Maria Calsamiglia

Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome

The aim of this work was to characterise the lesions and agents present in clinically normal and clinically affected pigs on a farm during an outbreak of postweaning multisystemic wasting syndrome (PMws), and to evaluate the diagnostic techniques for detecting porcine circovirus type 2 (PCV-2) and other microorganisms. Four pigs in the early stage and 11 pigs in the late stage of the disease, and eight clinically normal pigs were necropsied. Samples of lymphoid tissue and serum were also obtained from 12 slaughter pigs from the same farm. The tissues were examined histopathologically, and in situ hybridisation, serology and PCR were used to detect porcine circovirus type 1 (Pcv-1) and/or PCV-2 in tissues and/or sera. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszkys disease virus (ADv) and porcine parvovirus (PPv) were also investigated. Characteristic microscopical lesions of PMWS were observed in the lymphoid tissues of the pigs in all three necropsied groups; the lesions were most common and severe in the pigs in the early stage of the disease, less so in the pigs in the late stage of the disease, and least in the clinically normal pigs. PCV-2 infection was detected in all the necropsied pigs by in situ hybridisation and PCR. Only three pigs had the Pcv-i genome in serum or lymph node tissue. In contrast, the slaughter pigs had no microscopical lesions and no PCV-2 nucleic acid in their serum or tissues, and only one of them had the Pcv-i genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.

Application of a Nested Polymerase Chain Reaction Assay to Detect Mycoplasma Hyopneumoniae from Nasal Swabs

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.

Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome (PMWS).

The objective of the present study was to analyze, by flow cytometry, changes in PBMC subsets in pigs having postweaning multisystemic wasting syndrome (PMWS), a new condition associated to porcine circovirus type 2 (PCV2) infection. Thirteen acutely PMWS affected pigs were selected from a farm seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) and to Aujeszkys disease virus (ADV); 11 clinically healthy pigs were selected from a high health farm with no history of PMWS and free of the major swine pathogens, and used as a control group. All pigs were necropsied, and tissue samples were fixed in formalin; blood with EDTA anticoagulant was used to perform the flow cytometric analysis. PBMC were incubated with mAb against porcine CD3, CD4, CD8, CD25, CD45, IgM, SWC3, and SLA-Class II. Flow cytometric analysis showed substantial changes in leukocyte subsets in the peripheral blood of PMWS-affected pigs, which were characterized by an increase of monocytes, a reduction of T (mainly CD4(+)) and B-lymphocytes, and the presence of low-density immature granulocytes. Altogether, these changes would suggest an inability of acutely PMWS-affected pigs to mount an effective immune response.

Detection of Porcine Circovirus Types 1 and 2 in Serum and Tissue Samples of Pigs with and without Postweaning Multisystemic Wasting Syndrome

ABSTRACT Presence of porcine circovirus type 1 (PCV1) and PCV2 was studied in sera and superficial inguinal lymph nodes from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs by using in situ hybridization and PCR. PCV1 and PCV2 were found in less than 3% and more than 50% of the samples, respectively. The most sensitive technique and site was PCR in superficial inguinal lymph nodes, but in situ hybridization correlated better with presence of characteristic lesions.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection status in pigs naturally affected with post-weaning multisystemic wasting syndrome (PMWS) in Spain

The purpose of the study reported here was to determine the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in pigs affected with post-weaning multisystemic wasting syndrome (PMWS), a disease believed to be caused by porcine circovirus type 2 (PCV2). From May 1997 to February 2000, PMWS was diagnosed in 277 pigs (from 120 farms) submitted to the Veterinary Pathology Diagnostic Service, Veterinary School of Barcelona, Spain. In each case, the PMWS diagnosis was based on clinical history and the detection, by in situ hybridization, of nucleic acid of PCV2 in characteristic histologic lesions. Antigens for PRRSV were detected by immunohistochemistry in tissues of 66 (23.8%) of the same 277 pigs. Sera, which were available for 93 of the 277 pigs, were tested for PRRSV by a multiplex reverse transcription-polymerase chain reaction (RT-PCR). A total of 33 of these sera were RT-PCR positive, three for a North American strain(s) of PRRSV. In addition, 76 of the 93 sera were tested for antibodies to PCV2 (indirect immunoperoxidase) and PRRSV (enzyme-linked immunoassay). Antibodies for PCV2 and PRRSV were detected, respectively, in 56 (73.9%) and 43 (56.6%) of the 76 sera. Collectively, these results suggest that while infection with PRRSV may be common, it is not an essential component of PMWS.

Correlation between the presence of enzootic pneumonia lesions and detection of Mycoplasma hyopneumoniae in bronchial swabs by PCR.

In many diagnostic laboratories the diagnosis of mycoplasmal pneumonia in pigs is based on clinical signs and the presence of gross and histopathological lesions. The objective of this study was to evaluate the nested-PCR technique as an adjunct to the histopathological diagnosis of Mycoplasma hyopneumoniae infection. Respiratory disease of 184 swine cases submitted to the Minnesota Veterinary Diagnostic Laboratory between 1 January and 30 June 1998 were used. Bronchial swabs were collected and the nested-PCR performed. Lung samples were graded PCR positive or negative. Histopathological lesions were scored 0-4, depending on the mycoplasma-like characteristics of the lesions, with category 4 demonstrating strong evidence of mycoplasma infection.Nested-PCR correlated well with histopathological lesions characteristic of M. hyopneumoniae in categories 3 and 4 and approximately half of the histopathological categories 1 and 2 were nested-PCR positive. The results demonstrate that the nested-PCR is a valuable adjunct in the diagnosis of M. hyopneumoniae infection when non-diagnostic microscopic lesions of mycoplasmosis are found.

