bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Vitellogenin Gene Fragment in Boophilus microplus Ticks
Authors: Granjeno‐Colin Gabriela , Hernandez‐Ortiz Ruben , Mosqueda Juan , Estrada‐Mondaca Sandino , Figueroa Julio V. , Garcia‐Vazquez Zeferino
Published: August 2009

The objective of this study was to isolate, clone, and characterize a fragment of the vitellogenin (Vg) gene from a B. microplus tick strain from Mexico. Using cDNA and specific primers, an 1800‐bp fragment was amplified, cloned, and transformed in into E. coli, and then sequenced. Comparative analysis with a previously reported sequence showed 99% identity at both the nucleotide and amino acid level. The predicted amino acid sequence of the Mexican Vg has 6 positive mutations. There is an insertion of an aspartic acid on position 26 and a deletion on position 552 with respect to the reported sequence. There were 11 predicted glycosylation sites conserved in both strains. It is concluded that there is a high sequence homology of Vg in both strains.
Water Buffalos as Carriers of Babesia bovis in Argentina
Authors: Ferreri Lucas , Benitez Daniel , Dominguez Mariana , Rodriguez Anabel , Asenzo Gustavo , Mesplet Maria , Florin‐Christensen Monica , Schnittger Leonhard
Published: August 2009

The tick‐transmitted hemoprotozoan Babesia bovis is a major causative agent of bovine babesiosis, an often fatal disease of cattle. The disease is widespread in the northeastern region of Argentina, where an increasing part of the livestock is composed of water buffalos. Although clinical cases of buffalo babesiosis have not been reported so far, the pathogen‐transmitting tick vector has been occasionally observed by us to be feeding on water buffalos. We therefore set out to examine whether buffalos may constitute a reservoir of the parasite. Competitive enzyme‐linked immunosorbent assay (cELISA) detected B. bovis–specific antibodies in 20% of investigated buffalos (21/103), while direct detection of the pathogen by nested PCR was demonstrated in 34% of the animals (35/103). In one field, more than 60% of investigated animals (22/36) tested positive by nested PCR. These results are discussed in the context of buffalo babesiosis reported in other countries and in view of the currently effected control measures against bovine babesiosis in the region.
Effect of High‐ and Low‐Molecular‐Weight Components of Lactobacillus casei on Resistance against Babesia microti in NIH Mice
Authors: Bautista Carlos R. , Sandoval Alvaro , Aguilar Blanca R.
Published: August 2009

Three assays were carried out in order to assess the effect of low‐ and high‐molecular‐weight components (LMWc and HMWc, respectively) of Lactobacillus casei on the resistance of mice to Babesia microti infection. In the first study, 23 components were identified in a total extract of L. casei by SDS‐PAGE. In the second experiment, 15 components (ranging from 19 to 148 kDa) from L. casei (viable) and 12 other components (from 21 to 148 kDa) from L. casei (dead) were recognized by the sera of immunized mice in the extracts of sonicated L. casei. On the basis of these results, samples of sonicated L. casei were separated by preparative gel electrophoresis, and then those gel fragments containing the HMWc (range 63 to 111 kDa) and LMWc (range 19 to 59 kDa) were cut and eluted to constitute the LMWc and HMWc of L. casei. In the third study, five groups of 6 mice each received the following treatments: PBS in the control (C) group, L. casei LMWc in the LcL group, L. casei HMWc in the LcH group, total sonicated L. casei (LMWc + HMWc) in the LcT group, and viable L. casei in the LcV group. At day 0 each mouse in all groups was challenged with 2 × 104B. microti parasitized erythrocytes. At day 8 after challenge, the average percentage of parasitized erythrocytes ± SEM was significantly higher (P < 0.05) in the C group (11.8 ± 0.9) as compared with LcL (7.7 ± 1.5), LcH (10.3 ± 6.9), LcT (3.6 ± 0.7), and LcV (6.9 ± 1.9) groups. The results suggest that L. casei LMWc can induce an early protective immune response, similar to that generated by LcV in mice, against B. microti infection.
Immunophenotype of Dogs with Subclinical Ehrlichiosis
Authors: Lorente Carmen , Sainz Ángel , Tesouro Miguel A.
Published: August 2009

The purpose of this study was the characterization of the different subsets of lymphocyte cells in dogs with subclinical ehrlichiosis in order to contribute to the knowledge of the immune response developed in the course of this disease. Thirty‐eight dogs with subclinical ehrlichiosis and 15 unaffected dogs from two shelters in the area of Valencia (eastern Spain) were included in the study. The study of lymphocyte populations was made by flow cytometry. Monoclonal antibodies against CD3, CD4, CD8, and CD21 were used. Based on our results, the most common findings of the subclinical phase of canine ehrlichiosis were lymphocytosis, relative neutropenia, and a decrease in the neutrophil/lymphocyte ratio. Lymphocytosis in these dogs was from an increase of T lymphocyte counts. Tc cell counts in dogs with subclinical ehrlichiosis were higher than in healthy dogs. This rise in the number of Tc lymphocytes resulted in a reduced percentage of Th lymphocytes and in a decrease in the CD4/CD8 ratio.
In Vitro Fasciolicide Activity of Some Plant Extracts against Newly Excysted Flukes
Authors: Vera‐Montenegro Yolanda , Ibarra‐Velarde Froylán , Ramírez‐Avila Guillermo , Munguía‐Xochihua Javier
Published: August 2009

