Clinical signs

CT is present at birth and presents itself with tremor and, occasionally, ataxia. The tremor ranges from a slight shiver of the head to severe tremble of the entire piglet and may be complicated by ataxia or sometimes splay leg. In severe cases, the twitches can make the piglets jump off the floor (Kinsley, Reference Kinsley1922; Done et al., Reference Done, Woolley, Upcott and Hebert1986). There are no reports of fever or impaired demeanour associated with CT type A-II (Done, Reference Done1968; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Arruda et al., Reference Arruda, Arruda, Schwartz, Vannucci, Resende, Rovira, Sundberg, Nietfeld and Hause2017; Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017).

CT is an action tremor characterized by an increased tremor when the muscles are activated, e.g. the piglet is moving or stressed, and an absence of neurological signs when the muscles are relaxed, e.g. when the piglet is asleep or at rest (Done et al., Reference Done, Woolley, Upcott and Hebert1986). At present, there is no known medical treatment of CT, and whether a piglet will survive or not is dependent on its ability to grip the teat and suck as well as on its ability to avoid being crushed by the sow (Done, Reference Done1968, Reference Done1976). Most pigs recover spontaneously within 3 months of birth; however, some pigs might become chronic shakers (Done, Reference Done1976; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016). Interestingly, in pigs where the shaking is no longer evident in the unstressed animal, external stimuli such as a sudden noise or handling might trigger the tremor; thus, recovered pigs can sometimes start shaking when exposed to stress, e.g. at the abattoir (Done, Reference Done1976; Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017).

A variety of infectious agents, intoxications, and conditions may induce neurological signs similar to those of CT. However, CT may be differentiated from these conditions, as it is an action-tremor present at birth and is absent or reduced at sleep or rest. Hypoglycemia and hypothermia of neonatal pigs are the most likely differential diagnosis as they will induce shivering within a short time after birth, although in these cases the tremor will commonly be present at sleep or at rest as well (Curtis, Reference Curtis1974). Other conditions such as PCV-2 systemic disease, Aujeszky's disease, porcine reproductive and respiratory syndrome, otitis media, aflatoxicosis, classical and African swine fever, and salt poisoning, may present with a large number of neurological signs, including tremor and ataxia, although the neurologic signs are not congenital and are usually combined with severe multisystemic disease (Baskerville, Reference Baskerville1972; Liess, Reference Liess1987; Done and Paton, Reference Done and Paton1995; Segales and Domingo, Reference Segales and Domingo2002; Schulz et al., Reference Schulz, Staubach and Blome2017).

Epidemiology

The prevalence of CT type A-II is not well studied, but an estimate from China indicates that about 1–2% of all piglets are affected (Yuan et al., Reference Yuan, Han, Li, Huang, Yang, Ding, Zhang, Zhu, Zhang, Liao, Zhao and Chen2017). Typically, CT occurs on farms as outbreaks without any prior indication or clinical signs of infection among the sows (Done, Reference Done1976; Bolske et al., Reference Bolske, Kronevi and Lindgren1978; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016). The outbreak usually lasts for 2–3 months with a morbidity of 50–100%, being highest in litters born to first-parity sows. The mortality rates are generally low, but may occasionally approach 15–20% (Bolske et al., Reference Bolske, Kronevi and Lindgren1978; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016; Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017). In some farms, however, losses have been reported to be as high as 50% during outbreaks (Bolske et al., Reference Bolske, Kronevi and Lindgren1978; Done et al., Reference Done, Woolley, Upcott and Hebert1986; Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017).

There are a few within-farm epidemiological studies of outbreaks of CT associated with APPV.

When CT outbreaks caused by APPV occur on a farm, piglets born to primiparous sows are more commonly affected, whereas piglets born to multiparous sows usually remain unaffected (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016). The within-litter prevalence of APPV PCR-positive piglets, during an outbreak of CT, varies from <10 to 100%, which is evenly distributed between sexes and does not affect the average number of live-born piglets (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016).

