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Delayed-type hypersensitivity (DTH) is revealing cellular mediated immunity towards an antigen and can be adapted for vaccine compliance tests. This study aimed to confirm this potential for PCV2 vaccination under Italian field conditions. First farm: thirty three-week-old just-weaned piglets born from unvaccinated sows were either vaccinated (n=15) with CIRCOVAC IM 0.5mL or left unvaccinated (n=15). Five weeks later, the pigs were intradermally inoculated with 0.1-0.2mL of CIRCOVAC antigen solution in the lower abdomen area. The diameter of skin reaction (erythema) at the point of inoculation was visually inspected 24h post-inoculation. Second farm: 30 five-week-old piglets born from CIRCOVAC-routinely-vaccinated sows were either vaccinated with CIRCOVAC or left unvaccinated. The DTH reaction was analogously assessed, both before vaccination and 5 weeks post-vaccination. Piglets born from vaccinated sows were bled at weaning and all experimental animals at 5 weeks post-vaccination . Antibodies were titrated using an in-house ELISA or a seroneutralization technique. First farm: only 33% of vaccinated piglets showed high anti-PCV2 ELISA titres following vaccination. Vaccinated pigs exhibited consistent and significantly higher seroneutralizing titres than unvaccinated pigs indicating a proper vaccine take. The proportion of pigs showing erythema (100% vs. 33%) and the average diameter (25±15 mm vs. 4±7 mm) was significantly higher (p

Vaccination, allowing to contain infections and to prevent health problems is a crucial strategic management tool in today's pig industry. However, it also represents a cost item and therefore condition of vaccine application must be optimized. In the recent years needle free injection devices (NFID) have utilized various techniques for transcutaneous vaccine injections. Among the benefits to be highlighted there are: no broken needles in the meat, reduced inter animal transmission of diseases, respect of animal welfare. In this paper a scanographic (scanner photography) technique will be illustrated, for analyzing and quantifying transport / pattern of penetration and dispersion of a vaccine administered by NFID in piglets focussing on macroscopical visual injection outcome. Conventional piglets of 5-20 kg lbw were vaccinated on the left side of the neck with NFID Valery® (Giordano Poultry Plast, Caraglio Cuneo, Italy) with black china ink spiked CIRCOVAC® (MERIAL, Lyon, France), 1 cc ink/100 cc of vaccine), according to datasheet (0.5 ml/piglet) and immediately underwent euthanasia. Freezing of hanged piglets was carried out (48 h, -20°C) and subsequently frozen cross-sectional slices were obtained (thickness about 1 cm). Images were acquired by means of an ordinary flatbed scanner protected by a glass platen (hp, Palo Alto-CA, USA). Mechanism of liquid jet injectors relies on the principle of forcing fluids through a small orifice, generating a high pressured stream that penetrates into the skin with high velocity. There are numerous predictive “preskin” biophysical trials and predictive models, but there are rare models of evaluation of intramuscular penetration, and dispersion characteristics in vivo. Therefore the proposed method provides valuable and reliable information, is objective, repeatable and reproducible, not expensive, and allows the comparison of in vivo efficacy of various NFID.

Protection against viral infections is achieved through a large array of immune mechanisms. Priming pigs toward antigens is able to produce specific memory cells later responsible for a faster onset and a higher intensity of the immune response when animals are infected with the field viruses. The aim of the present study was to evaluate on a large scale and under Italian current field conditions the serological responses of pigs vaccinated or not vaccinated with a commercial inactivated Swine Influenza (SI) vaccine containing H1N1, H1N2 and H3N2 strains. A secondary objective was to assess if a common field off label use of the two vaccines, i.e. dilution of the dry pellet of a Pseudorabies (PR) MLV vaccine in the SI vaccine was not impairing the safety nor the immune response of the SI vaccine. Eleven farrow-to-finish farms in the North of Italy were studied during the year 2013: in each farm two groups of 12-14 pigs were vaccinated with GRIPOVAC®3 against SI concomitantly to PR vaccination (AKIPOR®6.3) scheme i.e. twice before turn-to fattening (70-90 days of age (DoA) and 3 weeks later) and at 180 DoA during fattening: one group received separated injections of the two vaccines and one group was vaccinated following dilution of the AKIPOR 6.3 dry pellet in GRIPOVAC 3. A control group of 12-14 pigs was also included in the study and were vaccinated only with AKIPOR 6.3 at the same dates. Blood samples were roughly collected at around 70, 125, 180 and 250 DoA (just before slaughter) for antibody titration against recent Italian SIV subtypes H1N1, H3N2, and H1N2 by inhibition of hemagglutination test (HI). A sample was considered positive for one specific subtype if the titre was ≥20; one farm was classified as positive against one given subtype if at least 2 sera showed an HI titre ≥20 against this subtype. Mitigation of the results was performed to account for cross-reactivity between strains. Statistical analyses were performed using nonparametric tests. No adverse event related to safety was seen in any of the farm for any of the vaccination schedules. No clinical signs evoking a flu passage was observed except in one farm in which a H3N2 flu passage was confirmed both virologically following nasal swabbing and serologically. However, a raise of the SIV antibodies titres in control pigs, induced a suspicion of a field infection in all herds: 2/11 herds for H1N1, 4/11 for H3N2, 4/11 for H1N2, and 1/11 for both H1N1 and H3N2, thus stressing a high incidence of swine influenza cases in fatteners. In this context, almost all pigs were negative towards SIV at 70 days of age before primo-immunization. One month after the second vaccination, a clear seroconversion was seen in both groups vaccinated against SI: the pigs vaccinated with GRIPOVAC 3 displayed indeed significantly higher HI titres as compared to the controls (p

