ВЫДЕЛЕНИЕ И ИДЕНТИФИКАЦИЯ ВИРУСА

ÑÅËÜÑÊÎÕÎÇßÉÑÒÂÅÍÍÀß ÁÈÎËÎÃÈß, 2013, ¹ 4, ñ. 65-69
ÓÄÊ 636.4:619:616.98:578:57.083
ÂÛÄÅËÅÍÈÅ È ÈÄÅÍÒÈÔÈÊÀÖÈß ÂÈÐÓÑÀ
ÝÏÈÇÎÎÒÈ×ÅÑÊÎÉ ÄÈÀÐÅÈ ÑÂÈÍÅÉ ÏÐÈ
ÂÑÏÛØÊÅ Â ÊÐÓÏÍÎÌ ÑÂÈÍÎÂÎÄ×ÅÑÊÎÌ ÕÎÇßÉÑÒÂÅ
Î.Ì. ÑÒÐÈÆÀÊÎÂÀ
Ýïèçîîòè÷åñêàÿ äèàðåÿ ñâèíåé (ÝÄÑ) õàðàêòåðèçóåòñÿ îñòðûì ýíòåðèòîì è ñèëüíûì
ïîíîñîì. Åå âîçáóäèòåëü îòíîñèòñÿ ê òîé æå ãðóïïå ðîäà Ñoronavirus ñåìåéñòâà Ñoronaviridae,
÷òî è âèðóñû òðàíñìèññèâíîãî ãàñòðîýíòåðèòà ñâèíåé (ÒÃÑ), êîðîíàâèðóñ êîøåê, ñîáàê è ÷åëîâåêà 229Å. Íà÷èíàÿ ñ 2005 ãîäà, íà òåððèòîðèè Ðîññèéñêîé Ôåäåðàöèè ðåãèñòðèðîâàëè âñïûøêè
áîëåçíè, êëèíè÷åñêè ñõîæåé ñ òðàíñìèññèâíûì ãàñòðîýíòåðèòîì ñâèíåé (ÒÃÑ) è âûçûâàþùåé ìàññîâûé ïàäåæ ïîäñîñíûõ ïîðîñÿò (â íåêîòîðûõ ñëó÷àÿõ äî 80 %). Ïðè ýòîì ñâèíîìàòêè â íåáëàãîïîëó÷íûõ õîçÿéñòâàõ áûëè ïðèâèòû ïðîòèâ ÒÃÑ. Öåëüþ íàøåé ðàáîòû áûëî âûäåëåíèå â êóëüòóðå êëåòîê è èäåíòèôèêàöèÿ èíôåêöèîííîãî àãåíòà, ñòàâøåãî ïðè÷èíîé âñïûøåê áîëåçíè ñ äèàðåéíûì ñèíäðîìîì ó ñâèíåé. Èç ïàòîëîãè÷åñêîãî ìàòåðèàëà îò áîëüíûõ ïîðîñÿò 3-5-ñóòî÷íîãî
âîçðàñòà âûäåëåí ÐÍÊ-ñîäåðæàùèé âèðóñ, èäåíòèôèöèðîâàííûé êàê âèðóñ ýïèçîîòè÷åñêîé áîëåçíè ñâèíåé. Òèòð èíôåêöèîííîñòè âèðóñà 5-ãî ïàññàæà â êóëüòóðå êëåòîê Vero ñîñòàâëÿë
103,50 ÒÖÄ50/ñì3. Âûäåëåííûé âèðóñ îáîçíà÷èëè êàê èçîëÿò ÁÑ-08. Ê íàñòîÿùåìó âðåìåíè ïðîâåäåíî 25 ïîñëåäîâàòåëüíûõ ïàññàæåé âèðóñà â êóëüòóðå êëåòîê; ñðîê ïðîÿâëåíèÿ öèòîïàòè÷åñêîãî äåéñòâèÿ ñîñòàâëÿåò 24-48 ÷, èíôåêöèîííûé òèòð — 104,75-105,50 ÒÖÄ50/ñì3.
Êëþ÷åâûå ñëîâà: ýïèçîîòè÷åñêàÿ äèàðåÿ ñâèíåé (ÝÄÑ), ïàòîëîãè÷åñêèé ìàòåðèàë, âûäåëåíèå âèðóñà, êóëüòóðû êëåòîê, öèòîïàòè÷åñêèé ýôôåêò.
