Application of the diagnostic protocol for PCV2 in experimental reproductive pathology of gilts

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.