Efficacy of nitroscanate against naturally acquired infection with Ancylostoma caninum, Dipylidium caninum, and Trichuris vulpis in dogs

Eighteen dogs with naturally acquired helminth infections were used to evaluate the efficacy of nitroscanate against Ancylostoma caninum, Dipylidium caninum, and Trichuris vulpis. Approximately 15 minutes before treatment, the dogs were given 100 to 200 g of canned dog food. Ten dogs were treated with nitroscanate (50 mg/kg of body weight, PO), and 8 dogs were given placebo tablets PO. The dogs were euthanatized and necropsied 10 days after treatment and helminths were recovered from the small intestine and cecum. On the basis of the number of worms recovered from treated dogs vs the number recovered from control dogs, we determined the efficacy of nitroscanate to be 99.6% against A caninum, 99.8% against D caninum, and 0% against T vulpis.     

Survival of Ancylostoma caninum on bare ground, pea gravel, and concrete.

Studies were done to determine the survival of infective Ancylostoma caninum 3rd-stage larvae on 3 ground covers commonly used in dog run construction: bare ground, pea gravel, and concrete. Changes in numbers of recovered larvae were compared to meterologic data and the most significant weather variables were determined. Larvae were recovered 1 to 7 days on bare ground. Larvae survived longer in the fecal mass (mean of 3 days) than on the bare ground (mean of 2 days). Rain was the most significant variable, in that it was positive in its effects (higher larval count) early in the experiment (causing fecal mass breakdown and release of larvae) and negative (lower larval count) later in the experiment (spreading larvae away from test site). Larvae were also recovered 1 to 7 days on pea gravel. They were recovered for a mean 2.6 days from the fecal sample, a mean of 1.5 days from the rocks directly below the fecal mass, and a mean of 1.3 days from the remaining rocks. Here also, rain was the most significant weather factor. It was negatively significant (lower larval count) for the fecal mass (spreading of the larvae) and positive for those in the pebbles (increasing the moisture in the pebbles). Survival time of larvae on concrete was shorter than that on the other 2 substrates: from 0 to 2 days. Larvae were recovered a mean of 1.3 days from the fecal mass and a mean of 0., days from the surrounding concrete. Rain was positively significant early in the experiments in that it released trapped larvae from the fecal mass. Sunlight consistently was negatively significant (lower larval count) due to its lethality to the unprotected larvae.     

RNA and protein synthesis is required for Ancylostoma caninum larval activation

The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 μM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 μM), cycloheximide (IC(50)=50 μM), and anisomycin (IC(50)=200 μM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h. None of the RNA or protein synthesis inhibitors prevented secretion of the activation-associated protein ASP-1, despite nearly complete inhibition of feeding. The results indicate that unlike dauer recovery in C. elegans, de novo gene expression is required for hookworm larval activation, and the critical genes are expressed within 12h of exposure to activating stimuli. However, secretion of infection-associated proteins is independent of gene expression, indicating that the proteins are pre-synthesized and stored for rapid release during the initial stages of infection. The genes that are inhibited represent a subset of those required for the transition to parasitism, and therefore represent interesting targets for further investigation. Furthermore, while dauer recovery provides a useful model for hookworm infection, the differences identified here highlight the importance of exercising caution before making generalizations about parasitic nematodes based on C. elegans biology.     

Dose-titration studies of ivermectin against experimental Ancylostoma caninum and Uncinaria stenocephala infections

Dose-titration trials of ivermectin were conducted on pups with dual experimental infections of 4th-stage larvae or adult Ancylostoma caninum and Uncinaria stenocephala. Ivermectin was administered orally or subcutaneously at dosages of 0.006, 0.012, or 0.024 mg/kg of body weight. Maximal or near maximal (greater than or equal to 96% to 100%) anthelmintic effect was observed for both stages of development for each hookworm species by either route of administration at a dosage of 0.024 mg/kg. Responses for all of the aforementioned categories were linearly related to increasing log dosage of ivermectin, with common slopes (regression coefficients). Regression analysis also provided estimates of the minimal dosages required to produce maximal reduction in worm burden for each stage, species, and route of administration. The estimated ivermectin dosages for maximal efficacy ranged from a low of 0.014 mg/kg for adult A caninum by oral treatment to 0.044 mg/kg for 4th-stage larvae of U stenocephala by oral treatment.     

