Mineralization in a Critical Size Bone-Gap in Sheep Tibia Improved by a Chitosan-Calcium Phosphate-Based Composite as Compared to Predicate Device.

Deacetylated chitin derivatives have been widely studied for tissue engineering purposes. This study aimed to compare the efficacy of an injectable product containing a 50% deacetylated chitin derivative (BoneReg-Inject™) and an existing product (chronOS Inject®) serving as a predicate device. A sheep model with a critical size drill hole in the tibial plateau was used. Holes of 8 mm diameter and 30 mm length were drilled bilaterally into the proximal area of the tibia and BoneReg-Inject™ or chronOS Inject® were injected into the right leg holes. Comparison of resorption and bone formation in vivo was made by X-ray micro-CT and histological evaluation after a live phase of 12 weeks. Long-term effects of BoneReg-Inject™ were studied using a 13-month live period. Significant differences were observed in (1) amount of new bone within implant (p < 0.001), higher in BoneReg-InjectTM, (2) signs of cartilage tissue (p = 0.003), more pronounced in BoneReg-InjectTM, and (3) signs of fibrous tissue (p < 0.001), less pronounced in BoneReg-InjectTM. Mineral content at 13 months postoperative was significantly higher than at 12 weeks (p < 0.001 and p < 0.05, for implant core and rim, respectively). The data demonstrate the potential of deacetylated chitin derivatives to stimulate bone formation.

Icelandic ovine Mycoplasma ovipneumoniae are variable bacteria that induce limited immune responses in vitro and in vivo.

PURPOSE: Mycoplasma ovipneumoniae is a pathogen that causes atypical pneumoniae in sheep and goats. While infection of lambs can induce strong immune responses, typically measured as serum antibodies, experimental vaccines appear to induce lower antibody titres. The purpose of this study was to better understand the bacterium and its interaction with the host, in order to improve the vaccination strategy. METHODOLOGY: We designed primers to compare seven M. ovipneumoniae gene sequences, in addition to the 16S sequence typically used, to estimate the variability between isolates. In addition, we labelled bacteria with a two-step process to examine whether bacteria could be intracellular as well as on the host surface in vitro. Finally, we vaccinated sheep four times and examined the induction of humoral and cellular responses. RESULTS: We were able to reliably amplify the seven housekeeping gene sequences to examine variability of the different isolates, and the bacteria could be found intracellularly, as well as on the host cell surface. Four vaccinations of sheep produced only modest humoral and cellular responses in this study, likely due to previous exposure of the animals to mycoplasmas. CONCLUSIONS: The moderate immune responses seen in this study indicate that previous exposure to mycoplasmas is a challenge for vaccination of lambs against M. ovipneumoniae. However, an alternative vaccination strategy, e.g. utilizing a recombinant vaccine, may overcome this vaccination hurdle in endemic regions and we suggest a possible vaccine candidate.

Genomic Dissection of an Icelandic Epidemic of Respiratory Disease in Horses and Associated Zoonotic Cases.

Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country's native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans.IMPORTANCE An epidemic of respiratory disease affected almost the entire native Icelandic horse population of 77,000 animals in 2010, resulting in a self-imposed ban on the export of horses and significant economic costs to associated industries. Although the speed of transmission suggested that a viral pathogen was responsible, only the presence of the opportunistic pathogen Streptococcus zooepidemicus was consistent with the observed clinical signs. We applied genomic sequencing to differentiate epidemic from endemic strains and to shed light on the rapid transmission of the epidemic strain throughout Iceland. We further highlight the ability of epidemic and endemic strains of S. zooepidemicus to infect other animals, including humans. This study represents the first time that whole-genome sequencing has been used to elucidate an outbreak on a national scale and identify the likely causative agent.

Variability of Pasteurella multocida isolated from Icelandic sheep and detection of the toxA gene.

