Porcine transmissible gastroenteritis

GENERAL INTRODUCTION: CORONAVIRIDAE

Introduction

Transmissible gastroenteritis (TGE) is a highly contagious disease of swine. It is characterized by vomiting, diarrhoea and high mortality (up to 100 per cent) in seronegative piglets less than two weeks of age. The causative agent (TGEV) is an enteric coronavirus that was first detected in the United States (US) in 1946 from outbreaks of acute diarrhoea with high mortality in piglets.¹ The disease has since been reported in most parts of the world where intensive pig farming is practised, including Europe, Asia (Japan, Korea, Malaysia, Taiwan), the Americas (North, Central and South) and Africa (Zaïre, Ghana). It was a major cause of disease and death in piglets in the US, Europe and elsewhere from the 1960s-1980s, notwithstanding the widespread application of vaccines. Since the emergence and rapid spread throughout Europe of porcine respiratory coronavirus (PRCV),² a deletion mutant of TGEV with respiratory tropism, a large proportion of the  pig population has become immune to that virus and, consequently, also to TGEV. The endemic status of PRCV has thus markedly decreased the clinical and economic importance of TGEV (see Porcine respiratory coronavirus infection).

Aetiology

Being a member of the family Coronaviridae and genus Alphacoronavirus,TGEV is a single-stranded RNA virus. Virions are pleomorphic (diameter of 60 to 160 nm) with a fringe of widely spaced, club-shaped projections 12 to 25 nm in length.³ They are composed of three structural proteins: the nucleocapsid protein (N), a small membrane glycoprotein (M, formerly labelled E1), and a large glycoprotein constituting the viral projections (S, formerly named E2). The S glycoprotein bears an epitope that induces neutralizing antibodies as well as several epitopes that induce non-neutralizing antibodies.⁴

There is only one serotype of TGEV, although some antigenic variation exists between field strains with different geographical origins, and between virulent and attenuated strains. Transmissible gastroenteritis virus is antigenically closely related to PRCV, canine coronavirus, feline enteric coronavirus and feline infectious peritonitis coronavirus,^{5, 6} and is distantly related to porcine epidemic diarrhoea virus (PEDV)⁷ and human coronavirus 229E.^{8, 9}

The virus is sensitive to lipid solvents and to various disinfectants, such as formaldehyde, sodium hypochloride, iodine and quaternary ammonium compounds. It is inactivated within a few days at 37°C and within a few hours when exposed to sunlight. Most strains of TGEV are only moderately sensitive to trypsin and are stable at pH3. The physicochemical properties of TGEV are provide elsewhere.^{10, 11}

Epidemiology

Pigs are the only animals for which TGEV is pathogenic. Other non-porcine hosts, such as dogs, cats and foxes, can be infected experimentally but do not develop signs of illness. Whether these or other species are natural hosts of this virus is uncertain.

Transmission of TGEV is direct, i.e. pigs excreting the virus come into contact with susceptible individuals or indirect through contact of susceptible animals with virus contained in faeces or contaminated fomites. In experimentally infected pigs, TGEV was shed for approximately 14 days in faeces.¹² TGEV can also be detected in lungs of some pigs.¹³

Herd immunity can be accomplished by intentionally exposing the sows to virulent TGEV to boost lactogenic (milk) immunity.^{14, 15} On isolated small farms, herd immunity is accomplished and TGEV infection is self-limiting. Conversely, on large farms (more than 200 sows) with a continuous farrowing system and continual influx of susceptible animals, TGEV can become endemic after the primary outbreak. Subsequent groups of pigs become infected after weaning when their maternal immunity wanes and they in turn infect other groups. The only clinical evidence of infection in these groups is mild diarrhoea at weaning. Typical TGE in young piglets that is associated with high mortality occurs when gilts or sows are TGEV seronegative or they fail to provide passive protection in the colostrum and milk to their offspring.