The aim of the present study was to evaluate, under in vitro conditions, the activity of 20 plant extracts against newly excysted Fasciola hepatica metacercariae. The plants were selected on the basis of anthelmintic activity, traditional use, availability, solubility, and permanence in the liver. For the in vitro evaluation, 24‐well Nunc‐immunoplates containing RPMI‐1640 culture medium bovine sera plus antibiotics were used. Extract concentrations of 500 and 50 mg/mL were placed in triplicate. Approximately 10 newly excysted flukes were placed in each well, and the plates were incubated in a CO2 incubator at 5% atmosphere for 3 days. In each plate four control wells containing nontreated flukes were included. All procedures were undertaken under an air‐flow cabinet. Efficacy was measured on days 1 and 3 by considering the motility of the live or dead trematodes evaluated with the aid of an inverted microscope. Results indicated that, from all extracts evaluated, Tilia mexicana showed an average efficacy for concentrations of 500 and 50 mg/mL, respectively, of 100 and 100%; Eriobotrya japonica, 100 and 95%; Coffea arabica, 100 and 100%; Crataegus mexicana, 95 and 90%; and Ternstroemia pringlei 100 and 100%. From this preliminary evaluation, we conclude that further and more detailed evaluations at lower concentrations should be undertaken to understand the real efficacy of these plant extracts.
The Caribbean Animal Health Network: New Tools for Harmonization and Reinforcement of Animal Disease Surveillance
Authors: Gongora Victor , Trotman Mark , Thomas Reginald , Max Millien , Zamora Pastor Alfonso , Lepoureau Maria Teresa Frias , Phanord Siméon , Quirico Jocelyn , Douglas Kirk , Pegram Rupert , Martinez Dominique , Petitclerc Martial , Chouin Emilie , Marchal Céline , Chavernac David , Doyen David , Vachiéry Nathalie , Molia Sophie , Hendrikx Pascal , Lefrançois Thierry
Published: August 2009

The Caribbean Animal Health Network (CaribVET) is a collaboration of veterinary services, diagnostic laboratories, research institutes, universities, and regional/international organizations to improve animal health in the Caribbean. New tools were used by the network to develop regional animal health activities: (1) A steering committee, a coordination unit, and working groups on specific diseases or activities were established. The working group on avian influenza used a collaborative Web site to develop a regionally harmonized avian influenza surveillance protocol and performance indicators. (2) A specific network was implemented on West Nile virus (WNV) to describe the WNV status of the Caribbean countries, to perform a technology transfer of WNV diagnostics, and to establish a surveillance system. (3) The CaribVET Web site (http://www.caribvet.net) encompasses information on surveillance systems, diagnostic laboratories, conferences, bibliography, and diseases of major concern in the region. It is a participatory Web site allowing registered users to add or edit information, pages, or data. An online notification system of sanitary information was set up for Guadeloupe to improve knowledge on animal diseases and facilitate early alert.
Defining the Role of Subolesin in Tick Cell Culture by Use of RNA Interference
Authors: Blouin Edmour F. , Manzano‐Roman Raúl , De La Fuente José , Kocan Katherine M.
Published: August 2009

Development of tick vaccines provides new opportunities for control of tick infestations and tick‐borne diseases. Recently, the tick‐protective protein, subolesin, was identified in a cell line derived from Ixodes scapularis by expression library immunization and a mouse model of tick infestations. While subolesin was conserved among ixodid tick species, the biological function of this gene is unknown. Subolesin expression in ticks was silenced by RNA interference (RNAi) to provide information on the gene's function, and silencing of subolesin profoundly impacted tick survival, feeding, and reproduction. In this research we used RNAi in the IDE8 tick cell line to further study the role of subolesin in development of cultured tick cells. The cells were incubated with subolesin double‐stranded (ds)RNA and cell growth was monitored. Incorporation of dsRNA by tick cells was monitored with Cy3‐labeled dsRNA. After 72 h cells were harvested for cell counts, morphology, and for confirmation of gene silencing by reverse transcriptase‐PCR. While the expression of subolesin in treated cells was reduced 80 ± 9% by RNAi as compared with mock‐treated cells, cell growth did not appear to be affected over the 72‐h period. This is the first report of the use of RNAi in tick cell culture. RNAi is a powerful tool for studying tick gene function and will likely contribute to our understanding of the role that tick genes play in cell development and infection with pathogens.
Molecular Cloning, Sequence Analysis, and Immune Recognition of Bm95 from Thai Strains of Rhipicephalus (Boophilus) microplus
Authors: Jittapalapong Sathaporn , Thanasilp Sarawan , Kengradomkit Chanya , Sirinarukmit Teeraphol , Kaewmongkol Gunn , Stich Roger W.
Published: August 2009

The efficacy of existing vaccines against Thai strains of Rhipicephalus (Boophilus) microplus remains to be determined, and these vaccine antigens should be compared to homologues derived from local strains. The purpose of this study was to compare Bm95 from Thai R. microplus to the previously reported sequence from Argentinean ticks. mRNA was isolated from R. microplus midgut samples, and cDNA was amplified with Bm95‐specific primers. The cDNA and protein sequences of Thai Bm95 were 94 and 92% identical, respectively, to Argentinean Bm95. Although the sequence was similar to Argentinean Bm95, there were 45‐amino acid differences among the homologues. Amplicons encoding Bm95 were cloned into pPICZαA and expressed in Pichia pastoris strain KM71H. The recombinant plasmid Bm95 (rBm95) was reactive to bovine hyperimmune serum to Thai R. microplus midgut, indicating that the Thai rBm95 was immunogenic. Further work is needed to compare the efficacies of Thai and Argentinean rBm95 for protection of cattle against infestation with Thai R. microplus.
Emergence and Potential of High‐Throughput and Integrative Approaches in Pathology
Authors: Frutos Roger , Viari Alain , Vachiéry Nathalie , Boyer Frédéric , Lefrançois Thierry , Martinez Dominique
Published: August 2009

