Occurrence, risk factors and genotypes of Enterocytozoon bieneusi in dogs and cats in Guangzhou, southern China: high genotype diversity and zoonotic concern.

BACKGROUND: Enterocytozoon bieneusi, a common opportunistic fungal pathogen, has a wide range of hosts. Limited epidemiological data on E. bieneusi intestinal infections in companion animals (dogs and cats) in China exists. In this study, fecal samples (651 from dogs and 389 from cats) in Guangzhou city, Guangdong Province, China, were collected, and the ribosomal internal transcribed (ITS) spacer region from the DNA extracted from them was Polymerase Chain Reaction (PCR)-amplified and sequenced. RESULTS: Based on the sequencing data, E. bieneusi was identified in the fecal samples collected from 149 (22.9%) and 79 (20.3%) dogs and cats. Of the factors investigated, poor living conditions appeared to be the major risk factor for contracting the pathogen. Eleven E. bieneusi genotypes, six known (PtEb IX, GD1, D, CD9, EbpC, I) and five novel (designated here as GD2- GD6), were found in dogs. Eight genotypes, six known (PtEb IX, GD1, D, CD9, EbpC, Type IV) and two novel (GD2 and GC1), were identified in cats. Genotype PtEb IX was most common in both dogs and cats, followed by genotype GD1. CONCLUSIONS: Although PtEb IX was the most common E. bieneusi genotype in dogs, this is the first report of this genotype dominating in cats. The same genotype distribution of the pathogen between the two different companion animals species in the same geographic area indicates that inter-species transmission is probable. The widespread existence of zoonotic E. bieneusi genotypes (D, EbpC, Type IV) in companion animals indicates that they are potential sources of environmental contamination and infections in humans.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

Seroprevalence of Epizootic Hemorrhagic Disease Virus in Guangdong Cattle Farms during 2013-2017, China.

Epizootic hemorrhagic disease (EHD) is an infectious viral disease caused by epizootic hemorrhagic disease virus (EHDV) and EHDV frequently circulates in wild and domestic ruminants. Sporadic outbreaks of EHD have caused thousands of deaths and stillbirths on cattle farms. However, not much is known about the circulating status of EHDV in Guangdong, southern China. To estimate the seroprevalence of EHDV in Guangdong province, 2886 cattle serum samples were collected from 2013 to 2017 and tested for antibodies against EHDV using a competitive ELISA. The overall seroprevalence of EHDV reached 57.87% and was highest in autumn (75.34%). A subset of positive samples were serotyped by a serum neutralization test, showing that EHDV serotypes 1 and 5-8 were circulating in Guangdong. In addition, EHDV prevalence always peaked in autumn, while eastern Guangdong had the highest EHDV seropositivity over the five-year period, displaying apparent temporal-spatial distribution of EHDV prevalence. A binary logistic model analysis indicated a significant association between cattle with BTV infections and seroprevalence of EHDV (OR = 1.70, p < 0.001). The co-infection of different serotypes of EHDV and BTV raises a high risk of potential genomic reassortment and is likely to pose a significant threat to cattle, thus urging more surveillance to monitor their circulating dynamics in China.

Ancylostoma ceylanicum Infection in a Miniature Schnauzer Dog Breed.

PURPOSE: Canine hookworm disease is a global zoonotic parasitic disease caused by a variety of nematodes in families Ancylostomatidae, including Ancylostoma spp., Necator spp., and Uncinaria spp., in the small intestine (mainly the duodenum) of dogs. The disease is widely distributed in China. The purpose of this study is to systematically diagnose and treat canine hookworm disease through the case of miniaturization Schnauzer dog feed infected with A. ceylanicum, so as to provide experimental basis for subsequent prevention and control of canine hookworm disease. METHODS: In the current study, we isolated hookworm eggs from a diseased miniature schnauzer, then the polymerase chain reaction (PCR) was used to amplify the ITS1-5.8S-ITS2 gene sequence from genomic DNA extracted from hookworms. Phylogenetic analysis based on ITS1-5.8S-ITS2 gene sequence sequences was inferred using MEGA-X. After phylogenetic analysis, etiologic and symptomatic therapies were used to treat the canine hookworm disease. RESULTS: The sequencing results showed that the length of the ITS1-5.8S-ITS2 gene sequence was approximately 960 bp, and ITS1 and ITS2 were extracted to analyze similarity with other hookworms to build a phylogenetic tree. After phylogenetic analysis, the results showed that the diseased miniature schnauzer was infected by A. ceylanicum. Using etiologic and symptomatic therapies, the sick dog with an A. ceylanicum infection was also treated for 5 days. CONCLUSIONS: To our knowledge, this is the first report of diagnosis and treatment for canine hookworm disease in Guangzhou city. In addition, with the improvement of economic level, the scale of pet dog breeding is also increasing. The diagnostic methods and treatment schemes adopted in this report will help to standardize the prevention and control of canine hookworm disease.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy.

