Detection rates of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and swine influenza virus in porcine proliferative and necrotizing pneumonia.

A retrospective study on pig lung tissues from 60 cases of proliferative and necrotizing pneumonia (PNP) was performed to determine the presence of porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine circovirus type 2 (PCV2) in these lesions. Cases selected included 30 cases diagnosed between 1988 and 1992 and 30 cases diagnosed between 1997 and 2001. In each group of 30 cases, 10 were from suckling piglets, whereas the other 20 were from postweaned animals representing either nursery or grower-finisher pigs. Immunohistochemistry using a monoclonal antibody to influenza virus type A was used to determine the presence of SIV, and in situ hybridization was used for the detection of PRRSV and PCV2 nucleic acids. PRRSV was detected in 55 of the 60 cases examined (92%), PCV2 in 25 cases (42%), and SIV in only 1 case (2%). In 30 cases (50%), PRRSV was the only virus detected, whereas in 25 other cases (42%), a combination of PRRSV and PCV2 could be detected in the lungs with PNP lesions. PCV2 could not be detected in the lungs of suckling pigs with PNP. All PCV2-positive cases were found in postweaned pigs and were always in combination with PRRSV. In this latter age group, PCV2 was detected in 63% of the cases (25/40). Data from our study indicate that SIV is rarely identified in PNP and that PCV2 infection is not essential for the development of PNP lesions. The results of the present study demonstrate that PRRSV is consistently and predominantly associated with PNP and should be considered the key etiologic agent for the condition.

Infectious agents identified in pigs with multifocal interstitial nephritis at slaughter.

One kidney was taken from each of 100 pigs at slaughter; 50 had gross lesions of multifocal interstitial nephritis and 50 had no gross lesions. Forty-nine of the affected kidneys had lesions that were characterised by the presence of either a few randomly distributed or numerous widely disseminated pale foci, 1 to 3 mm in diameter, on the cortical surface (white-dotted kidneys). Microscopically, these focal inflammatory lesions often had a distinct lymphofollicular pattern (follicular nephritis). Lesions of chronic vasculitis were observed in 21 of the affected kidneys. Histologically, the control kidneys had only small and sparse inflammatory foci. Standard bacterial cultures of kidneys of both groups were not significant, and cultures for the isolation of leptospires were all negative. Virological examination of the kidney homogenates by PCR did not reveal any porcine reproductive and respiratory syndrome virus and only a few cases were positive for the porcine circovirus type 1. However, porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) were detected in many kidneys of both groups but in a significantly higher proportion of the kidneys with interstitial nephritis. There was a significant association between the lesions and the presence of PPV and PCV-2 with odds ratios of 7.5 (P<0.0001) and 3.4 (P=0.0074), respectively, and the odds ratio increased to 22.7 (P<0.0001) when both viruses were identified in the same kidney. However, a subsample of kidneys taken from both groups were negative by immunohistochemistry for the presence of PPV and PCV-2 antigens.

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3-60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig(high)-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.

PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen.

An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.

Experimental transmission of porcine circovirus type 2 (PCV2) in weaned pigs: a sequential study.

Weaned specific pathogen-free pigs were inoculated intranasally with porcine circovirus type 2 (PCV2) and killed in groups of two or three animals at 6, 13, 20, 27 and 34 days post-inoculation (dpi), together with appropriate uninfected controls, for examination by histopathological, immunohistochemical (immunogold silver staining; IGSS), polymerase chain reaction (PCR) and viral isolation techniques. Serum samples were also collected for detection of antibodies. No major clinical signs were observed in infected pigs, and gross lesions were essentially limited to the lungs and lymph nodes of some of the animals. Histologically, no lesions were seen at 6 dpi, but bronchointerstitial pneumonia was invariably noted from 13 dpi onwards. Granulomatous inflammation, with or without intracytoplasmic inclusions, was present in lymphoid tissues (e.g. lymph nodes, thymus, spleen and tonsil) from day 20 onwards, being most severe at days 20 and 27 dpi. Liver inflammation was present at days 13, 20 and 27 dpi. Virus was demonstrated in the tissues by isolation and PCR methods throughout the experiment. PCV2 antigens were detected by IGSS in bronchial and bronchiolar epithelial cells, in mononuclear cells and multinucleated giant cells within inflammatory lesions, and in mononuclear cells of apparently normal tissues (e.glamina propria of the small intestine and the bronchus-associated lymphoid tissue). The lesions were consistent with those of postweaning multisystemic wasting syndrome (PMWS), although not all previously reported PMWS lesions were seen. PCV2 antibodies were detected in infected pigs from day 13 onwards. The results demonstrated widespread distribution of PCV2 after infection and persistence of the virus in vivo for at least 34 days. It would appear that PCV2 can induce PMWS lesions in weaned pigs in the absence of porcine parvovirus and other common swine pathogens.

