Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

Analysis of genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Central China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an epidemic etiology in pigs of all ages causing reproductive failure and respiratory manifestation. PRRSV has been circulating in Chinese pig farms for almost 20 years. The aim of the present study was to fully understand the extent of the genetic diversity and molecular characteristics of PRRSVs in Central China. A strain of PRRSV isolated from a recent outbreak farm in Hunan province in Central China, designated HUN-2014, was sequenced and analyzed with 39 other PRRSVs from 1998 to 2014 in Central China. Comparative results of genomic sequences revealed that all 40 PRRSVs belonged to the North American genotype (NA genotype) and shared 88.8-99.0% homology. Phylogenetic analysis showed three subgenotypes, namely conventional PRRSV (C-PRRSV), specially mutant PRRSV (S-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), in all 40 PRRSVs. Moreover, comparative analysis of amino acid (AA) sequences of NSP2, GP3, GP5 and ORF5a revealed the main evolution trend of PRRSVs in Central China from 1998 to 2014, which was from C-PRRSV to HP-PRRSV, accompanied by different evolving directions to S-PRRSV. In conclusion, both the major evolutionary trend and special features of genetic variation should be emphasized as theoretical basis for development of new vaccines and control strategies for PRRS.

Complete Genome Sequence of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant with a New Insertion in Glycoprotein 5, Isolated from a Stillborn Fetus.

Here, we report the complete genome sequence of strain GD-2011, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that was isolated from a stillborn fetus. The GD-2011 strain is characterized by a discontinuous 30-amino acid deletion in the nonstructural protein 2. In addition, GD-2011 had a 1-amino acid insertion in glycoprotein 5, which does not exist in any other HP-PRRSV strains.

Antimicrobial Resistance Profile and Genotypic Characteristics of Streptococcus suis Capsular Type 2 Isolated from Clinical Carrier Sows and Diseased Pigs in China.

Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for ß-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with MLSB phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2.

[Accurate titration of mycoplasma culture measured by 50% color change unit assay].

OBJECTIVE: A new method was introduced for precise determination of the live cell titer of mycoplasma culture, and would be a candidate to replace the commonly used CCU (color change unit) assay. METHODS: The CCU50 (50% color change unit ) was modified according to the method of TCID50 (50% tissue culture infective dose) assay used for viral titer assessment, and adopted to estimate the live cell titer of mycoplasma. Sensitivity and reproducibility of the CCU50 assay were assessed, and adaptability was checked with M. hyopneumoniae and M. synoviae. RESULTS: The CCU50 assay showed better reproducibility, sensibility and adaptability than traditional CCU assessment approaches. CONCLUSION: The method could be applied to accurate titration determination for mycoplasma, and might be considered as a useful tool for the research of high density fermentation of mycoplasma and development of vaccine.

Activation of c-Jun NH(2)-terminal kinase is required for porcine reproductive and respiratory syndrome virus-induced apoptosis but not for virus replication.

Apoptosis of host cells plays a critical role in pathogenesis of virus infection. MAPK kinases especially stress-activated protein kinases c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 are often involved in virus-mediated apoptosis. It has been shown that porcine reproductive and respiratory syndrome virus (PRRSV) infection resulted in apoptosis of the host cells both in vitro and in vivo. The current investigation was initiated to determine whether stress-activated protein kinases JNK and p38 play a role in apoptosis induction by PRRSV infection. We examined phosphorylation of JNK and p38, and found that JNK but not p38 was activated in response to PRRSV infection. We then examined effects of this kinase on apoptosis induction and virus replication by using specific inhibitor. We found that JNK inhibition by its inhibitor SP600125 led to the abolishment of PRRSV-mediated apoptosis, but did not suppress virus replication. Further studies demonstrated that ROS generation was involved in JNK activation, and Bcl-2 family anti-apoptotic proteins Mcl-1 and Bcl-xl were downstream targets of JNK to mediate apoptosis. We conclude that activation of JNK signaling pathway is essential for PRRSV-mediated apoptosis but not for virus replication.

Antimicrobial susceptibility and molecular subtypes of Staphylococcus aureus isolated from pig tonsils and cow's milk in China.

