Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Hantavirus-Leptospira coinfections in small mammals from central Germany.

European orthohantaviruses (Puumala orthohantavirus (PUUV); Dobrava-Belgrade orthohantavirus (DOBV), genotype Kurkino; Tula orthohantavirus (TULV)), and Leptospira spp. are small mammal-associated zoonotic pathogens that cause diseases with potentially similar symptoms in humans. We investigated the frequency of Leptospira spp. and hantavirus single and double infections in small mammals from 22 sites in Thuringia, central Germany, during 2017. TULV infections were detected at 18 of 22 sites (mean prevalence 13.8%, 93/674). PUUV infections were detected at four of 22 sites (mean prevalence 1.5%, 7/471), and respective PUUV sequences formed a novel phylogenetic clade, but DOBV infections were not detected at all. Leptospira infections were detected at 21 of 22 sites with the highest overall prevalence in field voles (Microtus agrestis) with 54.5% (6/11) and common voles (Microtus arvalis) with 30.3% (205/676). Leptospira-hantavirus coinfections were found in 6.6% (44/671) of common voles but only in two of 395 bank voles. TULV and Leptospira coinfection probability in common voles was driven by individual (age) and population-level factors. Coinfections seemed to be particularly associated with sites where Leptospira spp. prevalence exceeded 35%. Future investigations should evaluate public health consequences of this strong spatial clustering of coinfections.

Prophylactic irradiation to the contralateral breast for BRCA mutation carriers with early-stage breast cancer.

BACKGROUND: Women who carry germ-line mutations in BRCA1/2 are at very high risk of developing breast and ovarian cancer. Breast conserving therapy is associated with a similar risk of ipsilateral cancer recurrence in BRCA carriers compared with non-carriers. However, the risk of subsequent contralateral breast cancer in carriers is markedly increased. Therefore, mastectomy of the diseased breast along with risk reducing mastectomy of the contralateral breast is often advocated for BRCA carriers who are treated for early breast cancer. Yet, many BRCA carriers forgo this option for fear of harmful effects and choose breast conserving treatment and observation instead. In Israel, BRCA-associated breast cancer is relatively common. Accordingly, a national protocol was devised for this enriched population. PATIENTS AND METHODS: In this Institutional Review Board-approved phase II trial, the option of prophylactic irradiation to the contralateral breast, in addition to standard loco-regional treatment, was offered to BRCA carrier patients treated for early breast cancer who declined contralateral mastectomy. The primary end point was contralateral breast cancer. RESULTS: Between May 2007 and October 2017, 162 patients were enrolled. Eighty-one patients opted for standard loco-regional treatment including surgery and radiation to the involved side (control arm) and 81 patients chose additional contralateral breast irradiation (intervention arm). At a median follow-up of 58 months, 10 patients developed contralateral breast cancer in the control arm at a median of 32 months, as compared with 2 patients in the intervention arm who developed contralateral breast cancer 80 and 105 months after bilateral breast irradiation (log-rank P = 0.011). CONCLUSIONS: Among BRCA carrier patients treated for early breast cancer, the addition of contralateral breast irradiation was associated with a significant reduction of subsequent contralateral breast cancers and a delay in their onset. CLINICAL TRIAL: Phase II, comparative two-arm trial (NCT00496288).

Moving beyond the conventional stratified analysis to estimate an overall treatment efficacy with the data from a comparative randomized clinical study.

For a two-group comparative study, a stratified inference procedure is routinely used to estimate an overall group contrast to increase the precision of the simple two-sample estimator. Unfortunately, most commonly used methods including the Cochran-Mantel-Haenszel statistic for a binary outcome and the stratified Cox procedure for the event time endpoint do not serve this purpose well. In fact, these procedures may be worse than their two-sample counterparts even when the observed treatment allocations are imbalanced across strata. Various procedures beyond the conventional stratified methods have been proposed to increase the precision of estimation when the naive estimator is consistent. In this paper, we are interested in the case when the treatment allocation proportions vary markedly across strata. We study the stochastic properties of the two-sample naive estimator conditional on the ancillary statistics, the observed treatment allocation proportions and/or the stratum sizes, and present a biased-adjusted estimator. This adjusted estimator is asymptotically equivalent to the augmentation estimators proposed under the unconditional setting. Moreover, this consistent estimation procedure is also equivalent to a rather simple procedure, which estimates the mean response of each treatment group first via a stratum-size weighted average and then constructs the group contrast estimate. This simple procedure is flexible and readily applicable to any target patient population by choosing appropriate stratum weights. All the proposals are illustrated with the data from a cardiovascular clinical trial, whose treatment allocations are imbalanced.

Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management.

Bovine paratuberculosis (Johne's disease) is a bacterial, chronic, and wasting intestinal disease caused by Mycobacterium avium ssp. paratuberculosis (MAP). Johne's disease causes severe losses in dairy farm productivity and is also suspected to be a potential trigger for Crohn's disease in humans. The fecal-oral infection of MAP to neonates is recognized as an important within-herd transmission route. Our objective was to recommend diagnostic methods for herds with suspected paratuberculosis requiring fast results, as well as for herds with breeding programs or others that aim at being nonsuspected of paratuberculosis infection. We determined a period of 8 wk from sampling to diagnostic findings suitable for testing of cows during the dry period. We therefore tested environmental and individual fecal samples with one rapid and one highly sensitive diagnostic method. Environmental samples (boot swabs) were taken as a first step in 3 herds and tested using a DNA extraction protocol for feces and subsequent real-time PCR (referred to as fecal PCR). Additionally, cultivation in liquid medium for 6 wk was performed and verified with real-time PCR (referred to as liquid culture). Automation of DNA extraction based on magnetic beads and the PCR setup was performed with pipetting robots. As a result, we successfully detected MAP in boot swabs of all herds by both methods. In a second step, 245 individual fecal samples from the 3 herds were examined using also fecal PCR and liquid culture. The results obtained by fecal PCR were compared with detection of MAP using cultivation in liquid medium for 6 wk. Testing individual cows, we identified MAP-specific DNA in 53 fecal samples using the liquid culture. Using fecal PCR, we revealed 43 positive samples of which 39 also tested positive in the liquid culture, revealing MAP-positive cows in all 3 herds. The fecal PCR procedure allows rapid detection of MAP-specific DNA with 74% of the sensitivity of liquid culture. For the purpose of testing with maximal sensitivity, cultivation in liquid medium is recommended. Cultivation of MAP in liquid medium M7H9C means a significant time gain in comparison to cultivation on solid media, which requires twice as much time. Thus, this testing fits within the 6- to 8-wk dry period of gravid cows and provides test results before calving, a prerequisite to prevent fecal-oral transmission to newborn calves.

Urinary Markers of Fibrosis and Risk of Cardiovascular Events and Death in Kidney Transplant Recipients: The FAVORIT Trial.

Cardiovascular risk remains high in kidney transplant recipients (KTRs) despite improved kidney function after transplant. Urinary markers of kidney fibrosis and injury may help to reveal mechanisms of this risk. In a case-cohort study among stable KTRs who participated in the FAVORIT trial, we measured four urinary proteins known to correlate with kidney tubulointerstitial fibrosis on biopsy (urine alpha 1 microglobulin [α1m], monocyte chemoattractant protein-1 [MCP-1], procollagen type I [PINP] and type III [PIIINP] N-terminal amino peptide) and evaluated associations with cardiovascular disease (CVD) events (n = 300) and death (n = 371). In adjusted models, higher urine α1m (hazard ratio [HR] per doubling of biomarker 1.40 [95% confidence interval [CI] 1.21, 1.62]), MCP-1 (HR 1.18 [1.03, 1.36]), and PINP (HR 1.13 [95% CI 1.03, 1.23]) were associated with CVD events. These three markers were also associated with death (HR per doubling α1m 1.51 [95% CI 1.32, 1.72]; MCP-1 1.31 [95% CI 1.13, 1.51]; PINP 1.11 [95% CI 1.03, 1.20]). Higher concentrations of urine α1m, MCP-1, and PINP may identify KTRs at higher risk for CVD events and death. These markers may identify a systemic process of fibrosis involving both the kidney and cardiovascular system, and give new insights into mechanisms linking the kidney with CVD.

Filtration Markers, Cardiovascular Disease, Mortality, and Kidney Outcomes in Stable Kidney Transplant Recipients: The FAVORIT Trial.

