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Facultad de Medicina Veterinaria y Zootecnia. Email: marinconm unal. Background: porcine circovirus type 2 PCV2 is associated with reproductive disease in newly populated herds and in replacement breeding stock from new sources and is almost exclusively reported in gilts. Objective: the main purpose of this study was to assess the dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts and the effect on their piglets.
Methods: the study was conducted with 40 gilts selected at random from four breeding herds. Blood samples, nasal and vaginal swabs were obtained from the gilts at arrival, acclimatization, farrowing, and one day after farrowing. Colostrum samples were collected immediately after parturition and one day after farrowing. Blood, nasal swab, or tissue samples were collected from four piglets prior to suckling. All serums were analyzed by virus neutralization test VNT to establish the presence of antibodies.
Results: high levels of viremia and viral load of PCV2 in nasal and vaginal swabs were found in healthy gilts at arriving, confirming the introduction of infected animals into the farms.
PCV2 shedding was also observed in nasal and vaginal fluids and colostrum even in presence of serum neutralizing antibodies NA. Conclusion: replacement gilts can be infected with PCV2 before entering the farm and continuous exposure seems to occur horizontally in acclimatization and gestation units or before farrowing. Exposure and infection during gestation may result in infected but apparently healthy piglets.
De cuatro lechones neonatos, se colectaron muestras de sangre, hisopado nasal y tejidos antes de consumir calostro. Porcine circovirus type 2 PCV2 , a member of the Circoviridae family, is distributed worldwide and is considered an important emerging pathogen associated with several different syndromes and diseases in pigs, collectively grouped as porcine circovirus-associated diseases PCVAD; Chae, PCV2-associated systemic infection is clinically characterized by wasting, dyspnea, and lymphadenopathy and might be associated with diarrhea, pallor, and jaundice Harding, The most relevant histological lesions in this condition occur in lymphoid organs and consist of extensive lymphocytic depletion, macrophage infiltration, a few multinucleated giant cells, and botryoid basophilic cytoplasmic inclusion bodies Rosell et al.
Reproductive losses attributed to PCV2 infection are less commonly reported. In mature breeding animals, PCV2-associated reproductive failure can manifest as abortion, but it is more frequently associated with increased rates of mummified, macerated, stillborn and weak-born piglets West et al. Other case reports of reproductive failure implicated PCV2 either as the sole agent or in conjunction with other reproductive disease agents Brunborg et al. Gilts are fundamental to farm productivity and their introduction represents one of the most critical factors for the sanitary status of the farm.
Taking into account that PCV2-associated reproductive failure outbreaks are typically reported in gilt-start-ups or new farms and it is related to seronegative populations West et al. Since then, the diagnosis has focused on PCV2 systemic disease. However, it is unclear what is the role of gilts in PCV2 transmission occurring under field conditions, especially in farms subclinically infected with PCV2. The main objective of this study was to evaluate the dynamics of porcine circovirus type 2 infection and neutralizing antibodies in gilts subclinically infected and to determine the effect on their litters.
The study included four commercial breeding herds that volunteered to participate. The final number of participating farms was based on-farm availability to fulfill the sample criteria requirements strict supervision of farrowing and sufficient technical skills of farm workers to collect samples from sows and piglets. Farms B and C were multiplier units, with approximately 1, sows, producing replacement gilts. Farms A and B were commercial breeding herds.
At the time of sample collection none of the herds had reported PCVAD-associated problems and all herds were free from porcine reproductive and respiratory syndrome PRRS virus, classical swine fever CSF and Aujeszky's disease AD , as determined by periodically performed serological testing on sows of mixed parities or wean-to-finish pigs.
In the other farms, located in a CSF free zone, no vaccination was conducted. None of the other herds used PCV2 vaccination in the breeding animals. All gilts selected for the study were vaccinated against parvovirus PPV , Lestospira spp.
Characteristics of the herds are summarized in Table 1. Sample collection. Farm managers received a checklist with precise instructions on selection of sows and piglets, sample collection, and sample storage.
Regardless of breed, a total of 10 clinically healthy gilts from each farm were randomly selected from a group that was introduced to the acclimatization facilities on the same day. In farm D, the gilts arrived with 30 days of age in contrast to farms A, B, and C where they arrived with to days of age. Blood samples as well as nasal and vaginal swabs were collected from gilts at arrival, acclimatization, farrowing and one day after farrowing in order to establish their sanitary status.
Farrowing was supervised, and serum and colostrum samples were collected immediately after parturition and one day post-partum. Four normal looking piglets were arbitrarily selected from each litter, regardless of their weight. The piglets were put apart into a dry box to make sure they had no access to colostrum prior to blood and nasal swab collection.
Two piglets from each sow were euthanized on the first day of age and necropsied. Heart, lungs, liver, spleen, lymph node pool , brain, kidneys, serum, and nasal swab samples were collected from each piglet. Packaging complied with: a a leakproof primary receptacle plastic bags for tissues; plastic tubes for serum, colostrum and swabs , b a leak-proof secondary packaging with ice pads, and c a rigid outer packaging boxes. Each sample was assigned a unique identification number and sent via courier mail to the laboratory.
All sows were visually monitored weekly for clinical signs of PCV2- associated disease, including weight loss, diarrhea, and dyspnea. All sows were allowed to farrow naturally. Sow and piglet pre-suckle serum samples were tested for presence of anti-PCV2 antibodies using a VNT as described previously Fort et al.
DNA extraction. Five mL of colostrum samples were centrifuged at g for 10 min. The upper fat layer was removed and the middle aqueous layer milk whey and pellets were collected separately. DNA was extracted from the aqueous layer of the milk using the same commercial kit.
