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Introduction: Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques. Methods: In this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed. Results: We showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 μg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists. Discussion: In conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae. [ABSTRACT FROM AUTHOR]

Our study investigated the prevalence of feline herpesvirus‐1 (FHV‐1), feline calicivirus (FCV), feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) in captive Siberian tigers in Northeastern China. A total of 324 blood samples and 33 nasopharyngeal swab samples of Siberian tigers collected from 2019 to 2021 in three cities were investigated by nested PCR. The results showed that 28.1% (91/324) tigers were infected with at least one virus; the positive rates of FHV‐1, FCV, and FIV were 17.3%, 13.6%, and 0.9%, respectively; and the coinfection prevalence was 13.2%. No FeLV‐positive sample was detected. And we found that the blood is the best for FCV, FIV, and FeLV detection, but nasopharyngeal swabs for FHV‐1. By comparing the gD genes, TK gene, and gI gene of FHV‐1, the homology of the three FHV‐1 positive strains in this study was found to be 91.5%–99.9% shared with tigers and domestic cats. Based on a comparison of the nucleic acid sequences of 13 FCV strains, we found that the homology of strain HB‐1926 with the other strains in this study was only about 77.7%, but shared 99.3% and 98.6% homology with Urnaba strain in American cat and TG1 strain in Chinese tiger, respectively. However, the other 12 FCV strains shared 87.1%–87.5% homology compared with the Chinese domestic cats. Phylogenetic tree analysis showed that the HB‐1926 strain was not in the same clade as other strains. The fragments gag‐p26, pol‐RT, and pol‐RNAse of Siberian tiger FIV shared more than 99% homology than domestic cats FIV subtype A. This study demonstrated that captive Siberian tigers in Northeastern China were exposed to FHV‐1, FCV, and FIV, and it is necessary to develop more effective vaccines and improve daily management measures. [ABSTRACT FROM AUTHOR]

Equine Piroplasmosis (EP) is a tick‐borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real‐time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real‐time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real‐time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double‐quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single‐quenched probes. The newly developed real‐time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real‐time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real‐time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real‐time PCR assay and the nPCR assay or the previous developed real‐time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co‐infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses. [ABSTRACT FROM AUTHOR]

• The five subtypes of C. parvum we detected were all new subtypes. • All the positive samples were C. parvum. • The overall prevalence was 12.85% in diarrhea children. • There were significant differences in the prevalence at different ages. • Positive samples were detected throughout the year. Cryptosporidia (Cryptosporidium) is a protozoan that is widely parasitic in the intestinal cells of humans and animals, and it is also an important zoonotic parasite. However, there is no epidemiological investigation on Cryptosporidium spp. infection in infants with diarrhea of Inner Mongolia, the largest livestock region in China. To investigate the prevalence of Cryptosporidium , 2435 fresh fecal samples were collected from children with diarrhea in Inner Mongolia Maternal and Child Health Care Hospital. Molecular characterization of Cryptosporidium was carried out based on its 18S rRNA and gp60 gene sequences. The overall prevalence was 12.85% (313/2435), and in Hohhot (12.15%), it was lower than that in the surrounding city (14.87%) (P < 0.05). Moreover, Cryptosporidium was detected in different seasons and sexes. Concerning the age of children with diarrhea, the prevalence of those age groups between 0 and 1 was obviously lower than others, and there were significant differences in the prevalence at different ages (P < 0.001). Analysis of the 18S rRNA gene sequence revealed that all the positive samples were Cryptosporidium parvum , and there were 5 subtypes (IIdA23G3, IIdA24G3, IIdA24G4, IIdA25G3, and IIdA25G4). To the best of our knowledge, the above subtypes have not been reported. Our results provide a relevant basis for control and education on food safety and foodborne illness prevention. [ABSTRACT FROM AUTHOR]

