Salmonella enterica isolates from faeces of domestic reptiles and a study of their antimicrobial in vitro sensitivity

From October 2001 to February 2002, the faecal samples of 305 reptiles (165 saurians, 99 ophidians and 41 chelonians) were bacteriologically examined to detect Salmonella enterica. S. enterica was isolated from 73 (23.93%) faecal samples including 44 (60.27%) samples collected from saurians, 15 (20.55%) from chelonians and 14 (19.18%) from ophidians; considering the number of samples taken for each reptile group, S. enterica was isolated from the 36.58% of chelonians, 26.66% of saurians and 14.14% of ophidians. The isolates were distributed among 38 serotypes. Sixty-nine (94.52%) isolates were resistant to erythromycin. About one-third of the isolates was resistant to sulfisoxazole (35.61%), gentamycin (32.88%), amoxycillin (31.51%) and ampicillin (27.40%). All but one of the isolates were sensitive to chloramphenicol. A high percentages of isolates were sensitive to enrofloxacin (84.93%), nitrofurantoin (80.82%), trimethoprim (76.71%) and tetracycline (75.34%).     

Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.     

Prevalence of Salmonella spp. in pet reptiles in Japan

From November 2000 to July 2002, 112 fecal samples from pet reptiles, including 18 turtles, 71 lizards and 23 snakes, sold at a pet shop were examined for the prevalence of Salmonella spp. in Japan. Salmonella spp. were isolated from 83 (74.1%) of 112 samples, and a total of 112 Salmonella isolates were identified as subspecies I to IV. The majority of isolates (62.5%) belonged to subspecies I and 54 isolates could be identified as any of 28 serovars. The predominant serovars were found to be S. Bardo, S. Newport and S. Panama, which cause human salmonellosis. These results indicate that pet reptiles may be a potential infectious source of human salmonellosis in Japan.     

Distribution of integrons and SGI1 among antibiotic-resistant Salmonella enterica isolates of animal origin

In Salmonella enterica, resistance to antibiotics can be caused by the presence of SGI1, transposons or conjugative plasmids. In this study we were interested in the relative contribution of these genetic elements to the antibiotic resistance of S. enterica isolates collected within a single year in the Czech Republic from animal sources. Altogether 123 antibiotic-resistant isolates belonging to 16 different S. enterica serovars were classified into 3 groups according to the presence of SGI1 and the presence of integrons. The first group consisted of 62 strains in which neither SGI1 nor class 1 integron was detected. A high diversity among serovars and resistance phenotypes was found in this group. The second group consisted of 56 strains positive for both the SGI1 and class 1 integron, out of which 55 belonged to serovar Typhimurium and one to a nonmotile serovar [4,12] which harboured the SGI1-B variant. The third group comprised five strains which were positive for class 1 integron but negative for the SGI1. Sequencing of the integrons in these isolates identified integron with sat1 and aadA1 gene cassettes in S. Sandiego and S. Pullorum, dfrA1 and aadA1 gene cassettes in S. Typhimurium integron, and aadA21 gene cassette in S. Braenderub and S. Zanzibar.     

Sequence polymorphism of the Salmonella plasmid virulence factor D (SpvD) in Salmonella enterica isolates of animal origin

The nucleotide sequence encoding the Salmonella plasmid virulence factor D (SpvD) was determined in 17 Salmonella strains that were different in O and H antigen patterns, animal host and geographical origin, and year of isolation. Nucleotide sequence comparison revealed the existence of at least nine spvD alleles resulting in 8 SpvD protein variants although the nucleotide sequences were highly similar (identity 98.8-100%). The spvD gene products differed from each other in up to 4 amino acid residues only with the exception of the carboxy-terminally truncated SpvD variant of one S. Gallinarum field isolate. The highly conserved primary structure of SpvD in epidemiologically relevant salmonellae suggests that this virulence factor is a promising antigen candidate for diagnostic purposes (i.e. antibody detection in infected animals) but also for immunoprophylaxis in farm animal species.     

