Zengmin Miao

Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR.

Abstract Evidence is mounting that microorganisms originating from livestock impact the air quality of the animal houses themselves and the public in the surrounding neighborhoods. The aim of this study was to develop efficient bacterial source tracking capabilities to identify sources of Escherichia coli aerosol pollution caused by pigs. Airborne E. coli were isolated from indoor air, upwind air (10 and 50m away) and downwind air samples (10, 50, 100, 200 and 400m away) for five swine houses using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli strains from pig fecal samples were also collected simultaneously. The enterobacterial repetitive intergenic consensus polymerize chain reaction (ERIC-PCR) and the repetitive extragenic palindromic (REP-PCR) approaches were used to study the genetic variability and to determine the strain relationships among E. coli isolated from different sites in each swine house. Results showed that 35.1% (20/57) of the bacterial DNA fingerprints from the fecal isolates matched with the corresponding strains isolated from indoor and downwind air samples (similarity ⩾90%). E. coli strains from the indoor and downwind air samples were closely related to the E. coli strains isolated from feces, while those isolated from upwind air samples (swine house C) had low similarity (61–69%). Our results suggest that some strains isolated from downwind and indoor air originated in the swine feces. Effective hygienic measures should be taken in animal farms to prevent or minimize the downwind spread of microorganism aerosol.

ERIC-PCR identification of the spread of airborne Escherichia coli in pig houses.

To understand the spread of microbial aerosols in pig houses, with Escherichia coli (E. coli) as indicator, the airborne E. coli in 4 pig houses and their surroundings at different points 10, 50m upwind and 10, 50, 100, 200 and 400m downwind respectively from the pig houses were collected, and the concentrations were calculated at each sampling point. Furthermore, the feces of pigs were collected to separate E. coli. The ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction) technology was used to amplify the isolated E. coli DNA samples, then the amplified results were analyzed by NTSYS-pc (Version 2.10) to identify the similarity of isolated E. coli. The results showed that the airborne E. coli concentrations in indoor air of the 4 pig houses (21-35CFUm(-)(3) air) were much higher than those in upwind and downwind air (P<0.05), but there were no significant differences (P>0.05) at downwind distances. The ERIC-PCR results also showed that 52.4% of the fecal E. coli (four houses being respectively 2/4, 50%; 2/4, 50%; 3/6, 50%; 4/7, 57.1%) were identical to the indoor airborne E. coli isolates, and there was more than 90% similarity between the majority of E. coli (50%, 21/42) isolated from downwind air at 10, 50, 100 and 200m and those from indoor air or feces. It could be concluded that the aerosols in pig houses can spread to the surroundings, and thus effective measures should be taken to control and minimize the spread of microbial aerosols.

Occurrence and transmission of Newcastle disease virus aerosol originating from infected chickens under experimental conditions.

In order to better understand airborne transmission of Newcastle disease, a model system was established and two trials were conducted. Twenty-five principal specific pathogen free (SPF) chickens were inoculated with NDV and were housed in one isolator. 6 days after the chickens were challenged, 15 chickens were placed into another isolator which received its air supply from the first isolator. The NDV aerosol originating from inoculated chickens was collected with All Glass Impinger-30 (AGI-30) to study the occurrence and concentration of NDV aerosol. The antibody response to infection was assessed by the hemagglutination inhibition (HI) test and viral shedding was detected by RT-PCR and Dot-ELISA. NDV aerosol was initially detectable by RT-PCR and cell culture at day 2 or 3 post-inoculation (dpi). The aerosol concentration peaked at 1.69x10(4)PFU/m(3) air at 13dpi in trial 1, 9.14x10(3)PFU/m(3) air at 11dpi in trial 2 and was consistently detectable up to 40dpi. NDV shedding was detectable from 2 to 40dpi of inoculated chickens and from 6 days post-aerosol exposed infection (dpi) to 33dpi of aerosol exposed chickens. The viral strain induced high antibody level, both in inoculated and in aerosol exposed chickens. Airborne transmission did occur, as shown by NDV shedding and seroconversion to NDV in aerosol exposed chickens. The results indicated that viruses shed from infected chickens readily aerosolized and airborne transmission of NDV was possible.

