Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

Besnoitia besnoiti, an apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may dramatically affect body condition, and, in males, lead to irreversible infertility. While identification of clinical cases and their histopathological confirmation is relatively simple to carry out, finding subclinical forms of infection is more difficult, thus a more sensitive test for the identification of the etiological agent may be an appropriate diagnostic tool. We have developed the ITS1 rDNA-sequence-based conventional and real-time PCR which are highly sensitive and specific for the detection of B. besnoiti infection in cattle. A recombinant internal positive control was introduced to assess possible sample-related inhibitory effects during the amplification reaction and, in order to prevent false-positive results, a pre-PCR treatment of potentially contaminating dU-containing PCR product with uracil-DNA-glycosylase (UDG) was followed.

Screening Swiss water bodies for potentially pathogenic free-living amoebae

Free-ling amoebae (FLA) including Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia pedata, can cause opportunistic infections leading to severe brain pathologies. Human infections with pathogenic FLA have been increasingly documented in many countries. In Switzerland, thus far, the occurrence and distribution of potentially pathogenic FLA has not been investigated. Swiss water biotopes, including swimming pools, lakes, rivers and ponds, have now been screened for the presence of FLA, and assessment of their pathogenicity potential for a mammalian host has been undertaken. Thus, a total of 17 isolates were recovered by in vitro cultivation from these different aquatic sources. Characterization by sequence analysis of Acanthamoeba spp.-specific and 'FLA-specific PCR products amplified from 18s rDNA based on morphological traits, thermotolerance, and cytotoxicity towards murine fibroblasts yielded the following findings: Echinamoeba cf. exundans (3 isolates), Hartmannella spp. (3), Vannella spp. (4), Protacanthamoebica cf. bohemica (1), Acanthamoeba cf. castellanii (1) and Naegleria spp. (5). B. mandrillaris and N. fowleri did not range amongst these isolates. None of the isolates exhibited pronounced cytotoxicity and all failed to grow at 42 degrees C; therefore, they do not present any potential for CNS pathogenicity for humans.

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 degrees C, subsequently displayed a high thermotolerance, being able to grow at 42 degrees C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.

Is ITS-2 rDNA suitable marker for genetic characterization of Sarcoptes mites from different wild animals in different geographic areas?

The present study examined the relationship among individual Sarcoptes scabiei mites from 13 wild mammalian populations belonging to nine species in four European countries using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) as genetic marker. The ITS-2 plus primer flanking 5.8S and 28S rDNA (ITS-2+) was amplified from individual mites by polymerase chain reaction (PCR) and the amplicons were sequenced directly. A total of 148 ITS-2+ sequences of 404bp in length were obtained and 67 variable sites were identified (16.59%). UPGMA analyses did not show any geographical or host-specific clustering, and a similar outcome was obtained using population pairwise Fst statistics. These results demonstrated that ITS-2 rDNA does not appear to be suitable for examining genetic diversity among mite populations.

Suitability of low-dosage oxytocin treatment to induce milk ejection in dairy cows

Chronic use of high oxytocin (OT) dosages can cause a reduced response to endogenous OT. In this study the OT dosages used in the milking practice of 82 dairy cow farms were recorded. The OT dosages per cow used were high, especially when injected i.m. (23+/-2 IU) compared with i.v. (7+/-1 IU). In addition, the minimum OT dosages needed to obtain normal milk removal in cows with disturbed milk ejection were investigated. Seventeen cows routinely treated with OT during milking (group T) and 17 cows without previous OT treatment were used (group C). After cessation of spontaneous milk flow, both T and C groups were injected i.v. with a low dosage of OT (0.2 or 0.5 IU/cow). The time from injection until cessation of the OT-induced milk flow was recorded (response phase). The response phase and the amounts of removed milk by effect of the OT injection increased with increasing OT dosage. Values for 0.2 and 0.5 IU/cow of OT injected i.v. were (response phase and amount of milk removed) 198+/-27 and 302+/-18s and 3.4+/-0.7 kg and 6.5+/-1.3 kg, respectively, for the C group, and 157+/-15 and 221+/-16s and 3.2+/-0.5 and 5.5+/-1.0 kg, respectively, for the T group. Within 20 min of the OT injection, plasma concentrations returned to basal levels. The threshold OT concentration at cessation of milk flow after injection of 0.2 or 0.5 IU/cow of OT was calculated based on the OT plasma half-life. The threshold increased with increasing dosages of OT and was higher in group T (8+/-1 and 14+/-1 pg/mL for 0.2 and 0.5 IU/cow, respectively) than in group C (7+/-1 and 11+/-1 pg/mL for 0.2 and 0.5 IU/cow, respectively). In conclusion, desensitization of the udder toward OT occurs when the udder is exposed to elevated OT plasma concentrations, both short-term during the actual milking and long-term due to chronic high-dosage OT treatment. However, low-dosage OT treatments to induce normal milk removal can minimize the observed side effects.

