Streptococcus agalactiae in the environment of bovine dairy herds--rewriting the textbooks?

Many free-stall bovine dairy herds in Norway fail to eradicate Streptococcus agalactiae despite long-term control measures. In a longitudinal study of 4 free-stall herds with automatic milking systems (AMS), milk and extramammary sites were sampled 4 times with 1-2 month intervals. Composite milk, rectal- and vaginal swabs were collected from dairy cows; rectal swabs from heifers and young stock; rectal- and tonsillar swabs from calves; and environmental swabs from the AMS, the floors, cow beds, watering and feeding equipment. A cross sectional study of 37 herds was also conducted, with 1 visit for environmental sampling. Fifteen of the herds were known to be infected with S. agalactiae while the remaining 22 had not had evidence of S. agalactiae mastitis in the preceding 2 years. All samples were cultured for S. agalactiae, and selected isolates (n=54) from positive herds were genotyped by Multi Locus Sequence Typing (MLST). Results show that the bovine gastrointestinal tract and the dairy cow environment are reservoirs of S. agalactiae, and point to the existence of 2 transmission cycles; a contagious transmission cycle via the milking machine and an oro-fecal transmission cycle, with drinking water as the most likely vehicle for transmission. Ten sequence types were identified, and results suggest that strains differ in their ability to survive in the environment and transmit within dairy herds. Measures to eradicate S. agalactiae from bovine dairy herds should take into account the extra-mammary reservoirs and the potential for environmental transmission of this supposedly exclusively contagious pathogen.

Persistence of staphylococcal species and genotypes in the bovine udder.

Staphylococci are a major cause of intramammary infections (IMI) in ruminants. The main aim of this study was to investigate staphylococcal IMI in dairy cattle with emphasis on persistence and distribution of staphylococcal species and genotypes. With a sampling interval of 4-8 weeks, over a year, 4030 samples from 206 cows in 4 herds were collected. Coagulase-negative staphylococci (CNS) and Staphylococcus aureus were detected in 13.2% and 4.2% of the samples, respectively. Selected CNS isolates from quarter milk samples were identified to species level using sodA sequencing. Staphylococcus chromogenes (32%) and Staphylococcus simulans (25%) predominated. The proportion of S. chromogenes was greater in primiparous (52%) than in multiparous cows (12%), while the opposite was the case for Staphylococcus epidermidis (6% and 21%, respectively). Isolates from possibly persistent IMI were selected for pulsed-field gel electrophoresis (PFGE). Six staphylococcal species were found to cause persistent IMI; S. aureus, S. chromogenes, S. simulans, S. epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri. It was shown that several pulsotypes (PTs) within each species were associated with persistent infections, but only a few were spread and caused persistent IMI in multiple cows within a herd. Of special interest was the observation that only one, or a few, strains of each species caused persistent IMI in multiple cows within a same herd. This indicates strain differences with respect to transmissibility and pathogenicity.

Reservoirs of Staphylococcus aureus in meat sheep and dairy cattle.

The objective of the study was to investigate reservoirs and transmission of S. aureus in ewes and lambs in 3 meat sheep flocks. Repeated sampling of milk, teat skin, nasal- and vaginal mucous membranes was performed and samples were analysed for S. aureus. For comparison, samples were also collected from cows and young heifers in 3 dairy cattle herds. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE). S. aureus was detected in 8 (1.5%) of 520 milk samples from ewes and in 38 (6.4%) of 588 milk samples from cows. From body site swabs, S. aureus was found in 394 (32.6%) of 1208 samples from sheep and in 67 (16.0%) of 420 samples from cattle. The proportion of S. aureus-positive nasal swabs from ewes and cows were 56.7% and 13.9%, respectively. From lambs, 58.2% of the nasal swabs were S. aureus-positive. In each flock, one S. aureus pulsotype predominated. Identical S. aureus pulsotypes were found in milk and from body sites. Paired S. aureus isolates from the nasal cavity of (i) ewes and their lambs, (ii) twins and (iii) from repeated swabs of individual ewes were compared by PFGE, and in the majority of cases the two isolates were identical. The results contribute new knowledge indicating frequent transmission of S. aureus between the dam and her lambs and within animals in a flock. In contrast to cattle, S. aureus is frequently present in the nose of sheep which may represent the primary reservoir of S. aureus in sheep flocks.

