Collection and examination of faecal samples

This study was conducted from March 2011 to July 2012 in Guangzhou city, southern China, which is located in the central part of Guangdong Province (22°45′–23°05′N; 113°14′–113°34′E). Guangzhou city covers an area of about 8000 km² divided into ten geographical districts with an estimated population of approximately 12 million. This city contains a large number of stray and sheltered dogs, in five humane shelters (fig. 1). Shelters have different management conditions, but all shelters were cleaned twice daily and thus faecal samples were collected within 12 h of defecation. Stray dogs were captured and observed for 4–7 days before being housed in the nearest humane shelter. However, the early history of these dogs was unknown, except for geographical region, breed and gender.

Fig. 1

The location of the five humane shelters in Guangzhou city, South China.

A total of 254 dogs (135 stray dogs and 119 real shelter dogs) were captured in five humane shelters, including Conghua (106), Baiyun (58), Liwan (40), Haizhu (28) and Panyu (22). Fresh faecal samples from each dog were transported back to the Parasitology and Parasitic Diseases Laboratory, College of Veterinary Medicine, South China Agricultural University (SCAU) on the same day of collection, preserved in 2.5% potassium dichromate and kept at 4°C for microscopic examination. Data on geographical region, dog breed and gender were recorded and written clearly on sample bags.

Faecal samples were processed and examined for the presence of the hookworm eggs. Microscopic examination of stool samples was undertaken using saturated sodium chloride and glucose flotation as described previously (Henriksen & Christensen, Reference Henriksen and Christensen1992). Positive faecal samples were further characterized by molecular procedures.

Molecular analysis

DNA was extracted directly from faecal samples using a commercial DNA extraction kit (QIAamp DNA Stool Mini Kit, QIAgen, Hilden, Germany) according to the manufacturer's instructions. However, samples were pretreated with five cycles of heating at 100°C for 5 min, immediately followed by freezing at − 80°C for 5 min. A negative control (water) was used in each extraction group. DNAs were then stored at − 20°C.

Internal transcribed spacer (ITS) sequences of A. caninum (AM850106, DQ438071, EU159416), A. braziliense (DQ359149, DQ438056, JQ812692), A. ceylanicum (DQ381541, DQ780009, DQ831519) and U. stenocephala (AF194145, HQ262053, HQ262054) were aligned by Clustal X (Thompson et al., Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997). A pair of primers – AF (5′-CTTTGTCGGGAAGGTTGG-3′) and AR (5′-TTCACCACTCTAAGCGTCT-3′) – were designed from the conserved region of ITS sequences of four different hookworms by Primer Premier 5.0 to amplify the 404 bp region of A. caninum, the 408 bp region of A. braziliense, the 404 bp region of A. ceylanicum and the 406 bp region of U. stenocephala, which contain ITS1 and 5.8S rRNA sequences. The specificity of the primers was confirmed by polymerase chain reaction (PCR) using chromosomal DNA extracted from Toxocara canis, Giardia lambia, Cryptosporidium canis, Isospora canis and Dipylidium caninum.

Each test batch contained a positive control and negative control (water). Positive-control DNA was extracted from A. caninum or A. ceylanicum preserved in our laboratory. Each PCR was performed in 25 μl containing 2 μl of the DNA sample, 0.2 μl of Taq polymerase (TaKaRa, Dalian, China), 2.5 μl of 10 × Taq buffer (TaKaRa), 2 μl of deoxynucleoside triphosphate (dNTP, TaKaRa) mixture, 0.5 μl of each primer (AF/AR, 50 mm) and 17.3 μl of distilled water. PCR cycling parameters were as follows: 1 cycle of 96°C for 5 min; followed by 35 cycles of 96°C for 30 s, 60°C for 30 s and 72°C for 50 s; and 1 cycle of 72°C for 7 min.

Restriction fragment length polymorphism (RFLP) analysis was performed by digesting 7 μl of PCR product with 2 units of EcoRII (TaKaRa) in a total volume of 20 μl for 3 h at 37°C to distinguish between A. ceylanicum and A. braziliense, and with 2 units of BsuRI (TaKaRa) under the same conditions to distinguish between U. stenocephala and other hookworms. The theoretical cutting patterns of four different hookworm fragments treated by the two restriction enzymes are shown in table 1. PCR products and restriction fragments were analysed after electrophoresis in 2% and 3% agarose gels, stained with 0.2 μg/ml of ethidium bromide and visualized on a UV transilluminator.

Table 1

Predictive restriction loci in Ancylostoma caninum, A. ceylanicum, A. braziliense and Uncinaria stenocephala by endonuclease EcoRII and BsuRI at ITS1 and 5.8S loci; + indicates one digestive site and ++ two sites.

Positive amplicoms were purified and sent to Beijing Augct Co., Ltd. for sequencing using the ABI 3730 automated DNA sequencer (BigDye Terminator Chemistry, Oyster Bay, New York, USA). Nucleotide sequences were deposited in the GenBank database under accession numbers JX840456–JX840463. Obtained sequences were aligned with 25 ITS reference sequences using Clustal X programs (Thompson et al., Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997). A phylogenetic tree was constructed using MEGA version 5.1 (Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA). Bootstrap analyses were conducted using 1000 replicates to assess the reliability of inferred tree topologies. Neighbour-joining algorithms were conducted using the Kimura 2 parameter distance analysis.

Data analysis

Data were analysed using SPSS programmed for Windows version 11.5 (SPSS Inc., Chicago, Illinois, USA). The chi-square test was used to investigate the association between variables with significant differences expressed as P <  0.05.