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Journal of Clinical Microbiology, November 2004, p. 5047-5052, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5047-5052.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Routes of Transmission of Swine Hepatitis E Virus in Pigs

C. Kasorndorkbua,^1,2 D. K. Guenette,³ F. F. Huang,³ P. J. Thomas,¹ X.-J. Meng,³ and P. G. Halbur^1*

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa,¹ Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia,³ Department of Veterinary Pathology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand²

Received 13 May 2004/ Returned for modification 1 July 2004/ Accepted 31 July 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hepatitis E virus (HEV) is believed to be transmitted by the fecal-oral route in pigs. To date, in experiments, HEV has been transmitted successfully only by the intravenous or intrahepatic route. To assess the route of HEV transmission, 27 pigs were separated into nine groups of three pigs. Positive-control pigs were inoculated intravenously with swine HEV and served as the source of HEV for the other groups. Uninoculated contact pigs were placed in the positive-control group. On three consecutive days, naïve pigs were inoculated using samples collected from the positive-control pigs at 9, 10, and 11 days postinoculation. The tonsils and nasal mucosa of each positive-control pig were swabbed and that swab was used to rub the tonsils and nasal and ocular mucosa of naïve pigs. The positive-control pigs were also injected with bacterin, and the same needle was used to immediately inject naïve pigs. Feces were collected from positive controls and fed by oral gavage to naïve pigs. Weekly fecal and serum samples from each pig were tested for anti-HEV antibodies and HEV RNA. All positive-control pigs shed the virus in feces; two pigs were viremic and seroconverted to anti-HEV. All contact control pigs shed the virus in feces; two seroconverted and one became viremic. One of three pigs in the fecal-oral exposure group shed the virus in feces and seroconverted. Pigs exposed to the contaminated needles or the tonsil and nasal secretion swabs remained negative. This is the first report of experimental fecal-oral transmission of HEV in swine.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hepatitis E virus (HEV) is an important cause of enterically transmitted, non-A, non-B hepatitis in humans. The virus is a nonenveloped, single-stranded, positive-sense RNA virus (34). HEV has recently been classified as the prototype member in the Hepevirus genus of the family Hepeviridae (9). The disease caused by HEV is typically characterized as a self-limiting acute hepatitis with low mortality (20). However, severe hepatitis has been reported in pregnant women with up to 25% mortality (20).

HEV in humans is believed to be transmitted primarily by the fecal-oral route (21, 34). HEV is endemic or epidemic in certain regions of the world, including parts of Africa, Asia, and Mexico. Epidemics of HEV in regions where it is endemic are usually associated with heavy rains or flooding in areas that lack proper drinking water sanitation (19). Individuals from regions where HEV is not endemic who acquire HEV infection often have a history of traveling to developing countries where HEV is endemic (7, 14, 33).

Recent sporadic human HEV infections in people who had not traveled to countries where HEV is endemic led to the discovery of novel HEV isolates in industrialized regions, such as the United States, Europe, Taiwan, and Japan. Sequence analyses revealed that these HEV isolates are genetically divergent (11, 30, 31, 35, 36, 44, 48). The human and swine HEV isolates from industrialized countries are genetically clustered together in the same genotype (either genotype III or IV), raising concerns of hepatitis E as a zoonotic disease (3, 10, 15, 24, 29, 32, 35, 37, 40, 45, 47). Serological surveys of humans who are in close contact with pigs, such as swine veterinarians and pig handlers, showed an increased prevalence of anti-HEV antibodies in these occupational groups, suggesting potential pig-to-human HEV transmission (8, 27, 43).

Pigs have been experimentally infected with a genotype III human HEV and swine HEV, and the HEV-infected pigs shed the viruses in feces for several weeks (13). Direct evidence of zoonotic HEV transmission has recently been reported in Japanese patients who acquired hepatitis after consumption of uncooked pig livers (22) or consumption of raw meat from wild deer (38). On the basis of the sequence available, the swine HEV isolate detected in a raw pig liver sold in a grocery store was genetically identical to a human HEV isolate recovered from one of the Japanese hepatitis E patients (47).

