Swine polioencephalomyelitis in Brazil: identification of Teschovirus A, Sapelovirus A, and Enterovirus G in a farm from Southern Brazil

Abstract

Porcine encephalomyelitis can be associated with many etiologies, including viral agents, such as Porcine teschovirus (PTV), Porcine sapelovirus (PSV), and Porcine astrovirus (PoAstV). In this study, we investigated the presence of these viruses in a neurological disease outbreak in a swine farm in Southern Brazil. The piglet production farm unity had 1200 weaning piglets, and 40 piglets with neurological signs such as motor incoordination, paresis, and paralysis of hind limbs, with an evolution time of approximately 4 days. Among these, 10 piglets were submitted to postmortem examination. Gross lesions were restricted to a mild enlargement of the nerve roots and ganglia of spinal cord segments. The microscopic lesions were characterized by nonsuppurative encephalomyelitis and ganglioneuritis with evident neuronal degeneration and necrosis. Samples of the central nervous system (CNS), cerebrospinal fluid, and feces were collected and submitted to molecular analysis. PTV was identified in all samples of the CNS, while eight of the piglets were also positive for PSV, and seven were positive for Porcine enterovirus (EV-G). PoAstV was identified in a pool of feces of healthy animals used as controls. This study demonstrates the occurrence of encephalomyelitis associated with PTV on a swine farm in Southern Brazil, as well as the presence of other viruses such as PSV, EV-G, and PoAstV in the swineherd. Sequences of the fragments that were previously amplified by PCR showed a high similarity to PTV 6. Herein, we describe the first case report of severe swine polioencephalomyelitis associated with PTV in South America.

Viral encephalomyelitis in pigs is characterized by nonsuppurative polioencephalomyelitis, and its etiology can only be distinguishable via agent identification [1]. Many viruses can be implied as to the causative agent of these lesions, such as Teschovirus A (Porcine teschovirus/PTV), Sapelovirus A (Porcine sapelovirus/PSV), and Mammastrovirus 3 (Porcine astrovirus/PoAstV) [1–4]. The Picornaviridae family, which includes PTV, PSV, and also Enterovirus G (Porcine enterovirus/EV-G), comprises highly variable and heterogeneous enteric viruses, commonly found in fecal samples from domestic pigs and wild boars [5]. Previously, these porcine enteric picornaviruses were classified into the enterovirus genus; however, the molecular analysis demonstrated different features among these viruses, and reclassification was made [6].

Few descriptions regarding the prevalence of PTV, PSV, and EV-G are available in South America, and they include few Brazilian pig herds and watersheds near high pig density [7–9]. However, nervous clinical signs have not been described in these studies. Therefore, the aim of the present study was to describe the clinical, gross, histopathological, and molecular findings of an outbreak of polioencephalomyelitis with the identification of PTV, PSV, and EV-G in a swine farm in Southern Brazil. Additionally, we provide data confirming the presence of PoAstV circulating in healthy pigs in the same region.

Clinical and epidemiological data were obtained from the farm veterinarian in four technical visits. Piglets with severe nervous signs were euthanized, and tissue samples were collected and fixed in 10% buffered formalin. The tissues were routinely processed for histology and stained with hematoxylin and eosin (HE). Samples of the brain, spinal cord, cerebrospinal fluid (CSF), and feces from 10 sick pigs were collected and refrigerated for molecular assays and viral isolation in cell culture. All samples from the 10 piglets were individually tested and submitted to viral isolation. Feces of five weaning piglets from the same shed, without neurological signs, were also collected to serve as healthy controls and were stored in a pool.

The outbreak occurred in a piglet production farm with 2500 sows, located in Southern Brazil (29° 32′ 52″ S; 51° 25′ 32″ W). In a lot containing 1200 weaning piglets, with ages ranging from 40 to 65 days, 40 piglets were affected and 10 of these were selected for euthanasia. These piglets were distributed into three different sheds and several different stalls. Piglets with nervous signs were selected from different stalls. Clinical nervous signs were characterized by motor incoordination, paresis, and paralysis of hind limbs, with a duration of approximately 4 days. At postmortem examination, gross lesions were observed solely in three piglets and were characterized by a mild to moderate enlargement of the nerve roots and ganglia of the spinal cord, frequently in the cervical and sacral regions (Fig. 1a). No significant gross lesions were observed in the other organs. Histologically, all pigs had similar lesions, frequently detected in the spinal cord, brainstem, and spinal ganglia. The ganglia and spinal roots microscopically showed severe interstitial inflammatory infiltrate composed of lymphocytes, plasma cells, and macrophages (Fig. 1a Inset). In the brainstem, alterations were more prominent in the gray matter; however, an extension to the white matter was noticed, while the spinal cord lesions were restricted to the gray matter, and more severe in the ventral horns. These lesions were characterized as inflammatory infiltrate (Fig. 1b), mainly perivascular and meningeal, composed of lymphocytes and a small number of plasma cells and macrophages. Multifocal gliosis added to prominent neuron lesions characterized as chromatolysis, necrosis, satellitosis, and neuronophagia was observed. Lesions described in natural infections caused by PTV in piglets are characterized as nonsuppurative polioencephalomyelitis and ganglioneuritis [10, 11], which is in accordance with the findings of the present study.