Development of an oligonucleotide-specific capture plate hybridization assay for detection of Haemophilus parasuis

An oligonucleotide-specific capture plate hybridization assay has been developed to rapidly, specifically, and sensitively detect Haemophilus parasuis from nasal swabs. Several in vitro studies have been performed to determine the sensitivity and specificity of the test, and in vivo studies have validated this technique in pigs. Results suggest that the assay detects <100 colony-forming units/ml in a pure culture and gives a positive result when H. parasuis is present in a ratio of 1:103–104 in a mixed culture, and the probe does not hybridize with other related species found in the upper respiratory tract. This assay is more sensitive than culture for detection of the microorganism from nasal swabs and lesions.

Post-weaning multisystemic wasting syndrome (PMWS) clinical expression under field conditions is modulated by the pig genetic background

Post-weaning multisystemic wasting syndrome (PMWS) is a worldwide distributed disease of multifactorial origin and porcine circovirus type 2 (PCV2) has been identified as its essential infectious aetiology. Pig genetic background has been pointed to influence disease expression. In the present study, three different boar lines, namely A (100% Pietrain), B (50% Large White × 50% Pietrain) and C (25% Large White × 75% Duroc), were used to inseminate sows from the same genetic line (37.5% Large White × 37.5% Duroc × 25% Landrace) located on two PMWS-affected farms (farm-1 and farm-2). The PMWS clinical expression of their offspring was studied from weaning to slaughter, evaluating three parameters: total post-weaning mortality (PWM), PWM associated to PMWS (PMWS-PWM) and body weight (BW) evolution. The effect of other variables potentially related with PMWS, including sow and piglet PCV2 exposure, sow parity, piglet gender and piglet BW at weaning, were also considered in the study design. Overall, a total of 6.5% PWM and 4.3% PMWS-PWM occurred in the monitored farms. Pigs from boar line C showed the highest PWM (16.3%) and PMWS-PWM (12.4%), and the lowest BW; pigs from boar line A showed the lowest PWM (1.8%) and the highest BW. Furthermore, PWM was also higher in piglets from farm-2 and from multiparous sows. In farm-2, PMWS-PWM was higher in piglets from multiparous sows. Finally, BW was influenced by interactions between genetics and both farm and pig age, and was lower in piglets from farm-2. This study represents a consistent observation of the genetic background effect on PMWS clinical expression under field conditions.

Apoptosis in postweaning multisystemic wasting syndrome (PMWS) hepatitis in pigs naturally infected with porcine circovirus type 2 (PCV2).

The degree of apoptosis in the livers of pigs with hepatitis due to naturally-occurring postweaning multisystemic wasting syndrome (PMWS) was evaluated semi-quantitatively by immunohistochemical detection of the apoptotic marker cleaved caspase-3 (CCasp3). The amount and distribution of porcine circovirus type 2 (PCV2) virus in the liver was evaluated using in situ hybridisation. Livers with mild, stage I hepatitis exhibited similar degrees of apoptosis to controls; those with stage II lesions had variable apoptotic rates, ranging from mild to high, and in livers with more severe, stage III hepatitis, high levels of hepatocyte apoptosis was a feature. Statistical analyses indicated a positive association between the rate of apoptosis, the severity of the hepatitis and the amount of PCV2 DNA in the liver. Double immunolabelling for CCasp3 and PCV2 DNA revealed a predominance of cells labelling only for PCV2, followed by fewer cells labelling only for CCasp3, and the least number labelling for both. The findings suggest that apoptosis, possibly triggered by PCV2 infection and/or hepatic inflammation, plays a key role in the pathogenesis of hepatitis in pigs with naturally-occurring PMWS.

Exploratory study on the influence of climatological parameters on Mycoplasma hyopneumoniae infection dynamics

The objective of the present work was to elucidate the potential relationship between Mycoplasma hyopneumoniae infection and seroconversion dynamics and climatological conditions in four groups of pigs from the same farm born in different seasons of the year. Nasal swabs and blood samples were taken from 184 pigs at 1, 3, 6, 9, 12, 15, 18, 22 and 25 (slaughter age) weeks of age. Outside climatologic parameters, including temperature (°C), relative humidity (%), precipitation (l/m2) and wind speed (m/s) were recorded weekly from January 2003 to June 2004. Percentage of nPCR detection of M. hyopneumoniae in nasal swabs was associated significantly with the weekly precipitation rate [P = 0.0018, OR = 1.31 (IC = 1.11–1.55)]; the higher the precipitation rate, the higher the probability of being M. hyopneumoniae nPCR-positive. On the other hand, the percentage of seropositive pigs had a significant association with mean weekly temperature rate [P = 0.0012, OR = 0.89 [IC = 0.84–0.95]); the lower the temperature, the higher the probability of being M. hyopneumoniae seropositive. Animals born in autumn (when higher precipitations rates were recorded), entering finishing units in winter (when lower temperatures were recorded), and reaching slaughter in spring, had the highest probability of being infected by M. hyopneumoniae and the highest probability of being M. hyopneumoniae seropositive.