BACKGROUND
porcine circovirus type  <dig>  as an etiological agent of porcine circovirus diseases  was firstly identified in the 1990s in canada  <cit> , and was later detected and characterized in usa and europe  <cit> .
in the czech republic and slovakia pcv <dig> in pigs was confirmed and genetically typed a few years later .
over the last five years, due to the world financial depression, weak government support of farmers and infectious diseases, the number of pigs in slovakia has decreased dramatically while numbers in the czech republic have remained stable.
at the end of  <dig>  almost  <dig>  <dig> pigs, including up to  <dig>  <dig> sows were registered in slovakia, but little pig breeding is done and all-in/all-out swine fattening systems predominate.
beside the national husbandry, a few farms are managed by danish companies.
in the czech republic, approximately  <dig>  million of pigs are housed, including  <dig>  <dig> sows.
similar to slovakia, all-in/all-out systems are dominant.
pigs are mostly imported from denmark and netherlands to both countries.
post-weaning multisystemic wasting syndrome  as the most important pcvd is considered a multifactorial disease  <cit> .
in the syndrome development, several viruses and bacteria are involved  <cit> .
several scientific papers indicate that an increase of post-weaning mortality with significant impact on pig production worldwide is caused by the circulation of several viral pathogens on farms  <cit> .
during pmws outbreaks prrsv is frequently present in pig herds  <cit> .
it was considered that also torque teno sus virus  <dig>  and torque teno sus virus  <dig>  play a role in diseases associated with pcv <dig>  but controversial results were revealed .
the prevalence of porcine bocavirus  <dig>  in swine populations has been studied recently to see if the virus has any effect on pmws development  <cit> .
the aim of this study was to determine the presence of emerging , re-emerging  and newly-emerging  viruses in pigs at the terminal stage of pmws.
the occurrence of single and multiple viral infections in pigs of different age categories was also investigated.
the impact of vaccination against pcv <dig> was determined on the limited level on healthy fattening animals.
methods
none of the pigs were killed with the purpose to fulfill the objectives of the present study.
all diseased animals were naturally infected and their tissue samples were collected after the death as the consequence of the pathological process.
samples acquisition was performed with the agreement of the pig farmers.
pooled tissue samples  from domestic pigs originating from  <dig> farms in slovakia and  <dig> farms in the czech republic were collected during a period of six years between  <dig> and  <dig>  these samples were originally sent to diagnostic laboratories of the state veterinary institutes for the confirmation of diagnosis.
the samples were then passed to our laboratory for further investigation.
specimens from the healthy animals were obtained on the abattoir during official meat inspection.
sampling of these specimens adhered to the council regulation  no.
1099/ <dig> and the statutory order of the slovak republic no.
432/ <dig> on the protection of animals at the time of killing.
a collection of  <dig> tissue samples included in this study originated from diseased  and healthy  animals.
dead diseased pigs were divided into three groups: suckling piglets – between  <dig> and 6 weeks of age , post-weaning pigs – between  <dig> and 10 weeks of age  and fattening pigs – between  <dig> and 24 weeks of age .
clinical signs of pmws  were recorded in all  <dig> diseased pigs by veterinarian at the time of sampling.
nineteen of these animals also suffered from unspecified respiratory tract disorders.
to confirm pmws, the generally accepted criteria for diagnosis of pmws were applied: clinical manifestation, characteristic histopathological lesions in lymphoid tissues and detection of pcv <dig> within the tissues  <cit> .
the application of this complex diagnostic approach led to the confirmation of pmws in  <dig> pigs.
in the remaining  <dig> animals histopathological examination of lymphoid tissues was not performed but because the other criteria were fulfilled, these pigs were labeled as pwms-suspected.
pmws  and pmws-suspected  pigs formed a group of diseased pigs.
none of the diseased animals had been vaccinated against pcv <dig>
lymph node samples were collected from additional fifty nine apparently healthy  <dig> to 28 week old fattening pigs following slaughter in an abattoir.
all healthy animals originated from two slovak swine farms.
vaccination of piglets at the age of 3 weeks against pcv <dig> with ingelvac circoflex vaccine  was carried out on the first farm .
non-vaccinated animals were housed on the second farm .
tissue samples from younger healthy pigs were not available for ethical reasons, since it would have demanded killing of healthy animals.
clinical samples were prepared as 20% w/v tissue homogenates in pbs  or nuclease-free water .
total dna  and rna  were extracted from 300 μl or 200 μl of tissue homogenates using chelex resins  and trizol reagent , respectively, according to manufacturer’s recommendations.
for detection of rna viruses, reverse transcription using gene-specific primer  or random hexamers  followed by the pcr was performed.
dna viruses were analyzed using pcr or real-time pcr based on sybr green.
the references concerning the detection of specific pcr products are listed in table  <dig> table  <dig>
pcr primers used for amplification of pcv <dig>  prrsv, ttsuv <dig>  ttsuv <dig>  ptv and pbov1
virus
primers
lenght of amplification
genome region amplified
references
pcv2
prrsv
ttsuv1
ttsuv2
ptv
pbov1
pcr products were analyzed in 2% agarose gel and visualized under uv light after gelred staining .
results of real-time pcr employing sybr green  were considered positive when ct ≤  <dig> and specific peaks with expected melting temperature of amplicons were observed.
selected pcr products were sequenced by the sanger method employing fluorescently labeled ddntps.
the chromatograms were proofread by seqman pro .
sequences were aligned by clustalw method and nucleotide and deduced amino acid identities were calculated using megalign software .
program mega version  <dig>  <cit>  employing neighbor-joining method was used for the construction of phylogenetic trees.
statistical analyses of data were performed by chi-square  tests with confidence limits of 95%, p <  <dig>  using graphpad prism  <dig> for windows .