The fact that wild felines are carriers of pernicious infectious viruses should be a major concern due to the potential cross-species transmission between the felines and human or domestic animals. However, studies on the virus in the captive wild felines, especially in tigers, are thin on the ground. In this study, we screened four infectious viruses, namely, feline parvovirus (FPV), feline coronavirus (FCoV), canine distemper virus (CDV), and influenza A virus (IAV), in the blood samples of 285 captive Siberian tigers (Panthera tigris altaica) and in the spleen samples of two deceased lions (Panthera leo), which were collected from 2019 to 2021 in three Siberian Tiger Parks from the northeast of China. Nucleic acids isolated from the blood samples collected from tigers and the spleen samples collected from two deceased lions were positive for FPV by PCR, and the positive rate was 4.6% (13/285) in tigers. Furthermore, the VP2 gene of FPV was amplified by nested PCR, and the sequences of the VP2 gene from these six FPV positive strains shared 98.3–99.9% homology with the reference. The key amino acid sites of VP2 protein were consistent with that of FPV reference strains. Phylogenetic analysis based on the VP2 gene showed that in this study, FPV-positive strains were grouped within the FPV clade and closely related to the Asian strains clade. The results of this study showed that FPV circulated in the captive Siberian tigers and lions in northeastern China and provided valuable information for the study of FPV epidemiology in wild felines. Therefore, we suggest that regular antibody monitoring and booster immunization for tigers should be performed. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a regional infectious disease caused by the bite of Leishmania -carrying sandflies. The clinical symptoms include prolonged fever, spleen enlargement, anemia, emaciation, leukopenia, and increased serum globulin levels. If not appropriately treated, patients may die of complications caused by leishmaniasis within 1–2 years after the onset of the illness. Therefore, further investigation of the mechanisms of infection by this pathogen is required. Here, an epidemiological study of Leishmania carriers was conducted. The potential mechanism of infection through domestic animals as carriers of the parasite was investigated to identify potential reservoir hosts for Leishmania. Methods: The rK-39 strip test was performed on blood samples from previously infected patients. Blood samples were collected from the patients and their families. The blood, liver, spleen, and diaphragm muscle samples were collected from livestock. To perform nested polymerase chain reaction (PCR), DNA was extracted and the internal transcribed spacer sequence was used. The amplified products were then subjected to restriction fragment length polymorphism and phylogenetic analyses. Results: Among previously infected patients, 40% (12/30) showed positive results in the rK-39 strip test. The nested PCR positive rates for previously infected patients/relatives and livestock samples were 86% (77/90) and 80% (8/10), respectively. Moreover, the phylogenetic analysis showed that the pathogen was Leishmania infantum. Dogs, patients, and domesticated animals carrying Leishmania were found to be a potential source of infection for leishmaniasis. Conclusions: The results of this study provide a basis for developing disease prevention and control strategies for leishmaniasis. [ABSTRACT FROM AUTHOR]

Neospora caninum is a protozoan parasite which can infect many mammals and birds with a worldwide distribution. However, no molecular data are available about the occurrence of N. caninum in pigs. In this study, the serological and molecular prevalence of N. caninum infection in farmed pigs were investigated in Hunan province, China, between January 2017 and December 2018. A total of 1,500 serum samples collected from 10 herds in Hunan province were evaluated using a competitive‐inhibition enzyme‐linked immunoassay assay (cELISA). The overall seroprevalence of N. caninum in the examined pigs was 1.9%. The seroprevalence of N. caninum ranged from 0.3% to 4.6% among different regions in Hunan province of China (p <.05). DNA was extracted from brain samples, and the Nc‐5 gene and ITS‐1 region were amplified and then sequenced. Three (0.5%) of the examined 600 brain tissues were found to contain N. caninum DNA. Our phylogenetic analyses indicated that N. caninum samples were classified into two distinct groups. Although the prevalence is low within the pig groups investigated, our results revealed the emergence of N. caninum infection in pigs in China. The finding of the present study provides molecular evidence that the pigs are the natural intermediate host of N. caninum and may have major epidemiological importance. [ABSTRACT FROM AUTHOR]

The occurrence of Cryptosporidium and Giardia species in slaughter, sewage and river waters of the Qinghai Tibetan Plateau Area (QTPA), China, was investigated. A total of 456 samples were collected from different locations in the QTPA to study the contamination rates of Cryptosporidium spp. and Giardia via PCR and subsequent sequence analysis. Ten samples were Cryptosporidium positive, and 97 were Giardia positive, as confirmed by PCR amplification of the SSU rRNA gene. The percentages of positive Cryptosporidium and Giardia detection were 2.2% (10/456) and 21.3% (97/456), respectively. Cryptosporidium was detected in only sewage and river waters. Six species of Cryptosporidium were identified: Cryptosporidium hominis (n = 5), C. andersoni (n = 1), C. environmental (n = 1), C. struthionis (n = 1), C. canis (n = 1), and C. parvum (n = 1). G. duodenalis assemblage A was identified in almost all positive samples (n = 96), and one sample harboured G. duodenalis assemblage E. The results suggest that Cryptosporidium and Giardia species circulate through the aqueous environment and different hosts. Therefore, we strongly recommend that the local government and health authorities in China undertake control measures to reduce the contamination of water sources by these protozoa to protect the health of humans and animals. [ABSTRACT FROM AUTHOR]

