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The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.

Background: Blastocystis is an enteric protozoan infecting humans and animals all over the world. Domestic animals play an important role in transmitting parasites to other domestic animals and humans. In the present study, a survey was conducted on Blastocystis among humans and domestic animals sharing habitats in northeastern China’ s Heilongjiang Province, in order to investigate the prevalence, the subtype distribution, as well as to evaluate the risk of the zoonotic transmission of Blastocystis isolates.Methods: A total of 314 (57 humans, 257 domestic animals) fecal samples were collected from 33 family households in three villages. The corresponding sequences of the barcode region of the SSU rRNA gene obtained in this study were subject to molecular analysis for subtype and allele identification of Blastocystis sp.Results: The prevalence of Blastocystis was 14.0% (8/57) in humans and 17.9% (46/257) in domestic animals. Eight PCR-positive human samples, 100% (8/8) were successfully subtyped, allowing the identification of the ST3, followed by ST1 and ST2. The 46 PCR-positive animal samples, 65.2% (30/46) were successfully subtyped, ST5 followed by ST1 in pigs, ST5 in goats; ST10, ST14 followed by ST3 in cattle; ST7 in chickens. Phylogenetic analysis showed that Blastocystis ST3 sequences from humans in two geographical locations formed two distinct clades. Alleles were identified using the Blastocystis 18S database and a total of 10 different alleles were found in six STs.Conclusions: The present study is the first description of the prevalence and subtype (allele) distribution of Blastocystis sp. by molecular analysis in humans and domestic animals in family units in China. Blastocystis ST2 in humans and ST5 in goats were reported in Heilongjiang Province for the first time. ST1 (a4) and ST3 (a34) overlaps were observed in humans and some domestic animal species (pig and cattle). The findings of potentially zoonotic subtypes in domestic animals suggest that these animals may serve as reservoirs of human Blastocystis sp. infections, thus help develop more efficient, targeted control strategies against human blastocystosis.

Charging load prediction of electric vehicles (EVs) is an important prerequisite for studying the interaction between electric vehicles and power grid. Aiming at the influence of traffic road network information on the driving rule of EVs, a spatial-temporal distribution prediction method of charging load for EVs based on dynamic traffic information is presented. In this methodology, given the characteristic of multiple intersections in the urban road network, a dynamic road network model with the impedance of the road section and the impedance of the node are established firstly. The corresponding interactive model of transportation network-distribution network is determined according to the scale of road network. And then, the origin destination (OD) matrix analysis method and the real-time Dijkstra dynamic path search algorithm are introduced to assign start-stop nodes and plan driving paths for EVs and simulate their dynamic driving process and charging behavior. At last, the EV path planning experiment and the charging load prediction experiment in typical regions are designed to verify the effectiveness and feasibility of the proposed strategy.

Blastocystis is a common protozoan found in the surveys of human and animal fecal specimens. Extensive genetic diversity has been observed within the genus Blastocystis. At least 17 subtypes (ST) have been identified in mammals and birds, nine of which (ST1 to ST9) have been identified in humans. In China, although there have been a few reports on Blastocystis infection in humans and many animal species, no epidemiological data are available in either humans or animals in northeastern China's Heilongjiang Province. To determine infection rates and subtype distributions of Blastocystis in pigs, cattle, sheep and goats, to understand genetic characterizations and to assess zoonotic possibility of Blastocystis isolates, 337 fecal specimens from livestock (68 from pigs, 147 from cattle, 109 from sheep and 13 from goats) were collected in northeastern China's Heilongjiang Province. Each of them was detected for Blastocystis by PCR amplification of the partial SSU rRNA gene. An average infection rate of Blastocystis was 7.7% (26/337), and the highest infection rate was found in 9.5% (14/147) in cattle, followed by 8.8% (6/68) in pigs and 5.5% (6/109) in sheep. There was an absence of Blastocystis in goats. Five Blastocystis subtypes were identified: ST5 (n=6) in pigs; ST3 (n=2), ST10 (n=10) and ST14 (n=2) in cattle; ST1 (n=1), ST5 (n=1), ST10 (n=3) and ST14 (n=1) in sheep. 38.5% (10/26) of Blastocystis isolates belong to potentially zoonotic subtypes based on the previous findings of ST1, ST3 and ST5 in humans. This is the first report of Blastocystis in pigs, cattle and sheep in northeastern China's Heilongjiang Province. ST1, ST5 and ST14 were identified in sheep for the first time. Due to the low infection rate of Blastocystis and the small percentage of potentially zoonotic subtypes in these livestock, there is a minimal risk of zoonotic transmission of Blastocystis.

