Heartworm and Wolbachia: therapeutic implications

A safer, more effective adulticidal treatment and a safe method for reducing microfilaremia and breaking transmission of heartworm disease early in the treatment are needed. The present study evaluated efficacy of ivermectin (IVM) and doxycycline (DOXY) alone or together (with or without melarsomine [MEL]) in dogs with induced adult heartworm infection and assessed the ability of microfilariae from DOXY-treated dogs to develop to L3 in Aedes aegypti mosquitoes and subsequently to become reproductive adults in dogs. Thirty beagles were each infected with 16 adult heartworms by intravenous transplantation. Six weeks later, dogs were ranked by microfilarial count and randomly allocated to 6 groups of 5 dogs each. Beginning on Day 0, Group 1 received IVM (6 mcg/kg) weekly for 36 weeks. Group 2 received DOXY (10 mcg/(kgday)) orally Weeks 1-6, 10-11, 16-17, 22-25, and 28-33. Groups 3 and 5 received IVM and DOXY according to doses and schedules used for Groups 1 and 2. At Week 24, Groups 3 and 4 received an intramuscular injection of MEL (2.5 mg/kg), followed 1 month later by two injections 24h apart. Group 6 was not treated. Blood samples were collected for periodic microfilaria counts and antigen (Ag) testing (and later immunologic evaluation and molecular biology procedures). Radiographic and physical examinations, hematology/clinical chemistry testing, and urinalysis were done before infection, before Day 0, and periodically during the treatment period. At 36 weeks, the dogs were euthanized and necropsied for worm recovery, collection of lung, liver, kidney, and spleen samples for examination by immunohistochemistry and conventional histological methods. All dogs treated with IVM + DOXY (with or without MEL) were amicrofilaremic after Week 9. Microfilarial counts gradually decreased in dogs treated with IVM or DOXY, but most had a few microfilariae at necropsy. Microfilarial counts for dogs treated only with MEL were similar to those for controls. Antigen test scores gradually decreased with IVM + DOXY (with or without MEL) and after MEL. Antigen scores for IVM or DOXY alone were similar to controls throughout the study. Reduction of adult worms was 20.3% for IVM, 8.7% for DOXY, 92.8% for IVM + DOXY + MEL, 100% for MEL, and 78.3% for IVM + DOXY. Mosquitoes that fed on blood from DOXY-treated dogs had L3 normal in appearance but were not infective for dogs. Preliminary observations suggest that administration of DOXY+IVM for several months prior to (or without) MEL will eliminate adult HW with less potential for severe thromboembolism than MEL alone.     

Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0-6, 10-12, 16-18, 22-26, and 28-34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.     

Conservation genetic assessment of the paleback darter,Etheostoma pallididorsum,a narrowly distributed endemic in the Ouachita Highlands,Arkansas, USA

1. The paleback darter, Etheostoma pallididorsum, is considered imperilled and has recently been petitioned for listing under the Endangered Species Act. Previous allozyme-based studies found evidence of a small effective population size, warranting conservation concern. The objective of this study was to assess the population dynamics and the phylogeographical history of the paleback darter, using a multilocus microsatellite approach and mitochondrial DNA.
2. The predictions of this study were that: paleback darter populations will exhibit low genetic diversity and minimal gene flow; population structure will correspond to the river systems from which the samples are derived; reservoir dams impounding the reaches between the Caddo and Ouachita rivers would serve as effective barriers to gene flow; and the Caddo and Ouachita rivers are reciprocally monophyletic.
3. Microsatellite DNA loci revealed significant structure among sampled localities (global F_st = 0.17, P < 0.001), with evidence of two distinct populations representing the Caddo and Ouachita rivers. However, Bayesian phylogeographical analyses resulted in three distinct clades: Caddo River, Ouachita River, and Mazarn Creek. Divergence from the most recent ancestor shared among the river drainages was estimated at 60 Kya. Population genetic diversity was relatively low (H_e = 0.65; mean alleles per locus, A = 6.26), but was comparable with the population genetic diversity found in the close relatives slackwater darter, Etheostoma boschungi (H_e = 0.65; A = 6.74), and Tuscumbia darter, Etheostoma tuscumbia (H_e = 0.57; A = 5.53).
4. These results have conservation implications for paleback darter populations and can be informative for other headwater specialist species. Like other headwater species with population structuring and relatively low genetic diversity, the persistence of paleback darter populations is likely to be tied to the persistence and connectivity of local breeding and non-breeding habitat. These results do not raise conservation concern for a population decline; however, the restricted distribution and endemic status of the species still renders paleback darter populations vulnerable to extirpation or extinction.

