated. In addition, methods for targeted cell ablation have been successfully adapted from Caenorhabditis elegans methodology and applied to studies of neurobiology and behaviour in Strongyloides stercoralis. Together, these new technical developments offer exciting new tools to interrogate multiple aspects of the host-parasite interaction following nematode infection.

Activity of an injectable, sustained-release formulation of moxidectin administered prophylactically to mixed-breed dogs to prevent infection with Dirofilaria immitis.

OBJECTIVE: To test the ability of a single injection of a sustained-release formulation of moxidectin (moxidectin SR) to protect dogs against heartworm infection for 180 days after inoculation with infective third-stage larvae (L3) of Dirofilaria immitis. ANIMALS: 32 adult mixed-breed dogs. PROCEDURE: Dogs were allocated to 4 groups on the basis of weight and sex. Dogs were injected SC with saline (0.9% NaCl) solution or moxidectin SR at the rate of 0.06, 0.17, or 0.5 mg/kg of body weight (day 0). Each dog was inoculated SC with 50 D immitis L3 180 days later. On days 330 and 331, dogs were euthanatized. The heart, lungs, and thoracic cavity were examined, and number and sex of heartworms were determined. RESULTS: A mean of 35.9 heartworms was recovered from untreated control dogs. Fourteen worms were recovered from 1 of 8 dogs given moxidectin SR at the lowest dosage, and none of the dogs in the 2 highest moxidectin treatment groups were infected. Small barely palpable granulomas were detected at injection sites of moxidectin-treated dogs. Frequency and size of granulomas were positively correlated with dose of moxidectin administered. CONCLUSIONS AND CLINICAL RELEVANCE: A single dose of moxidectin SR at a dosage as low as 0.17 mg/kg can safely and reliably confer complete protection against infection after challenge-exposure with D. immitis L3, and protection lasts for at least 180 days. This mode of prophylactic treatment against infection with heartworms effectively eliminates failure of prophylaxis that results from erratic administration of medications designed for monthly administration.

Seasonal prevalence of third-stage larvae of Dirofilaria immitis in mosquitoes from Florida and Louisiana.

Heads of 109,597 mosquitoes collected during 1996 and 1997 from Gainesville, Florida (1996, n = 39,131; 1997, = 34,209), Bartow, Florida (1996, n = 12,000; 1997, n = 12,000), and Baton Rouge, Louisiana (1996, n = 12,257) were tested by a polymerase chain reaction and Southern hybridization-based test for the presence of third-stage larvae of the canine heartworm Dirofilaria immitis. Mosquito heads were pooled (1-200 heads) by month, locality, and species for testing. The test used was species specific for D. immitis and was capable of detecting DNA from a single larva in a pool of 200 mosquito heads. Specificity for the third larval stage was achieved by probing only mosquito heads. One or more D. immitis-infected mosquito heads were detected in each month of the year from Barrow in both 1996 and 1997. No infected mosquito heads were detected from Gainesville or Baton Rouge in December, January, February, or March. These results are in general agreement with previous sentinel dog and model prediction studies that showed heartworm transmission in the warm temperate Gulf coast region of the United States to be seasonal rather than continuous as previously believed.

PCR amplification of putative gpa-2 and gpa-3 orthologs from the (A+T)-rich genome of Strongyloides stercoralis.

Two G protein alpha subunit genes orthologous to gpa-2 and gpa-3 in Caenorhabditis elegans have been identified in the parasitic nematode, Strongyloides stercoralis. These genes mediate chemosensory signal transduction regulating dauer arrest in C. elegans. In the parasite, they represent candidate mediators for regulation of the choice between free-living and parasitic life cycles, the obligatory developmental arrest of infective larvae, and reactivation of development after infection. The (A+T) content of these genes is 72.2% for coding sequences, 90% for introns, and 84.1% for 5' and 3' flanking regions, requiring the use of low extension temperatures for long distance PCR. The possible significance of conserved structural motifs of these proteins is discussed.

Seasonality of heartworm infection and implications for chemoprophylaxis.

