Safety and efficacy of a new octavalent combined Erysipelas,Parvo and Leptospira vaccine in gilts against Leptospira interrogans serovar Pomona associated disease and foetal death

The safety and protective efficacy of a new octavalent combination vaccine containing inactivated Erysipelothrix rhusiopathiae, Parvovirus, and Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Australis (Bratislava), Grippotyphosa, Pomona and Tarassovi – Porcilis^® Ery + Parvo + Lepto – was evaluated in laboratory studies and under field conditions.The safety (2× overdose and repeated dose) was tested in 26 gilts. In this study, neither vaccine related temperature increase nor other systemic reactions were observed after intramuscular vaccination. No local reactions were observed except for one animal that had a small local reaction (2 cm diameter) that lasted for 5 days after the third vaccination.Efficacy was tested in 40 gilts. A group of 20 gilts was vaccinated at 20 and 24 weeks of age with Porcilis^® Ery + Parvo + Lepto and a group of 20 age- and source-matched animals served as the control group. The gilts were inseminated at 41 weeks or 66 weeks of age and were challenged with serovar Pomona 10 weeks after insemination, corresponding to 6 months (n = 2 × 10) and 12 months (n = 2 × 10) after the last vaccination. After both the 6- and 12-month challenges the control animals developed clinical signs (fever, lethargy and anorexia) and leptospiraemia as determined by positive blood culture. In addition, both the 6- and 12-month challenges resulted in death of 21% and 27% of the total number of foetuses in the control groups, respectively. Clinical signs and leptospiraemia were statistically significantly lower in vaccinated gilts after both the 6- and 12-month challenges. In addition, foetal death was statistically significantly lower (3% and 2%, respectively) in vaccinated gilts after both the 6- and 12 month challenges.The vaccine was tested further under field conditions on a Portuguese farm with a history of an increasing abortion rate associated with a Leptospira serovar Pomona infection (confirmed by PCR and serology). This study was designed as an observational-longitudinal field study. At the start of the study, all breeding sows and replacement gilts on the farm were vaccinated twice with Porcilis^® Ery + Parvo + Lepto at an interval of 4 weeks. Starting six months after the primary vaccination schedule, the animals were re-vaccinated during the second week of every subsequent lactation. New replacement gilts were vaccinated using the same schedule. After vaccination, the abortion rate reduced rapidly from 12.6% in winter months of 2012 (December 2011 to March 2012) to 0.5% in winter months of 2013, a statistical significant decrease of 96%. The total number of abortions on the farm decreased from 55 in 2012 to 6 in 2013. Thereafter, the abortion rate remained stable and in the period December 2013 to April 2014 was still low (0.6%).In conclusion, the present studies demonstrate that the octavalent Porcilis^® Ery + Parvo + Lepto vaccine can be safely used in gilts and sows and induces significant protection, for the duration of at least one year, against serovar Pomona induced clinical signs, leptospiraemia and foetal death. Protection against Pomona associated reproductive failure was confirmed under field conditions where a significant reduction in abortion rate was observed.     

Isolation and clinical sample typing of human leptospirosis cases in Argentina

Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.     

Multilocus sequence typing (MLST) of leptospiral strains isolated from two geographic locations of Tamil Nadu,India

Here the rodent carrier status for the transmission of human leptospirosis in Tiruchirappalli, district, Tamil Nadu, India was assessed. The predominantly circulating leptospiral STs were recognized by multilocus sequence typing (MLST). A total of 113 rodents were trapped from different provinces of the Tiruchirappalli district. The most prevalent rodent was Bandicota bengalensis (37.2%), and of the total, 52.2% (n = 59) rodents were found to be positive for leptospiral 16S rRNA. These results were validated with a leptospiral culture positivity of 45.8% (n = 27). Three isolates from Chennai (2 rodents and 1 human) and 1 human isolate from Tiruchirappalli were included to understand the spatial variations and to track the source of human leptospirosis. The serogroup, serovar, and species level identification of all 31 isolates identified 28 to be Leptospira borgpetersenii serovar Javanica and three as Leptospira interrogans serovar Autumnalis. MLST analysis defined all isolates to the existing ST profiles (ST145 and ST27) with the exception of 6 L. borgpetersenii (ST DR) isolates that showed variations in the sucA and pfkB loci. The DR ST was locally confined to Chatram province of Tiruchirappalli suggesting an epidemiological link. The predominant STs, ST145 and ST-DR form a group, indicating the presence of original strain that subsequently diverged evolutionarily into two STs. The variations between L. borgpetersenii in sucA and pfkB loci may be an indication that evolutionary changes transpired in Tiruchirappalli.     