In recent years a major revolution has occurred in the analysis and understanding of pathogenesis and host–pathogens/parasite interactions. This revolution has been achieved through the emergence of the high‐throughput integrative approaches used in the “omics” fields—such as genomics, transcriptomics, proteomics, interactomics, and metabolomics. The novelty of these approaches has resulted from the development of high‐throughput apparatus, assisted by the increasing power and software of computers that allow for high‐speed, multifactorial simultaneous analysis of numerous samples. This level of integration allows for in‐depth analysis of mechanisms, pace, and patterns of the evolution and adaptation of pathogens. This evolution from linear to multifactorial approaches has opened new ways of creating and characterizing new vaccines, diagnostic candidates, and drug targets.
Possible Differential Host Tropism in Anaplasma phagocytophilum Strains in the Western United States
Authors: Foley Janet , Nieto Nathan C. , Madigan John , Sykes Jane
Published: August 2009

Despite organization into a single species, Anaplasma phagocytophilum, of bacteria from diverse hosts, there is evidence that different hosts vary in susceptibility to local strains of this bacterial pathogen. In particular, there is evidence that clinical hosts (humans, horses, and dogs) in the western part of the United States may be infected with strains of bacteria that differ from those in the reported wildlife reservoir, the dusky‐footed woodrat. There is a lack of spatial overlap between clinical cases with woodrat exposure, genetic studies show highly distinctive strains in woodrats compared with those from clinical hosts, and experimental studies failed to transfer infection from woodrats into horses. These data suggest that multiple distinct A. phagocytophilum strains could circulate in western U.S. ecosystems. Host barriers to different bacterial strains would suggest the need for careful discrimination of A. phagocytophilum strains and further research to understand the host–pathogen interactions that result in differential outcomes in infection.
Enhancement of the Mexican Bovine Babesiosis Vaccine Efficacy by Using Lactobacillus casei
Authors: Bautista Carlos R. , Alvarez Jesus A. , Mosqueda Juan J. , Falcon Alfonso , Ramos Juan A. , Rojas Carmen , Figueroa Julio V. , Ku Magdalena
Published: August 2009

To evaluate the effect of Lactobacillus casei on the effectiveness of the Mexican bovine babesiosis mixed vaccine, 20 bovines were randomly allocated into four groups of five animals (I, II, III, and IV). At day −2 animals in groups I and II were inoculated with saline solution by intramuscular route (i.m.) and animals in groups III and IV were inoculated with L. casei. At day 0 bovines in groups I and III were inoculated i.m. with bovine normal erythrocytes and animals of groups II and IV were inoculated with the babesiosis vaccine. Twenty‐four days later each bovine was challenged with Babesia bovis‐ and B. bigemina‐infected erythrocytes. The average rectal temperature in groups I and III was higher (P < 0.05) than that in the vaccinated groups after challenge. The average packed cell volume was lower (P < 0.01) in the control groups than in the vaccinated groups. At day 10 after challenge, the average anti‐Babesia antibody level was higher in group IV than in group II. At day 7 after vaccination, the percentage of bovines positive to gamma interferon, as determined by real‐time PCR, was 20, 0, 40, and 80 for groups I, II, III, and IV, respectively. All animals in control groups (I and III) were treated against babesiosis to avoid their death because they showed signs of babesiosis. The results indicate that L. casei, inoculated 2 days before the inoculation of the Mexican bivalent bovine babesiosis vaccine, improves the vaccine's efficiency.
Expression Analysis of Heat Shock Protein 20 and Rhoptry‐associated Protein 1a in Sexual Stages and Kinetes of Babesia bigemina
Authors: Vichido Rodrigo , Falcon Alfonso , Ramos Juan A. , Alvarez Antonio , Figueroa Julio V. , Norimine Junzo , Brown Wendy C. , Castro Luis A. , Mosqueda Juan
Published: August 2009

Heat shock protein 20 (HSP‐20) and rhoptry‐associated protein 1a (RAP‐1a) are two proteins considered as candidates to be included in vaccines or diagnostics methods for the control of bovine babesiosis. It has been hypothesized that both genes have a basic function in the cellular physiology of erythrocyte‐infecting stages; it is not known if they have a functional role in tick stages. The objective of this work was to analyze whether hsp‐20 and rap‐1a are expressed in sexual stages and kinetes of Babesia bigemina. Purified RNA from sexual stages and kinetes was analyzed by reverse transcriptase (RT)‐PCR with specific primers for hsp‐20 or rap‐1a. Expression analysis was carried out using an indirect immunofluorescence test with specific antibodies against HSP‐20 and RAP‐1a. Results obtained by RT‐PCR showed amplicons for hsp‐20 and rap‐1a in sexual stages and kinetes. Positive signals were also detected when sexual stages and kinetes were analyzed with specific antibodies for HSP‐20 and RAP‐1a. The results obtained here confirm the hypothesis that the genes hsp‐20 and rap‐1a from B. bigemina are expressed in sexual stages and kinetes and stress the importance of these proteins in the cellular physiology of tick stages.
In Vitro Nematocidal Activity of Plant Extracts of Mexican Flora against Haemonchus contortus Fourth Larval Stage
Authors: Aguilar Hector Hugo Galicia , De Gives Pedro Mendoza , Sánchez David Osvaldo Salinas , Arellano María Eugenia López , Hernández Enrique Liébano , Aroche Ulises López , Valladares‐Cisneros Guadalupe
Published: August 2009