The current emphasis on heartworm prevention reflects the dependable protection provided by the monthly administered macrolide endectocides. This article reviews the prerequisites for heartworm transmission and the importance of daily temperature as a limiting factor in determining the seasonality of the transmission period. The practice of some veterinarians to continuously prescribe monthly chemoprophylaxis exaggerates the actual risk of heartworm transmission in most parts of the country and unnecessarily increases the cost of protection to their clients. Guidelines are provided for making an objective, conservative estimate of the earliest and latest dates for administering monthly chemoprophylaxis; and the use of seasonal projections for other clinical applications such as timing and interpretation of heartworm testing are discussed.

Strongyloides stercoralis: maintenance of exceedingly chronic infections.

Two hypotheses were tested to identify the mechanism(s) by which chronic Strongyloides stercoralis infections are maintained in experimental dogs as a model to explain delayed onset recrudescence in humans. Investigations tested the hypotheses that chronic infections result from 1) periodic reactivation of third-stage larvae from a reservoir of dormant parasites outside the gastrointestinal tract or 2) the periodic rejuvenation of postreproductive female worms remaining from a previous infection, lodged in the mucosal crypts. Populations of parenteral larvae survived in mature experimentally infected female dogs for 66 days; individual worms survived for 88 days, but there was no evidence that these larvae re-established patent, adult worm infections. Late in these infections, female worms were present in greater than predicted numbers with no evidence that autoinfection had occurred, suggesting that some postreproductive worms were long-lived. In separate trials, long-lived spent females were once again capable of producing viable larvae when the host was treated with corticosteroids.

Studies of reproductive competence in male Dirofilaria immitis treated with milbemycin oxime.

Normal adult Dirofilaria immitis from a microfilaremic donor dog and D. immitis from donors rendered microfilaria (MF) negative by seven consecutive monthly doses of milbemycin oxime (500 micrograms/kg) were transplanted into three previously uninfected and untreated dogs. Two dogs received reciprocal combinations of treated and untreated D. immitis and the third received untreated adults of both sexes. A fourth dog served as an infected, milbemycin treated, non-transplanted control. Eleven weeks after pairing treated female with untreated male worms, a low-level microfilaremia developed in the recipient. Two of the three treated female worms recovered from this dog were non-fertile, and the third contained a small number of elongate and coiled embryos but no mature intrauterine (stretched) microfilariae (MFF). The dog receiving treated male and untreated female worms became microfilaremic after two weeks. Microfilaremia peaked at 37,000/ml 16 weeks after transplantation and declined over the next 20 weeks to 7,200/ml. Untreated females paired with treated males either became non-fertile or exhibited low numbers of developing embryos and MFF scattered throughout their reproductive tracts. Pairing of untreated male and female worms produced a mcirofilaremia during the second post-operative week, which plateaued around 15,000 MFF per ml. Females recovered after this pairing contained a normal pattern of embryonic development, including stretched MFF. There were no significant differences in the percentage composition or absolute numbers of developing and mature sperm in the reproductive tracts of treated and untreated male worms. However, the resumption of MF production in one milbemycin treated female worm after pairing with normal males and failure of treated males to sustain MF production in untreated female worms suggest that milbemycin oxime impairs the sexual competence of male D. immitis. This may explain the ability of this drug to bring about long term suppression of microfilaremia without immediate adulticidal activity.

Induction of protective immunity against larval Onchocerca volvulus in a mouse model.

BALB/cBYJ mice were immunized against larval Onchocerca volvulus by subcutaneous injection of normal, irradiated, or freeze-thaw-killed Onchocerca sp. larvae. The mice received challenge infections of O. volvulus third-stage larva (L3) contained in diffusion chambers implanted subcutaneously. At two-weeks postinfection, the diffusion chambers were removed and larval survival was assessed. When mice were immunized a single time with 35-krad-irradiated or normal O. volvulus L3, there was a significant reduction in the survival of challenge parasites. However, there was little or no reduction in challenge worm survival when mice were immunized a single time with freeze-thaw-killed O. volvulus L3 or fourth-stage larva (L4), or irradiated O. lienalis L3. When a second dose of freeze-thaw killed O. volvulus L3 or irradiated O. lienalis L3 was administered, there was a significant reduction in parasite survival in immunized mice. Immunization with O. volvulus L4 or a combination of L3 and L4 failed to confer protection. These results demonstrate that mice can be immunized against larval O. volvulus and that diffusion chambers are an efficient method for studying protective immunity to this parasite in a mouse model.