Ýïèçîîòè÷åñêàÿ äèàðåÿ ñâèíåé (ÝÄÑ) — òÿæåëîå çàáîëåâàíèå, êîòîðîå õàðàêòåðèçóåòñÿ îñòðûì ýíòåðèòîì, ñèëüíûì ïîíîñîì è îáåçâîæèâàíèåì (1-3). Âîçáóäèòåëü îòíîñèòñÿ ê 1-é ãðóïïå ðîäà Ñoronavirus ñåìåéñòâà Ñoronaviridae. Ê òîé æå ãðóïïå ïðèíàäëåæàò âèðóñû òðàíñìèññèâíîãî
ãàñòðîýíòåðèòà ñâèíåé (ÒÃÑ), êîðîíàâèðóñ êîøåê, ñîáàê è ÷åëîâåêà
229Å. Âïåðâûå áîëåçíü çàðåãèñòðèðîâàíà â Àíãëèè â 1971 ãîäó, â äàëüíåéøåì âñïûøêè âîçíèêàëè â Åâðîïå è Àçèè (4-6). Ñîîáùåíèÿ î íàëè÷èè
âèðóñà ÝÄÑ â Ñåâåðíîé è Þæíîé Àìåðèêå îòñóòñòâóþò (7). Â Åâðîïå
âñïûøêè ÝÄÑ ðåãèñòðèðîâàëè ðåäêî, òîãäà êàê â Àçèè îíè ïðîèñõîäÿò
÷àñòî, íîñÿò ìàññîâûé õàðàêòåð è ñîïðîâîæäàþòñÿ âûñîêîé ñìåðòíîñòüþ
ïîðîñÿò (8, 9). Íà÷èíàÿ ñ 2005 ãîäà, íà òåððèòîðèè Ðîññèéñêîé Ôåäåðàöèè
ðåãèñòðèðîâàëè âñïûøêè áîëåçíè, êëèíè÷åñêè ñõîæåé ñ ÒÃÑ, âûçûâàþùåé ìàññîâûé ïàäåæ ïîäñîñíûõ ïîðîñÿò (â íåêîòîðûõ ñëó÷àÿõ äî 80 %).
Ïðè ýòîì ñâèíîìàòêè â íåáëàãîïîëó÷íûõ õîçÿéñòâàõ áûëè ïðèâèòû ïðîòèâ òðàíñìèññèâíîãî ãàñòðîýíòåðèòà ñâèíåé è ó íèõ òèòð íåéòðàëèçóþùèõ ÂÒÃÑ àíòèòåë ñîñòàâëÿë 1:64-1:256.
Öåëüþ íàøåé ðàáîòû áûëî âûäåëåíèå â êóëüòóðå êëåòîê è èäåíòèôèêàöèÿ èíôåêöèîííîãî àãåíòà, ñòàâøåãî ïðè÷èíîé âñïûøåê áîëåçíè ñ
äèàðåéíûì ñèíäðîìîì ó ñâèíåé.
