Zoonotic approach to Shiga toxin-producing Escherichia coli: integrated analysis of virulence and antimicrobial resistance in ruminants and humans.

In 2014-2016, we conducted a cross-sectional survey in 115 sheep, 104 beef and 82 dairy cattle herds to estimate Shiga toxin-producing Escherichia coli (STEC) prevalence, and collected data on human clinical cases of infection. Isolates were characterised (stx1, stx2, eae, ehxA) and serogroups O157 and O111 identified by PCR, and their antimicrobial resistance (AMR) profiles were determined by broth microdilution. STEC were more frequently isolated from beef cattle herds (63.5%) and sheep flocks (56.5%) than from dairy cattle herds (30.5%) (P < 0.001). A similar but non-significant trend was observed for O157:H7 STEC. In humans, mean annual incidence rate was 1.7 cases/100 000 inhabitants for O157 STEC and 4.7 for non-O157 STEC, but cases concentrated among younger patients. Distribution of virulence genes in STEC strains from ruminants differed from those from human clinical cases. Thus, stx2 was significantly associated with animal STEC isolates (O157 and non-O157), ehxA to ruminant O157 STEC (P = 0.004) and eae to human non-O157 STEC isolates (P < 0.001). Resistance was detected in 21.9% of human and 5.2% of animal O157 STEC isolates, whereas all non-O157 isolates were fully susceptible. In conclusion, STEC were widespread in ruminants, but only some carried virulence genes associated with severe disease in humans; AMR in ruminants was low but profiles were similar to those found in human isolates.

Incidence of ovine abortion by Coxiella burnetii in northern Spain.

The infectious causes of ovine abortion occurring in 148 farms in northern Spain between 1999 and 2003 were investigated. Laboratory analysis included microbiological, serological, pathological and molecular techniques. Border disease was diagnosed in 16% of the flocks, toxoplasmosis in 15%, chlamydiosis in 12%, salmonellosis in 10%, Q fever in 3%, miscellaneous infections in 7% (Yersinia spp., Listeria spp., Brucella spp.), and inflammatory lesions compatible with an infectious cause were seen in 7% of the flock. In an additional 1% of the flocks non-infectious causes were identified, and a diagnosis was not reached in 38% of the flocks. When a PCR retrospective study was carried out to investigate the possible implication of Coxiella burnetii in the cases without diagnosis, including those with inflammatory lesions, the prevalence of this pathogen increased from 3% up to 9% of the flocks, revealing the importance of this zoonotic pathogen as a small-ruminant abortifacient agent. Placenta was the most commonly positive sample, but other fetal tissues were also of value for C. burnetii DNA detection. The present results update information about the situation of abortion in sheep farms in northern Spain, and highlight the relevance of molecular diagnostic tools in routine laboratory analysis of abortions by C. burnetii.

Anaplasmataceae in wild ungulates and carnivores in northern Spain.

Wild vertebrates are essential hosts for tick-borne diseases but data on the prevalence and diversity of Anaplasma spp. in wildlife are scarce. In this study, we used real-time PCR to investigate the distribution of Anaplasma species in spleen samples collected from 625 wild animals (137 cervids, 227 wild boar, and 261 carnivores) in two regions in northern Spain. A first generic real-time PCR assay was used to screen for the presence of Anaplasma spp. followed by a second species-specific multiplex real-time PCR or partial sequencing of the 16S rRNA gene for species identification. Anaplasma phagocytophilum was highly prevalent in cervids (64.2%), but it was absent from wild boar and carnivores. Interestingly, Anaplasma marginale and Anaplasma ovis were not detected in cervids, but Anaplasma centrale was identified in 1 roe deer and 1 red deer, A. bovis in 4 roe deer, and a novel Ehrlichia sp. in one badger. These findings were highly associated with the tick burden identified in the different hosts. Thus, Ixodes ricinus, the recognized vector of A. phagocytophilum in Europe, was the main tick species parasitizing cervids (93.5%, 1674/1791), whereas Dermacentor reticulatus was the most abundant in wild boar (76.1%, 35/46) and Ixodes hexagonus in carnivores (58.4%, 265/454). More investigations are needed to assess the impact of the different Anaplasma species in wildlife and the risk of transmission to domestic animals.

Effect of propylthiouracil treatment on NADPH-cytochrome P450 reductase levels, oxygen consumption and hydroxyl radical formation in liver microsomes from rats fed ethanol or acetone chronically.

The antithyroid drug propylthiouracil (PTU) has been shown previously to reduce hepatic oxygen utilization and to protect the liver from ethanol-induced injury. The present study examined the effect of PTU on hepatic microsomal oxygen consumption and on the activities of NADPH-cytochrome P450 reductase (CYP-reductase) and cytochrome P4502E1 (CYP2E1) in rats receiving ethanol or acetone chronically. Liver microsomes from rats treated with ethanol for 29 days displayed increases in (i) O2 consumption (70%), (ii) hydroxyl radical (.OH) production (49%) and (iii) ethanol oxidation (50%). Microsomal CYP2E1 levels were increased markedly by chronic ethanol administration, while CYP-reductase was affected marginally, but not significantly (P = 0.06). Chronic treatment with acetone for 14 days, produced similar effects, except that .OH production was not enhanced. Administration of PTU (25 mg/kg/day) to ethanol- or acetone-fed rats, for 10 and 14 days, respectively, led to a marked reduction in the levels and activity of CYP-reductase, and to a decrease in the rates of microsomal O2 consumption, .OH production and ethanol oxidation, but did not lower the levels of CYP2E1 or the metabolism of the CYP2E1 substrate N,N-nitrosodimethylamine. These data suggest that the ability of PTU to protect the liver from ethanol-induced injury may be due to a reduction in the levels of CYP-reductase, thereby minimizing the enhancement of microsomal oxygen consumption and free radical generation associated with ethanol-induced CYP2E1 activity.