Salmonella in poultry flocks and humans--S. enterica subspecies enterica serovar Enteritidis in the past

After importing of breeder lines for laying flocks from Canada into the former GDR in 1966 the egg industry in this country was completely isolated from that in Western Germany or other Western European countries until opening the border in Germany in 1989. Because of this isolation from other countries, an analysis of the clonal diversity of Salmonella (S.) Enteritidis isolates originated from humans, chickens and food in the former GDR during the 1980s would provide a unique opportunity to obtain new insight into factors that may have triggered the S. Enteritidis epidemic. While isolates had previously been typed by the phage typing scheme of Lalko and Laszlo we applied for the first time the extended phage typing scheme by Ward for the retrospective analysis of the S. Enteritidis strains. Furthermore, isolates of phage type (PT) 4/6 (Ward / Lalko and Laszlo) from different livestocks were investigated by ribotyping. Although in total the PT4/6 dominated between 1986 and 1989 in poultry, other phage types have prevailed in the early 1980s and represented a considerable fraction of isolates until 1989. For instance, PT8/7 was isolated from one large layer farm (district Halle) from 1988 until 1989. During that time in another farm (district Cottbus) only PT8/7 was detected too. PT4/6 was isolated from neither of these two laying hen farms. The strains of PT4/6 could be distinguished by ribotyping in 19 different subtypes. The strains from the northern farms were distinct from those isolated in the southern regions. As farms which were harbouring either PT4/6 or PT8/7 had obtained laying hens from the same sources (breeder lines in Deersheim and Spreenhagen) it is highly probable that S. Enteritidis infection was acquired from the environment at each individual farm. This conclusion is also supported by the presence of different PT4/6 ribotypes in different farms. The presence of different phage types or PT4/6 ribotypes at different farms of laying hens suggests that in each case the S. Enteritidis strains present in the environment were able to enter chicken flocks.     

Risk factors for Salmonella enterica subsp. enterica infection in senegalese broiler-chicken flocks

Our objective was to assess the association of managerial practices, general hygiene and Salmonella infection in Senegalese broiler flocks. Seventy broilers farms were studied from January 2000 to December 2001 around Dakar. A questionnaire was submitted to the farmers and samples of fresh broiler droppings were taken. A 28.6% of the flocks were infected by Salmonella (mainly Hadar and Brancaster serovars). Salmonella infection of the previous flock (OR=6.82) and of day-old chicks (OR=3.73), frequent poultry farmers’ visits (OR=5.38) and keeping sick birds inside the farm (OR=5.32) increased the risk of Salmonella infection. But, using antibiotics on day-old chicks (OR=0.17) and a detergent for cleaning (OR=0.16) decreased the risk.     

Serovars of Salmonella from captive reptiles

The distribution on serovars of 60 Salmonella isolates from reptiles kept in captivity in Denmark during the period 1995–2006 was investigated. The isolates were all recovered from clinical specimens submitted to the National Veterinary Institute. A majority of the samples were from reptiles in zoological gardens or similar, while a minor number was from reptiles kept in private homes. A total of 43 serovars were detected, most of them being what is usually called exotic serotypes, and many not having a trivial name, while a few isolates belonged to well‐known human pathogenic serovars, such as S. Enteritidis, S. Typhimurium, S. Bovismorbificans. One isolate was rough and two were non‐typeable. Isolates from turtles belonged to the subspecies enterica, while many isolates from both sauria and snakes belonged to other subspecies. The findings underline the potential zoonotic risk by handling reptiles in zoological garden or other public settings, or keeping pet reptiles in private homes.     

Herd-level risk factors for Salmonella enterica subsp. enterica in U.S. market pigs