Gross and histological lesions

Gross examinations of the CNS, the peripheral nervous system or the skeletal muscles of affected piglets are, in general, without any significant findings (Done et al., Reference Done, Woolley, Upcott and Hebert1986; Blomstrom et al., Reference Blomstrom, Ley and Jacobson2014; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016). However, varying degrees of hypomyelination and mild vacuolization of the CNS are characteristic histologic findings, both in CT cases where APPV is detected and in CT cases where astrovirus is detected (Done et al., Reference Done, Woolley, Upcott and Hebert1986; Blomstrom et al., Reference Blomstrom, Ley and Jacobson2014; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016; Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017).

The hypomyelination of the CNS presumably causes impaired impulse transmission and saltatory conduction, resulting in neurologic signs such as tremor or ataxia (Patterson et al., Reference Patterson, Done, Foulkes and Sweasey1976; Done et al., Reference Done, Woolley, Upcott and Hebert1986). The hypomyelination is commonly characterized by thinly myelinated axons and occasionally nonmyelinated axons; any myelin that is present, however, has a normal histological appearance (Duncan, Reference Duncan1987). Interestingly, the extent of the hypomyelination of the CNS varies between individuals as well as between studies and outbreaks.

Done et al. (Reference Done, Woolley, Upcott and Hebert1986) used a light microscope to measure the diameter of the spinal cord from piglets born with CT, and reported that the spinal cord from a CT-affected piglet had, on average, a 12% decrease of myelin compared to a healthy control piglet. On the other hand, others report only a mild, unquantifiable hypomyelination, visualized as a mildly reduced staining intensity by Luxol fast blue (commonly used to stain myelin for light microscopy) in the spinal cord from severely affected CT-piglets (Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016). The hypomyelination could also be noted in the brain as numerous small vacuoles of the white matter (Blomstrom et al., Reference Blomstrom, Ley and Jacobson2014).

More prominent and extensive vacuolization due to hypomyelination of the white matter in the brain was documented in piglets during a CT type A-II outbreak in Austria, where the LINDA virus was discovered. The remaining myelin in the CNS, however, had a normal constitution and structure (Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017). This extensive vacuolization corresponded to the LINDA virus-affected piglets being more atactic and presenting with tremor of higher intensity than commonly described in other outbreaks of CT type A-II (Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017).

A recent study from China reported not only the discovery of a new virus, a porcine circovirus-like virus P1, in the brain of CT-piglets, but also previously unreported histological lesions in the brain. In this particular outbreak, the predominant lesion was the dissolution of the nucleus of Purkinje cells affecting coordination and balance (Wen et al., Reference Wen, Mao, Jiao, Zhang, Xie and He2018). If these cells are damaged, the piglet will present with neurologic signs such as tremor and ataxia (Wen et al., Reference Wen, Mao, Jiao, Zhang, Xie and He2018).

Causative agents

Soon after CT type A-II was first recognized, it was suggested to be caused by a virus (Larsson, Reference Larsson1955). Hence, when Done et al. (Reference Done, Woolley, Upcott and Hebert1986) were able to induce CT in piglets by injecting pregnant sows with tissue from CT-affected piglets, it was taken as evidence to support this hypothesis. Since then, several viruses have been associated with CT type A-II. At present, a virus first described in 2015, APPV, is proposed as a cause of CT type A-II (Hause et al., Reference Hause, Collin, Peddireddi, Yuan, Chen, Hesse, Gauger, Clement, Fang and Anderson2015; Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Postel et al., Reference Postel, Meyer, Cagatay, Feliziani, de Mia, Fischer, Grundhoff, Milicevic, Deng, Chang, Qiu, Sun, Wendt and Becher2017a). Interestingly, APPV has been detected in the brain of piglets with CT in Brazil as a co-infection together with porcine teschovirus (Possatti et al., Reference Possatti, Headley, Leme, Dall Agnol, Zotti, de Oliveira, Alfieri and Alfieri2018b).

In 1994, PCV-II was suggested as the causative agent of CT type A-II (Hines, Reference Hines1994), but since 2005 it is no longer generally regarded as a plausible causative agent. Because PCV-II is regularly detected in neurologically normal piglets, and rarely in piglets suffering from CT, it is usually considered as an incidental finding in piglets (Chae, Reference Chae2005; Ha et al., Reference Ha, Jung and Chae2005).

In 2014, astrovirus was demonstrated in the brain of piglets suffering from CT type A-II. However, astrovirus was also detected in the brain from healthy piglets. Hence, the hypothesis of astrovirus as a plausible cause of CT type A-II could be neither confirmed nor rejected (Blomstrom et al., Reference Blomstrom, Ley and Jacobson2014). However, 2 years later, astrovirus was described as an incidental finding together with APPV in brain-homogenate from piglets with CT (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016).