The natural route(s) of swine HEV transmission in pigs remains unknown. Swine HEV can be transmitted experimentally via direct contact with infected pigs (25). Repeated direct daily contact among pigs reared in confinement buildings may enhance the spread of swine HEV. Pigs housed in the same pen are exposed to saliva, nasal secretions, urine, and feces of multiple pen mates repeatedly each day. Experimental transmission of HEV to naïve pigs via feces collected from swine HEV-infected pigs was achieved when the pigs were inoculated intravenously, but not when the pigs were inoculated orally with an equivalent dose (17). Extrahepatic sites of HEV replication exist and include the intestinal tract (42). Therefore, it is logical to assume that, under natural conditions, swine HEV is transmitted via the fecal-oral route as is thought to be the case in human HEV infections (10, 46).

HEV viremia is transient and lasts only 1 to 2 weeks, whereas fecal virus shedding may persist for up to 7 weeks (13, 25). Repeated use of needles for drug administration or vaccination is commonly practiced in swine health management. Even though HEV viremia is transient (1 to 2 weeks), it is possible that blood contamination of needles from viremic pigs may also be a means for HEV to spread among pigs on and between farms. The objective of the present study was to determine whether swine HEV transmission occurs via exposure to (i) tonsil or nasal secretions from infected pigs, (ii) repeated use of contaminated needles, and (iii) oral consumption of feces from infected pigs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus inocula and pigs The swine HEV inocula used in the present study were prepared from feces collected from pigs intravenously infected with the prototype strain of swine HEV (24). The inocula contained a HEV titer of 10^4.5 50% pig infectious dose per ml, which is equivalent to approximately 10⁶ genome equivalents (GE) per ml (26). The inocula were kept at –80°C until used. Twenty-seven, 3-week-old, specific-pathogen-free pigs (Sus scrofa domesticus) were used in this study. All pigs were confirmed to be free of swine HEV in feces by nested reverse transcription-PCR (RT-PCR) prior to inoculation (26).

Experimental design The experimental design consisted of one negative-control, three sham-inoculated, and five exposure groups (see Table 3). Each group contained three pigs and was housed separately. In the exposure groups (groups 3, 4, and 5), each pig was in a different, individual pen to avoid direct contact with body secretions or excretions from other pigs in the same groups. Three pigs were intravenously inoculated with the swine HEV inocula and served as positive controls and virus shedders (group 1). Three pigs were not inoculated and served as negative controls (group 6) and as the source for swine HEV-negative inocula used in the three sham exposure groups. Three uninoculated pigs were housed in the same pen containing the three positive-control pigs to serve as direct-contact controls (group 2). The three exposure routes chosen in the study were designed to evaluate three potential transmission routes likely to occur under current swine production practices: (i) tonsil and nasal secretions, (ii) repeated use of needles, and (iii) fecal-oral route.

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TABLE 3. Detection of anti-HEV serum antibodies by ELISA and of HEV RNA by RT-PCR in pigs inoculated with biological samples from HEV-infected pigs

In the tonsil and nasal secretion exposure group (group 3), sterile plastic Dacron swab applicators (Medical Packaging Corp., Camarillo, Calif.) were used to collect tonsil and nasal secretions from each of the three pigs (a separate swab for each pig) in the positive-control group (virus shedders) by repeatedly and aggressively rubbing the palatine tonsil surface and nasal mucosal surfaces, respectively. Each of the contaminated swabs was then placed in a separate sterile plastic container and applied to one of the naïve pigs in the exposure group by rubbing the palatine tonsil surface, nasal mucosal surfaces, and palpebral and bulbar conjunctiva, respectively.

In the needle exposure group (group 4), a 1-in.-long, 18-gauge, hypodermic needle and a 3-ml syringe were used to vaccinate three positive-control pigs intramuscularly in the neck with 2 ml of Mycoplasma hyopneumoniae bacterin (Respisure; Pfizer). The needle was then removed and placed in a sterile plastic bag to repeat the administration of the vaccine to three naïve pigs in the needle exposure group.