RESULTS
evaluation of virus prevalence in each animal group
each of the five groups of pigs  was evaluated on the basis of the following parameters: 1) individual detection of six viral pathogens , 2) prevalence of significant co-infections in each pig and each group and 3) total number of viruses detected in each animal.
diseased suckling pigs
beside pcv <dig> , ttsuv <dig> was the most frequently detected virus .
prrsv and pbov <dig> infection was confirmed in  <dig> % and  <dig> % of suckling pigs, respectively.
no animal was found to be positive for ttsuv <dig> or ptv.
in accordance with these findings, the most frequent co-infection was with pcv <dig> and ttsuv <dig> .
the prevalence of co-infection with pcv <dig> and prrsv reached  <dig> %.
only one piglet was found to be simultaneously infected with pcv <dig>  prrsv and ttsuv <dig> and two piglets were co-infected with pcv <dig>  pbov <dig> and ttsuv <dig>
<dig> % of piglets were infected with two viruses  while in  <dig> % of animals the presence of one or three viruses was confirmed.
no piglet was found to be infected with more than three viral pathogens.
prevalence of viruses in individual groups of pigs.
viruses were detected in pooled tissue samples  of diseased pigs of different age and lymph nodes of healthy animals.
total dna or rna was isolated by chelex resins or trizol reagent, respectively.
rna was transcribed into cdna using random hexamers  or gene-specific primer  and superscript iii reverse transcriptase.
viral genomes were detected by pcr, rt-pcr or real-time pcr based on sybr green .
percentage of selected co-infections in each group of pigs
selected co-infections
diseased pigs
healthy pigs
suckling
post-weaning
fattening
fattening*
fattening
less than 6 weeks
7- <dig> weeks
11- <dig> weeks
28 weeks
22 weeks
n = 11
n = 14
n = 31
n = 30
n = 29
*pigs vaccinated against pcv <dig> using ingelvac circoflex vaccine .
number of viruses detected in each group of pigs.
number of viruses  detected by pcr, rt-pcr or real-time pcr in tissue samples obtained from diseased and healthy animals were compared between different groups of pigs.
diseased post-weaning pigs
all pigs were infected with pcv <dig> , and they were also frequently infected with ttsuv <dig>  or ttsuv <dig>  .
the prevalence of prrsv, ptv and pbov <dig> was also high .
most frequently, co-infections with pcv <dig> and ttsuv <dig> or ttsuv <dig> were found .
simultaneous detection of pcv <dig> and prrsv  was predominantly found in pigs suffering from the respiratory tract disorders.
<dig> % of post-weanlings co-infected with pcv <dig> and prrsv were found to be ttsuv <dig> positive as well.
among pigs simultaneously infected with pcv <dig> and ptv, almost  <dig> % were also infected with ttsuv <dig>   <dig> % with ttsuv <dig> and  <dig> % with prrsv.
pbov <dig> was detected only in pigs co-infected with pcv <dig> and ttsuv <dig>
<dig> %,  <dig> % and  <dig> % of post-weanlings were simultaneously infected with three, four and five viruses, respectively .
one pig  was affected with a single virus infection, i.e.
pcv <dig> and one animal was infected with all six viruses.
diseased fattening pigs
the most prevalent virus detected was pcv <dig> , followed by ttsuv <dig> that was detected in  <dig> % of the pigs .
prrsv and ttsuv <dig> were both detected in  <dig> % of the pigs, while  <dig> % and  <dig> % of pigs were infected with ptv and pbov <dig>  respectively .
simultaneous infection of pcv <dig> with ttsuv <dig> was most frequently observed , followed by pcv <dig> with prrsv and pcv <dig> with ttsuv <dig> co-infection at the same level .
the co-infections of pcv <dig>  pbov <dig> and ttsuv <dig> or pcv <dig>  ttsuv <dig> and ttsuv <dig> were detected less frequently .
triple virus infection was observed in  <dig> % of pigs, followed by double and single virus infection .
simultaneous detection of more than three viruses was rare in this group of pigs.
healthy fattening vaccinated pigs
all healthy vaccinated animals were pcv <dig> free and no prrsv, ptv and pbov <dig> were detected.
the prevalence of ttsuv <dig> and ttsuv <dig> in these animals reached the same value  and was lower compared to the healthy non-vaccinated pigs .
<dig> %  of these pigs were simultaneously infected with both ttsuvs.
most pigs  were not infected with any virus studied.
single virus infection was confirmed in  <dig> % and two viral pathogens in a single pig was rare .
healthy fattening non-vaccinated pigs
<dig> % of animals infected with pcv <dig> did not show clinical signs of pmws.
the animals were not infected with prrsv, ptv or pbov <dig> but ttsuv <dig> and ttsuv <dig> were found in  <dig> % and  <dig> % of samples analyzed, respectively .
co-infection of pcv <dig> with ttsuv <dig> was more frequently observed than co-infection of pcv <dig> with ttsuv <dig> .
simultaneous detection of pcv <dig>  ttsuv <dig> and ttsuv <dig> reached  <dig> % .
ttsuv <dig> or ttsuv <dig> was not confirmed in pcv2-free pigs.
infection with one, two and three viruses  was observed .