Emerging trends in the epidemiology of human astrovirus infection among infants,children and adults hospitalized with acute watery diarrhea in Kolkata,India

Human astroviruses (HAstVs) have now emerged as another common cause of non-bacterial acute gastroenteritis (AGE) in humans worldwide. This study investigated the epidemiology and genetic diversity of human astrovirus strains circulating among infants, younger children (up to 6 years), older children and adolescents (>6–17 years) and adults (18 years and above) hospitalized for diarrhea and their role in AGE in Kolkata, India. A total of 2535 fecal samples were screened for the presence of known enteric viral, bacterial and parasitic etiologies by conventional microbiological assays and molecular methods. The overall incidences of sole or mixed infection of HAstV with known enteric viral, bacterial and parasitic pathogens were detected in 60 cases (2.4%) among all age groups. The clinical symptoms of astrovirus-associated acute watery diarrhea cases were recorded for all sole and mixed infection cases. A high number of sole (n = 13/60 [21.7%]) and mixed infection cases (n = 22/60 [36.7%]) were observed in adults (18 years old or more). Considering all age groups, 18 sole infection cases (n = 18/60 [30%]) and 42 mixed infection cases (n = 42/60 [70%]) with Rotavirus (n = 11/25 [44%]), Vibrio cholerae O1 (n = 6/24 [25%]) Cryptosporidium spp and Giardia lamblia (n = 5/13 [38.4%]) were observed. Further, eleven HAstV samples from infants and children (up to 6 years), children and adolescents (>6–17 years) and adults (18 years and above) were analyzed for their sequences of overlap region between ORF1b (RdRp) and ORF2 (capsid). Among these, ten strains were found to have close genetic relatedness to the Japanese strain HAstV_G1 [AB009985]. Additionally, the IDH2211 Kolkata strain showed a close genetic match with the Thai HAstV_G3 strain [EU363889]. Our study reports show that HAstVs as the sole agent and as mixed infection with other known enteric viral, bacterial, parasitic pathogens are also responsible for AGE among infants, children, adolescents and adults in Kolkata, India.     

Parabens in 24 h urine samples of the German Environmental Specimen Bank from 1995 to 2012

Parabens are widely used as antimicrobial preservatives in personal care and consumer products, food and pharmaceuticals. Due to their ubiquity, humans are constantly exposed to these chemicals. We assessed exposure to nine parabens (methyl-, ethyl-, n- and iso-propyl-, n- and iso-butyl-, benzyl-, pentyl- and heptyl paraben) in the German population from 1995 to 2012 based on 660 24 h urine samples from the German Environmental Specimen Bank (ESB) using on-line HPLC coupled to isotope dilution tandem mass spectrometry. The limit of quantification (LOQ) was 0.5 μg/L for all parabens. We detected methyl-, ethyl- and n-propyl paraben in 79–99% of samples, followed by n-butyl paraben in 40% of samples. We infrequently detected iso-butyl-, iso-propyl- and benzyl paraben in 24%, 4% and 1.4% of samples, respectively. Urinary concentrations were highest for methyl paraben (median 39.8 μg/L; 95th percentile 319 μg/L) followed by n-propyl paraben (4.8 μg/L; 95th percentile 74.0 μg/L) and ethyl paraben (2.1 μg/L; 95th percentile 39.1 μg/L). Women had significantly higher urinary levels for all parabens than men, except for benzyl paraben. Samples from the ESB revealed that over the investigation period of nearly 20 years urinary paraben levels remained surprisingly constant; only methyl paraben had a significant increase, for both men and women. We found strong correlations between methyl- and n-propyl paraben and between n- and iso-butyl paraben. These results indicate that parabens are used in combination and arise from common sources of exposure. Urinary excretion factors are needed to extrapolate from individual urinary concentrations to actual doses.     

