Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan.

Porcine circovirus (PCV) is a small non-enveloped virus with a single-stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2-systemic disease (PCV2-SD), previously known as post-weaning multisystemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. Porcine circovirus type 2 genome was detected in 16 of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2-SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs.

The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.

Influence of long-term vaccination of a breeding herd of pigs against PCV2 on reproductive parameters.

The objective of the study was to evaluate an efficacy of sows vaccination protocols in the herd with serious problems affecting efficacy of reproduction. The study was performed in a large pig herd with about 1200 sows. Before vaccination against PCV2, farrowing rate in this farm was about 65%. Sows, boar and replacement gilts were immunized using Circovac vaccine (Merial, France) according to producer's recommendations. Parameters of production were analyzed since 2007 until 2010 in selected batches of sows inseminated at the same weeks of the year (17th, 18th, 19th and 20th) to eliminate seasonal variability. In total, 940 sows were subjected to the study. No significant changes in management during these years were introduced. The applied protocol of sow herd long-term vaccination proved to be very efficient. All measured production parameters: reproduction rate, number of piglets born alive, birth weight of piglets and number of piglets weaned per a litter improved after implementation of immunization program. Moreover, further improvement was observed with vaccination in the following reproduction cycles. The most spectacular effect of vaccination regarded average farrowing rate that increased from 64.76% in control group to 86.93% after basic vaccination. Two years after implementation of vaccination program this parameter reached 93.6%. Number of piglets weaned per sow per a litter improved from 10.31 to 11.74 after one year of vaccination and remained relatively stable through the following year. Simultaneously, the percentage of newborn piglets with birth weight < 1 kg decreased significantly (p < 0.05). To summarize, vaccination against PCV2 influenced positively the insemination rate, number of piglets born alive and weaned per litter as well as birth body weight and percentage of piglets weighing < 1 kg.

Comparison of PCR methods for detection of classical swine fever virus and other pestiviruses.

Classical swine fever (CSF) is a notifiable, highly contagious disease of swine controlled mainly with costly administrative methods. Swine may be infected not only with classical swine fever virus (CSFV), but also with other, non porcine, genetically and antigenically related pestiviruses. Differentiation of infections with CSFV and other pestiviruses is a crucial element of diagnostics. In the present study two real-time PCR methods and conventional one-tube nested PCR for specific detection of CSFV were compared. Additionally, two methods designed for detection of all pestivirus species real-time SYBR Green I and one-tube nested PCR were included into the study. Analyzed methods varied considerably regarding their sensitivity and specificity, what suggests that careful selection of diagnostic methods and their evaluation on a regular basis is necessary.

Linked outbreaks and control of porcine reproductive and respiratory syndrome and postweaning multisystemic wasting syndrome in a pig farm in Poland.

In a newly established farrow-to-finish farm (porcine reproductive and respiratory virus [PRRSV]-free, porcine circovirus type 2 [PCV-2]-infected), reproductive failure was seen seven months after population. The conception rate dropped from 89 to 51 per cent, and the abortion rate increased from 0.5 to 11 per cent. The following month, characteristic lesions of postweaning multisystemic wasting syndrome (PMWS) and elevated mortality were observed in weaned pigs. Laboratory examinations confirmed reproductive failure due to PRRSV and PMWS associated with apparent activation of the PCV-2 circulating in the farm. The herd was closed for replacement and a number of measures to improve hygiene, environmental conditions and feeding were applied. The abortion rate returned to preoutbreak levels four months after the beginning of the PRRS outbreak and the conception rate returned to normal four months later. Slower improvement was observed regarding the PMWS outbreak, with PMWS-related losses disappearing nine months after the detection of PMWS. Analysis of seroconversion profiles to PCV-2 and PRRSV during the outbreak and after its control indicated that while PRRSV was eliminated from sows and weaners by the control measures, the time of PCV-2 infection was unchanged and occurred at seven weeks of age during the PMWS outbreak as well as after its elimination. However, the elimination of PMWS from the herd coincided with increased levels of maternally derived antibodies to PCV-2 in one- to five-week-old pigs and faster serological responses to infection with PCV-2.

Porcine circovirus type 2 viremia and seroconversion in pigs from a farm affected by postweaning multisystemic wasting syndrome.

The aim of the study was to assess the usefulness of real-time PCR and serological methods as indicators of postweaning multisystemic wasting syndrome (PMWS) occurrence. Significantly higher level of porcine circovirus type 2 (PCV2) viral load in serum and significantly lower titre of specific antibodies in PMWS-affected pigs indicated that combination of quantitative PCR and serological methods may support diagnosis of PMWS.

Porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern Europe support the definition of new genetic subtypes.

Porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 and ORF7 sequences from Belarus were found to be of the European (EU) genotype, but grouped separately from all other EU genotype sequences described so far, including live-attenuated EU genotype PRRSV vaccines and Italian EU genotype sequences, some of which have been associated with reduced vaccine efficacy. Also, the Belarusian EU-PRRSV exhibited extreme ORF7 size polymorphism, ranging from 375 nt (the smallest EU genotype ORF7 yet described) to 393 nt (the largest ORF7 yet described for any arterivirus). With the Belarusian sequences, the diversity of EU genotype PRRSV now exceeds that of the North American (US) genotype PRRSV, suggesting a European origin of PRRSV. Finally, a very sharp geographical demarcation of highly diverse EU genotype PRRSV was observed along the eastern Polish border. The new Belarusian sequences have relevance for vaccine and diagnostic-antigen design and show that sequence analysis of PRRSV from more eastern parts of Europe may offer further insights into the emergence and evolution of PRRSV.