Molecular Epidemiology and Phyloevolutionary Analysis of Porcine Parvoviruses (PPV1 through PPV7) Detected in Replacement Gilts from Colombia.

Eight porcine parvovirus (PPV) species, designated as PPV1 through PPV8, have been identified in swine. Despite their similarities, knowledge about their distribution and genetic differences remains limited, resulting in a gap in the genetic classification of these viruses. In this study, we conducted a comprehensive analysis using PPV1 to PPV7 genome sequences from Colombia and others available in the GenBank database to propose a classification scheme for all PPVs. Sera from 234 gilts aged 180 to 200 days were collected from 40 herds in Colombia. Individual detection of each PPV (PPV1 through PPV7) was performed using end-point PCR. Complete nucleotide (nt) sequencing was performed on the PPV1 viral protein (VP), and near-complete genome (NCG) sequencing was carried out for novel porcine parvoviruses (nPPVs) (PPV2 through PPV7). Phylogenetic analyses were conducted by comparing PPV1-VP sequences to 94 available sequences and nPPVs with 565 NCG, 846 nPPV-VP, and 667 nPPV-nonstructural protein (NS) sequences. Bayesian phylogenetic analysis was used to estimate substitution rates and the time to the most recent common ancestor for each PPV. The highest prevalence was detected for PPV3 (40.1%), followed by PPV5 (20.5%), PPV6 (17%), PPV1 (14.5%), PPV2 (9.8%), PPV4 (4.2%), and PPV7 (1.3%). Notably, all tested sera were negative for PPV8 genomes. An analysis of the PPV1-VP sequences revealed two main clades (PPV1-I and PPV1-II), with the sequences recovered in this study grouped in the PPV1-II clade. Comparative analysis showed significant genetic distances for PPV2 to PPV7 at the NCG (>6.5%), NS (>6.3%), and VP (>7.5%) regions, particularly when compared to equivalent regions of PPV genomes recovered worldwide. This study highlights the endemic circulation of nPPVs in Colombian pig herds, specifically among gilts. Additionally, it contributes to the phylogenetic classification and evolutionary studies of these viruses. The proposed method aims to categorize and divide subtypes based on current knowledge and the genomes available in databanks.

Infection and Coinfection of Porcine-Selected Viruses (PPV1 to PPV8, PCV2 to PCV4, and PRRSV) in Gilts and Their Associations with Reproductive Performance.

Seven novel porcine parvoviruses (nPPVs) (PPV2 through PPV8) have been described, although their pathogenicity and possible effects on porcine reproductive failure (PRF) are undefined. In this study, these nPPVs were assessed in gilts from Colombia; their coinfections with PPV1, PCV2, PCV3, PCV4, and PRRSV and an association between the nPPVs and the reproductive performance parameters (RPPs) in sows were determined. For this, 234 serum samples were collected from healthy gilts from 40 herds in five Colombian regions, and the viruses were detected via real-time PCR. The results confirmed the circulation of PPV2 through PPV7 in Colombia, with PPV3 (40%), PPV5 (20%), and PPV6 (17%) being the most frequent. Additionally, no PCV4 or PPV8 was detected. PPV2 to PPV7 were detected in concurrence with each other and with the primary PRF viruses, and these coinfections varied from double to sextuple coinfections. Additionally, the association between nPPVs and PRF primary viruses was statistically significant for the presence of PPV6 in PCV3-positive (p < 0.01) and PPV5 in PPRSV-positive (p < 0.05) gilts; conversely, there was a significant presence of PPV3 in both PCV2-negative (p < 0.01) and PRRSV-negative (p < 0.05) gilts. Regarding the RPPs, the crude association between virus detection (positive or negative) and a high or low RPP was only statistically significant for PCV3 and the farrowing rate (FR), indicating that the crude odds of a low FR were 94% lower in herds with PCV3-positive gilts. This finding means that the detection of PCV3 in gilts (PCV3-positive by PCR) is associated with a higher FR in the farm or that these farms (with positive gilts) have lower odds (OR 0.06, p-value 0.0043) of a low FR. Additionally, a low FR tended to be associated with the detection of PPV4 and PPV5 (p-value < 0.20). This study is important for establishing the possible participation of nPPVs in PRF.

