Evidence for extensive genetic diversity and substructuring of the Babesia bovis metapopulation.

Babesia bovis is a tick-transmitted haemoprotozoan and a causative agent of bovine babesiosis, a cattle disease that causes significant economic loss in tropical and subtropical regions. A panel of nineteen micro- and minisatellite markers was used to estimate population genetic parameters of eighteen parasite isolates originating from different continents, countries and geographic regions including North America (Mexico, USA), South America (Argentina, Brazil), the Middle East (Israel) and Australia. For eleven of the eighteen isolates, a unique haplotype was inferred suggesting selection of a single genotype by either in vitro cultivation or amplification in splenectomized calves. Furthermore, a high genetic diversity (H = 0.780) over all marker loci was estimated. Linkage disequilibrium was observed in the total study group but also in sample subgroups from the Americas, Brazil, and Israel and Australia. In contrast, corresponding to their more confined geographic origin, samples from Israel and Argentina were each found to be in equilibrium suggestive of random mating and frequent genetic exchange. The genetic differentiation (F(ST)) of the total study group over all nineteen loci was estimated by analysis of variance (Θ) and Nei's estimation of heterozygosity (G(ST')) as 0.296 and 0.312, respectively. Thus, about 30% of the genetic diversity of the parasite population is associated with genetic differences between parasite isolates sampled from the different geographic regions. The pairwise similarity of multilocus genotypes (MLGs) was assessed and a neighbour-joining dendrogram generated. MLGs were found to cluster according to the country/continent of origin of isolates, but did not distinguish the attenuated from the pathogenic parasite state. The distant geographic origin of the isolates studied allows an initial glimpse into the large extent of genetic diversity and differentiation of the B. bovis population on a global scale.

Current advances in detection and treatment of babesiosis.

Babesiosis is a disease with a world-wide distribution affecting many species of mammals principally cattle and man. The major impact occurs in the cattle industry where bovine babesiosis has had a huge economic effect due to loss of meat and beef production of infected animals and death. Nowadays to those costs there must be added the high cost of tick control, disease detection, prevention and treatment. In almost a century and a quarter since the first report of the disease, the truth is: there is no a safe and efficient vaccine available, there are limited chemotherapeutic choices and few low-cost, reliable and fast detection methods. Detection and treatment of babesiosis are important tools to control babesiosis. Microscopy detection methods are still the cheapest and fastest methods used to identify Babesia parasites although their sensitivity and specificity are limited. Newer immunological methods are being developed and they offer faster, more sensitive and more specific options to conventional methods, although the direct immunological diagnoses of parasite antigens in host tissues are still missing. Detection methods based on nucleic acid identification and their amplification are the most sensitive and reliable techniques available today; importantly, most of those methodologies were developed before the genomics and bioinformatics era, which leaves ample room for optimization. For years, babesiosis treatment has been based on the use of very few drugs like imidocarb or diminazene aceturate. Recently, several pharmacological compounds were developed and evaluated, offering new options to control the disease. With the complete sequence of the Babesia bovis genome and the B. bigemina genome project in progress, the post-genomic era brings a new light on the development of diagnosis methods and new chemotherapy targets. In this review, we will present the current advances in detection and treatment of babesiosis in cattle and other animals, with additional reference to several apicomplexan parasites.

The cost of illness of atrial fibrillation: a systematic review of the recent literature.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, its prevalence increasing markedly with age. Atrial fibrillation is strongly associated with increased risk of morbidity, including stroke and thromboembolism. There is growing awareness of the economic burden of AF due to ageing populations and constrained public finances. A systematic review was performed (1990-2009). Cost studies for AF or atrial flutter were included; acute-onset and post-operative AF were excluded. Total, direct, and indirect costs were extracted. Of 875 records retrieved, 37 studies were included. The cost of managing individual AF patients is high. Direct-cost estimates ranged from $2000 to 14,200 per patient-year in the USA and from €450 to 3000 in Europe. This is comparable with other chronic conditions such as diabetes. The direct cost of AF represented 0.9-2.4% of the UK health-care budget in 2000 and had almost doubled over the previous 5 years. Inpatient care accounted for 50-70% of annual direct costs. In the USA, AF hospitalizations alone cost ∼$6.65 billion in 2005. In this first systematic review of the economic burden of AF, hospitalizations consistently represented the major cost driver. Costs and hospitalizations attributable to AF have increased markedly over recent decades and are expected to increase in future due to ageing populations.

The prevalence and abundance of helminth parasites in stray dogs from the city of Queretaro in central Mexico.