Retrospective serological survey of antibodies to porcine circovirus type 1 and type 2.

A retrospective serological survey was performed to determine the presence of antibodies to porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) in serum samples collected from sows at slaughterhouses in Canada in 1985, 1989, and 1997. Each serum sample was tested by indirect immunofluorescence on PCV-free PK15 cells, on PCV1-infected PK15 cells and on PCV2-infected PK15 cells. For the 3 years studied, sera positive to PCV1 and PCV2 were identified and the number of sera positive for PCV2 was greater than the number of sera positive for PCV1. The results indicated 1) that PCV2 appears to be the main PCV type circulating in the Canadian pig population, 2) that PCV2 had been circulating in the Canadian pig population at least 10 years before the postweaning multisystemic wasting syndrome (PMWS) was reported, and 3) that serological evaluation using PCV1 underestimates the seroprevalence of PCV2.

Typing of porcine circovirus in clinical specimens by multiplex PCR.

A multiplex PCR assay was developed to detect and differentiate between the porcine circovirus (PCV) infecting persistently the PK 15 cell line (PCV type I) and the PCV associated with postweaning multisystemic wasting syndrome (PMWS) (PCV type II). DNA products with unique sizes characteristic of each type of PCV were obtained. Sequencing of these products demonstrated that the nucleotide sequences were type-specific. Tissue samples from a total of 42 field cases from Québec were studied, among which 41 were collected in 1997-1998 and one which had been previously collected in 1994. These 42 cases found previously to be PCV-positive by PCR were tested in the present study by a multiplex PCR assay to determine the type of PCV in each case. From these 42 field cases, 40 cases were PCV type II-positive, one case was PCV type I-positive and one case was positive for both PCV types I and II. PCV type II was identified in typical PMWS field cases, but also in field cases submitted for various clinical histories, some of which were not suggestive of PMWS. In the field case where PCV type I was detected, there was no clinical evidence nor histological lesions suggestive of PMWS. The demonstration of PCV type II in a total of 41/42 field cases in the present study suggests that PCV type II may be the main type of PCV circulating in pigs. Furthermore the detection of PCV type II in a field case dating back to 1994 indicates that this PCV type was circulating in pigs in Québec several years before the report of clinical PMWS in this province.

Cutaneous and systemic necrotizing vasculitis in swine.

A systemic vasculitis involving particularly the skin and kidneys has been recently described in swine under the name dermatitis/nephropathy syndrome. Twelve pigs with gross cutaneous lesions typical of this condition were necropsied, and morphologic, immunohistochemical, microbiologic, and epidemiologic characteristics were studied. The pigs were divided into three groups comprising eight pigs with acute lesions, two with chronic lesions, and two with acute lesions kept for sequential skin biopsies. Acute skin lesions consisted of round to irregular, red to purple macules and papules that often coalesced to form large, irregular patches and plaques. With time, the lesions became covered by crusts and faded gradually, sometimes leaving scars. Characteristic distribution included the perineal area of the hindquarters, limbs, dependent parts of the abdomen and thorax, and margins of the ears. In the acute phase of the disease, necrotizing and leucocytoclastic vasculitis of small-caliber blood vessels were observed within the dermis and panniculus and in various extracutaneous locations such as the renal pelvis and synovial membranes. All pigs had macroscopic evidence of pneumonia and generalized lymphadenopathy. Microscopically, they had interstitial pneumonia and perivascular cuffing of mononuclear cells in various tissues including skin. The presence of immunoglobulins and complement was demonstrated by immunofluorescence in and around necrotic vessels of the skin in the early stages. Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) antigens were detected by immunohistochemistry in macrophages located around vessels of the tissues examined (skin and kidneys) in acute and chronic cases. PRRSV RNA was demonstrated by reverse transcription-polymerase chain reaction in lung and spleen homogenates from all pigs. The PRRSV was isolated in cell culture from 11 of the pigs. These findings suggest that PRRSV infection may play a role in the pathogenesis of this systemic vascular disease of swine.

