A histopathologic,immunohistochemical, and ultrastructural study of the intestine in pigs inoculated with classical swine fever virus

The aim of this study was to report on the lesions occurring in the intestine during experimental classical swine fever (CSF) and to clarify the nature of infected cells and the distribution of viral antigen. Thirty-two pigs were inoculated with the virulent CSF virus (CSFV) isolate Alfort 187 and slaughtered from 2 to 15 postinoculation days; four animals of similar background served as a control group. Immunohistochemistry, electron microscopy, and the transferase-mediated deoxyuridine triphosphate nick-end labeling method were used to detect viral antigens and apoptosis. The results showed progressive lymphoid depletion and mucosal necrosis. The lymphoid depletion could have been caused by apoptosis of lymphocytes but could not be directly attributed to the effect of CSFV on these cells. Vascular changes, pathogenic bacteria, and viral infection of epithelial cells were ruled out as causes of necrotic lesions. However, large virally infected monocytes-macrophages with ultrastructural changes indicative of activation were observed in the intestine. This suggests that monocytes-macrophages play an important role in the pathogenesis of intestinal lesions. An understanding of the function of these cells will require additional study.     

猪瘟病毒低毒力毒株FJFQ株的分离鉴定

从福建某猪场分离到 1 株病毒,其在PK 15细胞上的毒价为 106.5 TCID50/mL,该病毒能被猪瘟病毒高免血清所中和(效价为1∶8)。通过 RT -PCR 扩增出猪瘟病毒约250 bp的E2蛋白主要抗原编码区序列,其与几株已发表毒株序列的核苷酸及氨基酸同源性分别为79.9%~87.9%,77.7%~86.6%,与Alfort 株同属于基因二群。经本动物传3代均不表现明显的临床症状。用猪瘟兔化弱毒疫苗免疫后以此分离毒作强攻进行免疫保护相关实验,结果免疫组猪在攻毒前及攻毒后扁桃体 HCFA检测均为阴性,对照组猪扁桃体HCFA于攻毒后1周开始出现阳性结果,且一直持续到试验结束。用分离株免疫本动物后再攻石门毒, 2 头试验猪中 1 头死亡,1头出现临床症状。初步说明,所分离的病毒为猪瘟病毒(命名为CSFV- FJFQ株),可能是一株低毒力毒株,且其免疫原性不好。     

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.     

The sustainability, feasibility and desirability of breeding livestock for disease resistance

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak.     

Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs

The clinical diagnosis of classical swine fever (CSF) still caused problems to the veterinarians during the last decade. The primary CSF outbreak was often detected too late and, meanwhile, the virus had spread. Consequently, the recent classical swine fever virus isolates (CSFV) were suspected to be of low virulence. The purpose of the study was to quantify the virulence of four recent CSFV by evaluating the clinical and pathological signs caused by different CSFV. Pigs of the same breed and age group were inoculated intranasally with CSFV from recent epidemics in European Union (EU) member states. The CSFV used are registered in the data base of the EU Reference Laboratory for CSF and belong to different genotypes: 2.1, 2.2 and 2.3 respectively. Clinical signs of CSF were evaluated by using a score system suggested previously (Mittelholzer et al., 2000: Vet. Microbiol. 74, 293). For the evaluation of pathological lesions, a new pathological score was introduced. The four CSFV tested here were classified as moderately virulent in general, although one CSFV may cause different clinical courses, ranging from highly virulent to avirulent. This indicates the importance of additional factors in the host animal for virulence. Differences in the clinical and pathological signs between these four recent CSFV were rather minor, emphasizing that the genetic typing of CSFV is absolutely essential. Differences towards former CSFV (e.g. reference virus strain Alfort 187) were more pronounced, especially regarding the onset and duration of the disease, the occurrence of skin haemorrhages and pathological lesions of kidney, subcutis and serosae. It is concluded that clinical diagnosis of CSF is rather difficult in pigs up to 14 days post-CSFV infection using these four CSFV, emphasizing the need for careful differential diagnosis and the laboratory investigation for CSF at an early stage.     

Porcine circovirus type 2 (PCV2) infection decreases the efficacy of an attenuated classical swine fever virus (CSFV) vaccine

ABSTRACT: The Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), is an important tool for the prevention and control of CSFV infection and is widely and routinely used in most CSF endemic areas, including Taiwan. The aim of this study was to investigate whether PCV2 infection affects the efficacy of the LPC vaccine. Eighteen 6-week-old, cesarean-derived and colostrum-deprived (CDCD), crossbred pigs were randomly assigned to four groups. A total of 105.3 TCID50 of PCV2 was experimentally inoculated into pigs through both intranasal and intramuscular routes at 0 days post-inoculation (dpi) followed by LPC vaccination 12 days later. All the animals were challenged with wild-type CSFV (ALD stain) at 27 dpi and euthanized at 45 dpi. Following CSFV challenge, the LPC-vaccinated pigs pre-inoculated with PCV2 showed transient fever, viremia, and viral shedding in the saliva and feces. The number of IgM+, CD4+CD8-CD25+, CD4+CD8+CD25+, and CD4-CD8+CD25+ lymphocyte subsets and the level of neutralizing antibodies against CSFV were significantly higher in the animals with LPC vaccination alone than in the pigs with PCV2 inoculation/LPC vaccination. In addition, PCV2-derived inhibition of the CSFV-specific cell proliferative response of peripheral blood mononuclear cells (PBMCs) was demonstrated in an ex vivo experiment. These findings indicate that PCV2 infection decreases the efficacy of the LPC vaccine. This PCV2-derived interference may not only allow the invasion of wild-type CSFV in pig farms but also increases the difficulty of CSF prevention and control in CSF endemic areas.     

