Typhus Group Rickettsia in the North Mexico and the possible artropod vectors (Rickettsia do Grupo tfico no norte

do Mxico e seus possveis vetores)

Ing. Aaron Medina-Sanchez Phd Student and Fellow Laboratory of Rickettsial and Erlichial infectious diseases Research University of Texas Medical Branch. Galveston, Texas. 77550

Laboratorio de Entomologia Medica, Facultad de Biologia, Universidad Autonoma de Nuevo Leon, Monterrey Mexico. Department of Pathology, University Of Texas Medical Branch, Galveston, Texas . (E-mail:medisanch@yahoo.com)

Abstract Epidemic louse borne typhus was a scourge of Mexico from early colonial days until the mid-19th century, and Rocky Mountain spotted fever has caused highly fatal outbreaks in northern Mexico that have been recognized since the 1940s. An outbreak of 96 cases of typhus group rickettsiosis occurred in Montemorelos County in the state of Nuevo Leon in 1997. Thus, we initiated surveillance of the possible vectors of Rickettsia in this region of Mexico. The Universidad Autonoma de Nuevo Leon in collaboration with the University of Texas Medical Branch and the Department of Health of the state of Nuevo Leon analysed the sera of patients suspected to have dengue fever using the technique of immunofluoresence and Western blotting. We observed a substantial prevalence of antibodies to both the typhus and spotted fever groups of rickettsiae. In search for potential etiologic agents, we collected ticks and fleas at two sites in Nuevo Leon and examined them by cell culture and PCR for the genusspecific 17 kDa protein gene. Eighteen percent of the ticks contained rickettsial DNA, and a typhus group rickettsia was isolated from two Amblyomma ticks. Anti-typhus group LPS monoclonal antibody reactivity and ultrastructural characteristics identified a typhus group Rickettsia that is being characterized by DNA sequence analysis of the 17kDa, ompA, ompB and gltA genes. Introduction In 1910 Ricketts traveled to Mexico to investigate an outbreak of typhus and found that typhus was similar to Rocky Mountain spotted fever, and found that is caused by bacterial parasites transmitted by body lice. However, in his close work with typhus tabardillo patients, Ricketts himself became infected and died in Mexico City. Typhus has been considered to be present in Mexico since the sixteenth century. Two million deaths were ascribed to typhus in the epidemic of 1576, and the illness known as cocolixtle and matlazahuatl to the indigenous peoples likely were present prior to the arrival of Europeans. Public health records beginning in 1893 document numerous epidemics and a high incidence and mortality rate. During 1941, 1166 deaths caused by epidemic typhus were reported in Mexico with Nuevo Leon among the most affected states. Previous investigations of ticks as hosts of rickettsiae in Mexico identified spotted fever group rickettsiae presumed to be Rickettsia rickettsii in Rhipicephalus sanguineus and Amblyomma cajennense ticks. More recently in the state of Nuevo Leon, outbreaks of epidemic typhus in the municipality of Galeana in 1963 and of murine typhus in Montemorelos in 1996 were diagnosed based upon serologic and clinicoepidemiologic data. Between 1922 and 1930 and average of 670 typhus deaths were reported annually. Even more typhus deaths were recorded between 1931 and 1940. During 1941 1166 deaths caused by epidemic typhus were reported, and Nuevo Leon was among the more affected states Vector in different state of Mexico. .In 1944 Rhipicephalus sanguineus was identified like a vector of rickettsiosis in the Sonora, Sinaloa and was also reported in San Luis Potosi State. In Veracruz the tick vector of rickettsiosis in 1946 was amblyomma cajennense In 1963 was an outbreak of epidemic typhus in Los Ramones in the municipality of Galeana in Nuevo Leon. More recently between May and June of 1997 there was an outbreak of 96 cases of endemic typhus in Montemorelos, Nuevo Leon was demonstrated by Weil-Felix serology. The most affected age groups were 25-44 years old and females. (SSA, INDRE) Ctenocephalides felis was founded infected with R. felis in the State of Yucatan, and also patient have been confirmed. With this antecedents, and with the cooperation of the department of health of Nuevo Leon and the Universidad Autonoma de Nuevo Leon, we decide to realize a deeper investigation in order to identify the Rickettsiae and the possible arthropod vector in this zone of Mexico. MATERIALS AND METHODS Serology In 2001, the public health department of Nuevo Leon, Mexico, collected sera from febrile patients in Allende and Linares who were suspected to have dengue fever. IFA for IgG antibodies against Rickettsia prowazekii, R. typhi, and R. parkeri was performed as previously described and a titer of 1:64 or higher was considered positive. Positive samples were titrated to a dilution of 1:1024.(2) Western Immunoblotting Rickettsia prowazekii, R. typhi, and R. parkeri were cultivated in Vero cells, purified by 32% Percoll density gradient centrifugation, separated by SDS-PAGE, transferred to nitrocellulose membranes by semi-dry protein blotting, reacted with a 1:100 dilution of sera in blocking buffer, and detected with affinity-purified alkaline phosphatase-labeled goat anti-human IgG diluted 1:5000 in blocking buffer prior to development with BCIP-NBT (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Tick Collection Ticks were collected by flagging vegetation, dry ice capture, and removal from livestock. Some ticks were maintained alive and others stored at 80C. Ticks were

