Intensive antibiotic treatment of sows with parenteral crystalline ceftiofur and tulathromycin alters the composition of the nasal microbiota of their offspring.

The nasal microbiota plays an important role in animal health and the use of antibiotics is a major factor that influences its composition. Here, we studied the consequences of an intensive antibiotic treatment, applied to sows and/or their offspring, on the piglets' nasal microbiota. Four pregnant sows were treated with crystalline ceftiofur and tulathromycin (CTsows) while two other sows received only crystalline ceftiofur (Csows). Sow treatments were performed at D-4 (four days pre-farrowing), D3, D10 and D17 for ceftiofur and D-3, D4 and D11 for tulathromycin. Half of the piglets born to CTsows were treated at D1 with ceftiofur. Nasal swabs were taken from piglets at 22-24 days of age and bacterial load and nasal microbiota composition were defined by 16 s rRNA gene qPCR and amplicon sequencing. Antibiotic treatment of sows reduced their nasal bacterial load, as well as in their offspring, indicating a reduced bacterial transmission from the dams. In addition, nasal microbiota composition of the piglets exhibited signs of dysbiosis, showing unusual taxa. The addition of tulathromycin to the ceftiofur treatment seemed to enhance the deleterious effect on the microbiota diversity by diminishing some bacteria commonly found in the piglets' nasal cavity, such as Glaesserella, Streptococcus, Prevotella, Staphylococcus and several members of the Ruminococcaceae and Lachnospiraceae families. On the other hand, the additional treatment of piglets with ceftiofur resulted in no further effect beyond the treatment of the sows. Altogether, these results suggest that intensive antibiotic treatments of sows, especially the double antibiotic treatment, disrupt the nasal microbiota of their offspring and highlight the importance of sow-to-piglet microbiota transmission.

Efficacy Studies against PCV-2 of a New Trivalent Vaccine including PCV-2a and PCV-2b Genotypes and Mycoplasma hyopneumoniae When Administered at 3 Weeks of Age.

This study aimed to evaluate the efficacy of a new trivalent vaccine containing inactivated Porcine Circovirus 1-2a and 1-2b chimeras and a Mycoplasma hyopneumoniae bacterin administered to pigs around 3 weeks of age. This trivalent vaccine has already been proved as efficacious in a split-dose regimen but has not been tested in a single-dose scenario. For this purpose, a total of four studies including two pre-clinical and two clinical studies were performed. Globally, a significant reduction in PCV-2 viraemia and faecal excretion was detected in vaccinated pigs compared to non-vaccinated animals, as well as lower histopathological lymphoid lesion plus PCV-2 immunohistochemistry scorings, and incidence of PCV-2-subclinical infection. Moreover, in field trial B, a significant increase in body weight and in average daily weight gain were detected in vaccinated animals compared to the non-vaccinated ones. Circulation of PCV-2b in field trial A and PCV-2a plus PCV-2d in field trial B was confirmed by virus sequencing. Hence, the efficacy of this new trivalent vaccine against a natural PCV-2a, PCV-2b or PCV-2d challenge was demonstrated in terms of reduction of histopathological lymphoid lesions and PCV-2 detection in tissues, serum and faeces, as well as improvement of production parameters.

Retrospective assessment of porcine circovirus 3 (PCV-3) in formalin-fixed, paraffin-embedded tissues from pigs affected by different clinical-pathological conditions.