<dig> % of pigs were found to be negative for any virus tested.
comparison of virus prevalence between different groups of pigs
the results were further analyzed by comparing the different groups of diseased and healthy animals.
regarding the age, the post-weaning group of animals was found to be more infected with viruses than the other two age groups of diseased animals.
diseased pigs of all age groups were most frequently affected by triple virus infection , but simultaneous co-infection of more than four viruses was not rare either .
prevalence of multiple infections was not significantly different between diseased fattening pigs ) and healthy fattening non-vaccinated pigs ); χ2 =  <dig> ; p =  <dig> ).
this observation was mostly due to similar co-infection of both groups of pigs with ttsuv <dig> and ttsuv <dig> and high infection with pcv <dig> .
the comparison of healthy vaccinated fattening pigs with the same age category of diseased and healthy non-vaccinated pigs suggested strong benefit of vaccination against pcv <dig> .
the observations between diseased fattening pigs and healthy vaccinated fattening pigs clearly showed a significant difference in prevalence of multiple viral infections  and  <dig> % , respectively; χ2 =  <dig> ; p <  <dig> ).
a significantly lower number of animals co-infected with two or more viruses was also found between healthy vaccinated pigs when compared to the healthy non-vaccinated animals  and  <dig> % , respectively; χ2 =  <dig> ; p <  <dig> ).
genetic and phylogenetic analysis of pcr products
since pcv <dig> and prrsv circulating in slovakia and the czech republic were genetically typed earlier  <cit> , in the present study we focused on the newly identified viruses.
comparison of six ttsuv <dig> sequences from the untranslated region  revealed  <dig> - <dig> % nucleotide identity to each other.
five ttsuv <dig> sequences from the utr were  <dig> - <dig> % identical to each other.
genetic and phylogenetic analysis of viral strains detected in slovakia and the czech republic showed that the sequences clustered into ttsuvs lineages already identified by cortey et al.
<cit>  and li et al.
<cit>  .
two slovak ptv sequences from the 5’-utr were compared and a  <dig> % similarity to each other was observed.
moreover, sequences were almost identical to the prototype strains of the ptv- <dig> serotype.
an alignment of two pbov <dig> sequences of part of the vp1/vp <dig> genomic region obtained from isolates circulating in slovakia confirmed a  <dig> % identity at both the nucleotide and amino acid levels.
besides, nucleotide sequences were closely related to the first bocavirus isolated from pig sharing a  <dig> -100% similarity.
discussion
the hypothesis that co-infection of pcv <dig> with other viral or bacterial pathogens might lead to pmws development has been supported by several papers  <cit> .
our work was focused on the study of co-infections with six viral pathogens with the aim to extend information in this field.
we realize that co-infection in pigs is much more complex and not limited to six viruses studied in this work.
no doubt those animals could be infected with bacteria and other microorganisms which might influence investigated viral co-infections.
collection of samples obtained from dead animals suffering from pmws at the terminal stage of disease gives at least partial information about the occurrence of six viruses in those pigs.
determination of particular viruses in pigs with or without pmws revealed several interesting facts.
prrsv
the detection of prrsv  in pmws pigs in slovakia and czech republic  <cit>  was not surprising because prrsv is considered one of the major risk factors for a herd to be affected with pmws  <cit> .
simultaneous occurrence of pcv2/prrsv infection in pigs varies  <cit>  and our observations on slovak and czech farms support these findings.
the level of co-infection was the highest in post-weaning pigs and the lowest in suckling piglets.
detailed analysis revealed that all prrsv-infected animals suffered from prrsv/pcv <dig> co-infection.
dual infection of pigs with pcv <dig> and prrsv has consistently resulted in more severe disease than that caused by infection with either agent alone  <cit> .
in the present study, it was observed that pcv2/prrsv-infected pigs suffered from strong respiratory tract disorders on which an antibiotic treatment had no effect.
ttsuvs
although ttsuvs were frequently detected in pmws pigs their role in the development of the diseases is not clear  <cit> .
in general, contradictory results have been observed when co-infections of pcv <dig> and ttsuvs were studied.
in some pmws-affected pigs ttsuv <dig> was detected more frequently  <cit> , while in others  <cit>  and in the present study a higher prevalence of ttsuv <dig> was detected.
in our healthy pigs limited to the fattening group  the prevalence of ttsuv <dig> and ttsuv <dig> was similar.
de castro et al.
<cit>  also did not find any significant relationship between ttsuvs and pcv <dig> in pigs in brazil.
the overall controversial results indicate that ttsuvs are unlikely to play an important role in the development of pmws and our findings support this view.
it is not excluded that discrepancies in the results of different laboratories in the detection of ttsuvs in diseased and healthy pigs may also be related to the methods used for the detection of both viruses.
among molecular-genetic methods established for confirming ttsuvs in clinical samples, real-time pcr is often used because of its high detection sensitivity.
although a high prevalence of both ttsuvs was confirmed in our study also using real-time pcr, the lowest ct values of positive samples were  <dig>  and  <dig> .
most positive samples had ct values >  <dig>  which represents 102-101of viral copies.
it is really questionable if such amounts of virus can significantly contribute to the development of any disease, including pmws.
ptv
porcine teschovirus is widely distributed in pig populations around the world  <cit> .
pigs from the present study, in which ptv was detected, most frequently suffered from diarrhea and inappetence with a loss of weight.
although porcine teschovirus may cause an asymptomatic disease, no healthy animal was found to be infected in our collection of samples.
however, virus was frequently confirmed in clinically diseased post-weaning and fattening pigs.
in the last years, in almost all commercial pig herds ptv has been demonstrated in co-infections with other swine pathogens, most frequently pcv <dig> and prrsv  <cit> .
in the present study multiple infections of all three viruses was observed in  <dig> % of diseased pigs only .