identified in consultation with Dr. Pete Teel, Texas A&M University, College Station, Texas. Each tick was bisected, and placed in pools of halves of five ticks or stored as individual halves. Rickettsial Isolation Live ticks were sterilized by immersion in 70% alcohol for 10 min followed by a bleach solution and finally washed in sterile water, and dried on sterile filter paper in a biosafety class II cabinet. The pools were homogenized in a small Petri dish using a sterile scalpel, and a 200 L volume of tick homogenate was used for shell-vial isolation technique as described previously.(3) Molecular Detection of Rickettsiae DNA from the tick pools and rickettsial isolation cell cultures was extracted by using the Dneasy tissue kit (Qiagen, Chatsworth, Calif.) according to the manufacturers protocols. DNA was subsequently extracted from individual ticks from the positive pools using alkaline hydrolysis.(4) Approximately five microliters (60 ng) of extracted DNA from pools were used as templates for amplification of a 434 bp fragment of the rickettsial 17-kDa protein gene as previously described.(5) Molecular Characterization of Rickettsial Isolate The genes amplified for the phylogenetic analysis were gltA (citrate synthase), ompA and ompB (outer membrane proteins). PCR amplification was performed as described previously.6 These three genes are important molecular targets for taxonomy of rickettsiae. PCR products of the expected size were cloned using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) according to manufacturers protocol. Plasmids containing the DNA inserts of the expected size were sequenced at least three times using an ABI automated sequencer with M13 forward and M13 reverse sequencing primers (Invitrogen, Carlsbad CA). Electron Microscopy A T75 flask with infected Vero cell monolayers was fixed in Itos fixative, post fixed in osmium tetroxide for 1 h, and stained en bloc in 1% uranyl acetate0.1 M maleate buffer (pH 5.2) as described previously.7 Pellets were dehydrated in ethanol, embedded in epoxy resin (Poly/Bed 812), and polymerized at 60C overnight. Ultrathin sections (thickness, 70 nm) were prepared by using a Reichert Ultracut S ultramicrotome, placed on copper grids, stained with uranyl acetate and lead citrate, and examined in a Philips CM 100 and a Philips 201 electron microscope at 60 kV. RESULTS And DISCUSSION A total of 190 ticks in 38 pools of 5 half ticks were tested and all were negative by regular PCR using 17kD1-2 primers, for Nested PCR the results were 9 pools were positive. Phylogenies analysis: The results of the alignment in the nucleotide-nucleotide Blast web page for the gene gltA for a fragment of 1211bp showed a 99% of similarity for R. prowazekii strain Madrid E, same result for the rompB gene with 833 of 838 bp, this still need to do another two repetitions for assure that similarity. For the Gene 17kd the sequences 423 of 425 identities matched for a 99% of similarity. This analysis still need two more repetitions for better credibility. Ultrastructural Analisys: The rickettsia possessed typical bacillary morphology, also presents a surrounded halos characteristics when is free in the cytosol and also possessed a hallmark of the typhus group rickettsia the intracytosolic vacuoles, this vacuoles are present in the rickettsia isolated by shell-vial technique from the tick. Discussion This study indicate us that the possible vector of typhus in this region of mexico, is the tick and not the lice, like we use to think, furthermore the finding of Rickettsia prowazekii in Amblyomma cajennenses, can raise the question of the vectorial capacity of these ticks to transmit the etilogical agent. Also make us believe that the tick was the primary vector of Rickettsia prowazekii that infect a mammal infested with lice and these louse become infected and transmit the disease to more mammals, also considering that the louse, dont survive a infection of Rickettsia prowazekii. So in order to probe all these hypothesis, we need a good animal model that can serve us like a reservoir of Rickettsia prowazekii and observe if exist a transovarical transmission in A. cajennense. Reiss-Gutfreund described isolations of R. prowazekii from Amblyomma variegatum, Hyalomma marginatum rufipes, and H. truncatum in Ethiopia in 1957, 1961, and 1966, and one of these isolates (ZRS strain) was confirmed to be R. prowazekii at the Rocky Mountain Laboratory. (8) Studies of ticks from Ethiopia by Burgdorfer and Philip identified a single A. cohaerens tick that by hemolymph test with antiR. prowazekii serum was determined to contain a typhus group rickettsia that they were not able to isolate.(9) REFERENCES