Fourteen plant extracts were evaluated in vitro against the fourth larval stage of Haemonchus contortus. The plants species used were Tagetes erecta, Argemone mexicana, and Castela tortuosa. The assays were run in 24‐well cell culture plates at room temperature with three replicates. After exposure, aliquots were taken from the corresponding wells and transferred to a microscope for observation. Evaluation criteria were based on the average of live and/or dead larvae. ANOVA test and Tukey test were used to determine significant differences among the treatments. After 96 h, the T. erecta acetonic extract produced 99.7% lethal activity, followed by C. tortuosa hexanic extract (85.8%) and T. erecta methanolic extract (58.3%) (P < 0.0001).
The Little‐known Scenario of Anthelmintic Resistance in Equine Cyathostomes in Italy
Authors: Traversa Donato
Published: August 2009

Cyathostomes (Nematoda, Cyathostominae) cause colic, decreased performance and growth, peripheral edema, and dysorexia in equids. Also, the synchronous emergence of cyathostome larvae encysted in the colon and cecal wall causes larval cyathostominosis, which is characterized by protein‐depriving enteropathy, chronic diarrhea, edema, weight loss, colitis, and may be fatal. In the past few decades, drug resistance in cyathostomes has become widespread, especially for benzimidazoles. Resistance to tetrahydropyrimidines is still confined to a few areas, and the recent reports of reduced efficacy in macrocyclic lactones are of great concern. Although cyathostomes are widely distributed in Italy, with a prevalence up to 100%, information on the presence of drug‐resistant cyathostomes is scarce. The first study investigating drug resistance was conducted on a few farms in 1992 and showed the presence of cyathostome populations resistant to the (pro)benzimidazoles in northern Italy. A second survey was carried out 14 years later, with the focus on farms in central and southern regions. Cyathostomes resistant to fenbendazole and pyrantel pamoate were found, while macrocyclic lactones remained 98–100% effective. Recently, the efficacy of oxibendazole (97%) and moxidectin (100%) in removing fenbendazole‐resistant cyathostomes in Italy has also been demonstrated. These few studies indicate that drug‐resistant cyathostomes are present in multiple regions of Italy, but geographically and numerically broader investigations of farms are necessary. Finally, there is the undoubted need to use the remaining fully effective drugs (such as moxidectin) in a proper way to preserve their efficacy as much as possible.
Cytokine Response in the Intestinal Mucosa of Hamsters Infected with Taenia solium
Authors: Avila Guillermina , Aguilar Laura , Romero‐Valdovinos Mirza , Garcia‐Vazquez Francisco , Flisser Ana
Published: August 2009

Taenia solium grows in experimentally infected hamsters. An inflammatory reaction in the intestinal mucosa surrounding the scolex of the worms is produced. We searched for mRNA of Th1 and Th2 cytokines by in situ hybridization in intestinal biopsies. Hamsters were infected with T. solium cysticerci and necropsied on different days post infection (d.p.i.). Tissue from the small intestine was taken from the area surrounding the tapeworm scolex, fixed, and processed for histology. Antisense probes for the detection of interferon (IFN)‐γ, interleukin (IL)‐4, IL‐5, and IL‐13 were used. Kinetics of each cytokine was defined through detection on specific mRNA by counting the number of positive infected hamsters and of positive cells per 100 enterocytes on different d.p.i. IFN‐γ was detected as of d.p.i. 2; all animals were positive on d.p.i. 4 and 8; and on d.p.i. 16, only 20% were still positive. IL‐13 had a pattern similar to IFN‐γ, but all hamsters remained positive until d.p.i. 16 when the experiment was terminated. IL‐4 was positive in 40% of infected hamsters on d.p.i. 6. On d.p.i. 8, IL‐5 was only detected in 20% but increased to 100% by d.p.i. 16. These data suggest that tapeworms induce a mixed Th1/Th2 response with a polarization toward Th2 at 2 weeks post infection, which may influence the expulsion of worms.
The Effects of Global Change on the Threat of Exotic Arthropods and Arthropod‐borne Pathogens to Livestock in the United States
Authors: George John E.
Published: August 2009

Arthropod‐borne diseases are an important part of the group of foreign animal diseases that command attention from federal, state, and local animal health authorities in the United States because of the potential for adverse local and regional animal health impacts and also because of possible losses of export markets. Diseases of concern are listed by the US Animal Health Association and also by the Office International des Epizooties. Global change is causing the emergence of newly recognized diseases and altering enzootic and epizootic circumstances for known disease problems. Selected examples of arthropod‐borne diseases of importance are discussed in terms of their potential for introduction, spread, and impact on livestock and human health in the United States.
Pharmaceutical Proteins in Plants
Authors: Frutos Roger , Denise Hubert , Vivares Christian , Neuhaus Jean‐Marc , Vitale Sandro , Pedrazzini Emmanuela , Ma Julian , Dix Phil , Gray John , Pezzotti Mario , Conrad Udo , Robinson David
Published: August 2009

