Tesis doctoral de LlorenÁ§ Grau Roma
Postweaning multisystemic wasting syndrome (pmws) is considered a multifactorial pig disease in which porcine circovirus type 2 (pcv2) is the essential infectious agent. Pmws affects nursery and growing pigs, and causes severe economic losses to the swine industry worldwide. The most representative clinical sign is wasting, and affected pigs usually show a poor response to therapeutic treatments. Due to the ubiquitous nature of pcv2, pmws diagnosis is one of the most complex diagnoses within the currently known animal diseases. Thus, the individual pmws diagnosis is established when the suspected pig fulfils the following three conditions: (i) presence of compatible clinical signs; (ii) presence of moderate to severe characteristic microscopic lymphoid lesions; (iii) detection of moderate to high amount of pcv2 within these lesions. The lack of an effective and consistent experimental model to reproduce pmws makes especially relevant to carry out epidemiological studies within pmws affected farms. The present thesis aimed to expand the epidemiological knowledge on pcv2 infection and pmws through the realization of case-control field studies. Mainly, the potential influence of pcv2 genetics, the timing of pcv2 infection, the pcv2 maternal derived humoral immunity and the pig humoral response against pcv2 infection in pmws presentation were investigated. in the first study (study i) 87 open reading frame 2 (orf2) pcv2 sequences obtained from pigs with different clinical and pathological conditions were analyzed together with 148 pcv2 sequences available at the ncbi nucleotide database (http://www.Ncbi.Nlm.Nih.Gov) in september 2005. Results further confirmed the existence of two main genogroups and the definition of two pcv2 genotypes (1 and 2) was proposed. The suggested methodology to define pcv2 genotypes is based on the p-distance/frequency distribution of pcv2 sequences together with pcv2 phylogenetic analyses. Genotype 1 was shown to be predominant within pigs coming from pmws affected farms. Moreover, all sequences included in genotype 1 came from pigs from pmws affected farms, while all sequences obtained from non-pmws affected farms corresponded to genotype 2. Consequently, it was suggested that pcv2 genotype 1 might potentially be more pathogenic than pcv2 genotype 2. In addition, infection of single pigs from pmws affected farms harbouring both genotypes at the same time was described. the present thesis was developed within the european union (eu) project entitled control of porcine circovirus diseases (pcvd): towards improved food quality and safety (www.Pcvd.Org), which was funded by the eu sixth framework programme. Few months after the acceptation of the manuscript presented in the study i, the eu consortium on pcvd (www.Pcvd.Org) discussed and agreed in supporting the definition of pcv2 genotypes based on the proposed methodology. However, it was considered that the designation of genotype 1 and 2 could cause confusion with the current nomenclature. Thus, the mentioned consortium published the letter here presented as an addendum of study i. In this letter it was proposed the nomenclature of pcv2 genotype a (pcv2a) and genotype b (pcv2b), corresponding to genotypes 2 and 1, respectively. In addition, following this definition, a third pcv2 genotype was retrospectively detected in denmark, being initially denominated pcv2 genotype 3 and subsequently renamed as pcv2 genotype c (pcv2c) in the mentioned letter. in the second study (study ii) two different real-time quantitative pcr (qpcr) assays were compared on dna extracted from serum and nasal as well as rectal swabs. The two compared techniques were used routinely in two laboratories from two different countries, denmark and spain, both members of the above-mentioned eu consortium. This comparison was made in order to be able to interpret together the work performed in both countries, which is mainly presented in studies iii and iv. Results from study ii showed a significant linear association between the assays, and a systematic difference of 1.4 log10 copies of pcv2 per millilitre of sample. This difference indicated that the assay from the danish laboratory yielded a higher output than the assay from the spanish laboratory. Moreover, the danish assay had higher sensitivity than the spanish one. Results also showed that there was no linear association between the amount of pcv2 dna and the amount of total dna, neither in nasal nor in rectal swabs, suggesting that normaliziation of pcv2 dna load in swab samples to total dna concentration is not suitable to express the amount of pcv2 present in swab samples. in studies iii and iv, longitudinal case/control studies were performed in pmws affected farms from denmark and spain using similar designs. Fourteen independent batches of 100 to 154 pigs per batch were monitored from birth to pmws outbreak occurrence. Pigs displaying pmws-like signs and matched healthy cohorts were euthanized during the clinical outbreak. Pmws was diagnosed according to internationally accepted criteria and pigs were classified as: (i) pmws cases, (ii) wasted non-pmws cases and (iii) healthy pigs. Similar pcv2 infection dynamic patterns were observed in spain and denmark, with a delay in pmws age-presentation in spain compared to the one in denmark. Thus, all spanish pmws outbreaks occurred at the fattening phase, whereas all danish clinical outbreaks were observed at nurseries. Pmws diagnoses were confirmed by laboratorial tests in only half of pigs clinically suspected to suffer from pmws. Pcv2 qpcr and serological techniques were applied to analyse longitudinally collected sera. Moreover, pcv2 qpcr was also applied to nasal and rectal swabs. Results from these determinations were presented in study iii. Moreover, the evolution of two acute phase proteins (apps), pig-major acute phase protein (pig-map) and haptoglobin (hpt), in serum from studied pigs was also assessed. The corresponding results were presented in study iv. Overall, results showed that pcv2 load increased concomitantly to maternal antibody level waning, reaching the maximum viral load concurrently with the development of clinical signs. Interestingly, the acute phase response (apr) in pmws affected pigs occurred in parallel to pcv2 viremia, suggesting that pcv2 is the main responsible for the systemic inflammatory status suffered by diseased pigs. As a collective, pmws affected pigs harboured higher pcv2 loads and higher pig-map and hpt concentrations in sera, shed higher viral loads through both nasal secretions and faeces, and had lower level of maternal antibodies against pcv2 than non-pmws affected pigs. Moreover, in spain, pmws affected pigs showed also higher apr and higher pcv2 prevalence than non-affected ones at the sampling prior to pmws outbreak. Besides, in denmark, pmws affected pigs at the sampling prior to pmws outbreak showed higher pcv2 loads than non-affected ones. Furthermore, an impaired humoral response was observed in pmws affected pigs from spain at 11 weeks of age (prior to the appearence of clinical signs) and at the moment of necropsy, suggesting that this circumstance might be more a cause rather than a consequence of the disease. On the other hand, the lack of sensitivity and/or specificity observed from qpcr and/or serological techniques suggests that those techniques are not able to substitute histopathology plus detection of pcv2 in tissues for the individual pmws diagnosis. However, results indicated that qpcr might potentially be a reliable technique to diagnose pmws on a population basis. Additionally, obtained results supported the idea that although apps are unspecific markers of inflammation, they might be useful indicators of health, becoming a potentially interesting tool to monitor pmws development in epidemiological studies or in the assessment of the efficacy of pcv2 vaccines in the field.
Datos académicos de la tesis doctoral «New insights into the epidemiology of postweaning multisystemic wasting syndrome (pmws)«
- Título de la tesis: New insights into the epidemiology of postweaning multisystemic wasting syndrome (pmws)
- Autor: LlorenÁ§ Grau Roma
- Universidad: Autónoma de barcelona
- Fecha de lectura de la tesis: 28/04/2009
Dirección y tribunal
- Director de la tesis
- Joaquim Segalés I Coma
- Tribunal
- Presidente del tribunal: pedro Miguel Rubio nistal
- Francisco José Pallarés martínez (vocal)
- (vocal)
- (vocal)