Experimental transmission in guinea pigs of H9N2 avian influenza viruses from indoor air of chicken houses

This study aimed to determine the transmission characteristics of H9N2 avian influenza viruses (AIVs) derived from the air. Eight H9N2 AIVs were isolated from chicken houses between 2009 and 2010. We analyzed the phylogenic and pathogenic traits of these isolates. What is more, transmission characteristics in guinea pigs of two airborne isolates were determined in experimental conditions. Phylogenetic analyses indicated that the homologies of HA and NA genes of eight isolates were 95.4-99.7% and 86.6-99.8% respectively. They were able to duplicate in lung tissues of guinea pigs without prior adaptation. Two airborne isolates could both transmit among guinea pigs by direct contact. No infection was detected in aerosol contact animals while H9N2 AIV aerosols were detected in the air of isolators. Aerosol infection dose experiment showed that aerosol median infective dose (ID(50)) of H9N2 AIV to guinea pigs was 3.58×10(6)copies, demonstrating that the aerosols could infect guinea pigs at certain concentrations in experimental condition. In conclusion, H9N2 AIV aerosols were infectious to mammals, suggesting that urgent attention will need to be paid to its transmission.

Emissions of Escherichia coli Carrying Extended-Spectrum β-Lactamase Resistance from Pig Farms to the Surrounding Environment

The dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) from food-producing animals to the surrounding environment has attracted much attention. To determine the emissions of ESBL-producing E. coli from pig farms to the surrounding environment, fecal and environmental samples from six pig farms were collected. In total, 119 ESBL-producing E. coli were isolated from feces, air samples, water, sludge and soil samples. Antibiotic susceptibility testing showed that the ESBL-producing isolates were resistant to multiple antibiotics and isolates of different origin within the same farm showed similar resistance phenotypes. Both CTX-M and TEM ESBL-encoding genes were detected in these isolates. CTX-M-14 and CTX-M-15 were the predominant ESBL genes identified. ESBL producers from feces and environmental samples within the same farm carried similar CTX-M types. The results indicated that the ESBL-producing E. coli carrying multidrug resistance could readily disseminate to the surrounding environment.

REP-PCR tracking of the origin and spread of airborne Staphylococcus aureus in and around chicken house

Abstract  Staphylococcus aureus was used as an indicator to study the origin and spread of microbial aerosol in and around chicken houses. Air samples indoor, upwind (10 and 50 m), and downwind (10, 50, 100, 200, and 400 m) of four chicken houses were collected using Andersen‐6 stages sampler. The concentrations of S. aureus were determined for every sample site. Isolation of S. aureus from chicken feces was performed according to the standard method. The genetic relationship among the isolates was determined by profiles of PCR‐amplified repetitive extragenic palindromic (REP‐PCR) elements. The results showed that the concentrations of S. aureus indoor of four chicken houses were higher than those upwind and downwind sites (P < 0.05 or P < 0.01), but there were no significant concentration differences among downwind sites (P > 0.05). The fingerprints and the phylogenetic tree indicated that a part of the S. aureus (55.6%, 10/18) isolates from indoor air had the same REP‐PCR fingerprints as feces isolates. Consequently, most isolates (57.1%, 20/35) from downwind 10, 50, 100, 200, even 400 m had the same REP‐PCR fingerprints as those from indoor or feces. These data indicated that some isolates from downwind and indoor originated from the chicken feces. However, those isolates from upwind had low similarity (similarity index 0.6–0.87) to those from indoor or feces. Therefore, the isolates upwind were not from the chicken feces or indoor. These results suggest that microbes in chicken feces can be aerosolized and spread indoor and outdoor, especially to downwind of the chicken houses. It should have an important epidemiological and public health significance. Practical Implications Thus, the use of S. aureus as an indicator to study the origin and spread of airborne pathogens from chicken houses is potentially useful for enhancing public health and understanding the airborne epidemiology of this pathogen. Meanwhile it can provide evidence for studying the spreading model of airborne pathogens.

Avian influenza H9N2 seroprevalence among pig population and pig farm staff in Shandong, China.

BackgroundShandong province of China has a large number of pig farms with the semi-enclosed houses, allowing crowds of wild birds to seek food in the pig houses. As the carriers of avian influenza virus (AIV), these wild birds can easily pass the viruses to the pigs and even the occupational swine-exposed workers. However, thus far, serological investigation concerning H9N2 AIV in pig population and pig farm staff in Shandong is sparse.MethodsTo better understand the prevalence of H9N2 AIV in pig population and pig farm staff in Shandong, the serum samples of pigs and occupational pig-exposed workers were collected and tested for the antibodies for H9N2 AIV by both hemagglutination inhibition (HI) and micro-neutralization (MN) assays.ResultsWhen using the antibody titers ≥40 as cut-off value, 106 (HI: 106/2176, 4.87%) and 84 (MN: 84/2176, 3.86%) serum samples of pigs were tested positive, respectively; 6 (HI: 6/287, 2.09%) and 4 (MN: 4/287, 1.39%) serum samples of the pig farm staff were positive, respectively; however, serum samples from the control humans were tested negative in both HI and MN assays.ConclusionsThese findings revealed that there were H9N2 AIV infections in pig population and pig farm staff in Shandong, China. Therefore, it is of utmost importance to conduct the long-term surveillance of AIV in pig population and the pig farm staff.