Recurrent viremia in pigs naturally exposed to PCV2 infection and the shedding mainly through the fecal route are the two most important epidemiologic phenomena that allows both horizontal and vertical PCV2 transmission. One way to test the ability of a strategy to reduce PCV2 circulation is to measure its influence on viremia and on shedding through natural routes (nasal, fecal, vaginal). In this paper we present quantitative data on the effect of PCV2 vaccination in 2 trials run in the same conditions and aimed to assess PCV2 viremia and shedding in conventional gilts from a farm with high serological and viral prevalence of PCV2. Animals were purchased from a PCV2 infected farm in which no PCV2 vaccination was performed. Thirty pure Large White gilts were randomly divided in 3 groups and stabled in three different rooms as follows: vaccinated-infected (VI) group (n=6 trial 1 and n=6 trial 2), first vaccination at 16 weeks of age, the second one month later (CIRCOVAC®, MERIAL, France- 2 mL, IM), non-vaccinated infected (NVI) group (n=6 trial 1 and n=6 trial 2), and control (C) group (not-vaccinated not-infected) (n=3 trial 1 and n=3 trial 2). Hormonal estrus synchronization was followed by artificial insemination with a single (trial 1) or double (trial 2) semen dose (PCV2 negative by PCR) spiked with 0.2 ml of a suspension containing 103.9 TCID50/25 μl of PCV2 (VI and NVI groups), and one or two doses (trial 1 or 2, respectively) with no viral particles (C group). After ultrasonography at 29 days post-insemination (DPI), empty animals were euthanized at 30 DPI whilst pregnant ones between 52 and 56 DPI. Vaginal, nasal and rectal swabs, and blood samples were weekly collected from -2 DPI till the end of the experimental period and tested by real time-PCR for PCV2. During necropsy tissues from each fetus as well as placentas were collected and submitted to PCV2 real time-PCR (qPCR). Results of trials 1 and 2 were pooled since both the experimental conditions and the readouts were equivalent. In each of the VI and NVI groups respectively 7 and 6 out of the 12 gilts were pregnant at 29 DPI, while 4 out of 6 in the C group. VI group showed a significantly lower proportion of qPCR positive in vivo collected samples: 88/224 (VI) vs 174/306 (NVI) and 80/165 (C) (Chi Square =45.2.; P

Intestinal immune response plays an important defensive role for pathogens, particularly for those transmitted by the oro-faecal route or for foecal shedding modulation. This work examined three parts of intestine from twelve gilts experimentally infected with PCV2-spiked semen, six vaccinated (V group) and six unvaccinated (NV group) against PCV2, 29 and 53 days post infection (DPI). An immunohistochemical investigation for IgA-, IgG- and IgM-antibody bearing plasma cells (PCs) was run on intestinal samples coupled with a sandwich immunohistochemical method to reveal anti-PCV2 antibody-secreting PCs. Plasma cell density was compared in the two groups of animals at 29 and 53 DPI. The IgA, IgG and IgM PC density did not differ between groups but displayed an increase from the upper (villus) to the lower part of the crypts while a decreasing trend in PC density was identified from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling moment: the amounts of lamina propria PCV2-specific antibody-producing PCs remained constant, 10.55 ± 4.24 and 10.06 ± 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 ± 13.36 at 29 DPI to 18.76 ± 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered reference values in a population of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a "booster-like" response of the lamina propria in vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs containing specific anti-PCV2 antibodies at the level of intestinal mucosa.

