Effects of doxycycline on heartworm embryogenesis, transmission, circulating microfilaria, and adult worms in microfilaremic dogs.

Tetracycline treatment of animals or humans infected with filariae that harbor Wolbachia endosymbionts blocks further embryogenesis, and existing microfilariae gradually die. This treatment also kills developing larvae and has a slow-kill effect on adult filariae, all presumably due to elimination of the Wolbachia. Also, Dirofilaria immitis microfilariae in blood collected from dogs up to 25 days after the last dose of doxycycline developed to infective L3 that were normal in appearance and motility in mosquitoes but did not continue to develop or migrate normally after subcutaneous (SC) injection into dogs. The present study was designed to determine whether heartworm microfilariae collected at later times after treatment would regain the ability to continue normal development in a dog. The study also was expected to yield valuable data on the effects of treatment on microfilariae and antigen levels and adult worms. The study was conducted in 16 dogs as two separate replicates at different times. A total of five dogs (two in Replicate A and three in Replicate B) infected either by SC injection of L3 or intravenous transplantation of adult heartworms were given doxycycline orally at 10mg/kg twice daily for 30 days, with three untreated controls. Microfilarial counts in the five treated dogs gradually declined during the 12-13 months after treatment initiation. Two dogs were amicrofilaremic before necropsy and three had 13 or fewer microfilariae/ml. Only one treated dog was negative for heartworm antigen before necropsy. Overall, treated dogs generally had fewer live adult heartworms than controls, and most of their live worms were moribund. All three control dogs remained positive for microfilariae and antigen and had many live worms. L3 from mosquitoes fed on blood collected 73-77 or 161-164 days after initiation of doxycycline treatments were injected SC into five dogs. None of the dogs injected with L3 from mosquitoes fed on blood from doxycycline-treated dogs were ever positive for microfilariae or antigen, and none had worms at necropsy; three control dogs were positive for microfilariae and antigen and had many live worms. These data indicate that doxycycline treatment of microfilaremic dogs gradually reduces numbers of microfilariae and blocks further transmission of heartworms. This latter effect should be highly effective in reducing the rate of selection of heartworms with genes that confer resistance to macrocyclic lactone preventives and microfilaricides. The data also suggest that doxycycline has a slow-kill effect on adult heartworms.

Chitin synthase in the filarial parasite, Brugia malayi.

Fragments of putative chitin synthase (chs) genes from two filarial species (Brugia malayi and Dirofilaria immitis) were amplified by PCR using degenerate primers. The full genomic and cDNA sequences were obtained for the B. malayi chs gene (Bm-chs-1); the predicted amino acid sequence is highly similar, over a large region, to two CHS sequences of the nematode Caenorhabditis elegans and also to two insect CHS sequences. Bm-chs-1 is abundantly transcribed in B. malayi adult females, independent of their fertilization status, but is also expressed in males and microfilariae. Oocytes and early embryos contain large amounts of Bm-chs-1 transcript by in situ hybridization, but later stage embryos within the maternal uterus show little or no Bm-chs-1 transcript. No specific hybridization could be demonstrated in maternal somatic tissues. Polyclonal antibodies were raised against a peptide expressed from a recombinant cDNA fragment of Bm-chs-1; immunostaining detected CHS protein in oocytes and early to midstage embryos. These studies characterize a gene that is likely to be essential to oogenesis and embryonic development in a parasitic nematode. Because chitin synthesis and eggshell formation begin after fertilization, the presence of CHS protein in early oocytes suggests that the enzyme must be activated as a result of fertilization. These studies also demonstrate that chitin synthesis may not be restricted to eggshell formation in nematodes, as the Bm-chs-1 gene is transcribed in life cycle stages other than adult females.

Preliminary results on the effect of tetracycline on the embryogenesis and symbiotic bacteria (Wolbachia) of Dirofilaria immitis. An update and discussion.

The distribution and phylogeny of Wolbachia in filarial species suggests that these endosymbiotic bacteria may be important in the biology of their filarial hosts. An experiment to falsify this hypothesis would be to treat filarial worms with antibiotics which are active against intracellular bacteria. Indeed, it has already been shown that tetracycline treatment inhibits development in a model filarial species (Brugia pahangi) at different stages of the life cycle, in both mosquito and mammalian hosts. Here we discuss these previous data and present new results on the effect of tetracycline on the embryogenesis of the canine filaria Dirofilaria immitis.

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.