Clinical, virologic, and histopathologic observations of induced porcine parvovirus infection in boars

Twelve 8- to 12-month-old crossbred boars were inoculated with a virulent strain (NADL-8) of porcine parvovirus (PPV). Hemicastrations were performed on 6 boars 3, 7, 10, 14, 21, and 28 days after an IM injection of 10(8) median cell culture infectious dose (CCID50) of PPV (n = 3) or injection of 10(7.4) CCID50 given intratesticularly (IT, n = 3). Noninfected cell culture medium (0.25 ml) was injected into each testicle of a 7th boar (IT inoculated control). Virus or viral antigen was detected in testicular and epididymal tissues up to 14 days after inoculation. Direct immunofluorescence indicated that viral antigen was mainly associated with the vasculature of the interstitium. Microscopic lesions were not evident in the testicles and epididymides of IM inoculated boars. Acute-to-chronic testicular degeneration was evident in the IT inoculated boars, as well as in the IT inoculated control boar. Six boars were inoculated IM or orally/nasally with 10(7.9) CCID50 of PPV. Semen was collected twice weekly for 8 weeks after inoculation. Virus was not detected in any ejaculates. Semen also was collected from 4 boars for 5 weeks before inoculation, and preinoculation and post-inoculation semen quality was compared. Pronounced changes in sperm output, ejaculate volume, motility, or morphologic defects were not observed. The reproductive consequences of experimental PPV infection in boars were minimal because reproductive function was unaffected and venereal transmission of PPV was not detected.     

Transmission of classical swine fever virus by artificial insemination.

Classical swine fever (CSF) virus was introduced into an artificial insemination centre during the CSF epizootic of 1997-1998 in the Netherlands. The risk of further spread of CSF virus via contaminated semen was recognised, but could not be assessed because scientific data on this issue were not available. An animal experiment was performed to determine whether CSF virus could be transmitted via artificial insemination with contaminated semen. Three boars were inoculated with a CSF virus field isolate and from Day 5 till Day 18 thereafter, ejaculates were collected and prepared for insemination. Ruttish sows were inseminated with the extended semen from Day 5 till Day 18 after inoculation of the boars. All the inoculated boars remained healthy throughout the experiment and developed CSF neutralising antibodies between 14 and 21 days after inoculation. Virus was isolated from several semen samples collected from 5 till 11 days after inoculation. Two out of six sows inseminated with CSF contaminated semen seroconverted after insemination. All the other sows remained seronegative. In the foetuses of both the seropositive sows, CSF virus was detected at approximately 35 days post insemination. These results demonstrate that adult boars infected with CSF virus can excrete virus with semen and can, subsequently, transmit the virus to sows and their foetuses via artificial insemination.     

The effect of pseudorabies (Aujeszky''s) virus infection on young mature boars and boar fertility.

This study was designed to determine the effects of experimental inoculation with pseudorabies virus on the reproductive tracts of young adult boars. Pseudorabies virus was inoculated intranasally into 12 boars and intrapreputially into four boars. All animals seroconverted after nasal or preputial inoculation. Semen abnormalities were observed 21 days postinoculation with partial recovery by 50 days postinoculation. Virus was isolated from the preputial sheath of two intrapreputially inoculated boars 12 days postinoculation. It was concluded that pseudorabies virus infection can be established via preputial inoculation and that decreased spermatogenesis and infertility can result.     

