Isolation of Salmonella strains from reptile faeces and comparison of different culture media

AIMS: To provide information on epidemiology and isolation of Salmonella strains from reptiles. METHODS AND RESULTS: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50.5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport-Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58.7%), sulphamethoxazole (55.5%), streptomycin (32.6%), tetracycline (19.6%), ampicillin (17.4%) and nalidixic acid (13.1%). CONCLUSIONS: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.     

Sex and age differences in the avoidance of reptile exhibits by zoo visitors

Visitors at two zoos were observed as they approached the reptile exhibits at the respective institutions. Both age and sex differences were discovered among several variables designed to measure avoidance behavior. At most ages, females more often refused to enter the exhibits than males. Females also spent less time within the exhibits than males. With respect to age differences, teen aged visitors refused to enter less often and spent more time in the exhibits than did individuals of other ages.     

Isolation and characterization of bacteriophages infecting Salmonella spp

Bacteriophages infecting Salmonella spp. were isolated from sewage using soft agar overlays containing three Salmonella serovars and assessed with regard to their potential to control food-borne salmonellae. Two distinct phages, as defined by plaque morphology, structure and host range, were obtained from a single sample of screened sewage. Phage FGCSSa1 had the broadest host range infecting six of eight Salmonella isolates and neither of two Escherichia coli isolates. Under optimal growth conditions for S. Enteritidis PT160, phage infection resulted in a burst size of 139 PFU but was apparently inactive at a temperature typical of stored foods (5 degrees C), even at multiplicity of infection values in excess of 10 000. While neither isolate had characteristics that would make them candidates for biocontrol of Salmonella spp. in foods, phage FGCSSa1 behaved unusually when grown on two Salmonella serotypes at 37 degrees C in that the addition of phages appeared to retard growth of the host, presumably by the lysis of a fraction of the host cell population.     

Special wishes for the members of Antwerp Zoo; 65 years of history in pictures (1850-1914)

Antwerp Zoo in Belgium was founded in 1843 and is one of the oldest zoos in the world. It is part of the Royal Zoological Society of Antwerp (RZSA) which also manages Planckendael Wild Animal Park, De Zegge Nature Reserve, Flanders Congress and Concert Center and - since 2009 - Blankenberge Serpentarium - a reptile collection.During its almost 175 years existence the RZSA and Antwerp Zoo in particular went through periods with many highlights but also years with great difficulties. The early years till the start of World War I Antwerp Zoo went through a period that was important for the present zoological garden. The expansion and lay out of the park, the construction of buildings for visitors or to house animals, the offer for visitors to welcome them: much of it still exists or can still be noticed in the park or as part of the day to day operations of Antwerp Zoo and RZSA.For the young society, it was important to have a significant and satisfied membership. Members gave the RZSA not only a key appearance but their financial contributions were also needed for the further development of the gardens. To pamper the members from 1850 on a special annual wishing card was offered to them. This initiative came to an end in 1914. With a few exceptions all of them were very nicely illustrated with pictures of the animals, new constructions or other illustrations which illustrate the development of the zoological garden.The article describes in detail 62 (of 65) known wishing cards, analyzing the relevance of the illustrations for each year. Very often newly arrived species or new constructions are shown. As far as possible more details of these species were gathered. And the future existence of buildings and enclosures and the development of the gardens were also examined.We can conclude that the period before World War I was one with many highlights for Antwerp Zoo. The special whishing cards are very important historical documents and they continue to illustrate the evolution the Zoo went through during the first seventy years.     

A comparison of the efficacy of different disinfection methods in eliminating Salmonella contamination from turkey houses

