Immunoprophylactic effect of chicken egg yolk immunoglobulin (Ig Y) against porcine epidemic diarrhea virus (PEDV) in piglets.

Kweon CH, Kwon BJ, Woo SR, Kim JM, Woo GH, Son DH, Hur W, Lee YS J Vet Med Sci 2000 Sep 62:961-4 Abstract Porcine epidemic diarrhea virus (PEDV) is the causative agent of neonatal diarrhea in piglets, which causes high mortality rates. In this study, the immunoprophylactic effects of chicken egg yolk immunoglobulin (Ig Y) against PEDV were investigated in neonatal pigs. Ig Y was found to reduce the mortality in piglets after challenge exposures. The field application of Ig Y also revealed significant differences in survival rates of piglets given Ig Y, as compared with placebo or control. The results in this study indicated that Ig Y against PEDV could be an alternative way of supplementing prophylactic measures like colostral antibodies from sows.

RUSSIAN VETERINARY CONGRESS Section of the problems of the infectious pathology of pigs
EPIZOOTIC DIARRHEA OF THE PIGS
[V].[A].[SERGEEV], professor, the doctor of biological sciences, [O].[V].[SERGEEV], the candidate of biological sciences, NII (Scientific Research Institute) the virusologists [im]. Of the [d].[I].[Ivanovskogo] In 1971 in England among the feeder pigs and the nursing suckling-pigs they observed sharp outbreaks of diarrhea unknown to that. The clinical manifestations of disease were similar to transmissible gastroenteritis ([TGS]) besides that important difference, that in nursing sucklingpigs did not observe vomiting [2, 38]. Virus [TGS] and other known enteropathogenic agents were excluded. The analogous disease, which was being observed in other European Countries, was called name epizootic virus diarrhea (EVD). In 1978 was established the connection of the observed flashes with crown- such virus [6, 40]. Experimental infection by prototype strain CV777, called diarrhea in suckling-pigs and pigs during fattening [9]. Pathogen of disease was called name virus of epizootic diarrhea of pigs (PEDV) [39].

Etiology
Virus emf on the basis of antigenic and genetic criteria carries to the group 1 kinds Of [s]oronavirus of the family Of [s]oronaviridae together with the virus [TGS], the corona viruses of cats, dogs and man of 229[E]. The genome of prototype strain [S]V777 has a size of 28033 nucleotides [1, 17, 30, 50]. Similar to other corona viruses, virus emf contains 3 proteins: the [neglikolizirovannyy] protein N (57-58 cd), which together with the genomic RNA forms the nucleocapsid of virus; transmembrane glycoprotein M (27-32 cd) and [peplomernyy] glycoprotein S (180-200 cd) [of 11; 16; 50]. There is no proof of existence of more than one serotype of virus emf. Prototype strain CV777 on the nucleotide sequence of the gene N of [gomologichen] to Korean and Japanese isolates is more than to 90% [31,37,53]. The particles of virus emf on the morphology and the morphogenesis possess all characteristics of the family Of coronaviridae [6, 40]. Mean diameter 130 nm (95-190 nm). Club-shaped [peplomery] (shafts) have a length 18-23 nm and are arranged radially from the core, forming crown. Assembling particles occurs [putem] of budding through the internal cytoplasmic membranes [13, 48]. Virus emf is sensitive to ether and chloroform. Its density in the gradient of saccharose is equal to 1,18 [g]/[sm]3. The virus, adapted to the culture of cells, loses infectiousness with >60[S] during 30 min., but preserves stability with 50[S]. [pri] 4[S] virus it is stable with [rN] 4,0-9,0, with 37[S] it is stable with [rN] 6,5-7,5. Virus emf does not possess the hemagglutinating activity [36].

