Tongjie Chai

Simultaneous detection of airborne Aflatoxin, Ochratoxin and Zearalenone in a poultry house by immunoaffinity clean-up and high-performance liquid chromatography

An AOZ method, based on high-performance liquid chromatography (HPLC), was optimized on HPLC condition such as mobile phase and wavelength to simultaneously quantify six kinds of mycotoxins [four aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA)]. Conditions for immunoaffinity clean-up, HPLC and photo-derivatization were optimized in this study and successfully applied in assessment of airborne mycotoxins from a poultry house in Dalian, China. Fifty-two air samples were collected with AGI-30 air samplers using pure water as collection media. Twenty air samples (20/52, 38.46%) were positive for four toxins. Among the positive samples, airborne mycotoxin concentrations (mean+/-S.D.) for AFG(2), AFB(1), and ZEA were 0.189+/-0.024 (n=9), 0.080+/-0.003 (n=11) and 2.363+/-0.030 (n=5)ng/m(3) air, while the concentration for OTA was 8.530 (n=1)ng/m(3). No positive sample was found for either AFG(1) or AFB(2). A chicken may inhale 0.019-0.057 ng AFG(2), 0.013-0.019 ng AFB(1), 0.436-0.513 ng ZEA, and 1.706 ng OTA, respectively, in a day. A poultry worker may inhale 0.504-1.512 ng AFB(1), 0.752-2.28 ng AFG(2), 68.240 ng OTA, and 17.432-20.512 ng ZEA in a working day. This is the first report on airborne mycotoxins in poultry house. These data may have importance in animal and public health implications.

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.

Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma.

A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 μg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma.

Detection of Babesia divergens using molecular methods in anemic patients in Shandong Province, China

Babesiosis (piroplasmosis) is a zoonotic disease caused by an intraerythrocytic protozoan transmitted by Ixodes ticks. The aim of this study was to detect Babesia spp. infection using molecular methods in 377 blood samples from anemic patients. Sequence analysis showed that the 18S rRNA gene was 439 bases long by polymerase chain reaction (PCR) amplification and that the PCR products from the samples had an identical sequence (named Taian China, HM355854). BLAST search showed that the sequence was identical to the 18S rRNA sequences of Babesia divergens. The 18S rRNA sequence for Toxoplasma gondii was included as the outlier for phylogenetic analysis by using the program MEGA4.0 software. The results showed that the 18S rRNA gene sequences obtained from the present study was most closely related to B. divergens Switzerland (DQ312439) with 98.4% similarity (differing only by seven bases). The phylogenetic analysis also revealed that this sequence closely resembled B. divergens strains from other countries and belonged to the same clade. This is the first report of a human being infected by B. divergens in China.

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.

Immune responses of ducks infected with duck Tembusu virus

Duck Tembusu virus (DTMUV) can cause serious disease in ducks, characterized by reduced egg production. Although the virus has been isolated and detection methods developed, the host immune responses to DTMUV infection are unclear. Therefore, we systematically examined the expression of immune-related genes and the viral distribution in DTMUV-infected ducks, using quantitative real-time PCR. Our results show that DTMUV replicates quickly in many tissues early in infection, with the highest viral titers in the spleen 1 day after infection. Rig-1, Mda5, and Tlr3 are involved in the host immune response to DTMUV, and the expression of proinflammatory cytokines (Il-1β, –2, –6, Cxcl8) and antiviral proteins (Mx, Oas, etc.) are also upregulated early in infection. The expression of Il-6 increased most significantly in the tissues tested. The upregulation of Mhc-I was observed in the brain and spleen, but the expression of Mhc-II was upregulated in the brain and downregulated in the spleen. The expression of the interferons was also upregulated to different degrees in the spleen but that of the brain was various. Our study suggests that DTMUV replicates rapidly in various tissues and that the host immune responses are activated early in infection. However, the overexpression of cytokines may damage the host. These results extend our understanding of the immune responses of ducks to DTMUV infection, and provide insight into the pathogenesis of DTMUV attributable to host factors.

An investigation of duck circovirus and co-infection in Cherry Valley ducks in Shandong Province, China.

The co-infection of duck circovirus (DuCV) with Riemerella anatipestifer (RA) or/and Escherichia coli (E. coli) or/and duck hepatitis virus I (DHV-I) in Cherry Valley ducks in Chinas Shandong Province was investigated by using polymerase-chain-reaction (PCR)-based methods. For this study, 742 ducks sampled at random from 70 duck farms during 2006-2007 were examined using PCR and dot-blot hybridisation (DBH) tests. Overall the DuCV infection rate was 33.29%. Compared with those at 2 weeks of age, the ducks at 3-4 weeks of age were more susceptible to DuCV infection. Compared with the DuCV-negative ones, the DuCV-positive ducks had a higher rate of infection by DHV-I (25.5% vs. 7.475%), RA (23.48% vs. 8.28%) and E. coli (16.19% vs. 4.85%). This investigation shows that DuCV infection is common in Cherry Valley ducks on some farms in Shandong Province.

Adaptation of H9N2 AIV in guinea pigs enables efficient transmission by direct contact and inefficient transmission by respiratory droplets.

H9N2 avian influenza viruses circulate worldwide in poultry and have sporadically infected humans, raising concern whether H9N2 viruses have pandemic potential. Here, we use a guinea pig model to examine whether serial passage results in adaptive viral changes that confer a transmissible phenotype to a wild-type H9N2 virus. After nine serial passages of an H9N2 virus through guinea pigs, productive transmission by direct contact occurred in 2/3 guinea pig pairs. The efficiency of transmission by direct contact increased following the fifteenth passage and occurred in 3/3 guinea pig pairs. In contrast, airborne transmission of the passaged virus was less efficient and occurred in 1/6 guinea pig pairs and 0/6 ferret pairs after the fifteenth passage. Three amino acid substitutions, HA1-Q227P, HA2-D46E, and NP-E434K, were sufficient for contact transmission in guinea pigs (2/3 pairs). The two HA amino acid substitutions enhanced receptor binding to α2,3-linked sialic acid receptors. Additionally, the HA2-D46E substitution increased virus thermostability whereas the NP-E434K mutation enhanced viral RNA polymerase activity in vitro. Our findings suggest that adaptive changes that enhance viral receptor binding, thermostability, and replicative capacity in mammalian cells can collectively enhance the transmissibility of H9N2 AIVs by direct contact in the guinea pig model.

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.

Formation and transmission of Staphylococcus aureus (including MRSA) aerosols carrying antibiotic-resistant genes in a poultry farming environment.

There is a rather limited understanding concerning the antibiotic-resistance of the airborne S. aureus and the transmission of the antibiotic-resistant genes it carries Therefore, we isolated 149 S. aureus strains from the samples collected from the feces, the indoor air and the outdoor air of 6 chicken farms, and performed the research on them with 15 types of antibiotics and the REP-PCR trace identification. The 100% homologous strains were selected to conduct the research on the carrying and transmission status of the antibiotic-resistant genes. The results revealed that 5.37% strains (8/149) were resistant to methicillins (MRSA), and 94% strains (140/149) were resistant to compound sulfamethoxazole, etc. In addition, these strains displayed a resistance to multiple antibiotics (4, 5 or 6 types) and there were also 3 strains resistant to 9 antibiotics. It should be noted that the antibiotic-resistance of some strains isolated from the feces, the indoor and outdoor air was basically the same, and the strains with the same REP-PCR trace identification result carried the same type of antibiotic-resistant genes. The results showed that airborne transmission not only causes the spread of epidemic diseases but also exerts threats to the public health of a community.