Bacteriological and molecular investigations of Staphylococcus aureus in dairy goats.

In order to investigate reservoirs of Staphylococcus aureus in dairy goats, samples for bacteriological analyses were collected from seven herds. S. aureus was detected in 353 (6.2%) of 5671 milk samples, 53 (9.9%) of 535 teat skin swabs, 392 (68.9%) of 569 nasal swabs and in 180 (31.6%) of 569 vaginal swabs. Vaginal swabs were more often S. aureus-positive after kidding (44.9%) than before drying off (19.1%), while nasal swabs were more often positive before drying off (75.6%) than after kidding (62.0%). Retrieved S. aureus isolates were compared by pulsed-field gel electrophoresis (PFGE), and selected isolates were tested for enterotoxin genes (se) by PCR. By PFGE, 505 S. aureus isolates were divided into 33 pulsotypes (PTs). The five most prevalent PTs included 73.3% of the isolates and were found in 3-5 herds. Pairs of S. aureus isolates from persistent intramammary infections (IMI), repeated vaginal swabs, and from milk and teat skin from the same animal were usually identical. Paired isolates from other body sites of the same animal, including from bilateral IMI, were identical in less than 50% of the situations. The majority (71.9%) of analysed S. aureus isolates were se-positive. The genes sec, sell and tst were detected almost exclusively, but no correlation was observed between persistence of IMI and the enterotoxin gene profile of the causal S. aureus strains. The frequent presence of S. aureus on the mucous membranes may contribute to dispersal of the bacteria among dairy goats, hampering effective transmission control in dairy goat herds.

Intestinal parasites of the Arctic fox in relation to the abundance and distribution of intermediate hosts.

The intestinal parasite community of Arctic foxes (Vulpes lagopus) on the Svalbard archipelago in the High Arctic was investigated in relation to the abundance and distribution of intermediate hosts. Five species of cestodes (Echinococcus multilocularis, Taenia crassiceps, Taenia polyacantha, Taenia krabbei and Diphyllobothrium sp.), ascaridoid nematodes and one unidentified acanthocephalan species were found. The cestodes E. multilocularis, T. crassiceps and T. polyacantha all showed a decreasing prevalence in the fox population with increasing distance from their spatially restricted intermediate host population of sibling voles (Microtus levis). In addition, the prevalence of E. multilocularis in a sample from the vole population was directly related to the local vole abundance. The cestode T. krabbei uses reindeer as intermediate host, and its prevalence in female foxes was positively related to the density of reindeer (Rangifer tarandus platyrhyncus). Finally, the prevalence of the ascaridoid nematodes also decreased with increasing distance from the vole population, a finding that is consistent with the idea that voles are involved in transmission, most likely as paratenic hosts. The prevalence of the remaining species (Diphyllobothrium sp. and an unidentified acanthocephalan) was very low. We conclude that the distribution and abundance of intermediate host structure the gastrointestinal parasite community of the Arctic fox on the Svalbard archipelago.

Phylogenetic comparison of rabies viruses from disease outbreaks on the Svalbard Islands.

Periodic wildlife rabies epizootics occur in Arctic regions. The original sources of these outbreaks are rarely identified. In 1980, a wildlife epizootic of rabies occurred on the previously rabies-free Svalbard Islands, Norway. After this outbreak of rabies in the arctic fox population (Alopex lagopus), only single cases have been reported from the Islands over the following two decades. Phylogenetic characterization of four viruses isolated from infected arctic foxes from Svalbard from three different time periods suggest that the source of these epizootics could have been migration of this species from the Russian mainland. Arctic fox migration has likely contributed to the establishment of another zoonotic disease, Echinococcus multilocularis, on Svalbard in recent years.

