Cross-species infection of specific-pathogen-free pigs by a genotype 4 strain of human hepatitis E virus

Hepatitis E virus (HEV) is an important pathogen. The animal strain of HEV, swine HEV, is related to human HEV. The genotype 3 swine HEV can infect humans and genotype 3 human HEV can infect pigs. The genotype 4 swine and human HEV strains are genetically related, but it is unknown whether genotype 4 human HEV can infect pigs. A swine bioassay was utilized in this study to determine whether genotype 4 human HEV can infect pigs. Fifteen, 4-week-old, specific-pathogen-free pigs were divided into three groups of five each. Group 1 pigs were each inoculated intravenously with PBS buffer as negative controls, group 2 pigs similarly with genotype 3 human HEV (strain US-2), and group 3 pigs similarly with genotype 4 human HEV (strain TW6196E). Serum and fecal samples were collected at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days postinoculation (dpi) and tested for evidence of HEV infection. All pigs were necropsied at 56 dpi. As expected, the negative control pigs remained negative. The positive control pigs inoculated with genotype 3 human HEV all became infected as evidenced by detection of HEV antibodies, viremia and fecal virus shedding. All five pigs in group 3 inoculated with genotype 4 human HEV also became infected: fecal virus shedding and viremia were detected variably from 7 to 56 dpi, and seroconversion occurred by 28 dpi. The data indicated that genotype 4 human HEV has an expanded host range, and the results have important implications for understanding the natural history and zoonosis of HEV.     

Septicaemia and arthritis in pigs experimentally infected with Pasteurella multocida capsular serotype A

Twenty-five caesarean-derived, colostrum-deprived (CDCD) pigs and 18 specific pathogen-free pigs, aged 8 to 14 weeks, were inoculated intranasally or intratracheally with Pasteurella multocida capsular serotype A, isolated from a severe pneumonic lesion in a growing pig. The pigs were killed for necropsy on day 6 or 14 post-inoculation (PI) or, in the case of the only fatally infected animal, examined at the time of death. One CDCD pig, inoculated intratracheally with 5 ml of a bacterial suspension containing 1.7x10(9) colony-forming-units/ml, died of septicaemia on day 1 PI. Histological lesions such as severe pleuropneumonia, thrombi in glomerular capillaries, haemorrhage of the spleen, and abscesses in the tonsillar crypts were observed. The organism was recovered from a number of sites and its antigens were detected immunohistochemically in the pneumonic lesions, blood vessels of the tissues, and tonsillar crypts in the dead pig. Pneumonia, pleural adhesions and suppurative arthritis in the extremital joints were observed grossly in 3/29, 8/29 and 7/29 intratracheally inoculated pigs, respectively. In intranasally inoculated pigs, no macroscopical abnormalities were seen; histologically, however, exudative bronchopneumonia and fibrinous pleurisy were observed in 9/14 and 4/14 pigs, respectively. No significant changes were seen in the tissues of uninfected control pigs. The organism was recovered from the lesions and P. multocida type A antigen was demonstrated immunohistochemically. The organism was rarely recovered from the liver, spleen or lymph nodes (bronchopulmonary or mesenteric). The results suggest that P. multocida capsular serotype A alone can cause not only pneumonia in pigs but also septicaemia or arthritis.     

Pathogenesis of Korean type 1 (European genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs

The aim of this study was to elucidate the pathogenesis of experimental infection with Korean type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the virus distribution, sites of viral replication, viraemia and gross and microscopical lesions in conventional pigs studied for 28 days after intranasal inoculation. Mean rectal temperature was significantly higher in infected pigs than in negative control pigs at 2 days post inoculation (dpi) (P=0.004), 3 dpi (P<0.001), 4 dpi (P=0.003) and 5 dpi (P=0.034). The log(10)TCID(50)/ml of type 1 PRRSV increased significantly at 0-1 dpi (P=0.024) and 5-7 dpi (P=0.029), but decreased at 10-14 dpi (P=0.026) and 14-21 dpi (P=0.012) in infected pigs. Infected pigs developed multifocal, tan-mottled areas of lung tissue with irregular and indistinct borders. Microscopical lesions, when present, were multifocal, mild to moderate, generally most extensive at 5-7 dpi (P=0.036), and were nearly resolved at 28 dpi. Type 1 PRRSV nucleic acid and antigen were detected exclusively within the cytoplasm of macrophages and type I and II pneumocytes. The score for PRRSV-positive cells increased at 3-7 dpi (P<0.05) and decreased at 10-14 dpi (P=0.034) in infected pigs. Thus, respiratory disease was reproduced in conventional pigs by infection with Korean type 1 PRRSV.     

PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen

An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.     