Cryptosporidium skunk genotype is a zoonotic pathogen commonly identified in surface water. Thus far, no subtyping tool exists for characterizing its transmission in humans and animals and transport in environment. In this study, a subtyping tool based on the 60 kDa glycoprotein (gp60) gene previously developed for Cryptosporidium chipmunk genotype I was used in the characterization of Cryptosporidium skunk genotype in animal and storm runoff samples from a watershed in New York. Altogether, 17 positive samples from this watershed and 5 human and animal specimens from other areas were analyzed. We identified 14 subtypes of Cryptosporidium skunk genotype, 11 of which were seen in the watershed. In phylogenetic analysis, these subtypes belonged to 4 subtype families (XVIa, XVIb, XVIc, and XVId). No host-adapted subtypes were identified and the two subtypes in humans were genetically similar to some in raccoons, otters, and storm runoff samples from the watershed. The characteristics of gp60 protein sequences of the Cryptosporidium skunk genotype are similar to those of other Cryptosporidium species, but only its XVIb subtype family has a putative furin cleavage site. This subtyping tool might be useful in characterizing Cryptosporidium skunk genotype in clinical and environmental samples.

This is the protocol for the Nested PCR assay

Due to the rarity of human cases and the nonspecific clinical symptoms of dioctophymiasis, Dioctophyma renale infection is not well recognized and is easily neglected or misdiagnosed. Recently, we diagnosed a human case of dioctophymiasis accompanied by renal cancer. To enhance the understanding of human dioctophymiasis, this case is presented here, and a retrospective study of this disease was conducted based on relevant papers screened from PubMed and three Chinese databases. In the end, 32 papers describing 37 human cases of dioctophymiasis were assessed. These cases were distributed in ten countries of Asia, Europe, North America and Oceania, with the highest number in China (n = 22). The majority of the cases occurred in adults (91.9%, 34/37) and involved the kidneys (83.8%, 31/37). Ectopic parasitism mainly occurred in subcutaneous tissue (83.3%, 5/6). A proportion of 45.9% (17/37) of individuals had a history of eating raw or undercooked fish or frogs. The main clinical manifestations of human dioctophymiasis were loin pain (59.5%) and hematuria (59.5%). All the cases were diagnosed based on the morphological characteristics of eggs or adults in urine or tissue sections. Currently, there is no strictly defined therapeutic approach. This is the first retrospective analysis of human cases of dioctophymiasis. These review data will deepen our understanding of dioctophymiasis and help avoid misdiagnosis in clinical practice.