Measurement of volatile organic compounds in bottled and tap waters by purge and trap GC–MS: Are drinking water types different?

Volatile organic compounds (VOCs) including major trihalomethanes (THMs) in bottled and tap waters from Central Missouri were quantified and compared with drinking water guidelines. Tap (n = 34), spring (3 brands, n = 25), purified (8 brands, n = 58), distilled (3 brands, n = 12) and flavored waters (5 brands, n = 34) along with Missouri River water (n = 30), were analyzed by purge and trap gas chromatograph–mass spectrometry (GC–MS) using USEPA method 524.2. The mean recoveries of the internal and surrogates standards were fluorobenzene: 100%, benzene d5-, chloro: 100% and 1,4-dichlorobenzene d4: 96%. Approximately 97% of all drinking water types were found to be safe for human consumption. However, two tap water samples recorded bromodichloromethane concentrations above the 60 μg/L World Health Organization (WHO) limit. Moreover, six tap water samples exceeded both the 80 μg/L USEPA Stage I limit and the 100 μg/L European Community (EC) maximum contaminant concentration (MAC) for total THMs (TTHMs) in drinking water. TTHMs in the water types ranged from 0–322 μg/L (tap), 0.1–0.3 μg/L (spring), 0–7.6 μg/L (purified), 0.1–18.1 μg/L (distilled), and 0–5.7 μg/L (flavored). Kruskal–Wallis Chi square approximation showed significant differences (P < 0.001) in the levels of THMs found in the water types. Principal components (PC1 and PC2) with 97% total variance suggested similarities in most drinking waters except for Boonville and Columbia tap waters. Dendrogram plots of TTHMs and Σ VOCs revealed two principal clusters with Columbia and Boonville tap waters in a different cluster and also distant from other water types.     

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State,Nigeria

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12 months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n = 64% or 79% of clustered isolates, 2–32 isolates/cluster), with patients in the age group range 25–34 years being significantly associated with shared-type pattern SIT61 (p = 0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n = 42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n = 27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n = 52 isolates, 2–9 isolates per cluster), with a resulting recent n ? 1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p = 0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU–VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.     

Genetic variability of Rickettsia spp. in Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia,Russia: Identification of a new Candidatus Rickettsia species

Rickettsia spp. are the causative agents of a number of diseases in humans. These bacteria are transmitted by arthropods, including ixodid ticks. DNA of several Rickettsia spp. was identified in Ixodes persulcatus ticks, however, the association of Ixodes trianguliceps ticks with Rickettsia spp. is unknown. In our study, blood samples of small mammals (n = 108), unfed adult I. persulcatus ticks (n = 136), and I. persulcatus (n = 12) and I. trianguliceps (n = 34) ticks feeding on voles were collected in two I. persulcatus/I. trianguliceps sympatric areas in Western Siberia. Using nested PCR, ticks and blood samples were studied for the presence of Rickettsia spp. Three distinct Rickettsia species were found in ticks, but no Rickettsia species were found in the blood of examined voles. Candidatus Rickettsia tarasevichiae DNA was detected in 89.7% of unfed I. persulcatus, 91.7% of engorged I. persulcatus and 14.7% of I. trianguliceps ticks. Rickettsia helvetica DNA was detected in 5.9% of I. trianguliceps ticks. In addition, a new Rickettsia genetic variant was found in 32.4% of I. trianguliceps ticks. Sequence analysis of the 16S rRNA, gltA, ompA, оmpB and sca4 genes was performed and, in accordance with genetic criteria, a new Rickettsia genetic variant was classified as a new Candidatus Rickettsia species. We propose to name this species Candidatus Rickettsia uralica, according to the territory where this species was initially identified. Candidatus Rickettsia uralica was found to belong to the spotted fever group. The data obtained in this study leads us to propose that Candidatus Rickettsia uralica is associated with I. trianguliceps ticks.     