Pathogenesis of swine influenza virus subtype H1N2 infection in pigs

The purpose of this study was to elucidate pathogenesis and viral distribution in pigs infected with swine influenza virus subtype H1N2, over a period of 10 days, by morphometric analysis and in-situ hybridization. Fifteen colostrum-deprived pigs aged 3 weeks were inoculated intranasally with virus. Pneumonia was severe at 1 day post-inoculation (dpi), moderate at 3 and 5 dpi, and mild at 7 and 10 dpi. The pulmonary lesion score was correlated with the score of cells positive by in-situ hybridization for swine influenza virus (r(s)= 0.9114, P< 0.05). The distribution of swine influenza virus varied according to the duration of infection. At 1 and 3 dpi, hybridization signals were detected mainly in the bronchial and bronchiolar epithelial cells, but they were detected mainly in the pneumocytes and macrophages (alveolar and interstitial) at 7 and 10 dpi. The results confirmed that swine influenza virus subtype H1N2, isolated in Korea, is a virulent pathogen causing severe pneumonia.     

Immunohistochemical detection of interleukin-12 and interferon-gamma in pigs experimentally infected with Mycoplasma hyopneumoniae

The expression of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) was examined immunohistochemically in the lungs of pigs aged 21 days infected experimentally with Mycoplasma hyopneumoniae (Mh). Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 7 to 35 days post-infection (dpi). Immunolabelling for IL-12 and IFN-gamma was usually associated with inflammation, particularly in macrophages and lymphocytes in the thickened alveolar septa and in the hyperplastic bronchus-associated lymphoid tissue (BALT). Cells positive for both cytokines were detected at 7 dpi, their numbers increasing at 14 and 21 dpi, and slightly decreasing thereafter. The results suggest that IL-12 and IFN-gamma play a role in pulmonary defence mechanisms against Mh infection.     

In-situ hybridization for the detection of Haemophilus parasuis in naturally infected pigs

Detection of Haemophilus parasuis in naturally infected pigs was studied by in-situ hybridization with a non-radioactive digoxigenin-labelled DNA probe. Twenty pigs were selected on the basis of bacterial isolation and histopathological lesions. An 821 base pair DNA probe from the 16S small subunit ribosomal RNA (rRNA) was generated by the polymerase chain reaction. Hybridization signals were detected in formalin-fixed, paraffin-wax-embedded tissues (lung, heart, spleen and liver). Identification of the cell types containing H. parasuis was occasionally difficult, but examination of adjacent sections stained with haematoxylin and eosin confirmed that positive cells resembled either macrophages (large oval nuclei and abundant cytoplasm) or neutrophils (bilobed nuclei). In-situ hybridization would appear to be a valuable tool for the diagnosis of H. parasuis infection.     

Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus

Post-weaning multisystemic wasting syndrome (PMWS) has recently emerged as an important disease of pigs in North America, Europe and Asia. Porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) have been isolated from affected pigs. To investigate the pathogenicity of these isolates, groups of colostrum-deprived conventional pigs were inoculated with PCV2 alone (n=4), PPV alone (n=3) or dually with PCV2 and PPV (n=7) and examined post mortem between 21 and 26 days post-infection (dpi). Two control pigs were inoculated with an uninfected cell culture lysate. All pigs that received both viruses became dull at approximately 10-12 dpi and six of these animals subsequently developed jaundice. Hepatomegaly and enlarged kidneys were prominent post-mortem findings in these animals. Histopathological examination revealed severe macrophage infiltration, syncytia formation and numerous cytoplasmic and nuclear amphophilic inclusion bodies in lymphoid tissues. Granulomatous lesions were apparent in liver, lung, kidney, pancreas, myocardium, intestines, testis, brain and salivary, thyroid and adrenal glands. Abundant PCV2 antigen was detected in affected tissues. Only one of the four pigs inoculated with PCV2 alone developed clinical signs, but they all had histopathological lesions which, although less severe, were similar to those in the dually infected animals. The control pigs and those infected with PPV alone remained clinically normal and did not have gross lesions. The only histopathological lesion seen in these animals was mild interstitial nephritis in the pigs infected with PPV alone. These results indicate that lesions of PMWS can be induced by inoculating pigs with PCV2 alone, thereby fulfilling Koch's postulates. Concurrent infection with PPV increased the severity of the lesions, suggesting that co-factors are important in the pathogenesis of PMWS.     

Comparison of the mycobacteria growth indicator tube with solid culture for the detection of tuberculosis complex mycobacteria from blood

Aim of this study

The aim of this study was to compare the mycobacteria growth indicator tube and solid culture for recovery of complex tuberculosis mycobacteria from blood.

Patients and methods

One hundred and twenty-five specimens from 67 Djiboutian patients with a positive serologic diagnosis of HIV and fever were collected in an Isolator^® tube. After centrifugation and washing with phosphate-buffer, smears were prepared from the pellet for auramin staining. The remaining sediment was suspended in 1 ml of buffer. One half was inoculated into two MGIT (incubation at 30 and 37 °C into Bactec 960) and the other onto two Loewenstein–Jensen and two Coletsos medium (incubation at 30 and 37 °C).