pbov1
since the discovery of a boca-like virus in pmws-affected pig from sweden  <cit> , its presence has been confirmed in europe  <cit> , asia  <cit>  and africa  <cit>  with variable detection rates.
the role of pbov <dig> alone in the development of the disease is still not clear, especially when the virus was confirmed in both apparently healthy and sick pigs.
moreover, the virus was detected along with pcv <dig> regardless of whether or not pmws was diagnosed  <cit> .
pcv <dig> and pbov <dig> were simultaneously detected in  <dig> % of our samples  and only in pigs with clinical evidence of pmws.
moreover, an interesting phenomenon was observed.
in all pmws-infected pigs in which pbov <dig> alongside pcv <dig> was detected, ttsuv <dig> was also present.
of pmws-suspected pigs co-infected with pbov <dig>  60% were infected with pcv <dig> and ttsuv <dig>  on the other hand, no healthy pig was positive for pbov <dig>  simultaneous detection of pcv <dig>  ttsuvs and pbov <dig> in pmws-affected pigs has been described earlier  <cit> .
these findings indicate that pcv <dig> together with ttsuv <dig> might increase the susceptibility of pigs to bocavirus infection but this phenomenon has to be studied more deeply and with larger groups of pigs.
all single and multiple viral infections were related to the age of animals.
viruses were most frequently observed in pigs after weaning.
on the contrary, suckling piglets had the lowest prevalence of each virus and multiple co-infections and this was the only group in which ttsuv <dig> was not detected.
very similar findings were described by de castro et al.
<cit>  in simultaneous infections caused by pcv <dig>  ttsuv <dig> and ttsuv <dig>  thus, it is evident that maternal immunity due to colostrum feeding plays an important role in the protection of suckling pigs against viral infections.
the introduction of pcv <dig> vaccines has significantly changed the impact of pcv <dig> on global pig production .
in this study pcv <dig> was not found in tissues of healthy vaccinated pigs.
this finding might be explained by combination of careful performance of vaccination using the effective vaccine and following of correct vaccination scheme.
in addition, in slaughter-age pigs with a good health status their immune system is likely able to eliminate the virus.
since pcv <dig> detected in healthy non-vaccinated pigs reached  <dig> % it is supposed that good management or the absence of some co-factor protected the healthy swine from a pmws outbreak.
four of six viruses were not detected  in these animals and more than 56% of pigs were not infected with any virus tested.
by contrast, in the majority of healthy non-vaccinated pigs  simultaneous detection of two and three viruses was confirmed.
in addition, significantly lower number of animals co-infected with two and more viruses was found between vaccinated pigs compared to non-vaccinated ones .
according to observations of tshering et al.,  <cit>  ttsuvs seem not to be influenced by vaccination against pcv <dig>  lower number of ttsuv1-infected pigs was found in vaccinated animals when compared to the non-vaccinated ones, but the difference was not significant.
the prevalence of ttsuv <dig> was almost the same between these groups of pigs.
our study has shown that vaccination against pcv <dig> led to minimizing the risk of pmws development and contributed to the decrease in susceptibility of pigs to the other viral pathogens.
what is the role of viruses such as ttsuvs, pbov <dig>  ptv or other newly discovered viruses in the development of significant porcine diseases with high economical impact?
this question will become even more pertinent when novel pathogens are discovered by new molecular-genetic techniques, e.g.
next generation sequencing.
one may suggest that under certain conditions some viruses can cause significant disease, especially in an immunodeficient pig.
another possibility is based on the assumption that important pathogens, such as prrsv or bacteria depress the immune system of swine and allow the entry and replication of other viruses.
another point of view is that certain pathogens may re-activate a persistent virus, thus, facilitating the development of a pathological process.
our findings that a significantly higher prevalence of all viruses has been found in diseased pigs confirm that multiple infections are associated with the presence of disease.
vaccination of pigs against pcv <dig> was also associated with lack of infection with other viruses.
the role of individual viral pathogens in multiple infections and their participation in the development of diseases has to be systematically studied.
the first step, identification of multiple pathogens in diseased and healthy animals is just in progress.
the next step requires carefully designed biological experiments with controlled infections with virus, bacteria and parasites in presence and absence of stress factors with the aim of finding combinations able to induce pmws in pigs.
CONCLUSIONS
the following results have been achieved in this study: i/post-weaning diseased pigs were more often co-infected with viral pathogens than suckling or fattening pigs, ii/clinical signs were stronger in pmws pigs co-infected with prrsv while the other viruses studied did not considerably influence the clinical manifestation of pmws, iii/vaccination against pcv <dig> was associated with failure to detect pcv <dig> and significantly reduced viral co-infections in vaccinated pigs when compared to non-vaccinated pigs .
abbreviations
pcv2porcine circovirus type 2
pmwspost-weaning multisystemic wasting syndrome
pcvdporcine circovirus disease
prrsvporcine reproductive and respiratory syndrome virus
ptvporcine teschovirus
ttsuv1torque teno sus virus type 1
ttsuv2torque teno sus virus type 2
pbov1porcine bocavirus 1
pcrpolymerase chain reaction
rt-pcrreverse transcription-polymerase chain reaction
utruntranslated region
competing interests
the authors declare that they have no competing interests.
authors’ contributions
mv participated in the experimental work, in the molecular genetic studies, in the design of the study, performed statistical analyses and drafted the manuscript.
vl participated in the experimental work and molecular genetic studies.
is participated in the experimental work and statistical analyses.
aj participated in the experimental work, molecular genetic studies and in the design of the study.
sv as a project leader conceived the study, participated in the design and coordination of experiments and helped to draft the manuscript.
all authors read and approved the final manuscript.