Anaplasmosis is a highly prevalent tick-borne intracellular bacterial disease that affects various host species globally, particularly ruminants in tropical and subtropical regions. However, information regarding the distribution and epidemiology of anaplasmosis in small and large ruminants on Hainan Isalnd is limited. To address this knowledge gap, the present study aimed to assess the occurrence of Anaplasma spp. infections in goats (N = 731) and cattle (N = 176) blood samples using nested PCR and conventional PCR based assays. The results revealed an overall prevalence of 30.1% in goats and 14.8% in cattle. The infection rates of A. bovis, A. phagocytophilum, A. ovis and A. capra in goat samples were 22.7%, 13.8%, 2.0% and 3.4%, respectively, while the infection rates of A. bovis, A. phagocytophilum and A. marginale in cattle samples were 11.4%, 6.3% and 5.7%, respectively. A. bovis exhibited the highest prevalence among the Anaplasma spp. in both goat and cattle samples. In addition, the most frequent co-infection was the one with A. phagocytophilum and A. bovis. It was found that the age, sex and feeding habits of cattle and goats were considered to be important risk factors. Evaluation of the risk factor relating to the rearing system showed that the infection rate for the free-range goats and cattle was significantly higher when compared with stall-feeding system.This study represents one of the largest investigations on the distribution, prevalence, and risk factors associated with Anaplasma infection in ruminants on Hainan Island, highlighting a higher circulation of the infection in the region than previously anticipated. Further reasesrch is necessary to investigate tick vectors, reservoir animals, and the zoonotic potential of the Anaplasma spp. in this endemic region of Hainan Island., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Little is known about the presence and possible role of Porphyromonas gingivalis (P. gingivalis) in nasopharyngeal carcinoma (NPC), its co-infection with Epstein-Barr virus (EBV), or their association with clinical characteristics of patients with NPC in Central China, where NPC is non-endemic. A total of 45 NPC formalin-fixed paraffin-embedded (FFPE) tissues were retrospectively analyzed using immunohistochemistry (IHC) and a nested PCR combined with DNA sequencing to detect the presence of P. gingivalis, and using reverse transcription-quantitative PCR to detect the presence of EBV. Clinical data including EBV and P. gingivalis status were associated with overall survival (OS). All tumors were undifferentiated, non-keratinizing carcinomas, of which 40/45 (88.9%) were positive for EBV (EBV+), 26/45 (57.8%) were positive for P. gingivalis (by IHC), and 7/45 (15.6%) were positive for P. gingivalis DNA (P. gingivalis+). All seven P. gingivalis DNA-positive NPCs were co-infected with EBV. The 5-year survival rates of the patients with EBV-/P. gingivalis-, EBV+/P. gingivalis-, and EBV+/P. gingivalis+ tumors were 60.0% (3/5), 39.4% (13/33) and 42.9% (3/7), respectively. No significant difference was found between the OS of NPC patients among the different infection groups (P=0.793). In conclusion, to the best of our knowledge, this is the first study to describe and confirm the presence of P. gingivalis in FFPE tissues from patients with NPC. P. gingivalis was found to co-exist with EBV in NPC tumor tissues, but is not etiologically relevant to NPC in non-endemic areas, such as Central China. [ABSTRACT FROM AUTHOR]