Ìåòîäèêà. Ìàòåðèàëîì äëÿ èññëåäîâàíèÿ ñëóæèëè ïðîáû ïàòîëîãè÷åñêîãî ìàòåðèàëà (êèøå÷íèê, æåëóäîê, ïå÷åíü, ïî÷êè, ãîëîâíîé ìîçã)
îò âûíóæäåííî óáèòûõ è ïàâøèõ ïîðîñÿò 3-5-ñóòî÷íîãî âîçðàñòà, ïîëó÷åííûå ñ ñåíòÿáðÿ ïî äåêàáðü 2008 ãîäà èç ñâèíîâîä÷åñêèõ õîçÿéñòâ çàêðûòîãî òèïà â çàïàäíîì ðåãèîíå Ðîññèè. Äëÿ âûäåëåíèÿ âîçáóäèòåëÿ
èñïîëüçîâàëè ïåðåâèâàåìûå ëèíèè êóëüòóð êëåòîê ÐÊ-15, CV-1, MA104 è
Vero (ïðåäîñòàâëåíû ëàáîðàòîðèåé êóëüòóð êëåòîê Âñåðîññèéñêîãî ÍÈÈ
âåòåðèíàðíîé âèðóñîëîãèè è ìèêðîáèîëîãèè — ÂÍÈÈÂÂèÌ).  êà÷åñòâå ðîñòîâîé ïðèìåíÿëè ñðåäó Èãëà ñ äîáàâëåíèåì 10 % ôåòàëüíîé ñûâîðîòêè òåëÿò (ÔÑÒ), ïåíèöèëëèíà (100 åä/ìë), ñòðåïòîìèöèíà (100 ìêã/ìë),
àìôîòåðèöèíà Â (2,5 åä/ìë). Ïîääåðæèâàþùåé ñëóæèëà ñðåäà Èãëà áåç
65
ÔÑÒ ñ óêàçàííûìè àíòèáèîòèêàìè è òðèïñèíîì (5 ìêã/ñì3). Äëÿ ïðåäâàðèòåëüíîé îáðàáîòêè ãîìîãåíàòîâ ïàòîëîãè÷åñêîãî ìàòåðèàëà è êóëüòóðàëüíûõ ìàòåðèàëîâ, ïîëó÷åííûõ ïðè ïàññèðîâàíèè, èñïîëüçîâàëè ñðåäó Èãëà
ñ àíòèáèîòèêàìè è òðèïñèíîì â êîíöåíòðàöèè 5, 10 è 20 è 40 ìêã/ñì3.
Ïðè îáðàáîòêå ïàòîëîãè÷åñêîãî ìàòåðèàëà ãîòîâèëè 10 % ñóñïåíçèè èç ïðîá òêàíåé êèøå÷íèêà, æåëóäêà, ïå÷åíè, ïî÷åê, ãîëîâíîãî ìîçãà â
çàáóôåðåííîì ôèçèîëîãè÷åñêîì ðàñòâîðå. Ïîëó÷åííûå îáðàçöû îñâåòëÿëè
öåíòðèôóãèðîâàíèåì ïðè 2500-3000 îá/ìèí â òå÷åíèå 20 ìèí. Íàäîñàäî÷íóþ æèäêîñòü ïîñëå âíåñåíèÿ â íåå ïåíèöèëëèíà è ñòðåïòîìèöèíà äî
êîíöåíòðàöèè 100 åä(ìêã)/ìë âûäåðæèâàëè â òå÷åíèå 30 ìèí ïðè 37 °Ñ è
èñïîëüçîâàëè äëÿ èíîêóëÿöèè êóëüòóð êëåòîê (äàëåå ñóñïåíçèÿ ïàòîëîãè÷åñêîãî ìàòåðèàëà). Ïðè îöåíêå âëèÿíèÿ ðàçíûõ êîíöåíòðàöèé òðèïñèíà
íà ðåïðîäóêöèþ âèðóñà â êóëüòóðå êëåòîê ìàòåðèàë äëÿ çàðàæåíèÿ ïðåäâàðèòåëüíî îáðàáàòûâàëè èëè íå îáðàáàòûâàëè òðèïñèíîì (0; 5; 10; 20 è
40 ìêã/ñì3) è îñòàâëÿëè íà 30-40 ìèí ïðè 37 °Ñ.
Êóëüòóðû êëåòîê èíêóáèðîâàëè ïðè 37 °Ñ, ïîñëå ôîðìèðîâàíèÿ
ìîíîñëîÿ (÷åðåç 1-2 ñóò) ðîñòîâóþ ñðåäó óäàëÿëè, ìîíîñëîé 3-êðàòíî
ïðîìûâàëè áåññûâîðîòî÷íîé ñðåäîé Èãëà è èíîêóëèðîâàëè ñóñïåíçèÿìè
ïàòîëîãè÷åñêîãî ìàòåðèàëà èëè êóëüòóðàëüíûì ìàòåðèàëîì, ïîëó÷åííûì
ïðè ïðîâåäåíèè ïàññàæåé. Â êóëüòóðó êëåòîê âíîñèëè 5 ñì3 ìàòåðèàëà è
èíêóáèðîâàëè 60 ìèí ïðè 37 °Ñ. Çàòåì èíîêóëÿò óäàëÿëè è âî ôëàêîíû
ïîìåùàëè ïîääåðæèâàþùóþ ñðåäó Èãëà ñ òðèïñèíîì (â êîíöåíòðàöèè
5 ìêã/ñì3) èëè áåç òðèïñèíà. Èíîêóëèðîâàííûå êóëüòóðû èíêóáèðîâàëè
ïðè 37 °Ñ â òå÷åíèå 5-6 ñóò (ïðè ñëåïûõ ïàññàæàõ) ëèáî äî ïðîÿâëåíèÿ
öèòîïàòè÷åñêîãî äåéñòâèÿ (ÖÏÄ). Ïîñëå ýòîãî êóëüòóðó çàìîðàæèâàëè,
îòòàèâàëè, ñòåðèëüíî ñëèâàëè âî ôëàêîíû è õðàíèëè ïðè −70 °Ñ.