Environmental contaminants in arctic foxes (Alopex lagopus) in Svalbard: relationships with feeding ecology and body condition.

Adipose tissues from 20 arctic foxes (Alopex lagopus) of both sexes from Svalbard were analysed for polybrominated diphenyl ether (PBDE), polychlorinated biphenyl (PCB), p,p'-dichlorodiphenyltrichloroethane (DDE), chlordane, and hexachlorobenzene (HCB) concentrations. Gender (0.43

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese.

In recent years, the small-scale production of raw milk products has increased in Norway, and there is some concern that such foods may pose a risk of staphylococcal food poisoning to consumers. The aim of the study was to evaluate potential sources of contamination of raw milk cheese with Staphylococcus aureus on a bovine dairy farm with small-scale production. Samples for bacteriological analyses (n = 144) were collected from the animals, the environment, processing equipments, from humans, and from cheeses at various stages of production. Staphylococcus aureus was isolated from 10 of 11 cows, the farmer, equipment, the environment, and the cheese. Seventy-five Staph. aureus isolates were genotyped by pulsed-field gel electrophoresis, tested for enterotoxin (SE) production by reversed passive latex agglutination, for SE genes by multiplex polymerase chain reaction, and for penicillin resistance by the cloverleaf method. Five different pulsotypes were identified and SE gene fragments were identified in 11 isolates, but no isolates produced SE or were penicillin resistant. Staphylococcus aureus was found throughout the farm, and appeared to be spread with the milk to the environment, equipment, and to products. One pulsotype dominated and was identified from most sample sites on the farm. Raw milk products are vulnerable to contamination with Staph. aureus. Strategies to reduce the occurrence of Staph. aureus in bulk milk are of particular importance on farms where milk is used for raw milk products.

Comparison of Staphylococcus aureus genotypes recovered from cases of bovine, ovine, and caprine mastitis.

Staphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine mastitis. By pulsed-field gel electrophoresis, 160 different pulsotypes (PTs) were identified among 905 isolates recovered from 588 herds in 12 counties in Norway. Based on estimates of similarity, using an 80% cluster cutoff, the isolates were assigned to 47 clusters. One cluster included 62% of all the isolates and more than 45% of the isolates from each host species. Twenty-three PTs included isolates from more than one host species; these 23 PTs represented 72% of all the isolates. The six most prevalent PTs included isolates from all host species and contained 45% of the bovine isolates, 54% of the ovine isolates, and 37% of the caprine isolates. Antimicrobial susceptibility testing of 373 of the isolates revealed resistance to penicillin in 2.9% and to streptomycin in 2.4%; only 1.9% were resistant to 1 of the other 11 antimicrobials tested. The results of this study suggest that a small number of closely related genotypes are responsible for a great proportion of S. aureus mastitis cases in cows, ewes, and goats in Norway and that these genotypes exhibit little or no host preference among these species. Selection due to antimicrobial resistance appears not to have contributed to the predominance of these genotypes.

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway.

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.

Acute toxoplasmosis in three wild arctic foxes (Alopex lagopus) from Svalbard; one with co-infections of Salmonella Enteritidis PT1 and Yersinia pseudotuberculosis serotype 2b.