Cystatin C and beta-2-microglobulin (B2M) are filtration markers associated with adverse outcomes in nontransplant populations, sometimes with stronger associations than for creatinine. We evaluated associations of estimated glomerular filtration rate from cystatin C (eGFRcys ), B2M (eGFRB2M ), and creatinine (eGFRcr ) with cardiovascular outcomes, mortality, and kidney failure in stable kidney transplant recipients using a case-cohort study nested within the Folic Acid for Vascular Outcome Reduction in Transplantation (FAVORIT) Trial. A random subcohort was selected (N = 508; mean age 51.6 years, median transplant vintage 4 years, 38% women, 23.6% nonwhite race) with enrichment for cardiovascular events (N = 306; 54 within the subcohort), mortality (N = 208; 68 within the subcohort), and kidney failure (N = 208; 52 within the subcohort). Mean eGFRcr , eGFRcys , and eGFRB2M were 46.0, 43.8, and 48.8 mL/min/1.73m2 , respectively. After multivariable adjustment, hazard ratios for eGFRcys and eGFRB2M <30 versus 60+ were 2.02 (95% confidence interval [CI] 1.09-3.76; p = 0.03) and 2.56 (1.35-4.88; p = 0.004) for cardiovascular events; 3.92 (2.11-7.31) and 4.09 (2.21-7.54; both p < 0.001) for mortality; and 9.49 (4.28-21.00) and 15.53 (6.99-34.51; both p < 0.001) for kidney failure. Associations persisted with additional adjustment for baseline eGFRcr . We conclude that cystatin C and B2M are strongly associated with cardiovascular events, mortality, and kidney failure in stable kidney transplant recipients.

Is a reduction in albuminuria associated with renal and cardiovascular protection? A post hoc analysis of the ALTITUDE trial.

AIMS: To investigate whether the degree of albuminuria reduction observed in the ALTITUDE trial is associated with renal and cardiovascular protection, and secondly, whether the reduction in albuminuria was too small to afford clinical benefit. METHODS: In a post hoc analysis of the ALTITUDE trial in 8561 patients with type 2 diabetes and chronic kidney disease or cardiovascular disease we examined the effect of albuminuria changes at 6 months on renal and cardiovascular outcomes using Cox proportional hazard regression. RESULTS: The median change in albuminuria in the first 6 months in the aliskiren arm of the trial was -12% (25th to 75th percentile: -48.7_to_ +41.9%) and 0.0% (25th to 75th percentile: -40.2_to_55%) in the placebo arm. Changes in albuminuria in the first 6 months were linearly associated with renal and cardiovascular endpoints: a >30% reduction in albuminuria in the first 6 months was associated with a 62% reduction in renal risk and a 25% reduction in cardiovascular risk compared with an increase in albuminuria. The association between changes at 6 months in albuminuria and renal or cardiovascular endpoints was similar in the two treatment groups (p for interaction >0.1 for both endpoints). CONCLUSIONS: The addition of aliskiren to angiotensin-converting enzyme inhibitor/angiotensin receptor blocker therapy resulted in albuminuria changes that were associated with renal and cardiovascular risk changes. This did not translate into renal or cardiovascular protection because the overall reduction in albuminuria in the aliskiren arm was too small and nearly similar to that in the placebo arm.

Detection of Mycobacterium avium subspecies paratuberculosis in environmental samples by faecal culture and real-time PCR in relation to apparent within-herd prevalence as determined by individual faecal culture.

SUMMARY Environmental samples are a cost-saving and easy-to-use approach to diagnose paratuberculosis at the herd level. Detailed knowledge concerning its uncertainties in herds with a low prevalence of Mycobacterium avium spp. paratuberculosis (MAP) is required to design sampling strategies in control programmes. This study aimed to calculate a threshold level of the apparent within-herd prevalence (WHPapp) as determined by individual mycobacterial cultivation (faecal culture; FC) of all cows thus allowing the detection of a herd as MAP-positive at a certain probability level (P d). Out of 200 environmental samples taken twice from five predefined locations in a barn, 25 were positive by FC and 60 were positive by a quantitative real-time PCR method (qPCR). A logistic regression model was used to calculate the WHPapp threshold of detection. For 50% P d, a WHPapp threshold of 2.9% was calculated for the combination of three samples (milking area, main cow alleyways, holding pen) tested simultaneously both by FC and qPCR. The threshold increased to 6.2% for 90% P d. Repeated environmental sampling did not reduce the WHPapp threshold. Depending on the particular needs for prevalence estimation or in control programmes (single or repeated sampling) the provided WHPapp thresholds at different P d will enable decisions to be made about various sampling strategies.

Subcellular localization and transport kinetics of ruthenium organometallic anticancer compounds in living cells: a dose-dependent role for amino acid and iron transporters.