Pathogenicity of a skin isolate of porcine parvovirus in swine fetuses

The pathogenic properties of a skin isolate of porcine parvovirus (PPV), designated Kresse isolate, were compared with NADL-8 isolate, a prototype isolate of PPV, by in utero inoculation of mid-term and late-term gestation swine fetuses. Fetuses from pregnant sows of mid-gestation were inoculated with either NADL-8 or Kresse virus. Both isolates were highly pathogenic to mid-gestation fetuses. In contrast, dramatic differences in pathogenicity between these 2 isolates were observed in fetuses inoculated late in gestation. Such fetuses from each of 4 sows were inoculated with NADL-8 or Kresse virus isolate and sacrificed at 10, 18, 21, or 23 days postinoculation (PI). NADL-8-inoculated fetuses were grossly normal. The pathogenic effects of Kresse isolate were evident by gross pathology in fetuses collected at 18, 21, and 23 days PI, but not at 10 days PI. Hemagglutination (HA) and fluorescent antibody (FA) methods were used to identify virus in various tissues of late-gestation fetuses collected at 10 and 21 days PI. At 10 days PI, HA antigens were detected only in livers of NADL-8-inoculated fetuses, but in all tissues examined of Kresse-inoculated fetuses, including the brain. PPV specific fluorescence was demonstrated in tissues of fetuses inoculated with NADL-8 and Kresse virus. The major difference was that virus antigen was found in the brains of fetuses inoculated with Kresse virus, but not in NADL-8 infected fetuses. At 21 days PI, HA antigen was not detected in any of the tissues of fetuses inoculated with NADL-8 virus, with PPV specific fluorescence by FA being found only in the kidney. However, fetuses inoculated with Kresse virus displayed HA antigen in liver and PPV-specific fluorescence in all tissues tested including the brain. Both isolates induced similar antibody responses, 1:128 to 1:256 at 10 days and 1:512 to 1:1024 at 21 days PI. In addition, immunoglobulin G (IgG) deposits were demonstrated in kidneys and skin of fetuses inoculated with Kresse virus and IgM in brain, but not in tissues from fetuses inoculated with NADL-8 virus.     

Detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from Yorkshire, Hampshire, and Landrace boars.

Because transmission of porcine reproductive and respiratory syndrome virus (PRRSV) can occur through boar semen, it is important to identify persistently infected boars. However, even for boars given the same PRRSV strain and dose, variability in the duration of viral shedding in semen has been observed, suggesting that host factors are involved in PRRSV persistence. To determine whether there are host genetic factors, particularly litter and breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine breeds were used for this study. It was also determined whether PRRSV could be detected for a longer period of time in serum, semen, or peripheral blood mononuclear cells (PBMC) and if PRRSV could still be detected in tissues after these antemortem specimens were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire, and 2 Landrace PRRSV-naive boars were obtained and inoculated intranasally with a wild-type PRRSV isolate (SD-23983). All boars within each breed were from the same litter, and litters were within 9 days of age. Serum and PBMC were collected twice weekly from each boar and analyzed for the presence of PRRSV by virus isolation and the polymerase chain reaction (PCR). Serum was also used to obtain virus neutralization titers and enzyme-linked immunosorbent assay S/P values. Semen was collected twice weekly from 7 of 8 boars and analyzed by PCR. After all specimens were PRRSV negative for a minimum of 2-3 weeks, each boar was euthanized, and 21 tissues plus saliva, serum, feces, and urine were collected. All postmortem specimens were evaluated by virus isolation. Specimens that were PRRSV negative by virus isolation were then evaluated by PCR. The mean number of days (+/-SD) for the duration of PRRSV shedding in semen was 51+/-26.9 days, 7.5+/-4.9 days, and 28.3+/-17.5 days for Landrace, Yorkshire, and Hampshire boars, respectively. Because of small sample sizes and large SDs, the differences in duration of PRRSV shedding in semen between breeds were not considered significant. However, the trend suggested that Yorkshire boars were more resistant to PRRSV shedding in semen than were Landrace boars, requiring further investigation using a larger numbers of boars. PRRSV was detected for a longer period in semen than in serum or PBMC in 4 of 7 boars. Viremia could be detected for a longer period in serum than in PBMC in 6 of 8 boars. After a minimum of 2-3 weeks of PRRSV-negative serum, semen, and PBMC, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus isolation, indicating that boars still harbor PRRSV within the tonsil even though antemortem specimens are PRRSV negative.     

Effect of intratesticular inoculation with Aujeszky's disease virus on genital organs of boars

Ten 8-10-month-old Belgian Landrace boars were intratesticularly inoculated with 500 TCID50 of a virulent Belgian Aujeszky's disease virus (ADV) isolate (75V19) in 0.1 ml volume. One control boar was similarly inoculated with phosphate-buffered saline solution. The genital organs of six inoculated boars were examined by virus isolation and immunofluorescence. In spite of high virus titers, the fluorescence in the testicles remained limited to a few small foci in the interstitial connective tissue and tunica albuginea at or close to the inoculation site. Neither virus replication, necrosis nor inflammatory lesions could be demonstrated in the epithelium of the seminiferous tubules. However, virus replication was regularly demonstrated in the serosa covering testicles, plexus pampiniformis, ductus deferens and tunica vaginalis. Virus was also isolated from the scrotal fluid. It is suggested that the serosa is the primary target tissue for ADV. The other four boars were inoculated to study the effect of ADV on semen. Severe morphologic alteration and lowered sperm cell concentrations were observed during several weeks after inoculation or until slaughter at 47, 53 and 58 days post inoculation. Virus was isolated from semen of only two out of four boars examined at 9 and 10 days post inoculation.     

