Animals In Spain Research Articles

Microsporidia Detection and Genotyping Study of Human Pathogenic E. bieneusi in Animals from Spain

Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webeŕs stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination.

Primary and secondary poisoning by anticoagulant rodenticides of non-target animals in Spain

Anticoagulant rodenticide (AR) levels were studied in liver of 401 wild and domestic animals found dead in Spain with evidences of AR poisoning, including 2 species of reptiles (n=2), 42 species of birds (n=271) and 18 species of mammals (n=128). Baits (n=32) were also analyzed to detect the potential use of ARs in their intentional preparation to kill predators. AR residues were detected in 155 (38.7%) of the studied animals and 140 (34.9%) may have died by AR poisoning according to the clinical information, necropsy findings, residue levels and results of other toxicological analysis. Animals considered with sublethal AR exposure had total AR residues (geometric mean with 95% CI) in liver of 0.005 (0.003–0.007)μg/g wet weight (w.w.) and animals diagnosed as dead by AR poisoning had 0.706 (0.473–1.054)μg/g w.w. ARs were detected in 19% of baits illegally prepared to kill predators. In terms of the total incidents studied in our laboratory between 2005 and 2010 (n=1792 animals), confirmed poisonings represented 40.9% of the cases, and 21.1% of these were due to ARs (8.6% of the total sample). Nocturnal raptors (62%) and carnivorous mammals (38%) were amongst the secondary consumers with highest prevalence of AR exposure, especially to second generation ARs (SGARs). On the other hand, granivorous birds showed the highest prevalence of AR exposure (51%), especially to chlorophacinone in a region treated against a vole population peak in 2007. The presence of hemorrhages was significantly associated with AR levels in liver, but some animals (7.2%) with elevated residue levels (>0.2μg/g w.w.) showed no evidence of macroscopic bleeding. The use of accumulative SGARs and the application of baits on surface (i.e. treated grain by spreader machines) should be discontinued in future EU regulations on the use of rodenticides to prevent the poisoning of non-target wildlife species.

Ceratonia siliqua(carob) trees as natural habitat and source of infection byCryptococcus gattiiin the Mediterranean environment

Recent Cryptococcus gattii infections in humans and animals, including several outbreaks in goats, were the basis of this environmental survey in six provinces of Spain. A total of 479 samples from 20 tree species were studied. Cryptococcus gattii was found for the first time in autochthonous Mediterranean trees in Spain. Fourteen isolates of this pathogen were obtained from seven trees of three different species: 12 from carob (Ceratonia siliqua), one from Mediterranean stone pine (Pinus halepensis) and another from eucalyptus (Eucalyptus camaldulensis). All C. gattii isolates were genotype AFLP4/VGI and mating type alpha, and were found to be genetically identical with C. gattii strains isolated from humans and animals in Spain. This supports the hypothesis that these trees may be a natural source for infection of humans and mammals in the Mediterranean area.

Marine planarians (Platyhelminthes: Tricladida: Maricola) from the western Mediterranean Sea and the Cantabrian coast: new records, one new genus, and immunocytochemistry of the nervous system

Marine planarians were collected extensively from the Iberian Peninsula and Italy. As a result we provide new distributional records of six species of marine triclads, including the description of one new genus and species. The study increases substantially our knowledge of the distribution of this group of animals in Spain and reveals that even relatively well studied areas, such as Italy, still yield new species. In addition, we performed immunostaining studies of the nervous system of three of the species, which revealed the detailed organization of the main nerve cords and their branches. In the new species, the lateral nerve branches showed an arrangement that is different from that of the other species.

Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian Peninsula: new records and description of three new species

Little is known about the taxonomy and distribution of terrestrial planarians on the Iberian Peninsula. Few studies have tried to investigate the local diversity of these animals, due to both their lack of economic interest and their low abundance. In this study we have made extensive searches and collections of terrestrial planarians from the Iberian Peninsula, thus gathering new information on their taxonomy and biogeography. The study includes the description of three new species of the genus Microplana, viz. Microplana aixandrei sp. nov., Microplana grazalemica sp. nov., and Microplana gadesensis sp. nov. We present distribution maps summarizing published and new records of land planarians. The present work substantially increases our knowledge on this group of animals in Spain and Portugal and at the same time also evidences the scarcity of data and studies on the biology of these organisms.

Abundance and phenotypic diversity of Escherichia coli isolates with diminished susceptibility to expanded-spectrum cephalosporins in faeces from healthy food animals after slaughter