Ruthenium-based compounds are developed for anticancer treatment, but their mode of action including their import mechanism and subcellular localization remains elusive. Here, we used the intrinsic luminescent properties of cytotoxic organoruthenium (Ru(II)) compounds obtained with an anionic cyclometalated 2-phenylpyridine chelate and neutral aromatic chelating ligands (e.g., phenanthrolines) to follow their behavior in living cells. We established that the difference in sensitivity between cancer cells and noncancerous cells toward one of the compounds correlates with its import kinetics and follows a balance between active and passive transport. The active-transport mechanism involves iron and amino-acid transporters, which are transcriptionally regulated by the drug. We also demonstrated a correlation between the accumulation of these compounds in specific compartments (endoplasmic reticulum, nucleus, mitochondria) and the activation of specific cytotoxic mechanisms such as the mitochondrial stress pathway. Our study pinpoints a novel and complex mechanism of accumulation of ruthenium drugs in cancer cells.

Dynamics of Schmallenberg virus infection within a cattle herd in Germany, 2011.

In late 2011, the insect-transmitted Schmallenberg virus (SBV) emerged in Europe. In this study, a cattle farm located in the core region of the epidemic was closely monitored between May 2011 and January 2012. Up to the end of September every tested serum sample was negative by an SBV-specific antibody ELISA, suggesting the absence of an infection before autumn 2011. Around the end of September/beginning of October SBV genome was detected in blood samples of some animals, and a few cows exhibited fever during that period. Starting at the end of September the first cows seroconverted; the within-herd prevalence reached 100% within barely 1 month. Consequently, SBV spread rapidly in the tested herd during the vector season of 2011.

[Threat of transmission of infectious pathogens by Ixodes ricinus ticks in Germany]. / Gefahren der Übertragung von Krankheitserregern durch Schildzecken in Deutschland.

Tick-transmitted diseases are of great importance for the general health of the German population. Several viruses, such as tick-borne encephalitis virus (TBEV), Uukuniemi virus, Tribec virus, Eyach virus or bacteria, such as Borrelia, Rickettsiae, Francisella tularensis, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis (CNM) and Coxiella burnetii were detected in the most prominent tick in Germany, the hard tick Ixodes ricinus. While infections, such as TBE and Lyme disease are well known, other infections are hardly known even among experts. Although there have been a few descriptions of isolated cases in Germany, a systematic investigation regarding the distribution and the pathogenic potential of these pathogens is still lacking. In particular elderly people and people with underlying diseases seem to be mostly affected. The importance of new infectious disease agents, such as Candidatus Neoehrlichia mikurensis but also of long known pathogens, such as Rickettsiae still remains unclear, while some of them could be detected in 20 % of investigated ticks. Whether climate change contributes to the further distribution of these infectious agents remains unclear and requires further investigation. The increasing initiatives to create natural environments and the trend towards spending more time in nature for recreational activities will increase the danger of coming into contact with ticks and the respective infectious agents. Considering these circumstances an increase of diseases caused by these pathogens is to be expected.

Stereotactic MR-guided on-table adaptive radiation therapy (SMART) for borderline resectable and locally advanced pancreatic cancer: A multi-center, open-label phase 2 study.

BACKGROUND AND PURPOSE: Radiation dose escalation may improve local control (LC) and overall survival (OS) in select pancreatic ductal adenocarcinoma (PDAC) patients. We prospectively evaluated the safety and efficacy of ablative stereotactic magnetic resonance (MR)-guided adaptive radiation therapy (SMART) for borderline resectable (BRPC) and locally advanced pancreas cancer (LAPC). The primary endpoint of acute grade ≥ 3 gastrointestinal (GI) toxicity definitely related to SMART was previously published with median follow-up (FU) 8.8 months from SMART. We now present more mature outcomes including OS and late toxicity. MATERIALS AND METHODS: This prospective, multi-center, single-arm open-label phase 2 trial (NCT03621644) enrolled 136 patients (LAPC 56.6 %; BRPC 43.4 %) after ≥ 3 months of any chemotherapy without distant progression and CA19-9 ≤ 500 U/mL. SMART was delivered on a 0.35 T MR-guided system prescribed to 50 Gy in 5 fractions (biologically effective dose10 [BED10] = 100 Gy). Elective coverage was optional. Surgery and chemotherapy were permitted after SMART. RESULTS: Mean age was 65.7 years (range, 36-85), induction FOLFIRINOX was common (81.7 %), most received elective coverage (57.4 %), and 34.6 % had surgery after SMART. Median FU was 22.9 months from diagnosis and 14.2 months from SMART, respectively. 2-year OS from diagnosis and SMART were 53.6 % and 40.5 %, respectively. Late grade ≥ 3 toxicity definitely, probably, or possibly attributed to SMART were observed in 0 %, 4.6 %, and 11.5 % patients, respectively. CONCLUSIONS: Long-term outcomes from the phase 2 SMART trial demonstrate encouraging OS and limited severe toxicity. Additional prospective evaluation of this novel strategy is warranted.

