T. Sydler

Adhesive fimbriae produced in vivo by Escherichia coli O139:K12(B):H1 associated with enterotoxaemia in pigs

Two strains of E. coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment. Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E. coli (ETEC). Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC. It adhered in vitro to porcine enterocyte brush border fragments. Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments. However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy.

A new emerging genotype subgroup within PCV-2b dominates the PMWS epizooty in Switzerland

Postweaning multisystemic wasting syndrome (PMWS) is among the most important emerging pig diseases worldwide. Initially, the insidious nature of the disease made it difficult to pinpoint the pathogen. The presence of porcine circovirus type 2 (PCV2) in all PMWS diseased animals led to its acceptance, possibly together with an unknown factor, as the causative agent for PMWS. Also, presence of PCV2 in healthy individuals did not facilitate the understanding of the disease. Phylogenetic classification separates PCV2 viruses into at least two major groups. With the aid of a signature motif, a short amino acid motif encoded within the capsid protein, the viruses are determined as belonging to PCV-2a or PCV-2b. Recently, this classification received more attention, as it seemed to define PCV-2b to be more virulent. This simplification, however, could not be confirmed experimentally. Hence, we investigated whether virus genetic shift was an initiator for the PMWS epizooty in Switzerland. Piglet lymphoid tissues from 1973 to 2005 were investigated by histology, immunohistochemistry (IHC) and PCR. For genotype classification, a sequence amplificate of 137bp was used encompassing the signature motif. The onset of Swiss PMWS epizooty exhibited a marked shift in PMWS diseased and subclinically infected piglets to PCV-2b and specifically to one genotype subgroup. Complementary to these observations, healthy piglets also defined by IHC as negative are positive in the PCR reaction and are void of any PCV-2b virus during epizooty. Consequently, our data support PCV2 genome plasticity as a major contributing factor for PMWS disease manifestation.

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-Ilv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E. coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strains enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.

Intestinal Chlamydia in Finishing Pigs

Gut and blood samples from 119 finishing pigs derived from 11 farms were collected during routine slaughter at an abattoir. Sections of formalin-fixed, paraffin-embedded tissues were labeled immunohistochemically using genus-specific, mouse monoclonal antibody against chlamydial lipopolysaccharide; goat polyclonal antiserum against the major outer membrane protein of Chlamydia trachomatis; and mouse monoclonal antibody against the ovine abortion subtype of C. psittaci. Gut samples from 33 of 111 (29.7%) individual pigs stained positive with the genus-specific monoclonal antibody, and of these 30 of 32 (93.7%) also reacted with the C. trachomatis-specific antiserum. Labeled inclusions were restricted to mature enterocytes of the large intestine in 33 of 111 cases. Infection of small intestinal enterocytes was noted in only one of 82 ileal samples. The blood samples were tested for antichlamydial antibodies by enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT). With ELISA, 95 of the 115 sera tested (82.6%) yielded positive antichlamydial reactions. With CFT, 34 of the 119 sera tested (28.6%) were unequivocally positive (≥ 1: 10, 100% binding), and 10 (7.6%) yielded doubtful positive reactions (1: 10, 50-75% binding). Positive ELISA and CFT titers showed poor agreement (K = 0.112), whereas the agreement between positive findings by immunohistochemical labeling and CFT was fair (K = 0.205).

Diagnostic investigation into the role of Chlamydiae in cases of increased rates of return to oestrus in pigs.

Cervical swabs and serum samples were taken from Swiss herds of sows with high rates of irregular return to oestrus (group A) and from control herds without reproductive problems (group B. The genital tracts of 21 slaughtered sows of group A were also examined. The swabs and genital tracts were screened for Chlamydiae by a new 16S rRNA PCR and the sera by an ELISA for Chlamydiaceae lipopolysaccharide. Chlamydophila (Cp) abortus was isolated from seven of the 65 swabs taken from group A but from none of the 128 swabs taken from group B. Chlamydia suis was present in swabs from both groups A (1˙5 per cent) and B (2˙3 per cent). In addition, Cp abortus was detected in 33˙3 per cent of the genital tracts. Of the 193 sera tested, 61˙7 per cent were positive, with no significant difference between group A (52˙3 per cent) and group B (66˙4 per cent). Chlamydia-like organisms were detected in 28˙2 per cent of the swabs from group A and in 22 per cent of those from group B.