Aims: This study aimed to compare the efficacy of different disinfection methods in eliminating Salmonella contamination from turkey houses. Methods and Results: Fifty depopulated turkey houses which had all housed Salmonella‐positive flocks were visited after cleaning and disinfection. A minimum of 45 swab samples from different surfaces were taken per house and analysed for the presence of Salmonella. The sampled surfaces included intact floor surfaces, floor cracks, walls, feeders, drinkers, anteroom, nestboxes and miscellaneous items. Houses were grouped according to the disinfectant which had been used and the efficacy of the different groups of disinfectants was compared. Sixty‐eight % of houses tested positive for Salmonella after C&D. Out of 4440 samples, 207 tested positive for Salmonella, giving an overall sample prevalence of 4·7%. There was no significant difference in the level of residual contamination between breeding, rearing and finishing houses. Products containing a mixture of formaldehyde, glutaraldehyde and quaternary ammonium compounds (QAC) performed significantly better than products containing hydrogen peroxide and peracetic acid. Cleaning and disinfection was least effective in nestboxes and anterooms. Conclusions: Thorough cleaning and the choice of a suitable disinfectant are crucial if Salmonella contamination of turkey houses is to be eliminated. Significance and impact of the study: This study shows that disinfectants containing a mixture of formaldehyde, glutaraldehyde and QAC perform significantly better under field conditions than oxidising products and should therefore be the first choice for disinfection of turkey premises where Salmonella is present.     

Comparison of environmental sampling methods for detecting Salmonella in commercial laying flocks in the UK

Aims: To investigate the performance of the Salmonella National Control Programme (NCP) sampling/testing methods in laying flocks of domestic fowl. Methods and Results: Eighty‐five visits were made to 69 flocks representative of the main production systems (cage, barn and free‐range) infected with Salmonella. In each visit, three methodologies were compared: (i) the European Union (EU) baseline survey method (five faeces and two dust samples); (ii) an in‐house (Veterinary Laboratories Agency, VLA) ‘wet’ method that involved collecting 10 dust and 10 faeces samples into jars with buffered peptone water; and (iii) a method involving two samples of pooled faeces and one of dust (cultured as one sample of each type), which has been adopted for the NCP for laying flocks across the EU. Conclusions: The ‘wet’ method was the most sensitive, and the NCP the least, although individual NCP samples were the most sensitive ones. Significance and Impact of the Study: The apparent lower sensitivity of the NCP method may be compensated by repeated sampling of flocks (twice during rear and several times during lay). Sampling using VLA methodology should be advocated for farms aiming to disclose low‐level Salmonella before restrictions on the sale of eggs from Salmonella Enteritidis or Salmonella Typhimurium‐infected flocks are in place.     

Detection, selective isolation and characterisation of Dactylosporangium strains from diverse environmental samples

A culture-independent, nested PCR procedure based on genus-specific oligonucleotide primers detected the presence of members of the genus Dactylosporangium in 14 out of 21 diverse environmental samples. Clones generated from the 14 positive environmental samples formed distinct phyletic lines in the dactylosporangial 16S rRNA gene tree. Presumptive dactylosporangiae were isolated from 7 of these samples using a medium designed to be selective for members of the genus Dactylosporangium, namely Streptomyces Isolation Medium supplemented with gentamicin and antifungal antibiotics. One hundred and two out of 219 representative presumptive dactylosporangiae were considered as authentic members of the genus Dactylosporangium as they gave PCR amplification products using the genus-specific primers and had chemical features typical of dactylosporangiae. Representative of the Dactylosporangium isolates formed distinctive phyletic lines in the dactylosporangial 16S rRNA gene tree, contained the non-ribosomal peptide and type-I polyketide synthase genes and inhibited the growth of Bacillus subtilis, Kocuria rhizophila and Staphylococcus aureus strains. It is evident from these results that the genus Dactylosporangium is underspeciated, widely distributed in natural habitats and is a potentially rich source of novel secondary metabolites.     

Isolation of a cytotoxin from L-form Salmonella typhimurium

Abstract A cytotoxin protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 μg/ml, with a complete lysis obtained at 5 μg/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1–0.5 μg/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor α. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 μg/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.     