Background Enterocytozoon bieneusi is the most common microsporidian species causing diarrhea and other intestinal disorders in humans and animals. Like other infectious diseases, microsporidiosis usually disproportionately affects poor populations. In China, some ethnic minority areas remain poor. Currently, no information of E. bieneusi infection is available in minority populations. The present aims were to understand occurrence and genetic characterizations of E. bieneusi in ethnic minority groups from a poverty-stricken ethnic township in Yunnan Province, and to assess risk factors for E. bieneusi infection. Methodology/Principal findings 289 fecal specimens were collected from Yao people (one specimen each) with and without diarrhea, in Yunnan Province. E. bieneusi was identified and genotyped by PCR and sequence analysis of the ITS region of the rRNA gene. An average prevalence of 8.30% (24/289) was observed and four genotypes were identified—genotype Peru6 (n = 21) and three novel genotypes (one each). Genotype Peru6 was detected in two family members in each of three families. In a phylogenetic analysis, all of four genotypes fell into group 1 with zoonotic potential. The people owning individual pit toilets had a statistically higher prevalence of E. bieneusi (16.67%, 12/72) than those using public pit toilets (6.06%, 12/198). Conclusions/Significance This is the first report on occurrence and genetic characteristics of E. bieneusi in ethnic minority groups in China. Genotype Peru6 was found in humans in China for the first time and showed dominance in Yao people. The same genotype was found in some family members and all the genotypes fell into group 1, suggesting the possibility of anthroponotic and zoonotic transmissions. The majority (83.33%, 20/24) of E. bieneusi positive individuals did not present diarrhea. In any case, it is important to recognize their existence and the importance that asymptomatic individuals to E. bieneusi may have from an epidemiological point of view, as transmitters of this pathogen. The analysis of risk factors provides scientific evidence for the development of effective strategies for prevention and control of E. bieneusi infection., Author summary Enterocytozoon bieneusi is the most prevalent opportunistic microsporidian species causing diarrhea in humans and animals worldwide. Due to frequent occurrence of chronic or life-threatening diarrhea mainly in HIV/AIDS patients and others patients such as elderly people, transplant recipients and cancer patients, epidemiological studies of E. bieneusi focus on this susceptible population. Asymptomatic carriers of E. bieneusi are often neglected, and their role in the transmission of this microorganism is not paid enough attention. The prevalence of E. bieneusi is also actually related to the geographical locations, the populations and the socioeconomic conditions. The infectious diseases usually disproportionately affect poor populations. In China, although total economy has developed rapidly, there is the regional imbalance in economic development and some minority areas remain poor. Thus, we carried out a molecular epidemiology survey of E. bieneusi among 289 Yao people with and without diarrhea from a poverty-stricken ethnic township. We identified E. bieneusi in 8.30% (24/289) of people, with the majority (83.33%, 20/24) of positive individuals having no diarrhea. Meanwhile, zoonotic genotype Peru6 was found to be dominant in infected people. Our study emphasizes the importance and significance in epidemiological surveys of E. bieneusi in non-diarrheal people and assessment of risk factors for E. bieneusi infection.

Blastocystis is one of the most common intestinal parasites in humans worldwide. To understand its prevalence and to explore the risk factors for Blastocystis in humans in developing countries, a molecular epidemiological investigation of Blastocystis was conducted in ethnic minority groups on both sides of the China–Myanmar border. A total of 461 fecal specimens were collected from 289 Yao people in China and 172 Wa people in Myanmar, together with a questionnaire for each participant. Based on sequence analysis of the partial small subunit of ribosomal RNA (SSU rRNA) gene (barcode region or 260 bp region), an average prevalence of 6.29% (29/461) was observed, with 4.50% (13/289) in Yao people and 9.30% (16/172) in Wa people. Twenty-two Blastocystis isolates were successfully subtyped by sequence analysis of the barcode region. Three subtypes were identified: ST1 (n = 7), ST3 (n = 13) and ST4 (n = 2). A statistical difference in the prevalence of Blastocystis was only observed between children (12.37%, 12/97) and adults (4.95%, 16/323), and between not washing hands (11.02%, 14/127) and washing hands (4.76%, 15/315) after using toilets, emphasizing the importance and necessity of health education in people in the investigated areas, especially in children.

As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in av ariety of biomes, and the distribu- tion of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmis- sion of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi ,a n average infection rate of 31.9 % (29/91) was observed in deer, with 32.6 % (28/86) for sika deer, and 20 % (1/5) for red deer. Six ITS genotypes were identified: one known ge- notype BEB6 (n=20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n=5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD- II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans.

Cryptosporidium spp. and Enterocytozoon bieneusi are two prevalent opportunistic pathogens in humans and animals. Currently, few data are available on genetic characterization of both pathogens in rabbits in China. The aim of the present study was to understand prevalence and genetic characterization of Cryptosporidium spp. and E. bieneusi in rabbits. We collected 215 fecal samples from 150 Rex rabbits and 65 New Zealand White rabbits on two different farms in Heilongjiang Province, China. Cryptosporidium spp. and E. bieneusi were tested by polymerase chain reaction (PCR) and sequencing the partial small subunit of ribosomal DNA (SSU rDNA) and the internal transcribed spacer (ITS) region of rDNA, respectively. Cryptosporidium was detected in 3.3% (5/150) of Rex rabbits and 29.2% (19/65) of New Zealand White rabbits. All the 24 Cryptosporidium isolates were identified as C. cuniculus. Enterocytozoon bieneusi was only found in 14.7% (22/150) of Rex rabbits. Five known genotypes: CHN-RD1 (n = 12), D (n = 3), Type IV (n = 2), Peru6 (n = 1), and I (n = 1), and three novel ones CHN-RR1 to CHN-RR3 (one each) were detected. By analyzing the 60-kDa glycoprotein (gp60) gene sequences of C. cuniculus isolates, three subtypes were obtained: VbA28 (n = 2), VbA29 (n = 16), and VbA32 (n = 3). All these three C. cuniculus subtypes were reported previously in humans. Four known E. bieneusi genotypes have been found to be present in humans. The three novel ones fell into zoonotic group 1. The results suggest zoonotic potential of C. cuniculus and E. bieneusi isolates in rabbits.