BACKGROUND: Porcine circovirus 3 (PCV-3) is a recently discovered pathogen of swine that has been associated with several conditions. However, many questions remain unanswered regarding its infection, especially in terms of pathogenesis and disease impact. The aim of the present study was to retrospectively investigate the presence of PCV-3 genome by real time quantitative PCR (qPCR) and in situ hybridization (ISH) on selected formalin-fixed paraffin-embedded tissues of pigs affected by different clinical conditions and histological lesions. MATERIALS AND METHODS: Conditions investigated included porcine dermatitis and nephropathy syndrome (PDNS), periweaning failure-to-thrive syndrome (PFTS), congenital tremors type AII, reproductive disorders, and pigs affected by systemic periarteritis/arteritis, myocarditis, or encephalitis. Studied cases (n = 587) were investigated from a diagnostic database (n = 4162) that comprised samples collected within the period 1998-2021. From each condition/lesion, 10 to 12 cases were subsequently selected and tested by qPCR and ISH (72 cases total). RESULTS: A total of 587 cases fulfilled inclusion criteria of the different studied conditions and were distributed among the seven groups. For the further selected cases, PCV-3 genome was found by qPCR in 12/12 periarteritis, 5/10 reproductive disease, 5/10 PFTS, 3/10 myocarditis, 1/10 encephalitis and 1/10 congenital tremor cases. PCV-3 was not found in any of the PDNS cases assessed. In periarteritis cases, tissues more commonly affected were mesenteric arteries and kidney. Reproductive disease cases associated to PCV-3 genome consistently displayed myocarditis. The lesions and labelling distribution of PFTS cases with presence of PCV-3 genome were comparable to those of the periarteritis group. qPCR and ISH yielded similar results within each studied case and were statistically comparable. CONCLUSION: Our results suggest that periarteritis is the hallmark lesion of PCV-3-SD, and that mesenteric lymph node and kidney appeared to be the most reliable organs to confirm the presence of PCV-3 genome in cases with periarteritis.

Efficacy Studies of a Trivalent Vaccine Containing PCV-2a, PCV-2b Genotypes and Mycoplasma hyopneumoniae When Administered at 3 Days of Age and 3 Weeks Later against Porcine Circovirus 2 (PCV-2) Infection.

Four studies under preclinical and clinical conditions were performed to evaluate the efficacy of a new trivalent vaccine against Porcine circovirus 2 (PCV-2) infection. The product contained inactivated PCV-1/PCV-2a (cPCV-2a) and PCV-1/PCV-2b (cPCV-2b) chimeras, plus M. hyopneumoniae inactivated cell-free antigens, which was administered to piglets in a two-dose regime at 3 days of age and 3 weeks later. The overall results of preclinical and clinical studies show a significant reduction in PCV-2 viraemia and faecal excretion, and lower histopathological lymphoid lesions and PCV-2 immunohistochemistry scores in vaccinated pigs when compared to non-vaccinated ones. Furthermore, in field trial A, a statistically significant reduction in the incidence of PCV-2-subclinical infection, an increase in body weight from 16 weeks of age to slaughterhouse and an average daily weight gain over the whole period (from 3 days of age to slaughterhouse) was detected in the vaccinated group when compared to the non-vaccinated one. Circulation of PCV-2a in field trial A, and PCV-2b plus PCV-2d in field trial B was confirmed by virus sequencing. In conclusion, a double immunization with a cPCV-2a/cPCV-2b/M. hyopneumoniae vaccine was efficacious against PCV-2 infection by reducing the number of histopathological lymphoid lesions and PCV-2 detection in tissues, serum, and faeces, as well as reducing losses in productive parameters.

Exploratory Study of the Frequency of Detection and Tissue Distribution of Porcine Circovirus 3 (PCV-3) in Pig Fetuses at Different Gestational Ages.

Porcine circovirus 3 (PCV-3) has been associated with several pig diseases. Despite the pathogenicity of this virus has not been completely clarified, reproductive disorders are consistently associated with its infection. The aim of the present work was to analyze the presence of PCV-3 DNA in tissues from pig fetuses from different gestational timepoints. The fetuses were obtained either from farms with no reproductive problems (NRP, n = 249; all of them from the last third of gestation) or from a slaughterhouse (S, n = 51; 49 of the second-third of gestation and 2 from the third one). Tissues collected included brain, heart, lung, kidney, and/or spleen. Overall, the frequency of detection of PCV-3 was significantly higher in fetuses from the last third of the gestation (69/251, 27.5%) when compared to those from the second-third (5/49, 10.2%), although the viral loads were not significantly different. Moreover, the frequency of detection in NRP fetuses (69/249, 27.7%) was significantly higher than in S ones (5/51, 9.8%). Furthermore, PCV-3 DNA was detected in all tissue types analyzed. In conclusion, the present study demonstrates a higher frequency of PCV-3 DNA detection in fetuses from late periods of the gestation and highlights wide organ distributions of the virus in pig fetuses.

