Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.     

猪圆环病毒2型的PCR检测方法应用

本研究登录Genbank对猪圆环病毒2型基因的序列进行分析,用Primer 5.0设计了ORF2基因的扩增引物,试图选择一种比较合理的PCR方法检查PCV2感染的病原,以期这种PCR方法可以有效分析猪综合征障碍病毒(PRRSV)、猪细小病毒(PPV)、猪瘟病毒的扩增(HCV)、猪伪狂犬病毒(PRV)等比较常见的病原。研究结果表明,在PCV2进行扩增阳性样品检测中,发现一条异常447 bp的DNA条带,对产物测序结果进行扩增分析,证明其是PCV ORF2基因序列。敏感性检验分析表明检测样本DNA浓度时达到了9.8×10^-4ng/μL。PCR实验具有良好的稳定性和重复性。根据对66例临床病猪的分析表明,在PCV2的检测中阳性感染率是27.26%,这可能受到HCV、PRRSV、PRV、PPV等多种病毒的感染,其中混合感染的比例达到了72.23%。     

猪圆环病毒2型、猪繁殖与呼吸综合征病毒及猪细小病毒混合感染的流行病学调查

根据GenBank上发表的PRRSVORF7、PPVVP2及PCV的基因组序列设计合成引物,建立了分别用于检测PRRSV、PPV和PCV的RT-PCR、PCR及复合PCR方法。应用建立的复合PCR方法对送检的127份病料进行了PCV的检测,对鉴定为PCV2阳性的67份病料再分别进行PRRSV和PPV的检测,以确定猪群中PCV2与PRRSV和,或PPV混合感染情况,结果表明,35份样品表现为PRRSV与PCV2混合感染,占样品总数的52．3％;18份样品表现为PCV2与PPV混合感染,占26．9％。另外,还有一定比例的三重感染,共5个样品,占7．5％。由此可见,猪群中PCV2与PRRSV及PPV混合感染比较普遍。     

猪源胰酶中特异病毒和非特异病毒安全检测简

目的：对猪源胰酶样品进行病毒检测,以评价其病毒安全性。方法：非特异性病毒检测采用致细胞病变、血凝吸附试验和形态学检测方法;特异性病毒检测包括猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪圆环病毒（PCV）、猪细小病毒（PPV）、猪口蹄疫病毒（FMDV）和猪伪狂犬病病毒（PRV）特异性核酸检测,以及CSFV、PPV、PRV特定性抗原蛋白直接免疫荧光检测。结果：受检样品非特异性病毒检测中,未见可观察到的细胞病变产生和病毒粒子,对豚鼠、鸡和人的0型红细胞无凝集现象。特异性病毒检测中,PRRSV、CSFV、PCV、PPV、FMDV和PRV核酸检测均为阴性,CSFV、PPV、PRV免疫荧光检测均为阴性。结论：猪源胰酶样品经非特异性病毒检测和特异性病毒检测均无可检出的病毒存在。     

Prevalence of antibodies to selected viral pathogens in wild boars (Sus scrofa) in Croatia in 2005-06 and 2009-10

We determined prevalence of antibody to selected viral pathogens important for domestic pigs and livestock in 556 wild boar (Sus scrofa) sera collected during 2005-06 and 2009-10 in four counties in Croatia. These counties account for an important part of the Croatian commercial pig production and have a high density of wild boars. Samples were tested for antibodies to porcine parvovirus (PPV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2), swine influenza virus, porcine respiratory and reproductive syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, and swine vesicular disease virus (SVDV). Antibodies to all of the infectious pathogens except SVDV were detected. There was a statistically significant difference in prevalence between the two periods for PPV, ADV, PCV2, PRRSV, and PRCV, with a higher prevalence of PPV and ADV in the 2009-10 period (P<0.05). During the same period, the prevalence of PCV2, PRRSV, and PRCV was lower (P<0.05). Our results provide information on the current disease exposure and health status of wild boars in Croatia and suggest that wild boars may act as a reservoir for several pathogens and a source of infection for domestic pigs and other livestock as well as humans, especially for ADV.     