Acute disseminated toxoplasmosis was diagnosed in three wild arctic foxes (Alopex lagopus) that were found dead in the same locality on Svalbard (Norway). The animals included one adult female and two 4-months-old pups. The adult fox was severely jaundiced. Necropsy revealed multifocal, acute, necrotizing hepatitis, acute interstitial pneumonia, and scattered foci of brain gliosis, often associated with Toxoplasma tachyzoites. One pup also had Toxoplasma-associated meningitis. In addition, the latter animal was infected with Yersinia pseudotuberculosis serotype 2b and Salmonella Enteritidis phage type 1 (PT1), which may have contributed to the severity of the Toxoplasma infection in this animal. The diagnosis of toxoplasmosis was confirmed by positive immunohistochemistry and detection of anti-Toxoplasma gondii antibodies in serum of all foxes. The animals were negative for Neospora caninum, canine distemper virus, canine adenovirus, and rabies virus on immunolabelling of tissue sections and smears.

Spread of Staphylococcus aureus resistant to penicillin and tetracycline within and between dairy herds.

One hundred and seven bovine isolates of penicillin and tetracycline resistant Staphylococcus aureus, recovered from 25 different dairy herds in various parts of Norway, were characterized using antimicrobial susceptibility testing, multilocus enzyme electrophoresis, ribotyping, plasmid analysis and serotyping of capsular polysaccharide. Forty-one isolates from one particular herd, 37 isolates from 5 herds that used a common pasture and milking parlour in summer and 21 isolates from 12 herds in 8 different counties belonged to the same strain. The remaining 8 isolates, which originated from herds in 5 different counties, were assigned to 6 different strains. Seven out of these 8 isolates had the same plasmid restriction profile. In conclusion, penicillin and tetracycline resistant S. aureus occurring in dairy herds in Norway mainly seems to represent one particular strain that has achieved widespread distribution or belong to one of several different strains carrying identical plasmids.

The usage of veterinary antibacterial drugs for mastitis in cattle in Norway and Sweden during 1990-1997.

The prescribing patterns and annual incidence of use of antibacterial drugs for the treatment of mastitis in cattle in Norway and Sweden during the period 1990-1997 were estimated from drug wholesaler statistics. Although the drugs included in this study are also used in other species and/or other indications, mastitis in cattle is by far the most-common indication for their use. We used these sales figures to evaluate trends in the use of antibacterial drugs and changes in the incidence of treatment in bovine mastitis in Norway and Sweden. To facilitate comparisons (correcting for differences in activity and dosage) between the relative proportions of antibacterial drugs used in bovine mastitis, we introduced defined daily dose cow (DDDcow) as unit of measurement. Tentative DDDcow for the various injectable drugs were derived from doses recommended in Norway and Sweden. For intramammary drugs administered in the form of single-dose applicators, one applicator was defined as the DDDcow. The prescribing patterns of antibacterial drugs in bovine mastitis in Norway and Sweden during the study period seemed to be influenced by treatment policies, substances and formulations approved and treatment cost; length of the withdrawal period also seemed to play a role.

Direct digital radiography versus conventional radiography for estimation of canal length in curved canals.

A recent development in direct digital radiography (DDR) has made it possible to make additive multiple point (click) measurements of onscreen images. Canal length was examined using four estimation methods: measurement of a conventional D-speed film radiograph, and onscreen DDR allowing two clicks, six clicks, and an unlimited number of clicks of the measuring device. Thirty extracted human teeth with root curvatures ranging from 7 degrees to 47 degrees (Schneider method) were examined. Actual canal length was measured using a millimeter rule and x 2 magnification. Teeth were mounted and prepared for conventional and DDR imaging. Estimated canal length was determined by three board-certified endodontists using each of the four techniques. A two-way analysis of variance indicated that all radiographic techniques resulted in canal lengths that were significantly different from the true canal length, and there was no significant difference between experimental groups, regardless of the canal curvature.

Bacteria associated with clinical mastitis in dairy heifers.

