Using birth-death processes to infer tumor subpopulation structure from live-cell imaging drug screening data.

Tumor heterogeneity is a complex and widely recognized trait that poses significant challenges in developing effective cancer therapies. In particular, many tumors harbor a variety of subpopulations with distinct therapeutic response characteristics. Characterizing this heterogeneity by determining the subpopulation structure within a tumor enables more precise and successful treatment strategies. In our prior work, we developed PhenoPop, a computational framework for unravelling the drug-response subpopulation structure within a tumor from bulk high-throughput drug screening data. However, the deterministic nature of the underlying models driving PhenoPop restricts the model fit and the information it can extract from the data. As an advancement, we propose a stochastic model based on the linear birth-death process to address this limitation. Our model can formulate a dynamic variance along the horizon of the experiment so that the model uses more information from the data to provide a more robust estimation. In addition, the newly proposed model can be readily adapted to situations where the experimental data exhibits a positive time correlation. We test our model on simulated data (in silico) and experimental data (in vitro), which supports our argument about its advantages.

A national epidemic of campylobacteriosis in Iceland, lessons learned.

Epidemics often result in organizational, policy and technical changes within a country. In 1999, an epidemic of campylobacteriosis was reported in Iceland. The recent availability of fresh poultry products in the marketplace was suggested as the source of infection. This paper reports on the context of the epidemic, reviews interventions implemented to prevent campylobacteriosis, and discusses lessons learned. A retrospective study of interventions implemented in Iceland from June 1995 to December 2007 was conducted by interviewing key informants and reviewing Iceland's literature. Cumulative incidence rates of domestic campylobacteriosis by year and average incidence rates per epidemic period were calculated. Interventions included on-farm surveillance of Campylobacter, producer education, enhanced biosecurity measures, changes in poultry processing, a leak-proof packaging policy, a freezing policy for products from Campylobacter-positive poultry flocks, consumer education, and the creation of a legislated inter-organizational response committee. These interventions appear to have collectively contributed to a decrease in campylobacteriosis' incidence rate near pre-epidemic baseline levels. Expert consultations revealed that the implementation of a Campylobacter surveillance program in poultry and the freezing policy were critical to controlling the disease in the Icelandic population. It was also recognized that new multidisciplinary collaborations among public health, veterinary, and food safety authorities and a sustained co-operation from the poultry industry were integral factors to the mitigation of the epidemic. Iceland's response to the campylobacteriosis epidemic is a lesson learned of inter-disciplinary and inter-organizational precautionary public health action in the face of a complex public health issue.

Treatment of fresh poultry carcases with emulsions of glycerol monocaprate (monocaprin) to reduce contamination with Campylobacter and psychrotrophic bacteria.

1. A previous study has shown that emulsions of monocaprin in citrate lactate buffer at pH 4·1-4·3 are highly active in killing Campylobacter in water, where they reduce viable bacterial counts by more than 6 log(10) colony forming units (cfu) in 1 min at a concentration of 1·25 mM (0·03%). 2. The present study was carried out to evaluate whether monocaprin emulsions could be used to kill Campylobacter on raw poultry. 3. It was shown that immersion of naturally contaminated chicken legs in 20 mM (0·5%) monocaprin emulsion at pH 4·1 for 1 min at 20°C reduced the number of Campylobacter by 2·0 to 2·7 log(10) cfu. Pre-chill dipping of whole carcases into 20 mM monocaprin emulsion in the slaughterhouse also caused a significant reduction in Campylobacter contamination. 4. Immersion in monocaprin emulsions at pH 4·1 was also assessed as a means to reduce the number of psychrotrophic spoilage bacteria. There were lower psychrotrophic bacteria counts on treated chicken parts than on untreated controls after storage at 3°C for up to 14 d. 5. Immersion in emulsions of monocaprin, which is a natural lipid classified as GRAS, may be a feasible method to reduce the number of Campylobacter and spoilage bacteria on raw poultry. This method could reduce the risk of human exposure to Campylobacter, and at the same time increase the shelf-life of poultry products.

Prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli isolated from animals, foods and humans in Iceland.

