Papers by Maria Calsamiglia
Veterinary Microbiology, Feb 1, 2008
Veterinary Research, 2004
The objective of this study was to evaluate the effect of a commercial vaccine adjuvant on the cl... more The objective of this study was to evaluate the effect of a commercial vaccine adjuvant on the clinical and pathological outcome of PCV2 experimentally infected 8 to 9-week-old conventional pigs. Forty-four pigs were divided into four groups: non-infected control pigs, pigs that received a vaccine adjuvant, pigs inoculated with PCV2, and pigs inoculated with PCV2 together with the vaccine adjuvant. Infection was monitored until 69 days post-inoculation (PI). Some PCV2 inoculated pigs had hyperthermia, but no other clinical signs were recorded. No characteristic PMWS gross or microscopic lesions were observed in any of the pigs. PCV2 DNA was detected in lymphoid tissues by in situ hybridisation in 6 PCV2 inoculated pigs on day 69 PI. All PCV2 inoculated pigs seroconverted between days 21 and 49 PI, shortly after viremia detection. Moreover, viremia was detected between days 7 and 69 PI using PCR. A peak of the virus load was detected by real-time quantitative PCR between days 14 and 21 PI. There were no significant differences in the proportion of PCV2 positive serum and in the viral load between PCV2 and PCV2 + adjuvant inoculated pigs. Although PMWS was not reproduced in neither PCV2 nor PCV2 + adjuvant inoculated pigs, viremia detection and seroconversion indicated that all PCV2 inoculated pigs developed a chronic long-term asymptomatic infection. An increase of PCV2 replication was not observed in pigs inoculated with the adjuvant. These results indicate that the principle of immunostimulation may not be applicable under the experimental conditions used, suggesting that not all adjuvants used in commercial vaccines are capable of triggering mechanisms for PMWS development.
Journal of Veterinary Medical Science, 2004
Four swine facilities located in Northern Luzon, in the Philippines, showed lesions and clinical ... more Four swine facilities located in Northern Luzon, in the Philippines, showed lesions and clinical signs similar to those describe d in post weaning multisystemic wasting syndrome (PMWS). Post-mortem and histopathological examination revealed the presence of typical lymphoid lesions caused by porcine circovirus type 2 (PCV2). An in situ hybridization (ISH) technique allowed the detection of PCV2 nucleic acid associated with the lesions in target tissues. PCV2 DNA from paraffin embedded tissues was extracted, purified and sequenced. The phylogenetic analysis of the sequences obtained confirmed their identity, and grouped them into a PCV2 subgr oup, together with some Canadian, French and Dutch isolates. This report is the first description of the presence of PMWS in the Philippines.
Applied and Environmental Microbiology, Jun 1, 2006
Haemophilus parasuis is the cause of Glässer's disease and other clinical disorders in pigs. It c... more Haemophilus parasuis is the cause of Glässer's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains.
Veterinary Microbiology, Feb 1, 2002
The purpose of the study reported here was to determine the prevalence of porcine reproductive an... more 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
American Journal of Veterinary Research, 2004
Veterinary Microbiology, Dec 20, 2005
The present study focused on PCV2 quantification by TaqMan PCR in nasal (n=99), tonsillar (n=108)... more The present study focused on PCV2 quantification by TaqMan PCR in nasal (n=99), tonsillar (n=108), tracheo-bronchial (n=72), urinary (n=91) and faecal (n=42) swabs, as well as in serum (n=57), from a total of 146 pigs received at the Pathological Diagnostic Service at the Veterinary School of Barcelona (Spain). Animals were classified into three categories based on histopathological and in situ hybridisation (ISH) results: PMWS affected pigs (Group A, n=42), PCV2 subclinically infected pigs (Group B, n=29), and non-PMWS with PCV2 ISH negative pigs (Group C, n=75). Overall, tracheo-bronchial swabs had the higher PCV2 load followed by serum, tonsillar, nasal, faecal and, finally, urinary swabs. PCV2 genome was also detected in different proportions in all three categories of pigs; in all tested sites, viral load means were significantly higher (P&lt;or=0.02) in animals with PMWS (Group A pigs) than in animals without PMWS (Group B and C pigs). Therefore, the more severe the lesions, the higher amounts of viral genome by ISH, and the higher PCV2 load in serum and swab specimens. Except for the tracheo-bronchial swab, no significant differences (p&gt;0.05) were observed among tested specimens when age-groups (pigs younger than 1.5 months, and equal or older than 1.5 months of age) were compared. In summary, PCV2 is presumably excreted through respiratory (nasal and tracheo-bronchial) and oral (tonsillar) secretions, urine and faeces of both PMWS and non-PMWS affected pigs, with higher viral loads being associated with the presence of PMWS lesions.