Introduction: Mycoplasma pneumoniae (M. pneumoniae) is the most common atypical pathogen that causes respiratory infections in humans. Laboratory tests are important in the diagnosis of M. pneumoniae because of the atypical features in clinical signs and symptoms. Nowadays, both the P1 adhesin gene and 16S ribosomal (r) RNA (rRNA) gene of M. pneumoniae have been widely detected by polymerase chain reaction (PCR). The purpose of the present study was to evaluate the most suitable target in the detection of M. pneumonia via nested PCR. Methodology: Both the P1 adhesin gene and 16S rRNA gene for nested PCR reaction conditions were optimized through an orthogonal test and single-factor experiment. Then, the sensitivity of the two sets of targets was evaluated. Finally, based on the optimal conditions, 55 clinical samples of throat swabs collected from adult patients in 2013 were examined by established nested PCR. Result: The results revealed that PCR detection of the 16S rRNA gene was more sensitive than the P1 adhesin gene because the detection limits for both the P1 gene and 16S rRNA gene were at least 100 fg and 10 fg of M. pneumoniae DNA, respectively. Furthermore, the positive rate for detection of the 16S rRNA gene (30/55; 54.5%) was higher than that of the P1 adhesin gene (25/55; 45.5%). Conclusion: Our results indicate that the 16S rRNA gene is more suitable for diagnosis of M. pneumoniae infection than the P1 adhesin gene due to its higher sensitivity and positive rate in clinical samples. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp. and Enterocytozoon bieneusi are important protists in a wide range of vertebrate hosts, causing diarrheal diseases. Cattle are considered potential reservoirs of Cryptosporidium infection in humans, although their role in the transmission of E. bieneusi is not clear. In the present work, 793 fecal specimens from dairy cattle, native beef cattle, and water buffaloes on 11 farms in China were examined for the presence of Cryptosporidium spp. and E. bieneusi using nested PCR targeting the small subunit (SSU) rRNA gene of Cryptosporidium spp. and the internal transcribed spacer (ITS) of E. bieneusi . For Cryptosporidium , 144/446 (32.3%) dairy cattle, 44/166 (26.5%) beef cattle, and 43/181 (23.8%) water buffaloes were PCR-positive. Sequence analysis was successful for 213 of the 231 Cryptosporidium -positive isolates; among them 94 had Cryptosporidium andersoni , 61 had Cryptosporidium bovis , 54 had Cryptosporidium ryanae , 2 had a Cryptosporidium suis -like genotype, and 2 had mixed infections of C. bovis and C. ryanae . In dairy and beef cattle, C. andersoni and C. bovis were the most common species, whereas C. ryanae was the dominant species in water buffaloes. The latter species produced SSU rRNA sequences different between cattle and water buffaloes. For E. bieneusi , the infection rate of E. bieneusi in dairy cattle, beef cattle and water buffaloes was 4.9%, 5.4% and 2.2%, respectively. All 35 E. bieneusi -positive specimens were successfully sequenced, revealing the presence of four genotypes: three Group 2 genotypes previously reported in cattle as well as humans (I, J and BEB4) and one Group 1 genotype recently reported in yaks (CHN11). Genotypes I and J were the most common genotypes in dairy and beef cattle, while genotype CHN11 was the only genotype seen in water buffaloes. Thus, the distribution of Cryptosporidium spp. and E. bieneusi in water buffaloes might be different from in dairy and beef cattle in China. These findings indicate that some of the Cryptosporidium species and all four E. bieneusi genotypes identified in bovine animals in the study areas may have zoonotic potential. [ABSTRACT FROM AUTHOR]

Abstract: Compared with dairy and beef cattle, few data are available on the occurrence and distribution of Cryptosporidium species in yaks, which live in a very different habitat. In this study, 327 fecal specimens were collected from yaks in 4 counties in Qinghai Province of China and screened for Cryptosporidium by nested PCR analysis of the 18S rRNA gene. A total of 98 (30.0%) specimens were positive for Cryptosporidium. The occurrence of Cryptosporidium varied significantly among age groups; infection rates were 49.3% in weaned calves, 31.7% in yearlings, and 17.4% in adults. PCR products of all Cryptosporidium-positive specimens were successfully sequenced, with 56 specimens (57.1%) having C. bovis, 33 (33.7%) having C. ryanae, 2 (2.0%) having C. andersoni, 1 (1.0%) having C. ubiquitum, 1 (1.0%) having C. xiaoi, 2 (2.0%) having a novel genotype, and 3 (3.1%) having mixed infections of C. bovis and C. ryanae. There were some age-related differences in the distribution of Cryptosporidium species in post-weaned yaks examined. To our knowledge, this is the first report of C. andersoni, C. ubiquitum, C. xiaoi and a novel Cryptosporidium genotype in yaks. [Copyright &y& Elsevier]