Midwest U.S. herds (n = 63) were studied to identify risk factors for harboring Salmonella enterica among slaughter-weight pigs. Samples collected on farms (feces) and at slaughter (distal colonic content, cecal content and ileocolic lymph nodes) were cultured using conventional means. Approximately 15 pigs were studied per herd, for a total of 3754 samples. The proportion of pigs positive in one or more samples was calculated for each herd. Herd characteristics were described by a combination of interview and written survey. Logistic regression was used to detect relationships between the detection of Salmonella and potential herd-level risk factors. The mean individual pig prevalence was 5% for feces, 4% for distal colonic content, 15% for ileocolic lymph nodes, and 17% for cecal contents. One or more Salmonella isolates were detected in at least one sample type in every herd. The five most common serovars were S. Agona, S. Derby, S. Schwarzengrund, S. Typhimurium and S. Senftenberg, with 25 additional serovars detected. Salmonella prevalence estimates were positively correlated among all samples except distal colonic content and ileocolic lymph nodes. Pigs with culture positive fecal samples were at increased odds of being detected positive for each of the slaughter-collected samples examined, namely distal colonic content (OR = 30.5), ileocolic lymph nodes (OR = 12.9) and cecal content (OR = 23.2). Herds with positive fecal sample(s) had increased odds of having positive cecal content (OR > 1.5), distal colonic content (OR = 15.3) and ileocolic lymph nodes (OR = 12.7). Pigs from herds with at least some bowl drinkers had eight-fold higher odds of testing Salmonella positive than did pigs from herds with only nipple drinkers. Pigs from herds with only dry feeders had five-fold higher odds of testing Salmonella positive when compared with pigs from herds with combinations of wet/dry style feeders. Interventions at these two points should be considered when designing growing pig facilities to reduce Salmonella shedding.     

Phenotypic and genotypic differentiation of porcine Salmonella enterica subsp. enterica serovar Derby isolates

Sixty-two Salmonella enterica subsp. enterica serovar Derby isolates from slaughter pigs and meat products isolated in Southern Brazil were analyzed for their genomic relationships and for the presence of antimicrobial resistance genes. Twenty-four S. Derby isolates were indistinguishable by their subtracted restriction fingerprinting (SRF) pattern, XbaI- and BlnI-macrorestriction patterns, phage type, plasmid profile, and resistance pattern. In contrast to the BlnI-macrorestriction patterns, the XbaI-macrorestriction patterns were in good agreement with the results of SRF analysis and phage typing. Among the four phage types detected, PT10 and PT21 were the most common. The combination of all typing methods revealed a great diversity among the S. Derby isolates. All strains carried plasmids and the 60 resistant isolates showed at least tetracycline resistance. The resistance genes found were sul1 and/or sul2 (sulfonamide resistance), aadA2 (streptomycin/spectinomycin resistance), tet(A) (tetracycline resistance), tet(B) (tetracycline/minocycline resistance), bla(TEM) (ampicillin resistance), and dfrA14 (trimethoprim resistance). A correlation of the geno- and phenotypic characteristics with the origin of the isolates revealed a substantial temporal variation in the occurrence of specific S. Derby isolates in different independent pig production lines in Southern Brazil. The large number of resistant isolates underlined the potential risk that S. Derby isolates can pose to human health when they enter the food chain.     

Characterization of Salmonella enterica serovar Typhimurium strains of veterinary origin by molecular typing methods

Twenty-eight strains of Salmonella enterica serovar Typhimurium were characterized by three PCR-based methods. Ten strains harbored type I integrons and two different integron profiles were detected. Typing by amplified fragment length polymorphism (AFLP) resulted in observation of 10 profiles that differed by one to six bands. Salmonella strains were screened for presence of phage genes using a PCR-phage typing; five genes from P22 phage and genes encoding putative virulence factors from phages Gifsy-1, Gifsy-2 and Fels-1 were selected for testing. This set of genes was sufficient for dividing the strains into eight different PCR-phage profiles. Similar grouping of strains was observed in case of all the employed DNA techniques and they corresponded well with the phage type and antimicrobial resistance of the strains. The highest discriminating power was achieved with use of the AFLP, yet the detection of integrons and PCR-phage typing also proved to be valuable in typing the S. Typhimurium strains.     