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Occurrence and genotypes of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in household, shelter, breeding, and pet market dogs in Guangzhou, southern China.

Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. are common intestinal protozoans that infect humans and animals worldwide. A survey that assessed the prevalence, molecular characteristics, and zoonotic potential of these pathogens was conducted on a variety of dogs in Guangzhou, southern China. A total of 651 canine stool samples from household (n = 199), shelter (n = 149), breeding (n = 237), and pet market dogs (n = 66) were collected from eight districts in Guangzhou. Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. were detected by PCR amplification of the SSU rRNA gene. Giardia duodenalis-positive specimens were further assigned into assemblages using the glutamate dehydrogenase gene. Cryptosporidium spp., G. duodenalis, and Blastocystis sp. were found in 21 (3.2%), 20 (3.1%), and 35 (5.4%) samples, respectively. The overall prevalence of shelter dogs (40.28%, 60/149) was significantly higher than that of household (3.0%, 6/199), breeding (2.1%, 5/237), and pet market dogs (7.5%, 5/66) (χ2 = 154.72, df = 3, P < 0.001). Deworming in the past 12 months had a strong protective effect on the risk of contracting parasite infections (P < 0.001). No significant differences were detected between age or sex groups (P > 0.05). Dog-specific C. canis (n = 19) and zoonotic C. parvum (n = 2) were the only two Cryptosporidium species. Sequence analysis revealed the presence of three G. duodenalis assemblages: dog-specific assemblages D (n = 14) and C (n = 5), and cat-specific F (n = 1). Zoonotic Blastocystis ST3 (n = 28) was the dominant subtype, followed by ST1 (n = 6) and ST10 (n = 1). To our knowledge, this is the first large-scale investigation on the occurrence and molecular characteristics of Blastocystis sp. in dogs in China. Our results indicated that the dogs seemed to play a negligible role as reservoirs for Cryptosporidium spp. and G. duodenalis transmission to humans, but they are potential novel suitable hosts of Blastocystis sp. A strict sentinel surveillance system of dogs should be established to minimise the zoonotic risk of spreading blastocystosis among humans and dogs.

Autophagy induced by monensin serves as a mechanism for programmed death in Eimeria tenella.

Monensin (Mon), the first ionophoric antibiotic has widely been used for the treatment and prevention of coccidiosis in poultry until recently, however, at present; its efficacy has been compromised with the emergence of many Mon-resistant strains. Knowledge of the mode of the action of anti-parasitic agents is as important as for other antimicrobials, especially for discovery and long term use of the existing drugs. However, little is known about anti-parasitic drug: monensin's, mechanism of action and physiological alteration in Eimeria tenella. In this study, we explored Mon effects on the viability of Mon-Sensitive GZ (MonS-GZ) and Mon-Resistant GZ (MonR-GZ) Eimeria tenella strains using trypan blue staining and investigated Mon-induced autophagy using Western blotting, indirect immunofluorescence assay, and transmission electron microscopy. The results showed that monensin leads to programmed death of E. tenella parasites by inducing autophagy as a mechanism of anticoccidial action. Mon-induced autophagy was indicated by the decreased sporozoites survival rate, ATG8 over expression and localization, and intracellular vacuolar structures and autophagosomes formation in MonS-GZ strain while in MonR-GZ strains autophagy pathway was not triggered. The autophagy inhibitor 3-methyladenine (3-MA) effectively blocked programmed cell death and saved the MonS-GZ sporozoites. These findings indicated that autophagy serves as a potentially important mechanism of E. tenella cell death in response to Mon and disruption of the autophagy pathway may lead to emergence of drug resistance against this anti-parasitic drug.

The effect of autophagy on the survival and invasive activity of Eimeria tenella sporozoites.

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.