Immunohistochemical analysis of the distribution of classical swine fever (CSF) viral antigen in boar-pig hybrids and pigs four weeks after infection

We detected the classical swine fever virus (CSFV) antigen in three boar-pig hybrids (hybrids) and three pigs. All animals were experimentally infected with CSFV strain JPN/27/2019 to optimize diagnostic sampling and risk assessment of virus dissemination. Two hybrids died 17- and 19-days post-inoculation (dpi). The other animals were euthanized at 28 dpi. The detection of CSFV antigen at 28 dpi in epithelial cells of the apocrine sweat and sebaceous glands in the skin, salivary glands, mucosal epithelial cells in the rectum, and epithelial cells in the kidney and urinary bladder, suggests that CSFV persists in these tissues and spreads via sweat, saliva, feces, and urine for at least 4 weeks. These findings reveal that hybrids and pigs represent a high risk of virus dissemination four weeks after infection with CSFV strain JPN/27/2019. Prominent CSFV antigens were also detected in hair follicles of the skin. These results suggest that postmortem sampling of animal skin may be effective for CSF diagnosis and can be used to develop a rapid and easy diagnostic method using hair follicles.     

Coagulation changes in African swine fever virus infection

Pigs were infected with highly virulent (Tengani '62), with moderately virulent (DR '79) African swine fever (ASF) virus, or with virulent hog cholera (HC) virus. Changes in platelet counts, selected coagulation assays and concentrations of factor VIII-related antigen (VIIIR:Ag) were monitored. Permeability of aortic endothelium was studied after the injection of Evan's blue dye on various days after infection with DR '79 ASF virus. Virulent ASF virus caused prolongation of the activated partial thromboplastin time (APTT), 1-stage prothrombin time, and thrombin clotting time as early as postinoculation day (PID) 4. These changes became progressively more severe until death. Both virulent HC and DR'79 viruses induced an increase APPT and thrombin clotting time at PID 3 to 4, only occasionally did the prothrombin time increased significantly (P less than 0.01). The APPT began to decrease on PID 7 and 8, but only DR'79-infected pigs lived long enough to regain a normal APTT. Infection by ASF viruses caused acute thrombocytopenia after PID 6 and platelet counts of HC virus-infected pigs decreased progressively from the onset of fever to levels of 1 to 2 X 10(5)/mm3 at PID 6 to 7. All ASF virus-infected pigs had an increase in VIIIR:Ag beginning at PID 3, with maximum increases at PID 6 to 7. Hog cholera virus infection did not cause consistent changes in levels of VIIIR:Ag. Pigs infected with DR'79 virus did not have increased vascular permeability to Evan's blue dye during infection; however, there was markedly decreased staining of the aorta after pigs became thrombocytopenic.     

Respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines

In this study, the efficacy of two attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines was assessed. The virological protection in the lungs of vaccinated pigs upon challenge was studied. Also, challenged pigs were exposed to lipopolysaccharide (LPS) to evaluate clinical protection. Six-week-old pigs were immunized intramuscularly with commercial vaccines based on either an attenuated American or an attenuated European virus strain. Non-immunized pigs and pigs intramuscularly inoculated with the virulent Lelystad strain were included as controls. Six weeks after immunization, pigs were challenged either intratracheally or intranasally with the Lelystad strain, and 3 and 6 days later intratracheally exposed to Escherichia coli LPS. After LPS administration, pigs were monitored for clinical signs. At 4 and 7 days after challenge, pigs were euthanized to determine virus quantities in broncho-alveolar lavage (BAL) fluids and in lungs. Challenge virus was recovered from three out of eight pigs that had been primo-inoculated with the Lelystad strain with titers ranging between 0.3 and 3.1 log(10). Fifteen out of sixteen pigs vaccinated with the attenuated American strain were positive for challenge virus and their mean virus titers were similar to those of non-immunized challenge controls. Eleven out of 16 pigs vaccinated with the attenuated European strain were positive for challenge virus and their mean virus titers were 2.0-2.5 log(10) lower than those of non-immunized challenge controls. Thus, the virological protection in the lungs of vaccinated pigs upon challenge was incomplete, but was more pronounced in the homologous situation. Clinical signs upon LPS exposure in both vaccinated groups were not reproducible in two experiments.     

Application of competitive enzyme-linked immunosorbent assay for the serologic diagnosis of classical swine fever virus infection.

A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.     

Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection.