Antimicrobial resistance (AR) is an increasing phenomenon but its quantitative estimation remains controversial. The classical resistance percentage approach is not well suited to detect either emergence or low levels resistance. One option is to shift the focus from strains to hosts. This approach is applied to test for phenotypic diversity associated with diminished susceptibility to expanded-spectrum cephalosporins (DSESC) in faecal Escherichia coli from healthy food animals in Spain. We performed E. coli enumeration in faecal samples of broilers (82 pooled samples) and pigs (80 pooled samples) at the slaughterhouse level, using Coli-ID plates alone and supplemented with cefotaxime at two levels (1 and 8 μg/ml). Antimicrobial susceptibility of isolates was tested by the agar diffusion method. Clustering was carried out using these numerical values and Ward and UPGMA methods. When using plates supplemented with 1 μg/ml of cefotaxime for DSESC E. coli detection, 93% (76/82) of broiler pooled samples and 36% (29/80) pig pooled samples tested positive. When using 8 μg/ml of cefotaxime, 67% (55/82) of broilers and 13% (10/80) of pigs were positive. Nevertheless, the relative abundance of this phenotype was low in both animal species (range 0–4.3%). Irrespective of the clustering method (Ward or UPGMA), a noticeable phenotypic diversity was detected, especially from the plates containing 1 μg/ml of cefotaxime. We concluded that: (a) E. coli with phenotype DSESC are common in broilers and pigs but are less frequent in pigs, and (b) the host approach is the most appropriate method for antimicrobial resistance assessment when null or very low levels of antimicrobial resistant bacteria are expected.

Detection and characterization of extended-spectrum -lactamases in Salmonella enterica strains of healthy food animals in Spain

To carry out the characterization of the genes encoding extended-spectrum beta-lactamases (ESBLs) and their genetic environments in four expanded-spectrum cephalosporin-resistant Salmonella enterica isolates (serovars: two Virchow, one Enteritidis, one Rissen) recovered during the monitoring programmes performed in Spain by the VAV Network from faecal samples of pigs, poultry and laying hens at the slaughterhouse level. The presence and characterization of ESBL genes as well as their genetic environments in the four S. enterica isolates were investigated by PCR and sequencing. The presence of other resistance genes was also analysed by PCR and sequencing. Three avian S. enterica isolates (two Virchow and one Enteritidis) harboured the bla(CTX-M-9) gene combined with bla(TEM-1b). The bla(CTX-M-9) gene was included in these three isolates in a class 1 integron with the following 5'-->3' structure: integron 1 variable region (dfrA16-aadA2 gene cassettes)-qacEDelta1-sul1-orf513-bla(CTX-M-9)-orf3-like-orf1005. The sul2 gene was also detected in these three bla(CTX-M-9)-containing isolates and tet(A) in one of them. The two serovar Virchow isolates showed an indistinguishable PFGE pattern, although they were recovered from different animal species (broiler and laying hen). A porcine ESBL-positive isolate (serovar Rissen) harboured the bla(SHV-12) gene combined with bla(TEM-1b). This bla(SHV-12)-containing isolate also harboured the tet(A), aadA and sul1 genes. The emergence of ESBL-producing S. enterica isolates among food animals is described for the first time in Spain, with those of the CTX-M group being the predominant ESBLs detected.

Inter-country transfer of Gram-negative organisms carrying the VIM-4 and OXA-58 carbapenem-hydrolysing enzymes

1. Brinas L, Moreno MA, Teshager T et al. Monitoring and characterizationofextended-spectrumb-lactamases inEscherichiacolistrains from healthy and sick animals in Spain in 2003. Antimicrob Agents Chemother 2005; 49: 1262–4. 2. Olesen I, Hasman H, Aarestrup FM. Prevalence of b-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark. Microb Drug Resist 2004; 10: 334–40. 3. Hasman H, Mevius D, Veldman K et al. b-Lactamases among extended-spectrum b-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in the Netherlands. J Antimicrob Chemother 2005; 56: 115–21. 4. Liebana E, Gibbs M, Clouting C et al. Characterization of b-lactamases responsible for resistance to extended-spectrum cephalosporins in Escherichia coli and Salmonella enterica strains from food-producing animals in the United Kingdom. Microb Drug Resist 2004; 10: 1–9. 5. MulveyMR,BryceE,BoydDetal.AmblerclassAextended-spectrum b-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals. Antimicrob Agents Chemother 2004; 48: 1204–14. 6. Yates CM, Brown DJ, Edwards GFS et al. Detection of TEM-52 in Salmonella enterica serovar Enteritidis isolated in Scotland. J Antimicrob Chemother 2004; 53: 407–8.

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: A first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n=207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09-22.99, P<0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002-2003, O.R. 3.69; 95% CI 1.27-11.9, P<0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.

Monitoring and Characterization of Extended-Spectrum β-Lactamases in Escherichia coli Strains from Healthy and Sick Animals in Spain in 2003

Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 beta-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains) in Spain. Twelve of these strains harbored bla(CTX-M) genes and showed unrelated pulsed-field gel electrophoresis patterns.

Phage types and genotypes of shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in spain: identification and characterization of two predominating phage types (PT2 and PT8).

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.

Emerging nalidixic acid and ciprofloxacin resistance in non-typhoidal Salmonella isolated from patients having acute diarrhoeal disease.