 porcine circovirus type as an etiological agent of porcine circovirus diseases was firstly identified in the 1990s in canada, and was later detected and characterized in usa and europe. in the czech republic and slovakia pcv in pigs was confirmed and genetically typed a few years later. over the last five years, due to the world financial depression, weak government support of farmers and infectious diseases, the number of pigs in slovakia has decreased dramatically while numbers in the czech republic have remained stable. at the end of almost pigs, including up to sows were registered in slovakia, but little pig breeding is done and all-in/all-out swine fattening systems predominate. beside the national husbandry, a few farms are managed by danish companies. in the czech republic, approximately million of pigs are housed, including sows. similar to slovakia, all-in/all-out systems are dominant. pigs are mostly imported from denmark and netherlands to both countries. post-weaning multisystemic wasting syndrome as the most important pcvd is considered a multifactorial disease. in the syndrome development, several viruses and bacteria are involved. several scientific papers indicate that an increase of post-weaning mortality with significant impact on pig production worldwide is caused by the circulation of several viral pathogens on farms. during pmws outbreaks prrsv is frequently present in pig herds. it was considered that also torque teno sus virus and torque teno sus virus play a role in diseases associated with pcv but controversial results were revealed. the prevalence of porcine bocavirus in swine populations has been studied recently to see if the virus has any effect on pmws development. the aim of this study was to determine the presence of emerging, re-emerging and newly-emerging viruses in pigs at the terminal stage of pmws. the occurrence of single and multiple viral infections in pigs of different age categories was also investigated. the impact of vaccination against pcv was determined on the limited level on healthy fattening animals. methods none of the pigs were killed with the purpose to fulfill the objectives of the present study. all diseased animals were naturally infected and their tissue samples were collected after the death as the consequence of the pathological process. samples acquisition was performed with the agreement of the pig farmers. pooled tissue samples from domestic pigs originating from farms in slovakia and farms in the czech republic were collected during a period of six years between and these samples were originally sent to diagnostic laboratories of the state veterinary institutes for the confirmation of diagnosis. the samples were then passed to our laboratory for further investigation. specimens from the healthy animals were obtained on the abattoir during official meat inspection. sampling of these specimens adhered to the council regulation no. 1099/ and the statutory order of the slovak republic no. 432/ on the protection of animals at the time of killing. a collection of tissue samples included in this study originated from diseased and healthy animals. dead diseased pigs were divided into three groups: suckling piglets – between and 6 weeks of age, post-weaning pigs – between and 10 weeks of age and fattening pigs – between and 24 weeks of age. clinical signs of pmws were recorded in all diseased pigs by veterinarian at the time of sampling. nineteen of these animals also suffered from unspecified respiratory tract disorders. to confirm pmws, the generally accepted criteria for diagnosis of pmws were applied: clinical manifestation, characteristic histopathological lesions in lymphoid tissues and detection of pcv within the tissues. the application of this complex diagnostic approach led to the confirmation of pmws in pigs. in the remaining animals histopathological examination of lymphoid tissues was not performed but because the other criteria were fulfilled, these pigs were labeled as pwms-suspected. pmws and pmws-suspected pigs formed a group of diseased pigs. none of the diseased animals had been vaccinated against pcv lymph node samples were collected from additional fifty nine apparently healthy to 28 week old fattening pigs following slaughter in an abattoir. all healthy animals originated from two slovak swine farms. vaccination of piglets at the age of 3 weeks against pcv with ingelvac circoflex vaccine was carried out on the first farm. non-vaccinated animals were housed on the second farm. tissue samples from younger healthy pigs were not available for ethical reasons, since it would have demanded killing of healthy animals. clinical samples were prepared as 20% w/v tissue homogenates in pbs or nuclease-free water. total dna and rna were extracted from 300 μl or 200 μl of tissue homogenates using chelex resins and trizol reagent, respectively, according to manufacturer’s recommendations. for detection of rna viruses, reverse transcription using gene-specific primer or random hexamers followed by the pcr was performed. dna viruses were analyzed using pcr or real-time pcr based on sybr green. the references concerning the detection of specific pcr products are listed in table  table pcr primers used for amplification of pcv prrsv, ttsuv ttsuv ptv and pbov1 virus primers lenght of amplification genome region amplified references pcv2 prrsv ttsuv1 ttsuv2 ptv pbov1 pcr products were analyzed in 2% agarose gel and visualized under uv light after gelred staining. results of real-time pcr employing sybr green were considered positive when ct ≤  and specific peaks with expected melting temperature of amplicons were observed. selected pcr products were sequenced by the sanger method employing fluorescently labeled ddntps. the chromatograms were proofread by seqman pro. sequences were aligned by clustalw method and nucleotide and deduced amino acid identities were calculated using megalign software. program mega version employing neighbor-joining method was used for the construction of phylogenetic trees. statistical analyses of data were performed by chi-square tests with confidence limits of 95%, p <  using graphpad prism for windows.  