The prevalence of resistant bacteria in food products in Iceland is unknown, and little is known of the prevalence in production animals. The aim of this study was to investigate the prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli from healthy pigs and broiler chicken, pork, broiler meat, slaughterhouse personnel and outpatients in Iceland. A total of 419 E. coli isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC), and resistant strains were compared using pulsed-field gel electrophoresis (PFGE). All samples were screened for enrofloxacin-resistant strains with selective agar plates. The resistance rates among E. coli isolates were moderate to high from caecal and meat samples of pigs (54.1% and 28%), broilers (33.6% and 52%) and slaughterhouse personnel (39.1%), whereas isolates from outpatients showed moderate resistance rates (23.1%). Of notice was resistance to quinolones (minimum inhibitory concentrations: nalidixic acid > or = 32, ciprofloxacin > or = 0.12 and enrofloxacin > or = 0.5), particularly among broiler and broiler meat isolates (18.2% and 36%), as there is no known antimicrobial selection pressure in the broiler production in Iceland. The majority (78.6%) of the resistant E. coli isolates was genotypically different, based on PFGE fingerprint analyses and clustering was limited. However, the same resistance pattern and pulsotype were found among isolates from broiler meat and a slaughterhouse worker, indicating spread of antimicrobial-resistant E. coli from animals to humans. Diverse resistance patterns and pulsotypes suggest the presence of a large population of resistant E. coli in production animals in Iceland. This study gives baseline information on the prevalence of antimicrobial-resistant E. coli from production animals, and their food products in Iceland and the moderate to high resistance rates emphasize the need for continuing surveillance. Further studies on the origin of the resistant strains and the genetic relatedness of strains of different origin are needed.

Effect of glycerol monocaprate (monocaprin) on broiler chickens: an attempt at reducing intestinal Campylobacter infection.

Attempts have been made by several workers to prevent or to reduce colonization of Campylobacter in the intestines of broiler chickens by adding antibacterial agents to their food, but the results have varied. Monocaprin, the 1-monoglyceride of capric acid, has been found to be very active in killing Campylobacter in vitro. It was therefore studied whether Campylobacter infection in chickens would be affected by adding emulsions of monocaprin to their drinking water and feed. It was found that treatment with monocaprin in water and feed did not prevent spread of Campylobacter from artificially infected to noninfected 24-d-old chickens, but Campylobacter counts in cloacal swabs were significantly reduced, particularly during the first 2 d of treatment. There was also a significant reduction in the Campylobacter counts in cloacal swabs of naturally infected 36-d-old broilers that were treated for 3 d prior to slaughter. Addition of monocaprin to drinking water and feed 2 to 3 d before slaughter might therefore be considered as a means of reducing Campylobacter infection in broilers, possibly in conjunction with other antibacterial agents such as short-chain organic acids. Further studies are needed to determine whether this would reduce carcass contamination.

Occurrence of Campylobacter spp. in cecal contents among commercial broilers in iceland.

Campylobacter jejuni remains the most frequently reported bacterial cause of human gastroenteritis in Nordic countries. The primary source of transmission to humans is suggested as mishandled raw poultry or consuming improperly prepared chicken. The focus of this report was to characterize the prevalence and cell numbers of the organism within the commercial Icelandic poultry industry. Commercial broiler flocks were sampled from May 2001 through 2003 in a total population study. At the slaughter plant, 40 randomly selected ceca were obtained from each flock, pooled into four samples containing 10 ceca each, and analyzed. Cell numbers and prevalence of Campylobacter spp. were estimated by direct plating of dilutions onto Campy-Cefex agar and incubating the plates at 42 degrees C under microaerobic atmosphere; colonies were confirmed as Campylobacter spp. by microscopy and latex agglutination to provide quantification of cell numbers per gm of cecal material. A total of 15.4% of the flocks carried the organism at at a maximum cell number of 8.1 x 10(7) cfu/g, having a mean raw count of colonized birds at 1.3 x 10(7) cfu/g (geometric mean of 1.5 x 10(6)). During the 3 years of sampling, the prevalence ranged from 17.6% to 17.3% to 12.7% for slaughter years 2001, 2002, and 2003, respectively. Isolation rates varied with numbers of catch lots (groups of birds taken for slaughter)/flock; with one catch lot/flock, the prevalence was 13.7%, with two 17.5%, and with three 33.3%. With increased flock size, isolation rates also increased; flocks of greater than 5,000 birds had a prevalence of 12.0% positive, 14.0% of flocks with 5,000-10,000 birds were positive, and 25.5% of flocks with more than 10,000 birds were positive for Campylobacter spp. Isolation rates varied with the processing lines: M was positive at 17.3%, B was positive at 10.1%, and G at 17.2%. Flocks were more frequently colonized in the warmer months, and younger birds were less frequently colonized than were older slaughtered birds. This study provides descriptive microbiology pertaining to Iceland broilers in a total population study.

Listeria monocytogenes in horses in Iceland.