Cultivation. [Attenuatsiya]
Many types of the cultures of cells were unsuccessfully used for the cultivation of virus emf. Subsequently it was established that the culture of the cells Of vero (kidney of green marmoset) supports the series multiplication of virus emf. In the culture of the cells Of vero the virus was isolated from the small intestine both naturally sick and experimentally infected suckling-pigs. The field isolates of virus were passed to 100 series passages to the cells Of vero. The supporting medium in the period of the multiplication of virus contained trypsin in the concentration 1-2 g/ml. The multiplication of virus is accompanied by the expressed cytopathic effect ([TSPE]), which is evinced by the vacuolization of cells and shaping of syncytium (symplasts, which contain to 100 nuclei). With the isolation of virus from the feces of the sick suckling-pigs several blind passages were required to conduct before the appearance [TSPE] in the cells Of vero [19, 32, 33, 36, 43, 47]. During the series passivation the virus (105,0 [TTsD]50/[ml]) was introduced into the culture of the cells 48 hours after sowing of the cells, when monolayer still completely was not formed. After the development of expressed [TSPE], virus separated from the cells of [putem] of repeated freezing- thawing [19, 23]. The caption of virus reached peak (about 105,5 [BOE]/[ml]) 15 hours after inoculation [19, 36]. Isolate KPEDV-9 on 90 passages reached the caption of

106,5 [TTsD]50/[ml] [33], by a isolate DR13 on 55 passage reached the level of 106,0 [TTsD]50/[ml] and to 100 passages were accumulated in the limits 106,5 - 107,0 [TTsD]50/[ml] [47]. Virus emf, prolongedly adapted to the cells Vero successfully multiplied in the cultures of the cells both of the pig and [nesvinogo] origin: MA 104, CPK and ESK in the presence of trypsin [32]. In the lines of the cells of pig KSEK-6 and IBRS-2 the isolate P -5v in series multiplied even without the addition of trypsin [23], although the role of trypsin in an increase in the level of the reproduction of virus emf was shown experimentally [by 7]. There is information about the isolation of virus emf in the cultures of the cells of the bladder and kidney of pig, grown in the screw dies, covered with collagen, in the presence of trypsin in the supporting medium [43].

Epizootiology
In 1982 - 1990 of antibody to [VEDS] they were discovered in pigs in many countries of Europe and Asia. Communications about the presence of virus emf in North and South America are absent [41]. In Europe the flashes emf recorded rarely, whereas in Asia frequently occur mass flashes emf, which are accompanied by the high mortality of suckling-pigs. On the mass character, the sharpness and the severity of flow they cannot be clinically distinguished of the typical sharp flashes [TGS]. Fecal- oral way is the main thing, if not only, [putem] of the transfer of virus emf. The sharp flashes of infection in the receptive economies frequently occur through 4-5 days after sale or purchases of pigs. Virus emf does not differ from virus [TGS] with respect to the methods of transfer, but it seems that the first more easily causes persistent infection in the economy after the end of the sharp flash [of 41].

Clinical signs
Watery diarrhea is main and frequently only clinical sign emf. Flashes can considerably vary on the morbidity and the lethality. The pigs of all ages fall ill at some farms, and morbidity approaches 100%. Emf is very similar to [TGS] with exception of slower propagation and somewhat the lower mortality of the [novorozhdennykh] suckling-pigs. Suckling-pigs at the age to 1 week can perish from the dehydration after 3-4- day diarrhea. The average mortality of suckling-pigs usually composes 50%, but it can reach 100%. Adult pigs get better approximately in 1 week. After sharp flash diarrhea in suckling-pigs can be observed during 2-3 weeks after removal. In recent years typical sharp flashes with the high mortality of the [novorozhdennykh] suckling-pigs in Europe observe rarely, but they frequently occur in Korea [5] and Japan [48]. All feeder pigs fall ill with the development of diarrhea within a week with the sharp flash emf in the economy. In animals they observe the partial loss of appetite, depression and watery diarrhea. Toward the end feeder period emf frequently flows more heavily than [TGS]. Animals manifest large sickliness in the region of stomach and, as a rule, they get better in 7-10 days. Mortality composed 1-3%, usually at the early stage of diarrhea or even before the manifestation of

diarrhea. With [nekropsii] in such animals they reveal the sharp necrosis of spinal musculature. The highest mortality is observed in the pigs, sensitive to the stress. It is extended more slowly in comparison with [TGS] emf. The propagation of virus of one pigsty in another can engage 4-6 weeks, some pigsties, which do not have contact between themselves, can remain free from the infection.