Results

Eight cultures were contaminated: three on solid medium and MGIT simultaneously, five in MGIT only (three coagulase negative staphylococci, five enterobacteria). Fourteen strains of M. tuberculosis (six patients) and three M. canettii (two patients) (12 on solid media and MGIT, five in MGIT only) were recovered. The mean time to detection was 32.8 days for solid medium and 20.4 days for MGIT. Of a total of 25 patients with culture-proven tuberculosis, two patients had a positive blood culture only, six had blood and other specimens positive culture, 17 had a non blood specimens positive culture only.

Conclusion

MGIT processed into Bactec 960 is a viable tool for the detection of complexe tuberculosis mycobacteria from blood and the high-frequency of these mycobacteremia in HIV infected patients from country where the prevalence of tuberculosis is high is confirmed. However, the cost/benefit ratio of this bacteriologic diagnosis had to be evaluated in developping country.     

Detection by in-situ hybridization of Pasteurella multocida toxin (toxA) gene in the lungs of naturally infected pigs

In-situ hybridization with a non-radioactive digoxigenin-labelled probe was used to detect the Pasteurella multocida toxin (PMT) gene in tissue sections of pneumonic lung from pigs naturally infected with toxigenic P. multocida. The morphology of host cells was preserved despite the relatively high temperature used in the incubation procedure. Pulmonary abscessation was observed in 13 pigs naturally infected with toxigenic P. multocida type A (three pigs) or D (10 pigs). In these 13 pigs a strong hybridization signal for PMT DNA was detected, mainly in degenerate leucocytes in abscesses. Occasionally, PMT DNA was detected in degenerate neutrophils and macrophages in alveolar spaces. Detection of hybridization signals for PMT DNA would seem to be a potential indicator of the production of PMT. The study suggested that PMT plays an important role in pulmonary abscessation caused by P. multocida.     

Pathogenesis of renal dysfunction in chicks experimentally induced by avian nephritis virus

Renal dysfunction in chicks infected with avian nephritis virus (ANV) at 1 day or 1 week of age was studied. Two chicks inoculated per os and one inoculated intraperitoneally, both infected at 1 day of age, died with visceral urate deposits within 10 to 13 dpi, and the surviving infected chicks showed a markedly reduced weight gain. In a chronological study, ANV was consistently isolated from the kidney irrespective of the route of infection or age of chick. Fluorescent antigen to ANV was also detected as cytoplasmic granules in the tubular cells, accompanied by necrosis of tubular cells. A high concentration of serum uric acid was detected 4 to 13 dpi in chicks infected at 1 day of age and was frequently coincident with detection of the tubular cell necrosis. These results suggest that the increased serum uric acid concentration is caused by viral damage of kidney tubular cells.     

Experimental transmission of porcine circovirus type 2 (PCV2) in weaned pigs: a sequential study

Weaned specific pathogen-free pigs were inoculated intranasally with porcine circovirus type 2 (PCV2) and killed in groups of two or three animals at 6, 13, 20, 27 and 34 days post-inoculation (dpi), together with appropriate uninfected controls, for examination by histopathological, immunohistochemical (immunogold silver staining; IGSS), polymerase chain reaction (PCR) and viral isolation techniques. Serum samples were also collected for detection of antibodies. No major clinical signs were observed in infected pigs, and gross lesions were essentially limited to the lungs and lymph nodes of some of the animals. Histologically, no lesions were seen at 6 dpi, but bronchointerstitial pneumonia was invariably noted from 13 dpi onwards. Granulomatous inflammation, with or without intracytoplasmic inclusions, was present in lymphoid tissues (e.g. lymph nodes, thymus, spleen and tonsil) from day 20 onwards, being most severe at days 20 and 27 dpi. Liver inflammation was present at days 13, 20 and 27 dpi. Virus was demonstrated in the tissues by isolation and PCR methods throughout the experiment. PCV2 antigens were detected by IGSS in bronchial and bronchiolar epithelial cells, in mononuclear cells and multinucleated giant cells within inflammatory lesions, and in mononuclear cells of apparently normal tissues (e.glamina propria of the small intestine and the bronchus-associated lymphoid tissue). The lesions were consistent with those of postweaning multisystemic wasting syndrome (PMWS), although not all previously reported PMWS lesions were seen. PCV2 antibodies were detected in infected pigs from day 13 onwards. The results demonstrated widespread distribution of PCV2 after infection and persistence of the virus in vivo for at least 34 days. It would appear that PCV2 can induce PMWS lesions in weaned pigs in the absence of porcine parvovirus and other common swine pathogens.     

