題目：豬流行性腹瀉（PED）：對一種老疾病的新認識

摘要：

豬流行性腹瀉(PED)是由甲型冠狀病毒引起的豬腸道疾病。它引起所有年齡段豬只急性腹瀉，并可導(dǎo)致哺乳仔豬嚴(yán)重脫水和死亡。歐洲和亞洲分別在70年代和80年代首次認識到這種疾病，多年來一直是亞洲仔豬腹瀉爆發(fā)的一個原因，直至現(xiàn)在。自2013年美國和其他國家首次發(fā)現(xiàn)該病毒以來，已成為養(yǎng)豬生產(chǎn)中的一個主要問題，導(dǎo)致大量豬只死亡和重大經(jīng)濟損失。本綜述旨在使讀者了解PED的技術(shù)現(xiàn)狀，并回答一些疾病相關(guān)的問題。

表1：豬冠狀病毒感染的主要特征及分布

注釋：

Virus：病毒

Genus：基因型

Main characteristics of clinical disease：主要臨床癥狀特點

Distribution：分布

Porcine Epidemic Diarrhoea Virus(PEDV)：豬流行性腹瀉病毒

Alpha：α冠狀病毒

Acute and watery diarrhoea in pigs of all ages：所有年齡段豬只急性感染和水樣腹瀉

Mortality can reach up to 100 % in suckling piglets of less than 2 weeks due to severe dehydration：2周齡以下的哺乳仔豬因嚴(yán)重脫水，死亡率可達100%

only sporadic outbreaks in Europe during the last 10 years but a relevant cause of diarrhoea in pig farms in Asia since the 80s. Firstly described in America in 2013：過去10年歐洲僅零星爆發(fā)，但自80年代以來在亞洲豬場出現(xiàn)。2013年在美國首次爆發(fā)。

Transmissible Gastroenteritis Virus(TGEV)：傳染性胃腸炎病毒

Alpha：α冠狀病毒

Enteric disease clinically indistinguishable of porcine epidemic diarrhoea：與豬流行性腹瀉臨床上無法區(qū)分的腸道疾病

only very sporadic outbreaks in countries where PRCV is widespread：在出血熱廣泛流行的國家，只有零星的爆發(fā)

Porcine Respiratory Coronavirus (PRCV)：豬冠狀病毒

Alpha：α冠狀病毒

Self-limiting respiratory infection. Often subclinical but can exacerbate respiratory symptoms caused by other pathogens：自限性呼吸道感染。通常為亞臨床感染，但可加重由其他病原體引起的呼吸道癥狀

Endemic infection in many European swine herds：許多歐洲豬群的地方性感染

Hemagglutinating Encephalomyelitis Virus (HEV)：戊型肝炎病毒

Beta：β冠狀病毒

Neurotropic virus causing the typical vomiting and wasting disease or acute encephalomyelitis with motor disorders in piglets：引起仔豬典型嘔吐和消耗性疾病或急性腦脊髓炎并伴有運動障礙的嗜神經(jīng)病毒

Widespread infection although most of the cases remain subclinical：廣泛感染，盡管大多數(shù)病例仍處于亞臨床狀態(tài)

Porcine Delta Coronavirus (PDCoV)：德爾塔冠狀病毒

Delta：δ冠狀病毒

Mild to moderate enteric disease in young piglets similar to porcine epidemic diarrhoea or transmissible gastroenteritis：與豬流行性腹瀉、傳染性腸胃炎相似的仔豬輕度至中度腸道疾病

First identified in Hong Kong, China, in 2009 and North America in early 2014. However, a recent research detected anti-PDCoV IgG antibodies in serum samples collected in 2010, indicating an earlier undetected presence of PDCoV in the US pig population：2009年首次在中國香港發(fā)現(xiàn)，2014年初在北美發(fā)現(xiàn)。然而，最近的一項研究在2010年收集的血清樣本中檢測到了抗PDCoV IgG抗體，這表明在美國豬群中出現(xiàn)了更早的未被檢測到的PDCoV。