The prevalence of helminth species in stray dogs, from the capital city of the state of Queretaro, was evaluated. A total of 378 dogs were captured and examined for the presence of helminths from January to December 2008. The results showed that 275 (72.8%) of examined dogs were infected with one or more helminth species. Single infections were observed in 139 (50.5%) of infected dogs and 136 (49.5%) harboured mixed infections. Out of the 378 dogs examined, 208 (55.2%) presented nematodes and 182 (48.1%) cestodes. The prevalences (confidence interval) and mean intensities of infection ( ± SD) of nematodes and cestodes encountered were: Ancylostoma caninum 42.9% (37.9-47.8) and 22.1 ( ± 34.3); Toxocara canis 15.1% (11.8-19.0) and 8.3 ( ± 15.0); Spirocerca lupi 4.5% (2.7-7.1) and 3.9 ( ± 4.8); Toxascaris leonina 2.3% (1.1-4.5) and 4.8 ( ± 3.5); Physaloptera praeputialis 1.9% (0.8-3.8) and 9.7 ( ± 14.9); Dirofilaria immitis 1.3% (0.4-3.1) and 5.6 ( ± 2.1); Oslerus osleri 0.3% (0.0-1.6) and 5 ( ± 0.0); Dipylidium caninum 44.9% (40.0-50.0) and 18.1 ( ± 27.7); Taenia spp. 6.9% (4.7-9.9) and 6.9 ( ± 7.1). There were no significant differences in prevalences observed either between female (68.5%) and male (76.8%) or between young (70.6%) and adult (74.2%) animals. No differences were observed in the ANOVA test for the mean intensity of infection of any of the parasites (P>0.05).

Characterization of the apical membrane antigen-1 in Italian strains of Babesia bigemina.

Babesia bigemina is a parasite endemic in different parts of the world, including Europe and the Americas. One of the few genes characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a trans-membrane antigen recently identified. In this research, we characterized the ama-1 gene from three Italian B. bigemina strains, two B. bigemina strains obtained from Ragusa, Sicily (ITA1 and ITA3) and a third one obtained from Benevento, Campania (ITA2). Italian sequences were compared with those of the Australian strain obtained from the Sanger Institute web site and to strains from different parts of the world. The results obtained confirmed that this newly described ama-1 gene is highly conserved among Italian and foreign strains which has implications for vaccine development.

In silico predicted conserved B-cell epitopes in the merozoite surface antigen-2 family of B. bovis are neutralization sensitive.

The merozoite surface antigens MSA-2 of Babesia bovis constitute a family of polymorphic GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes. These are therefore putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a(1), a(2), b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with a moderate degree of polymorphism. The present work was aimed at defining neutralization-sensitive B-cell epitopes in the MSA-2 family, that are conserved among different B. bovis geographical isolates. To this end, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis by ClustalW alignments and B-cell epitope prediction algorithms performed on these sequences allowed the identification of several regions containing putative conserved B-cell epitopes. Four peptides representing these regions: (KDYKTMVKFCN from msa-2a(1); YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite extracts in all cases in ELISA tests. In addition, these sera reacted with the surface of merozoites of an Argentine and a Mexican B. bovis strains in immunofluorescence assays, and sera against two of the selected peptides inhibited invasion of erythrocytes by in vitro cultured merozoites. Taken together, the results show that the peptide sequences selected by bioinformatic analysis represent expressed and geographically conserved B. bovis B-cell epitopes that might be strong candidates for development of subunit vaccines.

A new set of molecular markers for the genotyping of Babesia bovis isolates.

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constraint for the development of cattle industries worldwide. The existence of different strains and subpopulations has long been described in this hemoparasite. However, few molecular markers have been reported for strain genotyping and characterization. Minisatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis. In this work we report a set of five molecular markers containing minisatellites that showed a variable degree of polymorphism in several American strains. We have used a bioinformatics approach to search for marker sequences contained in open reading frames. Five genes were chosen and primers were designed in conserved regions flanking the repeat region. Two of the genes were the previously described Bv80/Bb-1 and TRAP. The other three genes were named p200, Antigen 3 and Desmoyokin. Amplification by PCR, sequencing and comparative analysis of 11 strains from Argentina, Brazil, Uruguay, Mexico and USA determined that the tandem repeats varied in number and sequence among the isolates. Genome analysis of the five markers revealed that they were single copy and distributed across the four B. bovis chromosomes. When the new markers were analyzed in an experimental infection, absolute sequence conservation was found, indicating the stability of these markers during the course of infection. These markers were also stable during three syringe passages through calves. The application of this panel of molecular markers could provide new molecular tools for the genotyping of B. bovis isolates and analysis of changes in parasite populations following vaccination.

