Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia.

Porcine circovirus type 2 (PCV2) causes some of the most significant economic losses in pig production. Several multisystemic syndromes have been attributed to PCV2 infection, which are known as PCV2-associated diseases (PCVDs). This study investigated the origin and evolution of PCV2 sequences in domestic pigs and wild boars affected by PCVDs in Croatia. Viral sequences were recovered from three wild boars diagnosed with PCV2-systemic disease (PCV2-SD), 63 fetuses positive for PCV2 DNA as determined by PCR, 14 domestic pigs affected with PCV2-SD (displaying severe interstitial nephritis) and five domestic pigs with proliferative and necrotising pneumonia. Seventeen complete PCV2 genomes were recovered. Phylogenetic and evolutionary analyses based on median-joining phylogenetic networks, amino acid alignments and principal coordinate analysis were performed using complete genomes, as well as complete and partial ORF sequences for ORF1 and ORF2. Two of the 17 PCV2 sequences belonged to PCV2a, 14 to PCV2b and one was unclustered. PCV2b was the predominant genotype in Croatia and has been linked to international trade as a route of introduction. Correlation between particular viral strains with PCVDs is lacking.

Association of porcine circovirus type 2 with vascular lesions in porcine pneumonia.

The aim of this study was to evaluate the vasculature in porcine circovirus type 2-infected (PCV2-infected) lungs and to identify the PCV2 subtypes involved in porcine pneumonia. Pulmonary samples from 140 pigs, 2 weeks to 7 months of age, from 36 Hungarian commercial herds with clinical signs of respiratory disease were examined for the presence of respiratory pathogens, with bacterial culture, pathologic evaluation, and immunohistochemistry for PCV2, porcine reproductive respiratory syndrome virus, and swine influenza virus. PCV2 was the most commonly identified pathogen (49 cases) among the 74 of 140 cases (53%) with respiratory pathogens. PCV2 was detected immunohistochemically in the wall of 13% to 100% of pulmonary vessels (mean, 89%) in 38 of 49 cases (78%). Detection of PCV2 antigen was positively correlated with the presence of vascular lesions (P < .001, odds ratio [OR]: 159.54). Other pathogens capable of vascular injury in swine were found in 29 of 49 of the PCV2-positive cases (59%). The probability of detecting vascular lesions in PCV2-infected lung was higher than in infection with porcine reproductive respiratory syndrome virus (P < .002, OR: 14.63), Pasteurella multocida infection (P < .001, OR: 5.75), or Streptococcus spp. infection (not significant, OR: 1.45). Sequence analysis of open reading frame 2 amplicons was possible in 6 PCV2-positive cases, from which 5 cases proved to be PCV2b subtype and 1 case, PCV2a subtype. In conclusion, PCV2 antigen was commonly colocalized with pulmonary vascular lesions in pneumonia in Hungarian swine, and PCV2b was the dominant subtype.

Effect of different levels of mannan-oligosaccharide supplementation on some immunological variables in weaned piglets.

The effect of different doses of mannan-oligosaccharide (MOS) on specific and non-specific immune responses was studied in piglets, weaned at 28 days. A total of 58 piglets were used in six groups. Five groups were fed 0, 1, 2, 4 g MOS product per kg diet or with growth promoting antibiotics and immunized by inactivated Aujeszky's disease virus (AyV) vaccine at week 1 and 3 of the experiment (35 and 49 days). A sixth group, receiving the same non-supplemented diets was not immunized. Blood samples for lymphocyte stimulation (LST) and AyV neutralization (VN) tests were taken from all pigs on the first day of the experiment and at weekly intervals for 5 weeks. At week 8, the immunized piglets were infected orally with transmissible gastroenteritis virus. All piglets were weighed and slaughtered at week 10, digesta from small intestine were collected and tested for the presence of secretory (s)IgA. Feeding MOS supplementation resulted in enhanced specific and non-specific immune responses, however, a regressive dose-response of MOS was observed. Both the specific cellular (LST) and humoral responses (VN) were enhanced after 2 weeks of feeding 1 g/kg MOS and significantly differed from the antibiotic positive control. The same tendency was detected in case of the non-specific LSTs, although these started some weeks later showing significant differences by the fifth week. Higher doses of MOS had no further beneficial effect on systemic immunity. In addition, 1 g/kg MOS supplementation group also showed some advantage in local immune responsiveness. Therefore, based on the studied immune variables, 1 g/kg MOS product is suggested in the diet of weaned piglets.