Background: China is implementing a National Malaria Elimination Programme. A high proportion of clinically diagnosed malaria cases is reported in some provinces of China. In order to understand the exact situation and make clear the nature of these patients, it is of much importance to make case verifications, particularly from the pathogenic perspective. Methods: Guizhou Province was targeted because of its high proportion of clinically diagnosed malaria cases. After random selection of around 10% of malaria cases from 1 May 2011 to 30 April 2012, reported through the national web-based case reporting system from this province, field verifications were made on 14-17 May 2012 as follows. Firstly, the reported information of each case was rechecked with the onsite case registrations and investigation forms, and an in-depth interview was conducted with each patient. Secondly, the patient's blood smears kept by local CDC were cross-checked microscopically by a national experienced microscopist. Thirdly, two kinds of polymerase chain reaction (PCRs). including Tag-primer nested/multiplex PCR (UT-PCR) based on cytochrome oxidase gene (cox I) and nested PCR based on 18s rRNA gene were performed simultaneously using local CDC kept filter paper of dry blood samples to identify the Plasmodium spp. Results: Twelve out of 152 malaria cases were selected, including nine clinically diagnosed malaria cases, two confirmed falciparum malaria cases and one confirmed vivax malaria case. The original case documents on the site were completely in conformity with their reported data, and all the patients recalled their malaria symptoms and being cured only after consuming the corresponding anti-malarial drugs. Moreover, the re-examination results of microscopy and PCR were exactly in agreement with the original tests. Discussion: No inconsistent results were found against the reported case information in the present study and the reasons for clinically diagnosed patients remains unclear. Uniform and standardized sample collection and processing should be trained among clinicians, more sensitive and specific techniques should be explored to used in malaria diagnosis. A further study is needed in order to be more observationally focussed rather than retrospective. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is a widespread opportunistic pathogen found in humans and domestic animals, including cattle that poses a public health risk. This study was performed to evaluate the prevalence, genotypic diversity, and zoonotic potential of E. bieneusi among children and calves in Bangladesh. A total of 998 fecal samples were collected from children (n = 299) and calves (n = 699) and screened by nested PCR and sequencing of the ribosomal internal transcribed spacer (ITS). The overall prevalence of E. bieneusi infection was 6.4% in children and 7.9% in calves. ITS sequence analysis of 74 isolates revealed 10 genotypes, including eight known genotypes (A, D, Type IV, PigEBITS7, I, J, BEB4, and BEB6) and two new genotypes (BANEB1 and BANEB3). Specifically, genotypes A, D, Type IV, PigEBITS7, BANEB1, and BANEB3, and genotypes D, PigEBITS7, I, J, BEB4, and BEB6 were detected in children and calves, respectively. Among them, genotypes D and I were dominant genotypes in children and calves, respectively. The genotypes D and PigEBITS7 were found in both children and calves, with PigEBITS7 being observed for the first time in calves. In phylogenetic analysis, six genotypes (A, D, Type IV, PigEBITS7, BANEB1, and BANEB3), detected in 39.2% of the isolates, belonged to zoonotic Group 1. The remaining four genotypes I, J, BEB4, and BEB6 were clustered in Group 2 and are common members of the group with zoonotic potential. To the best of our knowledge, this study provides the first report of E. bieneusi infection in calves in Bangladesh and also the first molecular characterization of the parasite in children and calves in this country. Two new genotypes in children have been found, which is noteworthy. Furthermore, the presence of zoonotic genotypes indicates that cattle may serve as reservoirs for E. bieneusi, which can be a source of human microsporidiosis., (© 2021 Wiley-VCH GmbH.)

Abstract: Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20μL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong). [Copyright &y& Elsevier]

Background: Many studies have suggested that hepatitis B virus (HBV) genotypes show not only geographical distribution and race specificity, but also are associated with disease progression and response to interferon treatment. The objective of this study was to develop a nested polymerase chain reaction (nPCR) assay for genotypes A-D and subgenotypes B1, B2, C1 and C2 of hepatitis B virus (HBV) and to investigate the distribution characteristics of HBV genotypes/subgenotype in China. Methods: After redesigning the primers and optimizing the reaction conditions using common Taq polymerase, the sensitivity, specificity and reproducibility of the method were evaluated using plasmids and serum samples. In total, 642 serum samples from patients with chronic HBV infection were applied to investigate the distribution of HBV genotype and subgenotype in China. Results: The genotype and subgenotype could be identified when the HBV DNA load of a sample was ≥102.3 IU/mL. For the 639 successfully genotyped samples, the sequencing results of 130 randomly selected samples (20.3%, 130/639) were consistent with those of the nPCR method. The present study showed that HBV genotype B (11.2%, 72/642), C (68.2%, 438/642) and D (7.2%, 46/642) were circulating in China, while genotype C was the dominant strain except for western region where genotype D was the prevalent strain. The main subgenotypes of genotypes B and C were B2 (87.5%, 63/72) and C2 (92.9%, 407/438), respectively. Conclusions: The low-cost nPCR method would be a useful tool for clinical and epidemiological investigation in the regions where genotypes A-D are predominant. [ABSTRACT FROM AUTHOR]

