Control and eventual eradication of Trypanosoma evansi infection in dromedary camels after an episodic outbreak in mainland Spain: an example in a non-endemic area.

In 2008, Trypanosoma evansi was detected on a camel farm in mainland Spain. The animals were isolated, confined in a closed stable, and treated twice with melarsamine (Cymelarsan(®), Merial, Lyon, France) with an interval of 1 month. Clinical and laboratory examinations by means of parasitological, serological, and molecular procedures (polymerase chain reaction (PCR)) were carried out regularly for 6 years. After the treatment, all parasitemic camels were cleared of parasites, and in the seropositive camels a progressive decrease in antibody levels was observed, with complete disappearance of antibodies between 15 and 21 months, except in one animal which showed doubtful Ag-Ab reaction at 21 months post treatment. In the next assessment, 6 months later, the diagnostic tests conducted on all animals had a negative result. The diagnostic and therapeutic tools recently developed against T. evansi will evidence new and alternative approaches after the parasite is detected, particularly if outbreak occurs in geographically localized areas in territories free of the disease.

Cross-sectional study on prevalence of Trypanosoma evansi infection in domestic ruminants in an endemic area of the Canary Islands (Spain).

Trypanosoma evansi is the most widely spread of the pathogenic African trypanosomes of animals. The disease (surra) was first diagnosed in the Canary Islands in a dromedary camel in 1997; thus, a control plan was implemented achieving the eventual eradication of T. evansi from most of the infected areas in the Archipelago. However, a little area remains still infected despite the use of the same control measures. To evaluate possible reservoirs in the area a representative sample of domestic ruminants was examined by serological, parasitological and molecular tests. Of a total of 1228 ruminants assessed, 61 (5%) were serologically positive (7 cattle, 21 goats, 33 sheep), but T. evansi could be demonstrated in none of them. According to FreeCalc assessment, cattle and goat populations would be free from disease; however, the results from sheep are not adequate to conclude that the population would be free from disease. As a conclusion, surveillance must be exercised on ruminant farms in the surroundings of the infected area in order to evaluate the possible extension of the disease and their potential role as reservoirs of T. evansi.

Morphological and biometrical features of Trypanosoma evansi isolates from an outbreak in mainland Spain.

According to several authors, Trypanosoma evansi is a monomorphic trypanosome found exclusively in slender intermediate forms, although additional studies have revealed that many strains present stumpy forms on rare occasions. In a recent T. evansi outbreak in mainland Spain, several atypical forms were observed in blood smear examinations. Molecular procedures were then necessary to confirm the causal agent. Morphological and biometric measures were taken to characterize the different forms of T. evansi. In contrast to published information, the results of this study would indicate that biometrically distinct T. evansi could also be found in the same farm and even in the same animal species. These data could be useful for many trypanosomes endemic areas of the world where molecular methods are not commonly available.

The role of wild rodents in the transmission of Trypanosoma evansi infection in an endemic area of the Canary Islands (Spain).

Trypanosoma evansi was diagnosed for the first time in camels in the Canary Islands in 1997. Several sanitary measures including treatment of infected animals were taken; however, nowadays a little area is still infected. In order to determine possible reservoirs 138 wild rodents were trapped, 64 of them in the infected farms and the remaining 74 in other areas. The captured species were Rattus rattus (24), Rattus norvegicus (69) and Mus musculus domesticus (45). Serological (CATT/T. evansi), parasitological (micro-Hematocrit Centrifugation technique and stained smears) and molecular (PCR) methods for T. evansi and T. lewisi were used as diagnostic methods. None of the examined rodents was positive for T. evansi; 18, however, showed motile trypanosomes at micro-Hematocrit Centrifugation technique and resulted positive for T. lewisi by PCR. The results would suggest that the studied rodent species would not play a relevant role in the epidemiology of T. evansi infection in Canaries.

Antimicrobial susceptibility of Enterococcus strains isolated from poultry faeces.