Pathogenesis
Virus multiplied in the cytoplasm of the epithelial cells of [vorsinok] of thin and colons, which was shown by immunofluorescence and electron microscopy. The infected epithelial cells revealed after 12-18 hours, and they observed their maximum quantity 24-36 hours after infection. The replication of virus in the small intestine led to the destruction of epithelial cells and the shortening of [vorsinok]. The ratio of the height of [vorsinok] to the depth of crypts decreases to 3:1 in comparison with standard 7: 1. degeneration of the infected cells of the epithelium of colon did not observe [41]. The three-day suckling-pigs, [zarazhennye] with the orally prototype strain [S]V777 of virus emf, fell ill 22-36 hours after inoculation. Changes in the small intestine in suckling-pigs with emf are similar to changes with [TGS]. However, the replication of virus and the development of infectious process with emf flow more slowly than with [TGS] [9,10]. The replication of virus emf y of suckling-pigs was discovered only in the intestinal tract. Shibata et al [43] showed that the pigs at the age of 2 days - 12 weeks, [zarazhennye] by field virus emf, manifested stability to the infection depending on age. Only 2-7- day suckling-pigs perished. The clinical signs emf, described in Korea and Japan, are identical to those, which observed in Europe, with exception of the fact that there are no proofs of the replication of the Asian strains of virus in the colon [26, 48] and there were no cases of sudden death of feeder animals. [Patanatomicheskie] changes are expressed by the damage only of small intestine, which is filled up and are extended by yellow liquid [14, 15, 48]. Vacuolization and desquamation of the enterocytes of [vorsinok] of small intestine begin 24 hours after infection and coincide since the beginning of diarrhea. The infected fibers rapidly are shortened and their enzymatic activity sharply is reduced. Morphological changes in [vorsinok] are confirmed by scanning [EM] [12], which showed the great similarity of changes with emf and [TGS]. Histopathological changes in the colon were not revealed. The forming of virions in essence occurs inside the cells and completes [putem] of budding through the membranes of endoplasmic reticulum (EPR) [13, 21]. In the colon only some changes in the enterocytes observed. They contained virus particles, but they did not undergo desquamation. Diagnosis on emf it is not possible to place only on the basis clinical signs. Sharp flashes emf in the pigs of all ages cannot be clinically distinguished of the same with [TGS]. Etiological diagnosis can be established on the basis of the detection of virus emf, either its antigen or

specific antibodies. By the most sensitive, rapid and reliable methods appear straight immunofluorescence ([IF]) [19, 43] and the immunohistochemistry of the shear of the small intestine of the newly born suckling-pigs [18, 48]. However, these research can be conducted only on the suckling-pigs, killed during the sharp phase of diarrhea, preferably during the first 2 days after the beginning of disease. Given methods also are frequently unreliable with a study of bowels of the pigs, that perish naturally as a result of the sharp disturbance of digestion. The particles of virus emf can be revealed in the feces by the method of straight electron microscopy ([EM]), although to reveal them not easily, especially if the [peplomernaya] crown of virion is lost or visible illegibly. A quantity of positive fecal models, obtained from the experimentally [zarazhennykh] suckling-pigs during the first day of diarrhea, according to the data OF [EM], maximally composed 73%. , In order to distinguish viruses emf and [TGS], which have identical morphology, is required immunoelectronic microscopy. For the development of the antigens of virus emf in the feces, and also for the demonstration of specific antibodies in the blood serum was created a number of the collections of immunoenzyme analysis ([IF]A). They are sensitive and reliable for formulating the diagnosis, especially group. For the antigenic versions [IF]A used poly- and monoclonal antibodies and virus, multiplied in the organism of suckling-pigs [2, 3, by 51]. For [antitelnogo] [IF]A antigen is the partly cleaned virus, multiplied either in the organism of animals or in the culture of cells [2, 4, 33], or virus proteins S and N, isolated from the infected cells Of vero [29]. Antibodies also revealed in the milk of sows by the method of IFA [8]. Other diagnostic tests for the detection of virus emf in the feces include reverse transcription polymerase chain reaction (RT-PCR) [22, 25]. RT-PCR was developed for differential diagnostics of viruses emf and [TGS] in the models of bowels and feces of sick pigs [27, 28]. Specific antibodies to the virus emf can be revealed in the blood serum of pigs after natural or experimental infection, using [IF]A, blocking [IF]A, indirect [IF] and the neutralization of virus emf in the culture of the cells Of vero [2, 20, 41, 43]. The blood serum of the infected pigs should be obtained not earlier than 2 weeks after the beginning of diarrhea.