This study investigated and compared the antimicrobial resistance patterns and ribotypes of Staphylococcus aureus isolated from pig tonsils and cow's milk in China. A total of 90 isolates of S. aureus was included: 42 strains were isolated from tonsils of pigs and 48 from half-udder milk. The broth microdilution method and the double-disc diffusion test (D test) were used for antimicrobial susceptibility testing. The mecA gene for methicillin-resistant S. aureus (MRSA) and the ermA, ermB, ermC, and msrA genes for erythromycin-resistant strains were detected by polymerase chain reaction (PCR). The isolates were ribotyped with the Riboprinter system. The highest frequency of resistance was observed with clindamycin (91.1%), followed by penicillin (90.0%), and erythromycin (85.6%). All strains were susceptible to vancomycin and trimethoprim-sulfamethoxazole. The D test showed that 54.5% (42/77) of erythromycin-resistant isolates had the constitutive resistance phenotype and 45.5% (35/77) had the inducible resistance phenotype to clindamycin. A higher proportion of resistance to cephalosporins, macrolides, fluoroquinolones, and pleuromutilins was observed in pig isolates than in milk isolates (P < 0.05). The mecA gene was detected in all MRSA isolates; 89.6% of erythromycin-resistant strains harbored the ermC gene and 16.9% harbored the ermB gene. A total of 35 different ribogroups was found among the isolates investigated; 83.3% of pig strains belonged to 1 cluster with a similarity coefficient of 0.84. In contrast, 3 main clusters were observed among 68.8% of milk strains, which indicates a high degree of host specificity.

Ce travail visait à étudier et comparer les patrons de résistance antimicrobienne et les ribotypes d'isolats de Staphylococcus aureus provenant des amygdales de porc et du lait de vache en Chine. Un total de 90 isolats de S. aureus était inclus : 42 souches provenaient des amygdales de porcs et 48 provenant de lait de 2 des 4 quartiers. La méthode de microdilution en bouillon et l'épreuve de double diffusion en disque (test D) ont été utilisées pour déterminer la sensibilité aux antibiotiques. Le gène mecA des S. aureus résistants à la méthicilline (MRSA) et les gènes ermA, ermB, ermC, et msrA des souches résistantes à l'érythromycine ont été détectés par réaction d'amplification en chaîne par la polymérase (PCR). Les isolats ont été ribotypés avec le système Riboprinter. La fréquence de résistance la plus élevée a été observée avec la clindamycine (91,1 %), suivie de la pénicilline (90,0 %) et l'érytrhomycine (85,6 %). Toutes les souches étaient sensibles à la vancomycine et au trimethoprime-sulfaméthoxazole. Le test D a montré que 54,5 % (42/77) des isolats résistants à l'érythromycine avaient le phénotype constitutif de résistance et 45,5 % (35/77) avaient le phénotype de résistance inductible à la clindamycine. Une fréquence plus élevée de résistance aux céphalosporines, aux macrolides, aux fluoroquinolones, et aux pleuromutilines était observée chez les isolats porcins comparativement aux isolats laitiers (P < 0,05). Le gène mecA a été détecté à partir de tous les isolats MRSA; 89,6 % des souches résistantes à l'érythromycine portaient le gène ermC et 16,9 portaient le gène ermB. Un total de 35 ribogroupes différents a été trouvé parmi les isolats étudiés; 83,3 % des souches porcines appartenaient à 1 regroupement avec un coefficient de similarité de 0,84. En contrepartie, 3 regroupements principaux ont été observés parmi les 68,8 % d'isolats provenant de lait, ce qui indique un degré élevé de spécificité d'hôte.(Traduit par Docteur Serge Messier).

Rapid and sensitive detection of PRRSV by a reverse transcription-loop-mediated isothermal amplification assay.