A pestivirus provisionally named ‘LINDA virus’ was discovered in the brain of piglets from an Austrian farm, in connection with an outbreak of CT (Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017). By sequencing, the LINDA virus showed 60% identity to APPV and 68% identity to Bungowannh virus (Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017). The mortality rate during the LINDA virus outbreak was higher than in the average CT outbreak, due to the intense lateral shaking, impairing the piglets’ capability to suckle. Only 22.4 piglets per sow were weaned during the year of the outbreak, compared to on average 25.8 piglets per sow per year (Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017). The outbreak stopped abruptly when all sows had each given birth to one CT-affected litter, which may indicate that they developed immunity to the virus (Lamp et al., Reference Lamp, Schwarz, Hogler, Riedel, Sinn, Rebel-Bauder, Weissenbock, Ladinig and Rumenapf2017). No new reports of the LINDA virus have been published since this outbreak.

The latest report of a virus that might be a causative agent of CT type A-II is the recent finding from China of PCV-like virus P1, which has been found in the brains of piglets with CT type A-II (Wen et al., Reference Wen, Mao, Jiao, Zhang, Xie and He2018). No follow-up studies on this virus have yet been published.

Atypical porcine pestivirus (APPV)

APPV was first detected in the US in 2015, by high-throughput sequencing of porcine sera. In a few publications, it has also been referred to as ‘porcine pestivirus’ (Blomstrom et al., Reference Blomstrom, Fossum, Wallgren and Berg2016). This previously unknown virus was found to be widespread in the US pig population (Hause et al., Reference Hause, Collin, Peddireddi, Yuan, Chen, Hesse, Gauger, Clement, Fang and Anderson2015). Soon after the detection of the APPV, it was associated with CT type A-II. The virus was present in the CNS of newborn piglets with CT, whereas it was absent from the CNS in the vast majority of their healthy littermates and in the healthy control piglets (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016; Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017; Gatto et al., Reference Gatto, Arruda, Visek, Victoria, Patterson, Krull, Schwartz, de Oliveira and Arruda2018a, Reference Gatto, Harmon, Bradner, Silva, Linhares, Arruda, de Oliveira and Arruda2018b).

Researchers were also able to induce CT by injecting pregnant sows with sera containing APPV (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). Hence, Mokili's Metagenomic Koch's Postulates were fulfilled (Mokili et al., Reference Mokili, Rohwer and Dutilh2012) although Koch's postulates per se are yet to be fulfilled.

APPV is assigned to the family Flaviviridae, genus Pestivirus. It is an enveloped RNA virus with a positive sense, single-stranded genome of about 11–12 kb, and it has four structural proteins (C, Erns, E1, and E2) and eight non-structural proteins (Npro, P7, NS2, NS3, NS4B, NS5A, and NS5B) (Hause et al., Reference Hause, Collin, Peddireddi, Yuan, Chen, Hesse, Gauger, Clement, Fang and Anderson2015). The genome is highly variable, but, so far, genomic clustering of the virus related to geographic areas has not been possible, because almost every farm with clinical disease has a distinct strain of APPV (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Beer et al., Reference Beer, Wernike, Drager, Hoper, Pohlmann, Bergermann, Schroder, Klinkhammer, Blome and Hoffmann2017; Munoz-Gonzalez et al., Reference Munoz-Gonzalez, Canturri, Perez-Simo, Bohorquez, Rosell, Cabezon, Segales, Domingo and Ganges2017; Smith et al., Reference Smith, Meyers, Bukh, Gould, Monath, Scott Muerhoff, Pletnev, Rico-Hesse, Stapleton, Simmonds and Becher2017). Changes in pestivirus nomenclature have been suggested, with APPV being designated as pestivirus K (Smith et al., Reference Smith, Meyers, Bukh, Gould, Monath, Scott Muerhoff, Pletnev, Rico-Hesse, Stapleton, Simmonds and Becher2017).

