Characterisation of the course of Mycoplasma bovis infection in naturally infected dairy herds.

Mycoplasma bovis causes bovine respiratory disease, mastitis, arthritis and otitis. The importance of M. bovis has escalated because of recent outbreaks and introductions into countries previously free of M. bovis. We characterized the course of M. bovis infection on 19 recently infected dairy farms over 24 months. Our objective was to identify diagnostic tools to assess the efficacy of control measures to assess low risk infection status on M. bovis infected farms. PCR assays and culture were used to detect M. bovis, and in-house and BioX ELISAs were used to follow antibody responses. Cows and young stock were sampled on four separate occasions, and clinical cases were sampled when they arose. On 17 farms, a few cases of clinical mastitis were detected, mostly within the first eight weeks after the index case. Antibodies detected by in-house ELISA persisted in the serum of cows at least for 1.5 years on all farms, regardless of the M. bovis infection status or signs of clinical disease or subclinical mastitis on the farm. Six out of 19 farms became low risk as the infection was resolved. Our results suggest that, for biosecurity purposes, regular monitoring should be conducted on herds by screening for M. bovis in samples from cows with clinical mastitis and calves with pneumonia, in conjunction with testing young stock by screening longitudinally collected nasal swabs for M. bovis and sequential serum samples for antibody against recombinant antigen.

Cryptosporidium parvum Caused a Large Outbreak Linked to Frisée Salad in Finland, 2012.

Over 250 individuals fell ill in five outbreaks caused by Cryptosporidium parvum in Finland, October-November 2012. The cases were connected by lunch meals at restaurants in four different cities. In two outbreaks, the same C. parvumIIdA17G1 subtype was found in patients' stool samples which supports a single source of infection. Frisée salad was the only common food item served at the restaurants, and consumption of lunch salad containing the frisée salad was associated with the illness. Lunch customers who responded that they had eaten lunch salad were three times more likely to have become ill than those who had not answered whether they had eaten the salad or not (RR 2.66; 95% Cl 1.02-6.9, P-value <0.01). Cryptosporidiosis should be considered as a causal agent in long-lasting watery diarrhoea combined with abdominal cramps, and clinical samples should be tested for Cryptosporidium at the same time bacteria and viruses are tested. Measures to prevent contamination of 'ready-to-eat vegetables' with Cryptosporidium oocysts and methods to test frozen food samples should be developed.

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds.

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.

Atypical growth of Renibacterium salmoninarum in subclinical infections.

Two growth types of Renibacterium salmoninarum were isolated from subclinically infected rainbow trout, one producing the smooth colonies typical of R. salmoninarum and the other forming a thin film on the surface of the agar with no separate colonies. The atypical growth was present on kidney disease medium agar in primary cultures of the kidney but not on selective kidney disease medium (SKDM). Fluorescent antibody staining of the fresh isolate and polymerase chain reaction amplification were the most reliable techniques to identify the atypical growth of R. salmoninarum. The condition was reversible, with growth reverting from atypical to the smooth colony form in experimentally infected rainbow trout and under laboratory conditions. There was no mortality, or any clinical signs of bacterial kidney disease (BKD) in the fish challenged with the atypical growth, although small numbers of smooth colonies of R. salmoninarum were isolated from 8% of these fish. The atypical growth reported here may explain some of the failures of culture, when SKDM agar alone is used for the detection of BKD in subclinically infected fish.

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection.

A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.

The effect of antimicrobial growth promoter withdrawal on the health of weaned pigs in Finland.