A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic, prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains. As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR), the RT-LAMP method only used a turbidimeter, exhibited a detection limit corresponding to a 10(-4) dilution of template RNA extracted from 250 µL of 10(5) of the 50% tissue culture infective dose (TCID(50)) of PRRSV-containing cells, and no cross-reactivity was observed with other related viruses including porcine circovirus type 2, swine influenza virus, porcine rotavirus and classical swine fever virus. From forty-two field samples, 33 samples in the RT-LAMP assay was detected positive, whereas three of which were not detected by RT-PCR. Furthermore, in 33 strains of PRRSV, an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages. These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates, especially in developing countries.

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2.

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10(-5) ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.

Investigation of integrons/cassettes in antimicrobial-resistant Escherichia coli isolated from food animals in China.

In this study, 326 Escherichia coli isolates from food animals collected during the last four decades in China were characterized using antimicrobial susceptibility testing and screening for integrons/cassettes. Minimum inhibitory concentration (MIC) testing indicated that the antimicrobial resistance of E. coli has increased since the 1970s. The findings of this study present a warning to veterinary practitioners about the excessive use of antimicrobials, and suggest the necessity for surveillance and control of antimicrobial resistance in veterinary clinical medicine in China.

[Distributions of pathogenic capsular types and in vitro antimicrobial susceptibility of different serotypes of Streptococcus suis isolated from clinically healthy sows from 10 provinces in China].

OBJECTIVE: To determine the distributions of major pathogenic capsular types and in vitro antimicrobial susceptibility of different serotypes of Streptococcus suis isolated from clinically healthy sows in China. METHODS: Tonsil specimens of clinically healthy sows from 10 different provinces in China were collected, a total of 421 S. suis were isolated. Capsular types of S. suis were decided using the sera agglutination reaction. Antimicrobial susceptibility testing was performed using a broth microdilution method and the differences between serotypes were decided statistically. RESULTS: The prevalent capsular types of S. suis isolated from clinically healthy sows were 9 (26.6%), 3 (23.5%) and 7 (15.7%) types, respectively. 7.4% of isolates were confirmed to be S. suis type 2. Overall, differences in antimicrobial susceptibility among serotypes of S. suis were found. By comparison, lower resistance was observed for S. suis type 2 from clinically healthy sows. CONCLUSION: The prevalence of pathogenic S. suis serotypes from clinically healthy sows again indicates S. suis is a conditional pathogenic bacterium. Differential prevention and treatment regimes should be considered according to antimicrobial susceptibility of different serotypes of S. suis.

In vitro antimicrobial susceptibility of Streptococcus suis strains isolated from clinically healthy sows in China.

Streptococcus suis is an important pathogen in the swine industry. Because transmission is generally thought to occur between healthy carrier sows and their offspring, it is important to understand which antimicrobial agents are likely to be effective against the strains isolated. This study is the first to report on the antimicrobial susceptibility of S. suis isolated from clinical healthy sows. From 2005 to 2007 a total of 421 S. suis isolates were recovered from sows in China and subjected to antimicrobial susceptibility testing in accordance with the Clinical and Laboratory Standards Institute (CLSI) standards. High-level resistance were found with tetracycline (91.7%) and sulfisoxazole (86.7%), followed by clindamycin (68.4%), erythromycin (67.2%), tilmicosin (66.7%) and trimethoprim/sulfamethoxazole (59.1%). These six antimicrobial agents presented the highest MIC50 values and the antibiogram (19.2%) most frequently observed. Lower resistance rates among the beta-Lactams support their use as the primary drugs to treat the infection of S. suis. However, appropriate dosing or combination antibiotic therapeutic regimens should be adhered to in view of the resistant and intermediate strains to penicillin (9.5% and 42.3%), ampicillin (4.0% and 29.9%) and ceftiofur (22.1% and 37.3%), respectively.

Complete genome sequence of attenuated low-temperature Thiverval strain of classical swine fever virus.