Viral regulatory region effects on vertical transmission of polyomavirus SV40 in hamsters

Viral strain differences influence the oncogenic potential of polyomavirus simian virus 40 (SV40). We hypothesized that viral strain differences might also affect vertical transmission of SV40 in susceptible hosts. Pregnant Syrian golden hamsters were inoculated intraperitoneally with 10⁷ plaque-forming units of SV40 and offspring were sacrificed post-delivery (1-21 days, 6 months). Organ extracts were analyzed for SV40 DNA by polymerase chain reaction assay. Transmission of SV40 from mother to offspring was detected in over half of litters. Most placentas were virus-positive. Mothers inoculated with SV40 strains containing complex regulatory regions transmitted virus more frequently than those infected with simple enhancer viruses (p < 0.001). Virus was detected more often in progeny brain than in spleen (p < 0.05). Several progeny were virus-positive at 6 months of age, suggesting viral persistence. Maternal animals retained virus in several tissues through day 21 and developed T-antigen antibodies. These results indicate that SV40 replicates in hamsters, vertical transmission of SV40 can occur, and the viral regulatory region influences transmission.     

The relationship between Pepper mottle virus source leaf and spread of infection through the stem of <Emphasis Type="BoldItalic">Capsicum</Emphasis> sp.

Summary.  Pepper mottle virus (PepMoV) systemically infects Capsicum sp. in a typical source-to-sink manner with movement through the stem occurring in a predictable pattern. This study was carried out to determine the relationship between the inoculated leaf as a source of inoculum and the spread of PepMoV infection through the stem. C. annuum‘Early Calwonder’ plants were mechanically inoculated onto the first leaf with PepMoV and sets of 30 plants had their inoculated leaves removed each day from 1 through 7 days post-inoculation (dpi) with the inoculated leaves tested for infection by ELISA at the time of excision. Beginning at 2 dpi, PepMoV infection in the stem of plants with the inoculated leaf excised and plants of a nonexcision control treatment was determined using immuno-tissue blot analysis. PepMoV was detected in inoculated leaves beginning at 3 dpi with the percentage of infected leaves increasing each day through 7 dpi. PepMoV was first detected in the stem of inoculated plants of the 3 dpi excision treatment. The accumulation and extent of spread of infection in the stem was similar for plants that had their inoculated leaf removed at a time preceding detection by ELISA to plants in the nonexcision control treatment. These findings suggest that once virus is allowed to enter the stem from the inoculated leaf, subsequent spread of infection through the stem is a process independent from the source leaf. Received March 25, 2002; accepted May 17, 2002 Published online July 22, 2002     

Experimental reproduction of porcine circovirus type 2 (PCV2)-associated enteritis in pigs infected with PCV2 alone or concurrently with Lawsonia intracellularis or Salmonella typhimurium

Porcine circovirus (PCV)-associated disease (PCVAD) has emerged to become one of the most economically important pig diseases globally. One of the less commonly recognized clinical manifestations of PCVAD is PCV2 type 2 (PCV2)-associated enteritis in growing pigs; however, experimental confirmation of the ability of PCV2 alone or PCV2 coinfection with other agent(s) to induce enteritis is lacking. In this study, 120 specific-pathogen-free (SPF) pigs were divided randomly into six groups: controls (negative control pigs), PCV2 (inoculated with PCV2), LAW (inoculated with Lawsonia intracellularis), SALM (inoculated with Salmonella typhimurium), PCV2-LAW (concurrently inoculated with PCV2 and Lawsonia intracellularis) and PCV2-SALM (concurrently inoculated with PCV2 and Salmonella typhimurium). One half of the pigs in each group were subject to necropsy examination 14 days postinoculation (dpi) and the remaining pigs were examined at 28 dpi. The average daily weight gain was not different (P>0.05) between groups. Individual pigs inoculated orally with PCV2 regardless of coinfection status (2/10 PCV2, 1/10 PCV2-LAW, 3/10 PCV2-SALM) developed PCVAD with diarrhoea and reduced weight gain or weight loss between 14 and 28 dpi. Those pigs had characteristic microscopic lesions in lymphoid and enteric tissues associated with abundant PCV2 antigen. Enteric lesions were characterized by necrosuppurative and proliferative enteritis with crypt elongation and epithelial hyperplasia in LAW and PCV2-LAW pigs by 14 dpi, ulcerative and necrosuppurative colitis in SALM and PCV2-SALM pigs by 14 dpi, and lymphohistiocytic enteritis with depletion of Peyer's patches in PCV2, PCV2-SALM and PCV2-LAW pigs by 28 dpi. To the authors' knowledge, this is the first report documenting that under experimental conditions, PCV2 can induce enteritis independently from other enteric pathogens and that oral challenge is a potentially important route and perhaps the natural route of PCV2 transmission in growing pigs.