Òèï íóêëåèíîâîé êèñëîòû îïðåäåëÿëè ïî ìåòîäó Ìàéðà ñ ñîàâò.
(1974). ×óâñòâèòåëüíîñòü ê âîçäåéñòâèþ õëîðîôîðìà àíàëèçèðîâàëè â ñîîòâåòñòâèè ñ îïèñàíèåì Ìàéðà è Áåãåëÿ (1961).
Ïîëèìåðàçíóþ öåïíóþ ðåàêöèþ ïðîâîäèëè ñ èñïîëüçîâàíèåì êîììåð÷åñêîé òåñò-ñèñòåìû «ÝÄÑ» äëÿ âûÿâëåíèÿ ýïèäåìè÷åñêîé äèàðåè ñâèíåé ìåòîäîì ÏÖÐ, êàò. ¹ VET-58-FRT-S(RG,iQ) ñåðèÿ 18.08.12 (ïðîèçâîäèòåëü — ÔÁÓÍ ÖÍÈÈ ýïèäåìèîëîãèè Ðîñïîòðåáíàäçîðà, ã. Ìîñêâà) â
ñîîòâåòñòâèè ñ ïðèëàãàåìûìè ðåêîìåíäàöèÿìè.
Ðåàêöèþ íåéòðàëèçàöèè (ÐÍ) âûïîëíÿëè â ìèêðîâàðèàíòå ñ ïðèìåíåíèåì 96-ëóíî÷íûõ êóëüòóðàëüíûõ ïëàíøåòîâ («Corning», CØÀ). Â
ëóíêè ïîìåùàëè ïî 100 ìêë 2-êðàòíûõ ðàçâåäåíèé (ñ 1:2 äî 1:4096) íîðìàëüíîé, ñïåöèôè÷åñêîé è èññëåäóåìûõ ñûâîðîòîê â ñðåäå Èãëà ñ äîáàâëåíèåì ïåíèöèëëèíà (100 åä/ìë), ñòðåïòîìèöèíà (100 ìêã/ìë) è àìôîòåðèöèíà  (2,5 åä/ìë), çàòåì ïî 100 ìêë ðîñòîâîé ñðåäû Èãëà, ñîäåðæàùåé
âèðóñ â ðàñ÷åòíîé äîçå 100 ÅÄ. Ïëàíøåòû âûäåðæèâàëè â òå÷åíèå 1 ÷ ïðè
37 °Ñ, ïîñëå ÷åãî ïåðåíîñèëè îáðàçöû (ñìåñü ñûâîðîòêè ñ âèðóñîì) â ëóíêè
ïëàíøåòîâ ñ ìîíîñëîåì êóëüòóðû êëåòîê è èíêóáèðîâàëè â òå÷åíèå 4-5 ñóò
â àòìîñôåðå 5 % ÑÎ2 ïðè 37 °Ñ.  ÐÍ èñïîëüçîâàëè ðåôåðåíòíûå ñûâîðîòêè ê âèðóñó ÒÃÑ, ïîëó÷åííûå èç ìóçåÿ øòàììîâ ÂÍÈÈÂÂèÌ.
Ñòàòèñòè÷åñêóþ îáðàáîòêó äàííûõ âûïîëíÿëè ñ ïîìîùüþ ïðîãðàììû Statistica v. 17.