A 1-yr field investigation of clinical mastitis in heifers was carried out in 24 veterinary districts in Norway. Quarter lacteal secretions from cases that occurred prepartum or within 14 d postpartum were examined bacteriologically. The study included 1040 heifers with clinical mastitis, and the total number of quarters that were clinically affected was 1361. The organisms that were most frequently isolated from samples from these quarters were Staphylococcus aureus (44.3%), Streptococcus dysgalactiae (18.2%), Staph. aureus together with Strep. dysgalactiae (1.2%), coagulase-negative staphylococci (12.8%), Arcanobacterium pyogenes (3.5%), A. pyogenes together with Strep. dysgalactiae (0.5%) or Staph. aureus (0.4%), and Escherichia coli (6.4%). Of the coagulase-negative staphylococci, Staphylococcus simulans (53.7%), Staphylococcus hyicus (14.8%), and Staphylococcus chromogenes (14.8%) were the most prevalent species. Except for a higher relative percentage of A. pyogenes in cases that occurred before parturition (8.2%) than in cases that occurred after parturition (2.7%), no significant differences were observed in the distribution of the various organisms among prepartum and postpartum cases. Regional variations were observed in the distribution of organisms. The proportions of Staph. aureus and A. pyogenes were highest, and the proportion of coagulase-negative staphylococci was lowest, in late autumn and early winter. The proportion of E. coli was highest in summer. In heifers in which mastitis was associated with increased rectal temperature or other systemic signs, the proportion of clinically affected quarters that were infected with Staph. aureus was larger than that in heifers without systemic reaction.

Linear dye penetration of a calcium phosphate cement apical barrier.

Linear dye penetration was evaluated in teeth with open apices in which calcium phosphate cement was used as an apical barrier to facilitate obturation. The apical foramens of 42 extracted single-rooted human teeth were opened to a size 90 file. Half the teeth received apical barriers consisting of calcium phosphate cement (CPC) followed by obturation using a customized gutta-percha cone/ lateral condensation technique. The other half were obturated without benefit of apical barriers. Linear dye penetration was measured after 48 h exposure to India ink. The teeth receiving apical CPC barriers before obturation had significantly less dye penetration than teeth without apical barriers. Based on its proven biocompatibility and osteconductive potential, calcium phosphate cement may serve well as a replacement for calcium hydroxide in a single-visit immediate apical barrier apexification technique.

An in vitro study of furcation perforation repair using calcium phosphate cement.

Furcation perforations created in the pulpal floors of 30 extracted human molars were repaired with either light-cured glass ionomer cement (GI), calcium phosphate cement (CPC), or light-cured glass ionomer cement placed over a CPC matrix (M). After the cement was set, the teeth were immersed in India Ink for 48 h, dried, and sectioned longitudinally. Dye penetration into the furcation repair was independently evaluated by three board-certified endodontists. There was no significant difference in the mean extent of dye leakage among the three experimental groups. The use of CPC, with its enhanced biocompatibility, potential for osteoconduction, and sealing ability, may improve the prognosis of teeth with furcation perforations.

Mechanisms of nonopsonic phagocytosis of Pseudomonas aeruginosa.

The interaction of the macrophage cell line P388D1 with Pseudomonas aeruginosa in the absence of stimulators or opsonins led to substantial association of bacteria, as judged by visual counting and FACScan assays. This association was observable within 5 min of addition of bacteria, could not be disturbed by exhaustive washing, and occurred with pilus- or flagellum-deficient mutants but not with rpoN mutants, which have been proposed to lack a secondary adhesin. In contrast, specific antibody was capable of causing similar enhancement of bacterial uptake regardless of the rpoN phenotype. Fibronectin stimulated uptake of bacteria with the pilus as an adhesin, and stimulation was observable within 5 min. Both fibronectin-enhanced and antibody-opsonized uptake were susceptible to inhibition by pertussis toxin but not by cholera toxin. The influence of fibronectin on P388D1 cells was distinguishable from that of lipopolysaccharide, which caused substantial morphological changes in cells. Although lipopolysaccharide stimulated bacterial uptake, it actually suppressed fibronectin-mediated enhancement of uptake at high concentrations.