In the fecal-oral exposure group (group 5), 15 g of fresh feces collected from each of the shedder pigs was pooled and mixed, and then 15 g of pooled fecal samples was administered by oral gavage to each of the three naïve pigs in the fecal-oral exposure group. The pigs were monitored for 5 min to assure that no regurgitation occurred after the gavage.

The three inoculation procedures were repeated for three consecutive days (9 to 11 days postinoculation [dpi] of the positive-control shedder group). Three sham-inoculated controls (group 7, 8, and 9) were inoculated in similar manners to the corresponding exposure groups, except that the sham inocula were obtained from three swine HEV-free negative-control pigs (groups 6). To prevent potential cross contamination via people moving from the shedder group into exposure groups, two separate teams were involved in the collection of exposure inocula and inoculation.

The serum samples and fecal swabs from the positive-control shedders were collected daily from 3 to 14 dpi and stored at –80°C for the detection of HEV RNA by qualitative and quantitative RT-PCR. The tonsil or nasal swabs, serum samples, and fecal swabs collected on 9, 10, and 11 dpi were tested for HEV RNA by RT-PCR, and the positive samples were further tested by quantitative real-time RT-PCR. Fecal swab and blood samples were collected weekly from all pigs until the end of the study on 56 dpi. All fecal and serum samples were stored at –80°C until tested.

Detection of swine HEV RNA by nested RT-PCR RNA extraction was performed by the modified spin column method (QIAamp; QIAgen, Chatsworth, Calif.) as previously described (2). Portions (140 µl) of fecal suspension or serum samples were used for RNA extraction. The RNA extract was then immediately used for the reverse transcription reaction and cDNA synthesis in a reaction mixture containing 11.5 µl of RNA, R1 reverse primer, and Superscript II reverse transcriptase (GIBCO-BRL) at 42°C for 1 h. A nested RT-PCR assay specific for the prototype United States strain of swine HEV was performed as previously described (17). The first-round PCR primers were the forward primer F1 (5'-AGCTCCTGTACCTGATGTTGACTC-3') and the reverse primer R1 (5'-CTACAGAGCGCCAGCCTTGATTGC-3') and yielded a PCR product of 404 bp. The second-round PCR primers included the forward primer F2 (5'-GCTCACGTCATCTGTCGCTGCTGG-3') and the reverse primer R2 (5'-GGGCTGAACCAAAATCCTGACATC-3') and produced a PCR product size of 266 bp. Ten microliters of cDNA was used as the template for the first round of PCR in a 100-µl PCR mixture. The first round of PCR was initiated with the activation of ampliTaq Gold DNA polymerase at 95°C for 9 min. There were 39 cycles in the first round of PCR, with 1 cycle consisting of denaturation (1 min at 94°C), annealing (1 min at 52°C), and extension (1.5 min at 72°C). The first round concluded with a final incubation step (7 min at 72°C). Similar PCR parameters were used for the second round of PCR. The amplified PCR products were examined by 1% agarose gel electrophoresis. The PCR products were directly sequenced to confirm that they are indeed swine HEV sequences.

Detection of immunoglobulin G anti-HEV antibodies by ELISA Serum samples were tested by an enzyme-linked immunosorbent assay (ELISA) using a purified 55-kDa truncated recombinant capsid protein of human HEV strain Sar-55 as previously described (24, 26). Each serum sample was tested twice. The final optical density values were averages of the two results. Preimmune and hyperimmune swine sera were used as negative and positive controls, respectively.

Quantitative real-time RT-PCR Representative samples of feces, tonsil or nasal secretions, and sera were collected daily from three shedder pigs in the positive-control group for three consecutive days during the period of exposure inoculation (9, 10, and 11 dpi). Swabs of pooled fresh feces (used as inocula) were resuspended in 1-ml portions of sterile phosphate-buffered saline (PBS). Swabs of tonsil or nasal secretions were resuspended in 1-ml portions of sterile PBS.