     

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.     

Seroprevalence of psittacine beak and feather disease in wild psittacine birds in New South Wales

SUMMARY A haemagglutination inhibition assay was used to detect antibody to psittacine beak and feather disease virus in sera from wild sulphur crested cockatoos (Cacatua galerita), galahs (Eolophus roseicapillus), short-billed corellas (Cacatua sanguinea), eastern long-billed corellas (Cacatua tenuirostris) and other psittacine birds in New South Wales. The seroprevalence of psittacine beak and feather disease ranged from 41% to 94% in different flocks, indicating infection with the virus is widespread in wild populations.     

Identification of toxigenic Pasteurella multocida in atrophic rhinitis of pigs by in vitro characterisation

Toxigenic strains of Pasteurella multocida were readily differentiated from non-toxigenic strains by an agarose overlay method using bovine turbinate cells or bovine lung cells. Cells which were young and densely confluent were best suited to this assay. The incubation period required to distinguish toxigenic strains was dependent on the confluence of the monolayers, which was affected by the seeding rate, cell passage level and growth time prior to overlay. The agarose overlay method correctly identified 11 of 11 reference strains of Pasteurella multocida, and visible cytotoxic changes were present in the monolayers after 48 to 65 h. Outbreaks of the enzootic form of atrophic rhinitis in 2 New South Wales piggeries were associated with the isolation of toxigenic type D strains of P. multocida.     

Cloning and characterization of cDNAs encoding four different canine immunoglobulin γ chains

cDNAs encoding four different canine immunoglobulin G (caIgG) γ chains were identified in this study. One of these IgG γ chain cDNAs, (caIgG-A), represents 92.5% of the IgG γ chain cDNAs in a dog spleen cell cDNA library; a second partial IgG γ chain cDNA (caIgG-B) was also identified in the library. The other two IgG γ chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG γ chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG γ chains, the hinge regions were distinct, sharing only 19.0–35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG γ chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy γ chains (A, B, C and D), respectively. Specific primers were designed based on caIgG γ chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG γ chain mRNA levels in canine PBMC samples.     

The effects of antigenic competition on the efficacy of multivalent footrot vaccines

SUMMARY A multivalent footrot vaccine has been developed, containing pilus antigens produced in recombinant Pseudomonas aeruginosa and representing all nine serogroups of Dichelobacter (Bacteroides) nodosus commonly recognised in the field. The responses of sheep to the multivalent vaccine have been compared with those to monovalent vaccines representing only a single serogroup. Antigenie competition between serogroups occurred in sheep immunised with the multivalent formulation, but high levels of protection were still achieved. The study showed that in multivalent footrot vaccines, antigenic competition is predominantly due to the presence of a family of immunologically-related pilus antigens rather than to interference by extraneous proteins.     

Activity of flubendazole against developing stages of Dirofilaria immitis in dogs

A formulation of flubendazole was studied to determine the activity against developing stages of Dirofilaria immitis in artificially infected pups. Flubendazole suspension was administered subcutaneously at a dose rate of 50 mg/kg of body weight once a day for 1, 3, or 5 consecutive days at various times during the experimental period. The pups were necropsied 6 months after the experimental infection, and the heart and pulmonary arteries were examined for adult worms. Optimal activity was obtained when flubendazole suspension was administered subcutaneously for 5 consecutive days, either 1 or 2 months after infection. The formation of encapsulated deposits, containing white viscous fluid, was observed at the injection sites of all treated pups.