The Novel Porcine Parvoviruses: Current State of Knowledge and Their Possible Implications in Clinical Syndromes in Pigs.

Parvoviruses (PVs) affect various animal species causing different diseases. To date, eight different porcine parvoviruses (PPV1 through PPV8) are recognized in the swine population, all of which are distributed among subfamilies and genera of the Parvoviridae family. PPV1 is the oldest and is recognized as the primary agent of SMEDI, while the rest of the PPVs (PPV2 through PPV8) are called novel PPVs (nPPVs). The pathogenesis of nPPVs is still undefined, and whether these viruses are putative disease agents is unknown. Structurally, the PPVs are very similar; the differences occur mainly at the level of their genomes (ssDNA), where there is variation in the number and location of the coding genes. Additionally, it is considered that the genome of PVs has mutation rates similar to those of ssRNA viruses, that is, in the order of 10-5-10-4 nucleotide/substitution/year. These mutations manifest mainly in the VP protein, constituting the viral capsid, affecting virulence, tropism, and viral antigenicity. For nPPVs, mutation rates have already been established that are similar to those already described; however, within this group of viruses, the highest mutation rate has been reported for PPV7. In addition to the mutations, recombinations are also reported, mainly in PPV2, PPV3, and PPV7; these have been found between strains of domestic pigs and wild boars and in a more significant proportion in VP sequences. Regarding affinity for cell types, nPPVs have been detected with variable prevalence in different types of organs and tissues; this has led to the suggestion that they have a broad tropism, although proportionally more have been found in lung and lymphoid tissue such as spleen, tonsils, and lymph nodes. Regarding their epidemiology, nPPVs are present on all continents (except PPV8, only in Asia), and within pig farms, the highest prevalences detecting viral genomes have been seen in the fattener and finishing groups. The relationship between nPPVs and clinical manifestations has been complicated to establish. However, there is already some evidence that establishes associations. One of them is PPV2 with porcine respiratory disease complex (PRDC), where causality tests (PCR, ISH, and histopathology) lead to proposing the PPV2 virus as a possible agent involved in this syndrome. With the other nPPVs, there is still no clear association with any pathology. These have been detected in different systems (respiratory, reproductive, gastrointestinal, urinary, and nervous), and there is still insufficient evidence to classify them as disease-causing agents. In this regard, nPPVs (except PPV8) have been found to cause porcine reproductive failure (PRF), with the most prevalent being PPV4, PPV6, and PPV7. In the case of PRDC, nPPVs have also been detected, with PPV2 having the highest viral loads in the lungs of affected pigs. Regarding coinfections, nPPVs have been detected in concurrence in healthy and sick pigs, with primary PRDC and PRF viruses such as PCV2, PCV3, and PRRSV. The effect of these coinfections is not apparent; it is unknown whether they favor the replication of the primary agents, the severity of the clinical manifestations, or have no effect. The most significant limitation in the study of nPPVs is that their isolation has been impossible; therefore, there are no studies on their pathogenesis both in vitro and in vivo. For all of the above, it is necessary to propose basic and applied research on nPPVs to establish if they are putative disease agents, establish their effect on coinfections, and measure their impact on swine production.

The Prevalence and Genetic Diversity of PCV3 and PCV2 in Colombia and PCV4 Survey during 2015-2016 and 2018-2019.

Four genotypes of circovirus have been recognized in swine, with PCV2 and PCV3 being the most associated with clinical manifestations, while PCV4 does not have a defined disease. In addition, PCV2 is associated with different syndromes grouped as diseases associated with porcine circovirus (PCVAD), while PCV3 causes systemic and reproductive diseases. In the present study, we retrospectively detected PCV2, PCV3, and PCV4 in Colombia during two periods: A (2015-2016) and B (2018-2019). During period A, we evaluated stool pools from the 32 Colombian provinces, finding a higher prevalence of PCV3 compared to PCV2 as well as PCV2/PCV3 co-infection. Furthermore, we determined that PCV3 had been circulating since 2015 in Colombia. Regarding period B, we evaluated sera pools and tissues from abortions and stillborn piglets from the five provinces with the highest pig production. The highest prevalence found was for PCV3 in tissues followed by sera pools, while PCV2 was lower and only in sera pools. In addition, PCV2/PCV3 co-infection in sera pools was also found for this period. The complete genome sequences of PCV3 and PCV3-ORF2 placed the Colombian isolates within clade 1 as the majority in the world. For PCV2, the predominant genotype currently in Colombia is PCV2d. Likewise, in some PCV3-ORF2 sequences, a mutation (A24V) was found at the level of the Cap protein, which could be involved in PCV3 immunogenic recognition. Regarding PCV4, retrospective surveillance showed that there is no evidence of the presence of this virus in Colombia.