Prevalence of Cryptosporidium species and subtypes in paediatric oncology and non-oncology patients with diarrhoea in Jordan

Cryptosporidiosis is a protozoan parasitic disease which affects human and animals worldwide. In adult immunocompetent individuals, cryptosporidiosis usually results in acute and self-limited diarrhoea; however, it can cause life threatening diarrhoea in children and immunocompromised individuals. In the present study, we compared the prevalence of Cryptosporidium species and gp60 subtypes amongst paediatric oncology patients with diarrhoea (n = 160) from King Hussein Medical Centre for Cancer in Jordan, and non-oncology paediatric patients with diarrhoea (n = 137) from Al-Mafraq paediatric hospital. Microscopy results using modified acid fast staining identified a significantly (p ≤ 0.05) higher prevalence of Cryptosporidium in paediatric oncology patients with diarrhoea (14.4% - 23/160), compared to non-oncology paediatric patients with diarrhoea only (5.1% - 7/137). With the exception of one sample, all microscopy-positive samples (n = 29) and an additional 3/30 microscopy-negative controls were typed to species and subtype level at the 18S and gp60 loci, respectively. All Cryptosporidium positives were typed as C. parvum. Of the 22 typed Cryptosporidium positives from the paediatric oncology patients, 21 were subtyped as IIaA17G2R1 and one as IIaA16G2R1 C. parvum subtypes. The 7 typed positives from the paediatric patients from Al-Mafraq hospital were subtyped as IIaA17G2R1 (n = 5) and IIaA16G2R1 (n = 2). The 3 additional positives from the 30 microscopy negative control samples were subtyped as IIaA17G2R1. The high prevalence of the IIaA17G2R1 subtype, particularly amongst oncology patients, suggests that an outbreak of cryptosporidiosis may have been occurring in oncology patients during the collection period (April to December, 2016). New therapies for cryptosporidiosis in immunocompromised patients are urgently required.     

Detection of Leishmania infantum,Babesia canis,and rickettsiae in ticks removed from dogs living in Italy

The aims of this study were to determine natural infections by Anaplasma phagocytophilum/Anaplasma platys, Bartonella henselae, Ehrlichia canis, Leishmania infantum, Rickettsia spp., Babesia spp., and Hepatozoon spp. by molecular methods in ticks (n = 91) removed from dogs with clinical signs and laboratory abnormalities compatible with tick-borne diseases (n = 22) living in Italy and to assess the distribution and species of ticks encountered. Ticks from dogs living in southern Italy were all identified as Rhipicephalus sanguineus (n = 25), ticks from central Italy included Rh. sanguineus (n = 8) and Ixodes ricinus (n = 9), ticks from northern Italy included Rh. sanguineus (n = 45), Dermacentor marginatus (n = 3), and one I. ricinus. Leishmania infantum, Rickettsia spp., and Babesia canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. L. infantum was detected in I. ricinus from central Italy and in Rh. sanguineus from northern and central Italy. Rickettsia conorii and Ri. massiliae were detected in Rh. sanguineus ticks from central and southern Italy (Sicily), respectively. Bab. canis was detected in D. marginatus ticks from northern Italy.     

Single nucleotide polymorphisms in Plasmodium falciparum V type H+ pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms

BackgroundChloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H⁺). V type H⁺ pyrophosphatase (PfVP2) is thought to pump H⁺ into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms.MethodsBlood samples from 384 patients collected (1981–2009) in Honduras (n = 35), Colombia (n = 50), Liberia (n = 50), Guinea Bissau (n = 50), Tanzania (n = 50), Iran (n = 50), Thailand (n = 49) and Vanuatu (n = 50) were analysed. The pfcrt 72–76 haplotype, pfmdr1 copy numbers, pfmdr1 N86Y and pfvp2 V405I, K582R and P711S alleles were identified using PCR based methods.ResultsPfvp2 was amplified in 344 samples. The pfvp2 allele proportions were V405 (97%), 405I (3%), K582 (99%), 582R (1%), P711 (97%) and 711S (3%). The number of patients with any of pfvp2 405I, 582R and/or 711S were as follows: Honduras (2/30), Colombia (0/46), Liberia (7/48), Guinea-Bissau (4/50), Tanzania (3/48), Iran (3/50), Thailand (1/49) and Vanuatu (0/31). The alleles were most common in Liberia (P = 0.01) and Liberia + Guinea-Bissau (P = 0.01). The VKP haplotype was found in 189/194 (97%) and 131/145 (90%) samples harbouring pfcrt 76T and pfcrt K76 respectively (P = 0.007).ConclusionsThe VKP haplotype was dominant. Most pfvp2 405I, 582R and 711S SNPs were seen where CQ resistance was not highly prevalent at the time of blood sampling possibly due to greater genetic variation prior to the bottle neck event of spreading CQ resistance. The association between the pfvp2 VKP haplotype and pfcrt 76T, which may indicate that pfvp2 is involved in CQ resistance, should therefore be interpreted with caution.     

