Immunohistochemical detection of aetiological agents of proliferative and necrotizing pneumonia in italian pigs.

Proliferative and necrotizing pneumonia (PNP) is a form of interstitial pneumonia that occurs in weaning and post-weaning pigs. PNP is characterized by hypertrophy and hyperplasia of type II pneumocytes and coagulative necrosis and granular debris within alveolar spaces. Canadian and European studies suggest that the porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are the main causes of the disease, but Aujezsky's disease virus (ADV) and swine influenza virus (SIV) have also been considered as potential aetiological agents. An immunohistochemical study was carried out on the lungs of 28 Italian pigs with PNP in order to evaluate the role of PRRSV, PCV2 and ADV in PNP lesions. PRRSV infection was identified in the lungs of 11 pigs, PCV2 in the lungs of four pigs and coinfection with both viruses in the lungs of eight pigs. Neither virus was detected in the lungs of the remaining five pigs. ADV antigen was not detected in any sample. The principle aetiological agent of PNP in Italy therefore appears to be PRRSV. Coinfection with PRRSV and PCV2 is characterized by more severe microscopical changes in affected lungs.

Application of a protocol for the diagnosis of postweaning multisystemic wasting syndrome in Italy.

Samples of superficial inguinal and bronchial lymph nodes, ileum, tonsil and lung were taken from three to five pigs on each of 61 farms with a clinical history of postweaning multisystemic wasting syndrome (PMWS). The samples were examined histologically and by immunohistochemistry for porcine circovirus type 2 (PCV-2). PMWS was diagnosed in two stages: first, an evaluation of the haematoxylin and eosin-stained sections that identified the cases in which the characteristic PCV-2 cytoplasmic inclusion bodies were apparent, and secondly, a conclusive step in which immunohistochemistry was applied to confirm PMWS in the cases in which there were positive immunohistochemical results that coincided with lesions indicative of PMWS in at least one of the lymphoid and/or lung tissues. The location of PCV-2 in specific lesions (cell depletion in lymphoid organs and interstitial pneumonia) confirmed PMWS in 45 of the 61 farms, 31 of which were also infected with porcine reproductive and respiratory syndrome virus. The lymphoid tissues were more reliable than the lungs for the diagnosis of PMWS, both in individual pigs and in groups of pigs, and farm diagnoses based on a group of pigs were more reliable than diagnoses based on single pigs.

Longitudinal study of Salmonella infection in Italian farrow-to-finish swine herds.

A longitudinal study of Salmonella enterica infection was carried out in five Italian farrow-to-finish swine herds previously known to be infected by Salmonella. Five litters were randomly selected from each herd and in each litter six piglets were randomly selected and individually identified. Thus, the study included 30 pigs from each farm. At weaning, individual blood samples were collected for serological examination from all selected piglets and on the same day from all sows in the farrowing unit. Piglets were bled again at approximately 60, 90, 150, 210 and 270 days of life whereas the last blood sample was collected at slaughtering. In one of the herds, in which the duration of productive cycle was about 12 months, the last blood samples were collected at 350 days of life. With the same time scheduling, five pen pooled faecal samples were collected from each herd for bacteriological examination. At slaughtering, mesenteric lymph nodes were collected from each ear-tagged pig. Sero-prevalence (cut off S/P ratio 0.25) in sows varied from 93.8% to 100%. In four herds, sero-prevalence in piglets showed a similar profile with complete decline of maternal antibodies at day 60 and clear sero-conversion between day 90 and day 150. In one herd, sero-conversion was observed earlier and 56% of piglets were positive at day 90. The peak of sero-prevalence was observed between day 210 and day 270. Sero-prevalence at slaughtering varied from 66% to 100%. Salmonella was isolated from faecal samples in four of five herds. No Salmonella was isolated from mesenteric lymph nodes at slaughter in two of the herds. Culture prevalence from mesenteric lymph nodes in the other three herds ranged from 3.3% to 30%. This longitudinal study provides original information about epidemiological dynamics of Salmonella enterica infection in Italian swine herds in consideration of the unique extended fattening period typical of the Italian production.

Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe.

In the early 1970s, a human influenza A/Port Chalmers/1/73 (H3N2)-like virus colonized the European swine population. Analyses of swine influenza A (H3N2) viruses isolated in The Netherlands and Belgium revealed that in the early 1990s, antigenic drift had occurred, away from A/Port Chalmers/1/73, the strain commonly used in influenza vaccines for pigs. Here we show that Italian swine influenza A (H3N2) viruses displayed antigenic and genetic changes similar to those observed in Northern European viruses in the same period. We used antigenic cartography methods for quantitative analyses of the antigenic evolution of European swine H3N2 viruses and observed a clustered virus evolution as seen for human viruses. Although the antigenic drift of swine and human H3N2 viruses has followed distinct evolutionary paths, potential cluster-differentiating amino acid substitutions in the influenza virus surface protein hemagglutinin (HA) were in part the same. The antigenic evolution of swine viruses occurred at a rate approximately six times slower than the rate in human viruses, even though the rates of genetic evolution of the HA at the nucleotide and amino acid level were similar for human and swine H3N2 viruses. Continuous monitoring of antigenic changes is recommended to give a first indication as to whether vaccine strains may need updating. Our data suggest that humoral immunity in the population plays a smaller role in the evolutionary selection processes of swine H3N2 viruses than in human H3N2 viruses.

Influenza surveillance in birds in Italian wetlands (1992-1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots?

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.

Circulation of influenza viruses in wild waterfowl wintering in Italy during the 1993-99 period: evidence of virus shedding and seroconversion in wild ducks.

The mechanisms of perpetuation of influenza A viruses in aquatic birds, their main reservoir in nature, have not yet been completely clarified. One hypothesis is that they continue to circulate in waterfowl throughout the year, even though virus isolations during the winter months are rare. We analyzed influenza virus circulation in wild ducks in Italy during six winter seasons (1993-99), using virus isolations and serological analyses. It was apparent that influenza A viruses were constantly circulating in wild birds during all the seasons considered. Moreover, seroconversion rates (obtained from ducks recaptured during the same season) suggest a frequency of influenza infections higher than expected on the basis of the virus isolation rates.

Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe.

Three subtypes of influenza A viruses, H1N1, H1N2 and H3N2, co-evolve in pigs in Europe. H1N2 viruses isolated from pigs in France and Italy since 1997 were closely related to the H1N2 viruses which emerged in the UK in 1994. In particular, the close relationship of the neuraminidases (NAs) of these viruses to the NA of a previous UK H3N2 swine virus indicated that they had not acquired the NA from H3N2 swine viruses circulating in continental Europe. Moreover, antigenic and genetic heterogeneity among the H1N2 viruses appeared to be due in part to multiple introductions of viruses from the UK. On the other hand, comparisons of internal gene sequences indicated genetic exchange between the H1N2 viruses and co-circulating H1N1 and/or H3N2 subtypes. Most genes of the earlier (1997-1998) H1N2 isolates were more closely related to those of a contemporary French H1N1 isolate, whereas the genes of later (1999-2000) isolates, including the HAs of some H1N2 viruses, were closely related to those of a distinct H1N1 antigenic variant which emerged in France in 1999. In contrast, an H3N2 virus isolated in France in 1999 was closely related antigenically and genetically to contemporary human A/Sydney/5/97-like viruses. These studies reveal interesting parallels between genetic and antigenic drift of H1N1 viruses in pig and human populations, and provide further examples of the contribution of genetic reassortment to the antigenic and genetic diversity of swine influenza viruses and the importance of the complement of internal genes in the evolution of epizootic strains.

Antigenic and sequence analysis of H3 influenza virus haemagglutinins from pigs in Italy.