Since the first finding of APPV (Hause et al., Reference Hause, Collin, Peddireddi, Yuan, Chen, Hesse, Gauger, Clement, Fang and Anderson2015), the virus has been detected globally, e.g. in China, Sweden, Germany, Great Britain, Italy, Serbia, Switzerland, Taiwan, Brazil, Austria, Hungary, and Spain (Hause et al., Reference Hause, Collin, Peddireddi, Yuan, Chen, Hesse, Gauger, Clement, Fang and Anderson2015; Blomstrom et al., Reference Blomstrom, Fossum, Wallgren and Berg2016; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016; Munoz-Gonzalez et al., Reference Munoz-Gonzalez, Canturri, Perez-Simo, Bohorquez, Rosell, Cabezon, Segales, Domingo and Ganges2017; Denes et al., Reference Denes, Biksi, Albert, Szeredi, Knapp, Szilasi, Balint and Balka2018; Gatto et al., Reference Gatto, Harmon, Bradner, Silva, Linhares, Arruda, de Oliveira and Arruda2018b). Furthermore, the oldest detection of APPV is currently from samples stored in 1997, originating from Spanish piglets with CT (Munoz-Gonzalez et al., Reference Munoz-Gonzalez, Canturri, Perez-Simo, Bohorquez, Rosell, Cabezon, Segales, Domingo and Ganges2017).

Because APPV is a recent finding, the global prevalence has not been well defined, but both enzyme linked immunosorbent assay (ELISA) and PCR have been developed for diagnostic purposes (Hause et al., Reference Hause, Collin, Peddireddi, Yuan, Chen, Hesse, Gauger, Clement, Fang and Anderson2015; Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017; Yuan et al., Reference Yuan, Han, Li, Huang, Yang, Ding, Zhang, Zhu, Zhang, Liao, Zhao and Chen2017; Postel et al., Reference Postel, Meyer, Cagatay, Feliziani, de Mia, Fischer, Grundhoff, Milicevic, Deng, Chang, Qiu, Sun, Wendt and Becher2017a, Reference Postel, Meyer, Petrov and Becher2017b). No cross-reactivity with APPV-specific antibodies in the diagnosis of classical swine fever has been reported (Postel et al., Reference Postel, Meyer, Petrov and Becher2017b).

In 2017, serum samples from 1460 healthy pigs from Europe (Germany, Great Britain, Italy, Serbia, and Switzerland) and Asia (mainland China and Taiwan) were tested using an indirect APPV-ELISA and an APPV-specific PCR. The result indicated that 8.9% of the 1460 pigs were PCR-positive for APPV and 60% of the pigs were APPV-positive by serology (Postel et al., Reference Postel, Meyer, Cagatay, Feliziani, de Mia, Fischer, Grundhoff, Milicevic, Deng, Chang, Qiu, Sun, Wendt and Becher2017a). This is in line with other studies, e.g. from China, where 5.2% of 135 sampled pigs were PCR-positive for APPV (Yuan et al., Reference Yuan, Han, Li, Huang, Yang, Ding, Zhang, Zhu, Zhang, Liao, Zhao and Chen2017), and in a screening of semen where 15% of 597 breeding boars in the United States were PCR-positive for the virus (Gatto et al., Reference Gatto, Arruda, Visek, Victoria, Patterson, Krull, Schwartz, de Oliveira and Arruda2018a).

The highest viral load of APPV has been detected in lymphoid organs such as tonsils, lymph nodes, spleen, and thymus, which are also the tissues where APPV is most commonly detected. The virus is also frequently found in the brainstem, cerebrum, cerebellum, sera and in nasal and rectal swabs (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016; Munoz-Gonzalez et al., Reference Munoz-Gonzalez, Canturri, Perez-Simo, Bohorquez, Rosell, Cabezon, Segales, Domingo and Ganges2017; Yuan et al., Reference Yuan, Han, Li, Huang, Yang, Ding, Zhang, Zhu, Zhang, Liao, Zhao and Chen2017; Gatto et al., Reference Gatto, Arruda, Visek, Victoria, Patterson, Krull, Schwartz, de Oliveira and Arruda2018a).

The main histopathological findings associated with APPV-infection are the demyelination and/or hypomyelination of the CNS. Additional lesions commonly observed in the CNS consist of neuronal necrosis of the brain, gliosis, and neuronophagia with satellitosis, lesions that are more suggestive of necrosis than of inflammation (Possatti et al., Reference Possatti, Headley, Leme, Dall Agnol, Zotti, de Oliveira, Alfieri and Alfieri2018b).