圖1：基于地理和時間標(biāo)準(zhǔn)選擇PEDV全基因組(a)和S基因(b)對核苷酸序列進行遺傳進化樹分析

Infection sources and transmission

Direct and indirect PEDV transmission occurs mainly by faecal-oral route. Viral shedding in faeces starts on postinfection day one or two and continues for a period of 7 to 10 days [35, 36], although it can extend up to 36 weeks in some animals [37, 38]. The transmission of the infection is facilitated by the high viral load in faeces from infected animals [39, 40] as well as by the minimum infectious dose required to infect na?ve pigs [31]. Moreover, the resistance of the virus in the environment facilitates the faecal-oral transmission. PEDV is stable under low temperatures, while it is adversely affected by high temperatures. It survives between pH 5.0–9.0 at 4 °C while only between pH 6.5–7.5 at 37 °C. It can survive for at least 28 days in slurry at 4 °C, 7 days in contaminated dry feed at 25 °C or 14 days in contaminated wet feed at 25 °C [31]. This fact favours the indirect transmission by different faeces-contaminated fomites such as transport vehicles [41], feed [42], clothing or footwear.

Genetic and phylogenetic analyses of American PEDV isolates revealed a close relationship with Chinese isolates and their likely Chinese origin [43]. However, how the virus might have travelled from China to the USA is a matter of speculation.

The rapid spread of PEDV on swine farms in the USA raised questions regarding the possibility of airborne transmission of this infection. Although undoubtedly the faecal-oral route is the main source of PEDV transmission, it has been suggested [44] that PEDV may travel through the air for short distances on faecal dust particles, at least under certain conditions. However, airborne transmission of PEDV has only been shown under experimental conditions and up to now infectious PEDV has not been demonstrated in field air samples containing PEDV genetic material [44, 45]. The role that vectors play in the transmission of PEDV has also been investigated. So far, there has been no evidence of PEDV replication in non-porcine hosts, including rodents and starlings [46–48]. However, the potential role of vectors in the mechanic transmission of the virus from one farm to another cannot be ruled out, as has been described for TGEV [4].

Using highly sensitive molecular assays the presence of viral RNA has been reported in milk samples from infected lactating sows [28, 29] as well as in semen samples [29, 31]. However, infectious PEDV in these samples has not been demonstrated and their contamination with faecal material in the sampling cannot be excluded. Moreover, viral RNA has been detected in the serum fraction of whole blood samples from infected pigs [40, 49].

The role of spray-dried porcine plasma (SDPP), normally used as feed additive, as a potential vehicle of transmission of PEDV has been researched into. A number of experimental studies have demonstrated that spray-drying process as well as storage conditions are sufficient to inactivate infectious PEDV in SDPP [50, 51]. The infectivity of commercial SDPP positive for PEDV-RNA has also been investigated. A research group from Canada managed to reproduce PEDV infection in SDPP-inoculated piglets, although they failed to reproduce the infection in animals receiving feed supplemented with the same PEDV-positive SDPP [52]. Similarly, neither clinical signs nor PEDV RNA in faeces or PEDV specific antibodies were detected in pigs which were fed a diet containing 5 % SDPP confirmed positive for PEDV, in a bioassay experiment conducted by Opriessnig et al. [53]. According to this, there is no experimental evidence of PEDV transmission through PCR positive SDPP supplemented feed. This experimental data is corroborated by the fact that despite the use of large amounts of PEDV positive SDPP from the USA to feed pigs in Brazil or Western Canada, these areas remained free of PEDV infection [54].

感染源和傳播

PEDV主要通過糞口途徑直接和間接傳播。病毒在糞便中的 排毒 始于感染后的第1-2天，并持續(xù)7-10天，但在某些 豬只 中可延長至36周。受感染 豬只 糞便中的高病毒載量以及感染 陰性 豬所需的最低感染劑量，促進了感染的傳播。此外，病毒在環(huán)境中的耐性促進了糞口傳播。PEDV在低溫下是穩(wěn)定的，但在高溫下會受影響。在4 ℃ pH值5.0-9.0之間 、 37 ℃ pH值6.5-7.5之間存活。在4 ℃ 的 泥漿 中至少能存活28天，在25 ℃污染的干飼料中至少能存活 7天 ， 在25 ℃ 污染 的 濕飼料中至少能存活14天。這有利于通過交通工具、飼料、服裝 、 鞋類等糞便污染物的間接傳播。