PCV2 is involved in clinical and subclinical reproductive failure. In addition to clinical evidence, the clinical form is identified by microscopic lesions within foetal tissues (heart, liver or lymphoid tissues) and PCV2 antigen or PCV2 DNA within foetal tissues. The subclinical PCV2 in utero infection is only identified by the detection of PCV2 DNA in foetal tissues without the presence of microscopic lesions. The most used diagnostic algorithm in PCV2 diseases encompasses, after a screening by PCR, the in situ demonstration of PCV2 by in situ hybridization or immunohistochemistry (IHC). In this paper we present the results of IHC to PCV2 in cases of experimentally induced infections of fetuses. We conducted 3 trials that overall included 45 gilts of which: 9 (3 in each trial) were non-vaccinated and noninfected animals (controls; C); 18 (6 in one trial (vaccine A) and 12 in another trial (6 vaccine A and 6 vaccine B)) were gilts vaccinated against PCV2 and infected; 18 (6 for each trial) were non-vaccinated but infected gilts (NVI). Vaccine A (2 mL/dose, IM, administered twice) (group VAI) is a commercial inactivated PCV2 vaccine licensed for sows and piglets; Vaccine B (1 mL/dose, IM, administered twice) (group VBI) is a commercial vaccine based on an ORF2 capsid protein expressed in a baculovirus system, and licensed for use in piglets. Samples from fetuses and placentas were obtained at mid-gestation (55 days post-insemination) from conventional gilts experimentally infected with PCV2 spiked (103.9 TCID50/ 25 μl) semen. Tissues (heart, liver, spleen) from foetuses and the corresponding placenta were fixed in formalin, then underwent routine histology (haematoxylin-eosin stain H&E) and IHC according to a previous protocol. The same samples were also frozen for PCV2 genome assessment by real time PCR (RT-PCR). From the 3 trials were collected 233 fetuses (7 resorptions) and 230 placentas. From the 9 gilts of the group C were collected 51 fetuses and 51 placentas. From INV 107 placentas, 107 fetuses and 5 resorptions. From VAI group 53 placentas and 51 fetuses and 2 resorptions, while from VBI gilts 19 placentas and 19 fetuses. No gross lesions suggestive of PCV2 in fetuses and placentas were recorded. Histology allowed to disclose focal placental necrosis (1 case, group NVI) or necrotic placentitis (1 case, group VBI) both PCV2 positive by IHC. In fetal tissues IHC was positive in liver and heart of one fetus (group VBI) of which only the liver showed necrotic foci, and the liver in another fetus (group NVI) that not disclose changes by H&E stain. Presence of the viral genome is frequent, although in the absence of macro or microscopic lesions. The demonstration of PCV2 by IHC, even if sporadic, overlaps the histopathological identification of fetoplacental lesions. In the presented cases placental necrosis, placentitis and foetal hepatic necrosis were the lesions observed. It seems that 108 PCV2 genomic copies/ml is a good cut-off to predict fetoplacental tissues damage. The IHC is highly specific but not sensitive. The RT-PCR positivity of conceptuses can be interpreted as a risk factor for the PCV2 circulation in breeding sector, then the real time PCR assessment can represent a “risk assessment” and not only a diagnostic tool. Finally, the description of PCV2 induced placental lesion enrich the spectrum of PCVDs.

Galuppi, R., Avenoso, A.M., Leotti, G., Ostanello, F., Poglayen, G. and Tampieri, M.P., 2007. Diagnosis of sarcoptic mange in slaughtered swine. Veterinary Research Communications, 31(Suppl. 1), 233–236

L’influenza suina è una patologia endemica negli allevamenti intensivi ed è direttamente responsabile di gravi perdite economiche. Per la messa a punto di corrette strategie di diagnosi e di controllo aziendale dell’infezione, oltre alla corretta applicazione di misure di profilassi diretta e indiretta, sono necessarie delle attività di sorveglianza dei ceppi di SIV circolanti in grado di rilevare tempestivamente le loro modificazioni antigeniche e patogenetiche.

Post-weaning multisystemic wasting syndrome (PMWS) is causally associated with infection of pigs by Porcine Circovirus type 2 (PCV2). Lesions in lymphoid tissues of 52 PMWS-positive pigs sampled years 1998-2000 (epidemic) were compared with lesions from 127 PMWS-positive pigs sampled 2006-2008 (endemic). A variable degree of lymphoid depletion was a constant feature in tissues with microgranulomas formed by a scant number of centrofollicular epithelioid cells, or granulomas clustered in multiple undelimited nodules with little tendency to confluence, formed by epithelioid macrophages and a few multinucleate giant cells. Granulomatosis of lymphoid tissues is known in PMWS of pigs due to type IV hypersensitivity. Granulomatosis of lymphoid tissues was apparent in 1.07% during the 1998-2000 period and in 12.42% in the more recent period. Results suggest that frequency of lymphoid granulomatosis is influenced by the epidemiology of the disease.