Ðåçóëüòàòû. Íàáëþäàâøàÿñÿ êëèíè÷åñêàÿ êàðòèíà ïðè ðàçâèòèè ïàòîëîãèè áûëà ñëåäóþùåé: ó ïîðîñÿò ñ 2-4-ñóòî÷íîãî âîçðàñòà ïðîÿâëÿëàñü
äèàðåÿ ñ âûäåëåíèåì ñåðî-çåëåíûõ êàëîâûõ ìàññ, îáåçâîæèâàíèå, æàæäà,
àíåìè÷íîñòü âèäèìûõ ñëèçèñòûõ îáîëî÷åê, òåìïåðàòóðà òåëà ïîâûøàëàñü
èëè íàõîäèëàñü â ïðåäåëàõ ôèçèîëîãè÷åñêîé íîðìû (39,4 °Ñ), ó íåêîòîðûõ
æèâîòíûõ îòìå÷àëè ìûøå÷íóþ äðîæü. Ãèáåëü íàñòóïàëà ÷åðåç 2-5 ñóò ïî66
ñëå íà÷àëà äèàðåè. Êëèíè÷åñêèõ è ïàòîëîãîàíàòîìè÷åñêèõ ïðèçíàêîâ çàáîëåâàíèÿ îðãàíîâ äûõàíèÿ âûÿâëåíî íå áûëî. Ñâèíîìàòîê â õîçÿéñòâàõ
âàêöèíèðîâàëè ïðîòèâ ÒÃÑ, ðîòàâèðóñà ñâèíåé, êîëèáàêòåðèîçà, àíàýðîáíîé ýíòåðîòîêñåìèè, ðîæè, êëàññè÷åñêîé ÷óìû ñâèíåé.
Âåòåðèíàðíî-ñàíèòàðíîå ñîñòîÿíèå ôåðì â áîëüøèíñòâå ñëó÷àåâ ñîîòâåòñòâîâàëî ïðèíÿòûì òðåáîâàíèÿì. Òåððèòîðèÿ îãîðîæåíà ñïëîøíûì
çàáîðîì ïî ïåðèìåòðó, êîðïóñà íîâîãî è ñòàðîãî òèïà. Äåçáàðüåðû è äåçêîâðèêè èìåëèñü â âåòñàíïðîïóñêíèêàõ è íà ìåñòàõ âûåçäà òðàíñïîðòíûõ
ñðåäñòâ. Ñîäåðæàíèå áåçâûãóëüíîå, ïîãîëîâüå — îêîëî 2000 ñâèíîìàòîê.
Çàáîëåâàåìîñòü ïîðîñÿò çà ïåðèîä ðåãèñòðèðóåìîé âñïûøêè (îêîëî 3 ìåñ)
ñîñòàâèëà ïðèáëèçèòåëüíî 90 %, ëåòàëüíîñòü — ïðèìåðíî 85 %.
Ïðè ïîñòàíîâêå äèàãíîçà áûëè èñêëþ÷åíû áàêòåðèàëüíûå èíôåêöèè (êîëèáàêòåðèîç, àíàýðîáíàÿ ýíòåðîòîêñåìèÿ), à òàêæå âèðóñíûå èíôåêöèè — àôðèêàíñêàÿ ÷óìà ñâèíåé (À×Ñ), ðåïðîäóêòèâíî-ðåñïèðàòîðíûé ñèíäðîì (ÐÐÑÑ), òðàíñìèññèâíûé ãàñòðîýíòåðèò (ÒÃÑ), êëàññè÷åñêàÿ
÷óìà ñâèíåé (Ê×Ñ), áîëåçíü Àóåñêè, ðîòàâèðóñíàÿ èíôåêöèÿ ñâèíåé.
Ðèñ. 1. Öèòîïàòè÷åñêèé ýôôåêò â êóëüòóðå êëåòîê Vero, èíäóöèðîâàííûé ïîëó÷åííûì èçîëÿòîì
âèðóñà ýïèçîîòè÷åñêîé äèàðåè ñâèíåé: à, á, â — ñîîòâåòñòâåííî 24, 48, 72 ÷ ïîñëå çàðàæåíèÿ; ã — íåçàðàæåííàÿ êóëüòóðà (ìíîæåñòâåííîñòü çàðàæåíèÿ 0,1 åä. íà êëåòêó).