Generaly, outbreaks of TGE occur in winter suggesting that TGEV preservation in the frozen state in faeces or at cold temperatures can facilitate its transfer. Additionally, starlings (numerous in feedlots in winter) ¹⁶ and house flies ¹⁷ were indicated as potential mechanical vectors for TGEV transmission. It is not known how the virus survives the summer, but viral persistence on endemically- infected farms (in pigs, non-porcine reservoirs such as cats ¹⁸ and dogs ¹⁹ and mechanical vectors) is likely to be the main mechanism.¹⁰

Since the emergence of PRCV, typical and severe outbreaks of TGE with high mortality in new-born piglets have become rare in many European countries and in the US. In Belgium, no classical TGE has been reported since 1992 and only in the UK have occasional TGE outbreaks been reported (ca. 1990’s).²⁰ A 1993 serological survey revealed specific TGEV antibodies in only fifteen per cent of Belgian swine herds and in 7.6 per cent of the slaughterhouse sow sera ²¹ using competitive inhibition ELISA for differentiation of TGEV- and PRCV-specific antibodies ²². This contrasts with the US, where 45 per cent of swine herds in Iowa were TGEV seropositive in 1995.²³

Pathogenesis

The pathogenesis of TGE in the gastrointestinal tract has been studied in detail ^{12, 24} and has served as a model for coronavirus-associated diarrhoea in other animal species, such as cattle and dogs.

After ingestion, the virus passes intact through the stomach and replicates in enterocytes in multiple stages of differentiation along the entire small intestine, resulting in rapid and extensive epithelial cell necrosis, marked villous shortening or atrophy (reduction of the villous length/crypt depth ratio from 7:1 to 1:1) (Figure 1), accompanied by malabsorption and diarrhoea. Vomiting may be a consequence of the upper jejunum being affected. Dehydration can be severe, especially in piglets <1 week of age, and is frequently the immediate cause of death. As crypt epithelium is less affected, the villus epithelium regenerates early in the course of the disease. The lower mortality rate of older pigs following infection is likely due to the enterocytes being less susceptible to infection or to faster regenerative capacity. Additionally, older pigs may be more capable of compensation for the physiological effects of dehydration as well as having a more mature immune system. In pigs partially protected by lactogenic immunity, the proximal jejunum is usually spared from villous atrophy, explaining the milder course of the disease.

Pigs that recover from enteric infections with TGEV develop immunity manifested by the appearance of antibodies of all isotypes in the circulation and IgA [secretory IgA, (sIgA)] in intestinal secretions. Secretory IgA acquired from mother’s milk, provides protection rather than circulating antibodies (mostly IgG). Effective protection against enteric reinfection endures for at least six months after primary infection. Thereafter, if reinfection occurs, its effects are usually mild or subclinical and ephemeral. Although only indirect evidence exists concerning the role of cell mediated immunity in resistance to TGEV infection, it may play an important role in recovery or protection against reinfection.^{25, 26}

During TGEV infection of sows, lymphocytes are stimulated by viral antigens and plasmablasts (antibody-secreting immature plasma cells) migrate to the mammary gland (MG) where they secrete IgA antibodies against TGEV in milk, initiating the gut-MG-sIgA axis. Piglets that ingest milk from an immune mother are usually protected against TGEV for as long as they continue to ingest such milk regularly and are not exposed to high amounts of infectivity. Effective passive (lactogenic) protection is afforded only if the sow has undergone intestinal infection and the gut-MG-sIgA axis has been stimulated; viral antigens inoculated parenterally induce little or no IgA antibodies against TGEV in milk.¹⁵ This mechanism which protects pigs from enteric infections is therefore different from that which confers protection in cattle. In the latter, IgG1 (that is selectively transported from circulation to the lacteal secretions) is the principal immunoglobulin for passive immunity of calves. ^{27, 28}

Figure 1 Villous atrophy in the intestine of a pig suffering from transmissible gastroenteritis (lower section) compared to villi in a healthy pig (upper section)

Figure 2 Atrophied small intestinal villi covered by cuboidal epithelial cells in a pig that died naturally of transmissible gastroenteritis

Clinical signs

The clinical signs of TGE in fully susceptible pigs (epidemic situation) differ from those in swine in TGEV-endemically infected herds.

