Non-specific resistance to Babesia divergens in the Mongolian gerbil (Meriones unguiculatus)

Inoculation of mature gerbils with BCG gave protection to subsequent infection with B. divergens when inoculated by the intracardiac and intraperitoneal routes, the latter showing a dose dependent relationship. BCG vaccination was most effective in immature gerbils (less than 4 weeks old), which are innately resistant to B. divergens. Vaccination of gerbils with killed Propionesbacterium acne and zymosan A failed to elicit a protective response, which contrasts conspicuously with rodent babesia studies. Incubation of B. divergens-infected gerbil blood with hydrogen peroxide produced parasite inhibition only at the highest concentration and treatment of parasitized gerbils with the oxidative radical inducer, alloxan monohydrate, gave equivocal results so it is evident that, unlike Plasmodium spp., B. divergens is not significantly susceptible to the action of reactive oxygen forms.     

Morphological comparisons of the bovine piroplasm, Babesia divergens, in cattle and jird (Meriones unguiculatus) erythrocytes

In comparative studies of Babesia divergens in jirds and splenectomized calves several differences in the appearance of the parasite were found. Pyriform pairs of the parasite grew larger in jirds than in calves and also were larger than those of the original isolate when calves were infected after multiple jird passage. The parasites usually occupied a central intraerythrocytic position in jirds whereas in cattle they mainly occurred peripherally. The majority of pairs formed an obtuse angle relative to each other in both host species. The main difference in morphological types was found to be the rarity of single pyriforms in jirds and this, together with other small differences, was attributed to a faster replication rate of B. divergens in this host. Intraerythrocytic death of B. divergens was observed in both jirds and calves indicating similar defence mechanisms against primary infections in the 2 host species.     

Profiling the cellular immune response to multiple Brugia pahangi infections in a susceptible host

Human lymphatic filariasis is caused primarily by Brugia malayi and Wuchereria bancroffi. Unraveling this disease is complex, as people living in endemic areas exhibit a vast array of clinical states and immune responses. The Mongolian gerbil (Meriones unguiculatus)-B. pahangi model of human lymphatic filariasis has provided much information on immune parameters associated with filarial infection. Prior investigations in our laboratory have shown that gerbils closely mimic a subset of patients classified as microfilaremic but asymptomatic, a group that comprises the majority of people living in endemic areas. Worm recovery data suggest that gerbils carrying current B. pahangi infections do not show any resistance to subsequent subcutaneous B. pahangi infections. The aim of the present studies was to investigate the T cell cytokine response in gerbils receiving multiple infections of B. pahangi as a means of mimicking the conditions experienced by people in endemic areas. The T cell cytokine profile generated by multiply infected gerbils was not different from that previously generated by gerbils infected only once with B. pahangi. Gerbils infected multiple times with B. pahangi showed a transient increase in IL-5, which corresponded to the increased eosinophil levels previously reported from multiply infected gerbils. Chronically infected gerbils showed elevated IL-4 mRNA levels, as has been reported from gerbils infected only once with B. pahangi. Chronic infections were also associated with a state of immune hyporesponsiveness, as determined by the characterization of lymphatic thrombi and lymphoproliferation of spleen and renal lymph node cells to worm antigen.     

Acquired Resistance to Babesia divergens in Experimental Calves

SYNOPSIS. One intact and 2 splenectomized calves were infected with Babesia divergens and the persistence of the parasites in the blood was followed by periodic subinoculations into susceptible splenectomized calves. After periods varying from 3–7 years the parasites failed to be demonstrable by this method. When the immunity of the animals was challenged by the intravenous injection of blood containing Babesia divergens , they were all resistant to reinfection. These observations add support to the suggestion that sterile immunity may play a part in the resistance of cattle to reinfection with B. divergens.     