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal models for the study of immunity in human filariasis.

A major challenge to the development of vaccines against human lymphatic filariasis and onchocerciasis is to direct the immune response toward elimination of the early, prepathogenic larval stages and away from responses that mediate pathology. In this review, James Lok and David Abraham discuss the various animal models that have been used to investigate the pathways leading to immunity, immunological tolerance and chronic pathology in these diseases. Owing to the strict host specificities of the human-dwelling filariae, no single model serves to duplicate exactly all these aspects. Nevertheless, it has been possible to demonstrate a protective immune response invoked by and directed against incoming third-stage larvae of both lymphatic and skin-dwelling filariae. The fact that subsets of the sequelae of human filarial infection can be duplicated in animal systems should also aid in unravelling the mechanisms determining the course of infection and in ensuring that vaccine candidates do not produce an inappropriate immunopathological response. A proposed scheme for using animal models in screening candidates for a vaccine against Onchocerca volvulus is presented.

Experimental ocular onchocerciasis: local and systemic antibody and cell-mediated immune responses.

Hartley guinea pigs injected subconjunctivally with Onchocerca lienalis (OL) microfilariae (Mf) develop punctate corneal opacities resembling the punctate keratitis of human onchocerciasis. Antibody production and antigen-induced proliferative responses were studied in conjunctival-associated lymphoid tissues (CALT), spleens (SL) and peripheral blood lymphocytes (PBL) from experimentally infected guinea pigs. Cultured single cell suspensions of CALT, SL and PBL were assayed for IgG1, IgG2, IgA and IgE antibody production. IgG1, IgG2, and IgA Onchocerca-specific antibodies were found in culture supernatants of CALT, SL and PBL. When initiated 10 days after a challenge injection of OL, CALT cultures produced antibody levels equal to or less than those produced by the corresponding SL cultures. When initiated 66 days after the last injection of Mf, CALT cultures produced significantly more antibody than the corresponding SL cultures. Blastogenic responses to OL Mf antigen were observed in peripheral and splenic lymphocytes of OL-infected guinea pigs. Animals given subconjunctival injections of Mf followed by treatment with a microfilaricide had greater responses to OL antigen than those given Mf alone, while responses to phytomitogens were similar in drug-treated and non-treated animals. The CALT was locally immunologically responsive against the subconjunctivally injected OL Mf, with the capacity for localized memory responses. The local immunologic responses to conjunctival Onchocerca microfilariae may play a significant role in the immunopathological reactions of ocular onchocerciasis.

Synthetic and naturally occurring retinoids inhibit third- to fourth-stage larval development by Onchocerca lienalis in vitro.

A series of synthetic retinoids was screened for the ability to inhibit the third-to fourth-stage larval molt by Onchocerca lienalis in vitro. Of the 14 retinoids tested, eight gave significant inhibition of the molt at a concentration of 30.6 microM or less. Probit analysis of dose-response data collected for these active compounds indicated values for ED50 in the range of 3.7-17.1 microM. In general, the most active of these N-substituted retinamides were those with small alkyl or monohydroxy alkyl substituents. The most active of these was all-trans-N-(2-hydroxyethyl)retinamide with an ED50 of 3.7 microM. Both the all-trans and 13-cis isomers of the alkyl substituted derivatives were active, the all-trans-N-hydroxyethyl derivative being approximately 5 times as active as the corresponding 13-cis isomer. The N-2,3 dihydroxypropyl derivative, two derivatives with aromatic side chains and three N-(retinoyl)amino acids were inactive by the criteria set in the initial screening. There was no strict correlation between growth regulating activity against O. lienalis and binding affinity for a retinol binding protein from Onchocerca gibsoni.

Experimental ocular onchocerciasis in cynomolgus monkeys. II. Chorioretinitis elicited by intravitreal Onchocerca lienalis microfilariae.

