An Epidemiological Investigation and Drug-Resistant Strain Isolation of Nematodirus oiratianus in Sheep in Inner Mongolia, China

Abstract

Nematodirus causes major economic losses in the development of the livestock industry, as they are common gastrointestinal parasites of cattle, sheep, and other ruminants. The present study investigated Nematodirus infections in sheep in the Hulunbuir and Xilingol Grasslands, two well-known natural pastures in Inner Mongolia, China. The results indicated that the average infection rate was more than 50%. Furthermore, a naturally occurring strain of Nematodirus was obtained using numerous isolation methods in the survey region. Conventional morphology and molecular biology were used to identify the strain. The larval migration inhibition test was used to determine the ivermectin level in the isolated strain. The results indicated that the larvae belonged to the species Nematodirus oiratianus. The strain was found to be ivermectin-resistant. Thus, these results recorded the detailed development processes of Nematodirus eggs, providing novel insights regarding the prevention and control of nematodes by using conventional anthelmintic regimens and by offering valuable species resources for studying anthelmintic resistance in nematodes.

Keywords: ITS-rDNA; Nematodirus oiratianus; anthelmintic resistance; larval migration inhibition test; morphology.

Figure 1

Infection rate of Nematodirus in Hulunbuir (a) and Xilingol Grasslands (b) before the spring and autumn deworming.

Figure 2

Morphology of adult Nematodirus spp. (a) Anterior segment of adult Nematodirus spp. (100×). (b) Head vesicle of adult Nematodirus spp. (250×). (c) Eggs in the uterus of a female Nematodirus spp. (250×). (d) A thin transparent thorn in the tail of a female Nematodirus spp. (250×).

Figure 3

Development process of Nematodirus spp. (a) Each egg contained several egg cells, and the gap between the egg cells and the shell was large (400×). (b) Egg cells dividing (400×). (c,d) Egg cells begin to fuse (400×). (e) Embryo entering the tadpole stage (400×). (f,g) L1 larva forming (400×). (h–j) L1 larvae developing (400×). (k) L2 larva forming (250×). (l) L2 larva developing (400×). (m,m) The larva gradually developing into L3 larva (250×). (o) The larva hatching into mature L3 larva (100×).

Figure 4

Morphology of L3 of Nematodirus. spp. (a) L3 larva (100×). (b) Tail of L3 larva (100×). (c) Exsheathed L3 larva (250×). (d) Tail of exsheathed L3 larva (250×).

Figure 5

Homology alignment result based on ITS-rDNA sequences. The red box indicates that the isolated strain has high homology with N. oiratianus.

Figure 6

Phylogenetic tree of Nematodirus spp. based on ITS-rDNA sequences. The red box indicates that the isolated strain and N. oiratianus coalesced into one lineage.

Figure 7

The results of specific PCR for N. oiratianus. M: DNA Marker LD2000; 1: N. oiratianus; 2: N. longispiculata; 3: Ostertagia spp.; 4: Trichostrongylus spp.; 5: Trichuris spp.; 6: Haemonchus contortus; C: blank control.

Figure 8

Larva migration inhibition test results.