evaluation of virus prevalence in each animal group each of the five groups of pigs was evaluated on the basis of the following parameters: 1) individual detection of six viral pathogens, 2) prevalence of significant co-infections in each pig and each group and 3) total number of viruses detected in each animal. diseased suckling pigs beside pcv, ttsuv was the most frequently detected virus. prrsv and pbov infection was confirmed in % and % of suckling pigs, respectively. no animal was found to be positive for ttsuv or ptv. in accordance with these findings, the most frequent co-infection was with pcv and ttsuv. the prevalence of co-infection with pcv and prrsv reached %. only one piglet was found to be simultaneously infected with pcv prrsv and ttsuv and two piglets were co-infected with pcv pbov and ttsuv % of piglets were infected with two viruses while in % of animals the presence of one or three viruses was confirmed. no piglet was found to be infected with more than three viral pathogens. prevalence of viruses in individual groups of pigs. viruses were detected in pooled tissue samples of diseased pigs of different age and lymph nodes of healthy animals. total dna or rna was isolated by chelex resins or trizol reagent, respectively. rna was transcribed into cdna using random hexamers or gene-specific primer and superscript iii reverse transcriptase. viral genomes were detected by pcr, rt-pcr or real-time pcr based on sybr green. percentage of selected co-infections in each group of pigs selected co-infections diseased pigs healthy pigs suckling post-weaning fattening fattening* fattening less than 6 weeks 7- weeks 11- weeks 28 weeks 22 weeks n = 11 n = 14 n = 31 n = 30 n = 29 *pigs vaccinated against pcv using ingelvac circoflex vaccine. number of viruses detected in each group of pigs. number of viruses detected by pcr, rt-pcr or real-time pcr in tissue samples obtained from diseased and healthy animals were compared between different groups of pigs. diseased post-weaning pigs all pigs were infected with pcv, and they were also frequently infected with ttsuv or ttsuv. the prevalence of prrsv, ptv and pbov was also high. most frequently, co-infections with pcv and ttsuv or ttsuv were found. simultaneous detection of pcv and prrsv was predominantly found in pigs suffering from the respiratory tract disorders. % of post-weanlings co-infected with pcv and prrsv were found to be ttsuv positive as well. among pigs simultaneously infected with pcv and ptv, almost % were also infected with ttsuv % with ttsuv and % with prrsv. pbov was detected only in pigs co-infected with pcv and ttsuv %, % and % of post-weanlings were simultaneously infected with three, four and five viruses, respectively. one pig was affected with a single virus infection, i.e. pcv and one animal was infected with all six viruses. diseased fattening pigs the most prevalent virus detected was pcv, followed by ttsuv that was detected in % of the pigs. prrsv and ttsuv were both detected in % of the pigs, while % and % of pigs were infected with ptv and pbov respectively. simultaneous infection of pcv with ttsuv was most frequently observed, followed by pcv with prrsv and pcv with ttsuv co-infection at the same level. the co-infections of pcv pbov and ttsuv or pcv ttsuv and ttsuv were detected less frequently. triple virus infection was observed in % of pigs, followed by double and single virus infection. simultaneous detection of more than three viruses was rare in this group of pigs. healthy fattening vaccinated pigs all healthy vaccinated animals were pcv free and no prrsv, ptv and pbov were detected. the prevalence of ttsuv and ttsuv in these animals reached the same value and was lower compared to the healthy non-vaccinated pigs. % of these pigs were simultaneously infected with both ttsuvs. most pigs were not infected with any virus studied. single virus infection was confirmed in % and two viral pathogens in a single pig was rare. healthy fattening non-vaccinated pigs % of animals infected with pcv did not show clinical signs of pmws. the animals were not infected with prrsv, ptv or pbov but ttsuv and ttsuv were found in % and % of samples analyzed, respectively. co-infection of pcv with ttsuv was more frequently observed than co-infection of pcv with ttsuv. simultaneous detection of pcv ttsuv and ttsuv reached %. ttsuv or ttsuv was not confirmed in pcv2-free pigs. infection with one, two and three viruses was observed. % of pigs were found to be negative for any virus tested. comparison of virus prevalence between different groups of pigs the results were further analyzed by comparing the different groups of diseased and healthy animals. regarding the age, the post-weaning group of animals was found to be more infected with viruses than the other two age groups of diseased animals. diseased pigs of all age groups were most frequently affected by triple virus infection, but simultaneous co-infection of more than four viruses was not rare either. prevalence of multiple infections was not significantly different between diseased fattening pigs ) and healthy fattening non-vaccinated pigs ); χ2 = ; p =  ). this observation was mostly due to similar co-infection of both groups of pigs with ttsuv and ttsuv and high infection with pcv. the comparison of healthy vaccinated fattening pigs with the same age category of diseased and healthy non-vaccinated pigs suggested strong benefit of vaccination against pcv. the observations between diseased fattening pigs and healthy vaccinated fattening pigs clearly showed a significant difference in prevalence of multiple viral infections and %, respectively; χ2 = ; p <  ). a significantly lower number of animals co-infected with two or more viruses was also found between healthy vaccinated pigs when compared to the healthy non-vaccinated animals and %, respectively; χ2 = ; p <  ). genetic and phylogenetic analysis of pcr products since pcv and prrsv circulating in slovakia and the czech republic were genetically typed earlier, in the present study we focused on the newly identified viruses. comparison of six ttsuv sequences from the untranslated region revealed - % nucleotide identity to each other. five ttsuv sequences from the utr were - % identical to each other. genetic and phylogenetic analysis of viral strains detected in slovakia and the czech republic showed that the sequences clustered into ttsuvs lineages already identified by cortey et al. and li et al.. two slovak ptv sequences from the 5’-utr were compared and a % similarity to each other was observed. moreover, sequences were almost identical to the prototype strains of the ptv- serotype. an alignment of two pbov sequences of part of the vp1/vp genomic region obtained from isolates circulating in slovakia confirmed a % identity at both the nucleotide and amino acid levels. besides, nucleotide sequences were closely related to the first bocavirus isolated from pig sharing a -100% similarity. discussion the hypothesis that co-infection of pcv with other viral or bacterial pathogens might lead to pmws development has been supported by several papers. our work was focused on the study of co-infections with six viral pathogens with the aim to extend information in this field. we realize that co-infection in pigs is much more complex and not limited to