The geographical distribution of hookworms depends mainly on environmental temperatures that are optimal for the development, and survival of the parasitic larval stages. Ancylostoma caninum is mostly found in warm moist temperate regions, such as southern European countries.

There are two principal sources of infection: through the shedding of the parasite eggs in the faeces of infected animals (dogs or foxes) – which develop into infective larvae, living in the surrounding soil and vegetation only to be ingested orally or penetrate the skin; or in puppies through the ingestion of the mother’s milk.

Samples of infected dog faeces were sourced from an area of Italy where the hookworms A. caninum and Unicinaria stenocephala (U. stenocephala) are common. The eggs of these two hookworms cannot be reliably distinguished/identified microscopically – although the latter is on average considerably larger, the size ranges do overlap.  A. caninum eggs lie in the range of 56-65µm by 37-43µm and U. stenocephala 63-80 µm by 32-50µm.

Existing Literature

A literature search found an article (abstract below) detailing how the two species of larvae could be distinguished by culturing the eggs and larvae at 15°C, and examining the morphological differences in the resulting L3 larvae.

Differences in Lipid Granulation as the Basis for a Morphologic Differentiation between Third-Stage Larvae of Uncinaria stenocephala and Ancylostoma caninum

Robert L. Hill, Jr. and Edward L. Roberson

The Journal of Parasitology

Vol. 71, No. 6 (Dec., 1985), pp. 745-750



“Differences in the distribution of lipid granules between unstained third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum cultured at 15°C was found to be an effective means for differentiating these 2 species of canine hookworms. In contrast, larvae cultured at 22°C were less easily differentiated based on the distribution of lipid granules. After culturing at 15°C, third-stage larvae of U. stenocephala were motile and exhibited 32 well-demarcated intestinal cells which contained intracellular lipid granules. Intestinal cells were easily visualized due to the absence of extra intestinal lipid granulation. Ancylostoma caninum third-stage larvae cultured under similar conditions were significantly less motile and contained extra intestinal accumulations of lipid granules which obscured intestinal cells. Both species exhibited an overall decrease in lipid granulation during a 14-day observation period following culture at 15 C. Morphologic differentiation of these 2 species after 14 days was based on the absence of intra- and extra-intestinal lipid in U. stenocephala and the presence of some lipid granules in both these locations in A. caninum. The first- and second-stage larvae of both species cultured at 15°C exhibited dense accumulations of extra intestinal lipid granules and were morphologically indistinguishable. This suggests that the observed difference in lipid granulation between the third-stage larvae of U. stenocephala and A. caninum cultured at 15°C is due to differences in lipid utilization during the third stage rather than differences in lipid synthesis by the first- and second-stage larvae and is related to the adaptation of these parasites to their respective climatic regions.”

In Practice

(Recently our parasitology laboratory at Ridgeway Research was tasked with sourcing a field strain of  A. caninum L3 infective larvae, which is a zoonotic canine intestinal hookworm.)

Our parasitology laboratory, using the information from this article, successfully cultured hookworm larvae from the Italian dog faeces. This was incubated for 10 days at 15°C, the larvae were isolated using the Baermanns method, and observed microscopically the following day.

The motility of the larvae was observed. U. stenocephala were very active whereas A. caninum were very slow and lethargic; Lugol’s solution was used to render the larvae inactive so that figures 1 and 2 could be captured. 

Figure 1: A. caninum L3 larvae at X100 magnification, where the intestinal cells are obscured by extra-intestinal accumulations of lipid granules.

Figure 2: U. stenocephala L3 larvae at X100 magnification, where 32 well-demarcated intestinal cells containing intracellular lipid granules were visible.

Find this interesting? read more about dogs and parasites in agriculture here.

Written by Caroline Fenn