Genotyping of Porcine Circovirus 2 (PCV-2) in Vaccinated Pigs Suffering from PCV-2-Systemic Disease between 2009 and 2020 in Spain.

Vaccination against porcine circovirus 2 (PCV-2) is a common practice all over the world. Vaccines can prevent PCV-2-systemic disease (PCV-2-SD) outbreaks but not PCV-2 infection, which can be detectable in a percentage of vaccinated animals. Occasionally, PCV-2-SD is diagnosed in vaccinated farms. The objective of this study was to genotype the PCV-2 strains detected in vaccinated animals diagnosed with PCV-2-SD. Additionally, the evolution of the frequency of PCV-2 genotype detection at Spanish, European, and world levels was assessed. Fifty cases diagnosed as PCV-2-SD between 2009 and 2020 were included in this study. PCV-2 genotype was determined by sequencing the Cap gene region. Among them, only PCV-2b (23/50, 46%) and PCV-2d (27/50, 54%) genotypes were detected. Although the frequency of detection of these two genotypes was similar, their temporal distribution was different. Whereas most PCV-2b sequences (17/23, 74%) were detected between 2009 and 2012, PCV-2d sequences were obtained from 2013 to 2020. Indeed, a predominance of the PCV-2d genotype was observed from 2013 onwards, a trend also noticed at European and world levels. The results suggest that detection of particular genotypes in vaccinated animals probably reflects the general prevalence of the genotypes over time rather than genotype-specific vaccine-immunity escaping.

Exploratory field study on the effects of porcine circovirus 2 (PCV-2) sow vaccination at different physiological stages mimicking blanket vaccination.

BACKGROUND: The objective of the present study was to explore the benefits of Porcine circovirus 2 (PCV-2) blanket vaccination in a sow herd on productive parameters, PCV-2 infection and immune status in sows and their progeny. For this purpose, 288 sows were distributed among four balanced experimental groups. One group remained as negative control group and the other three received 1 mL of PCV-2 Ingelvac Circoflex® intramuscularly at different productive cycle moments: before mating, mid gestation (42-49 days post-insemination) or late gestation (86-93 days post-insemination); phosphate buffered saline (PBS) was used as negative control item. Reproductive parameters from sows during gestation and body weight of their progeny from birth to weaning were recorded. Additionally, blood was collected from sows at each vaccination time and piglets at 3 weeks of age. Moreover, up to 4 placental umbilical cords (PUC) per sow were taken at peri-partum. Sera from sows and piglets were analysed for PCV-2 antibody detection using an enzyme-linked immunosorbent assay (ELISA). Sera from sows and PUC were tested to quantify viraemia using a real time quantitative polymerase chain reaction (qPCR) assay. RESULTS: Globally, results indicated that vaccinated sows showed heavier piglets at birth and at weaning, less cross-fostered piglets, lower viral load at farrowing as well as in PUC, and higher antibody levels at farrowing, compared to non-vaccinated ones. When all groups were compared among them, sows vaccinated at mid or late gestation had heavier piglets at birth than non-vaccinated sows, and lower proportion of PCV-2 positive PUC. Also, cross-fostering was less frequently practiced in sows vaccinated at pre-mating or mid gestation compared to non-vaccinated ones. CONCLUSIONS: In conclusion, the present study points out that PCV-2 sow vaccination at different time points of their physiological status (mimicking blanket vaccination) offers benefits at production and serological and virological levels.