Veterinary Journal, Jul 1, 2011
The degree of apoptosis in the livers of pigs with hepatitis due to naturally-occurring postweani... more 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.
Journal of Virological Methods, Dec 1, 2007
Porcine circovirus type 2 (PCV2) is detected in high amounts within the characteristic microscopi... more Porcine circovirus type 2 (PCV2) is detected in high amounts within the characteristic microscopic lesions of postweaning multisystemic wasting syndrome (PMWS) affected pigs. In spite of recent advances on disease pathogenesis, the precise cell types that support viral replication are still a major issue of scientific discussion. In this study, a new methodology to detect cell types that support PCV2 replication was designed. For this purpose, two in situ hybridisation (ISH) methods were developed and applied on tissues of PMWS naturally affected pigs using two probes designed from the ORF1 sequence of the virus. While the complementary probe (CP) detected ssDNA, ORF1 mRNA and replicative form (RF) of PCV2, the RF probe (RFP) exclusively hybridised with the RF of the virus, thus, only labelling cells where PCV2 replication is taking place. Both probes demonstrated to be specific and equally sensitive by an in vitro Southern blot hybridisation assay. ISH labelling with the CP was extensive in lymphoid tissues and of variable amount in other non-lymphoid tissues. With this probe, mainly macrophage-like cells were labelled but also other cell types such as hepatocytes and other epithelial cells. Tissues in which RFP labelling was found more frequently were lung, inguinal and mesenteric lymph nodes, tonsil and liver. Labelling with the RFP was always nuclear, and found in the same cell types as with the CP, although in a relatively low proportion of them; labelling of macrophage-like cells was infrequent. Therefore, the results indicate that at least a certain proportion of macrophages may support PCV2 replication, but main cells where PCV2 replicates are of epithelial/endothelial origin. In summary, the present study permitted the study of cell types that support PCV2 replication by the use of ISH on formalin-fixed, paraffin-embedded tissues of PMWS affected pigs.
Journal of General Virology, Oct 1, 2004
The objective of the present study was to evaluate the involvement of apoptosis in the developmen... more The objective of the present study was to evaluate the involvement of apoptosis in the development of post-weaning multisystemic wasting syndrome (PMWS) lymphoid-depletion lesions. Twenty-one pigs that were categorized into three different lesional severity stages (S1, n=5; S2, n=7; S3, n=9) and five healthy control pigs (stage S0) were used. From all pigs, samples of thymus, spleen, tonsil, ileum and superficial inguinal lymph node were processed for histological examination, in situ hybridization for porcine circovirus type 2 (PCV2) detection and cleaved caspase-3 (CCasp3) immunohistochemistry for detection of apoptotic cells. PCV2 was quantified in serum samples by using TaqMan real-time PCR. CCasp3 labelling was measured in the different morphological compartments of all lymphoid tissues, using an automated system for quantification. Differences between each tissue compartment and lesional stage were assessed, as well as the correlation between apoptosis, lesional stage and viral load. Overall, the results indicated that the more intense the lymphoid depletion, the lower the rate of apoptosis. In the thymus, the cortex was the area where differences between PMWS-affected and control animals were more evident; it was found that all PMWS-affected pigs had significantly lower rates of apoptosis than the controls. In the secondary lymphoid organs, B-cell areas presented higher rates of apoptosis; similar apoptotic rates were found in this compartment in control and S1 pigs. In S2 and S3, B-cell areas were lost and the apoptotic pattern observed was a diffusely distributed low rate of positive cells. Significantly lower rates of apoptosis between PMWS-affected pigs and the control group were already evident in S1 for the thymus, spleen, superficial inguinal lymph node and Peyer's patches, but not for the tonsils. Apoptotic rates in lymphoid tissues were correlated inversely with viral load in serum and with severity of lesions. In conclusion, the results indicate that apoptosis is not a remarkable feature in PMWS lymphoid lesion development.
