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.

Clinical and economic impact of new trends in glaucoma treatment.

CONTEXT: Glaucoma is a chronic ophthalmic condition affecting approximately 15 million people. Several therapies are currently available (eg, beta-blockers, sympathomimetics, carbonic anhydrase inhibitors) but have side effects that may limit use. Over the last few years, new medications with improved efficacy and side-effect profiles have become available. This analysis evaluates 2 therapies, brimonidine and betaxolol, based on head-to-head clinical trial data to determine clinical consequences and their related expected costs. OBJECTIVE: To calculate comparative costs and the cost-effectiveness of brimonidine 0.2% and betaxolol 0.25% as first-line therapy for patients with primary open-angle glaucoma. DESIGN: Safety, efficacy, effectiveness, and quality-of-life data were collected in a multicenter, randomized, double-blind, head-to-head comparative effectiveness study, with a drug switch possibility. A disease-intervention model (decision tree) was developed with clinicians, academicians, and health economists. Components of care for each pathway in the model were identified and evaluated; their costs were applied at appropriate points throughout the tree. Expected outcomes and costs were computed and compared. PATIENTS: Participants were men (n = 76) and women (n = 112), 21 years of age or older, with newly diagnosed or currently untreated ocular hypertension or open-angle glaucoma. RESULTS: The clinical success rates of first-line brimonidine 0.2% and betaxolol 0.25% are 73.9% and 56.2%, respectively, as determined in a head-to-head comparative effectiveness trial. Total expected costs for patients receiving brimonidine and betaxolol as a primary therapy are $301.37 and $328.19, respectively, based on the model. Dividing costs by outcomes, the cost-effectiveness ratios for brimonidine and betaxolol are $407.81 ($301.37/0.739) and $583.97 ($328.19/0.562), respectively, representing the cost/unit outcome, or the cost to achieve clinical success. CONCLUSIONS: Brimonidine 0.2% is less costly and more cost-effective than betaxolol 0.25% when used as initial monotherapy with and without subsequent add-on therapies, including laser treatments and/or surgery, as needed.

Pharmacotherapies for attention-deficit/hyperactivity disorder: expected-cost analysis.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a common childhood neurobehavioral disorder characterized by inattention, hyperactivity, and impulsivity. Prevalence estimates in elementary school children generally range from 3% to 8%. ADHD is frequently treated with psychostimulant medications, which have been shown to improve both cognitive and behavioral outcomes for most children. OBJECTIVE: The goal of this study was to estimate the total expected costs for the treatment and management of school-age children with ADHD using 6 commonly prescribed pharmacotherapies: methylphenidate immediate-release/extended-release (MPH IR/ER), methylphenidate immediate-release (MPH IR), Metadate CD (branded MPH IR/ER), Concerta (branded MPH ER), Ritalin (branded MPH IR), and Adderall (a combination of dextroamphetamine and amphetamine salts). METHODS: A literature review and clinical assessment using a 27-question survey instrument were used to capture information on the clinical characteristics of ADHD, including common treatment regimens, clinical management of patients, pathways of care, and components of care. A meta-analysis provided response rates for 3 commonly used pharmacotherapies: Metadate CD, MPH IR, and Adderall. Information from the clinical assessment and the meta-analysis were used to populate a decision-analytic model to compute total expected cost for each comparator. RESULTS: The average total annual expected cost per patient was $1,487 for Metadate CD, $1,631 for Concerta. $1,792 for MPH IR/ER, $1,845 for MPH IR, $2,080 for Ritalin, and $2,232 for Adderall. CONCLUSIONS: Metadate CD had the lowest total expected cost and Adderall had the highest total expected cost among the ADHD pharmacotherapies evaluated. The differences were attributable to differences in drug-acquisition costs and the need for in-school dosing of twice-daily and thrice-daily medications.

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.

Management of patients with Bacilli Calmette-Guérin-refractory carcinoma in situ of the urinary bladder: cost implications of a clinical trial for valrubicin.

