Antti Sukura

ASSOCIATIONS BETWEEN TRICHINELLA SPECIES AND HOST SPECIES IN FINLAND

Examination of 627 wild animals—raccoon dogs (Nyctereutes procyonoides), red foxes (Vulpes vulpes), European lynxes (Lynx lynx), brown bears (Ursus arctos), wolves (Canis lupus), and badgers (Meles meles)—revealed Trichinella spp. The prevalence varied according to geographical region of Finland (north; southwest, SW; and southeast, SE) and was the highest among lynxes (70%, SW). The risk of trichinellosis was higher in the SE (odds ratio, OR, 19.4) and SW regions (OR 14.3), as compared with the northern region (OR 1), with no difference between the former 2 regions. Foxes (OR 2.1) and lynxes (OR 1.9) had a higher risk than raccoon dogs (OR 1) of being infected. The distribution of different Trichinella species was evaluated in 87 wild and domestic mammals by multiplex polymerase chain reaction. Trichinella spiralis was detected more often in domestic and synanthropic animals than in sylvatic hosts. Trichinella nativa was detected only in wildlife. Trichinella pseudospiralis was found both in sylvatic and synanthropic hosts. Trichinella britovi was detected only in mixed infections with other Trichinella species. The raccoon dog was the sole host for all 4 Trichinella species and also carried the most intense infections.

Lactobacillus oligofermentans sp. nov., Associated with Spoilage of Modified-Atmosphere-Packaged Poultry Products

ABSTRACT Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri group, which also includes L. durianis. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the L. durianis type strain confirming that these isolates belong to the same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743T (also known as DSM 15707T or AMKR18T) as the type strain.

Toxoplasma gondii in wild cervids and sheep in Finland: North-south gradient in seroprevalence

A nationwide seroepidemiological study was conducted to estimate the prevalence of Toxoplasma gondii in selected wild and domestic ruminants in Finland. Serum samples from 1367 game cervids collected during the hunting season in 2008-2009 and 1940 sheep sera collected in 2008 were screened with a commercial direct agglutination test at a serum dilution of 1:40. T. gondii-specific IgG antibodies were detected in 116 (9.6%) of 1215 moose (European elk, Alces alces), 36 (26.7%) of 135 white-tailed deer (Odocoileus virginianus), 3 (17.6%) of 17 roe deer (Capreolus capreolus), and 477 (24.6%) of 1940 domestic sheep. Seropositive sheep were found in 74 (76.3%) of the 97 flocks examined. The odds of seropositivity in the adult moose was 2.9 times higher than the odds in calves; in white-tailed deer, the odds ratio was 3.2. The male moose had a significantly lower seroprevalence than the female, whereas the seroprevalence in the male white-tailed deer was higher than in the female; the odds ratios were 0.6 and 2.5, respectively. A clear geographical gradient in the seroprevalence was revealed in moose and sheep. The seroprevalences were lowest (1.6 and 8.6%, respectively) in the north and highest (24.6 and 36.4%, respectively) in the south-western regions, and ranged between these values in the other regions. In fact, the seroprevalence in moose from the south-west was not significantly different from the prevalence in white-tailed deer from the same area. Thus, the Finnish wild cervids and sheep are commonly exposed to T. gondii, especially in the southern part of the country.

Genetic Divergence of the Dihydrofolate Reductase and Dihydropteroate Synthase Genes in Pneumocystis carinii from 7 Different Host Species

To investigate the phylogenetic and therapeutic implications of the genetic divergence in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes among different Pneumocystis carinii strains, these 2 genes in P. carinii obtained from 7 different host species were sequenced. Pairwise comparison of the DHPS sequences demonstrated 6%-24% and 6%-30% divergence in the nucleotide and deduced amino acid sequences, respectively. The DHFR gene was even more divergent, with differences of 15%-34% and 18%-42% in the nucleotide and deduced amino acid sequences, respectively. Phylogenetic analysis of DHFR and DHPS sequences revealed that all P. carinii strains were confined within a distinct group that was closely related to ascomycete fungi and that human-derived P. carinii was most closely related to monkey-derived P. carinii. Recognizing the substantial differences in the DHFR and DHPS genes among P. carinii from different host species has important implications for drug discovery and the development of new diagnostic methods.