Summary: This study explored the importance of hepatitis B virus infection in cholangiocarcinoma pathogenesis in northern China. The clinical data of 66 patients with cholangiocarcinoma were analyzed. The hepatitis B virus gene was amplified using nested polymerase chain reaction, and the hepatitis B virus–related antigen was detected using immunohistochemistry in formalin-fixed, paraffin-embedded tissue from patients with intrahepatic cholangiocarcinoma (n = 23) and extrahepatic cholangiocarcinoma (n = 43). Hepatitis B surface antigen seropositivity was found in 52.2% (12/23) of intrahepatic cholangiocarcinoma cases and 13.9% (6/43) of extrahepatic cholangiocarcinoma cases. Hepatitis B virus DNA (X region) was detectable in 34.8% (8/23) of intrahepatic cholangiocarcinoma cases. Hepatitis B surface antigen and/or hepatitis B core antigen was detectable in 30.4% (7/23) of intrahepatic cholangiocarcinoma cases. All cases with detected viral protein were also positive for hepatitis B virus DNA. In contrast, no hepatitis B virus antigens or hepatitis B virus gene was detected in any of the 43 extrahepatic cholangiocarcinoma cases. Our findings strongly suggest that chronic hepatitis B virus infection is a significant risk factor for intrahepatic cholangiocarcinoma, but not for extrahepatic cholangiocarcinoma, in northern China. Hepatitis B virus infection is potentially independently associated with intrahepatic cholangiocarcinoma. [Copyright &y& Elsevier]

Abstract: Infection with human papillomavirus (HPV) could be a suspected or potential modifiable risk factor in esophageal carcinoma (EC) but findings have not been consistent. We therefore investigated the epidemiology of HPV infection and integration in the pathogenesis of esophageal carcinoma (EC) in the Shantou region, China. This was a retrospective study involving nested PCR to evaluate HPV presence, HPV genotyping, and analyzing HPV-16 integration status in esophageal tumor tissues (n =106) and paired tumor-adjacent normal tissues, as well as normal esophagus tissue from control subjects (n =100). The detection rates of HPV DNA in EC and tumor-adjacent tissue were significantly higher than that in normal controls (77.4% and 80.2% vs. 33.0%). HPV infection was mainly found in adults, ages 35–47 years old, and the infection rate was negatively associated with the age of EC patients (P-trend<0.05). In addition, the HPV infection rates in patients who smoked was 3.27 times higher than in non-smoking patients (84.9% vs. 67.4%, P <0.05) but was not associated with gender, alcohol consumption, tumor grade or lymph-node metastasis of EC patients. The distribution of HPV genotypes in patients from high to low proportion was HPV-16, -58, -18, -33, -31 and -11. Infection with multiple HPV genotypes mainly included HPV-16/-18 and HPV-16/-33. The integration rate of HPV-16 in EC tissue was higher than that in tumor-adjacent and control tissues (93.4% vs. 50.9% and 45.5%). Our findings indicate that infection with HPV, especially the high-risk HPV, and their integration suggest an association in malignant transformation of EC in the high-incidence EC region in Shantou, China. [Copyright &y& Elsevier]

Clin Microbiol Infect 2010; 16: 477–481 The geographical distribution of hepatitis B virus (HBV) subgenotypes and their clinical implications in patients with acute and chronic hepatitis B in the Heilung-kiang province of northeast China were investigated. Nested PCR and multiplex PCR were performed with genotype-specific primers and with subgenotype-specific primers to identify genotypes and subgenotypes from serum samples of 412 HBV infections including 69 with acute self-limited hepatitis (ASH) and 343 with chronic hepatitis (CH). A total of 361 samples were genotyped and 304 were further subgenotyped. The most common HBV genotype was C (93.63%, 338/361), with subgenotype group C2 (83.73%, 283/338) predominating. Genotype B was also found and subgenotype B2 predominated within this genotype. Out of 69 infected patients with ASH, 48 were identified as genotype C and all belonged to subgenotype C2. Of 343 infected patients with CH, 313 were genotyped and 256 were subgenotyped; amongst these, C2 (91.80%, 235/256), B2 (7.42%, 19/256) and mixed subgenotypes B2 and C2 (0.78%, 2/256) were found. In HBV subgenotype C2 infections, ASH had a higher ratio of women than CH patients. These results show that HBV subgenotypes C2 and B2 were found in Heilung-kiang province of northeast China. In ASH and CH groups, the distributions of subgenotypes were coincident with C2, the predominant subgenotype. Analysis of the association between subgenotype and the outcomes of HBV infection was inconclusive in our study. [ABSTRACT FROM AUTHOR]