In this paper, porous mullite/corundum ceramics with high porosity, high strength and low thermal conductivity were fabricated by foam-gelcasting method using fused mullite powder as raw material. Effects of sintering temperature on linear shrinkage, bulk density, apparent porosity, compressive strength and thermal conductivity of the porous mullite/corundum ceramics were researched. As sintering temperature increased from 1350 to 1550 °C, linear shrinkage increased from 2.6 to 9.5%, porosity decreased from 81 to 78%, bulk density increased from 0.59 to 0.69 g/cm3, compressive strength increased from 3.1 to 11.7 MPa and thermal conductivity increased from 0.14 to 0.35 W/m·K. Porous microstructure was complex, where large spherical pores contained small cellular pores on their internal walls.

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea in humans and animals. However, epidemiological data on E. bieneusi infections in birds are relatively scare worldwide, especially in China. To understand the prevalence and genetic diversity of E. bieneusi in birds and to assess the zoonotic potential of bird-derived E. bieneusi isolates, 194 fecal specimens from Gruidae, Anatidae and Columbidae in Heilongjiang Province, China, were analyzed by PCR and sequencing of the single internal transcribed spacer region of the rRNA gene. The average prevalence of E. bieneusi was 22.2%, with 12.5% for Gruidae, 15.9% for Anatidae and 44.0% for Columbidae. Altogether seven genotypes of E. bieneusi were identified, including four known genotypes-Peru6 (n=29), BEB6 (n=5), D (n=3) and EbpA (n=1)-and three novel genotypes named CHN-B1 (n=1), CHN-B2 (n=3) and CHN-B3 (n=1). All the known genotypes obtained here were previously detected in humans. All the novel genotypes were clustered into the zoonotic group 1 in phylogenetic analysis. The results indicate that these birds may play a potential role in the transmission of E. bieneusi to humans.

Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans. It has been found in a wide range of animals and is considered an important zoonotic pathogen. To date, little information is available on the role that cattle play in the epidemiology of human microsporidiosis caused by E. bieneusi in China. In this study, 133 fecal specimens from dairy cattle were collected in Heilongjiang Province, China. Enterocytozoon bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA gene, with 30.1% positive. Nine ITS genotypes were identified: six known genotypes— O( n = 26), EbpA (n = 2), I (n = 2), J (n = 2), D (n = 1) and BEB4 (n = 1)—and three novel genotypes named as CC-I to CC-III (two each). Genotype O was identified in cattle for the first time. The observation of all the six known genotypes here reported previously in humans, and also the fact of all the three novel genotypes (CHNDC1 to CHN-DC3) falling into zoonotic group 1, indicate the possibility of cattle in the transmission of E. bieneusi to humans.

Enterocytozoon bieneusi is an emerging and clinically significant enteric parasite infecting humans and animals and can cause life-threatening diarrhea in immunocompromised people. Pigs are considered to be one of the main reservoir hosts of E. bieneusi based on their high prevalence rates and zoonotic genotypes in pigs. As an opportunistic pathogen, E. bieneusi infection of pigs can be inapparent, which leads to neglect in detecting this parasite in pigs and assessing the epidemiological role of pigs in the transmission of human microsporidiosis. In the present study, 95 healthy pigs aged 2 or 3 months were randomly selected from three areas in Heilongjiang Province, China. E. bieneusi isolates were identified and genotyped based on the small-subunit (SSU) rRNA and internal transcribed spacer (ITS) regions of the rRNA gene by PCR and sequencing. A high prevalence of E. bieneusi was observed, 83.2% (79/95) at the SSU rRNA locus versus 89.5% (85/95) at the ITS locus. Ten ITS genotypes were obtained, comprising six known genotypes—EbpA ( n = 30), D ( n = 19), H ( n = 18), O ( n = 11), CS-1 ( n = 1), and LW1 ( n = 1)—and four novel genotypes named HLJ-I to HLJ-IV; 70.6% (60/85) of E. bieneusi genotypes were zoonotic (genotypes EbpA, D, and O). The findings of a high prevalence of E. bieneusi in pigs and a large percentage of zoonotic genotypes indicate that pigs may play a role in the transmission of E. bieneusi to humans and may become an important source of water contamination in our investigated areas.