1. ALCANTARA, V.E., E.G. GALLARDO, C. HONG, et al.. Typhus group rickettsiaeantibodies in rural Mexico. Emerg. Infect. Dis. 10: 549550. 2004 2. ZAVALA-VELAZQUEZ, J.E., J. RUIZ-SOSA, I. VADO-SOLIS, et al.. Serologic study of the prevalence of rickettsiosis in Yucatan: evidence for a prevalent spotted fevergroup rickettsiosis. Am. J. Trop. Med. Hyg. 61: 405408. 3. KELLY, P.J., D. RAOULT & P.R. MASON. 1991. Isolation of spotted fever group rickettsias from triturated ticks using a modification of the centrifugation-shell vial technique.85: 397398. 1999 4. ALEKSEEV, A.N., H.V. DUBININA, I. VAN, et al. Identification of Ehrlichia spp.and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J. Clin.Microbiol. 39: 22372242. . 2001 5. WEBB, L., M. CARL, D.C. MALLOY, et al.. Detection of murine typhus infection in fleas by using the polymerase chain reaction. J. Clin. Microbiol. 28: 530534. 1990 6. BOUYER, D.H., J. STENOS, P. CROCQUET-VALDES, et al.. Rickettsia felis: molecularcharacterization of a new member of the spotted fever group. Int. J. Syst. Evol. Microbiol. 51: 339347. 332 ANNALS NEW YORK ACADEMY OF SCIENCES, 2001 7. ITO, S. & Y. RIKIHISA. Techniques for electron microscopy of rickettsiae. In Rickettsiae and Rickettsial Diseases. W. Burgdorfer & R.L. Anacker, Eds.: 213227. Academic Press. New York, NY. 1981. 8. REISS-GUTFREUND, R.J.. The isolation of Rickettsia prowazekii and mooseri from unusual sources. Am. J. Trop. Med. Hyg. 15: 943949, 1966. 9. BURGDORFER, W., R.A. ORMSBEE, M.L. SCHMIDT, et al.. A search for the epidemic typhus agent in Ethiopian ticks. Bull. World. Health Org. 48: 563569, 1973.

Ficha de confirmao Nome :Ing. Aaron Medina-Sanchez Aceito participar do evento X_ Sim ____ No

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