Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain.

Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic.

Use of the C3H/He Lyme disease mouse model for the recovery of a Spanish isolate of Borrelia garinii from erythema migrans lesions.

A skin biopsy from a patient with erythema migrans was inoculated into C3H/He mice and into culture medium. A Borrelia garinii strain named Rio1 was isolated from both a direct BSK medium culture and a mouse ear-punch biopsy culture. Inoculating human tissue into mice produced a disease resulting in severe inflammation of the left tibio-tarsal joint, development of perivascular infiltrates as seen in ear-punch biopsies and the spread of spirochaetes along the skin, far from the inoculation site. The isolation of this strain confirms the circulation of this Borrelia species in Spain as a human pathogen, as well as its arthrogenicity in an animal model. The method used to recover strain Rio1 from human tissue is described as rapid and sensitive compared to direct inoculation of tissue into BSK medium.

[Tick-borne recurrent fever. Description of 5 cases]. / Fiebre recurrente transmitida por garrapatas. Descripción de 5 casos.

BACKGROUND: Tick-borne relapsing fever (FRGT) is a worldwide zoonosis. The disease is caused by spirochetes of the genus Borrelia, and is transmitted to man by ticks of the genus Ornithodoros (O. erraticus in Spain). METHODS: 5 FRTG cases attended in our hospital in a 2 years period are described. Diagnosis was established in all cases by demonstration of borreliae in peripheral blood. Three Borrelia strains were isolated from blood of 3 of our patients and they are maintained by passages through mice. RESULTS: High fever, tachycardia and headache were noted in all cases accompanied in 2 of them by hepatosplenomegaly. Three of the patients complained of arthromyalgias or lumbar myalgia. Treatment with doxycicline was curative in all of them. CONCLUSIONS: FRGT incidence in our country is probably underestimated due to the low suspicion of the disease and the specificity of the clinical symptoms. The isolement of the causative Borrelia strain will allow the development of more specific serological tests necessary to establish the prevalence of this disease as well to assess its implication in chronic diseases, as is observed in other borreliosis (i.e. Lyme disease), that might be missed at this moment.

A new Borrelia species isolated from patients with relapsing fever in Spain.

BACKGROUND: Lyme disease and tick-borne relapsing fever are worldwide systemic borrelioses caused by several Borrelia species transmitted by hard ticks (family Ixodidae) and soft ticks (family Argasidae), respectively. A previous seroepidemiological study of Lyme borreliosis showed several serologically reactive patients with clinically atypical presentations, and this discovery led to the hypothesis that some of the cases of Lyme borreliosis had been caused by another borrelia organism. METHODS: Blood from patients in southern Spain who had suspected Lyme disease or relapsing-fever borreliosis was cultured before treatment began. Isolates of Borrelia spp were inoculated into several strains of mice of different ages. The 16S rRNA and flagellin in genes of Borrelia spp were sequenced by PCR and assessed by phylogenetic analyses. FINDINGS: We isolated a species of Borrelia from three patients with relapsing fever and from Ornithodorus spp ticks in southern Spain. This organism (refractory to in-vitro cultivation) caused a relapsing spirochaetaemia with multiple organ involvement in laboratory mice that recreated the human disease. Phylogenetic analysis showed that this organism is a previously unrecognised species. INTERPRETATION: We have discovered a new borrelia pathogen that is closely related to the other tick-borne agents of relapsing fever in Europe and Africa, and which causes a relapsing systemic disease with serological similarities to Lyme borreliosis.

A laboratory model of canine leishmaniasis: the inoculation of dogs with Leishmania infantum promastigotes from midguts of experimentally infected phlebotomine sandflies.

Twenty-five dogs (beagles) were infected with Leishmania infantum by the intradermal inoculation of an estimated 5-8,000 metacyclic promastigotes harvested from the midguts of 320 experimentally infected P. perniciosus. Details are given of the methods of infecting the flies and harvesting the parasites. All dogs developed small, self-healing chancres at the sites of inoculation. Parasites were isolated from lymph nodes, bone marrow or spleen of 21 dogs, 12 of which developed signs of disease and raised IFAT litres to Leishmania. Nine of the 21 remained healthy over a five-year observation period. Six of the nine were shown to have a cell mediated immune response to Leishmania. No parasites were isolated from four of the 25 dogs, two of which had a demonstrable cell mediated immunity and another had low transitory IFAT titres. The fourth had chancres at the sites of inoculation. The results show that dogs can be readily infected with promastigotes from the midguts of sandflies. However, a high proportion develop a cell mediated immunity and show on signs of disease. It is suggested that serological surveys of dogs for canine leishmaniasis reveal neither the true prevalence of infection nor the intensity of transmission. The efficacy of controlling human visceral leishmaniasis caused by L. infantum by destroying seropositive dogs is questioned.

Canine leishmaniasis: clinical, parasitological and entomological follow-up after chemotherapy.

Six naturally infected dogs [two with no signs of leishmaniasis ('asymptomatic'), two with a few signs ('oligosymptomatic') and two with many signs ('polysymptomatic')] were studied before and after chemotherapy. Another two, non-infected dogs were kept as controls. The dogs were studied clinically, haematologically and parasitologically five times over 11 months and their infectivity to sandflies was evaluated before and after the treatment. The 'asymptomatic' dogs were as infective to sandflies as the 'symptomatic' before treatment but all dogs were un-infective for at least a few months following chemotherapy. Treatment led to a temporary improvement in the clinical and biochemical condition of most of the dogs, the symptomatic dogs becoming asymptomatic, but parasitological cure was uncommon after 10 months' follow-up. There was often no correlation between clinical condition, parasitological condition and infectivity to sandflies. Some dogs from both the 'asymptomatic' and 'symptomatic' groups became infective to sandflies several months post-treatment.

Epidemiology of canine leishmaniasis: prevalence, incidence and basic reproduction number calculated from a cross-sectional serological survey on the island of Gozo, Malta.

Assessment of the resilience of canine leishmaniasis to control or, more ambitiously, the effort needed to eradicate infection, requires an estimate of the basic case reproduction number (R0). This paper applies the theoretical results of Hasibeder, Dye & Carpenter (1992) to data from a cross-sectional survey on the Maltese island of Gozo in which dogs of known age, sex and occupation (pet, guard etc) were subjected to three different serological tests for the presence of specific antibody (IFAT, DAT and ELISA). Difficulties in interpreting these test results, and hence of determining the proportion of dogs infected, present the main obstacle to estimating R0: estimates are critically dependent on the choice of threshold separating seropositives from seronegatives. The data do, however, allow a robust comparative analysis of risk which shows that the force of infection experienced by working dogs is about three times higher than that of pet dogs, a degree of non-homogeneous contact which actually has little effect on estimates of R0. We suggest a cautious point estimate of R0 congruent to 11, and comment briefly on its significance for leishmaniasis control.