six viruses studied in this work. no doubt those animals could be infected with bacteria and other microorganisms which might influence investigated viral co-infections. collection of samples obtained from dead animals suffering from pmws at the terminal stage of disease gives at least partial information about the occurrence of six viruses in those pigs. determination of particular viruses in pigs with or without pmws revealed several interesting facts. prrsv the detection of prrsv in pmws pigs in slovakia and czech republic was not surprising because prrsv is considered one of the major risk factors for a herd to be affected with pmws. simultaneous occurrence of pcv2/prrsv infection in pigs varies and our observations on slovak and czech farms support these findings. the level of co-infection was the highest in post-weaning pigs and the lowest in suckling piglets. detailed analysis revealed that all prrsv-infected animals suffered from prrsv/pcv co-infection. dual infection of pigs with pcv and prrsv has consistently resulted in more severe disease than that caused by infection with either agent alone. in the present study, it was observed that pcv2/prrsv-infected pigs suffered from strong respiratory tract disorders on which an antibiotic treatment had no effect. ttsuvs although ttsuvs were frequently detected in pmws pigs their role in the development of the diseases is not clear. in general, contradictory results have been observed when co-infections of pcv and ttsuvs were studied. in some pmws-affected pigs ttsuv was detected more frequently, while in others and in the present study a higher prevalence of ttsuv was detected. in our healthy pigs limited to the fattening group the prevalence of ttsuv and ttsuv was similar. de castro et al. also did not find any significant relationship between ttsuvs and pcv in pigs in brazil. the overall controversial results indicate that ttsuvs are unlikely to play an important role in the development of pmws and our findings support this view. it is not excluded that discrepancies in the results of different laboratories in the detection of ttsuvs in diseased and healthy pigs may also be related to the methods used for the detection of both viruses. among molecular-genetic methods established for confirming ttsuvs in clinical samples, real-time pcr is often used because of its high detection sensitivity. although a high prevalence of both ttsuvs was confirmed in our study also using real-time pcr, the lowest ct values of positive samples were and. most positive samples had ct values >  which represents 102-101of viral copies. it is really questionable if such amounts of virus can significantly contribute to the development of any disease, including pmws. ptv porcine teschovirus is widely distributed in pig populations around the world. pigs from the present study, in which ptv was detected, most frequently suffered from diarrhea and inappetence with a loss of weight. although porcine teschovirus may cause an asymptomatic disease, no healthy animal was found to be infected in our collection of samples. however, virus was frequently confirmed in clinically diseased post-weaning and fattening pigs. in the last years, in almost all commercial pig herds ptv has been demonstrated in co-infections with other swine pathogens, most frequently pcv and prrsv. in the present study multiple infections of all three viruses was observed in % of diseased pigs only. pbov1 since the discovery of a boca-like virus in pmws-affected pig from sweden, its presence has been confirmed in europe, asia and africa with variable detection rates. the role of pbov alone in the development of the disease is still not clear, especially when the virus was confirmed in both apparently healthy and sick pigs. moreover, the virus was detected along with pcv regardless of whether or not pmws was diagnosed. pcv and pbov were simultaneously detected in % of our samples and only in pigs with clinical evidence of pmws. moreover, an interesting phenomenon was observed. in all pmws-infected pigs in which pbov alongside pcv was detected, ttsuv was also present. of pmws-suspected pigs co-infected with pbov 60% were infected with pcv and ttsuv on the other hand, no healthy pig was positive for pbov simultaneous detection of pcv ttsuvs and pbov in pmws-affected pigs has been described earlier. these findings indicate that pcv together with ttsuv might increase the susceptibility of pigs to bocavirus infection but this phenomenon has to be studied more deeply and with larger groups of pigs. all single and multiple viral infections were related to the age of animals. viruses were most frequently observed in pigs after weaning. on the contrary, suckling piglets had the lowest prevalence of each virus and multiple co-infections and this was the only group in which ttsuv was not detected. very similar findings were described by de castro et al. in simultaneous infections caused by pcv ttsuv and ttsuv thus, it is evident that maternal immunity due to colostrum feeding plays an important role in the protection of suckling pigs against viral infections. the introduction of pcv vaccines has significantly changed the impact of pcv on global pig production. in this study pcv was not found in tissues of healthy vaccinated pigs. this finding might be explained by combination of careful performance of vaccination using the effective vaccine and following of correct vaccination scheme. in addition, in slaughter-age pigs with a good health status their immune system is likely able to eliminate the virus. since pcv detected in healthy non-vaccinated pigs reached % it is supposed that good management or the absence of some co-factor protected the healthy swine from a pmws outbreak. four of six viruses were not detected in these animals and more than 56% of pigs were not infected with any virus tested. by contrast, in the majority of healthy non-vaccinated pigs simultaneous detection of two and three viruses was confirmed. in addition, significantly lower number of animals co-infected with two and more viruses was found between vaccinated pigs compared to non-vaccinated ones. according to observations of tshering et al., ttsuvs seem not to be influenced by vaccination against pcv lower number of ttsuv1-infected pigs was found in vaccinated animals when compared to the non-vaccinated ones, but the difference was not significant. the prevalence of ttsuv was almost the same between these groups of pigs. our study has shown that vaccination against pcv led to minimizing the risk of pmws development and contributed to the decrease in susceptibility of pigs to the other viral pathogens. what is the role of viruses such as ttsuvs, pbov ptv or other newly discovered viruses in the