Influence of environmental factors on Argulus japonicus occurrence of Guangdong province, China.

To assess the effects of abiotic factors on Argulus japonicus occurrence in freshwater ecosystem, we sampled fish, free-swimming parasites, and submerged objects from rivers, fish farms, and ponds (reservoirs and lakes) at 27 locations in Guangdong province, China, from July, 2010, to March, 2013. Friedman's test and Spearman's correlation coefficient (r) were used to investigate relationship between A. japonicus occurrence and abiotic factors in three aquatic systems. Correlation of monthly density, mean density, prevalence, abundance, and intensity of A. japonicus with water temperature, biochemical oxygen demand of 5 days, chemical oxygen demand, and ammonia were very significant, but negative relation with pH, dissolved oxygen, and total phosphorus components. Nevertheless, total nitrogen components showed an insignificant impact on A. japonicus incidence. In addition, water temperature and stability were two key factors controlling A. japonicus infestation. Our study revealed the capability of A. japonicus to inhabit and prevail in heavily polluted freshwater. Results demonstrate that A. japonicus has good adaptability to the environmental factors' stress but cannot be used as a biological indicator of environmental pollution.

Genotyping of Giardia duodenalis isolates from dogs in Guangdong, China based on multi-locus sequence.

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ß-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.

The zoonotic risk of Ancylostoma ceylanicum isolated from stray dogs and cats in Guangzhou, South China.

Canine and feline hookworm infection is endemic in many countries with zoonotic transmission representing a potentially significant public health concern. However, there is limited data available on the zoonotic transmission of canine and feline hookworms in China. This study was conducted to evaluate the zoonotic risk of Ancylostoma ceylanicum isolated from stray dogs and cats in Guangzhou, south China. Primer pairs CAF/CAR were designed to amplify complete ITS sequences of obtained A. ceylanicum. The results were compared with fourteen ITS reference sequences of human-derived A. ceylanicum registered in GenBank, and phylogenetic trees were established by using NJ and ML methods. The sequence similarity of three dog-derived and five cat-derived A. ceylanicum with fourteen human-derived A. ceylanicum were 96.8%~100% and 97.8%~100%, respectively. Phylogenetic analysis placed A. ceylanicum isolated from dogs and cats in the same group with A. ceylanicum human isolates. Due to the ability of A. ceylanicum to cause a patent infection in humans, the zoonotic risk arising from dog and cat reservoirs to communities in this region should be determined.

Parasitism of Argulus japonicus in cultured and wild fish of Guangdong, China with new record of three hosts.

This study aimed to demonstrate the ability of Argulus japonicus to infect a wide range of freshwater fishes, as well as to understand the effects of fish origin and host body size on the incidence of A. japonicus. Samples of cultured and wild fish were collected randomly from July 2010 to March 2013, using angling, long-lining, gill-netting, and trapping from rivers and fish farms in Guangdong province, South China. Eight fish species were found to be heavily infected including the common carp, the goldfish, the black carp, the silver carp, the brown trout, the rainbow trout, the mandarin fish, and the perch. Furthermore, the black carp, the brown trout, and the mandarin fish were recorded as new hosts for the first time. During the present study, a total of 2,271 fishes were examined, out of which 712 fishes were found to be infected by a total of 1,443 A. japonicus. Abundance and intensity of A. japonicus infection were significantly influenced by origin of fishes (cultured and wild) and total length (class I, <250 mm; class II, 250-350 mm; and class III, >350 mm) of fish species, whereas varied impacts on prevalence of infection were observed. The correlation between total length of fishes and prevalence of A. japonicus infection was variable, where no significant correlation was observed in the black carp, the silver carp, the mandarin fish, and the perch. In spite of the weak negative correlation between body size of the silver carp and prevalence of infection, A. japonicus was the most abundant and intensive in the silver carp. Thus, aquaculturists should pay particular attention to the control of these fish lice due to its host biodiversity.

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.

Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63℃ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1α) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages.

A high-resolution melting (HRM) assay was applied to distinguish between Giardia duodenalis assemblages A and B from human and dog feces based on the triosephosphate isomerase gene (tpi). The genomic DNAs were selected from assemblages A (WB) and B (GS) as reference and plasmids were constructed. The reference plasmids and genomic DNAs from 15 Giardia-positive samples were analyzed by HRM assay. This was followed by separate real-time PCR assays specific for assemblages A and B using EvaGreen (EG) to identify PCR products by melting-point analysis. Our results indicate that PCR with HRM in a one-step closed-tube method is a reliable diagnostic method for G. duodenalis zoonotic assemblage identification and more rapid than restriction length polymorphism analysis and direct sequence analysis, HRM is specific, sensitive, reproducible, and rapid. This study is the first use of EG dye for Giardia genotyping. This assay is a promising approach to determine the presence and genotype of Giardia based on a highly variable gene.