Tuberculosis (TB) is a re‐emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle‐Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or “pharming,” is a promising emerging approach, and a zoonosis‐like TB is a good model for investigating the potential of this approach. Pharma‐Planta, a European Commission‐funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy.
Rickettsial Agents in Slovakian Ticks (Acarina, Ixodidae) and Their Ability to Grow in Vero and L929 Cell Lines
Authors: Boldiš V. , Kocianová E. , Štrus J. , Tušek‐Žnidarič M. , Sparagano Olivier A. E. , Štefanidesová K. , Špitalská E.
Published: August 2009

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty‐six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA‐based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR‐positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram‐negative cell wall, and a cytoplasmic membrane.
Toward Prevention of Cowdriosis
Authors: Pedregal Andreia , De Sousa Daniel Ribeiro , Nguyen Hoang Nam , Das Neves Eane Agapito , Lowagie Serge , Marique Thierry , Kagye Nathalie , Guerra Ines Teixeira , Kamba Yvette , Totte Philippe , Vachiéry Nathalie , Lefrançois Thierry , Martinez Dominique , Yourassowsky Catherine , Callens Natacha , Monnom Olivier , Dubois Frank , Wérenne John
Published: August 2009

Mass production of Ehrlichia ruminantium variants from different regions of sub‐Saharan Africa is one of the difficulties that must be overcome in producing a heartwater vaccine. Vaccine productivity can be limited by endogenous induction of interferon (IFN), which inhibits the propagation of Ehrlichia ruminantium (ER) in cell culture. Different kinds of endothelial cells, in which ER multiply efficiently, could be grown in a scalable way in VueLife Teflon bags on Cytodex 3 microcarriers where bead‐to‐bead transfer of cells occurs. The digital holographic microscope designed at the Université Libre de Bruxelles allows detection of the most appropriate time to harvest intracellular microorganisms for vaccine production.
Use of Cymelarsan in Goats Chronically Infected with Trypanosoma evansi
Authors: Gutierrez C. , Corbera J. A. , Bayou K. , Van Gool F.
Published: August 2009

Toxicity and therapeutic trials using Cymelarsan (an arsenical compound) against Trypanosoma evansi infection were carried out using chronically infected goats. For the toxicity trial, 40 goats were divided into four groups of 10 animals each; the first three groups received s.c. injections of 5, 10, and 15 mg/kg bw of Cymelarsan, respectively, and the last one served as control. No systemic reaction was observed in any goat throughout the experiment. For the therapeutic trial, 15 adult female goats were inoculated intravenously with at least 1 × 105T. evansi isolated in the Canary Islands. Six months after inoculation, the animals were treated with Cymelarsan at single dose of 0.3 mg/kg (5 animals), 0.5 mg/kg (5 animals), and 0.625 mg/kg (5 animals). At 4 and 6 weeks after treatment, two goats belonging to 0.3 mg/kg group showed recurrence of trypanosomes. Parasitemia, however, was negative in all animals belonging to 0.5 and 0.625 mg/kg groups until the end of the experiment (6 months after treatment). Thus, it can be concluded that Cymelarsan is a safe trypanocidal drug for goats and that the curative dose is 0.5 mg/kg or above.
Secretome of Animal Trypanosomes
Authors: Holzmuller Philippe , Grébaut Pascal , Peltier Jean‐Benoît , Brizard Jean‐Paul , Perrone Trina , Gonzatti Marisa , Bengaly Zakaria , Rossignol Michel , Aso Pedro María , Vincendeau Philippe , Cuny Gérard , Boulangé Alain , Frutos Roger
Published: August 2009

Animal trypanosomosis is one of the most severe constraints to agricultural development in sub‐Saharan Africa and is also an important disease of livestock in Latin America and Asia. The causative agents are various species of protozoan parasites belonging to the genus Trypanosoma, among which T. congolense and T. evansi are the major pathogenic species. The extracellular position of trypanosomes obliges us to consider both the parasite and its excreted/secreted factors in the course of the physiopathologic process. The advent of proteomics led us to propose a comparative approach of the proteome (i.e., the whole parasite content) and the secretome (i.e., naturally excreted/secreted molecules) of T. congolense and T. evansi with particular attention to common and specific molecules between strains of differing virulence and pathogenicity. The molecular identification of differentially expressed trypanosome molecules correlated with either the virulence process or the pathogenicity will provide new potential molecular targets for improved field diagnosis and chemotherapy of animal trypanosomosis.
Brain Region–Specific Neurodegenerative Profiles Showing the Relative Importance of Amphetamine Dose, Hyperthermia, Seizures, and the Blood–Brain Barrier
Authors: Bowyer John F. , Thomas Monzy , Schmued Larry C. , Ali Syed F.
Published: August 2009

Understanding the neurotoxic effects of acute high‐dose exposures of laboratory animals to methamphetamine (METH) and amphetamine (AMPH) is of relevance to understanding the neurotoxicity incurred in humans from overdose or abuse of these substances. We present recent findings on the neurodegenerative effects of both a single high dose of 40 mg/kg and a 4‐dose exposure to AMPH in the rat. Comparing these results with those we have previously observed in rodents exposed to either AMPH or METH helps further address how dose, hyperthermia, seizures and blood–brain barrier (BBB) disruption interact to produce neurodegeneration. With regard to the 4‐dose paradigm of AMPH exposure in the rat, our recent data, combined with previous findings, clearly show the importance of dose and hyperthermic interactions in producing neurodegeneration. The single high AMPH dose invariably resulted in extreme hyperthermia and brief episodes of clonic–tonic seizure activity in many rats. However, motor behavior indicative of status epilepticus was not observed in rats receiving the 40 mg/kg AMPH, which contrasts with what we have previously seen with 40 mg/kg METH dose in the mouse. This may explain why, unlike the mice given METH, there was minimal BBB disruption in the amygdala of rats. Nonetheless, in some of the surviving rats there was extensive neurodegeneration in the hippocampus and intralaminar and ventromedial/lateral thalamic nuclei. Early BBB disruption was seen in the hippocampus and may play an important role in the subsequent neurodegeneration. The fact that status epilepticus does not occur in rats that have major hippocampal and thalamic degeneration indicates that such damage may also occur in humans exposed to high doses of AMPH or METH in the absence of status epilepticus or prominent motor manifestations of seizure activity.
Addiction and Cognitive Functions
Authors: Spiga Saturnino , Lintas Alessandra , Diana Marco
Published: August 2009