Twenty isolates of Listeria monocytogenes associated with five confirmed and four suspected incidents of listeriosis in horses in Iceland were characterised by serotyping, pulsed-field gel electrophoresis and ribotyping. Semiquantitative estimates of the numbers of L monocytogenes were made on faeces from horses with clinical signs of listeriosis and on grass silage fed to them. Large numbers of L monocytogenes were often found in the faeces of horses with severe signs of disease. The 20 isolates could be divided into six genotypes, each incident involving only one genotype. One serovar 1/2a genotype was associated with three confirmed incidents of listeriosis in 1991, 1993 and 1997. In one incident, the same genotype was isolated from the organs of a horse with listeriosis and from the spoiled grass silage fed to it.

The improved survival of experimental animals fed with fish oil is suppressed by a leukotriene inhibitor.

Fish oil is believed to alter the immune response and improve survival after infections in experimental animals. This effect may be due to altered production of the leukotrienes (LT). We, therefore, performed a study in order to evaluate whether the effect of fish oil on the immune response of experimental animals is mediated through altered production of the LT. Female NMRI mice in four groups were fed with fish oil, fish oil with 5-lipoxygenase (5-LO) inhibitor (Zileuton, Abbott Laboratories, Chicago, IL, USA), corn oil or corn oil with 5-LO inhibitor. After 6 weeks, the mice were infected with Klebsiella pneumoniae and the survival was monitored. The experiment was performed twice. Analysis was performed mainly on data pooled from both experiments. The survival of the groups fed with fish oil was increased, compared to that of all the other groups and when compared to the groups fed with fish oil with 5-LO inhibitor (log-rank test) the difference was significant (P = 0.007). It has been postulated that the effect of fish oil on the immune system is mediated through altered production of LT. In our study, blocking of the production of the LT eliminated the beneficial effects of fish oil. Our results are in concord with the hypothesis that the effect of fish oil is, at least partly, mediated through altered production of LT.

The diversity of Listeria monocytogenes strains from 10 Icelandic sheep farms.

AIMS: The purpose of this study was to examine the diversity of Listeria monocytogenes strains from healthy sheep, winter feed and environment of sheep farms in Iceland. METHODS AND RESULTS: A total of 104 L. monocytogenes isolates from animals, winter feed and environment on 10 Icelandic sheep farms were compared by serotyping, ribotyping, and pulsed-field gel electrophoresis with ApaI and AscI. The isolates were divided into 24 genotypes, all identified as serovars 1/2a, 1/2b, or 4b. Nine genotypes were detected on more than one farm. On three of the farms there seemed to be a dominant strain of L. monocytogenes. Isolates from incidents of listeriosis in animals occurring on two of the farms belonged to the genotype most commonly found on the particular farm. Nine of the 24 genotypes found on the sheep farms have been associated with disease in animals and/or humans elsewhere in Iceland. CONCLUSIONS: Certain strains of L. monocytogenes seem to be widely distributed on Icelandic sheep farms. On some farms there appears to be a dominant strain of L. monocytogenes. Incidents of listeriosis in animals may tend to be associated with strains commonly found on the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the diversity of L. monocytogenes present in healthy sheep and their environment.

Determination of pertactin IgG antibodies for the diagnosis of pertussis.

OBJECTIVE: To compare increases in serum IgG antibody against pertactin with increases in IgG against pertussis toxin and filamentous hemagglutinin (FHA) in non-vaccinated children, children vaccinated with pertussis toxoid, and adults, all with culture-confirmed pertussis. METHODS: During a double-blind, placebo-controlled, efficacy trial of a monocomponent pertussis toxoid vaccine, acute and convalescent sera were obtained from study children and family members with suspected pertussis. In the present study, IgG antibodies against pertactin, pertussis toxin and FHA (determined by ELISA) were compared in 207 individuals with culture-verified pertussis and paroxysmal cough for >/= 21 days. RESULTS: Significant increases in geometric mean serum IgG against all antigens occurred in non-vaccinated children, but more children responded against pertussis toxin and FHA than against pertactin (96%, 97%, and 62%, respectively). Of the children who had pertussis even though they were vaccinated with the pertussis toxoid vaccine, 97% responded to FHA, while responses to pertussis toxin and pertactin were less common (68% and 61%, respectively). In the 20 adults, the proportions of responders to FHA, pertussis toxin and pertactin were 90%, 80% and 55%, respectively. CONCLUSION: Determination of IgG against pertussis toxin and FHA in paired sera in non-vaccinated children with pertussis is a more sensitive diagnostic tool than determination of IgG against pertactin. Pertactin IgG determinations might be of value as a complement to the other antibody assays in vaccinated children and in adults.