Vascular damage and lack of angiogenesis in systemic sclerosis skin

The aim of this study was to analyse microvascular damage and compensatory angiogenesis in skin from patients with systemic sclerosis (SSc) compared with systemic lupus erythematosus (SLE), Raynauds phenomenon (RP) and healthy controls. Immunohistochemistry was used for skin biopsies (9 SSc, 10 SLE, 9 RP and 12 healthy controls) using von Willebrand factor and β3 integrin subunit specific antibodies, TechMate immunostaining robot and biotin-streptavidin protocol. In the early stages of SSc, vWF was found in the perivascular space and interstitial matrix in papillary but not in the reticular dermis, in particular around small oedematous blood vessels infiltrated by mononuclear cells. The extravascular release of vWF in SSc specimens was associated with weak or even a total lack of immunoreactivity within the associated endothelial cells. Late stages of SSc were characterised by loss of the dermal papillae, subepidermal fibrosis, hypovascularity and strong endothelial vWF expression without extravascular leakage. In all SSc patients studied only a few vascular profiles were weakly immunostained for β3 integrin subunit. This work demonstrates that vWF is not only released into the systemic circulation, but is also leaked to the perivascular space/matrix. This local release and deposition of vWF is probably a sensitive and early marker of microvascular involvement in SSc pathogenesis. Local vWF release may play a role in platelet adhesion, aggregation, thrombogenesis and dermal connective tissue remodelling. In spite of some attempts towards compensatory angiogenesis in SSc, as evidenced by β3 integrin subunit expression, it was evident that the angiogenic response was not able to prevent the development of hypovascularity during the advanced stages of the disease.

A SEL1L mutation links a canine progressive early-onset cerebellar ataxia to the endoplasmic reticulum-associated protein degradation (ERAD) machinery.

Inherited ataxias are characterized by degeneration of the cerebellar structures, which results in progressive motor incoordination. Hereditary ataxias occur in many species, including humans and dogs. Several mutations have been found in humans, but the genetic background has remained elusive in dogs. The Finnish Hound suffers from an early-onset progressive cerebellar ataxia. We have performed clinical, pathological, and genetic studies to describe the disease phenotype and to identify its genetic cause. Neurological examinations on ten affected dogs revealed rapidly progressing generalized cerebellar ataxia, tremors, and failure to thrive. Clinical signs were present by the age of 3 months, and cerebellar shrinkage was detectable through MRI. Pathological and histological examinations indicated cerebellum-restricted neurodegeneration. Marked loss of Purkinje cells was detected in the cerebellar cortex with secondary changes in other cortical layers. A genome-wide association study in a cohort of 31 dogs mapped the ataxia gene to a 1.5 Mb locus on canine chromosome 8 (praw = 1.1×10−7, pgenome = 7.5×10−4). Sequencing of a functional candidate gene, sel-1 suppressor of lin-12-like (SEL1L), revealed a homozygous missense mutation, c.1972T>C; p.Ser658Pro, in a highly conserved protein domain. The mutation segregated fully in the recessive pedigree, and a 10% carrier frequency was indicated in a population cohort. SEL1L is a component of the endoplasmic reticulum (ER)–associated protein degradation (ERAD) machinery and has not been previously associated to inherited ataxias. Dysfunctional protein degradation is known to cause ER stress, and we found a significant increase in expression of nine ER stress responsive genes in the cerebellar cortex of affected dogs, supporting the pathogenicity of the mutation. Our study describes the first early-onset neurodegenerative ataxia mutation in dogs, establishes an ERAD–mediated neurodegenerative disease model, and proposes SEL1L as a new candidate gene in progressive childhood ataxias. Furthermore, our results have enabled the development of a genetic test for breeders.