Porcine circovirus 2 (PCV2) population study in experimentally infected pigs developing PCV2-systemic disease or a subclinical infection.

Porcine circovirus 2 (PCV2) is a single stranded DNA virus with one of the highest mutation rates among DNA viruses. This ability allows it to generate a cloud of mutants constantly providing new opportunities to adapt and evade the immune system. This pig pathogen is associated to many diseases, globally called porcine circovirus diseases (PCVD) and has been a threat to pig industry since its discovery in the early 90's. Although 11 ORFs have been predicted from its genome, only two main proteins have been deeply characterized, i.e. Rep and Cap. The structural Cap protein possesses the majority of the epitopic determinants of this non-enveloped virus. The evolution of PCV2 is affected by both natural and vaccine-induced immune responses, which enhances the genetic variability, especially in the most immunogenic Cap region. Intra-host variability has been also demonstrated in infected animals where long-lasting infections can take place. However, the association between this intra-host variability and pathogenesis has never been studied for this virus. Here, the within-host PCV2 variability was monitored over time by next generation sequencing during an experimental infection, demonstrating the presence of large heterogeneity. Remarkably, the level of quasispecies diversity, affecting particularly the Cap coding region, was statistically different depending on viremia levels and clinical signs detected after infection. Moreover, we proved the existence of hyper mutant subjects harboring a remarkably higher number of genetic variants. Altogether, these results suggest an interaction between genetic diversity, host immune system and disease severity.

Detection and genotyping of Porcine circovirus 2 (PCV-2) and detection of Porcine circovirus 3 (PCV-3) in sera from fattening pigs of different European countries.

PCV-2 is considered one of the most economically important viral agents in swine worldwide. Recently, PCV-3 has been found in pigs affected by different disorders and in healthy animals. The objective of this epidemiological work was to describe the frequency of detection of PCV-2 and PCV-3 in pig farms of 9 European countries. Moreover, a second aim was to assess the most frequent PCV-2 genotypes found in the studied farms. Sera from 5 to 10 pigs per farm were collected from 2 to 11 farms per studied country. A total of 624 sera of fattening pigs (10-25 week old) from 64 farms from Spain (n = 11), Belgium (n = 10), France (n = 8), Germany (n = 8), Italy (n = 7), Denmark (n = 8), the Netherlands (n = 5), Ireland (n = 5) and Sweden (n = 2) were analysed by conventional PCR. In addition, one or two PCV-2-positive samples per farm were genotyped by sequencing the ORF2 gene. PCV-3 PCR-positive samples with relatively low Ct values were also sequenced and phylogenetically analysed. PCV-2 DNA was detected in pig sera from all European tested countries, but Sweden. A total of 132 out of 624 (21%) sera were positive for PCV-2 PCR, corresponding to 30 out of the 64 (47%) tested farms. PCV-3 DNA was detected in 52 out of 624 (8%) sera, corresponding also to 30 out of the 64 (47%) studied farms from all tested countries. A total of 48 PCV-2 PCR-positive samples were successfully sequenced and genotyped, being PCV-2d the most frequently genotype found (n = 28), followed by PCV-2b (n = 11) and PCV-2a (n = 9). These results pointed out PCV-2d as the most prevalent genotype currently in Europe. The PCV-3 phylogenetic analysis showed high identity (>98%) among sequences from all the analysed countries. The relatively low co-infection (3%), likely suggest an independent circulation patterns of PCV-2 and PCV-3.

Detection of Porcine Circovirus 3 in Wildlife Species in Spain.

