Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus in China.

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.

First molecular detection of Plasmodium relictum in Anopheles sinensis and Armigeres subalbatus.

Background: Plasmodium relictum is one of the most important avian malaria species, which is mainly seen in wild birds, with infections reported in more than 70 different species and at high prevalence. Aim: The aim of this study was to determine the molecular prevalence of Plasmodium spp. in mosquitoes collected in China. Method: A Plasmodium -specific fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) was established in this study to analyze five species of mosquitoes (1,620 Culex pipiens pallens, 806 Aedes albopictus, 377 Armigeres subalbatus, 168 Anopheles sinensis, and 80 Culex tritaeniorhynchus) collected in hand nets from homes in 25 provinces of China. Results: Only females originated from six provinces were determined to be positive (0.6%, 10/1,809). Plasmodium species were detected in three mosquito species, such as C.pipiens pallens (0.5%, 8/1,620), A.sinensis (0.6%, 1/168), and A.subalbatus (0.3%, 1/377). Of the three mosquito species positive for P. relictum, only C. pipiens pallens is known to feed on birds and is recognized as the natural vector of P. relictum. Conclusion: This is the first time that P. relictum has been detected in A. sinensis and A. subalbatus. P. relictum, the agent of avian malaria, was present in mosquitoes in China, including mosquito species not previously thought to be the vectors.

Molecular Detection of Rickettsia felis and Rickettsia bellii in Mosquitoes.

To add to the limited information on Rickettsia in mosquitoes in China, we carried out a PCR survey on convenience samples of 3051 mosquitoes collected with hand nets in and around domestic dwellings in 25 provinces. Five species of mosquitoes were identified: Culex pipiens pallens (n = 1620), Aedes albopictus (806), Armigeres subalbatus (377), Anopheles sinensis (168), and Culex tritaeniorhynchus (80). A Rickettsia nested-PCR targeting the variable domain of gltA showed Rickettsia felis in four mosquito species of 16 provinces Cx. pipiens pallens (1.8%, 29/1620); Ae. albopictus (1.2%, 10/806); An. sinensis (1.2%, 2/168); and Ar. subalbatus (2.1%, 8/377). Rickettsia bellii was also widespread, occurring in 12 provinces and 2 species: Cx. pipiens pallens (4.3%, 69/1620) and An. sinensis (0.6%, 1/168). R. felis and R. bellii were found in almost similar numbers in female [1.5% (27/1809) and 1.2% (21/1809), respectively] as in male mosquitoes [1.8% (22/1242) and 4.0% (49/1242), respectively]. Our results indicated that mosquitoes in China are widely infected with R. felis, the agent of human flea-borne spotted fever, and that R. bellii can also occur outside of the Americas and its usual tick hosts.

Seasonal and Gender Differences in Presence of Rickettsia felis and Blood meals Provide Additional Evidence of a Vector Role for Mosquitoes.

Rickettsia felis belongs to spotted fever group Rickettsia and is an emerging human pathogen most commonly transmitted by a range of fleas and ticks. While recent evidence has suggested mosquitoes are infected with R. felis, there is little information about the role of mosquitoes in the organism's transmission. In this study, around 100 mosquitoes were collected monthly between 2013 and 2014 from the same residential dwelling at Yangzhou, China. The collected mosquitoes were identified for their species and gender, followed by gltA-based PCR and hydroxymethylbilane synthase-based PCR to determine the prevalence of Rickettsia and blood meal. Three mosquito species (Culex pipiens: 76%, 996/1,304; C. tritaeniorhynchus: 17%, 216/1,304; Aedes albopictus: 7%, 92/1,304) were identified. For 1,088 female mosquitoes, 31% of them (n=336) were positive for blood meal and 7% (n=77) carried R. felis DNA. In a strong contrast, none of the 216 male mosquitoes were positive for blood meal but two males were positive for Rickettsia. Interestingly, 63% of R. felis-positive mosquitoes (50/79) were negative for blood meal, being significantly higher than 37% of mosquitoes and being positive for both R. felis and blood meal (P=0.008). Furthermore, we compared the prevalence of Rickettsia and blood meal in the mosquitoes collected in the months with temperature below and above 23°C, the minimum temperature required for mosquito egg hatching. Mosquitoes captured in the months below 23°C showed significant higher positivity of R. felis(71/936, 7.6% vs. 8/368, 2.2%; P=0.002) and blood meal (294/936, 31.4% vs. 36/368, 9.8%; P < 10-4) than in the months above 23°C. Collectively, the seasonal and gender differences of R. felis and blood meal in mosquitoes add to the existing evidence, supporting a potential vector role of mosquitoes in the transmission of R. felis. Studies with a R. felis infection model covering the full life cycle of mosquitoes is necessary to unambiguously prove the transstadial and transovarial transmission of R. felis in mosquitoes.

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli.

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Identification and characterization of mcr mediated colistin resistance in extraintestinal Escherichia coli from poultry and livestock in China.

Antimicrobial resistance to colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of multidrug-resistant Enterobacteriaceae infection in humans. In this study, we investigated the presence of colistin resistance genes (mcr-1, mcr-2, mcr-3) in Escherichia coli strains isolated from poultry and livestock collected between 2004 and 2012 in China. Furthermore, we studied the maintenance and transfer of the mcr-1 gene in E. coli after serial passages. Overall, 2.7% (17/624) of the E. coli isolates were positive for the mcr-1 gene while none were positive for the mcr-2 and mcr-3 genes. The prevalences of mcr-1 were similar in E. coli isolates from chickens (3.2%; 13/404), pigs (0.9%; 1/113) and ducks (6.8%; 3/44) but were absent in isolates from cattle (0/63). The mcr-1 gene was maintained in the E. coli after six passages (equivalent to 60 generations). In vitro transfer of mcr-1 was evident even without colistin selection. Our data indicate the presence of mcr-1 in extraintestinal E. coli from food-producing animals in China, and suggest that high numbers of the mcr-1-positive bacteria in poultry and livestock do not appear to be readily lost after withdrawal of colistin as a food additive.