Molecular characterization of babesia bovis strains using PCR restriction fragment length polymorphism analysis of the msa2-a/b genes.

The merozoite surface antigen-2 (msa-2) family of Babesia bovis is a group of variable genes that share conserved 5' and 3' ends and encode for membrane-anchored glycoproteins that have been postulated as vaccine candidates. In this work, we analyzed the sequences of three of these genes (msa-2a1, a2, and 2b) from two geographically distant strains and detected a certain degree of genotypic diversity that could be exploited to work out new molecular tools for the discrimination of B. bovis field samples. Here we describe a PCR restriction assay that was developed based on this observation and tested on several B. bovis strains and isolates. The results show a strain-specific band pattern in geographically distant isolates, indicating the presence of differentially located BspMI restriction sites. This approach provides a simple method for the differentiation of American B. bovis strains.

Advances in the development of molecular tools for the control of bovine babesiosis in Mexico.

The severe negative impact that bovine babesiosis has in the Mexican cattle industry has not been ameliorated basically due to the lack of safe and effective commercially available vaccines and sensitive and reliable diagnostic tests. In recent years, the Bovine Babesiosis Laboratory at the National Center for Disciplinary Research in Veterinary Parasitology-INIFAP in Morelos State, Mexico has been directing efforts towards three main research areas: (1) The development of in vitro culture-derived, improved and safer live vaccines. This has been done in two ways: using gamma-irradiated bovine serum and erythrocytes for the in vitro culture of vaccine strains, which reduces the risk of contaminating pathogens, and improving the immune response, by the addition of L. casei, a strong stimulant of the innate immune system. (2) The study of antigens considered as vaccine candidates with the goal of developing a recombinant vaccine that suits the country's needs. Knowing their degree of conservation or variation in Mexican isolates, their phylogenetic relationship and their protective, immuno-stimulatory properties, are first steps towards that goal. (3) The development of new tools for diagnosis, detection and discrimination of bovine babesiosis is the third area. Developing variants of ELISA, which are more reliable than the currently used IFAT, are a priority, and finally, taking advantage of the genomes of Babesia bigemina, and B. bovis, we are identifying genes than allow us to discriminate isolates using molecular tools.

Taking advantage of the polymorphism of the MSA-2 family for Babesia bovis strain characterization.

The Merozoite Surface Antigen-2 (MSA-2) family of Babesia bovis is a group of variable genes which share conserved 5' and 3' conserved ends. These genes encode membrane anchored glycoproteins, named MSA-2a1, a2, b and c, which are immunodominant antigens located on the surface of sporozoites and merozoites. In this work, we have analyzed the sequences of the msa-2a1, a2 and 2b genes in two geographically distant strains from Mexico and Argentina and detected a certain degree of genotypic diversity that can be exploited for the development of a new molecular tool for the discrimination of B. bovis field samples. Here, we describe a PCR restriction assay based on the msa2-a1, -a2 and -2b genes of B. bovis. When field strains from Argentina, Mexico and USA were analyzed, the results showed a strain-specific band pattern indicating the presence of differentially located BspMI restriction sites. This approach provides a simple method for the genotyping/strain differentiation of B. bovis field samples.

Insomniac complaints interfere with quality of life but not with absenteeism: respective role of depressive and organic comorbidity.

BACKGROUND AND PURPOSE: Insomnia is common and associated with poor health status and quality of life. We designed a study to evaluate the impact of insomniac complaints with and without comorbidity on health status and absenteeism. PATIENTS AND METHODS: This is a cross-sectional study performed within a 1-year follow up study on a prospective cohort of French employees. Insomniac subjects (n=986) were compared to control subjects (n=584). Insomniacs suffering from self-reported depressive feelings and behavioral and organic sleep complaints were excluded. RESULTS: Subjects with insomniac complaints (whether with mood or behavioral and organic sleep complaints or not) reported poorer quality of life and had a higher absenteeism rate than controls (9.6+/-31 versus 5.8+/-19 days, P<0.01). A logistic regression model adjusting for depressive and behavioral and organic sleep complaints showed that insomniac complaints were no longer predictive of absenteeism. CONCLUSIONS: Insomniac complaints are strongly associated with deterioration in quality of life but not necessarily with higher absenteeism.

The treatment of mice with Lactobacillus casei induces protection against Babesia microti infection.