Pasteurella multocida can be part of the upper respiratory flora of animals, but under conditions of stress or immunocompromisation, the bacteria can cause severe respiratory symptoms. In this study, we compared 10 P. multocida isolates from Icelandic sheep with respiratory symptoms and 19 isolates from apparently healthy abattoir sheep. We examined capsule type, genetic variability and the presence of the toxA gene in the two groups. Surprisingly, we found that all ovine P. multocida isolates examined in this study carried the toxA gene, which markedly differs from what has been published from other studies. Interestingly, all isolates from abattoir animals were capsule type D, whilst bacteria isolated from animals with clinical respiratory symptoms had capsule type A, D or F. Examination of seven housekeeping genes indicated that the clinical respiratory isolates were significantly more heterogeneous than the abattoir isolates (P<0.05, two-tailed Mann-Whitney U test). The results suggest that there may be at least two groups of P. multocida in sheep - a genetically homogeneous group that resides in the respiratory tract and a genetically heterogeneous group that is the predominant cause of disease.

Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure.

The detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet - diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi.

Fatal infection in two Icelandic stallions caused by Halicephalobus gingivalis (Nematoda: Rhabditida).

Opportunistic infections with the free living nematode Halicephalobus gingivalis are infrequently reported in horses but the cases are widespread geographically. The nematodes are believed to penetrate wounds and subsequently reproduce within the host tissues. This paper reports two cases of a fatal disease in stallions of the Icelandic breed in Iceland. Case 1: a stallion, which sustained injuries to the mouth after an accident, developed severe neurological signs and had to be euthanatized. Histological examination revealed mild inflammation and malacia in the cerebellum associated with the presence of numerous H. gingivalis nematodes. Case 2: a stallion that started swerving to one side and lost balance was euthanatized due to lack of response to therapy and rapid deterioration. Histological examination revealed numerous H. gingivalis nematodes in the cerebellum, brain stem, cervical spinal cord and in the meninges, with minimal reactive changes. In case 1 the infection presumably was acquired by nematodes from soil penetrating through wounds in the mouth. The mode of the H. gingivalis infection in case 2 is uncertain. These are the first cases of H. gingivalis infection reported from Iceland and the second report from the Nordic countries.

Broiler chickens as source of human fluoroquinolone-resistant Escherichia coli, Iceland.

To investigate feed as a source for fluoroquinolone-resistant Escherichia coli in broiler chickens, we compared antimicrobial drug-resistant E. coli from broiler feed and broilers with ciprofloxacin-resistant human clinical isolates by using pulsed-field gel electrophoresis. Feed was implicated as a source for ciprofloxacin-resistant broiler-derived E. coli and broilers as a source for ciprofloxacin-resistant human-derived E. coli.

Broiler Campylobacter contamination and human campylobacteriosis in Iceland.

To examine whether there is a relationship between the degree of Campylobacter contamination observed in product lots of retail Icelandic broiler chicken carcasses and the incidence of human disease, 1,617 isolates from 327 individual product lots were genetically matched (using the flaA short variable region [SVR[) to 289 isolates from cases of human campylobacteriosis whose onset was within approximately 2 weeks from the date of processing. When there was genetic identity between broiler isolates and human isolates within the appropriate time frame, a retail product lot was classified as implicated in human disease. According to the results of this analysis, there were multiple clusters of human disease linked to the same process lot or lots. Implicated and nonimplicated retail product lots were compared for four lot descriptors: lot size, prevalence, mean contamination, and maximum contamination (as characterized by direct rinse plating). For retail product distributed fresh, Mann-Whitney U tests showed that implicated product lots had significantly (P = 0.0055) higher mean contamination than nonimplicated lots. The corresponding median values were 3.56 log CFU/carcass for implicated lots and 2.72 log CFU/carcass for nonimplicated lots. For frozen retail product, implicated lots were significantly (P = 0.0281) larger than nonimplicated lots. When the time frame was removed, retail product lots containing Campylobacter flaA SVR genotypes also seen in human disease had significantly higher mean and maximum contamination numbers than lots containing no genotypes seen in human disease for both fresh and frozen product. Our results suggest that cases of broiler-borne campylobacteriosis may occur in clusters and that the differences in mean contamination levels may provide a basis for regulatory action that is more specific than a presence-absence standard.