美國 PEDV分離株的遺傳和系統(tǒng)發(fā)育分析表明，該分離株與中國PEDV分離株關(guān)系密切，可能源自中國。然而，病毒是如何從中國傳播到美國 ？

PEDV在美國豬場的迅速傳播提出了關(guān)于這種感染可能通過空氣傳播的 可能 。盡管糞口途徑是PEDV傳播的主要來源，但有人認為PEDV至少在某些條件下可以通過糞便塵埃顆粒在空氣中短距離傳播。然而，PEDV的空氣傳播僅在實驗條件下被證明。調(diào)查了病媒在PEDV傳播中的作用。到目前為止，尚未發(fā)現(xiàn)PEDV在非豬宿主中復(fù)制的證據(jù)。但是，不能排除病媒在病毒從一個 豬場 機械傳播到另一個 豬 場 的 潛 在作用 。

使用高度敏感的分子檢測方法，已報道在感染哺乳母豬的常乳 和精液中存在病毒RNA。然而，這些樣本中傳染性PEDV尚未被證實，不能排除其被采樣中的糞便污染。此外，已在感染豬 只 全血 樣本 的血清中檢測到病毒RNA。

研究了豬噴霧干燥血漿蛋白 (SDPP)作為PEDV潛在傳播媒介的作用。大量實驗研究表明，噴霧干燥過程和儲存條件足以滅活SDPP中感染性PEDV。對PEDV陽性SDPP傳染性也進行了研究。來自加拿大的一個研究小組成功地在接種了SDPP的仔豬中復(fù)制了PEDV感染，盡管他們未能在喂食了相同的PEDV陽性SDPP 豬只 中復(fù)制感染。同樣，Opriessnig等人進行的一項生物測定實驗表明，在喂食含有5% SDPP飼糧 的 豬 只 中，證實PEDV陽性的豬 只 既 沒有出現(xiàn)臨床癥狀，也沒有發(fā)現(xiàn)糞便中 PEDV RNA，也沒有發(fā)現(xiàn)PEDV特異性抗體。由此可見，沒有實驗證據(jù)表明PEDV通過PCR陽性SDPP傳播。盡管巴西或加拿大西部地區(qū)使用了大量來自美國的PEDV陽性SDPP飼料喂養(yǎng)豬 只 ，但這些地區(qū)仍未發(fā)現(xiàn)PEDV感染，這一實驗數(shù)據(jù)得到了證實。

Pathogenesis, clinical signs and lesions

PEDV replicates in the cytoplasm of villous enterocytes of the small intestine and causes villous shortening and reduced enzymatic and absorptive capacity in the small intestine causing profuse watery diarrhoea, which lasts about a week [37, 55, 56]. Other clinical signs which are frequently associated to PEDV infection include vomiting, anorexia and fever. Although pigs of all ages are affected, the severity of PED is higher in suckling piglets of less than one week old which may die due to severe dehydration. The slower turnover of enterocytes in neonatal piglets (5–7 days) compared to three weeks-old piglets (2–3 days) could explain, at least partially, the higher susceptibility of these young piglets to PEDV [4].

PEDV has also been detected in epithelial cells of the colon in both experimentally and naturally infected pigs, although villous atrophy has not been demonstrated in the large intestine [40].

Replication of PEDV was classically circumscribed to the intestinal tract [3], until a recent research showed PEDV replication in alveolar macrophages of 3 day-old-colostrum-free piglets, which were experimentally inoculated with a Korean wild-type PEDV isolate [57]. Further studies are needed to confirm whether extra-intestinal replication also occurs with other PEDV isolates as well as to determine their clinical and epidemiological relevance. Two epidemiologic presentations of PED have been described on the farms. (a) Epidemic PED outbreaks occur when PEDV is introduced into a na?ve farm (where most of the animals are PEDV seronegative). The disease spreads rapidly affecting pigs of all ages with morbidity approaching 100 %. Moreover, PEDV can persist and become (b) endemic on the farm affecting post-weaning piglets that have lost their lactogenic immunity as well as newly introduced seronegative gilts.

Mortality associated with PED outbreaks is highly dependent on the age of the infected animals. Mortality can reach up to 80–100 % in suckling piglets of less than one week old, while in weaned pigs mortality rates are typically only 1 to 3 % [11, 30]. No mortality associated with PED is usually observed among adult pigs.