Differentiation of North American and European porcine reproductive and respiratory syndrome virus genotypes by in situ hybridization.

Non-radioactive probes that can detect specifically North American and European isolates of porcine reproductive and respiratory syndrome virus (PRRSV) in formalin-fixed paraffin-embedded tissues by in situ hybridization were developed. These probes allow the differentiation between North American and European genotypes of the PRRS virus as well as the detection of both genotypes. Two amplified cDNA products generated by polymerase chain reaction (PCR), one from the cDNA of the Canadian PRRSV LHVA-93-3 isolate and the second one from the European Lelystad isolate, and labelled with digoxigenin were utilized as probes. The LHVA-93-3 derived probe was found to detect Canadian and USA PRRSV isolates in infected cells, while the Lelystad derived probe hybridized only with European isolates. The specificity of both probes was also demonstrated on formalin-fixed tissues collected from PRRSV infected pigs. Furthermore, by combining the LHVA-93-3 (North American) probe and the Lelystad (European) probe, successful detection of both PRRSV genotypes in fixed tissues could be achieved.

Detection of porcine reproductive and respiratory syndrome virus in paraffin-embedded tissues: comparison of immunohistochemistry and in situ hybridization.

A non-radioactive in situ hybridization (ISH) method developed recently and an immunohistochemical method, the immunogold silver staining (IGSS), were compared for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in formalin-fixed paraffin-embedded tissues. Serial sections from 98 tissues, representing different organs from PRRSV experimentally infected pigs, and serial sections from 46 lung tissues from field cases were tested by both methods. Results obtained on tissues from experimentally infected pigs and tissues from field cases demonstrated that ISH was more sensitive than immunohistochemistry. More tissues were positive by ISH compared to IGSS and also a greater number of labelled cells and a stronger signal in stained cells were observed in ISH-treated sections. The ISH method described, using a 254 bp digoxigenin-labelled cDNA probe, is a rapid, highly specific and sensitive detection method which can be used for the diagnosis of PRRSV in routinely fixed and processed tissues.

Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay.

A rapid multiplex PCR assay was developed to distinguish between North American and European genotypes of porcine reproductive and respiratory syndrome (PRRS) virus after a portion of the polymerase gene (open reading frame 1b) was sequenced for two North American PRRS virus strains. DNA products with unique sizes characteristic of each genotype were obtained.

Differential reactivity of a monoclonal antibody directed to the membrane protein of porcine reproductive and respiratory syndrome virus.

A monoclonal antibody (2C12) against the 19 kDa membrane (M) protein of a Canadian isolate of porcine reproductive and respiratory syndrome (PRRS) virus was produced. By indirect immunofluorescence (IIF) cytoplasmic fluorescence was observed in infected cells, but the pattern of fluorescence was generally different and intensity was weaker than that observed using the nucleocapsid protein-directed monoclonal antibody SDOW17. When tested by IIF towards a total of 26 PRRS virus isolates from Canada, 122 isolates from the US and 13 isolates from Europe the 2C12 MAb reacted with all the North American isolates tested including the VR-2332 isolate and the vaccine (RespPRRS) isolate. However no reactivity was observed towards the European isolates tested including the Lelystad virus. This reactivity pattern suggests that the epitope recognized by this MAb on the M protein of PRRS virus appears highly conserved among North American isolates but absent or weakly expressed on European isolates of PRRS virus.

Evaluation of the presence of porcine reproductive and respiratory syndrome virus in packaged pig meat using virus isolation and polymerase chain reaction (PCR) method.