Porcine circovirus type 2 and associated diseases in Romania--short communication.

Porcine circovirus type 2 (PCV2) has been demonstrated to be the causal agent for postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). This report describes the first detection of PCV2 and associated diseases in a Romanian swine herd located in Transylvania. The clinical signs, pathological and histopathological changes observed in affected pigs were similar to those previously described for PDNS and PMWS. Polymerase chain reaction and in situ hybridisation were used for the detection of PCV2 nucleic acids from tissues and serum samples. Complete PCV2 genomes of both PMWS and PDNS cases were sequenced and analysed, and by comparing them with each other no genomic differences could be detected. The sequence analysis showed that the Romanian PCV2 was closely related to PCV2 identified in France and in Hungary.

Genetic characterization of type 2 porcine circoviruses detected in Hungarian wild boars.

Porcine circoviruses (PCV) are present in pigs worldwide; they are grouped into two types: PCV1 comprising non-pathogenic viruses and PCV2 responsible for several clinical manifestations. Both types are frequently detected in domestic pigs, the prevalence and role of PCV in wild boars however, is not well studied. During the years 2002-2003 over 2000 organ samples of Hungarian wild boars were collected, grouped and samples from 307 different animals were tested by polymerase chain reaction for the presence of PCV. 35.5% of the wild boars were positive for one or both PCV types and PCV2 was detected in 20.5% of the animals. The PCV2 viruses were divided into 7 groups (WB-H1-7) based on sequencing data and genomes representing these groups were sequenced completely. The wild boar PCV2 groups were distributed evenly in the geographical region, regardless of the time and place of collection. The phylogenetic analysis of the PCV2 sequences of wild boar and domestic pig origin showed the possibility of an epidemiological link between wild boar and domestic pig infections. Interestingly, the complete nucleotide sequence of the viruses and the predicted amino acid sequence of the replication associated protein (ORF1) grouped the viruses similarly, whereas the capsid protein (ORF2) comparisons revealed different relations among the groups, suggesting the possibility of genomic recombination in PCV2.

Sequence analysis and deletion of porcine adenovirus serotype 5 E3 region.

A 3000 basepair (bp) region corresponding to the E3 region, the flanking pVIII and part of the fiber protein genes, of the two prototype strains (HNF-61 and HNF-70) of porcine adenovirus serotype five (PAdV-5) was sequenced. A potential E3 promoter and poly-A signals were identified. The size of the E3 region was 2039 (strain HNF-61) and 2020 bp (strain HNF-70) the largest E3 so far reported among PAdVs. Three open reading frames (ORF2-4) were identified within the E3 region. Based on the predicted amino acid (aa) sequences ORF2 was similar to other adenovirus E3 ORFs, ORF3 showed some similarity to a bovine adenovirus (BAdV-1) ORF. ORF4 was unique to PAdV-5. E3 mRNA transcripts were detected early in infection by Northern blot analysis. Genomic clones of HNF-70 with a 1505 or 1237 bp deletions in the E3 region were constructed to map non-essential regions. After transfection of the DNA into swine testicle cells, virions were recovered for only the shorter 1237 bp deletion. At least 60% of the E3 region was not essential for virus replication, bringing the theoretical vector capacity of a helper independent PAdV-5 to 2.9 kb.

Characterization of early region 4 of porcine adenovirus serotype 5.