Campylobacter spp. in Icelandic poultry operations and human disease.

We describe the observed relationship of campylobacter in poultry operations to human cases in a closed environment. During 1999 in Iceland, domestic cases of campylobacteriosis reached peak levels at 116/100,000 and in 2000 dropped to 33/100,000. Approximately 62% of broiler carcass rinses were contaminated with Campylobacter spp. in 1999. During 2000, only 15% of the broiler flocks tested Campylobacter spp. positive. In 2000, carcasses from flocks which tested positive on the farms at 4 weeks of age were subsequently frozen prior to distribution. We suggest that public education, enhanced on-farm biological security measures, carcass freezing and other unidentified factors, such as variations in weather, contributed to the large reduction in poultry-borne campylobacteriosis. There is no immediate basis for assigning credit to any specific intervention. We continue to seek additional information to understand the decline in campylobacteriosis and to create a risk assessment model for Campylobacter spp. transmission through this well defined system.

Salmonella in sheep in Iceland.

In 1995 several outbreaks of food poisoning in humans occurred in Iceland, that were traced to salmonella contamination of singed sheep heads. This prompted us to study the prevalence of salmonella infection in sheep and to trace where and how infection might have occurred. Faecal, intestinal contents and tonsillar samples were collected in the spring and autumn from sheep on 50 farms in the southwestern part of the country, where salmonellosis had been detected and from 5 farms in the northwestern part of the country. All faecal samples from the southwest were negative, whereas samples from 3 farms obtained in the autumn in the northwest were positive. Tonsillae taken in the autumn were positive in sheep from 3 farms in the southwest and 2 in the northwest. Our results show that salmonella infection is rare in Icelandic sheep but healthy carriers my harbour the bacteria in tonsillae. Salmonella was not detected in drainage from slaughterhouses nor in singed sheep heads.

Paratuberculosis in Iceland: epidemiology and control measures, past and present.

Paratuberculosis as well as the slow virus infections maedi/visna and jaagsiekte came to Iceland in 1933 when 20 sheep of the Karakul breed were imported from Halle, Germany. At least five of these sheep were subclinical carriers of paratuberculosis. Within 16 years paratuberculosis together with the other Karakul diseases (maedi/visna and jaagsiekte) almost ruined sheep farming, the main agricultural industry in Iceland. The first clinical case of paratuberculosis in sheep was confirmed in 1938, and in cattle in 1944. The first cattle cases of paratuberculosis appeared on farms where the disease had been prevalent in sheep for years. The virulence in cattle appeared to be considerably lower than in sheep. Extensive measures were used to control the spread of paratuberculosis in sheep. Hundreds of kilometres of fences were put up and used together with natural geographic borders to restrict the movement of sheep from infected areas. Serological and other immunological tests were also used to detect and dispose of infected individuals. These measures proved inadequate and the disease could not be eradicated. Culling and restocking of uninfected sheep in endemic areas eradicated maedi/visna and jaagsiekte but not paratuberculosis. Experiments showed that vaccination against paratuberculosis could reduce mortality in sheep by 94%. Vaccination of sheep in endemic areas has been compulsory in Iceland since 1966 and as a result losses have been reduced considerably. Today, serology is used to detect and control infection in cattle herds. Furthermore, serology is used to control vaccination of sheep and screen for infection in non-endemic areas. The complement fixation (CF) test for paratuberculosis has been used until now, but recently we have started comparing the CF test with the CSL absorbed ELISA test.

Hydrogels containing monocaprin have potent microbicidal activities against sexually transmitted viruses and bacteria in vitro.

OBJECTIVE: To investigate the in vitro microbicidal and cytocidal potency of monocaprin dissolved in pharmaceutical hydrogel formulations and to evaluate their potential use as vaginal microbicides against sexually transmitted pathogens such as herpes simplex virus type 2 (HSV-2), human immunodeficiency virus type 1 (HIV-1), Chlamydia trachomatis, and Neisseria gonorrhoeae. METHODS: Gel formulations were mixed with equal volumes of virus/bacteria suspensions in culture medium and incubated for 1 and 5 minutes. The reduction in virus/bacteria titre was used as a measure of microbicidal activity. Similarly, gels were mixed with human semen to study their effect on leucocytes. The toxicity of the gels was tested in rabbits by the standard vaginal irritation test. RESULTS: Gels containing 20 mM of monocaprin caused a greater than 100,000-fold inactivation of HSV-2 and Neisseria in 1 minute and of Chlamydia in 5 minutes. Similarly, the gels caused a greater than 10,000-fold inactivation of HIV-1 in semen in 1 minute. They caused more than a 10,000-fold reduction in the number of viable leucocytes in semen in 1 minute. No toxic effect on the vaginal mucosa of rabbits was observed after daily exposure for 10 days. CONCLUSIONS: Hydrogels containing monocaprin are potent inactivators of sexually transmitted viruses and bacteria in vitro. This simple lipid seems to be a feasible choice as a mucosal microbicide for prevention of sexually transmitted infections. It is a natural compound found in certain foodstuffs such as milk and is therefore unlikely to cause harmful side effects in the concentrations used.

