Tsang Long Lin

Experimental Infection of Young Specific Pathogen-Free Cats with Bartonella henselae

Eighteen 12-week-old specific pathogen-free cats, blood culture- and serum antibody-negative for Bartonella henselae, were randomly allocated to groups and were intravenously inoculated with 10(10) (group 1), 10(8) (group 2), or 10(6) (group 3) B. henselae or with saline (group 4) or were not inoculated (group 5). Cats were humanely killed at 2, 4, 8, 16, and 32 weeks after inoculation. All B. henselae-inoculated cats were bacteremic by 2 weeks after infection. Bacteremia persisted until 32 weeks after infection in 1 cat. Cats in groups 1 and 2 had fever (>39.7 degrees C) and partial anorexia by 2 weeks after infection that lasted 2-7 days. All infected cats had Bartonella-specific IgM and IgG serum antibodies and lymphocyte blastogenic responses. Histopathologic lesions were observed in multiple organs of infected cats through 8 weeks after infection. Cats were readily infected with B. henselae by intravenous inoculation, developed histopathologic lesions that apparently resolved, and developed B and T lymphocyte responses to infection.

DNA-mediated vaccination against infectious bursal disease in chickens

The objective of the present study was to investigate the feasibility of a DNA vaccine to protect chickens against infectious bursal disease virus (IBDV) infection. A plasmid DNA carrying VP2, VP4, and VP3 genes of the standard challenge (STC) strain of IBDV was constructed and designated as pCR3.1-VP243-STC. One-day-old chickens were intramuscularly injected with the plasmid pCR3.1-VP243-STC once (group D1), twice (group D2), or three times (group D3) at weekly intervals. Chickens at 3 weeks old were orally inoculated with IBDV strain STC and observed for 10 days after challenge. Immunization twice (group D2) or three times (group D3) with the plasmid pCR3.1-VP243-STC conferred protection for 50-100 or 80-100% of chickens, respectively, as evidenced by the absence of clinical signs, mortality, and bursal atrophy. Although chickens vaccinated once (group D1) with the plasmid pCR3.1-VP243-STC did not have clinical signs, they exhibited varying degree of bursal atrophy after challenge. Enzyme-linked immunosorbent assay (ELISA) antibody titers in chickens protected by the plasmid pCR3.1-VP243-STC were significantly lower (P<0.05) than those not protected 10 days after challenge. IBDV antigen was not detected in the bursae of chickens that were protected by receiving the plasmid pCR3.1-VP243-STC twice or three times. The results indicate that the constructed plasmid pCR3.1-VP243-STC as a DNA vaccine provided efficacious protection for chickens against IBDV infection.

Molecular Detection and Differentiation of Infectious Bursal Disease Virus

Abstract SUMMARY. Vaccination of hens, with the subsequent maternal immunity imparted to chicks, is the primary means of controlling infectious bursal disease virus (IBDV). Effective vaccination depends on rapid and accurate diagnosis of the subtype present in a flock because vaccines based on the classic subtype of IBDV can fail to protect against challenge with a variant subtype. This review describes the various methods available to detect and differentiate between IBDV subtypes. Serotype 1 IBDV causes economically significant immunosuppressive disease in young chickens. Within serotype 1, two subtypes, classic and variant, can be differentiated by the virus neutralization assay. Antigen capture enzyme-linked immunosorbent assay (AC-ELISA) with MAbs has been successful at differentiating the very virulent IBDV phenotype (vvIBDV) from less pathogenic types. More rapid and sensitive molecular diagnostic methods based on reverse transcription–polymerase chain reaction (RT-PCR) for amplification of the IBDV VP2 gene have been a major focus of investigation in recent years. Conventional RT-PCR has been useful in detecting IBDV serotypes and, to a lesser extent, differentiating IBDV subtypes. One of the approaches has been the use of SspI and NgoM IV restriction enzymes, for restriction endonuclease (RE) analysis of RT-PCR products (RT-PCR-RE) and BstNI and MboI for restriction fragment length polymorphism (RFLP) analysis (RT-PCR-RFLP) to find unique banding patterns associated with antigenic variation within the variable region of the IBDV VP2 protein. However, these approaches were ultimately found to be unreliable because subtypes could not be consistently distinguished with restriction enzymes. These limitations led to studies in differentiating subtypes by detection of single nucleotide differences in sequence through real-time RT-PCR or DNA sequencing of RT-PCR products. Conventional RT-PCR, amplifying the VP2 hypervariable region, in combination with DNA sequencing of the PCR product, can differentiate classic, variant, and vvIBDV strains because variant and vvIBDV have characteristic nucleotide and amino acid substitutions. Real-time RT-PCR, targeting different regions of the IBDV genome, including VP1, VP2, and VP4 genes, in conjunction with melting-curve analysis is being investigated as a promising tool for molecular diagnosis of IBDV infection. These methods potentially allow for more rapid, sensitive, and specific detection and differentiation of IBDV classic, very virulent, and variant subtypes.

Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus.

Abstract The complete nucleotide sequence of polyprotein gene 1 and the assembled full-length genome sequence are presented for turkey coronavirus (TCoV) isolates 540 and ATCC. The TCoV polyprotein gene encoded two open reading frames (ORFs), which are translated into two products, pp1a and pp1ab, the latter being produced via −1 frameshift translation. TCoV polyprotein pp1a and pp1ab were predicted to be processed to 15 non-structure proteins (nsp2–nsp16), with nsp1 missing. ClustalW analysis revealed 88.99% identity and 96.99% similarity for pp1ab between TCoV and avian infectious bronchitis virus (IBV) at the amino acid level. The whole genome consists of 27,749nucleotides for 540 and 27,816nucleotides for ATCC, excluding the poly(A) tail. A total of 13 ORFs were predicted for TCoV. Five subgenomic RNAs were detected from ATCC-infected turkey small intestines by Northern blotting. The whole genome sequence had 86.9% identity between TCoV and IBV, supporting that TCoV is a group 3 coronavirus.

Immune response of neonatal specific pathogen-free cats to experimental infection with Bartonella henselae

The purpose of this study was to determine whether neonatal cats develop and maintain a persistent bacteremia for longer than do adult cats with a normal mature immune system, and whether neonatal cats are susceptible to infection with Bartonella henselae by oral inoculation. Neonatal specific pathogen-free (SPF) cats were inoculated with B. henselae intradermally (n = 4) or orally (n = 5) or with 0.9% NaCl (n = 2). Blood was collected periodically through 16 weeks post-inoculation (PI) for serology, bacteriology and complete blood count. Cats inoculated orally or intradermally at 3-5 days of age were bacteremic through 12-16 weeks PI, similar to what is documented for adult cats inoculated intradermally or intravenously. One cat inoculated at age 2 weeks was bacteremic through 10 weeks PI; the other was not bacteremic. Intradermally inoculated neonatal cats produced serum IgG antibodies to B. henselae but orally inoculated neonatal cats did not. Infected cats with and without serum IgG antibodies to B. henselae became blood-culture negative simultaneously, suggesting that IgG is not required to clear bacteremia.

Complete sequences of 3′ end coding region for structural protein genes of turkey coronavirus

Abstract Overlapping fragments of genomic RNA spanning 6963 nucleotides from 5′ end of spike (S) protein gene to 3′ end of nucleocapsid (N) protein gene of turkey coronavirus (TCoV) were amplified by reverse-transcription-polymerase chain reaction (RT-PCR). The primers were derived from the corresponding sequences of infectious bronchitis virus (IBV). The PCR products were cloned and sequenced and their nucleic acid structure and similarity to published sequences of other coronaviruses were analyzed. Sequencing and subsequent analysis revealed 9 open reading frames (ORFs) representing the entire S protein gene, tricistronic gene 3, membrane (M) protein gene, bicistronic gene 5, and N protein gene in the order of 5′–3′. The overall nucleic acid structures of these encoding regions of TCoV were very similar to the homologous regions of IBV. The consensus transcription-regulating sequence (TRS) of IBV, CT(T/G)AACAA, was highly conserved in TCoV genome at the levels of nucleotide sequence and location in regarding to the initiation codon of individual genes. Pair-wise comparison of gene 3, M gene, gene 5, or N gene sequences with their counterparts of IBV revealed high levels (82.1–92.0%) of similarity. Phylogenetic analysis based on the deduced amino acid sequences of S, M, or N protein demonstrated that TCoV was clustered within the same genomic lineage as the IBV strains while all the other mammalian coronaviruses were grouped into separate clusters corresponding to antigenic groups I or II. There were substantial differences of S protein sequence between TCoV and IBV with only 33.8–33.9% of similarity.

SipC multimerization promotes actin nucleation and contributes to Salmonella-induced inflammation.

Actin nucleation is the rate‐limiting step in actin assembly and is regulated by actin‐binding proteins and signal transduction molecules. Salmonella enterica serovar Typhimurium exploits actin dynamics by reorganizing the host actin cytoskeleton to facilitate its own uptake. SipC is a Salmonella actin‐binding protein that nucleates actin filament formation in vitro. The molecular mechanism by which SipC nucleates actin is not known. We show here that SipC199–409 forms multimers to promote actin nucleation. We found that wild‐type SipC199–409 forms dimers and multimers while SipC199–409#1, a nucleation mutant, is less efficient in dimer and multimer formation. Biochemical analysis suggested that SipC199–409 might form parallel dimers in an extended conformation. Furthermore, a mutant Salmonella strain that was defective in forming the SipC multimer and deficient in actin nucleation failed to cause severe colitis in a mouse model. These results allow us to present a model in which SipC forms multimers to promote actin nucleation.