All representative samples were subjected to a quantitative real-time RT-PCR assay. A standard curve for the quantification of swine HEV genome was generated using an in vitro swine HEV ORF2 cDNA plasmid as previously described (42). In vitro-transcribed RNA was quantitatively measured with a spectrophotometer. Serial 10-fold dilutions from 100 ng to 10 fg were made and subjected to quantitative PCR. All representative samples of three exposure inocula on 9, 10, and 11 dpi were tested in triplicate using the iCycler system (Bio-Rad). SYBR green 1 was used to detect PCR product. The amount of viral RNA in the samples was derived from the standard curve using the mean of the cycle threshold values generated. For the amount of viral RNA in a given sample, viral genome copy numbers were calculated according to an equation published elsewhere (5).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The three positive-control pigs inoculated intravenously with the swine HEV stock shed relatively large amounts of viruses in feces during the period (9 to 11 dpi) that the experimental transmission and exposure were performed (pigs A, B, and C in Tables 1 and 2). However, viremia was detected in only two of the pigs (pigs B and C) at the times tested, and these pigs seroconverted to immunoglobulin G anti-HEV antibodies by the end of the study (Table 1). Two of the three positive-control pigs exhibited fecal virus shedding during the entire exposure period (on 9 to 11 dpi) (Table 2). Pig A shed virus in feces on 4, 7, 8, and 10 to 14 dpi, pig B shed virus in feces on 4 and 7 to 14 dpi, and pig C shed virus in feces on 3 to 5 and 7 to 14 dpi. Only pig B had three consecutive days of viremia during the exposure period from 9 to 11 dpi (Table 2). Viremia was detected in pig B on 5, 6, and 8 to 14 dpi (Table 1). The HEV genomic titers in serum samples obtained from pig B were 2.8 x 10⁶, 4.4 x 10⁶, and 6.6 x 10⁶ copies per ml of sera on 9, 10, and 11 dpi, respectively (Table 2). Viremia was not detected in pig A or pig C during the exposure period (9 to 11 dpi) or at any time prior to 14 dpi (3 to 13 dpi daily). Pig A was not viremic at any time tested from 14 to 56 dpi; however, viremia was detected in pig C on 14 and 21 dpi (Table 1).

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TABLE 1. Viremia, fecal shedding, and seroconversion in positive-control (shedder) pigs inoculated intravenously with a swine HEV infectious stock

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TABLE 2. HEV genomic copy number in fecal and serum samples of positive-control pigs collected on the days when samples were taken from them for inoculation and exposure of naïve pigs

The results of quantitative real-time RT-PCR for HEV genome during the period of exposure are summarized in Table 2. The HEV genomic titers in fecal samples from pig A were 2.7 x 10⁶ and 10.5 x 10⁶ copies per g of feces on 10 and 11 dpi, respectively (fecal sample from this pig was negative for HEV RNA by RT-PCR on 9 dpi). The genomic titers in fecal samples from pig B were 2.9 x 10⁶, 3.7 x 10⁶, and 10.5 x 10⁶ copies per g of feces on 9, 10, and 11 dpi, respectively. Pig C had 3.8 x 10⁶, 6.2 x 10⁶, and 13.2 x 10⁶ copies per g of feces on 9, 10, and 11 dpi, respectively. The tonsil and nasal secretion swabs collected from the shedder pigs (pigs A, B, and C) were negative for HEV RNA during the period of exposure inoculation (9 to 11 dpi).

Although all three contact control pigs shed the virus in feces, only two of these pigs seroconverted by the end of the study on 56 dpi (Table 3). One of the two seropositive pigs was also viremic on 21 dpi. Only one of three pigs in the fecal-oral exposure group shed the virus in feces (from 21 to 35 dpi) and seroconverted by 56 dpi. Viremia was not detected in that pig on the days tested (14, 21, 28, 35, 42, 49, and 56 dpi). Pigs in the other exposure and sham inoculation groups remained negative for swine HEV RNA and anti-HEV antibodies throughout the study.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first report of successful transmission of HEV in pigs by the fecal-oral route. Of the three possible routes of HEV transmission (tonsil and nasal secretions, contaminated needles, and fecal-oral exposure) investigated under the conditions of this study, the fecal-oral route was the only route by which HEV transmission was achieved. However, it remains unclear whether the fecal-oral route of exposure accounts for the high incidence of HEV infection in the global pig population, since we achieved transmission in only one of three pigs by the fecal-oral route.