OBJECTIVE: This study was undertaken to identify the expected first- and second-year clinical costs associated with intravesical valrubicin therapy, using a decision analytic model, for patients with Bacilli Calmette-Guérin (BCG)-refractory carcinoma in situ (CIS) of the urinary bladder. BACKGROUND: Cancer of the urinary bladder is the fourth most common malignancy in men and the sixth most common noncutaneous carcinoma overall. One histopathologic stage of bladder cancer is CIS, for which BCG intravesical immunotherapy is the first-line therapy. Radical cystectomy has been recommended for patients with CIS who do not respond to or become refractory to therapy with BCG. Surgery, however, may not be appropriate for all patients, especially those who are ineligible for the lengthy procedure because of advanced age or comorbidities and those who prefer alternative nonsurgical management. For these groups, intravesical valrubicin therapy is a plausible alternative. METHODS: Models were developed and populated with data from 1 open-label study of 90 patients, information from the medical literature, and input from clinical experts. The analysis was conducted from the payor perspective for direct costs only. RESULTS: Our data indicate that first- and second-year expected costs for valrubicin therapy are $19,912 and $23,496, respectively. Expected cost for radical cystectomy was also evaluated, since some patients may have no other option if drug therapy fails. CONCLUSION: Our cost-consequence analysis and clinical data provide decision-makers with tools to aid in global budgetary projections of fractional and total expected health care costs associated with the management BCG-refractory CIS of the urinary bladder.

Duration of effect of botulinum toxin type A in adult patients with cervical dystonia: a retrospective chart review.

BACKGROUND: Clinical trials have established the efficacy and safety of botulinum toxin type A (BTX-A) in patients with cervical dystonia. To maintain the clinical benefits of BTX-A, injections need to be repeated whenever patients' symptoms begin to recur. OBJECTIVE: The purpose of this study was to determine, in clinical practice settings, the mean duration of effect of BTX-A in the treatment of adult patients with cervical dystonia. METHODS: A retrospective chart review was undertaken at an academic center and a private neurology practice. At each site, > or =50 patients being treated for cervical dystonia were identified and randomized for chart review. Patients had to have received the first assessable injection of BTX-A between January 1, 1998, and March 31, 1998, to coincide with the clinical availability of the most current formulation of the neurotoxin. A chart was eligible for review if the patient was aged > or =18 years, had a documented diagnosis of idiopathic cervical dystonia, was being treated with BTX-A, and had been under the continuous care of investigators from January 1, 1998, to August 31, 1999. Of the 102 patients initially identified, the first 30 from each site who met the study inclusion criteria were assessed for (1) age and sex; (2) severity of dystonia; (3) years of BTX-A use; (4) dates of first, second, third, and fourth BTX-A injections; (5) drug dose; (6) use of electromyography; (7) use of other prescribed therapies; (8) laboratory tests; and (9) adverse events. The mean interval between each visit and mean per-patient duration of effect were calculated and stratified by patient characteristics. RESULTS: The mean age of the patients was 56.4 years. Two thirds of the patients were women. Forty-one of the 60 patients (68.3%) had either moderate or severe disease, and 48 (80.0%) had experienced cervical dystonia for >5 years. The mean per-patient duration of effect across the 4 visits was 15.5 weeks (range, 12.2-24.3 weeks). The duration of effect did not differ significantly between study sites despite the differences in disease severity, drug dose, and use of adjunctive therapy. CONCLUSION: BTX-A the controls symptoms of cervical dystonia for 12 to 24 weeks, with a mean duration of effect per patient of 15.5 weeks.

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.

Impact of palivizumab on expected costs of respiratory syncytial virus infection in preterm infants: potential for savings.

In its clinical assessment of the respiratory syncytial virus (RSV)-specific monoclonal antibody palivizumab, the IMpact-RSV Study Group demonstrated a reduction in hospitalizations for RSV-related lower respiratory tract infection in infants who received prophylaxis compared with infants who did not receive prophylaxis. An assessment of the RSV-related expenses for managing both groups of infants is needed to provide insight into the value of prophylaxis. The present study was conducted to identify and compare RSV-related health care expenditures incurred by infants who did not receive prophylaxis throughout one RSV season and after. Using a decision-analytic model populated with data from the contemporary medical literature, a pharmacoeconomic study was conducted from the perspective of the payer. Probabilities for RSV-related hospitalizations of infants who did and did not receive prophylaxis were abstracted from several published studies. Components of inpatient and outpatient care were identified through examination of hospital records, reviews of the published literature, and consultation with expert clinicians. Charges related to prophylaxis and medical management of infection were abstracted from hospital billing records and published data. Appropriate charges were applied to decision-tree branches and multiplied by in-line probabilities for outcomes. Products at terminal nodes were summed to establish total expected charges for both groups of infants. Widespread clinical use of prophylactic palivizumab would result in incremental expenses < or =$3459 per infant or cost savings < or =$39,107 per infant. The variability in value of prophylaxis derives from the rate of RSV-related hospitalizations in the community and the total health care expense of managing infected infants.

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.