猪细小病毒LDR-PCR检测方法的建立和应用

为准确特异灵敏地检测猪细小病毒(PPV),建立一种新的LDR-PCR方法。首先在病毒的保守区内设计一对LDR探针,LDR探针两端各连有一段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。以标准质粒为模板,通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,可以特异地检测PPV,与猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)无交叉反应;最低检测限为102个拷贝。利用建立的方法对41例临床样本进行检测,14份样品PPV阳性,与普通PCR检测结果符合率为97.6%。     

Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.     

Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro

The perfection of in vitro maturation in the bitch has yet to be achieved, and is an essential prerequisite for gamete salvage programmes in endangered canine species. In contrast to most mammals, the bitch ovulates an immature oocyte which undergoes meiotic maturation within the oviduct. A model of the oviductal environment may therefore be useful for performing in vitro maturation. This study was performed to investigate the effect of introducing an oviductal element to the culture environment, first with the use of a synthetic oviductal fluid (SOF), and secondly, using coculture with isolated canine oviductal epithelial cells, upon the rate of oocyte maturation in vitro. It was found that there was no difference in the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) after 48 h in culture between SOF containing 0.3% bovine serum albumin (BSA, 45%), containing 4% BSA (36%) and control medium 199 (27%). There was also no difference in oocyte nuclear maturation to metaphase I/anaphase I/metaphase II (MI/AI/MII) after 48 h in culture between SOF containing 0.3% BSA (5%), containing 4% BSA (7%) and control medium 199 (6%). In addition, there was no difference in oocyte nuclear maturation to MI/AI/MII after 96 h between SOF containing 0.3% BSA (0), containing 4% BSA (7%) and control medium 199 (11%). In contrast, the proportion of oocytes undergoing GVBD after 96 h in culture was affected by the treatment used, with 27% in SOF + 0.3% BSA, 62% in SOF + 4% BSA and 63% in medium 199. It was found that there was no difference in the proportion of oocytes undergoing GVBD between the coculture treatments 199 (33%), 199 + cells (37%), coculture medium (30%) and coculture medium + cells (49%), and for oocyte nuclear maturation to MI/AI/MII, between medium 199 (2%), 199 + cells (0), coculture medium (6%) and coculture medium + cells (2%) after 48 h in culture. In addition, there was no difference in oocyte nuclear maturation to GVBD after 96 h between 199 (61%), 199 + cells (59%), coculture medium (65%) and coculture medium + cells (53%). In contrast, the proportion of oocytes maturing to MI/AI/MII after 96 h in culture was affected by the treatment used, with a significant difference between 199 (0), 199 + cells (9%), coculture medium (0) and coculture medium + cells (0). It was shown, therefore, that the culture of oocytes in the SOF improved oocyte nuclear maturation when supplemented with a high concentration of protein and that culture in the presence of oviductal epithelial cells improved oocyte maturation, but only after a prolonged period of time.     

Detection of porcine parvovirus using a taqman-based real-time pcr with primers and probe designed for the NS1 gene

A TaqMan-based real-time polymerase chain reaction (PCR) assay was devised for the detection of porcine parvovirus (PPV). Two primers and a TaqMan probe for the non-structural protein NS1 gene were designed. The detection limit was 1 x 102 DNA copies/μL, and the assay was linear in the range of 1 x 102 to 1 x 10? copies/μL. There was no cross-reaction with porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The assay was specific and reproducible. In 41 clinical samples, PPV was detected in 32 samples with the real-time PCR assay and in only 11 samples with a conventional PCR assay. The real-time assay using the TaqMan-system can therefore be practically used for studying the epidemiology and management of PPV.     