The Thiverval vaccine strain of classical swine fever virus (CSFV) was derived from virulent Alfort strain through the serial passages in cells at 29-30 degrees C. In this study, we determined the complete genome sequence of this strain and found that its genome contains one open reading frame (ORF) that encodes a polyprotein with 3,898 amino acids. The 5'-UTR of Thiverval is 373 nt long with only one mutation at position 220. In contrast, the length of 3'-UTR is highly heterogeneous ranging from 233 to 259 bp. The heterogeneity of length of the 3'-UTR was due to an insertion of a variable length of T-rich sequence ranging from 6 to 32 nt. The insertion may change the structure and free energy of the 3'-UTR, resulting in a destabilization of the 3'-UTR. Sequence alignment of Thiverval and other CSFV strains showed 85.2-99.6% identities at the nucleotide level and 92.5-99.5% at the amino acid level. The phylogenetic tree analysis of the complete ORF, partial region of E2, and NS5B suggests that the CSFV Thiverval strain belongs to genetic group 1 and subgroup 1.1. The results from this study provide insight into the molecular mechanism of the attenuation of Thiverval vaccine strain.

12-nt insertion in 3' untranslated region leads to attenuation of classic swine fever virus and protects host against lethal challenge.

We report here the discovery of an attenuation mechanism of classic swine fever virus (CSFV) induced by introduction of a continuous 12-nt (CUUUUUUCUUUU) insertion in viral 3' UTR. The 12-nt insertion sequence was first found in one attenuated vaccine strain HCLV (Hog Cholera Lapinized Virus) which did not exist in other CSFV strains. To address the function of the 12-nt insertion in viral replication and attenuation, we constructed and analyzed two chimeras stemmed from a highly virulent strain Shimen either with introduction of the 12-nt insertion in 3' UTR or the replacement of viral 3' UTR by the 3' UTR of HCLV. We found that the two chimeras' maximum titers declined approximately 100-fold than their parental strain Shimen in PK15 cells. An animal experiment showed that the two chimeras were both dramatically attenuated in pigs. All the chimera-infected pigs survived infection and remained clinically normal with the exception of a transient fever while the 100% mortality was observed for the Shimen-infected pigs. In addition, the two chimeras can induce neutralization antibody to completely protect the pigs against lethal challenge with highly virulent CSFV, which was similar to the vaccine strain HCLV. These data demonstrate that the 12-nt insertion in 3' UTR is sufficient for the attenuation of CSFV. Taken together, a novel attenuation mechanism of CSFV is found and may pave a way to further research for new attenuated vaccine.

[Research of real-time fluorescent PCR in the rapid differential detection of H5, H9, H7 subtype avian influenza inactivated vaccines].

Specific primers and TaqMan MGB probes were designed with Primer Express 2.0 software according to the conserved region of the H5, H9, H7 subtype AIV hemagglutinin gene to make research of real-time fluorescent one-step PCR in the differential detection of H5, H9, H7 subtype avian influenza inactivated vaccines. The result showed that the method was specific and reproducible. No cross-reaction was discovered with other avian disease vaccines. Real-time fluorescent PCR provided a specific, sensitive, rapid and convenient method for the subtype identification of avian influenza inactivated vaccines.

Induction of macrolide resistance in Mycoplasma gallisepticum in vitro and its resistance-related mutations within domain V of 23S rRNA.

Antibiotic-resistant mutants of Mycoplasma gallisepticum were selected in vitro from the susceptible strains S6 and BG44T by serial passages in stepwise concentrations of erythromycin, tylosin, or tilmicosin. High resistance to erythromycin or tilmicosin developed readily, whereas resistance to tylosin developed only after greater numbers of passages. Three mutants selected by each selector antibiotic were cloned and detected, and all cloned mutants exhibited cross-resistance to the three selector antibiotics as well as to lincomycin. Portions of the genes encoding domain V of 23S rRNA of the cloned mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible parent strains. Five of the six mutants selected by erythromycin harbored an A2058G (Escherichia coli numbering) mutation in one of the two 23S rRNA. One of the six mutants selected by erythromycin harbored a G2057A mutation and an A2059G mutation in the other 23S rRNA. In tilmicosin-selected mutants, two mutations, A2058G and A2503U, occurred in one of the two 23S rRNA. No mutation was detected in the two 23S rRNA of tylosin-selected mutants with low-level resistance. Mutations at homologous locations in the 23S rRNA of other macrolide-resistant bacteria indicate that the phenotype of macrolide resistance occurring in M. gallisepticum is strongly associated with point mutations in domain V of 23S rRNA.