Drug addiction is a compulsive behavioral abnormality. In spite of pharmacologic and psychosocial treatments to reduce or eliminate drug taking, addiction tends to persist over time. Preclinical and human observations have converged on the hypothesis that addiction represents the pathologic deterioration of neural processes that normally serve affective and cognitive functioning. The major elements of persistent compulsive drug use are hypothesized to be molecular and cellular mechanisms that underlie enduring changes in a number of forebrain circuits (involving the ventral striatum and prefrontal cortex) that receive input from midbrain dopamine neurons and are involved in affective and cognitive mechanisms, respectively. Here we review progress in identifying crucial elements useful in understanding the pathophysiology of the disease and its pharmacologic treatment. Pharmacologic targeting of K‐opiate receptors, with their discrete distribution within the dopaminergic system(s), and thus different actions on dopaminoceptive areas, may provide beneficial effects at the affective and cognitive level.
Crucial Role of Acetaldehyde in Alcohol Activation of the Mesolimbic Dopamine System
Authors: Diana Marco , Peana Alessandra Tiziana , Sirca Donatella , Lintas Alessandra , Melis Miriam , Enrico Paolo
Published: August 2009

Ethyl alcohol (EtOH), the main psychoactive ingredient of alcoholic drinks, is widely considered responsible for alcohol abuse and alcoholism through its positive motivational properties, which depend, at least partially, on the activation of the mesolimbic dopaminergic system. On the other hand, acetaldehyde (ACD), EtOH's first metabolite, has been classically considered aversive and useful in the pharmacologic therapy of alcoholics. Here we show that EtOH‐derived ACD is necessary for EtOH‐induced place preference, a preclinical test with high predictive validity for reward liability. We also found that ACD is essential for EtOH‐increased microdialysate dopamine (DA) levels in the nucleus accumbens (NAcc), and that this effect is mimicked by ACD administration to the intraventral tegmental area (VTA). Furthermore,in vitro, ACD enhances VTA DA neuronal firing. Coherently, EtOH‐stimulating properties on DA neurons are prevented by pharmacologic blockade of local catalase: the main metabolic step for biotransformation of EtOH into ACD in the central nervous system. These results providein vivoandin vitroevidence for a key role of ACD in EtOH motivational properties and its activation of the mesolimbic DA system. Additionally, these observations suggest that ACD, by increasing VTA DA neuronal activity, would oppose its well‐known peripherally originating aversive properties. These findings could help in devising new effective pharmacologic therapies in alcoholism.
Pathways of Methamphetamine Toxicity
Authors: Ferrucci Michela , Pasquali Livia , Paparelli Antonio , Ruggieri Stefano , Fornai Francesco
Published: August 2009

Methamphetamine (METH) is a drug of abuse which is neurotoxic for the nigrostriatal system. METH‐induced neurodegeneration involves production of reactive oxygen species, triggering autophagic vacuoles within nigral neurons of chronic abusers of METH. In fact, Cu,Zn‐superoxide dismutase 1 (SOD1) is a critical protein for the neurotoxic effects of METH on DA neurons. Moreover, mutations in the SOD1 gene cause amyotrophic lateral sclerosis, a dramatic neurodegenerative disorder. In the present paper we demonstrate that in G93A transgenic mice, overexpressing the ALS‐linked mutant form of SOD1, surviving motor neurons share common intracellular alterations with METH‐exposed DA neurons. We hypothesize that in mutant SOD1 transgenic mice, a defective autophagy might be responsible for the neurotoxic effects seen with in nigral neurons during METH toxicity.
MPTP‐ but Not Methamphetamine‐Induced Parkinsonism Extends to Catecholamine Neurons in the Gut
Authors: Natale Gianfranco , Kastsiuchenka Olga , Pasquali Livia , Ruggieri Stefano , Paparelli Antonio , Fornai Francesco
Published: August 2009

Methamphetamine (METH) produces nigrostriatal dopamine (DA) loss, partly resembling that which occurs in Parkinson's disease (PD). In PD there is also a marked alteration in the gut. Given the similarities between the central DA denervation produced by METH and PD, in the present study we evaluated the alterations in the gut following upon METH administration. To compare these effects with those occurring in PD, we also administered the parkinsonism‐inducing neurotoxin MPTP. METH and MPTP were administered to mice, and after 7 days we investigated the immunostaining for tyrosine hydroxylase in nervous plexuses. These data indicate that METH did not alter the catecholamine‐containing axons and autonomic neurons, while MPTP markedly reduced these components.
Role of Autophagy during Methamphetamine Neurotoxicity
Authors: Pasquali Livia , Lazzeri Gloria , Isidoro Ciro , Ruggieri Stefano , Paparelli Antonio , Fornai Francesco
Published: August 2009