Fig. 1.

Gross and histopathologic features observed in pigs with neurological disease. a Moderate enlargement of the nerve roots and ganglia. Inset: Severe inflammatory infiltrate composed of lymphocytes, plasma cells, and macrophages, around neuronal bodies of the cervical spinal ganglion. H&E, bar = 90 µm. b Gray matter of the spinal cord with multifocal mononuclear perivascular cuffs (arrow head) and gliosis (arrow). H&E, bar = 70 µm

The refrigerated samples were tested for PTV, PSV, EV-G, and PoAstV. RNA was extracted and PTV was detected using a nested reverse transcription-polymerase chain reaction (RT-PCR) with primers described by Krumbholz et al. [12] and modified by Donin et al. [13]. Primers used in the RT-PCR reactions targeted RNA-dependent RNA polymerase (RdRP) region of the PoAstV genome, and 5′UTR regions of PSV and EV-G. Primer pairs aimed at the 5′-NTR region, with a final amplification product of 158 base pairs (bp). Also, samples underwent nested RT-PCR for PSV and EV-G using primers described by Krumbholz et al. [12]. The expected product size for the PSV-nested RT-PCR amplified a final fragment of approximately 170 bp, and for the EV-G-nested RT-PCR, the expected product size was 270 bp. PoAstV testing occurred through RT-PCR using the forward primer described by Chu et al. [14] and the reverse primer as follows: 5′-GTGTCATAGTCCCTCCACA-3′, designed based on an alignment with several different Mammastrovirus sequences. The RT-PCR aimed to amplify a product of approximately 284 bp.

A larger fragment of the viral structural protein 1 (VP1) of the PTV coding region was amplified using RT-PCR with the primers described by La Rosa et al. [10] and Cano-Gómez et al. [5] for PTV sequencing [15] and classification. The RT-PCR product was sequenced, and a contig was generated using the BioEdit software. The contig was aligned with sequences from GenBank database (accession numbers: AF296088, AF296094, AM261024 to AM261027, GQ247878, GQ247879, GQ502318 to GQ502349, KX527849, JX069832, JX069833, JX069834, KY305430) with Clustal Omega software in the Ugene Package (Unipro UGENE vl. 27.0: A unified bioinformatics toolkit). Low-quality sequencing results were excluded from phylogenetic analysis. This data set used in the phylogenetic tree, inferred by the maximum likelihood method, was based on the general time reversible (GTR) model [16] in the MEGA X Software [17].

Viral isolation was attempted in cell cultures of baby hamster kidney (BHK-21), epithelial cells of human lung carcinoma (A549), African green monkey kidney cells (Vero), and porcine kidney cells (PK-15). All cells were maintained with Eagle’s Minimum Essential Medium (E-MEM) and supplemented with 10% of fetal bovine serum ((FBS) Gibco) and 1% of Penicillin–Streptomycin (10,000 IU/ml–10 mg/ml) antibiotic solution.

First, samples were macerated, diluted in E-MEM with 5% of antibiotics previously described, and incubated at 4 °C for 4 h. Next, samples were filtered and a volume of 250 µL was inoculated over on the cell monolayer in 6-well plates. Inoculated plates were maintained in incubators incubated at 37 °C for 2 h, being agitated every 15-min intervals. After this incubation period, the inoculum was removed and 2 mL of E-MEM with 1% antibiotics was added per well. Plates were maintained in the incubator at 37 °C with 5% CO₂ and observed daily for cytopathic effects (CPE) for 5 days. Supernatants were harvested and frozen for new passages and RT-PCR tests when CPE was observed that affected more than 50% of the cell monolayer.

Viral isolation from CNS samples belonging to pigs #2 and #3 was successful. Sequencing and alignment of the VP1 coding region fragments from these cases revealed a higher identity with PTV 6 and a larger distance of PTV5, PTV3, PTV10, and PTV11 (Fig. 2). Serotypes 1, 3, 6, 11, and 13 are linked to swine cases with nervous signs of natural occurrence [1, 4, 18, 19], while experimentally, serotypes 2 and 11 [20] can induce neurological disease. Different PTV serotypes have the potential to cause different clinical manifestations, such as reproductive disorders, pneumonia, diarrhea, and myocarditis [1].

Fig. 2.

Phylogenetic tree of the PTV viral structural protein 1 (VP1) region inferred by the maximum likelihood method, using 43 sequences of the GenBank database. The positive sample described in the present study is identified with a circle

The identification of PTV, PSV, EV-G, and PoAstV in different samples is demonstrated in Table 1. All animals with CNS lesions were positive for PTV. Polioencephalomyelitis associated with PTV was first described in Czechoslovakia and led to severe economic losses due to high morbidity and mortality in different age groups [1]. Previous molecular studies have suggested an endemic circulation of picornavirus, including PTV, in Brazilian swine farms [7, 8], watersheds [9], and wild boars, which can constitute a potential risk when considering cross-transmission [13]. Clinically, piglets in the outbreak described showed nervous clinical signs similar to those described previously, characterized mainly by hind limb paralysis [18, 20, 21]. Piglets were typically affected at age of 40–65 days, which was in accordance with what has already been described regarding nervous signs in PTV infection [18, 21].