Performance of ELISA antigens prepared from 8 isolates of porcine reproductive and respiratory syndrome virus with homologous and heterologous antisera.

Porcine reproductive and respiratory syndrome virus (PRRSV) ELISA antigens of high quality were produced using 8 different isolates of PRRSV: the European Lelystad virus (LV), the U.S. MN-1b, 89-46448, 93-44927, and 93-24025B, and the Canadian LHVA-93-3, PA-8 and GH-6 virus isolates. The performance of each of these 8 antigens and a commercial PRRSV antibody test kit (Idexx's HerdChek) were measured against antisera raised in 5 groups of 6 piglets inoculated with either LV, MN-1b, 89-46448, 93-44927, or 93-24025B. Among the 8 isolates, the 89-46448 isolate produced the broadest spectrum of antigen and resulted in earlier detection of antibodies to various North American PRRSV isolates, followed by MN-1b as the 2nd best ELISA antigen for the detection of North American PRRSV antibodies. The GH-6 and PA-8 viral antigens exhibited restricted detection of PRRSV antibodies. The LV and 89-46448 combined antigens produced the best performance for the detection of antibodies against both European and North American antigenic types of PRRSV. Using 173 panel samples collected at 11 to 60 d after intranasal inoculation with 1 of the 5 PRRSV isolates, the sensitivities of the indirect ELISA used were 73.4%, 98.3%, 90.8%, 98.3%, 83.2%, 93.1%, 77.1%, 64.2%, 98.8% and 95.9% for LV, MN-1b, LHVA-93-3, 89-46448, 93-44927, 93-24025B, PA-8, GH-6 antigens, 89-46448-LV combined antigens and Idexx's PRRSV antibody test kit, respectively. All 8 antigens gave negative results with preinfection porcine sera (n = 30); high background or nonspecific reactions were not observed with the antigens.

Comparative study of serological methods for the detection of antibodies to porcine reproductive and respiratory syndrome virus.

A comparison was made of serological diagnostic methods used for the detection of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus. In the "phase I" PRRS test panel comparison, a panel of sera collected from 135 pigs of various ages, from North American herds with and without PRRS histories, were sent to 4 different laboratories and tested by an indirect immunofluorescent assay (IFA), an immunoperoxidase monolayer assay (IPMA) and an indirect enzyme-linked immunosorbent assay (iELISA). In the "phase II" PRRS test panel comparison, a panel of 382 sera collected from pigs of various ages, PRRS histories, and from various locations in North America and France, were divided into 2 panels (A & B) and sent to 3 Canadian laboratories and tested by the IFA and iELISA. In the phase I comparison, agreement between the IFA of laboratory 4 and the iELISA and IPMA of laboratory 3 was excellent (kappa values of 95% and 98%, respectively). This contrasted with the poor agreement between these laboratories and the IFA results of laboratories 1 and 2 in the phase I trial. In the phase II comparison, the results demonstrated good agreement between various tests both within and between laboratories. The overall performance of the iELISA was superior in the combination of sensitivity (96.1%) and specificity (100%) relative to the reference classification of the serum samples and repeatability (kappa value 98%). The iELISA is technically superior to IFA and IPMA, time efficient, cost effective and suitable for testing of a large number of samples over a short period of time. Thus, the iELISA may be a better alternative to IFA or IPMA for routine detection of PRRS viral antibodies in swine sera.

Antigenic variability of porcine reproductive and respiratory syndrome (PRRS) virus isolates. Influence of virus passage in pig.

In order to study the antigenic variability of porcine reproductive and respiratory syndrome virus (PRRSV), 18 European and Canadian field isolates were analysed with a panel of 15 monoclonal antibodies (MAbs) raised against five different European and one Canadian PRRSV isolates. The antigenic pattern of these isolates was used to infer their phylogenetic relationships. Isolates which had the same reactivity pattern were gathered together so that five antigenic profiles were analysed. The pairwise distances between these groups were defined based on antigenic pattern differences. Two main antigenic groups were obtained, discriminating between the European and the Canadian populations, as illustrated by the great distance observed between European and Canadian isolates (D = 0.5619 +/- 0.0625) compared to the distance between European isolates (D = 0.1524 +/- 0.0735). The distance matrix allowed also the construction of a tree diagram. Bootstrap analysis was performed to test the confidence in the branching. The tree diagram confirmed the distinction between the European and the Canadian PRRSV populations. Antigenic variability between an isolate and its progeny recovered after one or two passages in vivo was examined on six isolates. It was restricted to the GP3 protein of the virus.

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.