Salmonella enterica subsp. arizonae-associated abortion in a mare

Abortion in mares can be a financially devastating event to horse owners and the equine industry. In this case report, we describe the gross, histopathological and microbiological findings associated with an abortion caused by Salmonella enterica subsp. arizonae, which should be considered in cases of abortion and metritis. Gross and histopathological evaluation of fetal and placental tissue were consistent with ascending septic placentitis resulting in fetal septicaemia. S. enterica subsp. arizonae was isolated from the placenta, lung and fetal gastric contents. Diagnosis was confirmed by MALDI-TOF MS. To our knowledge, this subspecies of S. enterica has not been previously reported as an isolate in a case of late-term equine abortion. A retrospective search of abortion cases at our institution was done, and no similar cases were identified for comparison. This case demonstrates S. enterica subsp. arizonae, although not previously reported as a cause of abortion in pregnant mares and uncommonly isolated from equids in the literature, may cause late-term abortions in susceptible animals. This pathogen should be considered in cases of abortion outbreaks on a farm and merits investigation of the carrier status of mares within a herd.     

Risk factors associated with Salmonella enterica subsp. enterica contamination of chicken carcases in Senegal

The objective of this study was to identify the risk factors for Salmonella spp. contamination of Senegalese chicken carcases during slaughtering. One hundred and twenty traditional slaughterhouses were studied from January 2000 to December 2002 in and around Dakar. A questionnaire was administered to the slaughterers and samples of breast skin were taken to assess the Salmonella spp. status of chicken carcases. Results showed that 43.3% of the chicken batches were contaminated with Salmonella spp., with Salmonella Hadar and Salmonella Brancaster as the two main serovars. Salmonella spp. contamination of the live birds before slaughtering was related to contamination of the carcases after slaughtering. Feed withdrawal before slaughtering and thorough cleaning and disinfection procedures decreased the risk of Salmonella contamination. One individual worker for each slaughtering stage was also associated with a decreased risk of Salmonella contamination. Using scalding water for plucking increased the risk of contamination. These results will help slaughterers to produce safer products for local consumers.     

Risk factors associated with Salmonella enterica subsp. enterica contamination of chicken carcases in Senegal

This study was to identify the risk factors for Salmonella spp. contamination of Senegalese chicken carcases during slaughtering. One hundred and twenty traditional slaughterhouses were studied from January 2000 to December 2002 in and around Dakar. A questionnaire was answered by the slaughterers, and samples of breast skin were taken to assess the Salmonella status of chicken carcases. Results showed that 43.3% of the batches were contaminated with Salmonella, indicating Salmonella Hadar and Salmonella Brancaster as the two main serovars. Salmonella contamination of the carcases after slaughtering was related to contamination of the live birds. Feed withdrawal before slaughtering and thorough cleaning and disinfection procedures decreased the risk of contamination. One individual worker for each slaughtering stage was also associated with a decreasing risk of contamination. Using scalding water for plucking the chicken carcases increased contamination risk. These results will help slaughters to produce safer products for local consumers.     

Evaluation of stocking density and subtherapeutic chlortetracycline on Salmonella enterica subsp. enterica shedding in growing swine

The objective of this research was to determine the effect of stocking density and inclusion of subtherapeutic chlortetracycline in the diet on Salmonella fecal prevalence and antimicrobial resistance in growing swine. A 2 x 2 factorial design was employed on a privately owned commercial swine farm. Four finisher rooms were included in the study. Two of the rooms received 50 g/tonnes of chlortetracycline in the ration, two rooms received no antimicrobials in the feed. In each room, alternate pens were assigned to either high stocking density (0.60 m2/pig) or low stocking density (0.74 m2/pig). Pigs were placed in the finisher rooms at 10 weeks of age and followed for 6 weeks. Individual fecal samples were collected from the floors of each pen and cultured once weekly. Antimicrobial resistance phenotypes were determined. Data were analyzed using multilevel, multivariable logistic regression. Pigs fed chlortetracycline were at increased odds (OR 6.88, 95% CI 2.77-17.12) to shed Salmonellae. No other associations between treatments (CTC and stocking density) and Salmonella prevalence or reduced susceptibility to antimicrobials were identified. Variance in the odds of a fecal sample to be positive was distributed mostly at the lowest level, the individual fecal sample. The increased risk of shedding associated with inclusion of subtherapeutic chlortetracycline in swine diets is discordant with previous results by our group, suggesting farm or strain specific factors may impact this association. Understanding this risk may provide a potential intervention for controlling Salmonella pre-harvest.     