To locate the E4 region on the genome of the HNF-70 prototype strain of the porcine adenovirus serotype 5 (PAdV-5), 4.5 kb at the extreme right end was sequenced. This area had 11 open reading frames (ORFs) on the left strand encoding more than 50 amino acids (aa). A Genbank homology search indicated that 8 of these ORFs were unique to PAdV-5. ORF5:253 aa, ORF6:245 aa and ORF11:146 aa showed similarities to known mammalian and avian adenovirus E4 ORFs. The putative E4 promoter sequences were also identified. Two TATA boxes, several GC boxes and two typical CCAAT boxes were located within this region. The presence of several potential poly-adenylation signals and the number of E4 transcripts detected by Northern blot analysis suggested the possibility of a complex mRNA transcription.

Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants.

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. Three transgenic plant lines were generated expressing the spike (S) protein of transmissible gastroenteritis virus (TGEV), a protein crucial for establishing mucosal immunity. All three of them were driven by a strong plant promoter. One construct contained the 3.7 kb 5' end of the native S gene sequence. In the second construct part of the S gene, from nucleotide 49 to 1785, was modified for optimal plant recognition and was fused to a plant signal peptide coding sequence. The third construct contained the D epitope-coding region of the S gene, from nucleotide 1201 to 1591, which was fused to the alfalfa beta-amylase gene. The S gene products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antigens from all three transgenic plant lines induced TGEV-specific immune responses in pigs as determined by virus neutralization and ELISA, and the resultant antibody titers for all three constructs were similar.

New pig disease in Hungary: postweaning multisystemic wasting syndrome caused by circovirus (short communication).

Postweaning multisystemic wasting syndrome (PMWS), a new disease in Hungary, was recognized in a swine herd located in Southeast Hungary, during the early winter of 1999. The first clinical signs of paleness, anaemia, and leanness appeared immediately after weaning, at the age of 40-50 days. Pustules were frequently observed on the skin of the trunk, and signs of necrotic dermatitis were also visible. A syndrome of poor growth and wasting was characteristic of the affected pigs. A porcine circovirus (PCV), the suspected causative agent, was detected by polymerase chain reaction (PCR). Sequencing data and restriction endonuclease (RE) analysis of the PCR products suggested that the virus belonged to the PCV-II group where all the causative agents of PMWS are also grouped.

Porcine adenoviruses: an update on genome analysis and vector development.

Although porcine adenoviruses (PAdV) are present in the swine populations worldwide, they usually do not cause any disease, or the infection is only manifested in a mild diarrhoea or respiratory signs. The importance of adenoviruses, however, is constantly growing as there is a possibility of developing them into viral vector vaccines against more significant swine pathogens. A short summary of the well-established facts of porcine adenoviruses is given and recent developments of the genetic analysis of these viruses are discussed in detail. The possibilities of vector development and examples of vector vaccines already reported in the literature are mentioned.

Application of the polymerase chain reaction to detect fowl adenoviruses.

The possibility of using the polymerase chain reaction (PCR) for the detection of fowl adenoviruses (FAdV) was tested. The optimal reaction parameters were evaluated and defined for purified genomic DNA of type 8 fowl adenovirus (FAdV-8), and then the same conditions were applied for nucleic acid extracted from infected cells. One hundred picograms of purified viral DNA, or 250 FAdV-8-infected cells, were detected by ethidium bromide staining of the PCR products in agarose gels. The sensitivity was increased to 10 pg purified viral DNA, or 25 infected cells, when the PCR products were hybridized with a specific labeled probe. Several field isolates of FAdV and the CELO virus (FAdV serotype 1) could be amplified by the same primers and conditions, but the size of the amplicons was smaller than that for the FAdV-8 PCR product. Other avian viruses and uninfected cell cultures tested negative.

Comparison of the inverted terminal repetition sequences from five porcine adenovirus serotypes.