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells.

Two different recombinant visna virus (VV) gag-baculoviruses were constructed for the expression of precursor VV Gag in insect cells. Both recombinant Gag viruses expressed proteins migrating on SDS PAGE at the predicted rate for VV Gag precursor, Pr50gag. However, differences were seen in the morphology of the virus-like particles produced. Monoclonal antibody directed against the VV Gag capsid protein (p25) and sera from sheep infected with ovine lentiviruses reacted to both 50-kDa proteins. A recombinant VV env-baculovirus was constructed, substituting sequences encoding the signal peptide of VV Env with the murine IFN-gamma analogue. Sera from ovine lentivirus infected sheep reacted in immunoblots with two proteins of approximately 100 and 200 kDa found in the plasma membrane of insect cells infected with env-recombinant virus. Sheep immunized with either the recombinant Gag or the Env proteins developed high antibody titers to VV in ELISA. The serum of sheep and ascitic fluid of mice immunized with the recombinant Gag reacted with native Pr50gag and the processed Gag proteins in immunoblots, whereas serum of the recombinant Env immunized sheep reacted with VV gp135 and a putative oligomer of gp135. The immunized sheep responded specifically to visna virus by lymphocyte proliferation in vitro.

Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis.

Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis. Biological activity was tested in vivo and in vitro. The lethal dose in mice was determined. Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed. Seven mutant proteins were produced. Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin. Mutations of two amino acids affected the lethal dose in mice. Both residues have counterparts in the membrane binding region of alpha-toxin. Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions.

Visna virus dUTPase is dispensable for neuropathogenicity.

The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.

Treatment of visna virus infection in lambs with the acyclic nucleoside phosphonate analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA).

Nucleoside and nucleotide analogues, which are inhibitors of human immunodeficiency virus reverse transcriptase, are highly active inhibitors of visna virus replication in cell cultures. One such analogue, the acyclic nucleoside phosphonate PMEA, has also been found to have a prophylactic effect on visna virus infection in lambs. In the present study, lambs were injected subcutaneously with 10 mg/kg PMEA three times a week starting 4 weeks after inoculation with visna virus, when brain infection had been established. After 3 weeks of treatment there was a reduction in the amount of virus isolated from blood cells of PMEA-treated lambs compared to controls and during the remaining 7 months of drug treatment there was significantly less virus isolated from the blood cells of treated lambs than of controls. Antibody response against visna virus was also slower in the treated than in the untreated control group. On the other hand, there was no difference in the amount of virus isolated from various organs of the two groups and the severity of CNS lesions in sheep treated with PMEA for 8 months was comparable to that found in untreated controls, even though PMEA reached concentrations in the cerebrospinal fluid which were well in excess of the EC50 value of the drug for visna virus.

Dietary fish oil supplementation increases survival in mice following Klebsiella pneumoniae infection.

The effect of dietary fish-oil supplementation on survival of NMRI mice after Klebsiella pneumoniae infection was investigated. 30 mice in each group were fed a fish-oil enriched diet, olive-oil enriched diet or standard chow diet. After 6 weeks, the mice were injected intramuscularly with Klebsiella pneumoniae. After 120 h the survival of the mice fed fish-oil enriched diet was 40%, while the survival for mice fed standard or olive-oil enriched diets was 20% and 25%, respectively. The survival curve over 120 h was significantly improved (p = 0.0034) for mice fed a fish-oil enriched diet, compared to the survival curves for mice fed the other 2 diets. The study was repeated by comparing the survival of mice fed a fish-oil enriched diet to those given a corn-oil enriched diet. After 120 h the survival curve for mice fed the fish-oil enriched diet was significantly better compared to the survival curve for mice given the corn-oil enriched diet (p = 0.01). A fish-oil enriched diet therefore increases survival in mice following Klebsiella pneumoniae infection, whether compared to a standard diet, olive-oil enriched diet or corn-oil enriched diet.