First virological and pathological study of Göttingen Minipigs with Dippity Pig Syndrome (DPS)

Abstract

Dippity Pig Syndrome (DPS) is a well-known but rare complex of clinical signs affecting minipigs, which has not been thoroughly investigated yet. Clinically affected animals show acute appearance of red, exudating lesions across the spine. The lesions are painful, evidenced by arching of the back (dipping), and the onset of clinical signs is generally sudden. In order to understand the pathogenesis, histological and virological investigations were performed in affected and unaffected Göttingen Minipigs (GöMPs). The following DNA viruses were screened for using PCR-based methods: Porcine cytomegalovirus (PCMV), which is a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3), porcine circoviruses (PCV1, PCV2, PCV3, PCV4), porcine parvovirus 1 (PPV1), and Torque Teno sus viruses (TTSuV1, TTSuV2). Screening was also performed for integrated porcine endogenous retroviruses (PERV-A, PERV-B, PERV-C) and recombinant PERV-A/C and their expression as well as for the RNA viruses hepatitis E virus (HEV) and SARS-CoV-2. Eight clinically affected and one unaffected GöMPs were analyzed. Additional unaffected minipigs had been analyzed in the past. The analyzed GöMPs contained PERV-A and PERV-B integrated in the genome, which are present in all pigs and PERV-C, which is present in most, but not all pigs. In one affected GöMPs recombinant PERV-A/C was detected in blood. In this animal a very high expression of PERV mRNA was observed. PCMV/PRV was found in three affected animals, PCV1 was found in three animals with DPS and in the unaffected minipig, and PCV3 was detected in two animals with DPS and in the unaffected minipig. Most importantly, in one animal only PLHV-3 was detected. It was found in the affected and unaffected skin, and in other organs. Unfortunately, PLHV-3 could not be studied in all other affected minipigs. None of the other viruses were detected and using electron microscopy, no virus particles were found in the affected skin. No porcine virus RNA with exception of PERV and astrovirus RNA were detected in the affected skin by next generation sequencing. This data identified some virus infections in GöMPs with DPS and assign a special role to PLHV-3. Since PCMV/PRV, PCV1, PCV3 and PLHV-3 were also found in unaffected animals, a multifactorial cause of DPS is suggested. However, elimination of the viruses from GöMPs may prevent DPS.

Fig 1. Lower back of GöMPs with acute dippity pig syndrome.

Pigs are suffering from red exudative skin alterations exemplary displayed in minipig # 8 (1, 2) and another minipig (3). Samples from different skin areas were included in the virological analyses: Affected skin (A), intermediate skin (B) and unaffected skin (C). Blue squares indicate excisional biopsy, green circles indicate punch biopsy.

Fig 2. Histological analyses of normal, affected and neighboring unaffected skin.

The pictures in the upper row had a magnification of 4x objective, that in the lower row of 10x objective.

Fig 3. PCR results of the screening of six Göttingen Minipigs.

Porcine viruses (PCMV, PCV1, PCV2, PCV3) were detected by real-time PCR and expressed as ct value (A-C) and conventional PCR expressed as detected/nondetected (D-F) in six minipigs suffering from dippity pig syndrome: pig # 1 (A), pig # 2 (B), pig # 3 (C) and pig # 6 (D), pig # 7 (E), pig # 8 (F), whereas pig # 4 (C) did not show any clinical signs.

Fig 4. Western blot analysis of serum and plasma of pigs with and without DPS for antibodies against PCMV/PRV.

Serum and plasma were tested at a dilution of 1:150 against the recombinant R2 fragment of the gB protein of PCMV/PRV, P, positive control serum; 1, serum # 5; 2, plasma # 5; 3, plasma # 3; 4, serum # 1; 5, serum # 4 (without clinical signs), N, negative control serum. Exposure time 1 sec.

Fig 5. Gene expression of PERV and PCMV in different organs of pigs with DPS.

Gene expression is displayed for two minipigs: minipig # 1 (A and B) and minipig # 2 (C). Skin A, B, C refer to the skin regions indicated in Fig 1. PHA-L, lymphocyte-specific phytohemagglutinin. The liver lobes are indicated as: LLL, left lateral lobe; LML, left medium lobe; RML, right medium lobe; RLL, right lateral lobe; CAL, caudate lobe. PERVpol indicated primer pairs used to detect the highly conserved polymerase gene of PERV. D, Increase of PERV expression after mitogen stimulation. PBMCs from pig # 2 and pig # 3 were incubated with the mitogen lymphocyte-specific phytohemagglutinin (PHA-L). Gene expression of PERVpol was referred to the expression in PK-15 cells which was set 100%.