The nucleotide sequences of the region of inverted terminal repetition from representative strains of all five porcine adenovirus (PAV) serotypes were determined and analyzed. The first 17 nucleotides of this region were identical in PAV-1 to 3 and PAV-5, and 10 bp of identical sequence was found in all the PAVs. The closest relationships were among PAV-1 to 3, which shared more common sequences than the other serotypes. PAV-4 had the longest inverted terminal repeat reported for any adenovirus. The proximal 54-bp AT-rich region was partially conserved and the distal GC-rich region was less well conserved among all five serotypes.

Restriction endonuclease analysis and physical mapping of the genome of porcine adenovirus type 5.

The HNF61 and HNF70 isolates of porcine adenovirus type 5 (PAV-5) were cultivated in PK-15 cells, and viral DNA was extracted from the infected cells by a modified Hirt procedure. The DNAs were digested by each of 9 restriction endonucleases, and fragments representing the entire genomes were cloned. Based on the sizes of the restriction enzyme fragments, the genome of each isolate was estimated to be 33.2 kb. Physical maps for the 9 restriction endonucleases were constructed. The physical maps of the two isolates were identical for 5 of the restriction endonucleases, but 4 enzymes revealed differences in restriction sites occurring mainly between map units 78 and 83, which may include the E3 region of the genome. There were no similarities between the physical maps of PAV-5 and those described for the other 4 serotypes of PAV.

Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies.

Sera of pigs immunized with parts of the transmissible gastroenteritis virus (TGEV) spike (S) protein expressed by recombinant baculoviruses were tested, together with a TGEV hyperimmune antiserum, for their abilities to protect three-day-old piglets against TGEV infection. The piglets were infected with virulent TGEV and the sera were given orally 3 h before infection, together with the virus, and every 6 h postinfection during the 30 h of the experiment. Virus shedding was monitored by TGEV isolation from rectal swab samples. The sera containing antibodies induced by the complete S protein or the amino terminal half of the S protein showed protective properties, indicated by delayed onset of clinical signs and virus shedding, similar to the TGEV hyperimmune serum. Those immune sera containing antibodies induced by shorter recombinant proteins were not protective.

Molecular cloning and physical mapping of porcine adenovirus types 1 and 2.

The 25R strain of porcine adenovirus type 1 (PAV-1) and the A47 strain of PAV-2 were propagated in ST cells, and DNA was extracted from the infected cells by a modified Hirt method. The DNA of each virus was digested by each of nine restriction endonucleases, and restriction enzyme fragments representing the entire genome were cloned. The genomic size of each virus was approximately 33 kb. Physical maps for the nine restriction endonucleases were constructed from the results of double digestion and Southern blot hybridization experiments, and oriented with respect to the PAV-3 genome. PAV-1 and PAV-2 were found to be related genetically to PAV-3, and there was a closer relationship between PAV-1 and PAV-3 than between PAV-1 and PAV-2 or between PAV-2 and PAV-3.

Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus.

Seven fragments of the spike (S) gene cDNA of transmissible gastroenteritis virus (TGEV), as well as the full length cDNA, were cloned and expressed in baculovirus vectors. Piglets were immunized with cells infected with the recombinant viruses. Each of the recombinants induced TGEV-specific antibodies detected in a fixed cell enzyme immunoassay. The amino terminal half of the S protein, containing all four major antigenic sites (A, B, C and D), and encoded by a 2.2 kb fragment of the S gene, induced virus neutralizing (VN) antibody titers comparable with those induced by the complete S protein. Recombinant proteins lacking the A antigenic site, or with a deletion including the putative receptor binding sites and the D antigenic site, were not capable of inducing levels of VN antibodies similar to those induced by the whole S protein.

Restriction endonuclease analysis of a porcine isolate of bovine herpesvirus type 1.