The impact of a pneumococcal conjugate vaccination program on the nasopharyngeal carriage,serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among healthy children in Turkey

BackgroundThe 7-valent conjugate pneumococcal vaccine (PCV7) was introduced by the Turkey National Immunization Program in 2008 and replaced by the PCV13 in 2011. We assessed the impact of PCV vaccination on the nasopharyngeal (NP) carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae (SP) among healthy Turkish children.MethodsA prospective surveillance study was performed between September 2011 and September 2013 in Istanbul, Turkey. NP swabs, demographic data, and vaccination statuses were obtained from 2165 healthy children aged 0–18 years. Pneumococcal carriage was defined by a positive culture; serotyping was performed via multiplex conventional PCR, and the antibiotic susceptibilities of the isolates were determined based on the minimum inhibitory concentration (MIC) values of the Clinical Laboratory Standards Institute (CLSI).ResultsThe prevalence of pneumococcal carriage was 6.4%. The carriage rates were 8%, 7%, and 5% in the following age groups: 0–24 months, 25–60 months, and >60 months, respectively. The carriage rate was significantly higher in the 0–24 month age group than in the >60 months age group (p = 0.03). Sixty percent of the children were not vaccinated with any PCV; 4%, 2%, and 4% received at least 1, 2 or 3 doses and 30% children received the full schedule (4 doses) of either PCV7 or PCV13. Among the isolated S. pneumoniae strains, 45% were of the non-vaccine type (NVT) and 55% were of the vaccine type (VT). The children who received at least a single PCV dose had significantly lower odds of colonization via VT serotypes than the non-vaccinated children [odds ratio: 0.61 (95% confidence interval = 0.41–0.91), p = 0.01]. The percentages of the serotypes covered by PCV7 and PCV13 were 51% and 56%, respectively. The most frequently isolated serotypes were 6A/B/C (n = 22, 16.5%), 19F (n = 18, 13.5%), 23F (n = 15, 11.2%), serotype 9V/A (n = 10, 7.5%), 12F (n = 5, 4.5%), 15A/F (n = 7, 4.5%) and 22 A/F (n = 6, 4.5%). Using the meningitis criteria and the MIC, 62% of the isolates were resistant to penicillin and 13% were non-sensitive to ceftriaxone. Erythromycin and clindamycin resistance were 43% and 31%, respectively.ConclusionWe shown that following nation-wide PCV vaccination, S. pneumoniae NP carriage was decreased.     

Genotyping of Brucella melitensis and Brucella abortus strains currently circulating in Xinjiang,China

Brucellosis is a well-known zoonotic disease that can cause severe economic and healthcare losses. Xinjiang, one of the biggest livestock husbandry sectors in China, has gone through increasing incidence of brucellosis in cattle and small ruminants recently. In this paper, 50 B. melitensis strains and 9 B. abortus strains collected from across Xinjiang area (from 2010 to 2015) were genotyped using multiple locus variable-number tandem-repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST). Based on 8 loci (MLVA-8), 50 B. melitensis strains were classified into three genotypes. Genotypes 42 (n = 38, 76%) and 63 (n = 11, 22%) were part of the East Mediterranean group, and one genotype with pattern of 1-5-3-13-2-4-3-2 represents a single-locus variant from genotype 63. MLVA-16 resolved 50 B. melitensis strains into 28 genotypes, of which 15 are unique to Xinjiang and 10 are in common with those in adjacent country Kazakhstan and neighboring provinces of China. Minimum Spanning Tree (MST) analysis implies that B. melitensis strains collected from across Kazakhstan, Xinjiang and China areas may share a common origin. Nine B. abortus strains were sorted into three genotypes by MLVA-8, genotypes 36 (n = 7, 77.8%), 86 (n = 1, 11.1%) and a new genotype with pattern of 4-5-3-13-2-2-3-1. Each B. abortus strain showed distinct MLVA-16 genotypes, suggesting that B. abortus species may possess more genetic diversity than B. melitensis. Using MLST, most B. melitensis strains (n = 49) were identified as sequence type ST8, and most B. abortus strains (n = 8) were recognized as ST2. Two new sequence types, ST37 and ST38, represented by single strain from B. melitensis and B. abortus species respectively, were also detected in this study. These results could facilitate the pathogen surveillance in the forthcoming eradication programs and serve as a guide in source tracking in case of new outbreaks occur.     