Cattle are the main reservoir host of C. andersoni, which shows a predominance in yearlings and adults of cattle. To understand the subtypes of C. andersoni and the population genetic structure in Heilongjiang Province, fecal specimens were collected from 420 dairy cattle and 405 beef cattle at the age of 12–14 months in eight cattle farms in five areas within this province and were screened for the presence of Cryptosporidium oocysts by microscopy after Sheather’s sugar flotation technique. The average prevalence of Cryptosporidium spp. was 19.15% (158/825) and all the Cryptosporidium isolates were identified as C. andersoni by the SSU rRNA gene nested PCR-RFLP using SspI, VspI and MboII restriction enzymes. A total of 50 C. andersoni isolates were randomly selected and sequenced to confirm the RFLP results before they were subtyped by multilocus sequence typing (MLST) at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16). Four, one, two and one haplotypes were obtained at the four loci, respectively. The MLST subtype A4,A4,A4,A1 showed an absolute predominance and a wide distribution among the six MLST subtypes obtained in the investigated areas. Linkage disequilibrium analysis showed the presence of a clonal population genetic structure of C. andersoni in cattle, suggesting the absence of recombination among lineages. The finding of a clonal population genetic structure indicated that the prevalence of C. andersoni in cattle in Heilongjiang Province is not attributed to the introduction of cattle. Thus, prevention and control strategies should be focused on making stricter measures to avoid the occurrence of cross-transmission and re-infection between cattle individuals. These molecular data will also be helpful to explore the source attribution of infection/contamination of C. andersoni and to elucidate its transmission dynamics in Heilongjiang Province, even in China.

Background Cryptosporidium spp. are common parasites of humans and animals. Farm animals, especially pre-weaned calves, are considered to be one of main animal reservoir hosts of Cryptosporidium in the transmission of human cryptosporidiosis. The aim of this study was to determine the distribution and genotypes of Cryptosporidium spp. in pre-weaned calves using molecular tools and to assess zoonotic transmission and elucidate the public health significance in northeastern China. Methodology/Principal Findings A total of 151 fecal specimens from pre-weaned calves were collected in Heilongjiang Province and were screened for Cryptosporidium by PCR. The average prevalence of Cryptosporidium was 47.68% (72/151). Cryptosporidium spp. were characterized by DNA sequencing of the small subunit (SSU) rRNA gene and the 60-kDa glycoprotein (gp60) gene. Based on the SSU rRNA gene, five Cryptosporidium spp. were identified, including C. bovis (n = 34), C. andersoni (n = 26), C. ryanae (n = 5), C. meleagridis (n = 5) and C. parvum (n = 2). The SSU rRNA nucleotide sequences were identical to each other, respectively, within C. ryanae, C. parvum, C. meleagridis and C. andersoni. Four types of C. bovis were found in the SSU rRNA gene, with two novel types. The gp60 gene was successfully sequenced in one C. parvum isolate and three C. meleagridis isolates, with IIdA19G1 for C. parvum and IIIeA22G2R1 for C. meleagridis. Conclusion/Significance Molecular analysis indicates that Cryptosporidium spp. are endemic in pre-weaned calves in Heilongjiang Province. The findings of C. parvum and C. meleagridis suggested the possibility of zoonotic transmission and public health significance. The transmission dynamics of C. parvum and C. meleagridis needed to be clarified by further molecular epidemiologic studies from humans and animals. Whether calves could act as the natural reservoirs of C. meleagridis needed to be confirmed by more systematic experimental infection studies.