The objective of this study is to investigate the infection and distribution of Myco plasma pneumoniae in autopsied pulmonary tissue of pediatric severe pneumonia. Mycoplasma pneumoniae nested polymerase chain reaction and immunohistochemistry were done on autopsy pulmonary tissue from 173 patients who died of severe pneumonia. Myco plasma pneumoniae was identified in 135/173 (78.03%) and 114/173 (65.89%) samples of autopsied pulmonary tissue of lethal severe pneumonia via nested polymerase chain reaction and immunohistochemistry, respectively. The coincidence of both assays was 92.4%. Mycoplasma pneumoniae associated fatal pneumonia has showed an increasing trend from 1988 to 2005 in South China, and the fatality rate of Mycoplasmcz pneumoniae associated fatal pneumonia in infants, 1 to 12 months, has risen to 66.9% (97/145). Mycoplasma pneumoniae is a significant cause of severe pneumonia, it is a universal event in infants, and children have died of severe pneumonia in South China. Myco plasma pneumoniae might be an important pathogen responsible for fatal pneumonia in Guangzhou area, South China. [ABSTRACT FROM AUTHOR]

Abstract: The effect of inoculation with arbuscular mycorrhizal fungi (AMF) on crop productivity under small-scale farming conditions and the persistence of the fungal inoculum in the field were investigated over 2 years. Sweet potato plants were inoculated with various combinations of AMF and grown under traditional Chinese farming procedures. Plantlets from germinated tubers were inoculated in a soil/sand mixture at the time of hand transplanting into the field. A technique for long-term preservation of the root samples and a fast, reliable DNA extraction method were developed to track and evaluate the persistence of the selected AMF in two field trials in China. The AMF rDNA was specifically amplified by nested PCR from colonized sweet potato roots collected from the field trials, and polymorphism of the 5′-end of the large ribosomal subunit was used to monitor fungi at the species level. AMF varied in their ability to establish after inoculation, and in their effect on yield and quality of sweet potato tubers. [Copyright &y& Elsevier]

Background: Feline and canine babesiosis is an important tick-borne disease caused by parasites of the genus Babesia. The disease has a worldwide distribution and causes serious health problems in domestic and wild canidae and felidae. Results: Genomic DNA was isolated from blood samples, which were randomly collected from pet dogs (n = 115) and cats (n = 25) in Changsha city of Hunan Province, China. Results of nested PCR assay targeting 18S rRNA gene and partial gene sequencing revealed that seven animals were infected with Babesia species, five dogs (5/115, 4.3%) and two cats (2/25, 8.0%). Sequence analysis showed that four dogs (3.5%) were positive for Babesia canis, and the other one for Babesia vogeli (0.87%). The two cats were infected by Babesia hongkongensis. Conclusions: The findings of this study will expand knowledge of the distribution of Babesia species and provide important epidemiological information for the control of animal babesiosis in China. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a serious public health problem globally. As the elimination of indigenous malaria continues in China, imported malaria has gradually become a major health hazard. Well-timed and accurate diagnoses could support the timely implementation of therapeutic schedules, reveal the prevalence of imported malaria and avoid transmission of the disease., Methods: Blood samples were collected in Wuhan, China, from August 2011 to December 2018. All patients accepted microscopy and rapid diagnosis test (RDT) examinations. Subsequently, each of the positive or suspected positive cases was tested for four human-infectious Plasmodium species by using 18S rRNA-based nested PCR and Taqman probe-based real-time PCR. The results of the microscopy and the two molecular diagnostic methods were analysed. Importation origins were traced by country, and the prevalence of Plasmodium species was analysed by year., Results: A total of 296 blood samples, including 288 that were microscopy and RDT positive, 7 RDT and Plasmodium falciparum positive, and 1 suspected case, were collected and reanalysed. After application of the two molecular methods and sequencing, 291 cases including 245 P. falciparum, 15 Plasmodium vivax, 20 Plasmodium ovale, 6 Plasmodium malariae and 5 mixed infections (3 P. falciparum + P. ovale, 2 P. vivax + P. ovale) were confirmed. These patients had returned from Africa (95.53%) and Asia (4.47%). Although the prevalence displayed a small-scale fluctuation, the overall trend of the imported cases increased yearly., Conclusions: These results emphasize the necessity of combined utilization of the four tools for malaria diagnosis in clinic and in field surveys of potential risk regions worldwide including Wuhan.

