Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), can induce 80–100% mortality in newborn piglets; therefore, specific and rapid detection methods are important for the prevention of this viral infection. In particular, methods for detecting neutralizing antibodies (nAbs) can be used to evaluate the immunization effect of PEDV vaccines. The spike protein of PEDV (PEDV-S) has been universally used as an antigen to develop immunoassays to detect nAbs. Nanobodies (Nbs) offer advantages such as ease of genetic engineering and low production costs, making them promising for diagnostic applications. In this study, PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels. A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase (HRP) in HEK293T cells. A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay (cELISA) to detect anti-PEDV nAbs. Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration. The optimal dilution of PEDV-S-Nb13-HRP was 1:200, and the optimal serum dilution was 1:10. The cutoff value of cELISA was determined as 28.1%, demonstrating high specificity, repeatability, stability, and good agreement rates with two commercial ELISA kits (93.6%) and a serum neutralization test (96.34%). Additionally, the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit. These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.

To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China, the complete genomes of six DuCV strains, which were detected from Cherry Valley ducks in China between 2007 and 2008, were sequenced. Sequence and phylogenetic analysis were carried out to compare these six strains with another 27 DuCV strains from Mulard duck, Muscovy duck, Pekin ducks and Mule duck. The analysis showed that the six DuCV strains exhibited typical genetic features of the family of DuCV, such as a stem-loop structure, three major open reading frames (Rep, Cap and ORF3), four intergenic repeats and the conserved motifs for rolling circle replication and for the dNTP binding domain located in the Rep protein. Phylogenetic analysis of the nucleotide sequences of the complete genome and Cap gene of these strains together with those that have been previously published demonstrated two distinct DuCV genotypes. The DuCV strains with complete genomes containing 1988 and 1989 nucleotides clustered in genotype A, whereas the strains with complete genomes containing 1991, 1992, 1995 and 1996 nucleotides lay in genotype B. The six DuCV strains from Cherry Valley ducks were divided into the two groups. The results of the study provides some insight into the variation of DuCVs in Cherry Valley ducks.

A full-length genome DNA of PCV-2 was amplified by PCR and sequenced,then named SDZQ-1.Sequence analysis of the complete genome of SDZQ-1 with that of twenty-one PCV-2 sequences published in GenBank indicated that the SDZQ-1 shows 95.0%-99.7%homology in complete nucleotide sequence compare with others.The two complete genome were connected into PUC19 subsequently in norientation to construct double copy plsmid pUC2PCV2.Transfect PK-15 cells with pUC2PCV2 in vitro.After four passages,the infectivity of the cloned chimeric PCV-2 genome was confirmed by PCR and IFA.

In the January of 2008,respiratory signs were observed in the chicks of a layer farm in Cangzhou,Hebei province.Necropsy of the dead chicks revealed that the kindey was swollen and distended with uric acid crystals.Kidneys and tracheas were collected for virus isolation.An IBV virus was identified by viral interference test,HA test,animal regressive research,cross-neutralization and RT-PCR.The virus was designated 0801 strain.The date of cross-neutralization indicated that the 0801 strain and Ma5 vaccine strain belong to the same serotype.The S1 gene of 0801 strain was amplified,cloned,sequenced and analyzed.The results showed that S1 gene was composed of 1620 nucleotides,homologies of nucleotide and amino acid sequences of S1 gene between 0801 strain and Massachusetts-type vaccine strains was both about 75%.

Rep gene was amplified from genomic DNA of Duck circovirus (DuCV) FJ0601 strain by polymerase chain reaction(PCR)and then cloned into pGEX-6P-1 according to the right open reading frame(ORF). The recombinant plasmid was transformed into E. coli BL21 strain for expression with the induction of IPTG. After SDS-PAGE, the expressed specific band was harvested and injected into four-week-old mice 3 times,then the antiserum was collected for IFA with the positive froze histological section of spleen,thymus and bursa tested by PCR. The focuses of infec-tion composed of positive staining cells were found in the three immune organs infected by DuCV,showing that the expressed fusion protein of Rep in vitro has some epitopes of natural Rep.

The tissues samples of 343 spontaneous diseased ducks from 36 duck flocks were collected in Shandong,Jiangsu,Sichuan,Fujian and Guangdong,and the DNA was extracted to detect duck circovirus(DuCV) by hybridization and PCR.The results showed that the samples of the 5 provinces existed DuCV,and the individual positive ratio of DuCV was 81.63%(280/343) and the flock positive ratio of DuCV was 94.44%(34/36),indicating that the infection of DuCV exists wide-ranging in duck flocks of China.