A Multi-Institutional Phase 2 Trial of Ablative 5-Fraction Stereotactic Magnetic Resonance-Guided On-Table Adaptive Radiation Therapy for Borderline Resectable and Locally Advanced Pancreatic Cancer.

PURPOSE: Magnetic resonance (MR) image guidance may facilitate safe ultrahypofractionated radiation dose escalation for inoperable pancreatic ductal adenocarcinoma. We conducted a prospective study evaluating the safety of 5-fraction Stereotactic MR-guided on-table Adaptive Radiation Therapy (SMART) for locally advanced (LAPC) and borderline resectable pancreatic cancer (BRPC). METHODS AND MATERIALS: Patients with LAPC or BRPC were eligible for this multi-institutional, single-arm, phase 2 trial after ≥3 months of systemic therapy without evidence of distant progression. Fifty gray in 5 fractions was prescribed on a 0.35T MR-guided radiation delivery system. The primary endpoint was acute grade ≥3 gastrointestinal (GI) toxicity definitely attributed to SMART. RESULTS: One hundred thirty-six patients (LAPC 56.6%, BRPC 43.4%) were enrolled between January 2019 and January 2022. Mean age was 65.7 (36-85) years. Head of pancreas lesions were most common (66.9%). Induction chemotherapy mostly consisted of (modified)FOLFIRINOX (65.4%) or gemcitabine/nab-paclitaxel (16.9%). Mean CA19-9 after induction chemotherapy and before SMART was 71.7 U/mL (0-468). On-table adaptive replanning was performed for 93.1% of all delivered fractions. Median follow-up from diagnosis and SMART was 16.4 and 8.8 months, respectively. The incidence of acute grade ≥3 GI toxicity possibly or probably attributed to SMART was 8.8%, including 2 postoperative deaths that were possibly related to SMART in patients who had surgery. There was no acute grade ≥3 GI toxicity definitely related to SMART. One-year overall survival from SMART was 65.0%. CONCLUSIONS: The primary endpoint of this study was met with no acute grade ≥3 GI toxicity definitely attributed to ablative 5-fraction SMART. Although it is unclear whether SMART contributed to postoperative toxicity, we recommend caution when pursuing surgery, especially with vascular resection after SMART. Additional follow-up is ongoing to evaluate late toxicity, quality of life, and long-term efficacy.

Relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central Europe.

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8×10(-4) substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus in zdár Kaplice proved for the first time that TBEV is indeed subject to local evolution.

Ketogenic diet as a potential intervention for lipedema.

Lipedema (LI) is a common yet misdiagnosed condition, often misconstrued with obesity. LI affects women almost exclusively, and its painful and life-changing symptoms have long been thought to be resistant to the lifestyle interventions such as diet and exercise. In this paper, we discuss possible mechanisms by which patients adopting a ketogenic diet (KD) can alleviate many of the unwanted clinical features of LI. This paper is also an effort to provide evidence for the hypothesis of the potency of this dietary intervention for addressing the symptoms of LI. Specifically, we examine the scientific evidence of effectiveness of adopting a KD by patients to alleviate clinical features associated with LI, including excessive and disproportionate lower body adipose tissue (AT) deposition, pain, and reduction in quality of life (QoL). We also explore several clinical features of LI currently under debate, including the potential existence and nature of edema, metabolic and hormonal dysfunction, inflammation, and fibrosis. The effectiveness of a KD on addressing clinical features of LI has been demonstrated in human studies, and shows promise as an intervention for LI. We hope this paper leads to an improved understanding of optimal nutritional management for patients with LI and stimulates future research in this area of study.

Chikungunya fever in two German tourists returning from the Maldives, September, 2009.

This report describes the first isolation and molecular characterisation of a chikungunya virus from two German tourists who became ill after a visit to the Maldives in September 2009. The virus contained the E1 A226V mutation, shown to be responsible for an adaptation to the Asian tiger mosquito Aedes albopictus. The E1 coding sequence was identical to chikungunya virus isolates from Sri Lanka and showed three nt-mismatches to the only available E1 nt sequence from the Maldives.

