Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection

An indirect, double-antibody sandwich-type ELISA for detection of transmissible gastroenteritis virus (TGEV) was developed, using a solid phase of rabbit hyperimmune serum and a pool of 3 antipeplomer monoclonal antibodies to trap and to detect the virus, respectively. The technique was used to detect viral antigen in feces of pigs that had been infected with the virulent Miller strain, the attenuated Purdue strain, or the Erica strain (a Dutch field isolate) of TGEV. The results were compared with those of a solid-phase immunosorbent electron microscopy (SPIEM) technique for virus detection. Both techniques detected shedding of virulent virus in feces obtained from pigs on the first or second day after infection, and virus excretion continued for 6 to 8 consecutive days. Virus shedding started later in pigs infected with the attenuated Purdue strain of TGEV and lasted only 2 to 4 days. In comparison with the 2 virulent strains, infection with the attenuated strain appeared to be limited to a smaller portion of the small intestine. Of 242 fecal specimens that were tested by use of ELISA and SPIEM, 119 had positive results in both tests. Additionally, virus could be detected by ELISA in 21 and by SPIEM in 16 specimens. Fecal specimens obtained from pigs before infection always reacted negatively by ELISA for TGEV antigen; there was no cross-reactivity with fecal specimens containing porcine rotavirus or porcine epidemic diarrhea virus. The ELISA and SPIEM were found to be specific and sensitive for the detection of TGEV in feces.     

Quantitative transmissible gastroenteritis virus shedding patterns in lactating sows.

To test the role of sows in spreading transmissible gastroenteritis (TGE), 11 sows were intravenously, intranasally, or intramammarily inoculated with virulent virus within 5 days of farrowing. Six of the sows were separated from their offspring, and 5 were allowed to nurse their litters. All sows became clinically ill with sign of anorexia, depression, and fever that persisted until postinoculation day 4 or 5. They shed virus through milk, nasal secretions, and feces, with individual variations occurring in degree and duration of shedding in the 1st week after inoculation. Of 40 pigs separately fed milk samples from the 6 inoculated sows, 19 pigs (47.5%) became sick in 24 to 40 hours, and virus was isolated from them at necropsy. Of 43 pigs in the 5 litters that nursed exposed dams, all became sick with typical signs of TGE, and 29 (67.4%) died in 2 to 9 days. Sows given the single intramammary inoculation of virus developed statistically significant higher levels of TGE virus-neutralizing antibodies than did sows inoculated intravenously or intranasally.     

Cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses

Cross-protection studies between the feline infectious peritonitis (FIP) and the porcine transmissible gastroenteritis (TGE) viruses were conducted in cats, pigs and pregnant gilts. Cats vaccinated with TGE virus developed neutralizing antibodies against TGE virus and low titer antibody against FIP virus detected by an indirect fluorescent antibody technique but were not protected against a virulent FIP virus challenge. Baby pigs and pregnant gilts vaccinated with FIP virus did not develop detectable antibodies to TGE virus. Nevertheless, it appeared that vaccination of swine with FIP virus conferred some immunity against TGE virus infection. Seventeen-day-old pigs vaccinated with two doses of FIP virus had a 67% survival rate following a virulent TGE virus challenge, and 75% of the 3-day-old pigs suckling either FIP or TGE-virus-vaccinated gilts survived virulent TGE virus infection in contrast to 0% survival of baby pigs suckling unvaccinated gilts.     