Ïðè ïîñëåäîâàòåëüíûõ ñëåïûõ ïàññàæàõ ïðîá ïàòîëîãè÷åñêîãî ìàòåðèàëà â êóëüòóðàõ êëåòîê ÐÊ-15, CV-1, MA104 è Vero ñ èñïîëüçîâàíèåì
äâóõ ìîäèôèêàöèé ìåòîäà (ñ ïðèìåíåíèåì òðèïñèíà è áåç åãî äîáàâëåíèÿ)
öèòîïàòîãåííûé âèðóñ áûë âûäåëåí èç òêàíè òîíêîãî îòäåëà êèøå÷íèêà
íà 5-ì ïàññàæå â êëåòêàõ Vero (ðèñ. 1) â ñëó÷àå, êîãäà èíîêóëÿò ïðåäâàðèòåëüíî îáðàáàòûâàëè ôåðìåíòîì. Òèòð èíôåêöèîííîñòè ïðè ýòîì ñîñòàâëÿë 103,50 ÒÖÄ50/ñì3.
Èç ïðîá ïàòîëîãè÷åñêîãî ìàòåðèàëà æåëóäêà, ïå÷åíè, ïî÷åê, ãîëîâíîãî ìîçãà â êóëüòóðå êëåòîê Vero èçîëèðîâàòü âèðóñ íå óäàëîñü.  êóëüòóðàõ êëåòîê ÐÊ-15, CV-1 è MA104 ïðè 8 ñëåïûõ ïîñëåäîâàòåëüíûõ ïàññà67
æàõ ñ ïðèìåíåíèåì îáîèõ âàðèàíòîâ âûäåëåíèÿ âèðóñ íå îáíàðóæèëè.
Íàêîïëåíèå âèðóñà ýïèçîîòè÷åñêîé äèàðåè ñâèíåé ïðè ïàññèðîâàíèè â êóëüòóðå
êëåòîê Vero â çàâèñèìîñòè îò ïðåäâàðèòåëüíîé îáðàáîòêè âèðóññîäåðæàùåãî
ìàòåðèàëà òðèïñèíîì (n = 4)
Êîíöåíòðàöèÿ
òðèïñèíà
1-é
ÒÖÄ50/ñì3
Áåç îáðàáîòêè (0 ìêã/ñì3) 3,50 (3,1-3,8)
5 ìêã/ñì3
3,50 (3,1-3,8)
3,50 (3,1-3,8)
10 ìêã/ñì3
3,75 (3,4-4,0)
20 ìêã/ñì3
40 ìêã/ñì3
3,75 (3,4-4,0)
Ï ð è ì å ÷ à í è å. Äîâåðèòåëüíûé èíòåðâàë
2-é
ÒÖÄ50/ñì3
3,50 (3,1-3,8)
3,75 (3,4-4,0)
4,00 (3,5-4,5)
4,50 (3,9-5,0)
4,50 (3,9-5,0)
95 %.
Ïàññàæ
3-é
ÒÖÄ50/ñì3
4-é
ÒÖÄ50/ñì3
5-é
ÒÖÄ50/ñì3
3,75
3,75
4,25
5,25
5,25
3,75
4,00
4,50
5,50
5,50
3,75
4,00
4,50
5,50
5,50
(3,4-4,0)
(3,4-4,0)
(3,7-4,7)
(4,9-5,5)
(4,9-5,5)
(3,4-4,0)
(3,5-4,5)
(3,9-5,0)
(5,1-5,8)
(5,1-5,8)
(3,4-4,0)
(3,5-4,5)
(3,9-5,0)
(5,1-5,8)
(5,1-5,8)
Ïðè îïðåäåëåíèè âëèÿíèÿ ðàçëè÷íûõ êîíöåíòðàöèé òðèïñèíà íà
ðåïðîäóêöèþ âèðóñà â êóëüòóðå êëåòîê Vero (â êà÷åñòâå êîíòðîëÿ êóëüòóðó
êëåòîê èíîêóëèðîâàëè íåîáðàáîòàííûì âèðóññîäåðæàùèì ìàòåðèàëîì)
ïðîâåëè 5 ïîñëåäîâàòåëüíûõ ïàññàæåé (òàáë.). Êàê ñâèäåòåëüñòâóþò ïðåäñòàâëåííûå äàííûå, ïðè îáðàáîòêå òðèïñèíîì â êîíöåíòðàöèè 5 ìêã/ñì3
òèòð âèðóñà âîçðàñòàë äî 4-ãî ïàññàæà íà 0,5 lg. Ìàêñèìàëüíîå óâåëè÷åíèå èíôåêöèîííîãî òèòðà áûëî óñòàíîâëåíî ïîñëå ïðåäâàðèòåëüíîé îáðàáîòêè âèðóññîäåðæàùåãî ìàòåðèàëà òðèïñèíîì â êîíå÷íîé êîíöåíòðàöèè 20 ìêã/ñì3.  ýòèõ óñëîâèÿõ òèòð âèðóñà ïîâûøàëñÿ äî 4-ãî ïàññàæà,
ñîñòàâèë 5,50 ÒÖÄ50/ñì3, è åãî âåëè÷èíà ñîõðàíÿëàñü äî 25-ãî ïàññàæà.
