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 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).

Determination of the maximum tolerated dose and the safety index of an experimental fasciolicide in cattle.

The aim of the present study was to determine the maximum tolerated dose (MTD) and the safety index (SI) of 5-chloro-2-methylthio-6-(1-napthyloxy)1H-benzimidazole, called compound alpha, in cattle. In addition, to search for possible adverse effects after treatment, the measurement of some biochemical, haematological and physiological parameters were also analysed. Eighteen crossbred heifers were divided into six groups of three animals each. Groups 1-5 received a single oral dose of 12, 36, 60, 120 and 180 mg/kg of body weight (bw) of compound alpha. Group 6 served as an untreated control. To determine the biochemical, haematological and enzymatic parameters, sera and blood samples were individually taken at 0, 4, 8, 16, 32, 128, and 720 h after treatment. Physiological parameters such as rectal temperature, heart rate (HR), respiration rate (RR) and ruminal motility were measured at the time intervals mentioned above. Estimation of the MTD and SI was obtained by using the formula reported by the Food and Drug Administration (FDA), the results showing an MTD of 180 mg/kg/bw and an SI of 15 times the recommended clinical dose. Some statistical differences were observed in a few of the biochemical, haematological and enzymatic parameters, the adverse effects being not highly representative. Alterations on HR and RR were statistically different (P<0.05) only in heifers treated with 180 mg.

Cloned lines of Babesia bovis differ in their ability to induce cerebral babesiosis in cattle.

Clones of a Babesia bovis isolate known to cause particularly severe cerebral babesiosis were tested for virulence phenotype by inoculation of cattle. Clones were selected for phenotyping by two criteria - rate of growth in culture and hybridization of a virulence-related probe to Southern blots. Largely on the basis of associated mortality, B. bovis clones were judged to vary in their pathogenic potential.

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.

Stage-specific surface antigens of the cattle lungworm Dictyocaulus viviparus.

Immunofluorescence on live Dictyocaulus viviparus parasites revealed a significant antibody response by vaccinated and patently infected bovine hosts to the sheath of infective larvae (L3), a structure which is generally thought to be shed from the parasite surface prior to invasion of host tissue. In contrast, surface-exposed antigens of the adult, egg and pulmonary L1 stages were recognized only by serum antibody from calves exposed to a patient lungworm infection. Radioiodination of sheathed L3 identified a restricted set of components while a more complex pattern of labelled material was observed with adult parasites. Many more components of adult worms were labelled by the Bolton-Hunter than by the Iodogen reagent, probably reflecting the more penetrative labelling propensities of the former. Stage-specificity of surface-associated antigens of adult parasites was demonstrated by their immunoprecipitation by antibody from patently-infected, but not from vaccinated, calves. There was no in vitro release of the major iodinatable surface-associated antigens of adult parasites not any binding of antibody raised against adult excretory-secretory (ES) products to the surface of living adult worms, suggesting that surface components do not contribute to adult ES products in this species. Antibody responses to the surface of adults, L1 and eggs were specific to patently-infected animals and may provide a useful indicator of exposure to patent infection.

Characterization of excretory-secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination.

In vitro released products of the adult stage of the bovine lungworm. Dictyocaulus viviparus, were characterized according to their SDS-PAGE profile, glycosylation pattern, in vitro synthesis and antigenicity in the context of infection and vaccination with irradiated larvae. Biosynthetic labelling experiments with 35S-methionine indicated active synthesis of ES throughout this time. There was, however, little incorporation of 3H-glucosamine into ES products, and lectin affinity chromatography and glycopeptidase F digestion identified only one glycosylated component. Immunoprecipitation of 125I-labelled ES products with sera from calves patently infected with D. viviparus demonstrated that all of these, with the exception of two components, are antigenic to the bovine host. One of those not immunoprecipitated was shown to be host serum albumin carried over into culture. A limited degree of cross-reactivity between nematode species was observed, with a D. viviparus female-specific antigen of 290 kDa being recognized by serum antibody from calves infected with the gastrointestinal nematodes Cooperia oncophora and Ostertagia ostertagi. Calves vaccinated with irradiated larvae of D. viviparus, despite not being exposed to the adult stage of the parasite, also showed some recognition of adult ES products. This might suggest that vaccination with irradiated larvae operates against both pre-pulmonary and pulmonary stages of the infection.