Table 1.

Identification of Porcine teschovirus (PTV), Porcine enterovirus (EV-G), Porcine sapelovirus (PSV), and Porcine Astrovirus (PoAstV) in the central nervous system, cerebrospinal fluid, and feces of pigs, collected during an outbreak of neurological disease in Southern Brazil

Pigs	Samples	RT-PCR
		PTV	EV	PSV	PoAstV
#1	CNS	+	+	+	ND
	CSF	ND	ND	ND	ND
#2	CNS	+	+	+	ND
	CSF	ND	ND	ND	ND
#3	CNS	+	ND	ND	ND
	CSF	ND	ND	ND	ND
#4	CNS	+	ND	ND	ND
	CSF	+	ND	ND	ND
#5	CNS	+	ND	+	ND
	CSF	+	ND	ND	ND
	Feces	+	+	+	ND
#6	CNS	+	ND	ND	ND
	CSF	+	ND	ND	ND
	Feces	+	+	+	ND
#7	CNS	+	ND	ND	ND
	CSF	+	ND	+	ND
	Feces	+	ND	+	ND
#8	CNS	+	ND	ND	ND
	CSF	ND	ND	ND	ND
	Feces	+	+	+	ND
#9	CNS	+	ND	+	ND
	CSF	ND	ND	ND	ND
	Feces	+	+	+	ND
#10	CNS	+	ND	ND	ND
	CSF	+	ND	ND	ND
	Feces	+	+	+	ND

CNS, central nervous system; CSF, cerebrospinal fluid; ND, not determined

The examined pigs from this outbreak were also positive for PSV (8/10) and EV-G (7/10). However, it is not possible to ensure the role of these viruses in the CNS lesions of these pigs, since their presence was not constant in all the samples from affected pigs. PSV has also been reported as the causative agent of severe polioencephalomyelitis in finishing pigs in the USA [2]. EV-G was detected in fecal samples of healthy herds in Asia and Europe and isolated from pigs with diarrhea; however, no association was identified between EV-G detection and the disease [22].

Astroviruses have been identified in a variety of mammals and birds, and their infections are usually asymptomatic. These viruses are implicated in cases of encephalomyelitis in humans, pigs, sheep, and cattle [23]. In our study, PoAstV was identified solely in the feces of healthy pigs. No pig with neurological signs was positive for astrovirus, which led to the conclusion that, in this case, the astrovirus was not involved in the disease. Swine alphaherpesvirus 1 and atypical porcine pestivirus, both viral diseases associated with neurological signs, have been ruled out. There have been no reports of Aujeszky’s disease in the studied region since 2004 [24], and atypical porcine pestivirus is more frequently associated with congenital tremors in piglets [25].

The presence of these viruses in swine herds does not affect only the herd itself but also causes contamination of the environment and watersheds since transmission usually occurs via the fecal–oral route and primarily infects and replicates in the gastrointestinal tract of the host [9, 26]. The dissemination of enteric viruses to the environment is facilitated by the mismanagement of animal manure due to bad practices when handling animal wastes. Although enteric virus infections are usually associated with diarrhea and self-limiting gastroenteritis in humans, in immunocompromised individuals, they can cause respiratory infections, hepatitis, and diseases that have high mortality rates, such as aseptic meningitis, encephalitis, and paralysis [26, 27]. Previous to this study, there was no description of severe polioencephalomyelitis caused by PTV in Brazil. This new data will allow more investigations regarding the frequency and prevalence of this virus, as well as other important enteroviruses in the Brazilian territory, and its impact on national swine production. More research is necessary to indicate the prevalence of PTV and other enteric viruses in watersheds near pig farms and its association with public health.

Author contribution

Márcia Elisa Hammerschmitt drafted the work and made substantial contributions to the interpretation of data. Paula Rodrigues de Almeida drafted the work and revised it critically for important intellectual content, made substantial contributions to the interpretation of data, and approved the version to be published. Bianca Santana de Cecco, Marina Paula Lorenzett, Claiton Ismael Schwertz, Raquel Aparecida Sales da Cruz, Rafaela Albuquerque Caprioli, Daniela Teresa Schuh, Meriane Demoliner, Ana Karolina Antunes Eisen, Fernando Rosado Spilki, Saulo Petinatti Pavarini, and David Driemeier made substantial contributions to the interpretation of data, revised it critically for important intellectual content, and approved the version to be published.

Funding

Pró-Reitoria de Pesquisa (Propesq/UFRGS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) supported this study.

Declarations

Ethics approval

All cases described herein occurred spontaneously, with no experimentation, inoculation, or treatment of live animals. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of interest

The authors declare no competing interests.