Methamphetamine causes nigrostriatal denervation and striatal dopamine loss, while sparing nigral cell bodies. Nigral dopamine neurons feature autophagic vacuoles and cytoplasmic α‐synuclein‐, ubiquitin‐ and parkin‐positive inclusion‐like bodies. On that basis, autophagy was considered essential in methamphetamine‐induced neurotoxicity, but its neurotoxic or protective role has never been addressed. Here we review the gap between the descriptive evidence on activation of autophagy and the lack of knowledge about its role during methamphetamine intoxication. Our preliminary findings rule out a detrimental role for autophagy; this represents the first step in understanding the consequence of activation of autophagy in methamphetamine toxicity.
Chronic Low‐Dose Oxidative Stress Induces Caspase‐3‐Dependent PKCδ Proteolytic Activation and Apoptosis in a Cell Culture Model of Dopaminergic Neurodegeneration
Authors: Carvour Martha , Song Chunjuan , Kaul Siddharth , Anantharam Vellareddy , Kanthasamy Anumantha , Kanthasamy Arthi
Published: August 2009

Oxidative stress has been implicated as a key event in the degenerative process of dopaminergic neurons; however, the cellular mechanisms underlying chronic oxidative stress–induced neurodegeneration remain to be established. In this study, N27 cells, a dopaminergic neuronal cell line derived from rat mesencephalon, exposed to low doses of H2O2 (0–30 μM for 12–24 hr) exhibited dose‐ and time‐dependent increases in cytotoxicity and ROS generation. In addition, the H2O2‐induced neurotoxicity was accompanied by increased caspase‐3 activity and PKCδ cleavage. Notably, treatment with antioxidants Trolox and MnTBAP or PKCδ cleavage inhibitor z‐DIPD‐fmk significantly protected against oxidative stress–induced apoptotic cell death. These results demonstrate that the N27 cell line is a useful model for the study of the chronic low‐dose oxidative stress–induced apoptotic cell death cascade and that caspase‐3‐dependent PKCδ proteolytic activation may be important in the apoptotic process in dopaminergic neurons undergoing chronic oxidative insult.
Association Study of the Calcineurin A Gamma Subunit Gene (PPP3CC) and Methamphetamine‐Use Disorder in a Japanese Population
Authors: Kinoshita Y. , Ikeda M. , Ujike H. , Kitajima T. , Yamanouchi Y. , Aleksic B. , Kishi T. , Kawashima K. , Ohkouchi T. , Ozaki N. , Inada T. , Harano M. , Komiyama T. , Hori T. , Yamada M. , Sekine Y. , Iyo M. , Sora I. , Iwata N.
Published: August 2009

Several lines of evidence from animal and genetic analyses showed that the calcineurin A gamma subunit gene (PPP3CC) plays an important role in the pathogenesis of schizophrenia. Moreover, a recent large Japanese case‐control study confirmed the genetic association of PPP3CC with schizophrenia. The symptoms of methamphetamine (MAP)‐induced psychosis are similar to those of schizophrenia, suggesting that PPP3CC is an attractive candidate gene not only for schizophrenia, but also for METH‐related disorders. In this study, we carried out a genetic association study of PPP3CC with MAP‐use disorder in a Japanese population. We selected five haplotype‐tagging SNPs from the aforementioned replication study and genotyped 393 samples (MAP abuse, 128; control, 265). We could not detect a significant association of all tagging SNPs with each condition. In conclusion, our data suggest that PPP3CC does not elevate the risk of MAP‐use disorder in the Japanese population.
A Role for mGluR5 Receptors in Intravenous Methamphetamine Self‐Administration
Authors: Osborne Megan P. H. , Olive M. Foster
Published: August 2009

Selective antagonists of the mGluR5 receptor attenuate rewarding and reinforcing effects of various drugs of abuse, including alcohol, nicotine, and cocaine. However, the ability of mGluR5 antagonists to alter the reinforcing effects of methamphetamine has not yet been explored. In this study, male Sprague‐Dawley rats were trained to perform an operant lever‐pressing task in order to obtain intravenous infusions of methamphetamine (0.2 mg/kg/infusion) or presentation of food pellets on a fixed ratio (FR1) schedule of reinforcement. After stabilization of methamphetamine or food self‐administration, the selective mGluR5 antagonist 3‐[(2‐methyl‐1,3‐thiazol‐4‐yl) ethynyl]pyridine (MTEP; 0.3, 1.0, or 3.0 mg/kg i.p.) or vehicle were administered to the animals in a randomized counterbalanced cross‐over design. MTEP at doses of 1.0 and 3.0 mg/kg significantly reduced methamphetamine self‐administration by 26 and 36%, respectively, but did not alter food reinforcement at any dose tested. These data suggest that mGluR5 receptors are involved in the reinforcing effects of methamphetamine, and that antagonists of this receptor may serve as novel pharmacologic agents for the treatment of addiction to methamphetamine.
Ultrastructural Localization of Neuronal Brain CB2 Cannabinoid Receptors
Authors: Brusco A. , Tagliaferro P. A. , Saez T. , Onaivi E. S.
Published: August 2009