The transmission route of APPV is yet to be determined. However, frequent detection of APPV in salivary glands, mandibular lymph nodes, duodenum, pancreas, and colon, together with prolonged shedding of virus in feces, might be suggestive of a fecal–oral route of transmission (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016; Postel et al., Reference Postel, Hansmann, Baechlein, Fischer, Alawi, Grundhoff, Derking, Tenhundfeld, Pfankuche, Herder, Baumgartner, Wendt and Becher2016).

Since APPV is difficult to propagate in cell culture, serum from piglets with clinical CT that were PCR-positive for APPV have been used to inject sows in two independent successful experiments (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). However, in 2017, APPV was successfully grown on Porcine Kidney cells-15 (Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017). This was done using an APPV-positive serum sample originating from a piglet with signs of CT, where colostral antibodies were avoided since the serum was obtained before suckling.

Arruda et al. (Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016) infected five sows at day 45 and 62 of pregnancy by simultaneous intravenous injection, intranasal inoculation, and injection of APPV directly into the amniotic vesicle, whereas de Groof et al. (Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016) infected three sows at day 32 of gestation via intramuscular injection. In both experiments, none of the sows that were inoculated with APPV developed any clinical signs of disease, nor did they develop a PCR-detectable viremia. However, these sows gave birth to piglets with typical signs of CT, and the piglets were also PCR-positive for APPV in multiple organs (e.g., CNS, spleen, tonsils, and serum) (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). In these studies, the within-litter prevalence of piglets born with signs consistent with CT was 57–100% (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016) and 0–100%, respectively (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). Interestingly, a proportion of the piglets in both studies also showed signs of splay-leg (0–40% of the pigs within the litter) in addition to CT (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). Splay leg is a condition characterized by impairment of the adducting muscles of the hindlimbs due to hypomyelination of the spinal cord and the nerves innervating the affected muscles (Thurley et al., Reference Thurley, Gilbert and Done1967; Szalay et al., Reference Szalay, Zsarnovszky, Fekete, Hullar, Jancsik and Hajos2001). In both studies, all piglets born with CT were positive for APPV; however, the majority of healthy piglets born to APPV-inoculated sows were also PCR-positive for APPV (Arruda et al., Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016; de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). Arruda et al. (Reference Arruda, Arruda, Magstadt, Schwartz, Dohlman, Schleining, Patterson, Visek and Victoria2016) reported that 10 out of 11 healthy piglets were APPV-positive and de Groof et al. (Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016) reported that 26 out of 28 healthy piglets were APPV-positive. Why APPV may be present in the CNS of healthy piglets is yet unknown.

Since it was hypothesized that APPV infection can result in persistently infected animals, two studies were performed to elucidate this proposal. A longitudinal follow-up study was done in piglets with CT, originating from the experimental infection (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). Most of these piglets were free from signs of CT, and 20 out of 27 were PCR-negative for APPV in serum at 4.5 months of age (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). Interestingly, five piglets were found to be asymptomatic carriers of APPV at 8.5 months of age. These pigs were PCR-negative for APPV in serum but were shedding high amounts of the virus in feces, saliva, and in preputial fluid (de Groof et al., Reference de Groof, Deijs, Guelen, Van Grinsven, Van OS-Galdos, Vogels, Derks, Cruijsen, Geurts, Vrijenhoek, Suijskens, Van Doorn, Van Leengoed, Schrier and Van Der Hoek2016). In the second study, two pigs, one female and one male, were born with CT and sampled over a 6-month period. They had APPV NS3H-specific antibodies present until 12 weeks of age and signs of CT until 14 weeks of age (Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017). At 6 months of age, the boar reached sexual maturity without any detectable levels of APPV NS3H-specific antibodies, but with high levels of APPV in both saliva and semen detected by the PCR. The viral concentration in serum, however, was low (Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017). At 6 months of age, the sow also had high APPV loads in the saliva; however, the serum level of APPV was below the PCR detection level (Schwarz et al., Reference Schwarz, Riedel, Hogler, Sinn, Voglmayr, Wochtl, Dinhopl, Rebel-Bauder, Weissenbock, Ladinig, Rumenapf and Lamp2017).