An investigation was carried out to assess the potential presence of porcine reproductive and respiratory syndrome virus (PRRSV) in packaged pig meat. Samples of meat were collected at the processing plants and were sent to the laboratory for testing by virus isolation and reverse transcription-polymerase chain reaction (RT-PCR). Samples collected at four plants were randomly selected from lots of packaged pig meat from different slaughtering days and were sent frozen to the laboratory. Homogenates of meat were prepared and were inoculated onto MARC-145 cells and after two passages the presence of PRRSV was monitored by indirect immunofluorescence staining using PRRSV specific monoclonal antibody. All pig meat samples (six pools of meat samples from 73 different lots = 438 total homogenates) tested were found negative by virus isolation. Primers from open reading frames 6 and 7 were designed and a RT-PCR assay was developed and was demonstrated to detect both North American and European PRRSV isolates. Using this assay virus was detected at a concentration as low as 0.355 infectious virions per ml in supernatant of PRRSV infected cells. This RT-PCR assay could detect PRRS viral nucleic acid from various tissue samples of experimentally infected pigs including muscle tissue, thus demonstrating its applicability on tissue samples. All meat sample homogenates tested by RT-PCR (one sample pool from the 73 lots) were also found negative for PRRS viral nucleic acid. The results suggest that pig meat does not retain detectable amounts of PRRSV and further support that the transmission of PRRSV through pig meat is unlikely.

Characterization of monoclonal antibodies against avian reovirus strain S1133.

Monoclonal antibodies (MAbs) were produced against a vaccinal S1133 strain of avian reovirus. Characterization of six MAbs in Western blotting, radioimmunoprecipitation and gold immunoelectron microscopy revealed that the MAbs were specific to the outer capsid proteins, mu2/mu2c, sigma2 and sigma3. Two of three MAbs, directed against sigma2 protein, neutralized the virus infectivity in a broadly specific manner, whereas the third had no neutralizing activity. The only MAb which reacted with sigma3 protein showed a type-specific neutralizing activity. Two MAbs recognizing the mu2/mu2c proteins failed to neutralize the virus infectivity.

Detection of porcine reproductive and respiratory syndrome virus in cell cultures and formalin-fixed tissues by in situ hybridization using a digoxigenin-labeled probe.

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.

Evaluation of the persistence of porcine reproductive and respiratory syndrome virus in pig carcases.

The presence of porcine reproductive and respiratory syndrome (PRRS) virus in pig meat was assessed in samples collected from experimentally infected pigs and from the carcases of pigs from infected herds at an abattoir. In the experimental study, pigs approximately six months old were inoculated with two isolates of PRRS virus and tissue samples were collected seven and 14 days after inoculation. At seven days, PRRS virus was recovered from lungs, tonsils, lymph nodes and muscle tissues, and viral antigens were detected by immunogold silver staining (IGSS) in formalin-fixed lungs, tonsils and scattered cells in muscle tissues. Neither PRRS virus nor antigens were detected in muscle tissue samples collected 14 days after inoculation. In the abattoir pigs, attempts were made to isolate PRRS virus from a total of 44 samples of muscle tissue, collected as pools, from the carcases of 44 pigs originating from seropositive herds. No PRRS virus could be isolated on porcine alveolar macrophages from these 44 muscle tissue samples and no PRRS virus antigens could be detected by IGSS in the formalin-fixed tissue samples. Although the results of experimental infections indicated that PRRS virus may be recovered from muscle tissues early after infection with the virus, the presence of PRRS virus in muscle tissues from carcases of slaughter pigs previously exposed to the PRRS virus could not be demonstrated.

Antigenic comparison of Canadian and US isolates of porcine reproductive and respiratory syndrome virus using monoclonal antibodies to the nucleocapsid protein.

Fifteen Canadian field isolates of porcine reproductive and respiratory syndrome (PRRS) virus from Quebec and Ontario were compared with 5 US PRRS virus (PRRSV) isolates and with the European Lelystad isolate using monoclonal antibodies (MAbs) SDOW17, EP147, and VO17 directed to the 15-kDa nucleocapsid protein of PRRSV. All Canadian and US isolates tested by indirect immunofluorescence were recognized by the 3 MAbs, and individual titers of MAbs towards Canadian and US PRRSV isolates were similar as well. In contrast, the Lelystad virus isolate reacted only with the SDOW17 MAb and showed no reactivity with either EP147 or VO17. The reactivity pattern with these MAbs suggests that the Canadian isolates of PRRSV tested are antigenically similar to US isolates of PRRSV, and that these North American isolates share highly conserved epitopes on the 15-kDa nucleocapsid protein that clearly differentiate them from the European Lelystad virus isolate.