Neospora caninum is an intracellular protozoan infecting many domestic and wild animals. In the present study, the brain tissues of wild birds collected in Hunan province of China were examined by N. caninum specific nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS-1) region of the nuclear ribosomal DNA. The prevalence of N. caninum was detected in 15.5% (37/239) of wild birds, including 20.5% (9/44) of the examined olive-backed pipit Anthus hodgsoni, 18.3% (24/131) of the examined tree sparrows Passer montanus, 7.9% (3/38) of the examined chestnut bunting Emberiza rutila and 3.8% (1/26) of the examined yellow-breasted bunting E. aureola. Phylogenetic analyses showed that N. caninum from different hosts and geographical origins are genetically diverse and can be further classified into two distinct groups. Our findings indicated that wild birds are potential source of N. caninum for other animals. To our knowledge, this is the first report of N. caninum infection in wild birds in China, which provides a foundation for the prevention and control of this parasite in China and elsewhere., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Since 2012, no indigenous malaria case have been reported in Shandong Province, China, whereas the number of imported cases and the genetic diversity of Plasmodium spp. have increased. Beginning in 2015, the number of Plasmodium ovale cases were increased and the P. ovale wallikeri malaria case began to arise. From 2015 to 2017, a total of 676 imported malaria cases were detected and 76 P. ovale spp. isolates were identified, of which 48 were P. ovale curtisi and 28 P. ovale wallikeri. The number of P. ovale wallikeri malaria cases increased yearly, six were identified in 2015, eight were identified in 2016, and 14 were identified in 2017. All cases with an attached travel history from Africa, with represented source countries of Equatorial Guinea (n = 9), Republic of the Congo (n = 4), and Democratic Republic of the Congo (n = 3). P. ovale wallikeri is increasing among travelers returning to Shandong Province from Africa. Although the P. ovale spp. infection rarely progressed to severity, this species is suspected to generate hypnozoites which have the potential to relapse. The ability to cause relapse is the threat to public health and should be concerned., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To identify 10 imported cases of Plasmodium ovale wallikeri infection in Sichuan Province from January to April in 2018, and to analyze the P. ovale wallikeri infection status of 1 079 malaria cases reported in this area from 2013 to 2017., Methods: The 10 cases collected in 2018 were examined by microscopy, RDT, nested PCR and sequencing. Meanwhile, the retrospective detections of P. ovale wallikeri of the 1 079 blood samples from past 5 years were conducted by nested PCR using wallikeri -specific primers., Results: For the 10 cases, the microscopic examinations were all positive for P. ovale , of which 2 were mixed infection with P. falciparum , and the results of RDT were all positive for Plasmodium . Except for 2 cases of P. falciparum infection, the routine nested PCR showed negative results for rest of the samples. The nested PCR for the specific detection of P. ovale wallikeri showed the 10 cases were all positive, and the following sequencing confirmed that the 10 cases were all infected with P. ovale wallikeri . The retrospective tests discovered 2 cases both reported in 2017 and recorded as simple P. falciparum infection tuned out to be mixed infection with P. falciparum and P. ovale wallikeri ., Conclusions: This is the first time that imported P. ovale wallikeri infection is founded in Sichuan Province.

Comprehensive epidemiological surveys for Lyme disease have not been conducted for the Bactrian camel in China. In this study, a total of 138 blood specimens collected from Bactrian camels from Zhangye City in Gansu Province and Yili and Aksu in Xinjiang Province, China, were examined for the presence of Borrelia spp. Species-specificity nested PCR based on the 5S-23S rRNA, OspA, flaB and 16S rRNA genes revealed that the total positive rate of Borrelia spp. was 3.6% (5/138, 95% CI = 0.2-17.9). These results were confirmed by sequence analysis of the positive PCR products or positive colonies. This is the first report of Borrelia pathogens in camels in China. Two Borrelia species that cause Lyme disease and one that causes relapsing fever were identified in the camel blood samples by sequencing. The findings of this study indicate that the Bactrian camel may serve as a potential natural host of Lyme disease and/or relapsing fever in China., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Domestic dogs and other canids function as definitive hosts, while other mammals serve as natural intermediate hosts. In the present study, the brain tissues of bats collected in Yunnan Province, Southern China were tested by N. caninum specific-nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA to determine whether bats could be infected with N. caninum. N. caninum DNA was detected in 1.8% (4/227) of bats, i.e., 1.7% (1/60) in Rousettus leschenaultia, 1.7% (1/58) in Hipposideros pomona, 2.9% (2/69) in Rhinolophus pusillus, and none (0/40) in Myotis daubentoniid. The findings of the present study are only the first indication that bats could serve as an intermediate host, and further studies are necessary to confirm whether bats are involved in the transmission of N. caninum infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research., (Copyright © 2014 Elsevier B.V. All rights reserved.)