Cell-mediated immune responses of suckling pigs inoculated with attenuated or virulent transmissible gastroenteritis virus

Two litters of suckling pigs seronegative for transmissible gastroenteritis (TGE) virus were orally inoculated with live attenuated (P115) or virulent (M5C) strains of TGE virus. A third seronegative litter (controls) was given cell culture fluids from uninfected cells. Lymphocytes were collected from blood, spleen, mesenteric lymph nodes, and Peyer patches of euthanatized pigs at 0 day and approximately weekly until 26 days after exposure and at approximately 45 days after exposure. Sera were tested for virus-neutralizing antibody titers by use of plaque reduction. Lymphocytes were tested in a lymphocyte proliferation assay for uptake of [3H]thymidine after incubation with the homologous or the heterologous strain of inactivated TGE virus or uninfected cell culture fluids. Only pigs inoculated with virulent TGE virus developed clinical signs of TGE and shed virus. However, all pigs inoculated with TGE virus seroconverted at 6 days after exposure. Responses of lymphocytes from all sources from TGE virus-inoculated pigs peaked between 6 and 14 days after exposure. Pigs inoculated with virulent TGE virus had higher lymphocyte proliferative responses and neutralizing antibody titers than did pigs inoculated with attenuated TGE virus. Cessation of virus shedding coincided with the peak of lymphocyte proliferative responses. The highest responses were with intestinal lymphocytes (mesenteric lymph nodes and Peyer patches) from pigs inoculated with virulent TGE virus. The responses of intestinal lymphocytes from pigs inoculated with attenuated virus were not significantly different from those of pigs inoculated with cell culture fluid. Lymphocytes collected from all sources, except blood from M5C-inoculated pigs, had significantly (P less than 0.05) higher responses to the homologous than to the heterologous TGE virus stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) using a detergent-solubilized antigen of purified virus was developed for detection of antibody against porcine transmissible gastroenteritis (TGE) virus in swine serum. The ELISA demonstrated antibody responses in pigs immunized intramuscularly with the attenuated TO-163 strain of TGE virus and in pigs orally infected with the virulent Shizuoka strain of the virus. The results of the ELISA were well correlated with those of the neutralization test. These results indicate the usefulness of the ELISA as a serological tool for TGE virus antibody.     

Pathogenesis of porcine enteric calicivirus-like virus in four-day-old gnotobiotic pigs

Eighteen 4-day-old gnotobiotic pigs were orally inoculated with porcine enteric calicivirus-like virus (C strain). Seven additional gnotobiotic pigs served as noninoculated controls. Mild diarrhea developed in all inoculated pigs by postinoculation day (PID) 3 and persisted for 3 to 7 days. Severe diarrhea developed in 2 inoculated pigs between PID 4 and 5. Twelve inoculated and 7 control pigs were euthanatized over a 7-day period. Small intestinal mucosal smears were stained with a fluorescein-conjugated anti-porcine enteric calicivirus-like virus serum. Immunofluorescence was observed in villous epithelial cells (primarily in the duodenum or jejunum) of all inoculated pigs, except for 1 pig euthanatized at PID 7. Villus length was determined in histologic sections of the small intestinal specimens from control and inoculated pigs. Statistically significant (P less than 0.01) villus atrophy was found in the duodenum and/or jejunum of inoculated pigs at PID 3 to 7. These observations were confirmed by scanning electron microscopy, which revealed shortening, blunting, fusion, or absence of villi in the duodenum and jejunum of inoculated pigs at PID 3 to 7. Lesions were not seen in control pigs. Calicivirus-like particles were detected by immune electron microscopy in the large intestinal contents and feces of inoculated pigs from PID 1 to 7.     

Concurrent porcine rotaviral and transmissible gastroenteritis viral infections in a three-day-old conventional pig.

A 3-day-old suckling pig with diarrhea was necropsied, and immunofluorescent microscopic examination of the small intestinal mucosa, together with immune electron microscopic examination of the large intestinal contents, provided a presumptive diagnosis of a concurrent infection with transmissible gastroenteritis (TGE) virus and porcine rotavirus. Immunofluorescent microscopic, immune electron microscopic, and serologic data obtained from gnotobiotic pigs experimentally inoculated with the large intestinal contents of the suckling pig confirmed this diagnosis. Two gnotobiotic pigs, convalescent from previous TGE viral infections, became infected with porcine rotavirus only. However, another gnotobiotic pig, convalescent from a previous porcine rotaviral infection, became infected with TGE virus only, following inoculation with the large intestinal contents of the suckling pig.     