Field infection of a gilt and its litter demonstrates vertical transmission and effect on reproductive failure caused by porcine circovirus type 3 (PCV3).

BACKGROUND: PCV3 is a member of the Circovirus family, associated with disease and mortality in pigs. It is not clear whether PCV3 putatively causes clinical symptoms and disease. In the present case, we reported a gilt infected with PCV3 associated with reproductive failures, vertical transmission, tissue lesions, viral replication by in situ hybridization, and the hypothesis that some strains of PCV3 clade one are associated with reproductive failures at the field level. CASE PRESENTATION: In May 2019, a pig farm in Colombia reported increased reproductive failures, and the presence of PCV3 in gilts and sows was established in a single form or coinfections, mainly with PCV2 and PPV7. Ten sows with a single infection with PCV3 were found, and one gilt with a pre-farrowing serum viral load above 103 was studied. This gilt was followed up during the pre-farrowing, farrowing period and on her litter for 6 weeks. During dystocic farrowing, a mummy and ten piglets were released, including two weak-born piglets. The highest viral loads for PCV3 were found in the mummy and the placenta. In the weak-born piglets, there were viral loads both in serum and in tissues, mainly in the mesenteric ganglia and lung. Replication of PCV3 in these tissues was demonstrated by in situ hybridizations. PCV3 was also found in the precolostrum sera of piglets and colostrum, showing vertical transmission. The viral load in piglets decreased gradually until week six of life. The viral genome's complete sequencing was made from the mummy, and its analysis classified it as PCV3 clade one. CONCLUSIONS: This report confirms that PCV3 can cause disease at the field level, and putatively, in this case, we find the generation of reproductive failures. The ability of PCV3 to cause disease as a putative pathogen may be associated with the viral load present in the pig and the strain that is affecting the farm. For this case, we found that viral loads above 103 (4.93 log genomic copies / mL) in the gilt were associated with clinical manifestation and that some PCV3 strains belonging to clade one are more associated with the reproductive presentation.

Longitudinal comparison of the humoral immune response and viral load of Porcine Circovirus Type 2 in pigs with different vaccination schemes under field conditions.

Background: Porcine Circovirus type 2 (PCV2) infections are distributed worldwide and cause Porcine Circovirus Associated Disease (PCVAD). To minimize the impact of PCV2 infection on swine health and production, different vaccination schemes have been used since 2006. However, the association between vaccination schemes, virus load and disease under field conditions are not completely understood. Therefore, the objective of this study was to compare the effect of two different PCV2 vaccination schemes on the humoral response and PCV2 load in pigs after weaning under field conditions. Methods: Two commercial pig farms (Farm A and B), endemically infected with PCV2, which were using two different PCV2 subunit vaccinations schemes for sow, gilts and piglets, were selected. We designed a longitudinal study and measured IgG levels by ELISA and virus load by quantitative PCR in pigs after weaning. Forty 3-week old piglets were randomly selected at weaning and followed for 20 weeks. IgG levels and virus loads were compared within and between farms and considered statistically different if the non-parametric Kruskal Wallis test p value was lower than 0.05. Results: We found that low virus loads were maintained in pigs from both farms regardless of the vaccination scheme used (p>0.05). However, there was significant difference in the mean IgG levels observed over time (p<0.05), suggesting that different humoral immune response are not necessarily associated with different virus loads observed over time. Conclusions: These results are important because they can help to prevent PCV2 infections using different vaccination schemes to minimize the effect of PCVAD on swine health and production.