Journal of Virological Methods, Apr 1, 2004
Postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on the presence of character... more Postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on the presence of characteristic histopathological lymphoid lesions and porcine circovirus type 2 (PCV2) within these lesions. Previous studies indicate that PCV2 load is higher in PMWS affected than in PCV2 infected, healthy pigs. On the other hand, PCV2 has been suggested to play a role in porcine dermatitis and nephropathy syndrome (PDNS) pathogenesis. This study describes a new TaqMan © real time PCR assay and its use to quantify viral load in serum samples. Serum viral loads were related with different degrees of PMWS characteristic lesions and PDNS cases. DNA extracted from serum samples from 75 animals with mild, moderate and severe PMWS lesions and 12 animals with PDNS was used as template. PCV2 DNA was quantified in 69 of 75 PMWS cases and in 11 of 12 PDNS cases. Significant differences in PCV2 load were observed between animals with severe, moderate and mild PMWS lesions, although variability within each group was high, probably due to heterogeneity in disease progression. These results suggest that high viral load is a major feature of PMWS affected pigs. PDNS affected animals had lower PCV2 loads. No significant differences in viral load were found between animals suffering from PDNS and animals with mild PMWS characteristic lesions, which were unaffected clinically.
International Journal of Biometeorology, Sep 9, 2011
The objective of the present work was to elucidate the potential relationship between Mycoplasma ... more 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/m(2)) 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.
Veterinary Journal, May 1, 2005
The objective of the present study was to determine the presence of recognised abortifacient viru... more The objective of the present study was to determine the presence of recognised abortifacient viruses such as porcine reproductive and respiratory virus (PRRSV), AujeszkyÕs disease virus (ADV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2), in tissues from aborted fetuses and stillborn neonates in cases of late reproductive failure in swine. A total of 293 specimens (fetuses aborted in the last third of gestation and stillborn piglets) from 100 different cases of late-term abortions and premature farrowing from 15 different Spanish provinces were studied. PRRSV was detected in 9/100 cases by RT-PCR. Only 1/100 cases analysed (corresponding to a late-term aborted fetus with a negative PRRSV RT-PCR result) was positive for PCV2 by PCR. Neither ADV (monitored by viral isolation plus antigen detection) nor PPV (monitored by ELISA antigen capture test) infection was identified. The results suggest that PRRSV is one of the most important infectious agents, if not the most relevant one, associated with fetal infection leading to abortion or premature farrowing in Spain. Moreover, other viral pathogens such as ADV, PPV and PCV2 seem to have a minor impact on reproductive disease.
Veterinary Microbiology, May 16, 2007
A field trial was conducted to study Mycoplasma hyopneumoniae (Mh) infection dynamics by nested p... more A field trial was conducted to study Mycoplasma hyopneumoniae (Mh) infection dynamics by nested polymerase chain reaction (nPCR) and serology in pigs of a farm affected by enzootic pneumonia (EP). Moreover, correlation of Mh detection at different respiratory tract sites with presence of EP gross and microscopic lung lesions was assessed. These parameters were studied and compared between vaccinated (two doses at 1 and 3 weeks of age versus one dose at 6 weeks of age) and non-vaccinated pigs. Animals were monitored from birth to slaughter by nPCR from nasal swabs and by serology. From 3 to 22 weeks of age, an average of 3 pigs per treatment and per batch were necropsied (n=302). The remaining pigs were sent to the slaughter (n=103). Nasal, bronchial and tonsillar swabs were taken from the necropsied/slaughtered pigs; gross and microscopic EP-suggestive lung lesions were also assessed. Single and double vaccination resulted in earlier seroconversion and higher percentage of Mh seropositive pigs compared to control group. At slaughter, double vaccinated pigs showed lower percentage of EP-compatible gross lung lesions and lower Mh prevalence at upper respiratory tract sites (nasal cavity and tonsil) than control pigs.
Veterinary Record, Sep 1, 2001
The aim of this work was to characterise the lesions and agents present in clinically normal and ... more 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), Aujeszky&#39;s 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-1 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-1 genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.
Journal of Veterinary Medical Science, May 25, 2004
Journal of Veterinary Medical Science, May 25, 2004
Veterinary Record, May 1, 2004
... scrofa Sus wasting syndrome in European wild boar ( Identification of postweaning multisystem... more ... scrofa Sus wasting syndrome in European wild boar ( Identification of postweaning multisystemic ... SHORT COMMUNICATIONS subtropical regions varies from 15 per cent to 85 per cent (Chung and others 1977, Taylor and Petri 1977, Bielsa and Greiner 1985, Foley 1994). ...
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Papers by Maria Calsamiglia