The functional expression of neuronal CB2 cannabinoid receptors (CB2‐Rs) in the brain has been controversial. We and others have now demonstrated that CB2‐Rs are expressed in neurons and glial cells in the brain. However, the subcellular localization of these receptors has not been characterized. In this study we used immunohistochemical electron microscopy to determine the subcellular distribution of CB2‐Rs in two brain regions. Brain sections from the CA1 hippocampal area and substantia nigra were immunostained for CB2‐Rs and analyzed by electron microscopy. In each region immunoperoxidase labeling for CB2‐Rs was detected in neurons as well as in glial and endothelial cells. In neuronal cells, CB2‐R immunoreactivity was observed in somata and large and medium‐sized dendrites. In the soma, the CB2‐R labeling was mainly associated with the rough endoplasmic reticulum and Golgi apparatus, suggesting its endogenous synthesis. In the dendrites, the CB2‐R labeling was observed in the cytoplasm and was associated with the plasma membrane near the area of synaptic contact with axon terminals, indicating a postsynaptic distribution of these receptors. In CB2‐Rs in immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. In the substantia nigra, some unmyelinated axons were immunoreactive for CB2‐Rs, but we rarely found CB2‐R‐labeled axon terminals. These results extend our previous detection of postsynaptic cortical CB2‐Rs and provide additional ultrastructural evidence that CB2‐Rs are mainly postsynaptic in the CA1 area of the hippocampus and substantia nigra. The functional implication of pre‐ and/or postsynaptic localization of CB2‐Rs remains to be determined.
Serotonin (5‐HT) Transporter Ligands Affect Plasma 5‐HT in Rats
Authors: Rothman Richard B. , Zolkowska Dorota , Baumann Michael H.
Published: August 2009

Dual dopamine (DA)/serotonin (5‐HT)–releasing agents are promising candidate medications for stimulant addiction and other disorders. However, certain 5‐HT transporter (SERT) substrates are associated with development of idiopathic pulmonary arterial hypertension (IPAH) and valvular heart disease (VHD). According to the “5‐HT hypothesis,” SERT substrates increase the risk for developing IPAH and VHD by increasing plasma 5‐HT. To test this hypothesis directly, we determined the effects of acute and chronic fenfluramine, and other SERT ligands, on plasma 5‐HT in male rats. For acute treatments, rats received i.v. vehicle or test drug (0.3 and 1.0 mg/kg), and serial blood samples were withdrawn. For chronic treatments, vehicle or test drug was infused via osmotic minipump (3 and 10 mg/kg/d) for 2 weeks. On the last day of infusion, rats received i.v. fenfluramine challenge (1 mg/kg), and serial blood samples were withdrawn. Plasma 5‐HT was measured usingex vivomicrodialysis in whole‐blood samples. Baseline plasma 5‐HT was <1.0 nM. Acute injection of fenfluramine or other SERT substrates caused large (up to 24‐fold) dose‐dependent increases in plasma 5‐HT. Chronic fenfluramine at 3 and 10 mg/kg/d produced 1.7‐ and 3.5‐fold increases in baseline plasma 5‐HT, while chronic fluoxetine had no effect. Chronic infusions of fenfluramine or fluoxetine diminished the ability of acute fenfluramine to elevate dialysate 5‐HT, and both drugs markedly reduced whole‐blood 5‐HT. Acute fenfluramine increases plasma 5‐HT to concentrations that are below the micromolar levels necessary to produce adverse cardiovascular effects. Chronic fenfluramine and fluoxetine have minimal effects on plasma 5‐HT, suggesting that the increased risk for IPAH associated with fenfluramine does not depend upon elevations in plasma 5‐HT.
Prenatal Nicotine and/or Cocaine Differentially Alters Nicotine‐Induced Sensitization in Aging Offspring
Authors: Sobrian Sonya K. , Johnston Matthew , Wright Jewel , Kuhn Daniela , Ameis Kamal
Published: August 2009

Repeated exposure to psychostimulant drugs can result in behavioral sensitization, an amplified response in locomotor activity and stereotypy, which is used to model aspects of drug addiction. The expression of behavioral sensitization, induced by i.p. injections of nicotine once daily for 5 days, was examined in 450‐day‐old male rats exposed prenatally on GD 8–20 to one of the following conditions: (1) low nicotine: 2.5 mg/kg/day nicotine [LN]; (2) high nicotine: 5.0 mg/kg/day nicotine [HN]; (3) low nicotine/high cocaine: 2.5 mg/kg/day nicotine plus 40 mg/kg/day cocaine [LN/HC]; (4) high nicotine/low cocaine: 5.0 mg/kg/day nicotine plus 20 mg/kg/day cocaine [HN/LC]; (5) pair‐fed controls: food intake yoked to HC dams [PF]; and (6) saline controls: daily injections of 0.9% NaCl solution[SAL]. Initial injection of nicotine did not alter activity or stereotypy in comparison to saline injections, with offspring in all prenatal treatment groups showing a desensitization to nicotine. Five consecutive daily nicotine injections resulted in behavioral sensitization in HN and HN/LC prenatal drug groups. Offspring exhibited an increase in horizontal activity that was evident on day 3, and still present after a 1.0 mg/kg i.p. nicotine challenge 72 hours after the last injection (day 8). SAL offspring exhibited attenuated sensitization. In contrast, nicotine sensitization was not seen in the LN, HC/LN, and the PF offspring; activity remained at the level seen after the initial injection of nicotine. Moreover, nicotine significantly reduced total activity in the LN and PF groups in comparison with their saline‐injected counterparts. These data suggest that gestational exposure to high‐dose nicotine, either alone or in combination with cocaine, may carry a greater risk than low‐dose nicotine exposure of stimulant abuse in later life.