We have investigated the resistance of Enterococcus isolated from poultry faeces to antibiotics commonly used as therapy of enterococcal infections. Identification was made by the method of Facklam and Collins. Minimal inhibitory concentrations of penicillin, ampicillin, vancomycin and teicoplanin were determined and high level aminoglycoside resistance was investigated. Genes codifying high level aminoglycoside resistance (HLAR) were determined by PCR. Fifty five Enterococcus strains were isolated (63.6% E. faecalis, 12.7% E. mundtii, 9.1% E. faecium, 7.3% E. casseliflavus, 3.7% E. durans and 3.6% E. hirae). None of the strains were resistant to VAN, TEC, P or AM. HLAR was found in 34.5% of strains for SM, 27.3% for KM and 7.3% for GM. The gene for the bifunctional enzyme was found only in one strain, that showed HLAR to GM and KM. Fourteen strains harboured the gene aph(3')-III, being 11 resistant to KM and STR, and three resistant to GM, KM and STR. The remaining six strains showed HLAR to STR, but were negative for the three genes tested by PCR. The gene ant(4'4") was not detected in any of the strains. No unexpected vancomycin resistance was detected. The resistance rates among poultry strains were lower than those found among human strains isolated from hospital patients in recent Canary studies.

Group 1 beta-lactamases of Aeromonas caviae and their resistance to beta-lactam antibiotics.

The contribution of beta-lactamase production to beta-lactam antibiotic resistance was examined in an Aeromonas caviae mutant strain, selected in vitro by cefotaxime and derived from a wild-type strain isolated in our laboratory from crude sewage. Both strains produced beta-lactamase. The mutant strain (AC7m) produced beta-lactamase constitutively, in contrast to the parental strain (AC7), which was inducible by cefoxitin. AC7m was regarded as a mutant from AC7, which over-expressed beta-lactamase. The mutant strain showed a remarkable reduction in sensitivity to most of the beta-lactam antibiotics tested, such as (i) aminopenicillins and their combinations with clavulanic acid and sulbactam, (ii) carboxypenicillins, (iii) ureidopenicillins, and (iv) cephalosporins. This strain remained susceptible to ceftazidime, imipenem, and aztreonam. Isoelectric focusing of sonic extracts revealed that both strains AC7 and AC7m shared a common major beta-lactamase band at pI 6.5. The plasmid DNA assays showed that the beta-lactamases expressed by each A. caviae strain were chromosomally encoded. Based on substrate and inhibitor profiles determined in sonic extracts for AC7 and AC7m, the enzymes displayed on isoelectric focusing at pI 6.5 were assigned to chromosomal Group 1 beta-lactamases. Imipenem would therefore be the appropriate choice for therapy of infections caused by A. caviae beta-lactamase over-expressing mutants.

Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa.

A total of 67 strains of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa were studied. Twenty-two isolates were from healthy dogs (five from hound dogs and 17 from companion dogs) and 45 from dogs suffering otitis externa (14 from hound dogs and 31 from companion dogs). Presumptive identification was attempted using the following tests: production of acetoin, anaerobic utilization of mannitol, acid production from mannitol, presence of beta-galactosidase, and growth on P agar supplemented with different concentrations of acriflavine. Susceptibility of staphylococci to 16 antibiotics was determined. Most effective antibiotics were imipenem, amoxycillin/clavulanic acid, ciprofloxacin, tobramycin, gentamicin and marbofloxacin. Penicillin, ampicillin and polymyxin B showed the lowest activity. There were no significant differences in antimicrobial susceptibility among isolates from healthy dogs and dogs suffering from otitis externa.

Identification and antibiotic resistance of faecal enterococci isolated from water samples.

We have isolated 47 strains of presumptive faecal streptococci from different water samples. Identification was made by the method of Facklam et al. (1989). Antibiotic resistance was studied on Mueller-Hinton Agar. Twelve antibiotics were tested. High-level aminoglycoside resistance (HLAR) and resistance to glycopeptides were studied. Biochemical identification of presumptive faecal streptococci isolates gave the following results: 19 Enterococcus faecalis, 12 E. faecium, 8 E. hirae, 4 E. durans and 4 E. mundtii. E. mundtii is not included among faecal enterococci. None of the strains were resistant to glycopeptides (vancomycin and teicoplanin). Three strains of Enterococci showed HLAR. Two of them were isolated from coastal bathing waters and the other from wastewater. This suggests that water could contribute to spread of HLAR enterococci and it should be a matter of concern for public health authorities.

Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa.

Development of antibiotic resistance in bacteria is a problem of great concern. It is important to establish the convenience of antimicrobial susceptibility tests in animal infections. The aim of this study was to test the susceptibility to antibiotics of Pseudomonas strains isolated from chronic canine otitis externa. We tested 23 strains of Pseudomonas: 19 Ps. aeruginosa, three Ps. fluorescens and one Pseudomonas spp. The most effective antibiotics were tobramycin (100% susceptible), marbofloxacin (91.3%) and ceftazidime (91.3%). Ticarcillin and gentamicin, commonly used for the treatment of otitis externa also showed good results (susceptibility of strains was 86 and 65.2% respectively). Lower susceptibility was found using enrofloxacin (52.1%) probably due to its indiscriminate use. We emphasize the need for a rational policy of antibiotic prescribing in order to prevent the selection of resistant strains.

Aminoglycoside resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from the Canary Islands.

Strains of Pseudomonas aeruginosa resistant to aminoglycoside antibiotics were selected from 152 clinical isolates. We identified two patterns of resistance correlating with the resistance mechanism characterized by changes in permeability, enzymatic modification due to the acetylating enzyme, AAC(6')-II, or a combination of both. We detected enzymatic activity of the phosphorylase enzyme, APH(3'), in all the isolates. We compared the mechanisms of resistance detected by three methods i.e., radioenzymatic assay, phenotype of resistance and DNA probes. The phenotype of resistance was tested using a kit developed by Schering-Plough Corp. Hybridization was made with 18 DNA probes for the most frequent genes encoding for aminoglycoside-modifying enzymes. All isolates with AAC(6') activity hybridized with the aac(6')-Ib probes and to a minor degree, with the aac(6')-IIb probe. None of the isolates showed hybridization with aph(3')-I, aph(3')-II, or aph(3')-III DNA probes. Serotyping of the strains showed that the O:11 serotype was the most frequent one in strains whose resistance was due to the AAC(6') enzyme. The O:6 serotype was associated with changes in permeability. Encoding of the resistance mechanism seemed to be chromosomal in all the strains.

Type I beta-lactamases of Enterobacter cloacae and resistance to beta-lactam antibiotics.

The interaction of type-I beta-lactamases from Enterobacter cloacae with diverse beta-lactam compounds was examined. The ability of penicillin and cefoxitin to induce beta-lactamase production in this strain was assessed. The effect of beta-lactamase inhibitors was measured on beta-lactamase extracts and on intact cells. E. cloacae 78 strain is a stably derepressed mutant showing limited susceptibility to a number of antibiotics except imipenem. Imipenem would therefore be the appropriate choice for therapy of infections caused by stably derepressed mutants of Enterobacter sp. producing type-I beta-lactamases.

Selection of subpopulations resistnat to amikacin and netilmicin of gentamicin-resistant clinical strains of Staphylococcus aureus and Staphylococcus epidermidis.

Recently we have found several strains of Staphylococcus aureus and Staphylococcus epidermidis, which in spite of containing aminoglycoside-modifying enzymes (AMEs) remained susceptible to antibiotics such as netilmicin (NET) and amikacin (AN). Assuming an interest in this agent from a clinical point of view, the aim of this study was to determine if these strains became resistant after prolonged contact with such antibiotics. We found that minimal inhibitory concentrations (MICs) of the bacterial strains not only increased when using these two agents, but also when using other aminoglycosides such as gentamicin (GM), tobramycin (TM), amikacin (AN) and isepamicin (ISE). In order to see the effect of prolonged use of NET on enzyme production, three strains containing AMEs were selected and we could observe an increase in the enzyme levels after successive passages through media containing NET.