Antimicrobial resistance in clinical Escherichia coli isolates from poultry and livestock, China.

Poultry and livestock are the most important reservoirs for pathogenic Escherichia coli and use of antimicrobials in animal farming is considered the most important factor promoting the emergence, selection and dissemination of antimicrobial-resistant microorganisms. The aim of our study was to investigate antimicrobial resistance in E. coli isolated from food animals in Jiangsu, China. The disc diffusion method was used to determine susceptibility to 18 antimicrobial agents in 862 clinical isolates collected from chickens, ducks, pigs, and cows between 2004 and 2012. Overall, 94% of the isolates showed resistance to at least one drug with 83% being resistance to at least three different classes of antimicrobials. The isolates from the different species were most commonly resistant to tetracycline, nalidixic acid, sulfamethoxazole, trimethoprim/sulfamethoxazole and ampicillin, and showed increasing resistance to amikacin, aztreonam, ceftazidime, cefotaxime, chloramphenicol, ciprofloxacin. They were least resistant to amoxicillin/clavulanic acid (3.4%) and ertapenem (0.2%). MDR was most common in isolates from ducks (44/44, 100%), followed by chickens (568/644, 88.2%), pigs (93/113, 82.3%) and cows (13/61, 21.3%). Our finding that clinical E. coli isolates from poultry and livestock are commonly resistant to multiple antibiotics should alert public health and veterinary authorities to limit and rationalize antimicrobial use in China.

Chlamydia pecorum is the endemic intestinal species in cattle while C. gallinacea, C. psittaci and C. pneumoniae associate with sporadic systemic infection.

To investigate the prevalence and diversity of bovine Chlamydia spp. in cattle, whole blood from dairy and beef cattle in 11 provinces of China (n=2003) and vaginal swabs, whole blood samples, feces, milk samples from cows in a Yangzhou dairy farm (n=108) were examined using genus- and species-specific PCRs. In cattle from 11 provinces, 2.4% (48/2003) of whole-blood samples were positive for Chlamydia spp., and four Chlamydia species (C. pneumoniae, 41.7%, 20/48; C. psittaci, 22.9%, 11/48; C. gallinacea, 20.8%, 10/48; C. pecorum, 6.3%, 3/48) were identified. In a further study on a Yangzhou dairy farm, 64.8% (70/108) of the cows were positive for Chlamydia spp. C. pecorum was the intestinal endemic species (51/51, 100%), and C. gallinacea was the most frequent species in vaginal swabs (24/27, 88.9%), whole blood buffy coats (5/8, 62.5%) and milk (4/6, 66.7%). C. psittaci and C. pneumoniae were infrequently detected. DNA sequencing of the ompA gene demonstrated the presence of multiple in-herd C. pecorum serovars and single C. gallinacea and C. psittaci serovars which were identical with those of poultry from Yangzhou. This is the first report of C. gallinacea and C. pneumoniae in cattle. Further study is required to address the transmission of Chlamydia spp., in particular of C. gallinacea and C. pneumoniae from their natural hosts, and their potential pathogenic effect on health and production of cattle.

Highly Drug-Resistant Salmonella enterica Serovar Indiana Clinical Isolates Recovered from Broilers and Poultry Workers with Diarrhea in China.

Highly drug-resistant Salmonella enterica serovar Indiana became the most common serovar in broilers with diarrhea in China over the course of this study (15% in 2010 to 70% in 2014). While most S. Indiana isolates (87%, 384/440) were resistant to 13 to 16 of the 16 antibiotics tested, 89% of non-S. Indiana isolates (528/595) were resistant to 0 to 6 antibiotics. Class 1 integrons and IncHI2-type plasmids were detected in all S. Indiana isolates, but only in 39% and 1% of non-S. Indiana isolates.

One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species.

Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct Tm (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255 bp, Sudan 211 bp, Reston 192 bp, Taï Forest 166 bp, Bundibugyo 146 bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

First report of Rickettsia felis in China.

BACKGROUND: Rickettsia felis is a recently described flea-borne spotted fever group Rickettsia that is an emerging human pathogen. Although there is information on the organism from around the world, there is no information on the organism in China. METHODS: We used a commercial ELISA to detect antibodies reactive against R. felis in blood samples and developed a PCR to detect the gltA of the organism in blood samples and external parasites. RESULTS: We found reactive antibodies in people (16%; 28/180), dogs (47%; 128/271) and cats (21%; 19/90) and positive PCRs with DNA from people (0.1%; 1/822), dogs (0.8%; 8/1,059), mice (10%; 1/10), ticks (Rhipicephalus sanguineus; 10%; 15/146), lice (Linognathus setosus; 16%; 6/37), fleas (Ctenocephalides felis felis; 95%; 57/60) and mosquitoes (Anopheles sinensis, Culex pipiens pallens; 6%; 25/428), but not from cats (0/135) or canine fecal swabs (0/43). CONCLUSIONS: This is the first report of R. felis in China where there is serological and/ or PCR evidence of the organism in previously reported [people, dogs, cats, ticks (Rhipicephalus sanguineus), fleas (Ctenocephalides felis felis) and mosquitoes (Anopheles sinensis, Culex pipiens pallens)] and novel species [mice and lice (Linognathus setosus)].