Clinical cases and sample collection
All animal studies were approved by the ethical committee of Uppsala 2017-02-10 (Dnr 5.8.10-00431/2017) and the owners of the herds gave informed consent prior to the start of the study.
During the period from June 2017 to June 2018, 15 piglets were obtained from four Swedish farms with ongoing outbreaks of congenital tremor. Of these piglets, 13 piglets were aged 1-2 days and two piglets were aged 5 days. All piglets were in good general condition with moderate to severe signs of congenital tremor. Three of the four farms were located in the central part of Sweden, with the remaining farm being located in the south of Sweden. The farms are marked on the map in Figure 3. During the same period, 13 piglets aged 1-2 days old suffering from splay leg were obtained from four different farms located in the central part of Sweden. Most of these piglets had decreased demeanour. Piglets from the same farms and sows at their next farrowing were included as healthy controls; eight 1-day-old piglets in good condition were obtained. In cases where the original sow was unavailable, a piglet born to a sow from the same farrowing group was sampled. None of the sampled farms had any documented contact with each other and the outbreaks were separated in time, or had simultaneous outbreaks of congenital tremor and splay leg.
The piglets were transported to the pathology section at the Swedish University of Agricultural Sciences in Uppsala, Sweden. The piglets were sedated with an intramuscular injection of tiletamine and zolazepam (ZoletilÒ, Virbac, Carros, France) and a blood sample was obtained from the jugular vein. From the five piglets originating from farm E urine and saliva were also collected during sedation using commercial ESwabs (Copan Italia Via Perotti, Italy). All piglets were euthanized by an intraperitoneal injection of pentobarbital (Allfatal vet. Apotek Produktion & Laboratorier AB, Malmö, Sweden) with necropsy being performed within minutes.
Samples from the brain, spinal cord, saliva, urine, hearth, lung, quadriceps muscle, kidney, liver, spleen, ventricle, duodenum, jejunum, ileum, caecum, and colon were sampled and immediately put on dry ice. The tissue samples were then stored at -80 °C. Corresponding tissue samples were fixed in 10% formaldehyde for future studies.
A retrospective study was carried out on material from piglets sampled in 2004 (n=11) and 2011/2012 (n=6). The samples from 2004 consisted of serum originating from eleven piglets affected by congenital tremor. The samples were collected from one litter of piglets originating from a farm located in the central part of Sweden. Necropsies were performed on all 11 piglets from 2004 with no records of gross lesions. These 11 piglets all tested negative for PCV-2.
The samples from 2012 consisted of brain tissue collected at the end of 2011 and beginning of 2012 from piglets on one farm during an ongoing outbreak of congenital tremor. Three newborn piglets with congenital tremor were euthanised and sampled at the farm. When the outbreak had ceased, three healthy newborn control animals from the same farm were euthanised similarly. The brains were collected and subjected to histopathological investigation, but no complete necropsies of the bodies were performed. Brain tissue from all six piglets tested PCR-positive with respect to porcine astrovirus (29). In the piglets with clinical signs of congenital tremor, mild to moderate vacuolar changes of the white matter were observed in the cerebrum, brain stem, and cerebellum (29).
Both the serum and brain samples were stored at -80 °C for future investigations.
Sample preparation, RNA isolation, qRT-PCR (quantitative reverse transcription-PCR) and sequence analysis
The brain samples were cryolyzed using a Precellys tissue homogenizer (Bertin Corp. Rockville, MD, USA), RNA was extracted from all samples through a trizol-phenol-chloroform protocol, and cleaned using the GeneJET RNA kit (ThermoFisher Scientific, Waltham, MA, USA). In addition, RNA from the sera was extracted using the same protocol but without homogenization. The APPV genome was detected using an APPV-specific RT-qPCR protocol based on the QuantiTect Probe RT-PCR kit (Qiagen, Hilden, Germany) as described by (6) with a primer-pair targeting the NS3 encoding region of the APPV genome. The assay was run in duplicate under standard conditions on a Bio-Rad CFX96ä Real-time system in a C1000 Touchä thermal cycler (Bio‐Rad, Hercules, CA, USA) with a plasmid containing the NS3 encoding region of the APPV genome as a positive control. One primer and one probe, denoted “Swe” in Table 2, were slightly modified as compared to the protocol by (6) in accordance with (13), to better match the only described sequence of Porcine pestivirus in Sweden. All the samples were also analysed by an APPV‐specific RT‐qPCR targeting the non‐structural protein NS5B in accordance with (12). The RT‐qPCR was run in duplicate under standard conditions using the qScript XLT One‐Step RT‐qPCR ToughMix (Quanta Biosciences, Gaithersburg, USA) on the above-mentioned Bio-Rad CFX96ä Real-time system in a C1000 Touchä thermal cycler (Bio‐Rad, Hercules, CA, USA).
From each APPV-positive farm, the sample with the lowest Ct-value was selected for sequencing. A part of the NS3-gene was PCR-amplified using the primers APPV_5087-fw and APPV_5703_Swe-rev with the Invitrogenä SuperScriptä IV One-Step RT-PCR-System, using the ezDNaseä Enzyme protocol (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. The product was run on a 2% agarose gel stained with GelRed, visualized by UV-transillumination (GelDoc, Bio-Rad Laboratories, Inc., Richmond CA, US), purified using the Thermo Fisher Scientific GeneJET Gel Extraction Kit) and Sanger-sequenced at Macrogen Inc. Europe (Amsterdam, NL).
To get a clear and readily understood format of the tree, the phylogenetic analysis was performed on 26 full and partial genome sequences covering the APPV NS3 sequences extracted from the GenBank. The tree was constructed using the MAFFT alignment tool and the PHYLIP Neighbor-Joining method with a bootstrap value of 1000 using the UGENE software (38). A bayesian tree were also made using the MR Bayes tool within the UGENE software (38). To confirm the tree’s constitution and clustering, additional Neighbor-Joining trees were constructed, as well as Bayesian trees made with the MR Bayes tool within the UGENE software (38). The bayesian trees and the Neighbor-Joining trees were consistent with each other.