Molecular Identification of Ancylostoma caninum Isolated from Cats in Southern China Based on Complete ITS Sequence

Ancylostoma caninum is a blood-feeding parasitic intestinal nematode which infects dogs, cats, and other mammals throughout the world. A highly sensitive and species-specific PCR-RFLP technique was utilised to detect the prevalence of A. caninum in cats in Guangzhou, southern China. Of the 102 fecal samples examined, the prevalence of A. caninum in cats was 95.1% and 83.3% using PCR-RFLP and microscopy, respectively. Among them, the prevalence of single hookworm infection with A. caninum was 54.90%, while mixed infections with both A. caninum and A. ceylanicum were 40.20%. Comparative analysis of three complete ITS sequences obtained from cat-derived A. caninum showed the same length (738 bp) as that of dog-derived A. caninum. However, the sequence variation range was 98.6%–100%, where only one cat isolate (M63) showed 100% sequence similarity in comparison with two dog-derived A. caninum isolates (AM850106, EU159416) in the same studied area. The phylogenetic tree revealed A. caninum derived from both cats and dogs in single cluster. Results suggest that cats could be the main host of A. caninum in China, which may cause cross-infection between dogs and cats in the same area.


Introduction
Ancylostoma caninum is a blood-feeding parasitic intestinal nematode which infects dogs, cats, and other mammals throughout the temperate and tropical areas in the world [1][2][3]. In addition to the veterinary importance, A. caninum can also cause zoonotic disease in humans. The larvae of A. caninum hatch from eggs and develop into infective larvae via two molts. The infective larvae then infect host animals such as dogs and cats, migrate into the intestine, and develop into adult worms following two more molts. If the infective larvae invade humans, they can cause cutaneous larvae migrans (CLM) or "creeping eruptions, " which are hypersensitive reactions in response to the migration of A. caninum larvae; however, they cannot develop into adult worms just by migrating under the skin [4].
Although some cases recorded that A. caninum was found in cats [5][6][7][8], this species has been still regarded as an uncommon parasite of cats. Therefore, A. caninum was described as "dog hookworm" [9] and was supposed as a host-specific parasite for canids [2,10], while Palmer stated that A. caninum was the predominant species of hookworm in dogs [11], and A. tubaeforme was the predominant species of hookworm in cats. In China, high occurrence of A. caninum has been reported with prevalence of 1.04%-73%, but without significant area differences [12,13]. Although A. caninum in cats has been reported in Thailand (23%) [14], Australia (30%) [11], and Sichuan province in China (25% and 51%) [15,16], data on prevalence of A. caninum in cats are still scarce.
Herein, this study presents the first molecular identification based on complete ITS sequence, as well as it describes a simple and effective detection method for A. caninum from cats in southern China.  Another pair of primers CAF (5 -GACTGCGGACTG-CTGTAT-3 ) and CAR (5 -AAGTTCAGCGGGTAGTCA-3 ) was designed by Primer Premier 5.0 based on ITS sequences (JQ812694, AJ920347, and AM039739) of A. caninum to amplify the complete ITS sequence of cat-derived A. caninum.

PCR-RFLP.
Both PCRs were performed in 25 L volume containing 2 L of the DNA sample, 0.2 L of Taq polymerase (TaKaRa, Dalian, China), 2.5 L of 10×Taq buffer (TaKaRa), 2 L of diethylnitrophenyl thiophosphate (dNTP, TaKaRa) mixture, 0.5 L of each primer (AF/AR or CAF/CAR, 50 mM), and 17.3 L of distilled water. PCR cycling parameters were as follows: 1 cycle at 96 ∘ C for 5 minutes, then 35 cycles of 96 ∘ C for 30 seconds, at 60 ∘ C for 30 seconds, and at 72 ∘ C for 90 seconds, followed by 1 cycle at 72 ∘ C for 7 minutes. RFLP analysis was performed by digesting 7 L of PCR product with 2 U of each restriction endonuclease (TaKaRa) in a final volume of 20 L for 3 hours at 37 ∘ C. PCR products and restriction fragments were analyzed after electrophoresis in 2% and 3% agarose gels with 0.2 g/mL of ethidium bromide staining and were visualized on a UV transilluminator.

Sequence Confirmation and Phylogenetic Analysis.
Positive amplicons were purified and sequenced using ABI 3730 automated DNA sequencer (BigDye Terminator Chemistry). Obtained sequences were aligned with 15 ITS reference sequences using Clustal X programs [17]. Phylogenetic trees were constructed using MEGA version 5.1 (MEGA5.1: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA). Bootstrap analyses were conducted using 1,000 replicates to assess the reliability of inferred tree topologies. Neighbor-joining algorithms were conducted using the Kimura 2 parameter distance analysis. Obtained nucleotide sequences have been deposited in the GenBank database under accession numbers KC755015 and KC755025.
The results showed that U. stenocephala was absent in the 97 examined PCR samples, where there was no enzymatic digestion by restriction endonucleases BsuRI (not shown). Cat
In China, A. caninum was reported twice in Sichuan province, southwest China, with prevalence of 25% and 51%, respectively, [15,16], while in our survey, overall higher prevalence (95.1%) of A. caninum infections in cats was detected in Guangzhou (southern China). Our results suggested that the predominant species of hookworms in cats was A. caninum in China, while A. tubaeforme was considered to be the predominant species in Australia [11], which strongly supports our suggestion that the prevalent species is related to its geographical distribution, as well as cats could be the main host for A. caninum in China.
The first cat-derived A. caninum complete ITS sequences (GenBank: KC755026, KC755028, and KC755029) are presented in our study. The length of obtained sequences (738 bp) was identical to the dog-derived A. caninum ITS sequence, as well as the sequence similarity range was 98.6%-100%. In addition, A. caninum from both cats and dogs was connected in a single cluster in the constructed phylogenetic tree. This finding indicated that there could be a crossinfection of A. caninum between dogs and cats in the studied areas.
In conclusion, the results of this study demonstrated that The PCR-RFLP technique described in this study was a rapid and straightforward method for the identification and discrimination of A. caninum. Moreover, the ITS sequences could be used to identify this hookworm species from different local hosts. Current information regarding the prevalence of A. caninum showed possible cross-infections between different hosts. Therefore, it is imperative to have current information regarding the prevalence of this hookworm and the associated risk factors of this infection. This will allow a more effective implementation of strategic control programmes for hookworm infections.