Antimicrobial resistance of Campylobacter spp. isolated from broiler flocks in Iceland 2001-2005.

Minimum inhibitory concentrations of six antimicrobial agents were determined for one Campylobacter sp. isolate from each of the 362 Campylobacter-positive commercial chicken flocks in Iceland in the years 2001-2005. Of all isolates tested, 6.9% were resistant, although none were multiresistant. Resistance to ampicillin was most commonly observed (3.6%) followed by resistance to enrofloxacin (3%), nalidixic acid (1.9%), and oxytetracyclin (0.3%), with cross-resistance between enrofloxacin and nalidixic acid. All isolates were susceptible to erythromycin and gentamicin. Resistance rates among Campylobacter coli isolates (7/13 or 53.8%) were much higher than among Campylobacter jejuni isolates (18/349 or 5.2%), and resistance patterns differed. Resistant strains were compared using pulsed field gel electrophoresis. Macrorestriction with SmaI and KpnI restriction enzymes yielded 13 different pulsotypes, none of which indicated a predominant genotype. Specific pulsotypes with uniform resistance patterns arising on geographically separated farms indicate clonal dissemination. Although resistance levels were low and similar to that seen in the other Nordic countries, further research on this matter is needed as there is no antimicrobial selective pressure in chicken farming in Iceland.

Antimicrobial resistance and serotype distribution among Salmonella spp. in pigs and poultry in Iceland, 2001-2005.

Little information is available on antimicrobial resistance of bacteria isolated from animals and animal products in Iceland. The objective of this study was to analyze serotype distribution and antimicrobial resistance patterns of Salmonella spp. isolated from healthy chickens and pigs in Iceland during 2001-2005. A total of 163 Salmonella strains, isolated in the national Salmonella surveillance program, were available for study. Isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC) and resistant strains were compared using pulsed-field gel electrophoresis (PFGE) and phage typing. The most commonly isolated serotypes were Salmonella Infantis (61%) and S. Typhimurium (33%); other serotypes were less prevalent. The overall prevalence of resistance was 13.6% in chickens and 12.8% in pigs. Twenty one isolates (12.8%) were resistant to one or more antimicrobials, 19 S. Typhimurium strains, one S. Infantis strain, and one S. Worthington strain. Sixteen out of the 19 resistant S. Typhimurium strains were multiresistant (to > or =3 antimicrobial agents), and, of these, 15 had identical or closely related PFGE patterns (previously phage typed as DT104). The prevalence of antimicrobial resistance in Salmonella spp. in pigs and poultry in Iceland is low; however, we found a multiresistant S. Typhimurium clone that causes concern. Continuous resistance surveillance is important, and further research on the source of resistant clones and possible transmission to humans is needed.

Lack of evidence for vertical transmission of Campylobacter spp. in chickens.

Campylobacter jejuni is a major cause of bacterial food-borne infection in the industrial world. There is evidence that C. jejuni is present in eggs and hatchery fluff, opening the possibility for vertical transmission from hens to progeny. Poultry operations in Iceland provide an excellent opportunity to study this possibility, since breeding flocks are established solely from eggs imported from grandparent flocks in Sweden. This leaves limited opportunity for grandparents and their progeny to share isolates through horizontal transmission. While Campylobacter was not detected in all grandparent flocks, 13 of the 16 egg import lots consisted of eggs gathered from one or more Campylobacter-positive grandparent flocks. No evidence of Campylobacter was found by PCR in any of the 10 relevant quarantine hatchery fluff samples examined, and no Campylobacter was isolated from the parent birds through 8 weeks, while they were still in quarantine rearing facilities. After the birds were moved to less biosecure rearing facilities, Campylobacter was isolated, and 29 alleles were observed among the 224 isolates studied. While three alleles were found in both Sweden and Iceland, in no case was the same allele found both in a particular grandparent flock and in its progeny. We could find no evidence for vertical transmission of Campylobacter to the approximately 60,000 progeny parent breeders that were hatched from eggs coming from Campylobacter-positive grandparent flocks. If vertical transmission is occurring, it is not a significant source for the contamination of chicken flocks with Campylobacter spp.