Detection of duck plague virus by reverse passive hemagglutination test

A reverse passive hemagglutination (RPHA) test was developed to detect duck plague virus (DPV). The technique used sheep erythrocytes stabilized with formaldehyde and pyruvaldehyde and coated with immunoglobulin G (IgG) containing anti-DPV antibody prepared from antiserum produced in sheep. Optimum coating of stabilized erythrocytes occurred at 25 C and pH 4.0 with a concentration of IgG of 20-40 micrograms/ml and a 90-min incubation period. The coated cells were stable for 40 days when stored at 4 C or for at least 4 months (the longest period tested) when frozen at -70 C or -196 C. The RPHA test was conducted at 25 C and read after 3 hours. The high specificity of the test is indicated by the absence of cross-reactions with heterologous virus strains, with specimens prepared from normal duck livers, and with normal chicken embryo chorioallantoic fluid, as well as by the inhibition of hemagglutination only with DPV antiserum. The RPHA test detected six strains of DPV in all virus-containing specimens as well as the immunofluorescence (IF) test did; however, conventional plaque assays (PA) failed to detect virus in five specimens that contained three non-plaque-forming strains of DPV. The mean quantity of DPV that could be detected in the RPHA test was 25 plaque-forming units or 65 fluorescent units per ml. Although the RPHA test was less sensitive than either the PA or the IF test, there was a positive correlation in the titers of DPV antigens between all three tests. The RPHA test is a rapid, simple procedure that is sufficiently sensitive for diagnostic detection of DPV in acute infections, especially in tissues of ducks dying of the disease.     

The Pattern of Enteroviral Infections in a Herd of Swine

Twenty-six pigs from four litters in a healthy herd of swine were examined periodically for the fecal excretion of viruses by the use of porcine kidney cell cultures. Viruses were initially isolated from all pigs between 34 to 64 days of age. The pigs within each litter began shedding virus in their feces approximately at the same time, usually within one week, and the type of virus initially recovered was usually the same. Subsequently, waves of infection with different enteroviruses appeared to occur during the observation period of six months. At least six antigenically different viruses were isolated from this herd over a 26-month period. Most, if not all, of these viruses were considered to belong to the enterovirus group. No disease was associated with these enteroviral infections.

The colostrum and milk of sows contained significant amounts of enteroviral antibodies. Prior to nursing, the serum of new-born pig contained no enteroviral antibodies but, shortly after nursing, high titers of such antibodies were present in the serum. Antibodies were detected in the feces of suckling pigs.

     

Intrafetal inoculation of swine with transmissible gastroenteritis virus.

Fetuses in 3 sows were inoculated (intramuscularly) with transmissible gastroenteritis (TGE) virus on 95th, 77th, and 74th days of the gestation. At 15, 14, and 37 days later (or days when pigs were obtained by hysterectomy), there was evidence of intestinal localization of virus, with villous atrophy and subsequent repair. All intrafetal-inoculated pigs became serologic-positive for TGE. A noninoculated pig shown to be seropositive for TGE at 15 days of age (after hysterectomy) was resistant to challenge exposure with virulent TGE virus given on the 32nd day, in contrast to 3 seronegative littermates that developed typical disease when challenge exposed.     

Isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter.

A virologic survey was conducted to determine the frequency of transmissible gastroenteritis (TGE) virus infection in farm-raised sows. Pharyngeal swab specimens collected in an abattoir were examined for TGE virus by inoculation onto swine-testes cell culures. The virus was detected in 61 (3%) of a sample of 2,058 Iowa sows after slaughter. All TGE viral isolates, given orally to 2- or 3-day-old pigs, caused acute gastroenteritis and in some cases death. All pigs that recovered from illness had serum antibody to TGE virus.     

Comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites A and X of the S glycoprotein.

