The vaccine efficacy of a genetically engineered deletion mutant strain of pseudorabies virus, strain 783, was compared with that of the conventionally attenuated Bartha strain. Strain 783 has deletions in the genes coding for glycoprotein I and thymidine kinase. In experiment 1, which had a 3-month interval between vaccination and challenge exposure, strain 783 protected pigs significantly (P < 0.05) better against virulent virus challenge exposure than did the Bartha strain. The growth of pigs vaccinated with strain 783 was not arrested, whereas that of pigs vaccinated with the Bartha strain was arrested for 7 days. Of 8 pigs given strain 783, 4 were fully protected against challenge exposure; none of the pigs given strain Bartha was fully protected. In experiment 2, which had a 3-week interval between vaccination and challenge exposure, the growth of pigs vaccinated with strain 783 was arrested for 3.5 days, whereas that of pigs vaccinated with the Bartha strain was arrested for 6 days. In experiment 3, pigs with moderate titer of maternal antibodies were vaccinated twice IM or once intranasally with either strain 783 or Bartha and were challenge-exposed 3 months after vaccination. Pigs given strain 783 twice IM were significantly (P < 0.05) better protected than were the other pigs. They had growth arrest of only 6 days, compared with 9 days for pigs of other groups, and shed less virus after challenge exposure. Results of this study indicate that the vaccine based on the deletion mutant strain 783 is more efficacious than is the Bartha strain of pseudorabies virus.

Introduction: Avian pathogenic Escherichia coli (APEC) is a leading cause of extraintestinal infection and heavy economic losses. Imparting immunity after vaccination with live attenuated strain vaccination is an ideal strategy for infection control. This study considers an FtsK knockout mutant strain as a candidate. Material and Methods: An FtsK knockout mutant of APEC strain E058 was constructed and the pathogenicity of the mutant and wild-type strains was further evaluated in chickens. Results: The 50% lethal doses of each strain for one-day-old specific-pathogen-free (SPF) chickens challenged experimentally via trachea were 10⁵.⁵ and 10⁷.⁰ colony-forming units (CFU) respectively. Chickens challenged with the wild-type strain exhibited typical signs and lesions of avian colibacillosis, while those inoculated with the mutant strain showed mild pericarditis and pulmonary congestion. The growth rate of the FtsK mutant strain was much slower than the wild-type strain in the heart, spleen, liver, and lung of infected chickens. Conclusion: These results indicated that the APEC FtsK mutant can be attenuated for chickens, and that this mutant has the potential for the development of an APEC vaccine.

Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection. Material and Methods: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined. Results: The core centres (Ser³² and Cys⁴⁷) of Prdx6 were successfully mutated by changing the 94ᵗʰ nucleotide from T to G and the 140ᵗʰ nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit. Conclusion: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

Brucellosis is one of the common zoonoses caused by Brucella abortus (B. abortus). However, little has been reported on factors affecting invasion of B. abortus into host cells. To investigate cell-type dependent invasion of B. abortus, phagocytic RAW 264.7 and THP-1 cells and non-phagocytic HeLa cells were infected with wild-type and mutant B. abortus, and their invasion efficiencies were compared. The invasion efficiencies of the strains were cell-type dependent. Wild-type B. abortus invasion efficiency was greater in phagocytic cells than in epithelial cells. The results also indicated that there are different factors involved in the invasion of B. abortus into phagocytic cells.

Studies on virulence factors and pathogenecity of Haemophilus parasuis have long been hindered by a lack of a consistent system for genetic manipulation. In this study, competence was induced by transferring H. parasuis from rich medium to starvation medium media-IV (M-IV) and iscR gene deficient mutants of H. parasuis were generated efficiently. Transformation frequency varied from 4.1 × 10(−5) to 1.1 × 10(−8) when using circular plasmid, and increased to about 2- to 31-fold when transformed using linearized plasmid. Allele replacement occurred efficiently in 6 strains, which are transformable using both circular and linearized pTRU, but not in another 2 strains which could only be transformed using linearized plasmid. The iscR mutants were stable for at least 20 passages in vitro. Haemophilus parasuis strains vary extensively in natural transformation efficiency and the method established here allows for transformation of a larger spectrum of strains with an easily accessed plasmid. This provides important tools for genetic manipulation of H. parasuis.

OBJECTIVE To compare mitochondrial oxygen consumption rate (OCR) of fibroblasts from Doberman Pinschers with and without dilated cardiomyopathy (DCM) and mutation of the gene for pyruvate dehydrogenase kinase isozyme 4 (PDK4) and to evaluate in vitro whether treatment with adeno-associated virus (AAV) vector (ie, gene therapy) would alter metabolic efficiency. ANIMALS 10 Doberman Pinschers screened for DCM and PDK4 mutation. PROCEDURES Fibroblasts were harvested from skin biopsy specimens obtained from Doberman Pinschers, and dogs were classified as without DCM or PDK4 mutation (n = 3) or with occult DCM and heterozygous (4) or homozygous (3) for PDK4 mutation. Fibroblasts were or were not treated with tyrosine mutant AAV type 2 vector containing PDK4 at multiplicities of infection of 1,000. Mitochondrial OCR was measured to evaluate mitochondrial metabolism. The OCR was compared among dog groups and between untreated and treated fibroblasts within groups. RESULTS Mean ± SD basal OCR of fibroblasts from heterozygous (74 ± 8 pmol of O2/min) and homozygous (58 ± 12 pmol of O2/min) dogs was significantly lower than that for dogs without PDK4 mutation (115 ± 9 pmol of O2/min). After AAV transduction, OCR did not increase significantly in any group (mutation-free group, 121 ± 26 pmol of O2/min; heterozygous group, 88 ± 6 pmol of O2/min; homozygous group, 59 ± 3 pmol of O2/min). CONCLUSIONS AND CLINICAL RELEVANCE Mitochondrial function was altered in skin fibroblasts of Doberman Pinschers with DCM and PDK4 mutation. Change in mitochondrial function after in vitro gene therapy at the multiplicities of infection used in this study was not significant.

OBJECTIVE To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. SAMPLE 4 unique isolates of M haemolytica recovered from clinically infected cattle. PROCEDURES Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. RESULTS Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.

Previously we described the development of an attenuated Pasteurella multocida mutant that expresses only the N-terminal truncated fragment of P. multocida toxin (N-PMT) and its protective effects in a mouse model. This paper details our evaluation of the vaccine potential of this mutant strain in pigs. Pigs vaccinated with the mutant showed significantly higher rates of antibody induction and lower nasal conchal (turbinate) scores for atrophic rhinitis than controls, which suggests that this mutant strain may be a good candidate for a live attenuated vaccine.

Kittens are the principal disseminators of Toxoplasma gondii. They can shed > 10(8) oocysts in the feces after initial infection with bradyzoites in tissue cysts. Thereafter, most kittens develop protective immunity and do not shed oocysts again if they are reinfected. Bradyzoites of a T gondii mutant, designated T-263, were used to vaccinate kittens. Their use did not result in oocyst shedding, but successfully prevented 84% (31/37) of the kittens from shedding oocysts when challenge exposed with a normal isolate of T gondii. Vaccination of outdoor-roaming cats and kittens would be a useful public health measure to prevent transmission of toxoplasmosis near homes, on farms, and in zoos. It is anticipated that several years will be required for a lyophilized bradyzoite vaccine to be ready for licensing and possible commercial availability.