Disease patterns in field and bank vole populations during a cyclic decline in central Finland

Declining field vole (Microtus agrestis) and bank vole (Clethrionomys glareolus) populations were sampled (117 field voles and 34 bank voles) in south-central Finland during the winter of 1988-89. The last surviving field voles were caught in April and bank voles in February. A subsample (16) of the April field voles were taken live to the laboratory for immunosuppression. The histopathology of the main internal organs and the presence of aerobic bacteria and certain parasites were studied. In the lungs, an increase in lymphoid tissue, probably caused by infections, was the most common finding (52% of all individuals). The prevalences in the voles, in the whole material, of Chrysosporium sp. and Pneumocystis carinii in lungs were 13 and 10% in field voles, and 9 and 0% in bank voles, respectively. Cysts of Taenia mustelae (9 and 27%) were the most common pathological changes in the liver. Enteritis was also rather common (14 and 34%). In field voles the prevalences of Frenkelia sp. in the brain and Sarcocystis sp. in leg muscles were low (both 6%). Bordetella bronchiseptica was commonly (31%) isolated from field vole lungs and Listeria monocytogenes from the intestines (34%). Salmonella spp. could not be found. The dynamics and abundance of inflammations in the lungs and intestines, as well as B. bronchiseptica isolations from the lungs, indicate that obvious epidemics took place in declining vole populations. Of the Luhanka subsample of 16 field voles brought to the laboratory in April, one died of listeriosis, two of Bordetella, and five died for unknown reasons. Even if small mustelids are the driving force in microtine cycles, it is possible that diseases also contribute to the decline.

New insights into Staphylococcus aureus stress tolerance and virulence regulation from an analysis of the role of the ClpP protease in the strains Newman, COL, and SA564.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.

Feline toxoplasmosis in Finland cross-sectional epidemiological study and case series study

Three subgroups of the Finnish cat population underwent investigation for different aspects of feline toxoplasmosis. Blood samples of 445 purebred pet cats and 45 shelter cats were screened for Toxoplasma gondii–specific immunoglobulin G antibodies with a direct agglutination test. The overall seroprevalence was 48.4%; older cats and cats receiving raw meat in their diet were more often seropositive. Fecal samples were obtained from 131 shelters cats; 2 of the cats were found shedding T. gondii–like oocysts, and the oocysts shed by 1 of the 2 were confirmed as T. gondii with polymerase chain reaction. Among 193 cats submitted for necropsy during a 3.5-year period, 6 (3.1%) had been diagnosed with generalized toxoplasmosis and were retrospectively further investigated. The main pathological lesions included acute interstitial pneumonia, acute necrotizing hepatitis, and nonsuppurative meningoencephalitis with glial granulomas. Immunohistochemical staining demonstrated a mild to massive parasite burden in tissues with pathological lesions as well as in unaffected tissues. The results of the direct multilocus genotyping of T. gondii parasites detected were consistent with endemic genotype II, and the causative parasite strains were isolated from 2 of the generalized toxoplasmosis cases. The results indicate that cats in Finland commonly encounter T. gondii and contribute to the environmental oocyst burden, while the endemic genotype II can also prove fatal to the parasite’s definitive host. Preventing feline T. gondii infections is not only of public health importance but also a welfare issue for the cats themselves.

Alpha- and β-casein components of host milk induce biofilm formation in the mastitis bacterium Streptococcus uberis.

Streptococcus uberis is an environmental udder pathogen that infects cattle and can cause persistent intramammary infection (IMI), despite the fact that isolates are mainly susceptible to antibiotics. As biofilm growth can cause persistent infection, the ability of ten S. uberis isolates from clinical and subclinical IMIs to form biofilms on the polystyrene surface of a conventional 96-microplates model was examined. Biofilm formation was judged by different staining methods (crystal violet and resazurin) and by atomic force and fluorescence microscopy. These analyses revealed that two out of ten S. uberis strains tested were able to form biofilms. Upon treatment with Proteinase K, biofilms of S. uberis were completely disintegrated, which indicates that biofilm formation is protein-mediated in these strains. Addition of trace amounts of milk, the natural growth medium of S. uberis, significantly increased biofilm formation by most of the strains initially classified as non-biofilm producers. Alpha-casein and β-casein were the primary inducers of biofilm growth, and casein degradation by serine protease activity was required to achieve maximal biofilm production. These results suggest that the extracellular proteolytic activity of S. uberis contributes to an increased biofilm formation. Such a mode of growth induced by host proteins might help to explain the persistence of IMIs caused by this pathogen.