二乙烯亚胺对猪细小病毒的灭活作用

使用新型灭活剂二乙烯亚胺(binary ethylenimine,BEI)对猪细小病毒(Porcine parvovirus,PPV)进行了灭活试验,通过ST传代细胞接种法观察病毒灭活后是否出现细胞病变,并结合血凝试验检测灭活效果,确定最佳灭活方法。用3～5日龄乳鼠检测BEI灭活后的病毒培养物和相应制备疫苗的安全性,并用豚鼠检测该灭活工艺制备疫苗的效果,与传统甲醛灭活进行了比较。结果显示,终浓度为1‰的BEI在32℃情况下经20 h即可彻底灭活PPV病毒;BEI灭活的病毒制备的疫苗免疫豚鼠较甲醛灭活病毒产生较高的血凝抑制抗体。     

Testicular changes observed in boars following experimental inoculation with pseudorabies (Aujeszky''s) virus.

Four boars were inoculated intranasally with pseudorabies virus to determine if microscopic testicular changes occurred as a result of infection. Testicular biopsies and semen samples were taken at two, four and six weeks postinoculation and the boars were castrated immediately after the last sample collection. Testicular samples and semen were cultured to determine if the virus was present. Pseudorabies virus was not isolated from the semen or testicular tissue. Virus was isolated from trigeminal ganglia at necropsy and from nasal swabs taken one day after castration. Consequently, a time of high risk for shed of the virus from clinically normal carrier animals is immediately following castration. Gross changes were not observed in testicular tissues and microscopic changes in the testicles were the result of biopsy. Lesions consistent with pseudorabies virus infection were observed in the central nervous system of all inoculated boars. Temporary lowered fertility may result from the effects of elevated body temperature on spermatogenesis during acute clinical disease. However, it appears that the strain of pseudorabies virus used, lacked the ability to infect and/or replicate in the boars' reproductive tracts.     

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.     

Infectivity of porcine circovirus type 2 DNA in semen from experimentally-infected boars

Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.     

种公猪精液中与繁殖障碍有关的6种病毒的检测

采用PCR和RT-PCR技术,于2006年4月～10月对上海及其周边地区的30个猪场和人工授精站的生产公猪精液样品355份进行了猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒(PCV)、猪瘟病毒(CSFV)和日本脑炎病毒(JEV)等6种与猪繁殖障碍有关的病毒的检测,结果表明,日本脑炎病毒检测为阴性,检出PRRSV、PRV、CSFV、PPV和PCV阳性数和阳性率分别为6份(1.69%)、9份(2.54%)、5份(1.41%)、75份(21.1%)、6份(1.69%),有一个猪场的5份样品存在PRV和PPV混合感染.     

猪细小病毒病毒样颗粒研究进展

猪细小病毒(PPV)是引起母猪繁殖障碍和仔猪死亡的主要病原,疫苗免疫预防是控制该病的主要手段。由于对生物安全的担心,目前国内使用的疫苗仍以灭活苗为主。病毒样颗粒(VLPs)疫苗以其安全性高、免疫原性好成为各类病毒疫苗研究的热门方向。猪细小病毒病毒样颗粒(PPV-VLPs)是不含PPV DNA的空衣壳结构,由PPV VP2结构蛋白体外自行组装形成,形态上与天然病毒粒子相似,具有很强的免疫原性和生物学活性。论文就VLPs疫苗的免疫机制及PPV-VLPs的组装及其在国内外的研究进行综述,为PPV-VLPs研究提供参考。     

Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus.