Comparison of sampling methods to detect Salmonella infection of turkey flocks

Aims:  To compare the efficiency of various sampling methods for detection of Salmonella in turkey flocks.
Methods and Results:  In a field study that compared various sampling methods one pair of boot swabs taken from the whole turkey house provided suitably sensitive results for fattening and rearing flocks and was no less sensitive than two pairs, each from half the house, tested as a pooled sample. The sensitivity was further enhanced by adding a dust sample. The dust sample appeared to be particularly useful in flocks with a low prevalence, especially in breeding flocks, and was more sensitive than a method which used five pairs of boot swabs per flock. Combined incubation of a boot swab and a dust sample showed no interference between the two sample types and a maximum sensitivity of detection. Litter samples and commercial sponge drag swabs provided a lower level of detection.
Conclusions:  A single pair of boot swabs taken from the whole house is recommended for routine sampling of commercial rearing or fattening flocks. An additional dust sample could be added to increase detection in flocks with a low prevalence or in breeding flocks, but adding an additional pair of boot swabs would not increase detection compared with a single pair.
Significance and Impact of the Study:  This study demonstrates that significant efficiencies can be made in sampling programmes for detection of Salmonella in turkey flocks without detracting from the sensitivity. Similar studies are recommended for other poultry sectors, particularly in chicken breeding flocks.     

Isolation of Legionella from water samples using various culture methods

The efficacy of a non-selective medium and two selective media were compared for the isolation of legionellas from water samples. The effect of acid wash treatment for decontamination of the water samples on the isolation frequency of legionellas was also studied. The 236 samples were taken from cooling, humidifying and drinking water systems; 21% were legionella-positive when inoculated directly on modified Wadowsky-Yee (MWY) medium and 26% were positive when concentrated (x 200) before cultivation on MWY or CCVC media. Inoculation on MWY medium after concentration followed by decontamination by the acid-wash technique gave the highest isolation frequency (31%). The lowest frequency (8%) was found with the non-selective BCYEα medium. An isolation frequency of 28% was achieved with the BCYEα medium after concentration and acid-wash treatment of the samples. Forty per cent of the samples were positive for legionellas when the results from all the culture methods were combined. Not all the legionella-positive samples were identified by a single culture method. Ninety-three of the 95 positive samples were detected with the two best combinations of three culture methods. The best culture method for detecting legionellas depended on the source of the water sample. Some water quality characteristics, like temperature and organic matter content, affected the isolation frequency of Legionella spp.     

2013-2014年湖北省外环境中禽流感病毒污染情况分析

为了解2013-2014年湖北省外环境中禽流感病毒污染情况,采用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)对全省7个监测点1 642份活禽相关外环境标本进行禽流感病毒型别与阳性率监测.结果显示,2013-2014年湖北省外环境中禽流感病毒平均阳性检出率为19.1％,检出的禽流感病毒型别以H9亚型为主(占57.3％),未检出H7亚型.不同地区和季节禽流感病毒阳性率有所差异,鄂西北冬春季病毒检出阳性率高于鄂西南和江汉平原;家禽屠宰加工厂检出阳性率最高;阳性率最高的标本来自清洗禽类的污水.结果提示,湖北地区外环境中禽流感病毒阳性率较高,且型别多样,家禽屠宰加工人员对禽流感病毒有较高的暴露风险,需加强健康宣教,提高个人防护意识.     

The marine diapsid reptile Endennasaurus from the Upper Triassic of Italy

Abstract:  The marine reptile Endennasaurus from the Upper Triassic Zorzino Limestone of northern Italy is redescribed and reassessed. New details of the skull and postcranial skeleton are revealed, confirming the attribution of this genus to the diapsid reptile clade Thalattosauriformes. Phylogenetic analysis suggests that Endennasaurus was related to the European genus Askeptosaurus and the Chinese Anshunsaurus . Despite a rather conservative postcranial morphology, Endennasaurus clearly occupied a highly specialized dietary niche as it combined a slender tapering premaxillary rostrum with a complete absence of either marginal or palatal teeth.     

Improved bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken meat samples

AIMS: To evaluate an improved bioluminescent enzyme immunoassay (BEIA) using biotinylated firefly luciferase for the rapid detection of Salmonella in naturally contaminated chicken meat samples. METHODS AND RESULTS: Capture agents and lipopolysaccharide (LPS) extraction reagents for Salmonella were investigated to improve the sensitivity of the BEIA. Also, the use of Oxoid SPRINT (Simple Pre-enrichment and Rapid Isolation New Technology) as a pre-enrichment and selective medium for 26-h BEIA detection of Salmonella in chicken meat samples was examined. The use of polymyxin B as a capture agent on solid support and 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) for extraction of the LPS facilitated sensitive detection of Salmonella. Of 120 chicken meat samples, 25 samples were positive using the improved BEIA with the SPRINT and 25 samples were positive using the SPRINT followed by the standard isolation methods. CONCLUSIONS: The improved BEIA, in which polymxin B was used as a capture agent and CHAPS was used for extraction of the antigen, had a sensitivity of 96.0% and a specificity of 98.9% for the detection of Salmonella in chicken meat. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved BEIA combined with the SPRINT medium for the detection of Salmonella in chicken meat samples produced comparable results to the culture methods in 26 h.     