Genotype identification and prevalence of Giardia duodenalis in pet dogs of Guangzhou, Southern China.

Giardia duodenalis is a flagellated parasite and is considered one of the most common causes of protozoal diarrhea in both humans and animals worldwide. This paper represents the first study of the prevalence of G. duodenalis in pet dogs in Guangzhou, China. Faecal samples (209 specimens) were obtained from young (<6 months old), adult (6 months to 3 years) and elder dogs (>3 years old). 8.61% (18/209) faecal samples were recorded positive using microscopy examination, and 11.00% (23/209) using PCR. The prevalence was significantly higher in diarrheic dogs (26.31%) compared with non-diarrheic dogs (5.10%), while it was higher in young (25.58%) than both adult (7.37%) and elder (7.04%) dogs and the difference was statistically significant (P<0.05). The prevalence in male dogs 11.30% (13/115) was higher than females 10.87% (10/92), and in suburban dogs (12.15%) higher than urban 9.80%, but the difference was not statistically significant (P>0.05). Sequence analysis of the 23 PCR-positive samples revealed the presence of Assemblage D (18/23), and zoonotic Assemblage A (5/23). The present investigation reported a high infection rate of G. duodenalis in pet dogs, especially in young dogs. Genotypic characterization demonstrated that the zoonotic Assemblage A was found, a fact that poses a potential risk of G. duodenalis transmission from pet dogs to humans. It is suggested that pet owners should take appropriate hygiene measures to prevent and control giardiasis in this region.

Freshwater abiotic components' impact on the viability of fish lice, Argulus sp., in Guangdong province, China.

Parasite intensity dynamics can be strongly affected by ecosystem abiotic components. We conducted this study to investigate how the intensity of Argulus sp. is influenced by physicochemical factors in three water bodies - river, fish farm, and reservoir - and to find the occurrence of Argulus sp. under different conditions. Argulus sp. specimens were collected using different methods - gill-netting 30 × 1.5 m, 40 mm mesh monofilament net and set from a small boat, beach seining and plankton net - from July 2010 until Jun 2011 at Guangdong province, China. Main water parameters taken for the assessment consisting of temperature, pH, DO, biochemical oxygen demand of 5 days (BOD5), chemical oxygen demand (COD), NH (4) (+) , total nitrogen (TN) and total phosphorus (TP). The results show that the ratio of female to male Argulus sp. during the studied period was 2.3:1. The presence and intensity of Argulus sp. were temperature-dependent and variable; pH values were lower than standers with high intensity of Argulus sp. Argulus sp. has the ability to live in low dissolved oxygen (DO) concentration. Relatively high concentrations of BOD5, COD, NH (4) (+) , TN and TP were detected with related variable intensity of Argulus sp., which is supposed to be a freshwater fish ectoparasite found in polluted ecosystems. It is concluded that the fish ectoparasite Argulus sp. have a strong potency of genetic adaptation impacted by environmental factors. However, this needs further study in order to gain insight into the question of the likelihood of adaptation to abiotic factors' variation.

Analysis of genetic variability within Argulus japonicus from representatives of Africa, Middle East, and Asia revealed by sequences of three mitochondrial DNA genes.

This study investigated the genetic variability within fish louse Argulus japonicus (Crustacea: Branchiura) from Africa, Middle East, and Asia by polymerase chain reaction in three mitochondrial DNA (mtDNA) regions, namely, cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4). Six different sequences from a portion of the cox1 gene (pcox1) and a portion of the nad1 and nad4 genes (pnad1 and pnad4) for ten adult specimens from infected fish in China, Egypt, and Syria were amplified separately from individual and the amplicons were subjected to direct sequencing. A + T percentages were 68.8-69% for pcox1, 77.1-77.6% for pnad1, and 60.4-60.9% for pnad4. Among all the collected parasites, A. japonicus sequence variations were 0.0-1.9% for cox1, 0.0-2.3% for nad1, and 0.0-0.8% for nad4. In rivers, sequence variations among all individuals were 0.4-0.8% for cox1, 1.0-2.3% for nad1, and 0.4-0.8% for nad4, while sequence variations among all the collected parasites in fish farms were 0.6-1.9% for cox1, 0.0-1.7% for nad1, and 0.2-0.6% for nad4. The nad1 was the most variable gene among selected markers, while nad4 was a more conserved gene than cox1. All isolates of A. japonicus were sister to Argulus americanus in phylogenetic tree and they grouped together in one sub-clade, while isolates from China and Egypt fish farms were closely clustered together. However, moderate genetic drift and slight mutation could be observed among A. japonicus individuals. These findings demonstrated the convenience and attributes of the three selected mtDNA sequences for population genetic studies of A. japonicus where nad1 is a new and reliable marker to detect the sequence variation among A. japonicus individuals.