Differences in Soil Fungal Communities between European Beech (Fagus sylvatica L.) Dominated Forests Are Related to Soil and Understory Vegetation

Fungi are important members of soil microbial communities with a crucial role in biogeochemical processes. Although soil fungi are known to be highly diverse, little is known about factors influencing variations in their diversity and community structure among forests dominated by the same tree species but spread over different regions and under different managements. We analyzed the soil fungal diversity and community composition of managed and unmanaged European beech dominated forests located in three German regions, the Schwäbische Alb in Southwestern, the Hainich-Dün in Central and the Schorfheide Chorin in the Northeastern Germany, using internal transcribed spacer (ITS) rDNA pyrotag sequencing. Multiple sequence quality filtering followed by sequence data normalization revealed 1655 fungal operational taxonomic units. Further analysis based on 722 abundant fungal OTUs revealed the phylum Basidiomycota to be dominant (54%) and its community to comprise 71.4% of ectomycorrhizal taxa. Fungal community structure differed significantly (p≤0.001) among the three regions and was characterized by non-random fungal OTUs co-occurrence. Soil parameters, herbaceous understory vegetation, and litter cover affected fungal community structure. However, within each study region we found no difference in fungal community structure between management types. Our results also showed region specific significant correlation patterns between the dominant ectomycorrhizal fungal genera. This suggests that soil fungal communities are region-specific but nevertheless composed of functionally diverse and complementary taxa.

mRNA expression and binding sites for alpha2-adrenergic receptor subtypes in muscle layers of the ileum and spiral colon of dairy cows

OBJECTIVE: To measure maximum binding capacity (B(max)) and levels of mRNA expression for alpha(2)-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows. SAMPLE POPULATION: Ileal and colonic muscle specimens from 6 freshly slaughtered cows. PROCEDURES: Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for alpha(2)-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 ((3)H-RX821002) and subtype-selective ligands as competitors. RESULTS: mRNA expression for alpha(2AD)-, alpha(2B)-, and alpha(2C)-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for alpha(2AD)-AR was significantly greater than that for alpha(2B)- and alpha(2C)-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of (3)H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The B(max) of alpha(2AD)- and alpha(2C)-AR sub-types was greater than that of alpha(2B)-AR. The B(max) and level of mRNA expression were only correlated (r = 0.8) for alpha(2AD)-AR. Ratio of B(max) to mRNA expression for alpha(2C)-AR was similar to that for alpha(2B)-AR, but significantly greater than for alpha(2AD)-AR. CONCLUSIONS AND CLINICAL RELEVANCE: Subtypes of alpha(2)-AR in bovine intestinal muscle layers are represented by a mixture of alpha(2AD)- and alpha(2C)-ARs and of alpha(2B)-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in alpha(2)-adrenergic mechanisms regulating bovine intestinal motility.

PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections

We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.

Biofilm-related infections of cerebrospinal fluid shunts

Cerebrospinal fluid (CSF) shunts carry a high risk of complications. Infections represent a major cause of shunt failure. Diagnosis and therapy of such infections are complicated by the formation of bacterial biofilms attached to shunt surfaces. This study correlated the pathophysiology and clinical course of biofilm infections with microscopical findings on the respective shunts. Surface irregularities, an important risk-factor for shunt colonisation with bacteria, were found to increase over time because of silicone degradation. Scanning electron-microscopy (SEM) documented residual biological material (dead biofilm), which can further promote extant bacterial adhesion, on newly manufactured shunts. Clinical course and SEM both documented bacterial dissemination against CSF flow and the monodirectional valve. In all cases, biofilms grew on both the inner and outer surfaces of the shunts. Microscopy and conventional culture detected all bacterial shunt infections. Analyses of 16S rDNA sequences using conserved primers identified bacteria in only one of three cases, probably because of previous formalin fixation of the samples.

Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland

We have designed and performed a new PCR method based on the 18S rRNA in order to individuate the presence and the identity of Babesia parasites. Out of 1159 Ixodes ricinus (Acari: Ixodidae) ticks collected in four areas of Switzerland, nine were found to contain Babesia DNA. Sequencing of the short amplicon obtained (411-452 bp) allowed the identification of three human pathogenic species: Babesia microti, B. divergens, for the first time in Switzerland, Babesia sp. EU1. We also report coinfections with B. sp. EU1-Borrelia burgdorferi sensu stricto and Babesia sp. EU1-B. afzelii.

Babesiosis in free-ranging chamois (Rupicapra r. rupicapra) from Switzerland

Pathological examination of five adult chamois (Rupicapra r. rupicapra) found dead in two different regions from the Swiss Alps revealed pale mucous membranes and musculature, swollen spleen and haemoglobinuria. Histologically, haemosiderosis in the spleen and centrilobular hepatic necrosis were the predominant findings. On blood smears, small (approximately 0.84-1.47 microm), round to pyriform, peripherally located inclusions were present in the erythrocytes. PCR followed by sequencing of DNA extracted from blood or spleen of the infected animals revealed 99-100% identity of the amplified part of the 18S rRNA gene with GenBank entries attributed to Babesia divergens/Babesia capreoli. This is the first report of fatal Babesia infections in chamois raising the question of an emerging disease in this species.