Phylloquinone content of herbs,spices and seasonings

Culinary herbs and spices contain a variety of bioactive compounds including phylloquinone, the most common dietary form of vitamin K. In this study, we analyzed the phylloquinone content of a large number of commonly available culinary herbs, spices, and seasonings. Samples of fresh herbs (n = 19), dried herbs (n = 28), spices and seeds (n = 26), seasoning blends (n = 9), and other flavor enhancers (n = 11) were purchased in Montreal (Quebec, Canada) and Washington (DC, USA). All samples were analyzed in quadruplicate using standardized HPLC procedures. Most fresh herbs contained between 450 and 1200 μg of phylloquinone/100 g. Dried herbs were even richer with some (cilantro, marjoram, parsley) showing concentrations of ≈3000 μg/100 g. Phylloquinone content of spices and seeds was relatively low (5–250 μg/100 g), while being highly variable among seasoning blends (2.3–1878 μg/100 g). According to our results, portions of only 3 g of herbs can increase daily intakes of phylloquinone by up to 100 μg, contributing significantly to the daily vitamin K intake. Herbs can thus be important food sources of phylloquinone and should be accounted for when assessing vitamin K intakes in research or in patients treated with vitamin K antagonists. Future research should focus on the bioavailability of phylloquinone in these products.     

Nonhuman primate retroviruses from Cambodia: High simian foamy virus prevalence,identification of divergent STLV-1 strains and no evidence of SIV infection

Nonhuman primates (NHPs) carry retroviruses such as simian immunodeficiency viruses (SIV), simian T-cell lymphotropic viruses (STLV) and simian foamy viruses (SFV). Here, we revisited NHPs from Cambodia to assess the prevalence and diversity of these retroviruses using updated viral detection tools. We screened blood from 118 NHPs consisting of six species (Macaca fascicularis (n = 91), Macaca leonine (n = 8), Presbytis cristata (n = 3), Nycticebus coucang (n = 1), Hylobates pileatus (n = 14), and Pongo pygmaeus) (n = 1) by using a Luminex-based multiplex serology assay that allows the detection of all known SIV/HIV and SFV lineages. We also used highly sensitive PCR assays to detect each simian retrovirus group. Positive PCR products were sequenced and phylogenetically analyzed to infer evolutionary histories.Fifty-three of 118 (44.9%) NHPs tested positive for SFV by serology and 8/52 (15.4%), all from M. fascicularis, were PCR-confirmed. The 8 novel SFV sequences formed a highly supported distinct lineage within a clade composed of other macaque SFV. We observed no serological or molecular evidence of SIV infection among the 118 NHP samples tested. Four of 118 (3.3%) NHPs were PCR-positive for STLV, including one M. fascicularis, one P. cristata, and two H. pileatus. Phylogenetic analyses revealed that the four novel STLV belonged to the PTLV-1 lineage, outside the African radiation of PTLV-1, like all Asian PTLV identified so far. Sequence analysis of the whole STLV-1 genome from a H. pileatus (C578_Hp) revealed a genetic structure characteristic of PTLV. Similarity analysis comparing the STLV-1 (C578_Hp) sequence with prototype PTLVs showed that C578_Hp is closer to PTLV-1s than to all other types across the entire genome. In conclusion, we showed a high frequency of SFV infection but found no evidence of SIV infection in NHPs from Cambodia. We identified for the first time STLV-1 in a P. cristata and in two H. pileatus.     