Vaccination of Dams Increases Antibody Titer and Improves Growth Parameters in Finisher Pigs Subclinically Infected with Porcine Circovirus Type 2

ABSTRACT Porcine circovirus type 2 (PCV2) is the obligate infectious agent in postweaning multisystemic wasting syndrome (PMWS) of pigs. To control PMWS, we vaccinated dams at 4 and 2 weeks before pregnancy and again in the 12th week of gestation with an inactivated PCV2 vaccine (Circovac). Two producer farms run under the control of Swiss Swine Health Organization were selected for the experiment. Previously, in one farm PMWS was diagnosed on pigs after weaning, whereas in the other farm, pigs wasted during the fattening period. For the experiments 113 dams were randomly vaccinated, and 111 dams were sham injected. Vaccination increased serum antibodies in dams 3- to 9-fold, accompanied by serum antibody titer increases in their offspring. In the sixth week of life, progeny from vaccinated dams had about the same IgG antibody titers as progeny of unvaccinated dams at the third day of life. In sera of vaccinated dams only low concentrations of PCV2 DNA were detected, and no progeny developed PMWS. Interestingly, at day 56 four progeny of unvaccinated dams tested positive for anti-PCV2 IgM antibodies, indicating a primary infection with PCV2. Of economic importance is the observation that progeny of vaccinated dams had a significantly higher daily weight gain in the fattening period (farm X, +51 g/day; farm Y, +30 g/day) and thus a shortened fattening period of about 6 days compared to progeny of controls. To our knowledge this is the first demonstration of subclinical circovirus infection and its effects on growth performance of fattening pigs by vaccination of dams.

Bovine besnoitiosis in Switzerland: Imported cases and local transmission

Bovine besnoitiosis is an economically important disease of cattle, caused by Besnoitia besnoiti (Protozoa, Apicomplexa). A considerable spreading of this parasitic infection has been observed in Europe in the last ten years, mainly related to animal trade. In order to investigate the possibility of B. besnoiti being unnoticed introduced and getting established in Switzerland through the import of breeding cattle from France, a total of 767 animals (650 cattle imported from France and 117 cattle that had contact with B. besnoiti positive cattle in Swiss farms) were screened for antibodies against B. besnoiti by both a commercial ELISA and by the indirect fluorescent antibody test (IFAT). A total of 101 (13.17%) samples showed a positive reaction in ELISA (cut-off: percent of positivity [PP] ≥ 15) and 16 (2.09%) samples had IFAT titers ≥ 1:100. Eight of those samples reacted positive in Western blot (WB), corresponding to five imported Limousin cattle (two cows and one bull from France and two cows from Germany) and to three cattle born in Switzerland (one Limousin heifer born from one of the positive German cows, and two adult Braunvieh cows, that had been in contact with one of the French cows at a Swiss farm). Seven of those animals were subclinically infected and one animal showed only very mild signs. They were subsequently slaughtered, and the serological diagnosis could be confirmed by real-time PCR and/or histopathology in seven animals. The most frequent parasite localizations were the tendons and surrounding connective tissue of the distal limbs and the skin of the head region. Furthermore, B. besnoiti could be successfully isolated in vitro from one French, one German and one Swiss cattle (isolates Bb-IPZ-1-CH, Bb-IPZ-2-CH and Bb-IPZ-3-CH). In the current situation in Switzerland, prophylactic and control measures should include a serological examination of cattle to be imported from endemic areas and the culling of all confirmed positive animals from the herd. The evidence of B. besnoiti infection in both imported and locally born cattle shows that the conditions for the establishment and dissemination of this parasite in Switzerland seem to be adequate.

Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45

Enteric chlamydial infections of pigs with Chlamydia (C.) suis are frequent and often subclinical. The enteric pathogenicity of C. suis strain S45 was investigated in gnotobiotic piglets. Piglets from three litters (n=31) were inoculated with egg-grown chlamydiae at 2-3 days of age (n=17) or used as controls (n=14). They were observed for clinical signs, killed and necropsied sequentially at 2-13 days postinoculation (DPI). Feces were collected daily and investigated with an ELISA for chlamydial antigen. At necropsy, specimens were collected for histopathology and for immunohistochemical, PCR-based, and serological (complement fixation test, ELISA) detection of chlamydiae. Chlamydial replication and associated symptoms and lesions were observed from 2 to 13 DPI and were particularly pronounced within the first week PI. Clinical symptoms consisted of moderate-to-severe diarrhea, slight and transient anorexia, weakness and body weight loss. Immunohistochemistry and ELISA revealed that chlamydial replication was particularly marked at 2-4 DPI and primarily located in the small intestinal villus enterocytes. Further sites of replication included large intestinal enterocytes, the lamina propria and Tunica submucosa, and the mesenteric lymphnodes. Histopathological changes included moderate-to-severe villus atrophy with flattened enterocytes and focal villus tip erosions, and moderate mucosal inflammatory cell infiltrates and lymphangitis in the small intestine. PCR of spleen tissue and blood was mostly negative for chlamydiae, indicating that they did not substantially disseminate into the host up to 13 DPI. All sera were negative for anti-chlamydial antibodies. In conclusion, C. suis strain S45 elicited significant enteric disease and lesions in gnotobiotic piglets indicating its pathogenic potential for swine.

Metastatic Calcification in a Dog Attributable to Ingestion of a Tacalcitol Ointment

A 22-week-old 21-kg female Bernese Mountain Dog ingested a topical antipsoriatic preparation containing the synthetic vitamin D analog tacalcitol. The dog died after a history of lethargy, recumbency, paresis of the hindlimbs, increased rectal temperature, dyspnea, and hematemesis. Histologic examination revealed metastatic calcification in the kidneys, lungs, myocardium, brain, stomach, and tear glands. The appearance of soft tissue mineralization in multiple organs is consistent with hypercalcemia derived from excessive vitamin D uptake. Oral toxicity studies for tacalcitol in the dog are not available, but the present report emphasizes the extraordinary toxic risk of drugs containing this vitamin D analog to dogs.

Coreplication of the Major Genotype Group Members of Porcine Circovirus Type 2 as a Prerequisite to Coevolution May Explain the Variable Disease Manifestations

ABSTRACT A member of the family Circoviridae, porcine circovirus type 2 (PCV2), is associated with postweaning multisystemic wasting syndrome (PMWS), a recent emerging disease worldwide. PCV2 is also found in clinically asymptomatic animals. This paradoxical finding makes the syndrome etiology challenging. We developed new assays to study PCV2 with links to syndrome etiology. For analysis, we used PCV2-infected tissues from subclinically infected and diseased piglets. We compared antigen- and PCV2 DNA-derived signals for tissue localization and intensity. Oligonucleotides were designed to the signature motif of the PCV2 capsid open reading frame to discriminate experimentally between PCV2 genotype groups by PCR, in situ hybridization (ISH), and fluorescence in situ hybridization (FISH). Unexpectedly, all PCV2-infected animals carried both PCV2a and PCV2b genotype group members. Using confocal microscopy, genotype single-cell infections and cell superinfections were visible. Additionally, we discriminated replicative DNA from total PCV2 DNA isoforms with FISH. This aided in our inquiry into cellular genotype-specific replication. Importantly, single-genotype-group replication was not observed. In infected cells with replicating virus, both genotype groups were equally present. These findings suggest PCV2 genotype group members relaxed replication regulation requirements and may even point to PCV2 replication cooperativity in vivo. These observations explain the readily seen PCV2 DNA recombinations and the high overall PCV2 genome plasticity. Hence, we suggest a novel mechanism of syndrome etiology that consists of a continuously changing PCV2 genome pool in hosts and pig herds, posing a constant challenge to the individual maturing immune system.