Horacio Gil

Rickettsia monacensis and Human Disease, Spain

We identified Rickettsia monacensis as a cause of acute tickborne rickettsiosis in 2 humans. Its pathogenic role was assessed by culture and detection of the organism in patients’ blood samples. This finding increases the number of recognized human rickettsial pathogens and expands the known geographic distribution of Mediterranean spotted fever–like cases.

Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia. Interest in this zoonotic pathogen has increased due to its classification as a category A agent of bioterrorism, but little is known about the molecular mechanisms underlying its virulence, and especially what secretion systems and virulence factors are present. In this study, we characterized two genes in the F. tularensis genome, tolC and a gene we term ftlC, whose products have high homology with the Escherichia coli TolC protein. TolC functions as the outer membrane channel component for both type I secretion and multidrug efflux systems. We constructed deletion mutations of these genes in the F. tularensis live vaccine strain by allelic replacement. Deletion of either tolC or ftlC caused increased sensitivity to various antibiotics, detergents, and dyes, indicating both genes are involved in the multidrug resistance machinery of F. tularensis. Complementation of the deletion mutations in trans restored drug resistance. Neither tolC nor ftlC was required for replication of the live vaccine strain in murine bone marrow-derived macrophages. However, deletion of tolC, but not ftlC, caused a significant attenuation of virulence in a mouse model of tularemia that could be complemented by addition of tolC in trans. Thus, tolC is a critical virulence factor of F. tularensis in addition to its role in multidrug resistance, which suggests the presence of a functional type I secretion system.

Presence of pili on the surface of Francisella tularensis.

ABSTRACT Francisella tularensis is a highly infectious gram-negative bacterium with potential for use as a bioweapon. Analysis of the F. tularensis live vaccine strain (LVS) ultrastructure by electron microscopy revealed the presence of long, thin fibers, similar in appearance to type 4 pili. The highly virulent F. tularensis Schu S4 strain was found to contain type 4 pilus genes, and we confirmed that these genes are present and expressed in the LVS.

Progress in control of cystic echinococcosis in La Rioja, Spain: decline in infection prevalences in human and animal hosts and economic costs and benefits

The Autonomous Community of La Rioja is a region in the north of Spain where the mean annual number of surgical cases of cystic echinococcosis (CE) was 50 (19 per 100000 inhabitants) during the years 1984-1987. This high clinical incidence prompted local authorities to implement a control program in 1986, whose methods and results are reported here. Initially, the program consisted in documenting the prevalence of CE in sheep and humans, communicating an awareness of the disease risks among the population, and treating all registered dogs with praziquantel at intervals of 45 days. Stray dogs were collected systematically, euthanized, and their intestines were examined for Echinococcus granulosus infection. Epidemiological data collected during the course of the program demonstrated that the major reservoirs of E. granulosus were the stray dogs, precisely the ones not receiving periodic praziquantel treatment. Therefore, the program emphasis was shifted to targeting critical points in the transmission of E. granulosus, including improved control of stray dogs, echinococcidal treatments of working sheep dogs, providing means for safe disposal of slaughtered sheep offal and safe disposal of dead sheep in sanitary pits. These measures led to a decline in prevalence of E. granulosus in dogs from 7.0% at the beginning of the program to 0.2% in 2000, i.e. a reduction of 97.2%. Prevalence of infection in adult sheep declined from 82.3 to 20.3%, i.e. a reduction of 75.4%, while the mean number of cysts per infected animal decreased from 6.5% to 0.58 (91% reduction). The rate of diagnoses of new cases in humans between these two dates dropped by 78.9%, from 19 to 4 per 100000 population. In terms of economic costs, these reductions were estimated to yield an increasing cumulative cost/benefit balance that was already positive on year 8 of the program (1994), and that reached 1.96 in year 2000.

Tick-Borne Zoonotic Bacteria in Wild and Domestic Small Mammals in Northern Spain

ABSTRACT The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.

Molecular Method for Identification of Rickettsia Species in Clinical and Environmental Samples

ABSTRACT We present a PCR method targeting the 23S-5S internal transcribed spacer coupled with reverse line blotting that allows Rickettsia species detection and identification in a single step. The method is highly sensitive and specific in identifying Rickettsia species from both patient and environmental samples. The generic approach used allowed us to identify new pathogens.

Photochemical inactivation with amotosalen and long-wavelength ultraviolet light of Plasmodium and Babesia in platelet and plasma components.

BACKGROUND: Transfusion‐transmitted cases of malaria and babesiosis have been well documented. Current efforts to screen out contaminated blood products result in component wastage due to the lack of specific detection methods while donor deferral does not always guarantee safe blood products. This study evaluated the efficacy of a photochemical treatment (PCT) method with amotosalen and long‐wavelength ultraviolet light (UVA) to inactivate these agents in red blood cells (RBCs) contaminating platelet (PLT) and plasma components.

Molecular Method for Bartonella Species Identification in Clinical and Environmental Samples

ABSTRACT A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.

Mac-1+ cells are the predominant subset in the early hepatic lesions of mice infected with Francisella tularensis.

ABSTRACT The cell composition of early hepatic lesions of experimental murine tularemia has not been characterized with specific markers. The appearance of multiple granulomatous-necrotic lesions in the liver correlates with a marked increase in the levels of serum alanine transferase and lactate dehydrogenase. Francisella tularensis, detected by specific antibodies, can be first noted by day 1 and becomes associated with the lesions by 5 days postinoculation. These lesions become necrotic, with some evidence of in situ apoptosis. The lesions do not contain B, T, or NK cells. Rather, the lesions are largely composed of two subpopulations of Mac-1+ cells that are associated with the bacteria. Gr-1+ Mac-1+ immature myeloid cells and major histocompatibility complex class II-positive (MHC-II+) Mac-1+ macrophages were the most abundant cell phenotypes found in the granuloma and are likely major contributors in controlling the infection in its early stages. Our findings have shown that there is an early development of hepatic lesions where F. tularensis colocalizes with both Gr-1+ Mac-1+ and MHC-II+ Mac-1+ cells.

Variability of Bartonella Genotypes among Small Mammals in Spain

ABSTRACT In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas—247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)—by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.