Background Giardia duodenalis is a widespread intestinal protozoan of both humans and mammals. To date, few epidemiological studies have assessed the potential and importance of zoonotic transmission; and the human giardiasis burden attributable to G. duodenalis of animal origin is unclear. No information about occurrence and genotyping data of sheep and goat giardiasis is available in China. The aim of the present study was to determine prevalence and distribution of G. duodenalis in sheep and goats in Heilongjiang Province, China, and to characterize G. duodenalis isolates and assess the possibility of zoonotic transmission. Methodology/Principal Findings A total of 678 fecal specimens were collected from sheep and goats on six farms ranging in age from one month to four years in Heilongjiang Province, China. The average prevalence of G. duodenalis infection was 5.0% (34/678) by microscopy after Lugol's iodine staining, with 5.6% (30/539) for the sheep versus 2.9% (4/139) for the goats. Molecular analysis was conducted on 34 G. duodenalis isolates based on the triosephosphate isomerase (tpi) gene. 29 tpi gene sequences were successfully obtained and identified as assemblages A (n = 4), B (n = 2) and E (n = 23). High heterogeneity was observed within assemblage E at the tpi locus, with five novel subtypes found out of seven subtypes. Two subtypes of assemblage A were detected, including subtype AI (n = 3) and a novel subtype (designated as subtype AIV) (n = 1). Two assemblage B isolates were identical to each other in the tpi gene sequences. Conclusions/Significance This is the first report of G. duodenalis infections in sheep and goats in China. The present data revealed the unique endemicity on prevalence, distribution and genetic characterization of G. duodenalis in sheep and goats in Heilongjiang Province. The findings of assemblages A and B in sheep and goats implied the potential of zoonotic transmission., Author Summary Giardiasis is a kind of zoonotic disease with global distribution. Due to the great number of asymptomatic giardiasis cases, human giardiasis is often underreported. The sources of infection of giardiasis are feces of humans and mammals with the pathogen being transmitted by the fecal-oral route. In this study, we described the occurrence of sheep and goat giardiasis and genetic charaterizations of G. duodenalis isolates in Heilongjiang Province, China. The average infection rate was 5.0% (34/678), with 5.6% (30/539) for the sheep versus 2.9% (4/139) for the goats. G. duodenalis assemblages and subtypes were genetically diagnosed by sequence analysis. Three assemblages were successfully identified out of 29 tpi gene sequences, with the percentages 13.8% (4/29), 6.9% (2/29), 79.3% (23/29) for assemblages A, B and E, respectively. Five novel subtypes were found out of seven subtypes of assemblage E. Two subtypes of assemblage A were detected, with one belonging to a novel subtype and the other belonging to assemblage AI. Two assemblage B isolates were identical to each other at the tpi locus. Prevalence, distribution and genetic characteristics of subtypes of G. duodenalis in sheep and goats appear to be unique in the areas examined. The sheep and goats infected with assemblages A and B have important public heath significance.

Background In the enteropathogenic Yersinia species, RovA regulates the expression of invasin, which is important for enteropathogenic pathogenesis but is inactivated in Yersinia pestis. Investigation of the RovA regulon in Y. pestis at 26°C has revealed that RovA is a global regulator that contributes to virulence in part by the direct regulation of psaEFABC. However, the regulatory roles of RovA in Y. pestis at 37°C, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood. Methodology/Principal Findings The transcriptional profile of an in-frame rovA mutant of Y. pestis biovar Microtus strain 201 was analyzed under type III secretion system (T3SS) induction conditions using microarray techniques, and it was revealed that many cell-envelope and transport/binding proteins were differentially expressed in the ΔrovA mutant. Most noticeably, many of the T3SS genes, including operons encoding the translocon, needle and Yop (Yersinia outer protein) effectors, were significantly up-regulated. Analysis of Yop proteins confirmed that YopE and YopJ were also expressed in greater amounts in the mutant. However, electrophoresis mobility shift assay results demonstrated that the His-RovA protein could not bind to the promoter sequences of the T3SS genes, suggesting that an indirect regulatory mechanism is involved. Transmission electron microscopy analysis indicated that there are small loose electron dense particle-like structures that surround the outer membrane of the mutant cells. The bacterial membrane permeability to CFSE (carboxyfluorescein diacetate succinimidyl ester) was significantly decreased in the ΔrovA mutant compared to the wild-type strain. Taken together, these results revealed the improper construction and dysfunction of the membrane in the ΔrovA mutant. Conclusions/Significance We demonstrated that the RovA regulator plays critical roles in the construction and functioning of the bacterial membrane, which sheds considerable light on the regulatory functions of RovA in antibiotic resistance and environmental adaptation. The expression of T3SS was upregulated in the ΔrovA mutant through an indirect regulatory mechanism, which is possibly related to the altered membrane construction in the mutant.

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.