development of significant porcine diseases with high economical impact? this question will become even more pertinent when novel pathogens are discovered by new molecular-genetic techniques, e.g. next generation sequencing. one may suggest that under certain conditions some viruses can cause significant disease, especially in an immunodeficient pig. another possibility is based on the assumption that important pathogens, such as prrsv or bacteria depress the immune system of swine and allow the entry and replication of other viruses. another point of view is that certain pathogens may re-activate a persistent virus, thus, facilitating the development of a pathological process. our findings that a significantly higher prevalence of all viruses has been found in diseased pigs confirm that multiple infections are associated with the presence of disease. vaccination of pigs against pcv was also associated with lack of infection with other viruses. the role of individual viral pathogens in multiple infections and their participation in the development of diseases has to be systematically studied. the first step, identification of multiple pathogens in diseased and healthy animals is just in progress. the next step requires carefully designed biological experiments with controlled infections with virus, bacteria and parasites in presence and absence of stress factors with the aim of finding combinations able to induce pmws in pigs.  the following results have been achieved in this study: i/post-weaning diseased pigs were more often co-infected with viral pathogens than suckling or fattening pigs, ii/clinical signs were stronger in pmws pigs co-infected with prrsv while the other viruses studied did not considerably influence the clinical manifestation of pmws, iii/vaccination against pcv was associated with failure to detect pcv and significantly reduced viral co-infections in vaccinated pigs when compared to non-vaccinated pigs. abbreviations pcv2porcine circovirus type 2 pmwspost-weaning multisystemic wasting syndrome pcvdporcine circovirus disease prrsvporcine reproductive and respiratory syndrome virus ptvporcine teschovirus ttsuv1torque teno sus virus type 1 ttsuv2torque teno sus virus type 2 pbov1porcine bocavirus 1 pcrpolymerase chain reaction rt-pcrreverse transcription-polymerase chain reaction utruntranslated region competing interests the authors declare that they have no competing interests. authors’ contributions mv participated in the experimental work, in the molecular genetic studies, in the design of the study, performed statistical analyses and drafted the manuscript. vl participated in the experimental work and molecular genetic studies. is participated in the experimental work and statistical analyses. aj participated in the experimental work, molecular genetic studies and in the design of the study. sv as a project leader conceived the study, participated in the design and coordination of experiments and helped to draft the manuscript. all authors read and approved the final manuscript. 
the effect of vaccination against pcv with ingelvac circoflex vaccine was also investigated. viruses were detected by pcr, rt-pcr and real-time pcr in the pooled tissue samples of pigs with pmws which were divided into three groups: suckling piglets, post-weaning pigs and fattening pigs. porcine circovirus type is an etiological agent of porcine circovirus diseases. no healthy pig was found to be infected with prrsv, ptv or pbov vaccination against pcv did not influence the prevalence of ttsuvs, but significantly protected pigs against multiple viral infections. studies of viral co-infections in pmws pigs led often to controversial results. simultaneous infection with ttsuv and pbov was frequently confirmed in pigs with pmws. post-weaning pmws pigs were more often co-infected with viral pathogens than suckling or fattening pigs. post-weaning multisystemic wasting syndrome as the most important pcvd is considered a multifactorial disease. co-infection with prrsv enforces clinical signs of pmws, the influence of other viral co-infections is not clear. vaccination against pcv significantly reduced viral co-infections in pigs. the impact of vaccination against pcv and the influence of age on the occurrence of single and multiple viral infections in pigs were also investigated. between non-vaccinated pigs, the highest prevalence of individual viruses and multiple viral infections were found in diseased post-weaning and fattening animals with pmws.
 porcine circovirus type is an etiological agent of porcine circovirus diseases. post-weaning multisystemic wasting syndrome as the most important pcvd is considered a multifactorial disease. it was demonstrated that not only pcv but several viruses are associated with pmws. studies of viral co-infections in pmws pigs led often to controversial results. the aim of this work was to determine the presence of emerging, re-emerging and newly-emerging viruses in samples of dead pigs suffering from pmws. the impact of vaccination against pcv and the influence of age on the occurrence of single and multiple viral infections in pigs were also investigated.  viruses were detected by pcr, rt-pcr and real-time pcr in the pooled tissue samples of pigs with pmws which were divided into three groups: suckling piglets, post-weaning pigs and fattening pigs. in addition, lymph node samples were collected from apparently healthy fattening pigs. the effect of vaccination against pcv with ingelvac circoflex vaccine was also investigated. between non-vaccinated pigs, the highest prevalence of individual viruses and multiple viral infections were found in diseased post-weaning and fattening animals with pmws. severe clinical disease was observed in swine co-infected with pcv and prrsv. the prevalence of ttsuv and ttsuv was high in all groups of pigs and did not appear to have a significant effect on the syndrome. simultaneous infection with ttsuv and pbov was frequently confirmed in pigs with pmws. no healthy pig was found to be infected with prrsv, ptv or pbov vaccination against pcv did not influence the prevalence of ttsuvs, but significantly protected pigs against multiple viral infections.  post-weaning pmws pigs were more often co-infected with viral pathogens than suckling or fattening pigs. co-infection with prrsv enforces clinical signs of pmws, the influence of other viral co-infections is not clear. vaccination against pcv significantly reduced viral co-infections in pigs. keywords post-weaning multisystemic wasting syndrome multiple infectionsvaccinationporcine circovirus type porcine reproductive and respiratory syndrome virus torque teno sus virus porcine teschovirus porcine bocavirus issue-copyright-statement© the author 2014 