Pigs were inoculated with various strains of transmissible gastroenteritis virus (TGEV) or with porcine respiratory coronavirus (PRCV), and antigenic site-specific antibody responses were compared. A blocking-ELISA was used to study to what extent antibodies in convalescent sera interfered with the binding of monoclonal antibodies (MAB) 57.16 or 57.110 to the attenuated TGEV/Purdue virus. Monoclonal antibody 57.16 is directed against the A site on the peplomer, neutralizes virus, and recognizes TGEV and PRCV. Monoclonal antibody 57.110 is directed against the X site on the peplomer, but does not neutralize virus, and recognizes only TGEV. Antibodies directed against TGEV and PRCV could be detected in a blocking ELISA, using MAB 57.16 as a conjugate. Antibodies directed against both viruses were detectable as early as 1 week after inoculation. Antibody titers correlated well with those in a virus-neutralization test. Antibodies against TGEV could be detected in a blocking ELISA, using MAB 57.110 as a conjugate. Such antibodies were not induced by a PRCV infection. In the blocking ELISA, using MAB 57.110 as a conjugate, antibodies were detectable as early as 2 weeks after inoculation. There was a significant difference between antibody titers reached after infection with various TGEV strains, however. This difference is ascribed to a variation of the antigenic site defined by MAB 57.110 in TGEV strains. Conditions for a differential test for TGE serodiagnosis, and for serologic discrimination between TGEV- and PRCV-infected pigs, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

伪狂犬病弱毒株的分离鉴定及生物学特性的研究

在流行病学调查中分离到1株病毒,经鉴定为伪狂犬病弱毒株,定名为F971株。分离病毒经克隆纯化后测得其毒价为10^7.59TCID50/ml,通过细胞中和试验表明分离病毒能也有效地被猪伪狂犬病毒闽A株阳性血清中和。病毒在电镜下可以清楚地观察到囊膜及外周纤突。分离株对3日龄乳鼠有一定的致病力,但对家兔、3日龄乳猪及妊娠母猪都有很高的安全性。用不同的剂量10^0、10^-1、10^-2肌肉注射3日龄乳猪后14天用10^5.7TCID50伪狂犬病强毒攻击,所有试验仔猪均得到保护。用分离株免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵御10^5.7TCID50强毒的攻击。用ELISA普查试剂盒测定免疫猪抗体,结果均为阳性,而用g^1-ELISA试剂盒测定抗体时,结果均为阴性。证明分离株具有缺损g^1糖蛋白的特性。综合上述特性,确定F971为1株g^1糖蛋白缺损的猪伪狂犬病弱毒株。     

Efficacy of vaccination of sows with serologically related coronaviruses for control of transmissible gastroenteritis in nursing pigs

Three groups of pregnant sows were vaccinated at 8 and 2 weeks before parturition with tissue culture-adapted feline infectious peritonitis (FIP) virus, porcine transmissible gastroenteritis (TGE) small-plaque (SP) virus from a persistently infected cell line, or noninfected cell culture fluids (controls). Pigs nursing vaccinated sows were orally challenge exposed with virulent TGE virus when they were 1 to 3 days old. The morbidity of the nursing pigs was 48% in the SP-TGE group, 82% in the FIP group, and 93% in the controls. The survival rate among the nursing pigs was 77% in the SP-TGE groups, 48% in the FIP group, and 14% in the controls. Virus-neutralizing antibodies of immunoglobulin A were detected in colostrum and milk of the SP-TGE group, but not in the FIP or control groups.     

Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages

This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.     

A Lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model

Transmission of Lawsonia intracellularis from experimentally inoculated pigs to naive swine was demonstrated in this study. The study was conducted using conventional pigs divided into three groups as follows: principles inoculated with L. intracellularis, sentinels, and controls. The pigs were inoculated and paired on 13 and 9 days post-inoculation with a sentinel pig for 7 days. Fecal samples and serum samples were collected throughout the study for polymerase chain reaction (PCR) and antibody testing by indirect fluorescent antibody techniques. After co-mingling, the inoculated group was necropsied; sentinel and control pigs were necropsied 7-14 days later. The intestinal tracts were evaluated grossly and microscopically for lesions. PCR was performed on intestinal mucosal scrapings and feces. Warthin-Starry and fluorescent antibody staining procedures were conducted to confirm colonization with L. intracellularis. Gross and microscopic lesions typical of porcine proliferative enteropathy (PPE) were observed in both the inoculated and sentinel groups. Transmission was demonstrated from inoculated principle pigs to sentinel pigs. PCR results detected cyclical shedding of L. intracellularis in the feces. Seroconversion occurred in pigs that were exposed to L. intracellularis. From this study, it was demonstrated that transmission of L. intracellularis can occur easily in an environment with experimentally infected pigs and that PCR can be a useful tool to monitor fecal shedding of the organism.     

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.     

利用宏基因组学鉴定藏猪腹泻粪便病毒种群

为进一步了解藏猪腹泻粪便中病毒种群，从青藏高原地区16个藏猪场采集腹泻粪便样本146份，将样本混合成一个pool样，提取RNA反转录成cDNA，建库后利用TruSeq Illumina测序，对测序的序列进行整理分析及相关病毒的分子特征进行研究。数据分析结果表明：藏猪腹泻粪便样本病毒种群包括11个病毒科的19种病毒，主要以线性和环状的小DNA病毒为主，如猪粪相关环状病毒7型、猪腺病毒和猪博卡病毒（PBoV）等；与腹泻相关的病原有猪流行性腹泻病毒（PEDV）、猪圆环病毒2型（PCV-2）和牛病毒性腹泻病毒1型（BVDV-1）3种；与腹泻相关的新发病原有porcine bufavirus、rabovirus和pasivirus 3种。利用SOAP软件组装出猪细小病毒6型（PPV-6）、PCV-2、PBoV-2完整或接近全长的基因组和戊型肝炎病毒（HEV）完整的ORF2基因序列，系统发育结果显示PPV-6和PCV-2与参考毒株有较近的遗传进化关系，PBoV-2与参考毒株有较远的遗传进化关系，单独聚为一簇，可能是一个全新的基因型。为进一步调查PCV-2在藏猪腹泻粪便中的检出率，对采集146份样本进行检测，检出率为10.96%（16/146，95% CI：6.4%~17.2%），且与四川地区PCV-2流行株有较近的亲缘关系。本研究发现藏猪腹泻粪便中病毒种类复杂多样，且可能存在与其他动物和人交叉感染情况，具有重要的公共卫生学意义，也为藏猪腹泻病防控提供一定的理论依据。     

3种非猪瘟病毒单独或混合感染对猪瘟弱毒疫苗免疫效力的影响

将 2 0头 9月龄左右猪瘟、伪狂犬、猪繁殖与呼吸障碍综合征抗原、抗体阴性猪分成 6组 ,分别利用猪细小病毒(PPV)、猪伪狂犬病毒 (PRV)和猪繁殖与呼吸障碍综合征病毒 (PRRSV)单独或混合感染。 7d后连同对照猪 4头 ,免疫接种猪瘟兔化弱毒疫苗 (HCL V) ,13d后连同 4头阴性对照猪一起攻击猪瘟石门强毒。整个试验期间分别每天测温 ,观察临床症状 ,每周采集扁桃体和血样做各种病毒抗原及抗体检测。结果表明 ,非猪瘟病毒感染 7d后 ,所有各组猪均从体内检测到了相应感染的病原 ,表明 3种非猪瘟病毒感染成功。在攻击猪瘟石门强毒后 2周 ,感染了非猪瘟病毒后接种 HCL V疫苗的 4个免疫组 12头猪除 1头外 ,11头全为猪瘟病毒 (HCV)抗原检测阳性 ,且多呈强阳性 ;而单一 HCL V疫苗免疫组在猪瘟强毒攻击后检测不到 HCV;所有 HCL V疫苗免疫猪均存活 ,而非免疫对照组 4头猪全部在攻毒 16 d内死亡。