Depletion in the number of lymphocytes and viral persistence are thought to be the most important outcomes of classical swine fever virus (CSFV) infection. To define the change in peripheral blood mononuclear cells (PBMC) and virus replication in leukocytes after CSFV infection, 8-week old pigs were infected with the LPC vaccine strain or virulent CSFV (HCV-YL strain). Changes in the relative number of PBMCs were analyzed by flow cytometry. The results showed a significant increase in the relative percentage of monocytes in PBMCs during acute CSFV infection of naive pigs (p < 0.05). Monocyte frequencies were not changed in LPC-vaccinated pigs and control pigs. There was also a significant decrease in the number of IgM+ cells (p < 0.05) and a slight decrease in the number of CD4+ lymphocytes after 5 days of infection. There was no change in the frequency of CD8+ lymphocytes in PBMCs after infection. To define which subpopulation of PBMCs was the target for CSFV infection, PBMC populations from CSFV infected pigs were separated and stained for virus antigen expression. Alveolar macrophages (AM) were also studied. The results showed that CSFV replicated in all PBMC subpopulations: CD4+, CD8+, and IgM+ lymphocytes, and monocytes as well as AMs. However, virus antigen expression was more intense in monocytes and AMs. The infection of lymphocytes may, therefore, contribute to the depletion in their numbers after infection and lead to defective antibody production during virulent CSFV infection.     

Experimental inoculation study indicates swine as a potential host for Hendra virus

Hendra virus (HeV) is a zoonotic virus from the family Paramyxoviridae causing fatal disease in humans and horses. Five-week-old Landrace pigs and 5-month-old Gottingen minipigs were inoculated with approximately 10⁷ plaque forming units per animal. In addition to fever and depression exhibited in all infected pigs, one of the two Landrace pigs developed respiratory signs at 5 days post-inoculation (dpi) and one of the Gottingen minipigs developed respiratory signs at 5 dpi and mild neurological signs at 7 dpi. Virus was detected in all infected pigs at 2–5 dpi from oral, nasal, and rectal swabs and at 3–5 dpi from ocular swabs by real-time RT-PCR targeting the HeV M gene. Virus titers in nasal swab samples were as high as 10^4.6 TCID₅₀/mL. The viral RNA was mainly distributed in tissues from respiratory and lymphoid systems at an early stage of infection and the presence of virus was confirmed by virus isolation. Pathological changes and immunohistochemical staining for viral antigen were consistent with the tissue distribution of the virus. This new finding indicates that pigs are susceptible to HeV infections and could potentially play a role as an intermediate host in transmission to humans.     

Classical swine fever virus: clinical, virological, serological and hematological findings after infection of domestic pigs and wild boars with the field isolate "Spante" originating from wild boar

A classical swine fever virus (CSFV) field isolate originating from wild boar was investigated on its virulence in domestic pigs and wild boar. Three weaner pigs and two wild boars (yearlings) were intranasally inoculated with the isolate "Spante" and tested for clinical, virological, hematological and serological findings until day 31 after infection (p. i.). One day p. i. the piglets were put in contact to three sentinel pigs. During a period of 31 d neither the domestic pigs nor the wild boars showed clinical signs specific for CSF. Two infected weaner pigs became transiently viraemic, transmitted CSFV in nasal secretions, showed a slight leukopenia and reacted serologically positive. The contact infection resulted in a viraemia in two sentinel piglets on day 30. Only one contact animal developed antibodies. None of the wild boars became viraemic, excreted CSFV in nasal secretions or developed antibodies. The CSFV isolate "Spante" represents a low virulent virus. Referring to a significant higher percentage of virologically positive tissue samples after nested PCR compared with the virus isolation, persistence of CSFV is discussed.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Histopathologic and immunohistochemical characterization of Nipah virus infection in the guinea pig

Mortality rate in humans infected with Nipah virus (NiV) has been reported as high as 92%. Humans infected with NiV show a widespread multisystemic vasculitis with most severe clinical and pathologic manifestations in the brain, lungs, and spleen. The purpose of this study was to study pathologic and immunohistochemical findings in guinea pigs infected with NiV. Of 28 animals inoculated intraperitoneally, only 2 survived the infection, and most died between 4 and 8 days postinoculation (dpi). Viral antigen with minimal pathologic changes was first detected 2 dpi in lymph nodes and spleen. More severe changes were noted in these organs 4-8 dpi, where pathologic damage had a vasocentric distribution and viral antigen was abundant in vascular endothelium, tunica media, adventitia, as well as in macrophages lining sinuses. The urinary bladder, uterus, and ovaries were also affected with necrosis and acute inflammation. In these organs, immunohistochemical positive staining was intense in blood vessels, epithelial cells, and ovarian follicles. Approximately 50% of the animals that died or were euthanized in extremis had evidence of viral antigen and histopathologic changes in brain, especially involving meninges and ependymal cells, with lesser changes in the neural parenchyma. A unifying feature of the damage for all affected tissues was necrosis and inflammation of the vasculature, chiefly in arterioles, capillaries, and venules. Inoculation of guinea pigs intraperitoneally with NiV produces a disease with considerable resemblance to the disease in humans, but with reduced pulmonary involvement and marked infection of urinary bladder and the female reproductive tract.