Äàëüíåéøåå óâåëè÷åíèå êîíöåíòðàöèè òðèïñèíà â âèðóññîäåðæàùåì ìàòåðèàëå íå ïðèâîäèëî ê ïîâûøåíèþ èíôåêöèîííîãî òèòðà âèðóñà
ïðè ïàññèðîâàíèè íà âîñïðèèì÷èâîé êóëüòóðå êëåòîê.
Ïðè îïðåäåëåíèè òèïà íóêëåèíîâîé êèñëîòû ïîñðåäñòâîì
îáðàáîòêè çàðàæåííîé êóëüòóðû
êëåòîê 5-éîä-2´-äåçîêñèóðèäèíîì
íå îòìå÷àëîñü çàòðóäíåíèÿ ðåïëèêàöèè, ÷òî ïîçâîëèëî íàì îòíåñòè âûäåëåííûé âèðóñ ê ÐÍÊñîäåðæàùèì. Ïðè îáðàáîòêå õëîðîôîðìîì âèðóñ ïîòåðÿë èíôåêÐèñ. 2. Ýëåêòðîôîðåç â àãàðîâîì ãåëå ïðîäóêòîâ
öèîííóþ àêòèâíîñòü, ÷òî óêàçûÏÖÐ ÐÍÊ âèðóñà ýïèçîîòè÷åñêîé äèàðåè ñâèíåé: 1,
2 — ýêñïåðòèçíûå òêàíåâûå ñóñïåíçèè; 3-8 — âàåò íà íàëè÷èå ó íåãî ëèïèäíîé
ïàññàæè âèðóñà â êóëüòóðå êëåòîê (ñîîòâåòñòâåí- îáîëî÷êè.
íî 1, 5, 10, 15, 20 è 25 ïàññàæåé); 9, 10 — êîíÑëåäóåò îòìåòèòü, ÷òî èíöåíòðèðîâàííûé âèðóñ; 11 — ïîëîæèòåëüíûé êîíôåêöèîííîñòü âèðóñà íå ñíèæàòðîëü; 12 — îòðèöàòåëüíûé êîíòðîëü.
ëàñü ïîñëå îáðàáîòêè óëüòðàçâóêîì è ïðè ìíîãîêðàòíîì çàìîðàæèâàíèè/îòòàèâàíèè. Â ÐÍ ñ ñûâîðîòêàìè
ïðîòèâ ÂÒÃÑ áûëî óñòàíîâëåíî, ÷òî îíè íå íåéòðàëèçóþò èçîëÿò.
Ïðè èçó÷åíèè ñ ïîìîùüþ ÏÖÐ ñóñïåíçèè ïàòîëîãè÷åñêîãî ìàòåðèàëà, à òàêæå êàæäîãî 5-ãî ïàññàæà âûäåëåííîãî âèðóñà â êóëüòóðå êëåòîê ìû ïîäòâåðäèëè íàëè÷èå âèðóñà ÝÄÑ êàê â èñõîäíîì ïàòîëîãè÷åñêîì
ìàòåðèàëå, òàê è â ïîñëåäóþùèõ 25 ïàññàæàõ (ðèñ. 2).