Infectivity of Ancylostoma caninum in dogs by different routes of inoculation

Infectivity of A. caninum in dogs inoculated with 500 larvae by different routes (percutaneous, subcutaneous, intravenous, per os, through a stomach tube and foot pad) was studied by ascertaining the worm burden at necropsy 21-27 days post-infection. Infection through foot-pads yielded the maximum number of worms. The percentage of worm establishment using foot-pad inoculation was 73.8%, followed by subcutaneous (49.0%), percutaneous (45.0%) and per os (35.0%) routes. These results show that active penetration of the larvae through skin and particularly foot-pad is the most favourable mode of infection of dogs.     

Mitochondrial DNA variation of the dog hookworm Ancylostoma caninum in Brazilian populations

The mitochondrial cytochrome oxidase I gene was partially sequenced for 164 Ancylostoma caninum individuals, originating from five different localities in Brazil, with the aim of describing the genetic diversity and genetic structure of Brazilian hookworm populations. Allelic and nucleotide diversity were moderate (overall h=0.88 and pi=0.016) and were similar among cities. There was moderate genetic differentiation among the populations sampled (approximately Phi(ST)=0.12) and a weak but nonsignificant correlation between geographical and genetic distance. This genetic structure was similar to that observed among populations of the human hookworm, Necator americanus, but distinct from that typically found in trichostrongylid nematode parasites of livestock. Thus, a pattern of different genetic structures among different groups of nematodes is emerging. We also observed a few individuals that had a highly divergent mtDNA sequence (almost 7% sequence divergence from the other sequences). These results in combination with data from other studies suggest that A. caninum populations worldwide consist of a mix of previously differentiated populations, or perhaps even cryptic species. This study contributes to the knowledge of genetic structure and diversity of hookworms, which in turn will be useful in developing methods for their control.     

Antibody responses in pregnancy-induced transmammary transmission of Ancylostoma caninum hookworm larvae.

Third stage larvae of the Ancylostoma caninum hookworm nematode have the capacity to infect a dog, abort the normal maturation pathway to become blood-feeding intestinal worms, and instead distribute throughout the body in a developmentally arrested state that is relatively resilient to most chemotherapeutic agents. During pregnancy, a percentage of the arrested larvae reactivate and transmit via the mammary glands to infect the nursing puppies with resulting iron-deficiency anemia and potential mortality. To determine if the suppression of parasite-specific antibody responses during pregnancy facilitates the reactivation and transmammary transfer of hookworm larvae, a murine model of A. caninum infection was used to compare the infected versus uninfected animals that were either bred or not bred. Initial comparisons of genetically divergent BALB/c versus C57BL/6 mice showed that both the strains mounted strong Th2 biased IgG1and IgE antibody responses to A. caninum infection. Using the BALB/c strain for the breeding analyses, it was confirmed that larval transfer to the mouse pups only occurred during the post-partum lactational period. In the dams, levels of total and antigen-specific IgG1 and total IgE were highly correlated with parasite burden. During most phases of pregnancy and lactation, infected dams had lower total IgG1, IgG2a and IgE levels as compared to unbred mice at comparable times post-infection; this downward modulation of antibody responses supports the established dogma of a generalized immunosuppression associated with pregnancy. However, at parturition and post-partum lactation, antigen-specific IgG1 levels measured at 1 : 5000 serum dilutions were comparable between bred and unbred mice, and antigen-specific IgG2a levels at 1 : 100 serum dilutions were also not significantly different except for a marginal reduction in the bred mice at the lactational timepoint. The comparable anti-A. caninum IgG1 levels between bred and unbred mice, and low correlation between IgG2a levels and larval burden suggest that parasite-specific antibody responses do not play a major role in the pregnancy-associated transmammary transmission of A. caninum larvae. This conclusion does not rule out the possibility that underlying fluxes in the levels of specific cytokines associated with pregnancy and infection may be involved in the process of larval reactivation and transmission.     

Characterisation of humoral immune responses in dogs vaccinated with irradiated Ancylostoma caninum

Infection with Ancylostoma caninum, an intestinal hookworm of dogs, can cause debilitating and potentially life-threatening disease. In the current study, protective immunity to hookworm infection was induced in dogs following vaccination with irradiation-attenuated third-stage larvae (L3) with significant reductions in both worm (P<0.03) and faecal egg counts (P<0.0004) following a challenge infection. Vaccination with irradiated L3 and challenge with infective L3 stimulated a dominant antibody response to antigens of less than 20 kDa in an excretory/secretory extract from adult parasites. Immunoscreening of an adult A. caninum cDNA library with antisera from the vaccine trial identified a number of clones. The three clones with the strongest immunoreactivity proved to be identical and encoded a peptide with similarity to the N-terminal domain of the tissue matrix metalloproteinase inhibitor (TIMP)-2 mammalian tissue metalloproteinase inhibitor family.