Cesarean-derived, colostrum-deprived pigs (n = 23) were inoculated intranasally and subcutaneously with a low cell culture passage of type 2 porcine circovirus. In 11 pigs, a persistent fever that lasted 7-17 days began 12-15 days after inoculation with virus. Additional signs of disease in those 11 pigs included depression (11 of 11 pigs), palpable enlargement of inguinal, prefemoral, and popliteal lymph nodes (11 of 11), icterus (6 of 11), and hyperpnea (2 of 11). The remaining 12 pigs had fever that occurred intermittently for 2-4 days between days 12 and 20 postinoculation. Overt signs of disease in those pigs were limited to palpable enlargement of inguinal and popliteal lymph nodes (9 of 12 pigs). When compared with control pigs of similar age, the average daily rate of weight gain for all pigs inoculated with virus was less over a 2-week period that began 2 weeks post inoculation. At postmortem examination, lymph node enlargement was seen in 14 of 14 pigs euthanized between days 20 and 28 postinoculation. Lymph node enlargement was especially prominent in pigs that developed a persistent fever. Microscopic lesions noted in pigs that developed a persistent fever included cellular depletion in lymphoid tissues; hepatic cell necrosis; and lymphogranulomatous inflammation of lymph nodes, Peyer's patches of the intestine, liver, kidney, and heart. Virus was isolated with varying frequency from nasal, rectal, or tonsil swab specimens, buffy coat, serum, urine, and lung lavage fluid obtained antemortem or postmortem. Virus was isolated from or viral DNA was detected in a variety of tissues obtained postmortem up to 125 days postinoculation. Antibody against type 2 porcine circovirus usually was detected in serum between 15 and 20 days postinoculation; however, antibody against virus was not detected in serum from 4 pigs euthanized 20-24 days postinoculation. Direct contact with pigs inoculated with virus 42 days previously resulted in transmission of virus to 3 of 3 control pigs.     

Semen alterations in porcine rubulavirus-infected boars are related to viral excretion and have implications for artificial insemination

Porcine rubulavirus (PoRV), also known as blue eye disease (BED) of swine, causes respiratory and reproductive problems in pigs at several developmental stages. To study the effect of PoRV infection on semen production, five boars were infected with 1 x 10(6) TCID(50)/ml of PoRV strain PAC-3 and evaluated for 59 days post inoculation (DPI). Infected boars developed reproductive tract pathology that included swelling of the testes and epididymides. Analysis of the semen showed that the infection had little effect on semen production in four animals, but semen from one boar showed severe alterations in sperm concentration, motility, and morphology. When motility was analyzed in BTS-diluted semen after 24, 48, or 72 h, alterations were detected in all boars. Furthermore, viral antigen was detected in semen, the seminal plasma fraction, or sperm fraction from all boars. These results showed that PoRV is excreted via semen and, therefore, artificial insemination is a potential route of dissemination.     

Detection of mink enteritis virus in mink feces, using enzyme-linked immunosorbent assay, hemagglutination, and electron microscopy

Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA.     

Hormonal changes in sows after induced porcine parvovirus infection in early pregnancy

Hormonal changes, lesions, and virus isolation studies were determined in sows after uterine artery inoculation with porcine parvovirus [( PPV], strain NADL-8) in early pregnancy. Two sows were given PPV on days 14 or 16 and were euthanatized and necropsied on day 35 after twice daily plasma collection for hormone measurement. Parvovirus was given to 4 sows on day 14 and to 4 sows on day 21 with 5 times daily plasma samples collected for 1 week. Sows were examined on days 21 and 28, respectively. Four control sows in each group on days 14 and 21 were given a placebo injection and were similarly studied. All embryos in all but 1 sow given PPV were in various stages of resorption at necropsy. Normal embryos were present in all control sows. Estrone sulfate values increased logarithmically, progesterone values remained stable, and concentrations of 13, 14-dihydro-15-keto-prostaglandin (PG) F2 alpha (PGFM), a PGF2 alpha metabolite, were less than 200 ng/ml for sows given a placebo. In contrast, sows with resorbing embryos did not have an increase in estrone sulfate values. A decrease in plasma progesterone values occurred in 9 of 10 sows inoculated with PPV; this decrease was accompanied by greater than or equal to 1 marked increase in PGFM concentrations. Quantitative assessment of the uterus revealed significantly greater cytoplasmic density in endometrial and glandular cell (P less than 0.01), a greater glandular epithelium height (P less than 0.05), and twice the number of glands (P less than 0.05) in control sows, compared with values in sows inoculated with PPV.(ABSTRACT TRUNCATED AT 250 WORDS)     

一例猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染的诊断

山东德州某猪场发生猪高热、呼吸系统疾病甚至死亡的疫情。采集病料提取病变组织总DNA或RNA进行猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒、猪瘟病毒的PCR或RT-PCR检测。PCR扩增出353 bp的猪圆环病毒2型特异性条带。同时进行细菌分离培养、生化鉴定等试验,诊断为猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染。