Risk factors for Campylobacter spp. colonization in broiler flocks in Iceland.

We sampled 1,091 Icelandic broiler flocks at slaughter from May 2001 to December 2003 to determine the prevalence of, and investigate risk factors for the presence of, Campylobacter spp. at the flock level. Approximately 15% of the flocks were positive for Campylobacter spp.; most (95%) of the infected flocks being raised during the months of April-September. Based on the data from the latter months, and using multivariable logistic regression with random effects for herd, we found that the odds of a flock being positive for Campylobacter spp. increased with age and flock size. Additionally, vertical ventilation systems were strongly associated with positive flocks (OR=5.3). After controlling for these variables, we found no evidence of an effect of: year; company; Campylobacter being carried over from one flock to the next; time interval between flocks; using (at the hatcheries) eggs laid on the floor; density of bird housing, or the number of catch lots a flock was divided into for slaughtering purposes on the risk of a Campylobacter-positive flock.

Vaccinia virus complement control protein diminishes formation of atherosclerotic lesions: complement is centrally involved in atherosclerotic disease.

Complement is known to be activated in atherosclerotic lesions, but the importance of this event in disease pathology is a matter of debate. Studies of rabbits fed a high-fat diet have indicated complement activation as a rate-limiting step, whereas results from genetically modified mouse strains (ApoE-/- or LDLR-/-) have failed to support this finding. To resolve whether this reflects differences between species or between genetically driven and diet-induced disease, we studied the effect of a complement inhibitor, vaccinia virus complement control protein (VCP), on C57BL/6 mice, the background strain of ApoE-/- and LDLR-/- mice. Atherosclerosis was induced by a high-fat diet, and VCP (20 mg/kg) was injected once per week after the eighth week. Fatty streak development was monitored at 15 weeks by microscopic examination of oil red-O-stained sections from the root of the aorta. VCP injections led to significant (50%) reduction of lesion size (P = 0.004). Lesions were marked by gradual accumulation of lipids and macrophages but did not develop beyond the fatty streak stage. VCP activity disappeared from serum in 4 days, and the possibility therefore exists that a higher level of protection may be achieved by more frequent injections. We conclude that the development of fatty streaks in diet-induced atherosclerotic disease can be significantly retarded by prophylactic treatment with a complement inhibitor. These results support previous findings from complement-deficient rabbits and suggest that the pathogenesis of atherosclerosis in diet-induced disease differs from that induced by major defects in lipid metabolism.

The effect of dietary fish oil on survival after infection with Klebsiella pneumoniae or Streptococcus pneumoniae.

Dietary fish oil is believed to have a beneficial effect in various infections and in autoimmune disorders. This effect may correspond to an altered immune response. In order to discover whether the effect of dietary fish oil is different in various infections, we studied the survival of mice fed fish oil or corn oil supplemented diets and infected in the lungs with either Klebsiella pneumoniae or Streptococcus pneumoniae. 120 NMRI mice were divided into 4 groups, of which 2 groups were fed a fish oil supplemented diet and 2 a corn oil supplemented diet. After 6 weeks the mice were infected in the lungs with Klebsiella pneumoniae (fish oil groups and corn oil groups) or with Streptococcus pneumoniae serotype 3 (both groups). The survival rate was monitored. The experiment was performed twice. The survival of the mice fed fish oil enriched diet and infected with Klebsiella pneumoniae was significantly better compared with the mice fed corn oil enriched diet (p = 0.0001 and p = 0.0013). No difference was found between the mice fed corn oil enriched diet or fish oil enriched diet and infected with Streptococcus pneumoniae serotype 3 (p = 0.74 and p = 0.15). Our results indicate that dietary fish oil has a beneficial effect on survival of mice after experimental pneumoniae when infected with Klebsiella pneumoniae, but not after infection with Streptococcus pneumoniae serotype 3.