猪圆环病毒2型与猪细小病毒共感染对猪肺泡巨噬细胞吞噬功能及其干扰素表达水平的影响

【目的】分析猪圆环病毒2型(PCV2)与猪细小病毒(PPV)共感染对猪肺泡巨噬细胞(PAM)吞噬功能及其干扰素表达水平的影响,为进一步阐明猪断奶后多系统衰减综合征的发病机制提供实验依据。【方法】将48头5周龄健康仔猪随机分为PCV2组、PPV组、PCV2/PPV组和对照组,每组12头。PCV2和PPV组经口鼻途径分别接种PCV2或PPV,PCV2/PPV组同时接种PCV2和PPV,对照组接种细胞营养液。感染后3、7、14和35d(dpi)从每组随机选择3头剖杀,检测PAM的存活率、吞噬活性及其α1型干扰素(IFN-α1)、γ干扰素(IFN-γ)mRNA的表达水平。【结果】PCV2组和PCV2/PPV组PAM的存活率于3、7、14dpi均低于对照组,35dpi均与对照组PAM的存活率接近,PCV2与PCV2/PPV两组之间差异不显著(P>0.05);3个病毒感染组PAM的吞噬功能均显著下降(P<0.01),其中PCV2/PPV组的吞噬功能低于PCV2组(P<0.05);PCV2/PPV组PAM中IFN-α1和IFN-γ mRNA的水平持续低于PCV2、PPV组和对照组的水平(P<0.01)。【结论】PCV2与PPV共感染没有引起PAM的活力进一步下降,但导致其吞噬功能及IFN-α1和IFN-γmRNA的表达水平持续降低。     

Antibodies to selected pathogens in wild boar (Sus scrofa) from Catalonia (NE Spain)

From 2004 to 2007, blood samples from 273 healthy wild boars (Sus scrofa), culled during the hunting season, were obtained in three areas of Catalonia (NE Spain): Pyrenees, Sant Llorenç del Munt i l’Obac Natural Park (SLM), and Ports de Tortosa i Beseit National Hunting Reserve (PTB). We investigated the presence of antibodies against classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine vesicular disease virus (PVDV), porcine respiratory and reproductive syndrome virus (PRRSV), Aujeszky’s disease virus (ADV), porcine influenza A virus (PIV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Salmonella spp., and Toxoplasma gondii. Four wild boars were suspicious for CSFV, but the infection was discarded with a virus neutralization test, and infection with a border disease virus was confirmed. Negative results were obtained against ASFV and PVDV. Antibodies were detected against PRRSV (3%), ADV (0.8%), PIV (6.4%), PCV2 (64.6%), PPV (54.7%), M. hyopneumoniae (26.6%), E. rhusiopathiae (5.3%), Salmonella spp. (11.3%), and T. gondii (43.5%). In SLM, we detected a higher seroprevalence for PIV and M. hyopneumoniae and a lower seroprevalence for E. rhusiopathiae than in the other two areas. In PTB, seroprevalence was higher for PPV, Salmonella spp., and PCV2. Adult wild boar displayed higher seroprevalence for PPV, PIV, and M. hyopneumoniae, whereas presence of antibodies for Salmonella spp. was higher in juveniles compared with adults and piglets.

     

Improvement of virus safety of an antihemophilc factor IX by virus filtration process

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized productionscale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were (i)v.12 for HAV, (i)t.28 for PPV, (i)u.33 for EMCV, (i)u.51 for HIV, (i)u.17 for BVDV, and (i)u.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.     

Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV were reduced from the initial titer of 6.06, 7.72, and 6.92 log₁₀ TCID₅₀, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67, and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and ≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49, ≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient virus reduction capacity for achievement of a high margin of virus safety.     

Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.     

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.     

Dry-heat treatment process for enhancing viral safety of an antihemophilic factor VIII concentrate prepared from human plasma

Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment (100 degrees for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at 4oC. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were > or =5.55 for HAV, > or =5.87 for EMCV, > or =5.15 for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.     