To investigate the possible mechanism of maintenance of old human influenza A (H3N2) viruses in pigs, the haemagglutinins (HAs) of seven isolates from swine were studied by analysis of nucleotide and deduced primary amino acid sequences, as well as reactivity of the HA molecule to chicken antisera and monoclonal antibodies. The swine HAs were closely similar to the HA of the A/Victoria/3/75 human variant as regards antigenic and molecular characteristics. These findings are consistent with the hypothesis that the swine HA genes were transmitted from an early human H3 virus to pigs, where they survived with limited mutations over a period of 5 years. The sequence data were also compared with swine H3 sequences to investigate genetic relationships between the H3 genes from swine viruses isolated in different geographical areas. An evolutionary tree, constructed from the nucleotide sequences of viruses isolated from pigs in China and in Italy, illustrated that, depending on the country of their isolation, the HA genes of swine influenza A (H3N2) viruses have different origins, e.g. human and avian, and evolved independently in different lineages. The study provides direct support for the hypothesis that pigs might serve as a 'mixing vessel' for the generation of pandemic strains of human influenza viruses.

Experimental encephalomyocarditis virus infection in pigs.

A field isolate of Encephalomyocarditis (EMC) virus was inoculated intravenously into 8 pigs. Four animals died at post inoculation day (PID) 2, the remaining being sacrificed at PID 5, 7, 11 and 15. Two control, in-contact pigs were sacrificed at PID 19. Virus was isolated from leucocytes and nasal swabs until PID 4, from rectal swabs until PID 2 and, in the pigs found dead at PID 2, from several organs. EMC virus was further isolated from brain and spleen of the pig sacrificed at PID 7. One of the 2 control pigs became infected: virus was isolated from nasal swabs at days 6 and 7 and from leucocytes at day 4 of the experiment. Serum-neutralizing (SN) antibody was detected in the injected pigs starting from PID 4; two days later, it was also revealed in the infected, in-contact control. To our knowledge, this is the first report of an experimental transmission of EMC virus infection in pigs by contact exposure.

Genetic reassortment between avian and human influenza A viruses in Italian pigs.

Pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses. To examine the possibility that pigs serve as "mixing vessels" for such reassortment events (Scholtissek et al., Virology 147, 287-294, 1985), we phylogenetically analyzed the internal protein genes of classic H1N1, avian-like H1N1, and human-like H3N2 viruses circulating among Italian pigs. The results show that human-like H3N2 strains isolated from 1985 to 1989 contained the internal protein genes of avian-like H1N1 viruses, whereas those isolated in 1977 and 1983 did not. Thus, at some time between 1983 and 1985, genetic reassortment took place between avian- and human-like viruses in Italian pigs. This study provides the first evidence supporting genetic reassortment between human and avian viruses in a natural swine environment.

Ultrastructural study of experimental myocarditis induced by cardiovirus (EMCV-M) in swine.

Eight 6-week-old piglets were inoculated with a strain of encephalomyocarditis virus (EMCV) isolated from an outbreak which occurred naturally in the Po Valley in 1988. Two non-infected animals, kept in the same cage, were used as controls. Out of the eight inoculated piglets, two died and two were suppressed on the 2nd post infection day (PID), the four remaining were killed on the 5th, 7th, 11th and 15th PIDs. Control animals were killed at the end of the experiment. The pathogenesis of myocarditis has been studied using routine methods (Alcian-PAS, Masson's trichrome, Gomori's for reticulin and Mallory's stain), histochemical techniques (ATPase and NADH-TR reactions) and ultrastructural observations (TEM). All the inoculated piglets showed macro and/or microscopic lesions of lymphocytic myocarditis, only in one case associated with fibrinous exudation. One control piglet also showed myocarditic lesions, probably due to a contact infection. An early myocardial fibrosis was already present on the 5th PID. Ultrastructurally the cardiac muscle cells showed severe myofibrillar losses and other regressive alterations. Only on the 15th PID did we observe calcification of the degenerating myocytes, while ultrastructurally we detected needle-like calcium deposits in the mitochondria from the 5th PID. From the 5th PID in the areas of myocarditis the myocytes showed a reduction and/or absence of ATPase and NADH-TR reactions. On TEM, one or more aggregates of viral particles in crystalline array were detected in the cytoplasm of many endothelial cells.