The use of the antimicrobial growth promoters (AGPs) carbadox and olaquindox has been banned in the European Union (EU) since September 1999. We studied the effects of the withdrawal on the health of weaned piglets on two types of piglet-producing farms (farrowing herds and farrow-to-finish herds) from the different regions of Finland. Farms with no major problems with post-weaning diarrhoea were selected for the study to better evaluate the effect of AGPs alone. Data on production, medication and incidence of diarrhoea were collected from 73 farms during 1 year after the withdrawal. On 29 of these farms, the data collection began 4 months before the withdrawal. The health management of the pigs is considered good in Finland, and special attention has been paid to improve the husbandry practices and management of the farms. Eighty-two percent of the farms in the study were free of both Mycoplasma hyopneumoniae and Sarcoptes scabiei infection. Brachyspira hyodysenteriae infection was not detected in any of the farms. The median number of sows in the herds was 56.0 (IQR=43.0; 72.5) in 2000. The level of antimicrobial use in each herd was classified as low, moderate and high when the percentage of weaned pigs treated for diarrhoea during a 4-month period was 0-5%, 6-19% and > or =20%, respectively. Only on four herds (14%), there was an increase in the level of antibiotic use after the AGP withdrawal, when seasonally corresponding 4-month periods were compared. Fourty-one percent of these 29 farms were categorized as low users of antimicrobials, 38% as moderate users and 21% as high users. The level of antimicrobial use for treatment of diarrhoea after weaning (and the incidence of diarrhoea in weaned piglets) did not increase significantly after the withdrawal of AGPs from weaner feeds according to farmers' evaluations. In this study, the Escherichia coli infection was the most-common cause of diarrhoea in weaned pigs. The age at weaning did not change after the withdrawal of AGPs.

Molecular epidemiological study of Brachyspira pilosicoli in Finnish sow herds.

Brachyspira (B.) pilosicoli, the causative agent of intestinal spirochaetosis in pigs, is a quite common laboratory finding from faecal samples of weaned and growing pigs in Finland. A better understanding of the epidemiology of B. pilosicoli in and between Finnish pig farms is needed. Altogether 131 B. pilosicoli isolates from 49 Finnish sow herds were studied by pulsed-field gel electrophoresis. MluI was used as a restriction enzyme for all the isolates, and SmaI for 70 isolates. The isolates were divided into 54 different macrorestriction profiles (MRP) by MluI. Most farms had distinct B. pilosicoli genotypes, and common genotypes among herds were rare. B. pilosicoli was re-isolated after 3 years in three herds; the same MRP persisted in each of these herds. A genetic clustering of B. pilosicoli isolates between two major pork production areas was not detected.

Putative virulence factors of atypical Aeromonas salmonicida isolated from Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.).

Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.

Mapping the binding domain of the F18 fimbrial adhesin.

F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.

Virulence factors of Escherichia coli isolated from bovine clinical mastitis.

Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors. The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E. coli, clinical pictures of coliform mastitis, environmental conditions and herd management. The genes of nine virulence factors were detected by polymerase chain reaction. Presence of K1 and K5 capsules was assessed by use of specific bacteriophages. Serum resistance was tested by a turbidimetric assay. Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E. Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17. Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants. Genes for F17a, CS31A, Afa7D and Afa7E were not detected. Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates. Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113). K1 and K5 capsules were not detected.

Eradication of endemic Brachyspira pilosicoli infection from a farrowing herd: a case report.

Brachyspira pilosicoli and B. innocens were isolated repeatedly from a herd of 60 sows which mostly produced feeder pigs but also raised some fattening pigs. Postweaning diarrhea had been a severe problem in this herd for years. The B. pilosicoli eradication plan was based on the general guidelines for elimination of B. hyodysenteriae, with some modifications. The eradication measures were run in August 1997. In-feed medication with 200 p.p.m. tiamulin lasted for 18-30 days, depending on the age group. The piggery unit was emptied, cleaned, disinfected and dried, and all worn surfaces were repaired. The animals were removed to temporary sheds situated 0-100 m from the piggery unit. Only the sows and the boar returned to the piggery unit. All other pigs were sold from the sheds within 3 months after the eradication. Immediately after the eradication, the clinical postweaning diarrhea disappeared. The success of the program was monitored four times bacteriologically, and the last control sampling was in December 1999, 7 months after the total withdrawal of antimicrobial feed additives. The primary cultures from the last three samplings were also analysed with B. pilosicoli-specific PCR. All the samples were negative for B. pilosicoli. However, B. innocens could be isolated from each batch of samples. The analysis of B. innocens isolates by pulsed-field gel electrophoresis indicated that at least one genotype persisted in the herd. The clinical and laboratory findings suggest that the eradication of B. pilosicoli had succeeded in this herd.

Prevalence and characteristics of shiga toxin-producing Escherichia coli from healthy cattle in Japan.

The prevalence of Shiga toxin-producing Escherichia coli (STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with stx-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested stx-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony stx hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic E. coli (EHEC) hlyA genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.