In epidemic situations, TGE spreads rapidly and is characterized by the sudden onset of acute diarrhoea in pigs of all ages, with neonatal animals being most severely affected. All the animals on a farm become infected within a week unless precautions are taken to prevent transmission of the virus. After an incubation period of 24 hours, the pigs may first vomit and then develop watery diarrhoea. After two to three days young pigs become severely dehydrated and most succumb to the infection. In suckling piglets infected during the first week of life the mortality rate may approach 100 per cent, but the rate decreases to almost zero in pigs that are older than two weeks of age. Due to the high levels of TGEV shed by infected piglets, all litters in the farrowing house will likely become infected within a few days. Sows may be sub-clinically infected or may become anorexic with diarrhoea and yield less milk or suffer agalactia. In growing and finishing pigs, watery diarrhoea usually develops, lasting five to seven days.

Sows in edemically infected herds show no signs of illness because they are immune to the virus. Piglets are also protected by antibodies in their dam’s milk as indicated previously. The advent of weaning results in the cessation of antibody supplementation with the milk, and the piglet’s intestine is again susceptible to infection (usually within one to three weeks of weaning). Infection in such animals is manifested by the development of diarrhoea and the spread of the virus to subsequent groups of recently weaned pigs. In endemic situations, diarrhoea may also occasionally develop in three to eight day-old piglets born to non-immune or partially immune sows/gilts. The mortality rate in partially protected piglets is variable.

Pathology

The gross lesions in pigs that die of TGE are essentially those of dehydration accompanied by thin-walled, translucent small intestines. Histologically, villous atrophy is the main lesion, and the shortened villi are usually covered with flat to cuboidal epithelium (Figure 2), with the number of mitotic cells in the crypts markedly increased.²⁹ In typical cases, these lesions are present throughout the small intestine, except for the first few centimetres of the duodenum. Lymphocyte infiltration is marked in the lamina propria and between the epithelial cells of the villi during recovery, when regeneration of the villous epithelium is evident.

Diagnosis

The diagnosis of TGE in neonatal piglets is best achieved by the detection of viral antigens or viral genome in enterocytes or faeces using immunofluorescence or immunohistochemistry on cryostat sections or mucosal impression smears of the small intestine or by real-time polymerase chain reaction (RT-PCR).^{30, 31} Ideally, specimens should be collected from pigs killed soon after the onset of diarrhoea. Faeces or intestinal contents may be tested, preferably before severe dehydration occurs, because extensive enterocyte necrosis (and therefore lack of cells susceptible to TGEV), decreased faecal output and electrolyte imbalance may interfere with viral detection.

Demonstration of TGEV in the faeces of pigs with diarrhoea is not always successful due to increased volume of the intestinal contents (and thus decreased TGEV concentration) and accelerated intestinal peristalsis. The continuous pig testis cell line ST is generally the most sensitive system for virus isolation.³² Virus replication in inoculated cell cultures is indicated by the occurrence of cytopathic effects or immunofluorescence. The latter is aimed at the detection of viral antigen although one or two passages in cell culture may be necessary to overcome toxicity of the inoculum and to increase viral replication. Coronavirus particles can be detected in negatively-stained preparations of faeces from piglets with diarrhoea by transmission electron microscopy. This technique is time and labour consuming, but has the advantage of also detecting other viruses. Additionally, TGE and porcine endemic diarrhoea (PED) coronaviruses and porcine deltacoronavirus (PDCoV) cannot be differentiated unless immunoelectron microscopy is applied.