Phylogenetic Analysis of Hemagglutinin Genes of H9N2 Avian Influenza Viruses Isolated from Chickens in Shandong, China, between 1998 and 2013

Since H9N2 avian influenza virus (AIV) was first isolated in Guangdong province of China, the virus has been circulating in chicken flocks in mainland China. However, a systematic phylogenetic analysis of H9N2 AIV from chickens in Shandong of China has not been conducted. Based on hemagglutinin (HA) gene sequences of H9N2 AIVs isolated from chickens in Shandong of China between 1998 and 2013, genetic evolution of 35 HA gene sequences was systematically analyzed in this study. Our findings showed that the majority of H9N2 AIVs (21 out of 35) belonged to the lineage h9.4.2.5. Most of isolates (33 out of 35) had a PSRSSR↓GLF motif in HA cleavage site. Importantly, 29 out of these 35 isolates had an amino acid exchange (Q226L) in the receptor-binding site. The substitution showed that H9N2 AIVs had the potential affinity to bind to human-like receptor. The currently prevalent H9N2 AIVs in Shandong belonged to the lineage h9.4.2.5 which are different from the vaccine strain SS/94 clade h9.4.2.3. Therefore, the long-term surveillance of H9N2 AIVs is of significance to combat the possible H9N2 AIV outbreaks.

Prevalence and Antibiotic Resistance Profiles of Extended-Spectrum β-Lactamase–Producing Escherichia coli Isolated from Healthy Broilers in Shandong Province, China

Food-producing animals carrying extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) have posed a potential threat to human and animal health. However, information regarding ESBL-EC in the intensive broiler breeding areas of Shandong Province, Peoples Republic of China, is very limited. The goal of our study was to investigate the prevalence and drug resistance characteristics of ESBL-EC in healthy broilers from Shandong Province. A total of 142 ESBL-EC isolates were collected from four prefectures in Shandong Province from October 2014 to February 2015. ESBL-EC isolates were frequently detected (142 of 160 samples, 88.8%) in healthy broilers. Antibiotic susceptibility testing showed that all 142 ESBL-EC isolates were resistant to ampicillin, piperacillin, and cefazolin but were sensitive to imipenem and meropenem. All ESBL-EC isolates carried one or more of the bla genes, in which blaCTX-M, blaTEM-1, and blaSHV-5 genes were identified in 142, 106, and 5 isolates, respectively. The blaCTX-M gene includes blaCTX-M-15 (56), blaCTX-M-65 (42), blaCTX-M-55 (36), blaCTX-M-14 (21), blaCTX-M-79 (1), blaCTX-M-3 (1), blaCTX-M-123 (1), and blaCTX-M-132 (1). In addition, 17 ESBL-EC isolates cocarried the genes of the CTX-M-1 and CTX-M-9 groups. Our findings indicate that healthy broiler flocks in Shandong Province in China are an important reservoir for ESBL-EC, with blaCTX-M and blaTEM-1 being the prevalent resistance genes identified.

Airborne spread and infection of a novel swine-origin influenza A (H1N1) virus

BackgroundThe novel swine-origin influenza A (H1N1) virus (S-O 2009 IV) can cause respiratory infectious diseases in humans and pigs, but there are few studies investigating the airborne spread of the virus. In January 2011, a swine-origin H1N1 epidemic emerged in eastern China that rapidly spread to neighboring farms, likely by aerosols carried by the wind.MethodsIn this study, quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect viruses in air samples from pig farms. Based on two aerosol infection models (Pig and guinea pig), we evaluated aerosol transmission and infection of the novel S-O 2009 IV isolate.ResultsThree novel S-O 2009 IV were isolated from the diseased pig. The positive rate and viral loads of air samples were 26.1% and 3.14-5.72 log10copies/m3 air, respectively. In both pig and guinea pig infection models, the isolate (A/swine/Shandong/07/2011) was capable of forming aerosols and infected experimental animals at a range of 2.0-4.2 m by aerosols, but aerosol route was less efficient than direct contact.ConclusionsThe results indicated that S-O 2009 IV is able to be aerosolized by infected animals and to be transmitted to susceptible animals by airborne routes.