Determination of viral copy number. Hyacinthe, Quebec, Canada were used in the assay. The primers amplified a bp region of the cap gene ORF2 of PCV2 corresponded to nucleotides to as reported. All realtime PCR reactions unknown samples, and controls were performed by duplicate in neighboring wells on the sample plate. Results are reported as an average of the duplicates.
Real-time PCR performance. The linear portion of the standard curve was found to span 5. This cutoff corresponded to a threshold cycle Ct of The dilution factor was taken into consideration when calculating copies of PCV2 in the original sample.
The viral concentration was expressed as the mean log 10 viral DNA copy numbers by mL of serum or swab sample. Tissue quantifications were also expressed by mg of tissue sample. A difference between the two melting temperatures of about 1.
Comparisons of means were conducted using Duncan's multiple range test. PCV2 neutralizing antibodies were identified in all groups at their arrival. With the exception of farm D, VN titers in sows were similar at arrival, acclimatization, farrowing and post-farrowing. Taking into account the distribution of titers in piglets, positive live-born pre-suckle piglet serum samples were detected in The distribution of VN titers within each farm ranged from to and there was no significant difference between VN serum titers VNT50 between farms.
The analysis of samples from different stages showed differences in the number of PCV2 DNA positive gilts among farms. However, viremic sows were found in each farm at arrival although gilts themselves showed no clinical signs of disease. There was no significant difference between virus level in serum on the four stages for farms A, B, and D. Nasal and vaginal swabs from sows were examined to determine if the virus was present in secretions, which could then shed into the environment.
None of the farms showed statistically significant differences when comparing viral load on nasal swabs among them. However, there was difference on viral load in vaginal swabs collected at arrival and acclimatization as compared with those collected at farrowing in the farm A. In addition, colostrum PCV2 DNA positive samples were detected only in farms B and C and the number of positive colostrum samples within each farm was consistent for both days examined.
The viral load was low compared to other fluids tested and there was no significant difference between virus levels in the times studied. Results of virus load in serum, nasal swabs and pool tissues of live-born piglets at farrow and one day post-farrow are presented in Table 4.
There was no significant difference between pre-suckle serum virus level in the four farms at farrow. In farm A, tissue viral load was the lowest compared with the other farms.
PCV2 genotypes in primiparous sows and piglets. Additionally, PCV2 b was the predominant genotype in all types of analyzed samples regardless of the phase evaluated. The data presented here showed that PCV2 infection occurs in gilts from their introduction to breeding herds until their first parity. A high level of viremia and a high viral load of PCV2 in nasal and vaginal swabs detected at the arrival in healthy gilts in this study allows to establish the risk of incorporating subclinically infected animals or new PCV2 virus strains to the breeding herd.
In terms of PCV2 DVA virus load in serum at arrival, there was no relationship between virus level and age; weaning and pre-puberty females showed viremia and furthermore, most gilts were positive for PCV2 DNA in nasal and vaginal swabs, which suggests that they came in contact with PCV2 while in the genetic farm. By contrast, very high VN titers were found in farms A, B, and C at arrival age - days and low VN titers were detected in farm D gilts at 30 days of age when maternal immunity waned.
In addition, it can be speculated that humoral immune response in Farm D sows was not detected after vaccination due to the short time period between vaccination and serum sample collection. Later, the gilts were under constant infection challenge through contact with viremic sows and contaminated environment in the acclimatization, gestation or farrowing units.
These observations indicate that PCV2 infection in gilts is persistent before the first parity. According to the data presented here and field investigations Opriessnig et al.
Nevertheless, the introduction of PCV2- seronegative gilts may have been an important factor associated with reproductive inefficiency in the herd that may have predisposed groups of gilts to enter the breeding herd with no active immunity to PCV2. Immunity of replacement gilts to PCV2 should be considered when changing gilt sources and also in low-immunity, high-risk herds, such as all-gilt or low-parity herds, recently populated herds, and herds with high replacement rates Pittman, In the present study, coexistence of NA with viral DNA was observed in gilts subclinically infected at arrival, during the acclimatization period and at farrowing.
A possible explanation could be that PCV2 infection prolonged impairment of humoral response. In a previous study by Meerts et al. This hypothesis was based on observations made under experimental and field conditions in which PMWS pigs lacked NA but developed total antibody titers in a similar way to subclinically infected animals. Viral persistence despite high levels of NA was also observed in the experimentally PCV2-infected pigs and has been also reported for chicken anaemia virus, a related virus of the Circoviridae family Brentano et al.
In contrast to other experimental and field observations of PCV2-associated reproductive failure West et al.
[Circovirus and porcine circovirus infection: history of a controversy] 
The diagnosis of PMWS is a difficult task and must follow three criteria: i the presence of compatible clinical signs, ii the presence of characteristic microscopic histopathological lesions, and iii the presence of PCV2 within these lesions in moderate or high. The presence of PCV2 in lymphoid tissues must be demonstrated by in situ hybridization or immunohistochemical methods. The in situ hybridization is a more complex and expense compared to other diagnostic tools, on the other hand, one problem concerning the immunohistochemical methods for PMWS diagnostic is the lack of a commercial anti-PCV2 peroxidase conjugate; therefore, the aim of this work was to obtain a polyclonal-antibody-immunoperoxidase-conjugate for the PCV2 specific detection. An anti-PCV2-peroxidase conjugated for the PCV2 specific detection was obtained based on the use of the available commercial vaccine against PCV2 as immunogenic inoculation for producing a polyclonal antibody in rabbits.
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