In contrast to the contact control pigs and to the way pigs are typically raised in the field, the three pigs in the fecal-oral exposure group were placed in individual pens in the same room to decrease the likelihood of exposure to feces and urine from other pigs in the room. Aerosol transmission would not have been prevented by the room and pen design. Since transmission did not occur from pig to pig within the room, aerosol transmission is not likely. The flooring of the animal pens used in the study was raised, plastic-coated metal wire decks which accumulate minimal amounts of feces. In contrast, under field conditions, most pigs are raised in confinement facilities that have 25 to 200 pigs per pen and a combination of solid and slatted concrete flooring, which generally allows for more feces to accumulate and thus for repeated exposure to feces from multiple pigs.

Experimental HEV infection by oral inoculation in nonhuman primates has reportedly been less reproducible than intravenous inoculation (1). Furthermore, an attempt to reproduce swine HEV infection by oral inoculation in pigs was unsuccessful with a single dose of a standard swine HEV stock that was infectious when given intravenously (17). This suggests that swine HEV infection in pigs requires either a higher titer or repeated exposure to initiate infection via the fecal-oral route. Direct and repeated contact among large numbers of pigs in the same pen would increase the likelihood of repeated exposure to pigs excreting a high dose of HEV in feces. It is obvious that the infectious dose of HEV required for successful HEV transmission is different for the intravenous and oral routes of exposure. It has been demonstrated that a HEV titer of 10² GE was sufficient to induce HEV infection in pigs when the virus was inoculated intravenously on a single day (17); however, a higher titer of more than 10⁶ GE and repeated exposure (for three consecutive days) was essential for transmission via the fecal-oral route, and this was successful in only one of three pigs in the present study.

Others have reported that HEV infection in nonhuman primates is dose dependent (20, 39). All three shedder pigs in the present study shed what is considered a high titer of HEV (2.7 x 10⁶ to 13.2 x 10⁶ virus genomic copies per g of feces) in their feces, particularly during the third day of the exposure inoculation (11 dpi). Feces from all three positive-control pigs were pooled prior to inoculation. However, only one pig in the fecal-oral exposure group became infected. The reason for the discrepancy in HEV transmission among the three pigs in the fecal-oral exposure group remains obscure.

HEV is a temperature-sensitive, labile virus (4). The infectivity of HEV is reportedly diminished after a rapid change in temperature (4). In this study, the length of time between the collection of fresh feces from the rectum of HEV-infected pigs and the exposure inoculation was less than 5 min. The pooled fresh feces collected were at ambient temperature until subsequently given orally to naïve pigs. Inactivation of the virus in such a short period under these conditions should be minimal.

In contrast to the fresh feces used in this experiment, a suspension (10% [wt/vol] in PBS) of feces was used as inocula in our previous study in which we failed to transmit HEV by the fecal-oral route (17). In that study, the fecal suspensions were clarified by centrifugation at 1,100 x g for 10 min, and the supernatant was stored frozen at –80°C for up to 4 years (17). The same frozen fecal suspension was used as the intravenous inocula for the shedder pigs in the current and previous experiments (13, 17, 18). The freeze-thaw process might have affected the ratio of infectious to defective HEV particles (4, 23) and contributed to the unsuccessful transmission of the HEV via the oral route in a previous experiment (17).

Proteolytic degradation of the infectious HEV particles in the intestinal tract of the host may also inactivate infectious HEV particles (4). However, inactivation of the HEV particles in the intestinal tract may be offset by newly assembled HEV virions originating from in situ replication in the intestines, thus maintaining a high level of infectious virus particles in feces (42). HEV infection in the intestinal mucosa without a systemic HEV infection may explain why one positive-control pig and some contact control pigs shed HEV in feces but did not develop HEV viremia (one of three pigs) or anti-HEV antibodies (two of three pigs). The lack of systemic HEV infection may explain the inability to detect HEV viremia and the insufficient serum antibody response in some pigs.