Isolation of Sporothrix schenckii from environmental samples in Venezuela

The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis frequently found in Latin America. The isolation of this fungus from the environment and other sources has been widely reported. Nevertheless, to our knowledge this fungus has not been isolated from the endemic areas of Venezuela. In studies related to a clinical case of sporotrichosis in "Colonia Tovar", produced by traumatism after manipulating soil samples, the fungus was isolated from the soil of that particular area. This is the first report of the isolation of S. schenckii from environmental sources in an endemic area of Venezuela.     

Isolation of Cryptococcus neoformans from environmental samples in Alicante

A study of the incidence of Cryptococcus neoformans in Alicante was carried out in environmental samples from the cities of Alicante and Santa Pola. The samples were pigeons faeces and Eucalyptus camaldulensis tissues. This study shows that the prevalence of the yeast in faeces from captive pigeons is higher (81.5%) than in the same samples from urban pigeons (16.3%). Regarding the biotype and varieties, a 79.3% of the isolates belonged to C. neoformans, and all of them were C. neoformans var. neoformans. None of the E. camaldulensis samples showed C. neoformans growth, although some Cryptococcus laurentii strains were isolated from flowers.     

Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples

An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.     

Isolation of lipids from biological samples

Abstract

Isolation of the lipid fraction from biological samples has been a crucial part of countless studies over the last century. This considerable research interest has led to the development of a number of methods for isolating a range of molecular species that fall under the umbrella term “lipid”. Such methods vary in popularity, complexity, specificity and even toxicity. In this review, we explore examples of published methods (1952–2014) for isolating lipids from biological samples and attempt to assess the limits of techniques both from a chemical and biological perspective. We also suggest how a suitable method might be chosen for a novel application.     

A hypothetico-deductive analysis of the environmental factors involved in the current reptile distribution pattern in the Canary Islands

Aim We analyse the influence of different factors on the inter‐insular colonization processes that yielded the current distribution pattern of endemic reptile species in the Canary Islands. Location The seven main islands of the Canary Islands. Methods We tested 11 hypotheses about factors that might have affected the colonization processes. We used Mantel and partial Mantel tests to establish the statistical significance of the relationship between the islands predicted by each hypothesis, and a parsimony analysis to detect the most parsimonious hypothesis. Results The only significant relationships were those predicted by inter‐island distance, also taking area into account, and habitat similarity, considering current vegetation in the islands. The latter was the best hypothesis according to the parsimony criterion. Main conclusions Our results support the hypothesis that habitat similarity between islands is the main factor affecting the colonization processes of the whole group, although inter‐insular distance seems to play a separate role in colonization events.     

Direct isolation of Francisella spp. from environmental samples

Aims:  To develop a selective medium for isolation of F. tularensis, F. novicida and F. philomiragia from environmental samples.
Methods and Results:  A selective media, cysteine heart agar with 9% chocolatized sheep blood, containing polymyxin B, amphotericin B, cyclohexamide, cefepime and vancomycin (CHAB-PACCV) was developed and evaluated for growth of Francisella spp. No differences were observed in recovered colony forming units (CFUs) for F. tularensis , F. novicida and F. philomiragia on CHAB-PACCV vs nonselective CHAB. Growth of non- Francisella species was inhibited on CHAB-PACCV. When environmental samples were cultured on CHAB and CHAB-PACCV, only CHAB-PACCV allowed isolation of Francisella spp. Three new Francisella strains were isolated directly from seawater and seaweed samples by culture on CHAB-PACCV.
Conclusions:  CHAB-PACCV can be used for direct isolation of Francisella spp from environmental samples.
Significance and Impact of the Study:  Francisella spp. show a close association with environmental sources. Future utilization of CHAB-PACCV for isolation of Francisella spp. directly from environmental samples should prove valuable for investigating outbreaks and human infections attributed to environmental exposure.