In this study, we report that administration of Lactobacillus casei confers protection to mice against the intracellular protozoan Babesia microti. Mice treated with L. casei orally or intraperitoneally were inoculated 7 days later with an infectious dose of B. microti. Mice treated with lactobacilli showed significant reduction in the percentage of parasitized erythrocytes (PPE) compared to untreated mice. When mice were inoculated intraperitoneally with L. casei 3 or 0 days before challenge with B. microti, the PPE was significantly lower compared to untreated mice and there were no differences between treated mice and mice immune to B. microti infection. When mice treated with live or dead L. casei were compared to mice inoculated with Freund Complete Adjuvant before a B. microti infection, a significant reduction of PPE was observed. These results show the protective effect of L. casei administered to mice against a B. microti infection and suggest that it might act by stimulating the innate immune system.

Bovine babesiosis and anaplasmosis follow-up on cattle relocated in an endemic area for hemoparasitic diseases.

A multiplex PCR/DNA probe assay was used to monitor Babesia bovis, B. bigemina and Anaplasma marginale infection in cattle introduced to a Boophilus microplus-infested area in Veracruz, Mexico. Eight intact, 18-month-old, cross-bred beef cattle (four naive, Group A; four Babesia species--premunized, Group B) were immediately exposed to ticks after arrival and were clinically monitored from day 6 to day 98 post-exposure (PE) to ticks. Blood sample analysis for DNA detection by the MPCR/DNA probe assay showed that Group A animals were infected with B. bovis from day 11 up to day 22 PE, requiring treatment on days 17-20. Group B animals were detected positive to B. bovis on days 17-20, did not require treatment and remained persistently infected from days 70 to 84 PE. Treatment of Group A animals delayed the infection with B. bigemina. These animals became positive to the parasite on days 63-77 PE. In contrast, Group B animals (untreated) showed B. bigemina infection on days 21-26 and 63-84 PE. One animal was positive for A. marginale infection on days 63-66 PE, the rest of the animals became so on days 80-98 PE. All infected animals required treatment with oxytetracycline. Monitoring the triple hemoparasite infection with the MPCR/DNA probe assay provided important epidemiological information. Thus, precautionary measures can be established when cattle are moved to a babesiosis/anaplasmosis risk area.

Use of a duplex PCR/DNA probe assay to monitor Babesia bovis and Babesia bigemina in cattle during a vaccination trial.

A Duplex Polymerase Chain Reaction (DPCR)/DNA probe assay was used to detect Babesia bovis and B. bigemina DNA in cattle undergoing immunization trials. Blood samples were collected from 15 non-splenectomized, 1-2 years old bulls, inoculated with 1 x 10(7) each of culture-derived B. bovis- and B. bigemina-infected erythrocytes. 15 bulls inoculated with normal erythrocytes served as a control group. All cattle were field exposed to tick-transmitted Babesia 21 days (20 animals, Group I) and 60 days (10 animals, Group II) post-inoculation (PI). After immunization, the DPCR/DNA probe assay detected B. bigemina and B. bovis parasite DNA in all inoculated animals from days 4 to 14 PI. At challenge, B. bovis DNA was detected in all control animals as early as day 8 (Group I), or day 11 (Group II) post-introduction to a tick-infested area. The immunized bulls showed B. bovis positive PCR/DNA probe signals from day 0 (Group II) and day 8 (group I), up to day 32 post-exposure to ticks. Positive B. bigemina signals were detected from day 0 (Group I) and day 8 (Group II), up to day 36 post-exposure to ticks. During challenge, it was not possible to clearly define whether the PCR/DNA probe signals detected in the blood from immunized cattle were a result of amplified DNA from the culture-derived parasites, from the tick-transmitted parasites, or both.

Comparative sensitivity of two tests for the diagnosis of multiple hemoparasite infection of cattle.

The purpose of this study was to evaluate the efficacy of light microscopy (LM) examination of blood smears and a multiplex polymerase chain reaction (MPCR) assay, in terms of their ability to detect cattle experimentally infected with Babesia bovis, Babesia bigemina, and Anaplasma marginale. Blood samples were collected from 32 intact, 1-2 year old, Holstein bulls, previous to and after simultaneous inoculation of culture-derived or field isolates of B. bovis- and B. bigemina-infected erythrocytes. To establish the triple hemoparasite infection, 16 of the bulls were also inoculated with a calf-derived isolate of A. marginale. The results showed that both tests had 100% specificity. In contrast, the sensitivities of the MPCR assay against the LM test were 93.5% and 70.9%; 96.7% and 100%; and 93.8% and 93.8% for B. bovis, B. bigemina, and A. marginale infection, respectively. The advantages and disadvantages of the MPCR assay to differentially diagnose cattle with multiple hemoparasite infection are discussed.