HEV RNA was not detected in the tonsil and nasal cavity secretions tested in this experiment during the period of viremia. There is no evidence that tonsils and salivary glands support replication of swine HEV (42), so it is not surprising that the pigs in the tonsil or nasal exposure group failed to become infected. This is further supported by the lack of evidence of transmission to noncontact pigs breathing the same air as the pigs in the fecal-oral exposure group. It is unlikely that the nasal mucosa or respiratory tracts are the portals of virus shedding or routes of HEV infection. Urine is also less likely to be a source of HEV transmission in pigs, since there is also no evidence that swine HEV replicates in the kidney (42).

Repeated use of the same needle and syringe is common when vaccines and drugs are administered to growing pigs on commercial pig farms. We chose to use a Mycoplasma hyopneumoniae vaccine, since this type of vaccine is commonly administered to pigs between 5 and 12 weeks of age, when HEV transmission likely occurs (24). The volume of residual blood in used needles varies depending on the route of injection, the size of the hypodermic needle, and the volume and type of syringe. Up to 7.5 µl of residual blood was transferred during sharing of needles by intravenous drug users. In needle sharing simulations, the residual blood volume was increased when a 2-ml syringe was used than when a 1-ml syringe was used (12). In the present study, we used an 18-gauge, 1-in.-long needle on a 3-ml syringe for three consecutive days of the exposure inoculation process. Much larger multiple-dose syringes (i.e., 50 ml and larger) are typically used in the swine industry. The doses were administered into muscle in the present study, so residual blood in the needle would be less than that of the needles used for intravenous injection. Furthermore, there is no evidence that skeletal muscle is the site of replication for HEV (42) or that pork meat contains a detectable amount of HEV (17).

The HEV viremic period is variable and transient and generally lasts only about 1 to 2 weeks (13, 16, 25). A recent survey of 99 pigs from several regions in Indonesia reported a sharp contrast between the prevalence of anti-HEV seroconversion (72%) and the number of pigs whose serum samples tested positive for HEV RNA (1%) (41). The transient and/or intermittent characteristic of HEV viremia may contribute in part to the ineffective transmission of HEV through contaminated needles. From a comparative perspective, the injection of therapeutic drugs was not a risk factor for the high prevalence of anti-HEV antibodies in Danish patients who may have contracted HEV infection via the blood-borne route (6). Swine veterinarians in the United States who reported experiencing needle sticks or cuts with blood-to-blood contact were not at a significantly higher risk of anti-HEV seropositivity compared to those who had no history of such exposure (28). The inability to transmit HEV infection with used needle exposure in this experiment and the lack of epidemiological evidence of transmission of swine HEV to humans with exposure to pig blood may be due to an inadequate amount of infectious HEV particles in the blood on the contaminated needle or necropsy knives.

In summary, the findings of the present study indicate that experimental fecal-oral transmission of swine HEV in pigs did occur but was not efficient. Efficient transmission of swine HEV in pigs via the fecal-oral route may require repeated exposure and high doses. Evidence of the transmission of swine HEV through tonsil and nasal secretions was lacking. It also appears unlikely that the repeated use of needles represents a common means of swine HEV transmission in pigs. The ubiquitous nature of swine HEV in the swine population and the presence of swine HEV in pig feces for a considerably longer period than the length of HEV viremia also favors the fecal-oral route as the primary route of swine HEV transmission. However, it remains unknown whether there are undetermined factors that may facilitate swine HEV transmission through the fecal-oral route to the degree that HEV infection is reported to be widespread in global pig populations.

 
We thank Robert Purcell and Suzanne Emerson for providing the human HEV Sar-55 antigen and the swine HEV stock.

This work was supported in part by a grant (to P.G.H.) from the Iowa Livestock Health Advisory Council and by grants (AI01653 and AI46505 to X.-J.M.) from the National Institutes of Health.

 
* Corresponding author. Mailing address: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011. Phone: (515) 294-1173. Fax: (515) 294-1072. E-mail: pghalbur{at}iastate.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, November 2004, p. 5047-5052, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5047-5052.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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