A serological survey of selected pathogens in wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) in northern Turkey

During the hunting season in March 2012, a total of 93 blood samples were collected from wild boars (Sus scrofa) shot in the area of northern Turkey (Samsun and Gumushane provinces). These blood samples were examined by enzyme immunoassay (ELISA) for the presence of antibodies to classical swine fever virus (CSFV), Aujeszky’s disease virus (ADV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), swine influenza virus (SIV), porcine parvovirus (PPV), swine vesicular disease virus (SVDV), hepatitis E virus (HEV), African swine fever virus (ASFV), porcine rotavirus (PRV), transmissible gastroenteritis virus (TGEV) and bovine viral diarrhoea virus (BVDV). Out of 93 serum samples examined, 65 (69.9 %) were positive for PRV, 22 (23.7 %) were positive for ADV, 5 (5.4 %) were positive for BVDV, 4 (4.3 %) were positive for PPV and 2 (2.2 %) were positive for PRRSV. All sera were negative for ASFV, SVDV, HEV, SIV, PRCV, TGEV and CSFV. The results, recorded for the first time in Turkey, supported the hypothesis that wild boar act as a potential reservoir of selected viruses and thus have a role in the epidemiology of these diseases.     

Retrospective serological survey on selected viral pathogens in wild boar populations in Germany

The objective of this study was to retrospectively evaluate the occurrence of porcine parvovirus (PPV), Aujeszky’s disease virus (ADV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV) in selected wild boar populations in Germany (n  = 1,221). Commercial enzyme linked immunosorbent assay and hemagglutination inhibition tests were used for serological monitoring. The serosurvey revealed seroprevalence rates of 64.28%, 11.26%, 7.87%, 7.84%, 3.82% and 1.59% for PPV, ADV, PRCV, SIV, PRRSV and TGEV, respectively. The seroprevalence rates differed between populations and age classes with the highest number of antibody-positive wild boars in older animals (>1 year old). No antibodies to TGEV were found in Baden–Wuerttemberg and in Mecklenburg–Western Pomerania (investigation period 1997/1998). In addition, sera collected in Mecklenburg–Western Pomerania in 1997/1998 were negative for SIV. Even though the seroprevalence rates established for these viruses, except for PPV, were relatively low, wild boars may act as a reservoir for pathogens and a source of infection for domestic pigs and humans. Based on the epidemiological situation, no risk of a spread of these viruses should emanate from wild boars, neither for wildlife nor for livestock. However, effective and science-based disease monitoring programmes should continuously be carried out in wild boar populations.     

猪圆环病毒2型及猪繁殖与呼吸综合症病毒的快速检测

According to the published genome sequences of Porcine circovirus type 2(PCV2)and Porcine reproductive and respiratory syndrome virus(PRRSV),primers were designed and PCR,RT-PCR were set up for the detection of PCV2 and PRRSV,respectively,With the established methods,38 clinical samples from the respiratory disease pigs were detected for the presence of PCV2 and PRRSV. The results demonstrated that 22 samples were positive for PCV2,27 samples were positive for PRRSV and among the above positive samples,18 samples were positive for both viruses,The data obtained in the present study indicated that PCV2 and PRRSV maybe play an important role in the course of the development of respiratiory diseases.     

Reduction of infectious epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos by non-specific protease

Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced bovine embryos than to zonae of in vivo derived embryos. Currently, the International Embryo Transfer Society recommends that all in vivo derived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contrast to in vivo derived embryos, this method is not effective for disinfecting in vitro produced embryos. Our hypothesis was that a more potent, non-specific protease from Streptomyces griseus (S. griseus) would provide a more effective treatment for virus removal from in vitro produced bovine embryos. Bovine oocytes were matured, fertilized, and cultured in completely defined in vitro conditions. Zygotes were washed according to the procedure outlined by the International Embryo Transfer Society, replacing trypsin with the experimental protease. Experimental incubations were with 0.1% (4 units/ml) protease for 0, 30, 45, 60 and 75s intervals. Embryos were able to withstand exposure to this enzymatic treatment for only 45s before their developmental potential was significantly reduced; 60s exposure was detrimental (P<0.05). Oocytes were exposed to epizootic hemorrhagic disease virus serotype 2 (EHDV-2, 10(6) TCID(50)/ml) during in vitro maturation. Resulting zygotes were washed according to the International Embryo Transfer Society procedure and either exposed to trypsin or protease. Exposure to EHDV-2 prevented cumulus expansion and markedly reduced embryonic development (P<0.05). There were no differences in development among virus exposed groups receiving no treatment or treatment with trypsin or protease. However, proportions of infected embryos were reduced after protease treatment versus positive controls and trypsin treated embryos.