Deoxyribonucleic acid (DNA) was extracted from bovine herpesvirus type 1 (BHV-1) isolated from a stillborn porcine fetus, from the Cooper reference strain of BHV-1, and from an Ontario bovine respiratory isolate. Each DNA was digested with the restriction endonucleases HindIII, EcoRI, HpaI and BamHI. Except for very minor differences in the patterns produced after digestion with EcoRI and HindIII, the DNA of the porcine isolate reacted in a similar manner to the bovine viruses, and it was concluded that the porcine virus is genetically similar to bovine isolates of BHV-1.

Monoclonal antibodies to Mycoplasma gallisepticum membrane proteins.

Monoclonal antibodies (MAbs) were prepared to study the immunogenesis of Mycoplasma gallisepticum. Balb/c mice were immunized with M. gallisepticum immunostimulating complexes and the supernatant of heterokaryotes screened with M. gallisepticum and closely related M. synoviae as antigens in indirect enzyme-linked immunosorbent assay. All selected MAbs proved to be M. gallisepticum species-specific when they were tested against 10 different avian Mycoplasma species. After immunoblotting analysis, five polypeptides were identified with estimated molecular weights of 110,000, 66,000, 64,000, 56,000, and 50,000. Cell membrane localization of the recognized polypeptides was studied by immunoelectron microscopy. None of the MAbs inhibited the hemagglutinating activity of freshly prepared M. gallisepticum. However, one MAb (B3) specific for p56 agglutinated the stained M. gallisepticum antigen in the slide agglutination test. Results seemed to correlate with published information on the protein composition and agglutinating activity of Mycoplasma gallisepticum.

Evaluation of a monoclonal blocking enzyme-linked immunosorbent assay for the detection of Mycoplasma gallisepticum-specific antibodies.

A monoclonal blocking enzyme-linked immunosorbent assay (blocking-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in poultry sera with the help of a peroxidase-labeled monoclonal antibody (MAb) recognizing an epitope of a 56-kilodalton polypeptide (p56) of MG. Immunoglobulins from undiluted MG-positive sera prevent the MAb conjugate from attaching to its specific binding site on p56, which results in no color development. The opposite result--a strong color reaction--was obtained after incubation with MG-negative sera (or when no serum was added before the MAb conjugate). Results were expressed in percent inhibition or ELISA titers. The blocking-ELISA detected 84.7% positive chickens in an experimentally infected flock and 72.6% of chickens in naturally infected flocks, whereas the hemagglutination-inhibition (HI) test was positive only with 68.4% and 48.6% of these serum samples, respectively. All HI-positive serum samples reacted positively in blocking-ELISA. Of sera negative by the HI test, 51.6% and 46.8% proved to be positive when examined with the blocking-ELISA. Overall agreement between the ELISA and HI test was 76.8%. Infection with closely related M. synoviae did not induce any false-positive reactions in blocking-ELISA. There was a strong positive correlation between HI and blocking-ELISA titers (r = 0.83).

Potential viral vectors for the stimulation of mucosal antibody responses against enteric viral antigens in pigs.

Four viruses were compared for their ability to induce an intestinal antibody response in piglets. Antibodies were not detected in response to oral vaccination with either fowlpox virus or a baculovirus (BV). Simultaneous oral dosing and parenteral inoculation with high concentrations of BV in an oil emulsion adjuvant induced high levels of circulating virus neutralising (VN) antibodies, and also low levels of intestinal antibodies when booster doses of virus were given. In response to oral vaccination with swinepox virus (SPV), low levels of circulating and intestinal VN antibodies, and higher titres of antibodies reactive in an enzyme immunoassay, including intestinal antibodies of the IgA class, were detected. Oral vaccination with porcine adenovirus type 3 (PAV-3) stimulated both circulating and intestinal VN antibodies, and IgA antibodies were demonstrated in the intestinal contents. It was concluded that SPV and PAV-3 might be suitable vectors for the expression of genes encoding the protective antigens of porcine enteric viruses.