[Pr]o[filaktika] and the control

Since emf is not extended very rapidly, it is possible to use general health preventive measures and to the period to detain the penetration of virus into the [reproduktornye] pigsties with the farrows and the [novorozhdennymi] suckling-pigs. The delay of the natural infection of sucklingpigs before reaching of more elder age can considerably decrease morbidity and loss of pigs. At the same time by analogy with [TGS] the early contact of pregnant sows with the feces infected with virus emf or the feeding of the suspension of bowels of sick suckling-pigs stimulates the [laktogennyy] immunity and is reduced flash emf in the economy. If virus circulates in the sequential dungs of nursing suckling-pigs, it is possible to attempt to end its permanent transfer of [putem] of the displacement of pigs immediately after the end of nursing period to another place not less than to 4 weeks. Simultaneously the entering of new animals into the economy must be temporarily ended with this.

B of Asia, in contrast to Europe, flashes emf proved to be such severe that arose the need of the creation of vaccine against emf. Possible of [putem] of the creation of vaccine is [attenuatsiya] of virus prolonged passivation in the culture of cells. C[eriynoe] passivation leads to reduction in the pathogenicity of virus for the newly born suckling-pigs and the sows, but preserve its ability to cause protective immune response in the inculcated pigs [34, 47]. The attenuated strain KPEDV-9 on 93 passages in the culture of the cells Of vero was used for the oral and intramuscular introduction to [bezmolozivnym] suckling-pigs at the age of 1-4 days. Of 8 suckling-pigs, that received 10 ml of the suspension of virus in the concentration of 108 [TTsD]50/[ml] 3 they orally, only developed soft diarrhea. All suckling-pigs, inculcated by virus at doses 106 and 107 [TTsD]50/[ml], did not show any signs of infection [34]. Strain DR13 on 100 passages in the cells Of vero did not cause diarrhea, anorexia and viremia in the [novorozhdennykh] suckling-pigs and the pregnant sows. Only 1 of 14 suckling-pigs it manifested weak diarrhea [47]. These data were confirmed in the subsequent experiments [44, 45]. The pregnant sows, inculcated by the intramuscularly attenuated strain KPEDV-9 at the dose of 107 [TTSD]50, developed the high captions of antiviral antibodies in the blood and the colostrum, which easily were revealed in IFA. The [novorozhdennykh] suckling-pigs, that were being born from the vaccinated sows, infected by orally field virulent virus emf at doses 10 and 5 [LD]50 they observed during 2 weeks. With 10 [LD]50 the mortality was 20% in comparison with 100% in the control group, with 5 [LD]50 all immune suckling-pigs survived in comparison with loss 60% of control suckling-pigs [34]. Analogous results were obtained in the experiences with the strain DR13, attenuated by a similar method [47]. It was also shown that the oral introduction of attenuated DR13 leads to the more expressed immunity in sows and the best protection of posterity from the infection in comparison with the intramuscular introduction [46]. However, the effectiveness of the application of the attenuated strains under the field conditions is in prospect to set. In Japan from 1997 for the preventive maintenance of sows commercial live vaccine from the strain R -5v, attenuated in the culture of cells, uses. Vaccine is considered effective, although not all sows develop the expressed [laktogennyy] immunity [49]. Are noted for the experiences of the passive protection of suckling-pigs the immunoglobulins of [nesvinogo] origin. Oral introduction to the [novorozhdennym] suckling-pigs of the yolk of chicken eggs or cow's colostrum, that contain immunoglobulins to the virus emf, rendered immunopreventive effect, preventing disease or decreasing the mortality [35, 42]. Obtaining the antigen of virus emf in the transgenic plants is also of interest. The synthesis of recombinant glycoprotein S of virus in the transgenic tobacco composed 2,1% of total dissoluble protein, which makes this plant with potential object for creating stern vaccine [24].

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