Proliferative and necrotizing pneumonia (PNP) is a particular form of interstitial pneumonia in weaning and post-weaning pigs characterized by hypertrophy and hyperplasia of type II pneumocytes and presence of coagulative necrosis and granular debris within alveolar spaces. American and European studies suggest PRRSV and PCV2 as the probable causes of the disease. An immunohistochemical study was carried out on 24 PNP lungs to evaluate the role of PRRSV and PCV2 in PNP lesions in Italy. Results were as follows: co-infection (PCV2 and PRRSV) in 7 PNP lungs, PRRSV only in 9, PCV2 only in 4, and neither of the two antigens was found in the remaining 4. These findings support the major role of PRRSV as cause of PNP in Italy, even if co-infection is characterized by more severe histological aspects.

Background Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen. Results Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen. Conclusions PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.

Proliferative and necrotizing pneumonia (PNP) is a form of interstitial pneumonia that occurs in weaning and post-weaning pigs. PNP is characterized by hypertrophy and hyperplasia of type II pneumocytes and coagulative necrosis and granular debris within alveolar spaces. Canadian and European studies suggest that the porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are the main causes of the disease, but Aujezsky’s disease virus (ADV) and swine influenza virus (SIV) have also been considered as potential aetiological agents. An immunohistochemical study was carried out on the lungs of 28 Italian pigs with PNP in order to evaluate the role of PRRSV, PCV2 and ADV in PNP lesions. PRRSV infection was identified in the lungs of 11 pigs, PCV2 in the lungs of four pigs and coinfection with both viruses in the lungs of eight pigs. Neither virus was detected in the lungs of the remaining five pigs. ADV antigen was not detected in any sample. The principle aetiological agent of PNP in Italy therefore appears to be PRRSV. Coinfection with PRRSV and PCV2 is characterized by more severe microscopical changes in affected lungs.

The aims of this study were the evaluation of a quantitative method for the assessment of pneumonia lesions applied to heavy-weight slaughtered pigs, the identification of risk factors connected with the increase in the prevalence and severity of the lesions and the evaluation of a possible correlation between the presence of pneumonia lesions and the decrease in the carcass quality. The lungs of 10 041 pigs (109 slaughtered batches) coming from 91 farms located in Northern Italy were examined. Lung lesions were scored using the method developed by Madec and Kobisch (Journ. Rech. Porc. Fr., 14, 1982, 405). Before the scoring, anamnestic information regarding the farm of origin of each batch were collected. For 41 batches (3603 pigs), information about carcass quality were also collected. Pneumonia lesions were found in 59.6% of the lungs (range 3-91%), and the average batch score was 2.11 (range 0.03-7.15). We identified as farm risk factors those related to an increase in the severity of the lung lesions, the presence of breeders within the herd, the starting of a growing cycle during the winter season and the lack of vaccination programmes to Mycoplasma hyopneumoniae. Moreover, we also found a statistically significant association between the increase in the mean lung score of the batch and the decrease of the carcass quality.

Samples of superficial inguinal and bronchial lymph nodes, ileum, tonsil and lung were taken from three to five pigs on each of 61 farms with a clinical history of postweaning multisystemic wasting syndrome (PMWS). The samples were examined histologically and by immunohistochemistry for porcine circovirus type 2 (PCV-2). PMWS was diagnosed in two stages: first, an evaluation of the haematoxylin and eosin-stained sections that identified the cases in which the characteristic PCV-2 cytoplasmic inclusion bodies were apparent, and secondly, a conclusive step in which immunohistochemistry was applied to confirm PMWS in the cases in which there were positive immunohistochemical results that coincided with lesions indicative of PMWS in at least one of the lymphoid and/or lung tissues. The location of PCV-2 in specific lesions (cell depletion in lymphoid organs and interstitial pneumonia) confirmed PMWS in 45 of the 61 farms, 31 of which were also infected with porcine reproductive and respiratory syndrome virus. The lymphoid tissues were more reliable than the lungs for the diagnosis of PMWS, both in individual pigs and in groups of pigs, and farm diagnoses based on a group of pigs were more reliable than diagnoses based on single pigs.