Distribution and function of plasmids in Salmonella enterica

Plasmids of Salmonella enterica vary in size from 2 to more than 200 kb. The best described group of plasmids are the virulence plasmids (50-100 kb in size) present in serovars Enteritidis, Typhimurium, Dublin, Cholerae-suis, Gallinarum, Pullorum and Abortus-ovis. They all encode spvRABCD genes involved in intra-macrophage survival of Salmonella. Another group of high molecular weight plasmids are plasmids responsible for antibiotic resistance. Since most of these plasmids are conjugative, besides storage of genetic information, they contribute to the spread of genes in bacterial populations. The low molecular weight plasmids are the last group of plasmids found in S. enterica. Some of them have been shown to increase resistance to phage infection due to the presence of restriction modification systems. Despite limited knowledge on their function, their presence or absence is frequently used for strain differentiation in epidemiological studies.     

Herd-level diagnosis for Salmonella enterica subsp. enterica serovar Dublin infection in bovine dairy herds

Herd-level sensitivities of bacteriological and serological methods were compared in 79 bovine dairy herds, recently infected with Salmonella enterica subsp. enterica serovar Dublin. All farms experienced clinical signs of salmonellosis for the first time and had no history of vaccination against salmonellosis. At the start of the study, infection with serovar Dublin was confirmed with at least one positive bacteriologic culture for serovar Dublin from a clinical case (gold standard for herd infection).Bacteriological culture was done on samples of dung-pits, drinking water, bulk-milk filters, and faeces of animals with current or earlier clinical signs of salmonellosis. Blood samples of all animals and bulk-milk samples were tested using an ELISA.Herd-level sensitivity (HSe) of culture of dung-pits, drinking water, bulk-milk filters, and faeces of animals with current or earlier signs of salmonellosis was 45, 5, 7, and 38%, respectively. HSe for serology of all animals was 100%. If blood samples of all calves 4-6 months old were examined, at least one calf was seropositive on 91% of the infected farms. If serology was performed on samples of animals with current or earlier signs of salmonellosis, at least one animal was seropositive on 80% of the infected farms. HSe for bulk-milk samples was 54%. However, if clinical signs of salmonellosis were observed only in lactating animals, sensitivity of bulk-milk serology was 79%.Interesting combinations of methods were the combination of serology of bulk milk with either serology of animals with current or earlier signs of salmonellosis (HSe=91%), or serology of all calves of 4-6 months old (HSe=99%).     

Molecular analysis of Salmonella enterica subsp. enterica serovar Agona isolated from slaughter pigs

Salmonella enterica subsp. enterica (S.) serovar Agona plays an important role in Brazil as causative agent of salmonellosis in food-producing animals - in particular, pigs and poultry - as well as in humans. A total of 45 S. Agona isolates collected from slaughter pigs at three different slaughterhouses in Southern Brazil was investigated in this study for their phenotypic and genotypic relatedness. For this, the antimicrobial susceptibility patterns and the phage types were determined. Molecular analysis included the determination of plasmid profiles as well as the analysis of XbaI- and BlnI-generated macro-restriction patterns. Moreover, a novel typing method called subtracted restriction fingerprinting (SRF) was successfully applied to the S. Agona isolates. Based on all properties determined, a dominant clonal group comprising 33 of the 45 isolates was identified. Members of this group were susceptible to all antimicrobials tested, did not carry plasmids, shared the same phage type and were closely related or even indistinguishable by their EcoRI-PauI SRF patterns as well as their XbaI and BlnI macro-restriction patterns. Members of this clonal group were identified at all 3 slaughterhouses at variable frequencies and originated from pig herds raised in 15 different cities in Southern Brazil which were located up to 450 km apart from each other. Since the S. Agona-carrying slaughter pigs were from various integrated production lines, the results of this study suggest that a specific clonal group of S. Agona had entered numerous pig production lines. This observation supports the requirement for the establishment of monitoring and control programmes in Brazil which should also include molecular techniques to better trace the dissemination of S. Agona and other Salmonella serovars in pigs and other food-producing animals.