As has already been mentioned, differences in the severity of PED outbreaks have been reported. Particularly severe PED outbreaks have been described in Asia since 2010 and also in the USA. Differences in the virulence of PEDV isolates have been proposed to explain this variability [28, 58, 59]. From our point of view, this is one of the most relevant questions to face regarding PED nowadays: the reason or reasons which could explain variations in the clinical outcome of an outbreak. Although some reports have suggested that they could be associated with differences in the virulence of PEDV isolates, exhaustive challenge studies using pig adapted virus (not cell culture adapted isolates) in suckling piglets are needed to elucidate the role of the strain.

Some insights have been obtained related to the virulence of different strains. In the USA, at least two main variants of PEDV have been recently identified using molecular methods. The first one seems to be a highly virulent virus and similar to viruses described in several Asian countries after 2010 while the second, the S INDEL variant, has been associated to mild clinical outbreaks [59]. This S INDEL variant includes some particular insertions and deletions in the S gene and is also similar to some Asian isolates, part of which were recovered before 2010. The classical European reference strain of PEDV CV777 is also an S INDEL isolate although it is located in a different cluster and well differentiated from American INDEL isolates (Fig. 1a and b). PEDV isolates recovered in European countries (Germany, Italy, Belgium, the Netherlands and France) in 2014 and 2015 have been characterized and all of them were found to be INDEL isolates similar to the variant described in the USA [13–19]. Most of these recent PED outbreaks in Europe occurred in fattening farms and, as expected, no mortality was observed. However, PEDV isolates recently recovered in severe outbreaks of PEDV in Ukraine have shown a genome nucleotide similarity reaching 99.8 % with non-INDEL isolates from the United States and Mexico [20]. So far, this has been the only report of PEDV non-INDEL isolates in Europe. Apart from differences in the virulence of the PEDV strains, many other parameters including management, immune status of the population and herd sanitary status could also explain variations in the clinical outcome of PED outbreaks [31]. Thus, the contribution of co-infections with other viruses, particularly with other enteric viruses such as porcine delta coronavirus (PDCoV) or the recently described mammalian orthoreovirus 3 (MRV3) has also been pointed out. Both viruses have been detected in faecal samples collected from PEDV positive farms in the USA. PDCoV has been associated with mild to moderate diarrhoea in experimentally inoculated na?ve suckling piglets [33] while MRV3 caused severe diarrhoea with 100 % mortality in 3-day-old piglets [60].

發(fā)病機制、臨床體征及病變

PEDV在小腸絨毛腸細胞的細胞質(zhì)中復(fù)制，導(dǎo)致小腸絨毛縮短，酶和吸收能力降低，導(dǎo)致大量水樣腹瀉，持續(xù)約一周。通常與PEDV感染有關(guān)的其他臨床癥狀包括嘔吐、厭食和發(fā)燒。盡管所有年齡段的豬都受感染，但PED的嚴(yán)重程度在不到一周的哺乳仔豬中較高，這些仔豬可能會因嚴(yán)重脫水而死亡。與3周齡仔豬(2-3日齡)相比，新生仔豬(5-7天)腸上皮細胞更替較慢，這至少部分解釋了這些仔豬對PEDV的較高易感性。

在實驗和自然感染豬的結(jié)腸上皮細胞中也檢測到PEDV，盡管在大腸中未發(fā)現(xiàn)絨毛萎縮。

PEDV的復(fù)制通常局限于腸道，直到最近的一項研究表明，PEDV在3日齡未吃初乳的仔豬肺泡巨噬細胞中復(fù)制，實驗中接種了韓國PEDV野毒株。需要進一步的研究來確認是否其他PEDV分離株也會發(fā)生腸外復(fù)制，以及確定它們的臨床和流行病學(xué)相關(guān)性。PED的兩種流行病學(xué)表現(xiàn)已在豬場上進行了描述。(a)當(dāng)PEDV被引入陰性豬場時，就會發(fā)生流行性PEDV爆發(fā)。該疾病傳播迅速，對所有年齡的豬均有影響，發(fā)病率接近100%。此外，PEDV可持續(xù)存在并在豬場流行，影響斷奶后失去乳源性免疫的仔豬以及新引進的血清陰性母豬。