The clinical symptoms of infectious coryza are multiple and include nasal discharge, facial swelling, lacrimation, and anorexia. In general, the disease is not fatal to chicken; so, in experiments where animals are infected with Avibacterium paragallinarum, there have been debates about conducting the challenge model and evaluating the clinical signs. In this experiment, 150 chickens, aged 30 days, were randomly divided into different groups. Some groups were infected with the ‘in-contact’ challenge model and others with the artificial intrasinus-injection-route model. The bacterial isolates used were three field isolates of different serogroups of A. paragallinarum, including Hpg-8 (Page serovar A), CCM6075 (Page serovar B) and Hpg-668 (Page serovar C). During this study, a scoring system was used to record the clinical signs of the infected birds and evaluate the pathogenic diversity of the two models. The final results indicated that the ‘in-contact’ challenge model of the three isolates showed a more reliable representation of the natural infection under field conditions than the artificial intrasinus-injection-route model. Thus, on carrying out animal experiments, the effect of ‘in-contact’ challenge model is more accurate than the artificial intrasinus-injection-route model.

The isolate GN52 of Riemerrella anatipestifer was passaged on the Martin Medium successively according to the optimum condition. The experiments included Gram staining, biochemical test, drug sensitivity test and animal experiments were carried out on the bacteria of 3rd, 11th, 21st, 31st, 41st, 51st and 61st generations. It indicated that the bacterial morphs, biochemical character, drug resistance of the strain had no obvious change, but the virulence showed a trend of reduction.

The duck circovirus (DuCV) infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province. Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%∼97.5% nucleotide sequence identity to all the four Taiwan isolates (TC1/2002, TC2/2002, TC3/2002, TC4/2002), 82.9% identity to the America (33753-52) isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.

In order to analyze sequences of OmpA of Chinese strains of Riemerella anatipestifer(RA),according to outer membrane protein A(OmpA) gene sequences from R.anatipestifer strains in the GenBank,a pair of specific primers was designed.Eight R.anatipestifer strains from Fujian,Guangdong and Shandong were amplified in the whole open reading frame(ORF) by PCR and then cloned and sequenced.The comparative analysis of the sequences with twenty-six R.anatipestifer strains in GenBank indicated all OmpA gene had a long complete ORF composed of 1164 nucleotides which encoded a protein of 387 amino acids,and all initiation codon were ATG and end codon were TAA.The sequence of OmpA were relatively conservative which identity was 88.2%～100% and the identity of the amino acids was 88.95%~100%.The results showed that the sequence of OmpA relatively conservative and the identity of the nucleotide and amino acid had no positive relation with the isolate serotype,the time and location of isolation.

The genomic DNA extracted from chicken embryo fibroblasts (CEF) of SPF chickens from three chicken farms was used as template to amplify the ALV proviral DNA by PCR with four pairs of primers, high positive detection rates of gag - gene (29/46), pol - gene (27/46), env - gene (24/46) and LTR fragment (31/46) were achieved. Eight continuous and overlapping fragments were amplified from one DNA sample with 8 pairs of primers according to published sequences, then cloned into the TA vector and se quenced. The complete sequence of the whole genome of ALV strain SD0501 was established and analyzed with DNAstar software. Comparisons of SD0501 sequence with that of other representative endogenous avian virus strains demonstrated that the genomes of ALV were relatively conservative, the nucleotide identity of all the strains was over 99.1%, and env - gene was over 98.5%. However, a low identity was demonstrated among the representative strains of different subgroups, especially, the env - gene showed obvious difference, the corresponding identity was as low as 56.3% - 91.5%.

The homologous antisera had been gotten by utilizing the representative strains of the Avibacterium paragallinarum such as Hpg-8(serotype A),CCM6075(serotype B) and Hpg-68 (serotype C),and then A,B and C mono-specific antisera had been acquired by cross absorption test and all mono-specific antisera showed serotype-specificity.Further investigations indicated that the serotype of the six isolates of Avibacterium paragallinarum all belonged to serotype A identified by using the three mono-specific antisera.

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 China's 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.

For studing the biological characteristics of three strandard strains of Listeria monocytogene(L208,L128-9,219/60),the operation of pure culture,dye and observation,AST(antimicrobial susceptibility test) and biochemical experiment were done.The results showed that the cultural characteristics,patterns of structure and basic biological characteristics are basically consistent with the published information.But the result of AST showed that the bacteria are susceptible for most antibiotics,and are resistant medical for only a few special antibiotics. The study provided the necessary foundation for the future to in light of this strain′s other relevant studies.