Common occurrence of Cryptosporidium hominis in horses and donkeys

Extensive genetic variation is observed within the genus Cryptosporidium and the distribution of Cryptosporidium species/genotypes in humans and animals appears to vary by geography and host species. To better understand the genetic diversity of Cryptosporidium spp. in horses and donkeys, we characterized five horse-derived and 82 donkey-derived Cryptosporidium isolates from five provinces or autonomous regions (Sichuan, Gansu, Henan, Inner Mongolia and Shandong) in China at the species/genotype and subtype levels. Three Cryptosporidium species/genotypes were identified based on the analysis of the SSU rRNA gene, including Cryptosporidium parvum (n = 22), the Cryptosporidium horse genotype (n = 4), and Cryptosporidium hominis (n = 61). The identification of C. hominis was confirmed by sequence analysis of the HSP70 and actin genes. Subtyping using sequence analysis of the 60 kDa glycoprotein gene identified 21 C. parvum isolates as subtype IIdA19G1, the four horse genotype isolates as subtypes VIaA15G4 (n = 2) and VIaA11G3 (n = 2), and the 61 C. hominis isolates as IkA16G1 (n = 59) and IkA16 (n = 2). The common finding of C. hominis reaffirms the heterogeneity of Cryptosporidium spp. in horses and donkeys and is possibly a reflection of endemic transmission of C. hominis in these animals. Data of the study suggest that horses and donkeys as companion animals may potentially transmit Cryptosporidium infections to humans.     

Use of ORAC to assess antioxidant capacity of human milk

The oxygen radical absorbance capacity (ORAC–FL) assay, a useful measure of the antioxidant capacity (AC) reported for some biological samples, supplements, and food samples, was standardized and validated for measuring AC of human milk. Limits of linearity, precision and accuracy of the ORAC–FL assay were made by constructing a Trolox Calibrator which included the addition of human milk as the sample matrix. AC assay results indicated excellent linearity (R² = 0.990 ± 0.005), precision (2.2%) and accuracy on recovery (94.8 ± 3.2%) over a wide range of Trolox concentrations. To validate the assay further, mature milk samples were collected from 100 lactating mothers in Vancouver and Winnipeg, and were measured for vitamin E isomers by HPLC and AC using the standardized ORAC–FL assay. Alpha-tocopherol (α-Toc) was the major vitamin E isomer detected in human milk, established using both ultra-violet and fluorescent detection methods. Milk α-Toc concentrations were found to correlate significantly (P < 0.01) with ORAC–FL assay values in milk obtained from mothers in both Vancouver (R = 0.439, n = 60) and Winnipeg (R = 0.408, n = 40). Although milk is a complex matrix with multiple components possessing potential antioxidant activity, our results indicated that the ORAC–FL assay is a very useful indicator for assessing the antioxidant capacity of human milk.     

Rapid determination of nine parabens and seven other environmental phenols in urine samples of German children and adults

We developed a fast, selective and sensitive on-line LC/LC–MS/MS method for the simultaneous determination of nine parabens and seven environmental phenols in urine. Parabens are widely used as antimicrobial preservatives. Bisphenol A, triclosan, triclocarban, 2-phenylphenol, and benzophenones are used inter alia in disinfectants, sunscreens and in polymers. Some of these substances are suspected endocrine disruptors.Limits of quantification and analytical quality criteria fully met the needs for determining exposure levels occurring in the general population. We analyzed 157 spot urine samples from the general German population (59 females, 39 males and 59 children). For the parabens, we found methyl, ethyl and n-propyl paraben with high detection rates (77–98%), followed by n-butyl (36%), iso-butyl (17%), iso-propyl (3%) and benzyl paraben (3%). We detected no pentyl and heptyl paraben. Urinary concentrations were highest for methyl paraben (median 24.5 μg/L; 95th percentile 379 μg/L) followed by ethyl (1.4 μg/L; 35.2 μg/L) and n-propyl paraben (1.2 μg/L; 68.1 μg/L). Other environmental phenols with high detection rates were BPA (95%), triclosan (45%) and benzophenone 1 and 3 (26%). For most of the parabens/environmental phenols we found higher urinary levels in females than in males or children, probably due to differences in (personal care) product use. However, high levels (in the mg/L range) were also observed in children.Exposure to the above substances is occurring worldwide. Differences between countries do seem to exist and might be caused by different product compositions or different use habits. Human metabolism data is urgently needed to extrapolate from urinary biomarker levels to doses actually taken up.     