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR.

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.

Genetic diversity among Campylobacter jejuni isolates from healthy livestock and their links to human isolates in Spain.

This study was aimed at determining the genetic diversity of Campylobacter jejuni from healthy ruminants and poultry, and study by multilocus sequence typing (MLST) their links to human isolates in Spain. MLST analysis of 160 animal isolates generated 45 sequence types (STs, nine of them new to this study), that clustered into 18 clonal complexes (CC) and nine singletons. The 71 isolates from humans generated 28 STs (13 CC plus four singletons). Only 11 STs and nine CCs were shared by humans and animals (particularly from dairy cattle and sheep), mainly corresponding to sporadic cases rather than outbreaks, probably as an adaptation of the general human population to the types commonly circulating in livestock. PCR analysis of the distribution of four virulence-associated genes detected the cdtABC gene cluster in all 160 isolates but with a 700-bp deletion in four of them, and amplified the virB11, cgtB and wlaN genes in 4.7%, 21.3% and 21.9% respectively. A subset of 87 C. jejuni animal isolates analysed using flaA PCR-RFLP, MLST and pulsed-field gel electrophoresis generated 31, 38 and 55 types respectively. The combined typing approach used provided reliable inter-strain relationships, confirming the co-existence of several strains in some farms, but also identifying identical genotypes sampled over a wide temporal span in different environments and hosts. Typing results confirmed a high genetic diversity of C. jejuni in our region and suggested that ruminants are also important sources for human infection. MLST data provided will help to obtain a more comprehensive image of the population structure of C. jejuni and establish reliable source attribution schemes.

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain.

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms.

AIMS: To determine prevalence and strain diversity of thermophilic campylobacters in healthy ruminants and swine. METHODS AND RESULTS: Faecal samples collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country were screened in pools for thermophilic campylobacters. Two hundred and three herds were positive (67.1% dairy cattle, 58.9% beef cattle, 55.0% sheep and 52.9% pig), and species-specific PCR identified Campylobacter jejuni in 20.7% of the herds and Campylobacter coli in 6.4%. Campylobacter coli was isolated from the four production systems and was the most prevalent species in swine, where C. jejuni was not found. Other thermophilic campylobacters were found in all production systems. Four hundred and ninety-three animals from 11 positive herds were individually analysed, detecting significantly higher within-herd prevalences in dairy cattle (66.7%) and swine (57.8%) than in sheep (8.8%) or beef cattle (5.4%). flaA PCR-RFLP and pulsed-field gel electrophoresis analysis of a selection of isolates showed high genetic diversity. CONCLUSIONS: Healthy swine, cattle and sheep are important reservoirs of thermophilic campylobacters of different species and high genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient farm-based intervention measures are needed to reduce risk of infection. Non-C. jejuni/C. coli species should be monitored to investigate their significance for infection.

Simultaneous pair-feeding system for the administration of alcohol-containing liquid diets.

We present studies in which a new, simultaneous pair-feeding system is utilized to administer alcohol-containing liquid diets to rats. In this system, consumption of an alcohol-containing diet by an animal leads to the corresponding delivery of the same amount of control diet to a control animal, which avidly consumes its diet upon presentation. Thus, circadian nutritional pairing is possible, avoiding the long period of fasting to which control animals are exposed, on a daily basis, when the diets are administered by the conventional pair-feeding system with Richter tubes. The new system does not require actual daily measurement of the diet consumed by the ethanol-fed animal and allows the conservation of the diet for prolonged periods, thus not requiring daily diet changes. The feeding system also allows the pair-feeding of more than one simultaneous control and can be adapted to a number of other fluids and to other animals species, when paired delivery of the fluid is desired.

Circulating neutrophils and liver injury in rat models of experimental alcoholic liver disease.

The present study examined the relationship between circulating neutrophils and liver injury in two widely used rat models of chronic ethanol administration. Hematological alterations, liver histopathology, and biochemical indices of liver injury were assessed in rats receiving chronic ethanol by oral liquid diet feeding (Lieber-DeCarli method) or by continuous intragastric infusion (Tsukamoto-French method). Oral administration of ethanol did not affect circulating neutrophil counts, but resulted in minimal liver injury characterized by elevated serum alanine aminotransferase (79%), increased liver mass (15%), and moderate steatosis. In contrast, rats receiving ethanol by continuous intragastric infusion showed an approximately 2-fold increase in circulating neutrophils, and a moderate degree of liver injury, indicated by a 169% elevation of serum alanine aminotransferase and a 2-fold increase in liver mass. Liver biopsies from these rats showed severe steatosis and scattered necrotic hepatocytes, and some neutrophil infiltrates. To determine whether an increase in the number of circulating neutrophils could potentiate liver injury induced by oral ethanol feeding, rats were treated with human recombinant granulocyte colony-stimulating factor at a dose of 100 microg/kg/day (s.c.) for 4 days. Treatment with granulocyte colony-stimulating factor resulted in a 6- to 9-fold increase in circulating neutrophil counts. Nevertheless, this change did not enhance the minor degree of ethanol-induced liver injury in this model. Our results indicate that, whereas neutrophil leukocytosis accompanies more severe manifestations of ethanol hepatotoxicity in rats, this condition per se does not directly induce or exacerbate ethanol-induced liver injury.

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl.

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal beta1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac alpha2,3Gal beta1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.