Viral antigens can be demonstrated by ELISA systems (including those commercially available) in the faeces or intestinal contents of infected animals. Ideally, faecal samples collected from several animals at an early stage in the course of the disease should be examined.³³

Serological diagnosis using TGEV- specific ELISA tests (including those commercially available ³⁴ such as SVANOVIR® TGEV/PRCV-Ab) is often advantageous in older animals. ²² These antibody ELISA tests possess high sensitivity and specificity (>0.93) and do not cross-react with other swine coronaviruses.

Transmissible gastroenteritis virus can be propagated in primary, secondary, continuous or transformed cultures of porcine kidney (including PK-15 and SK-6), swine testis (ST) and thyroid cells. A continuous ST cell line permits the replication of most field strains. Virus neutralization tests using ST and SK-6 cells and a well-adapted virus strain in microplate systems are apt for routine serology. Cytopathic effects comprise syncytium formation and rounding of affected cells.³² Susceptible animals seroconvert within 14 days of initial infection.^{22, 35}

However, in countries where TGEV, PRCV, PEDV or PDCoV occur, the serodiagnosis of TGE requires the use of an ELISA system that differentiates among them (see Porcine respiratory coronavirus infection).

Differential diagnosis

Typical in epidemics of TGE pigs of all ages may show acute diarrhoea with neonatal piglets being most severely affected while with outbreaks of diarrhoea caused by other enteropathogens (e.g. rotavirus, PEDV, PDCoV, Coccidia spp.and Escherichia coli)  suckling or recently weaned pigs usually affected.

Epidemic and endemic TGE cannot be clinically differentiated from them,³⁶ and laboratory tests (noted earlier) are required to confirm the diagnosis.³⁷

Control

Multiple methods for immunizing sows with the aim of inducing lactogenic immunity and consequent protection of neonatal piglets have been attempted.^{15, 35, 38} A variety of viral vaccines (virulent, attenuated, inactivated and subunit) and routes of administration (oral, intranasal, intramuscular, subcutaneous, and intramammary).^{10, 15, 39} have been used for induction of lactogenic immunity. Intramuscular or intramammary inoculation of sows with live attenuated, inactivated or subunit vaccines during pregnancy does not affect complete protection, although vaccination reduced mortality rates. Attenuated virus does not stimulate the gut-MG-sIgA axis sufficiently for the induction of immunity similar to that observed following natural intestinal infection with virulent virus. At least two commercial vaccines against TGEV are available from Merck Animal Health (PROSYSTEM^® TGE/Rota and PROSYSTEM^® TREC) both contain a live-modified TGEV strain for combined oral-intramuscular administration. However, independent evaluation of the efficacy of the commercial TGEV vaccines is lacking.

Feeding virulent virus to sows is effective ininducing lactogenic immunity, but it also propagates the disease on the farm unless all the sows are immunized. This practice results in the rapid development of immunity in pregnant sows (particularly in those due to farrow two weeks or more after the start of the outbreak) and minimizes losses in new-born piglets. However, it may also result in dissemination of the virus or other pathogens to adjacent herds.

Elimination of endemic TGE in a herd can be attempted by deliberately and simultaneously exposing all the sows and weaned pigs to virulent autogenous virus, such as slurry of minced intestines of acutely infected pigs. If this is done, no weaning of piglets should occur during the following three to four weeks, so that there are no susceptible animals present in the herd while TGEV circulates on the farm.

Treatment of clinically affected new-born suckling piglets with electrolyte/glucose solutions is usually ineffective in field situations, but may reduce mortality in piglets that are one week or older.

Precautions should be taken to prevent the introduction of TGEV to susceptible herds by avoiding the introduction of animals and the access to the farm by potentially contaminated vehicles from TGEV affected farms.

Countries that are free of TGE should apply stringent measures to prevent the importation of the disease. This applies not only to the importation of live animals, but also, as has recently been shown, to the unprocessed tissues of slaughtered TGEV-infected animals.⁴⁰

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