Chorioretinitis due to onchocerciasis is a major cause of blindness, and the pathogenesis is poorly understood. We have developed an experimental model for onchocercal chorioretinitis using cynomolgus monkeys (Macaca fascicularis). Two normal monkeys and two monkeys which had received prior sensitization with subcutaneous injections of live Onchocerca lienalis microfilariae were given intravitreal injections of either 0, 10, 50 or 500 live microfilariae. Posterior segment changes included disc edema, venous engorgement, retinal vasculitis, intraretinal hemorrhage, and progressive retinal pigment epithelial (RPE) disturbances. Histopathological findings included perivascular infiltrates with eosinophils, eosinophilic choroiditis, and RPE hypertrophy, hyperplasia and loss of pigment. Microfilariae in the retina had no surrounding inflammation but were found adjacent to areas of RPE alterations. Overall the inflammatory reaction in the two unsensitized monkeys was more severe than that seen in the sensitized monkeys. The retinal appearance of the monkeys resembled that found in human onchocerciasis, and this model appears to be a promising one for future investigations.

Occurrence of some blood and intestinal parasites in dogs in Curaçao, Netherlands Antilles.

In August 1986, 133 dogs at the Veterinary Service of the Netherlands Antilles and the SPCA of Curaçao were examined for microfilaremia and for evidence of gastrointestinal parasitism. Microfilariae of Dipetalonema reconditum were present in 27.8% of the dogs examined with no significant difference in the infection rate between domestic and feral dogs. Microfilariae of the canine heartworm Dirofilaria immitis were found in 9% of the dogs with a significantly higher rate of infection in domestic (pet) dogs (13.5%) than in feral dogs (3.4%). Of the intestinal parasites observed Ancylostoma sp. was present in the highest percentage of dogs (68.4%) followed by Toxocara sp. (7.5%). Other parasites were present in less than 5% of the dogs examined and included, in decreasing order of prevalence, Spirocerca sp., Giardia sp., coccidia, Taenia sp. and Trichuris sp. The present paper presents the first evidence of Di. reconditum on Curaçao and suggests the introduction of D. immitis to the island within the 10 years preceeding this report. The persistently high rate of infection with Ancylostoma underscores the continuing risk of cutaneous larva migrans to human beings in the region.

Abnormal patterns of embryogenesis in Dirofilaria immitis treated with ivermectin.

The percentage composition and spatial distribution of embryogenic stages in the uteri of female Dirofilaria immitis were examined at various times after treatment with a microfilaricidal dose of ivermectin and compared to nontreated parasites. Worms sampled 42 days post-treatment (PT) exhibited an increased proportion of stretched microfilariae in the distal portion of the uterus. A decreased proportion of developed embryos was noted in the mid body region of worms sampled 42 days PT, and these forms were completely absent from the proximal area of the uterus. Relative numbers and spatial distribution of other stages remained virtually identical to controls. Radical changes in the composition and spatial distribution of embryogenic forms were noted in the uteri of a single worm sampled 80 days PT. Unlike nontreated parasites and worms sampled 42 days PT, stretched microfilariae constituted the predominant form in the distal uterus of this worm, and these stages were found in decreasing numbers throughout the proximal segments. Also, the intermediate embryogenic stages were either rare or absent.

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro.

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.

Autoantibody induced by experimental Onchocerca infection. Effect of different routes of administration of microfilariae and of treatment with diethylcarbamazine citrate and ivermectin.

Hartley guinea pigs were injected with microfilariae (Mf) of Onchocerca lienalis as a model for acute inflammatory responses to Mf in human Onchocerca volvulus infection. IgG autoantibody reactive with a 3 M KCl extract of guinea pig cornea was detected by ELISA in the serum of guinea pigs injected with O. lienalis Mf three or more times sub-conjunctivally, or two or more times subcutaneously. Administration of the microfilaricides diethylcarbamazine citrate and ivermectin did not alter the proportion of animals expressing autoantibody or the mean autoantibody titer. The severity of acute corneal inflammatory reactions to Mf was similar in animals with and without circulating autoantibody. Although autoantibody responses did not correlate with acute corneal inflammatory reactions to dead Mf, the ability of Mf to induce formation of an antibody reactive with a component of autologous cornea suggests that autoimmune mechanisms might participate in chronic onchocercal lesions in the cornea, eg, sclerosing keratitis.