PED爆發(fā)相關(guān)的死亡率高度依賴于受感染豬只日齡。不到一周的哺乳仔豬死亡率可達80%-100%，而斷奶仔豬的死亡率通常只有1%-3%。在成年豬中，通常未觀察到與PED相關(guān)的死亡。

如前所述，PED疫情的嚴(yán)重程度有所不同。自2010年以來，在亞洲和美國都出現(xiàn)了特別嚴(yán)重的PED疫情。PEDV分離株毒力的差異被認為可以解釋這種差異。從我們的觀點來看，這是當(dāng)今PED面臨的最相關(guān)的問題之一：一個或多個可以解釋爆發(fā)臨床結(jié)果差異的原因。盡管一些報告表明，它們可能與PEDV分離株的毒力差異有關(guān)，但需要在哺乳仔豬中使用豬適應(yīng)病毒(而不是細胞培養(yǎng)適應(yīng)的分離株)進行全面的攻毒研究，以闡明該毒株的作用。

已經(jīng)獲得了一些與不同毒株毒力有關(guān)的見解。

在美國，最近至少有兩種主要的PEDV變種被分子方法鑒定出來。第一種病毒似乎是一種高毒力病毒，與2010年后在幾個亞洲國家的病毒相似，而第二種病毒，即S INDEL變種，與溫和的臨床爆發(fā)有關(guān)。這種S INDEL變異包括S基因的一些特定的插入和缺失，也類似于一些亞洲分離株，其中部分在2010年前被發(fā)現(xiàn)。PEDV CV777也是INDEL分離株雖然處于不同分支(圖1a和b)。對PEDV分離株在歐洲國家(德國、意大利、比利時、2014年和2015年對荷蘭和法國)進行了鑒定，發(fā)現(xiàn)它們均為INDEL分離株，與美國變異株相似。歐洲最近爆發(fā)的PED大多發(fā)生在育肥場，如預(yù)期的那樣，沒有觀察到死亡。然而，最近在烏克蘭嚴(yán)重爆發(fā)的PEDV中發(fā)現(xiàn)的PEDV分離株與來自美國和墨西哥的非INDEL分離株的基因組核苷酸相似性達到99.8%。到目前為止，這是歐洲唯一一份關(guān)于PEDV非INDEL分離株的報告。除了PEDV毒株的毒力差異外，許多其他參數(shù)，包括管理、豬群免疫狀況和群體衛(wèi)生狀況也可以解釋PED爆發(fā)的臨床結(jié)局差異。因此，也有人指出了與其他病毒，特別是與其他腸道病毒(如PDCoV或MRV3)混合感染。從美國PEDV陽性豬場收集的糞便樣本中已檢測到這兩種病毒。在實驗接種的陰性哺乳仔豬中，PDCoV與輕度至中度腹瀉有關(guān)，而MRV3在3日齡仔豬中引起嚴(yán)重腹瀉，死亡率為100%。

Control and prevention

There is no specific treatment for PEDV other than supportive care and symptomatic treatment. Mortality occurs in suckling piglets as a result of dehydration which should be corrected using oral electrolyte solutions. In adult pigs, dry feed should be withdrawn for a period of 12–24 h and then, carefully reintroduced while water should be kept freely available [3, 4]. In order to increase passive immunity to piglets and minimize losses, sows due to farrow in at least 2 weeks can be deliberately exposed to virulent virus by the oral route. A recent study revealed that morbidity was reduced from 100 to 43 % in litters exposed to virulent PEDV when their sows were previously exposed to a mild virulent strain (S INDEL variant) of PEDV [63]. Oral administration of chicken egg-yolk or cow colostrum containing PEDV immunoglobulins could offer an immunoprophylactic defence [64, 65]. The increase in lactogenic immunity is also the aim of PEDV vaccines which are used in pregnant sows. Attenuated or killed vaccines against PEDV have been used in several Asian countries for years [66].However, it has been suggested that live vaccines can revert to virulence and their use and usefulness under field conditions have been questioned [5, 27, 67]. Recently, a PEDV subunit vaccine based on the S protein gene of PEDV as well as a vaccine with killed virus have been licensed in the USA [68], although there are still no studies which prove their efficacy. However, PEDV vaccines have never been used in Europe as the disease was not of sufficient economic importance in this area. In general, PEDV vaccines have been reported to be useful to booster antibody response in animals that have already been infected by PEDV.