BackgroundNon-typhoidal Salmonella are one of the key etiological agents of diarrhoeal disease. The appearance of multiple drug resistance along with resistance to quinolones in this bacterium poses a serious therapeutic problem, We determined the prevalence of nalidixic acid and ciprofloxacin resistance in non-typhoidal Salmonella isolated from faecal samples of patients with acute diarrhoeal disease attending the outpatient and inpatient departments of a hospital in Saudi Arabia during the years 1999 to 2002.MethodsNon-typhoidal Salmonella were isolated from faecal samples. Antimicrobial susceptibility was tested by the disc diffusion test MICs to nalidixic acid and ciprofloxacin were determined by the agar dilution method.ResultsDuring the study period, 524 strains of non-typhoidal Salmonella were isolated Strains belonging to serogroup C1 were the commonest (41.4%) followed by serogroups B and D (15.6% and 14.5%, respectively), Resistance to ampicillin was observed in 22.9% and to trimethoprim/sulfamethoxazole in 18.5% of the strains. Nalidixic acid resistance was encountered in 9.9% and ciprofloxacin resistance in 23% of the strains. Resistance to nalidixic acid significantly increased from 0.1% in 1999 to 5.5% in 2002 (P=0.0007) and ciprofloxacin resistance increased significantly from 0.1% in 1999 to 0.9% in 2002 (P=0.0001). MICs to nalidixic acid and ciprofloxacin were determined among 29 nalidixic acid-resistant strains of non-typhoidal Salmonella isolated during 2002. The MIC was >256 μg/mL to nalidixic acid and 8 to 16 μg/mL to ciprofloxacin.ConclusionThe increasing rates of antimicrobial resistance encountered among non-typhoidal Salmonella necessitate the judicious use of these drugs in humans. Moreover, these findings support the concern that the use of quinolones in animal feed may lead to an increase in resistance and should be restricted.

Intestinal colonization by vanA- or vanB2-containing enterococcal isolates of healthy animals in Spain.

Fecal samples of healthy animals (66 pigs, 22 pets) recovered during 1998 in La Rioja, Spain, were analyzed for vancomycin-resistant enterococci colonization. Vancomycin resistance mechanisms were analyzed by PCR and sequencing. vanA-containing enterococci were detected in 3 of 66 samples (4.5%) and 5 of 22 samples (22.7%) of the pig and pet samples, respectively. Seven unrelated pulsed-field gel electrophoresis (PFGE) patterns were detected among the 8 vanA isolates (7 Enterococcus faecium, 1 E. faecalis). The tet(M) gene was present in all eight vanA enterococcal isolates, while the erm(B) and aac(6')-Ie-aph(2")-Ia genes were detected in 6 and 3 isolates, respectively. Colonization by vanC-1-containing enterococci (E. gallinarum) was demonstrated in 3% and 4.5% of the pig and pet samples. The aac(6')-Ie-aph(2")-Ia, ant(6)-Ia, aph(3')-IIIa, erm(B) and tet(M) genes were identified in one of the E. gallinarum isolates from a pig fecal sample. One vanB2-containing E. hirae strain was detected in the fecal sample of a healthy pig. In this isolate, the vanB2 gene cluster was integrated into the Tn5382-like element, as demonstrated by specific PCRs and sequencing. The tet(M) and erm(B) genes were also detected in this isolate. This is the first report in which a vanB2-containing enterococci is detected in animals and in E. hirae.

Molecular characterization of Cryptosporidium sp. from animals in Spain.

LUIS NAVARRO-i-MARTINEZ, FERNANDO J. BORNAY-LLINARES, CRISTINA RUEDA, CARMEN del AGUILA, ALEXANDRE J. da SILVA, ANA OLEAGA, VICENTE RAMAJO, SOLEDAD FENOY and NORMAN J. PIENIAZEK Universidad Miguel Hernandez de Elche, Alicante, Spain, and Universidad San Pablo-CEU, Madrid, Spain, and Instituto de Recursos Naturales y Agrobiologia-CSIC, Salamanca, Spain, and Centers for Disease Control and Prevention, Atlanta, GA, USA

Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.

Antimicrobial resistance in salmonellae from humans, food and animals in Spain in 1998.

We studied 1710 Salmonella: spp. isolates from human (1051), food (421) and animal (238) sources. They were tested by the disc diffusion method for susceptibility to 12 different antimicrobial agents. The incidence of resistance and multiple resistance (MR) among the salmonella strains of different origins, the relationship between their most frequent serotypes and phage types (PTs) and their antimicrobial resistance patterns were determined. In general, the incidence of resistance and MR was significantly higher in animal isolates than in human and food isolates (P < 0.05). Resistance to each individual drug among the human isolates and food isolates was very similar, with resistance to ampicillin, tetracycline, streptomycin and sulphonamides most frequently observed. MR has remained uncommon in Salmonella enteritidis. Nevertheless, 90% of PT6A of the human isolates and 100% of the food isolates were ampicillin resistant and 80 and 60%, respectively, of the PT1 isolates were nalidixic acid resistant. Salmonella typhimurium was the most multiresistant serotype in the three sample populations and ten different patterns of MR were seen. Almost 100% of the Salmonella hadar isolates, from human and food sources, were resistant. We recommend restriction of the use of antibiotics in veterinary medicine in order to reduce the selection and spread of multiresistant strains.

Predatory Animals in Spain

Predatory Animals in Spain