Molecular genetic analysis of Plasmodium vivax isolates from Eastern and Central Sudan using pvcsp and pvmsp-3α genes as molecular markers

In Sudan, Plasmodium vivax accounts for approximately 5–10% of malaria cases. This study was carried out to determine the genetic diversity of P. vivax population from Sudan by analyzing the polymorphism of P. vivax csp (pvcsp) and pvmsp-3α genes. Blood samples (n = 76) were taken from suspected malaria cases from 2012–2013 in three health centers of Eastern and Central Sudan. Parasite detection was performed by microscopy and molecular techniques, and genotyping of both genes was performed by PCR-RFLP followed by DNA sequence for only pvcsp gene (n = 30). Based on microscopy analysis, 76 (%100) patients were infected with P. vivax, whereas nested-PCR results showed that 86.8% (n = 66), 3.9% (n = 3), and 3.9% (n = 3) of tested samples had P. vivax as well as Plasmodium falciparum mono- and mixed infections, respectively. Four out of 76 samples had no results in molecular diagnosis. All sequenced samples were found to be of VK210 (100%) genotype with six distinct amino acid haplotypes, and 210A (66.7%) was the most prevalent haplotype. The Sudanese isolates displayed variations in the peptide repeat motifs (PRMs) ranging from 17 to 19 with GDRADGQPA (PRM1), GDRAAGQPA (PRM2) and DDRAAGQPA (PRM3). Also, 54 polymorphic sites with 56 mutations were found in repeat and post-repeat regions of the pvcsp and the overall nucleotide diversity (π) was 0.02149 ± 0.00539. A negative value of dN − dS (−0.0344) was found that suggested a significant purifying selection of Sudanese pvcsp, (Z test, P < 0.05). Regarding pvmsp-3α, three types were detected: types A (94.6%, 52/55), type C (3.6%, 2/55), and type B (1.8%, 1/55). No multiclonal infections were detected, and RFLP analysis identified 13 (Hha I, A1-A11, B1, and C1) and 16 (Alu I, A1-A14, B1, and C1) distinct allelic forms. In conclusion, genetic investigation among Sudanese P. vivax isolates indicated that this antigen showed limited antigenic diversity.     

A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Bogotá, Colombia

With an incidence of 25.6/100,000 in 2008, tuberculosis (TB) remains an important public health problem in Colombia. In this study, a total of 152 Mycobacterium tuberculosis complex strains isolated in Bogotá, Colombia between years 1995 and 2007 were genotyped by spoligotyping and 12-loci MIRU-VNTRs. The various spoligotyping-based genotypic lineages in our sample were: Latin American & Mediterranean (LAM) n = 75, 49.34%; Haarlem, n = 38, 25.0%; ill-defined T group, n = 21, 13.82%; S family, n = 5, 3.29%; X clade, n = 2, 1.32%; Beijing, n = 1, 0.65%, while strains with unknown signatures (n = 10) represented 6.58% of isolates. Using spoligotyping as a first molecular marker and MIRU-VNTRs as second marker, we obtained 102 single patterns and 14 clustered patterns (n = 52 strains from 49 patients, 2–8 strains per cluster). The MIRU-VNTRs patterns corresponded to 50 MITs for 109 strains and 43 orphan patterns. The most frequent patterns were MIT190 (n = 12), MIT45 (n = 10), and MIT25 (n = 9). The Hunter & Gaston discriminatory index (HGDI) of both methodologies used together showed a value of 0.992. In our setting, the HGDI of five loci subset (MIRU10, 16, 23, 26 and 40) contributed most to the discriminatory power of 12-loci format used (HGDI = 0.977). The lineage distribution of M. tuberculosis showed that more than 3/4 of strains in Bogotá are commonly found in Latin America, Caribbean, and Europe. This observation might reflect the shared post-Columbus history of Colombia and its Latin-American neighbors as well as strains brought in by 20th century immigrants from Europe. We also demonstrate the usefulness of MIRU-VNTR to detect suspected links among patients and polyclonal infections.