José A. Orden

Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain

Abstract Faeces samples from 218, 1 to 30-day-old, diarrheic dairy calves in 65 dairy herds were screened for the presence of Cryptosporidium and concurrent infections with rotavirus, coronavirus, F5+ Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1–7, 8–14, 15–21 and 22–30 days. Cryptosporidium infection was detected in 43.8%, 71.9%, 63.2% and 6.9% of the calves in the respective age groups. Significant differences in the detection rate of Cryptosporidium were found between the age group 22–30 days and all other age groups, and between the age group 1–7 days and the age groups 8–14 days and 15–21 days. Cryptosporidium was the only enteropathogen detected in 60 of the 114 (52.6%) diarrheic calves. Concurrent infections with other enteropathogen(s) were detected in 64.3%, 46.3%, 39.5% and 0% of the Cryptosporidium-infected calves in the age groups 1–7, 8–14, 15–21 and 22–30 days, respectively. A significant age-associated decrease in the detection rate of mixed infections (p <0.05) was found. The detection rates of the other enteropathogens considered in calves with Cryptosporidium infection were 87% for rotavirus, 11.1% for coronavirus, 27.8% for F5+ E. coli and 1.8% for Salmonella.

Verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC in 1-30-days-old diarrhoeic dairy calves

Faecal samples from 221, 1-30-days-old, diarrhoeic dairy calves were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC. Calves were grouped according to their age (1-7, 8-14, 15-21 and 22-30 days) and analyses of prevalences were done by Mantel-Haenzsel chi 2-test for trend. VTEC and eae-positive non-VTEC were detected in 20 (9.0%) and 18 (8.1%) of the diarrhoeic calves, respectively. A significant age-associated increase in the prevalence of VTEC (p = 0.0001), but not in the prevalence of eae-positive non-VTEC (p = 0.381), was found. Significant differences in VTEC prevalence were found between the age-group 22-30 days and in all other age-groups. 43 (5.0%) of the 861 E. coli isolates from the 221 diarrhoeic calves were VTEC, and 30 (69.8%) of these strains produced VT1 only. More than one-half of the VTEC strains (55.8%) were positive for the eae gene and all these eae-positive VTEC strains produced VT1 only. A high percentage (76.7%) of VTEC strains belonged to E. coli serogroups (O4, O26, O39, O91, O113, O128 and O145) associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. 51 (5.9%) of the E. coli strains studied were eae-positive non-VTEC and the serogroups most prevalent among these strains were O4, O14, O26 and O123. Only four of the eae-positive strains were also espB-positive by hybridization with a probe from a human EPEC isolate and none of these strains produced VT.

Rotavirus and concurrent infections with other enteropathogens in neonatal diarrheic dairy calves in Spain

Abstract Faeces samples from 218, one to 30 days old, diarrheic dairy calves in 65 dairy herds were screened for the presence of rotavirus and concurrent infections with coronavirus, Cryptosporidium, F5+ Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1–7, 8–14, 15–21 and 22–30 days. Rotavirus infection was detected in 46.9%, 45.6%, 33.8% and 48.3% of the calves in the respective age-groups. No significant differences in the detection rate of rotavirus were found among calves on the different age-groups. Rotavirus was the only enteropathogen detected in 39 of the 93 (41.9%) diarrheic calves positive to this agent. Concurrent infections with other enteropathogen(s) were detected in 31.3%, 33.3%, 20.6% and 3.4% of the rotavirus infected calves in the age-groups 1–7, 8–14, 15–21 and 22–30 d, respectively. A significant age-associated decrease in the detection rate of mixed infections (p<0.01) was found. The detection rates of the other enteropathogens considered in calves with rotavirus infection were 20.4% for coronavirus, 85.2% for Cryptosporidium, 16.7% for F5+ E. coli and 1.8% for Salmonella.

Verotoxin-producing Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) isolated from healthy cattle in Spain

Aims: To determine the prevalence and characteristics of verotoxigenic Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) in healthy cattle.

Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids

Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus syrian hamster model

Several animal models have been established to study visceral leishmaniosis (VL), a worldwide vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem. BALB/c mice and Syrian hamsters are the most widely used experimental models. In this paper, we summarize the advantages and disadvantages of these two experimental models and discuss the results obtained using these models in different studies of VL. Studies using the BALB/c mouse model have underscored differences between the liver and spleen in the course of VL, indicating that pathological evaluation of the visceral organs is essential for understanding the immune mechanisms induced by Leishmania infantum infection. The main goal of this review is to collate the relevant literature on Leishmania pathogenesis into a sequence of events, providing a schematic view of the main components of adaptive and innate immunity in the liver and spleen after experimental infection with L. infantum or L. donovani. This review also presents several viewpoints and reflections about some controversial aspects of Leishmania research, including the choice of experimental model, route of administration, inoculum size and the relevance of pathology (intimately linked to parasite persistence): a thorough understanding of which is essential for future VL research and the successful development of efficient control strategies for Leishmania spp.

Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants.