Subtyping of Salmonella enterica serovar typhimurium outbreak strains isolated from humans and animals in Iceland.

A total of 75 Salmonella enterica serovar Typhimurium strains of various (mainly human and animal) origins were typed by pulsed-field gel electrophoresis (PFGE) and phage typing. These strains were collected during an outbreak in Iceland in 1999 and 2000. The typing revealed that 84% of the strains belonged to the same PFGE and phage type (PT), namely, PFGE type 1Aa and PT 1.

Harmonization of antimicrobial susceptibility testing among veterinary diagnostic laboratories in the five Nordic countries.

A total of 100 bacterial strains (25 Escherichia coli, 25 Salmonella enterica, 25 Staphylococcus aureus, and 25 Enterococcus strains) and four reference strains were tested for susceptibility toward 8-12 antimicrobial agents in 12 veterinary diagnostic laboratories in the five Nordic countries using routine methodology. In addition, the 25 Enterococcus strains were identified to species level. A total of 22,598 (97.2%) out of 23,259 test results were in accordance when the data were categorized as susceptible or resistant. When the reported results were categorized according to the National Committee of Clinical Laboratory Standards breakpoints, the percentage of concordant results increased to 98.4% and the performance between laboratories varied between 94.2 and 99.4% concordant results. For E. coli, S. aureus, and Salmonella, all laboratories except one had more than 97% concordant results, whereas for Enterococcus spp., two laboratories had less than 90% concordant results. Susceptibility testing of Salmonella to fluoroquinolones gave rise to almost 0.5% nonconcordant results and susceptibility testing of S. aureus to vancomycin resulted in that 1.8% of the strains were incorrectly reported as vancomycin resistant. Ten laboratories identified the Enterococcus spp. to species level. All five Enterococcus faecium and 10 Enterococcus faecalis selected from the strain collection at the Danish Veterinary Institute were correctly identified by all laboratories, whereas some problems were observed identifying other enterococcal species. This study showed a good performance and agreement in antimicrobial susceptibility testing at the 12 participating laboratories and that surveillance data covering susceptibility test results of E. coli, S. aureus, and Salmonella from animals in the Nordic countries are comparable. But it also showed that some aspects can be improved. In addition, the study showed that the different laboratories are capable of identifying E. faecalis and E. faecium.

[Beneficial effect of dietary fish-oil is independent of the infection site.].

OBJECTIVE: Fish-oil enriched diet has a protective effect on experimental animals infected intramuscularly with Klebsiella pneumoniae. It also has beneficial effect in several other diseases, including autoimmune disorders. The pathophysiological effects of dietary fish-oil have still not been revealed although it is expected to influence the immune response. We have previously shown that dietary fish-oil has beneficial effect in mice infected intramuscularly with Klebsiella pneumoniae. If the beneficial effect of dietary fish-oil is due to influence on the immune response it should be independent of the infection site. We therefore investigated whether dietary fish-oil has beneficial effect in intrapulmonary infection with Klebsiella pneumoniae as it has on intramuscular infection with the same bacteria. MATERIALS AND METHODS: Sixty NMRI mice were fed diets enriched with fish-oil (30 mice) or corn-oil (30 mice) for six weeks. The mice were then infected in the lungs with Klebsiella pneumoniae and the survival was monitored. The experiment was performed twice. The results were compared to our earlier results with intramuscular infections. RESULTS: The survival of the mice fed the fish-oil enriched diet and infected in the lungs with Klebsiella pneumoniae was significantly better compared to the survival of mice fed the corn-oil enriched diet in both experiments (p=0.0001 and p=0.0013). These results are similar to our earlier findings when the mice were infected intramuscularly. CONCLUSIONS: These results indicate that the beneficial effect of dietary fish-oil on infection is independent of the site of infection. These results are in accordance with the hypothesis that dietary fish-oil influences the immune response.