Porcine circovirus 3 (PCV-3) is the third member of the family Circoviridae, genus Circovirus, able to infect swine. A high prevalence of viral DNA has been recorded in wild boars. Recently, PCV-3 DNA was identified in Italian wild ruminants. Based on these previous results, this study assessed the frequency of PCV-3 DNA detection in free-ranging ruminants and Lagomorpha species in Spain. In addition, the genetic characterization of the PCV-3 PCR-positive samples was performed. A total of 801 serum samples, including red deer (Cervus elaphus, [CE]; n = 108), roe deer (Capreolus capreolus, [CC]; n = 87), Pyrenean chamois (Rupicapra pyrenaica, [RP]; n = 133), Iberian ibex (Capra pyrenaica, [CP]; n = 92), mouflon (Ovis aries, [OA]; n = 91), fallow deer (Dama dama, [DD]; n = 104), European rabbit (Oryctolagus cuniculus, [OC]; n = 101), and European hare (Lepus europaeus, [LE]; n = 85) from Catalonia (northeast Spain) were tested by conventional polymerase chain reaction (PCR) and, when positive, sequenced. Overall, PCV-3 DNA was found in three out of 801 analyzed sera (0.37%) corresponding to one red deer (1/108, 0.9%), one mouflon (1/91, 1.1%), and one fallow deer (1/104, 0.96%). None of the samples collected from Lagomorpha species resulted PCR positive. The partial genome sequences detected in positive samples displayed high identity with some PCV-3 sequences detected in wild boars and domestic pigs (99.7% and 100%, respectively). In conclusion, the present study indicated that free-ranging ruminant and Lagomorpha species are not relevant in the epidemiology of PCV-3 in Spain.

Effect of sow and piglet porcine circovirus type 2 (PCV2) vaccination on piglet mortality, viraemia, antibody titre and production parameters.

The present study describes the effects of sow and/or piglet porcine circovirus type 2 (PCV2) vaccination on viraemia, antibody response and production parameters (average daily weight gain [ADWG] and mortality) of piglets from a PCV2 subclinically infected farm. Four hundred seventy-six piglets born from vaccinated (V) or non-vaccinated (NV) sows were further subdivided in a total of four groups: NV sows-NV pigs (NV-NV, n=134), NV sows-V pigs (NV-V, n=135);V sows-NV pigs (V-NV, n=104) and V sows-V pigs (V-V, n=103). A single vaccination of sows before mating was able to confer significantly higher antibody titres to their piglets at 4 weeks of age and a different PCV2 dynamics infection compared to piglets coming from NV sows. Piglet vaccination (independently of sow treatment) caused an earlier seroconversion and lower percentages of PCV2 infected pigs compared to the NV ones throughout their life. The double PCV2 vaccination strategy was able to reduce PCV2 infection but apparently caused some interference in piglet humoral response development. PCV2 vaccination was able to overcome this interference since the ADWG was improved in both groups of vaccinated piglets, independently of the sow treatment, being the highest ones obtained in the double vaccination group.

Retrospective study on swine Torque teno virus genogroups 1 and 2 infection from 1985 to 2005 in Spain.

A retrospective study to detect evidence of swine Torque teno virus (TTV) genogroups 1 and 2 infection in sera of pigs from the Spanish livestock between years 1985 and 2005 was carried out by means of PCR. Also, the molecular evolution of TTV genogroups 1 and 2 during the 20-year period studied using a 250-base sequence of the non-coding region of the viral genome was assessed. Both TTV genogroup genomes were found in pig sera from the first year of study. Taking into account the whole study period, 113 out of 162 animals (69.8%) were infected with one or the other genogroup, while 38 out of 162 pigs (23.5%) were co-infected with both genogroups. Moreover, TTV genogroup 2 (90 out of 162, 55.6%) was significantly more prevalent than genogroup 1 (54 out of 162, 33.3%). The non-coding region of swine TTV genome sequenced showed its adequacy as a molecular marker in swine TTV. This study represents the earliest evidence of TTV infection in pigs to date, 14 years before the initial description of this virus. Moreover, this is also the earliest evidence of TTV infection in any species.

Mujer de 66 años con dolor en el hombro / A woman with a painful shoulder

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