Âûäåëåííûé âèðóñ îáîçíà÷èëè êàê èçîëÿò ÁÑ-08. Ê íàñòîÿùåìó
âðåìåíè áûëî ïðîâåäåíî 25 ïîñëåäîâàòåëüíûõ ïàññàæåé âèðóñà â êóëüòóðå
êëåòîê; ñðîê ïîÿâëåíèÿ öèòîïàòè÷åñêîãî äåéñòâèÿ ñîñòàâëÿë ïðè ýòîì 2448 ÷, èíôåêöèîííûé òèòð — 104,75-105,50 ÒÖÄ50/ñì3
Èòàê, èç ïàòîëîãè÷åñêîãî ìàòåðèàëà îò áîëüíûõ 3-5-ñóòî÷íûõ ïîðîñÿò, îòîáðàííîãî ïðè âñïûøêå áîëåçíè ñ äèàðåéíûì ñèíäðîìîì è äîñòàâëåííîãî ñ ñåíòÿáðÿ ïî äåêàáðü 2008 ãîäà èç çàïàäíîãî ðåãèîíà ÐÔ, âû68
äåëåí ÐÍÊ-ñîäåðæàùèé âèðóñ, ÷óâñòâèòåëüíûé ê ðàñòâîðèòåëÿì ëèïèäîâ.
Ïî ðåçóëüòàòàì ÏÖÐ âîçáóäèòåëü èäåíòèôèöèðîâàí êàê âèðóñ ýïèçîîòè÷åñêîé äèàðåè ñâèíåé (ÝÄÑ).  íàñòîÿùåå âðåìÿ ñ èñïîëüçîâàíèåì ýòîãî
èçîëÿòà ïðîâîäèòñÿ ðàáîòà ïî ñîâåðøåíñòâîâàíèþ äèàãíîñòèêè ÝÄÑ, ñîçäàíèþ ñðåäñòâ äëÿ ñïåöèôè÷åñêîé èììóíîïðîôèëàêòèêè áîëåçíè è îïðåäåëåíèÿ íàïðÿæåííîñòè ïîñòâàêöèíàëüíîãî èììóíèòåòà.
Àâòîð âûðàæàåò áëàãîäàðíîñòü È.Þ. Åãîðîâîé (Âñåðîññèéñêèé ÍÈÈ âåòåðèíàðíîé âèðóñîëîãèè è ìèêðîáèîëîãèè) çà ïîìîùü ïðè âûïîëíåíèè ÏÖÐ.
ÃÍÓ Âñåðîññèéñêèé ÍÈÈ âåòåðèíàðíîé âèðóñîëîãèè
è ìèêðîáèîëîãèè Ðîññåëüõîçàêàäåìèè,
Ïîñòóïèëà â ðåäàêöèþ
5 ìàðòà 2013 ãîäà
601120 Ðîññèÿ, Âëàäèìèðñêàÿ îáë., Ïåòóøèíñêèé ð-í, ã. Ïîêðîâ,
e-mail: [email protected]
ISOLATION AND IDENTIFICATION OF EPIZOOTIC DIARRHEA
VIRUS IN PIGS UNDER OUTBREAK AT A LARGE FARM
O.M. Strizhakova
All-Russian Research Institute of Veterinary Virology and Miñrobiology, Russian Academy of Agricultural Sciences, Pokrov, Petushinskii Region, Vladimir Province, 601120 Russia, e-mail [email protected]
Received March 5, 2013
Abstract
Epizootic diarrhea fever (EDF), characterized by acute enteritis and hard intestinal disorders, is caused by the virus of the same group of Ñoronavirus (Coronaviridae) as the virus of transmissible gastroenteritis of pigs, coronavirus of cats and dogs, and human 229E strain. From 2005, at the
territory of the Russian Federation the outbreaks of the disease were recorded, clinically similar to
pig transmissible gastroenteritis. The infections caused a loss of piglets, in some cases up to 80 %.
However, the sows in such farms have been vaccinated against the transmissible gastroenteritis of
pigs. The aim of our study was to isolate in cell culture and identify an infectious agent that causes
the outbreaks with diarrheal syndrome in pigs. From the pathological material of 3-5-day-old piglets
with the pathology a RNA virus was isolated and identified as the virus of epizootic diseases of pigs.
Virus infectivity in the fifth passage in Vero cell culture was 103,50 ÒCID50/sm3. The isolate was
named as BS-08. To date, there are 25 successive passages of the virus in cell culture. A citopatic effect is registered in 24-48 hours, and the infectious titer (titer of citopatic effect — TCE) makes
104,75-105,50 TCID50/sm3.
Keywords: porcine epidemic diarrhea virus (PEDV), pathological material, virus isolate,
cell culture, cytopathic effect.
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