Survival time of Leptospira kirschneri serovar Grippotyphosa under different environmental conditions.

Leptospirosis is a re-emerging zoonotic disease of high medical importance that affects humans worldwide. Humans or animals acquire an infection with pathogenic leptospires either by direct contact with infected animals or by indirect contact to contaminated environment. Survival of Leptospira spp. in the environment after having been shed via animal urine is thus a key factor to estimate the risk of infection, but not much is known about the tenacity of pathogenic leptospires. Here, the survival time of both a laboratory strain and a field strain of L. kirschneri serovar Grippotyphosa in animal urine and their tenacity while drying was investigated and compared at different temperatures (15°C-37°C). Leptospira spp. are also often found in rivers and ponds. As the infection risk for humans and animals also depends on the spreading and survival of Leptospira spp. in these environments, the survival of L. kirschneri serovar Grippotyphosa was investigated using a 50-meter-long hose system simulating a water stream. Both strains did not survive in undiluted cattle or dog urine. Comparing different temperatures and dilution media, the laboratory strain survived the longest in diluted cattle urine with a slightly alkaline pH value (3 days), whilst the field strain survived in diluted dog urine with a slightly acid pH value up to a maximum of 24 h. Both strains did not survive drying on a solid surface. In a water stream, leptospires were able to move faster or slower than the average velocity of the water due to their intrinsic mobility but were not able to survive the mechanical damage caused by running water in the hose system. From our results we conclude, that once excreted via animal urine, the leptospires immediately need moisture or a water body to survive and stay infectious.

Tumor resistance to alkylating agents conferred by mechanisms operative only in vivo.

EMT-6 murine mammary tumors were made resistant to cis-diamminedichloroplatinum (II) (CDDP), carboplatin, cyclophosphamide (CTX), or thiotepa in vivo by treatment of tumor-bearing animals with the drug during a 6-month period. In spite of high levels of in vivo resistance, no significant resistance was observed when the cells from these tumors were exposed to the drugs in vitro. The pharmacokinetics of CDDP and CTX were altered in animals bearing the respective resistant tumors. The resistance of all tumor lines except for the EMT-6/thiotepa decreased during 3 to 6 months in vivo passage in the absence of drugs. These results indicate that very high levels of resistance to anticancer drugs can develop through mechanisms that are expressed only in vivo.

Enhanced sensitivity and fast turnaround time in laboratory diagnosis for bovine paratuberculosis in faecal samples.

Bovine paratuberculosis (Johne's disease in cattle) is caused by the pathogen Mycobacterium avium subsp. paratuberculosis (MAP) and is a widespread chronic bacterial infectious disease in cattle. Due to the peculiarities of the pathogen, detection of MAP in faeces remains difficult. DNA extraction and real-time PCR for detection of MAP in bovine faeces (direct PCR) have been refined and feasible procedures for rapid, sensitive and automatable detection of the pathogen agent have been developed. Accordingly, in a first step we tested 20 faecal samples using two MAP complete kits (DNA extraction kits based on magnetic beads combined with real-time PCR assays) and six other DNA extraction kits for faeces. MAP-specific DNA was detected by real-time PCR assays. Cultivation of MAP on the solid medium HEYM and in the liquid medium M7H9C served as reference standards. The two complete kits detected significantly more MAP-DNA positive samples than the other procedures applied (p < 0.04). Ct values of 37 and 38 served as cut-off for the respective real-time PCR assays calculated on the basis of standard curves and droplet digital PCR (ddPCR). In a second step, the two MAP complete kits were employed for a comprehensive study including 107 positive and 50 negative faecal samples which had been previously tested on HEYM cultivation. The MAP complete kits yielded sensitivity values of 86% and 89% and specificity values of 100% compared to cultivation of MAP in the liquid medium M7H9C. In detail, cultivation of MAP in M7H9C detected the pathogen in 97% and 100% of the samples tested after an incubation period of six and twelve weeks, respectively. However, the cultivation of MAP on HEYM succeeded in only 74% after twelve weeks of incubation. In all these solid culture positive samples, MAP was also detected using the two complete kits. Additionally, the impact of repeated freezing and thawing of samples on re-cultivation of MAP was tested using 20 faecal samples and resulted in a reduction to 75% and 25% of bacterial growth when using liquid medium M7H9C and solid medium HEYM, respectively. The results of this study show that complete kits with refined automatable protocols for DNA extraction in combination with real-time PCR assays for detection of MAP can compete with sensitive cultivation of the pathogen in liquid medium.