As there are no specific treatments for the control and potential eradication of the disease from the herd, preventive measures which preclude the introduction of the virus or new PEDV strains in the area, country or farm are of paramount importance. Supported by the detection methods mentioned in the diagnosis, surveillance should be used to certify that trading of swine or related derivatives do not cause the spread of new strains of the virus. Lorries used in transport have been highlighted as a relevant source of transmission [41] and special attention should be paid in the effectiveness of the cleaning and disinfecting protocols to inactivate and remove the virus. At herd level, basic external biosecurity rules such as quarantine of reposition, ban the entrance of unwashed vehicles, strict visitor policies (time interval between visiting two farms, provide footwear and appropriate clothing, showers and so on) should be carried out without exception and internal biosecurity such as controlling the slurry level, carcasses disposal and carcass bin cleaning, movement of the caretakers on the farm and so on could prevent the establishment of an endemic form of the disease. Finally, many virucidal disinfectants have been shown to be effective in inactivating PEDV. Phenol, quaternary ammonium compounds, glutaraldehyde and bleach are examples of such disinfectants. Water temperature is a crucial factor and temperatures over 60 °C help to inactivate the virus. Proper cleaning and disinfecting of facilities and equipment is crucial to control PEDV.

控制和預(yù)防

除了護理和對癥治療外，PEDV沒有特定的治療方法。哺乳仔豬因脫水而死亡，應(yīng)口服電解質(zhì)溶液。在成年豬中，應(yīng)停用干飼料12-24小時，同時應(yīng)保持水的自由供應(yīng)。為了提高仔豬的被動免疫力，減少損失，可以對至少2周內(nèi)分娩母豬進行返飼。最近的一項研究表明，如果母豬之前接觸過PEDV的一種弱化強毒株(S INDEL變種)，那么暴露于強毒株的仔豬發(fā)病率從100%下降到43%?？诜蠵EDV免疫球蛋白的雞蛋黃或牛初乳可以提供免疫預(yù)防防御。提高乳源性免疫也是PEDV疫苗的目的，該疫苗用于妊娠母豬。針對PEDV的弱毒或滅活疫苗已在一些亞洲國家使用多年。然而，有人提出，弱毒疫苗可以毒力返強，它們在臨床條件下的使用和有效性受到質(zhì)疑。

最近，一種基于PEDV S蛋白基因的PEDV亞單位疫苗和一種滅活病毒疫苗在美國獲得了許可，但仍沒有研究證明其有效性。然而，PEDV疫苗從未在歐洲使用過，因為該疾病沒有足夠的經(jīng)濟重要性。一般來說，PEDV疫苗已被報道對已感染PEDV的豬只增強抗體反應(yīng)是有用的。

截止目前，沒有特定的治療方法來控制和從豬群中根除這種疾病，因此，在該地區(qū)、國家或豬場防止病毒或新的PEDV毒株傳入的預(yù)防措施至關(guān)重要。監(jiān)測應(yīng)用于證明豬或相關(guān)衍生物的交易不會導(dǎo)致新的病毒株的傳播。用于運輸?shù)目ㄜ嚤粡娬{(diào)為相關(guān)的傳播源，應(yīng)特別注意清潔和消毒程序的有效性，以滅活和清除病毒。

在豬群層面，基本的外部生物安全措施，如檢疫、禁止未經(jīng)清洗的車輛進入、嚴(yán)格的訪客政策(訪問兩個豬場之間的時間間隔、提供鞋和適當(dāng)?shù)囊路?、淋浴?應(yīng)毫無例外地進行，而內(nèi)部生物安全措施，如控制泥漿水平、尸體處置和尸體箱清潔、飼養(yǎng)員在豬場的移動等。最后要強調(diào)的是，許多消毒劑已被證明對滅活PEDV是有效的。如苯酚、季銨鹽化合物、戊二醛和漂白劑。消毒液的水溫是一個關(guān)鍵因素，超過60℃的水溫有助于滅活病毒。正確清潔和消毒設(shè)施和設(shè)備是控制PEDV的關(guān)鍵。