Causas de mortalidad o descarte de cerdas en dos granjas de producción intensiva en Colombia / Causes of culling or mortality in sows from two intensive swine production farms in Colombia

Con el propósito de determinar las causas de muerte o descarte de madres en dos granjas de producción intensiva en Colombia, se realizó un estudio prospectivo mediante la evaluación histopatológica de tejidos colectados en la necropsia de cerdas muertas o eliminadas en el periodo comprendido entre el 15 de noviembre de 2004 y el 15 de septiembre de 2005. Además, entre los años 2003 y 2005 se realizó un estudio retrospectivo de datos registrados en el programa informática Pig-CHAMP de madres muertas o descartadas en una de estas granjas. Las principales causas de eliminación o muerte en las dos granjas fueron de origen gastrointestinal(21,05-22,86 por ciento), y cardiovascular (8,57-10,53 por ciento). El resto de causas variaron entre las dos granjas. En la granja uno se encontraron casos con lesiones músculo-esqueléticas (22,86 por ciento), urinarias (14,29 por ciento), y respiratorias (11,43 por ciento). En la granja dos, causas respiratorias (31,58 por ciento) y urinarias (21,05 por ciento). Las cerdas muertas o descartadas estaban entre uno y diez días del periodo de lactancia: para la granja uno, 25,73 por ciento, y para la dos, 47,4 por ciento. La edad de estas cerdas correspondió al primer parto; en la granja uno, 34 por ciento, y en la dos, 42 por ciento. Se detectó la presencia del virus del síndrome reproductivo y respiratorio porcino por la técnica de inmunohistoquímica en las dos granjas. En el estudio retrospectivo realizado en la granja uno, los principales motivos de el imitación correspondieron a causas reproductivas 43 por ciento (2003), 33,57 por ciento (2004), y 28,42 por ciento (2005). Otras fueron lesiones músculo-esqueléticas (11 por ciento al 14 por ciento). Durante los tres años evaluados el mayor número de animales muertos o descartados estaban entre uno y tres partos. Se discutió la importancia de estos hallazgos...

Estandarización y aplicación de la técnica de PCR – anidado para la detección de Mycoplasma hyopneumoniae / Standardization and application of the PCR – nested technique for the detection of Mycoplasma hyopneumoniae

El Mycoplasma hyopneumoniae es el agente causal de la Neumonía enzoótica porcina, una de las enfermedades más importantes en la industria porcina. En la actualidad, el proceso de diagnóstico ante-mortem es una de las principales dificultades, dadas la poca sensibilidad y especificidad de las técnicas que se utilizan. El propósito de esta investigación fue estandarizarla técnica de Reacción en Cadena de la Polimerasa (PCR) anidada para la detección de Mycoplasma hyopneumoniae, utilizando diferentes métodos de extracción del ADN, tanto comerciales como convencionales. Para determinar la sensibilidad de la técnica se realizaron diluciones seriadas al cultivo de Mycoplasma hyopneumoniae, cepa J, mantenida en medioFriis. También se evaluaron algunas modificaciones hechas a la mezcla de la reacción dePCR tradicional reportadas en la literatura y, una vez ajustada, la técnica fue aplicada enmuestras clínicas como hisopados nasales y lavados traqueobronquiales. Los mejores resultados fueron obtenidos cuando el ADN se extrajo de la bacteria por calor yse trató con proteinasa K para luego ser utilizado en una reacción de PCR - anidado que nocontenía estabilizadores de la Taq-polimerasa, como el glicerol o la seroalbúmina bovina.Bajo estas condiciones, se logró detectar M. hyopneumoniae hasta la dilución de 10-5 de uncultivo puro de la cepa J de referencia y también a partir de algunas muestras clínicas...

Phylogenetic analysis of recent isolates of classical swine fever virus from Colombia.

The ability to discriminate between different classical Swine fever virus (CSFV) isolates is a prerequisite for identifying the possible origin of an outbreak. To determine the relatedness between Colombian isolates from different geographical regions, genetic sequences of the glycoprotein E2 and the 5'UTR of CSFV were amplified by PCR, sequenced and compared with reference strains of different genetic grouping. The viruses originated from classical swine fever (CSF) outbreaks in Colombia during 1998-2002. All viruses characterized belonged to genogroup 1 and were members of the subgroup 1.1. The results indicate that the outbreaks from the year 2002 are caused by a strain related to the virus CSF/Santander, isolated in 1980, suggesting that the current CSF outbreaks are the consequence of a single strain that continues to circulate in the field. For the first time, an association between isolates from outbreaks in Colombia in the 1990s was established with a virus isolate from Brazil, indicating a possible origin of the virus causing the outbreak.