The secreted and somatic proteinases of the bovine lungworm Dictyocaulus viviparus and their inhibition by antibody from infected and vaccinated animals.

Proteinase activities were examined in extracts and excretory-secretory (ES) products of the infective and adult stages of the cattle lungworm, Dictyocaulus viviparus. Multiple enzyme activities were identified from each source, as defined by pH optima, substrate specificities, inhibitor effects and substrate gel electrophoresis. Serine-, cysteine- and metalloproteinases were identified, secreted materials being more active against protein substrates per unit protein than were extracts, and the particular proteinases produced varied with the developmental stage of the parasite. The antigenicity of these parasite proteinases was demonstrated by the inhibition of enzymic activity with Protein G-purified serum IgG antibody from both infected and vaccinated hosts and in the retardation of enzyme migration on electrophoresis of enzyme-antibody complexes. For the adult products, this confirmed that the enzymes concerned were of parasite origin, and not host-derived. These results argue for investigation of D. viviparus proteinases as targets for the antibody response in the limitation of parasite-mediated tissue damage and as the active principle behind the anti-D. viviparus vaccine.

Antibody response to Babesia bigemina infection in calves measured by ELISA and immunoblotting techniques.

To measure the antibody response to Babesia bigemina with an ELISA test, three groups of cattle were experimentally infected with two isolates of the parasite. It was possible to demonstrate specific antibody binding directed against the parasite as early as the 7 days postinfection (PI). The highest level of antibody was obtained around day 1 to 23 and remained detectable for 260 days. Challenge of the animals 260 days PI with a tick-induced B. bigemina infection depicted that homologous strain-challenged calves did not show an increase of IgG antibody levels, where as those challenged with the heterologous isolate did. In the latter groups the resulting level of antibodies was even higher than after the primary infection. The immunoblotting technique showed that the antibody response is probably directed against groups of B. bigemina components with a relative mobilities of 68-64 kDa, 62-54 kDa and 52-42 kDa, which appear to be major components of the protozoa. By observing the cross-reacting antigenicity among seven B. bigemina isolates, it was demonstrated that these components are not isolate-restricted.

The antibody repertoire in infection and vaccination with Dictyocaulus viviparus: heterogeneity in infected cattle and genetic control in guinea pigs.

The antigen recognition profiles of serum antibody from calves infected or vaccinated with irradiated Dictyocaulus viviparus larvae were analysed by immunoprecipitation of radio-iodinated in vitro-released excretory-secretory materials from live adult parasites. Immunoprecipitates were analysed by SDS-PAGE and considerable heterogeneity in antigen recognition between individual animals was observed, regardless of infection regimen. This heterogeneity was also found to occur amongst outbred guinea pigs infected with the parasite and permitted an examination of the genetics of the effect using inbred guinea pigs (Strains 2 and 13). The antibody repertoires of the two strains were distinct, with only slight variation occurring between individuals within a strain. Previous work on nematode infections in rodents has demonstrated a role for the major histocompatibility complex (MHC) in the control of the immune repertoire. If this, as is probable, holds for the guinea pig, then it can be ascribed to the MHC Class II region because Strain 2 and Strain 13 bear identical Class I alleles but disparate Class II alleles. Whilst there is no evidence to date that the efficiency of vaccination of cattle is influenced by genetic factors, the operation of vaccines based on a single or a few molecularly cloned parasite antigens might be seriously compromised by the kind of genetic restriction to the immune repertoire described here.

Serological diversity in Haemophilus somnus.

Serological interrelations among 46 strains of Haemophilus somnus of various geographical and pathological origins were studied by agglutination and agglutinin adsorption procedures. Sera prepared against nine representative American and Swiss isolates agglutinated all other strains to various titers. Adsorption of antisera to American strains by Swiss cultures tended to remove mainly the reactivity with Swiss antigens, whereas adsorption of such antisera to American strains in most instances abolished all agglutinating activity. Analogous observations were made when sera against Swiss cultures were adsorbed with cells of American and Swiss origin, respectively. Results of tests involving unadsorbed and adsorbed sera suggest the existence of at least three sets of antigen in H. somnus: American, Swiss, and common, which appear to exist in various combinations, accounting for four agglutination groups. These reflect to a considerable degree the geographical, although not the pathological or anatomical, origin of the respective strains.