The types of the eae and espB genes of 178 attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic and healthy ruminants were investigated by PCR. Six types of the eae gene: beta (beta), gamma1 (gamma-1), gamma2 (gamma-2), epsilon (epsilon), zeta (zeta) and iota (iota), and three types of the espB gene: alpha, beta and gamma were identified in the strains studied. Moreover, three strains were negative to all the types of the eae gene tested. The types beta and gamma2 in healthy cattle, beta, gamma2 and epsilon in healthy sheep and goats, and beta in diarrhoeic calves, lambs and goat kids were the most frequent types of the eae gene among the strains studied. Although the eaebeta gene was the most prevalent among AEEC from healthy and diarrhoeic ruminants, the percentages of AEEC strains with this type found in this study in diarrhoeic animals (66.7-100%) were higher than those found in healthy animals (33.3-40.6%). Thus, these data suggest that AEEC strains with the eaebeta gene are associated with neonatal diarrhoea in ruminants. The eaegamma1, eaezeta and eaeiota genes were found in low percentages in the strains studied (4.5, 2.8 and 7.3%, respectively). All the types of the eae gene, except the type iota, showed a close correlation with the types of the espB gene: the eaebeta and eae epsilon genes with the espBbeta gene, the eaegamma2 and eaezeta genes with the espBalpha gene and the eaegamma1 gene with the espBgamma gene.

Prevalence and characterization of Vero cytotoxin-producing Escherichia coli isolated from diarrhoeic and healthy sheep and goats

Faecal samples from 146 diarrhoeic lambs and goat kids, and from 511 healthy sheep and goats were screened for the presence of Vero cytotoxin-producing Escherichia coli (VTEC). In healthy sheep and goats, VTEC were isolated in 24.4 and 16.2% of the animals, respectively. Moreover, VTEC were detected in 3.1 and 5.9% of the diarrhoeic lambs and goat kids, respectively. These data suggest that VTEC seems not to be associated with diarrhoea in lambs and goat kids. Only four VTEC strains were eae-positive. The absence of the eae gene in most of these VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, almost half (42.9%) and 12.2% of VTEC strains isolated from healthy sheep and goats, respectively, belonged to serotypes associated with severe diseases in humans.

Catalase deficiency in Staphylococcus aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene

Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified Staphylococcus aureus catalase were used to locate the S. aureus and S. aureus subsp. anaerobius kat regions by PCR. Southern hybridization analysis with a probe derived from a 1.1 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the S. aureus and S. aureus subsp. anaerobius chromosome. The nucleotide sequence of S. aureus katA revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas S. aureus subsp. anaerobius katB is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52 584 Da. These catalases are highly homologous to typical monofunctional catalases from prokaryotes. The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in S. aureus KatA. Escherichia coli cells carrying cloned katA had a catalase activity approximately 1000 times that of untransformed E. coli, but no detectable increase in catalase activity was observed with E. coli carrying cloned katB. Northern blotting showed the presence of a kat-specific transcript in S. aureus subsp. anaerobius, suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration. Compared to the nucleotide sequence of katA, katB showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other S. aureus subsp. anaerobius strains analysed. The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA. Moreover, four of the mis-sense mutations present in katB lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA-->Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site. Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in S. aureus subsp. anaerobius, as deduced from results obtained with chimeric catalase constructs.

Prevalence and characteristics of necrotoxigenic Escherichia coli (NTEC) strains isolated from diarrhoeic dairy calves.

Fecal samples from 246, 1-90-days old diarrhoeic dairy calves in 72 herds were screened for the presence of cytotoxic necrotizing factors (CNF)-producing Escherichia coli (NTEC). NTEC were detected by tissue culture assays and PCR in 39 (15.8%) of the diarrheic calves, and the majority of these animals (34 of 39, ca. 87.2%) were infected by NTEC producing CNF2. Calves were grouped according to their age (1-7 days, 8-14 days, 15-21 days, 22-30 days and 31-90 days) and analyses of prevalence were done by the Mantel-Haenzsel chi2-test for trend. A significant age-associated increase in the prevalence of NTEC producing CNF2 (p<0.0001) was found. Eighty-one (8.4%) of the 958 E. coli isolates from the 246 diarrheic calves were positive for CNF in the tissue culture assays. These strains were analyzed by PCR and this technique showed that three (3.7%) strains were CNF1-positive and 75 (92.6%) were CNF2-positive. Moreover, three of the strains positive in the tissue culture assays were negative by PCR. These strains were subsequently assayed in several biological tests (rabbit skin test, mouse intraperitoneal test and mouse footpad test) which showed that they were really NTEC, probably producing CNF2, but with some different properties to classical strains producing CNF2. NTEC strains producing CNF2 belonged to different serogroups (O2, O7, O9, O14, O15, O41, O43, O45, O55, O76, O86, O88, O109, O115, O123, O128, O153 and O159) than strains producing CNF1 (O11 and O32) or PCR-negative strains (O111). Moreover, a strong association between CNF2 and F17 fimbriae was found (78.6% of CNF2-positive strains were F17-positive, whereas only 22.9% of CNF2-negative strains were F17-positive).