Antimicrobial susceptibility of Mycoplasma hyorhinis

A broth microdilution technique was used to determine the antimicrobial susceptibility of 15 field isolates of Mycoplasma hyorhinis to 10 antimicrobial agents, representative of different classes, and contrasting newer agents to existing ones. For the macrolides, the MIC(90) for tylosin and tilmicosin was 1 and 4 microg/ml, respectively, but was > or = 16 microg/ml for erythromycin. Tetracycline, lincomycin and enrofloxacin each had an MIC(90) of 2 microg/ml. The mycoplasma had similar levels of susceptibility to the aminoglycoside and aminocyclictol classes exhibiting an MIC(90) of 4 microg/ml for gentamicin and 2 microg/ml for spectinomycin. The isolates exhibited high MICs to trimethoprim/sulfamethoxazole with an MIC(90) > or = 16/304 microg/ml. In summary, M. hyorhinis isolates from the US had low MICs against a variety of antimicrobials tested, with the exception of erythromycin and trimethoprim/sulfamethoxazole.

DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody

The objective of the present study was to determine if a DNA vaccine carrying large segment gene of infectious bursal disease virus (IBDV) could confer protection against infectious bursal disease (IBD) in broiler chickens in the presence of maternal antibody. Broiler chickens with maternal antibody titers to IBDV were intramuscularly injected with a DNA plasmid coding for VP2, VP3, and VP4 genes of IBDV strain variant E (VE) (P/VP243/E) at 1-day, 1-week, and/or 2 weeks old. The dose of P/VP243/E used ranging from 400microg to 10mg. Broiler chickens at 3 weeks old were orally challenged with IBDV strain (VE) and observed for 10 days. Only broiler chickens vaccinated with 7.5 or 10mg of P/VP243/E 3 times had 90 or 100% protection against challenge by IBDV strain VE and protected broiler chickens had significantly higher (P<0.05) bursa weight/body weight (B/B) ratios, significantly lower (P<0.05) bursal lesion scores, and the absence of IBDV antigens in bursae determined by immunofluorescent antibody assay (IFA). Antibody titers to IBDV as determined by enzyme-linked immunosorbent assay (ELISA) or virus neutralization (VN) assay in chickens of each group in each trial were gradually decreased prior to challenge. There was no significant difference (P>0.05) in ELISA or VN titers to IBDV among all groups of broiler chickens or among the groups of broiler chickens vaccinated with various dose of P/VP243/E before challenge. Broiler chickens in the groups receiving 7.5 or 10mg of P/VP243/E had significantly lower (P<0.05) ELISA or VN titers to IBDV than those in the challenge control (CC) groups or the other groups vaccinated with various dose of P/VP243/E after challenge. Broiler chickens in the groups vaccinated with 10mg of P/VP243/E 3 times had significantly higher (P<0.05) stimulation indices for IBDV-stimulated lymphocyte proliferation response than those in the vector control (VC) or CC group at 14, 21, 24, or 31 days after first DNA vaccination. The results indicated that DNA vaccination with DNA encoding large segment gene of IBDV confers protection against challenge by IBDV in broiler chickens with maternal antibody to IBDV.

Antimicrobial susceptibility and resistance of chicken Escherichia coli, Salmonella spp., and Pasteurella multocida isolates.

Abstract Escherichia coli, Salmonella species, and Pasteurella multocida are the major bacterial pathogens isolated from poultry. Difference in susceptibility to antibiotics by microorganisms has become a major factor in drug choice and success of treatment. Great concerns have been raised Regarding emerging antimicrobial resistance among bacteria that may result in unpredictable antimicrobial susceptibility and failure of therapy. The primary objective of the present study was to determine the levels of antimicrobial susceptibility/resistance of E. coli, Salmonella species, and P. multocida isolated from diseased chickens. A total of 445 E. coli isolates, 387 Salmonella spp. isolates, and 80 P. multocida isolates from diseased chickens during the period ranging from 2001 to 2003 were obtained. Minimal inhibitory concentrations of 14 antimicrobial agents against each bacterial isolate were determined using a microbroth dilution assay described by the Clinical Laboratory Standards Institute. Resistance of E. coli isolates measured as follows: 98.20% were resistant to tilmicosin, 79.33% to tetracycline, 51.46% to spectinomycin, 44.04% to gentamicin, and 40% to ampicillin. Resistance to tetracycline was found in 72.61% of Salmonella spp. isolated, followed by resistance to spectinomycin (68.48%), ampicillin (63.57%), gentamicin (63.31%), and ticarcillin (61.76%). The resistance rate of P. multocida isolates to all antimicrobials tested was less than 5%, except for tetracycline (6.25%). In summary, E. coli and Salmonella isolates were sensitive to ceftiofur and fluoroquinolones but were resistant to other antimicrobials tested, while P. multocida isolates remained sensitive to all the antimicrobial agents tested in a 3-yr analysis.