Clinical and Serological Response after Experimental Inoculation with Babesia Divergens of Newborn Calves with and without Maternal Antibodies

The influence of maternal antibodies on clinical and serological response after experimental inoculation with Babesia divergens of newborn calves was studied. Five calves, born to dams seropositive for B.divergens, (Group 1) had specific maternal antibodies when tested 12 h after their first feeding of colostrum. At that point they were inoculated i.v. with B.divergens infected erythrocytes. Five other calves, born to dams seronegative for B.divergens, (Group 2) had no Babesia specific maternal antibodies when inoculated at the same age. Babesia divergens organisms were demonstrated in blood smears from calves in both groups at some point 5 to 10 days p.i. All calves in both groups had B.divergens specific IgM antibodies at 7 to 17 days p.i. as shown by a modified IF-test. Specific IgG antibodies, transferred by colostrum, were found in all calves of Group 1 before inoculation of B.divergens. The IgG titre of these animals increased by a doubling dilution step at 11–25 days p.i. Among calves of Group 2 specific IgG antibodies were found at first between day 9 and 15 p.i. Both IgM and IgG antibody titres had to be investigated since demonstrated IgG antibodies can originate both from maternally transferred antibodies and from actively produced antibodies after an infection. There was no difference in clinical parameters; parasitaemia, PCV, Hb, and rectal temperature between the groups. This experiment gives evidence that there can be a resistance to bovine babesiosis in newborn calves independent of maternal antibodies.     

A conserved subtilisin protease identified in Babesia divergens merozoites

Invasion of erythrocytes is an integral part of the Babesia divergens life cycle. Serine proteases have been shown to play an important role in invasion by related Apicomplexan parasites such as the malaria parasite Plasmodium falciparum. Here we demonstrate the presence of two dominant serine proteases in asexual B. divergens using a biotinylated fluorophosphonate probe. One of these active serine proteases (p48) and its precursors were recognized by anti-PfSUB1 antibodies. These antibodies were used to clone the gene encoding a serine protease using a B. divergens cDNA library. BdSub-1 is a single copy gene with no introns. The deduced gene product (BdSUB-1) clearly belongs to the subtilisin superfamily and shows significant homology to Plasmodium subtilisins, with the highest degree of sequence identity around the four catalytic residues. Like subtilisin proteases in other Apicomplexan parasites, BdSUB-1 undergoes two steps of processing during activation in the secretory pathway being finally converted to an active form (p48). The mature protease is concentrated in merozoite dense granules, apical secretory organelles involved in erythrocyte invasion. Anti-PfSUB1 antibodies have a potent inhibitory effect on erythrocyte invasion by B. divergens merozoites in vitro. This report demonstrates conservation of the molecular machinery involved in erythrocyte invasion by these two Apicomplexan parasites and paves the way for a comparative analysis of other molecules that participate in this process in the two parasites.     

Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua)

A growing concern for the high consumption of antibiotics in aquaculture has initiated a search for alternative methods of disease control. Improved resistance against infectious diseases can be achieved by the use of probiotics. Probiotics are live microorganisms supplemented in food or feed which give beneficial effects on the intestinal microbial balance. In the present study a dry feed containing lactic acid bacteria (Carnobacterium divergens) isolated from Atlantic cod (Gadus morhua)intestines was given to cod fry. After three weeks of feeding the fry was exposed to a virulent strain of Vibrio anguillarum. The death rate was recorded during further three weeks of feeding with lactic acid bacteria supplemented feed. A certain improvement of disease resistance was obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in surviving fish given feed supplemented with lactic acid bacteria. No obvious growth inhibition of V. anguillarum was observed in an in vitro mixed culture of this bacterium and the C. divergens isolated from cod intestines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Heat shock response of Babesia divergens and identification of the hsp70 as an immunodominant early antigen during ox, gerbil and human babesiosis.

Using antisera (alpha-R and alpha-C7Ag) directed against the conserved Gly-Gly-Met-Pro-epitope of the hsp70 family, a single antigen was identified in the human Babesia divergens Rouen 1987 isolate by Western immunoblotting and immunoprecipitation experiments. This B divergens hsp70 is highly conserved as shown by the analysis of five other geographical B divergens isolates from different hosts (human and bovine). Indirect immunofluorescence assay performed on the asexual intraerythrocytic stages showed that the hsp70 is mainly cytoplasmic and stage-independent. Heat-shock experiments, with 20 min incubation at 40 degrees C followed by a 10 to 50 min shift to 37 degrees C in the presence of [35S]-methionine, led to an increase of two hsp of 85 and 70 kDa while protein synthesis in general decreased within 10 min. Immunoprecipitations of [35S]-methionine radiolabelled proteins with human, ox and gerbil antisera raised against various B divergens isolates, showed the presence of a B divergens 70 kDa protein which was demonstrated to be a hsp70 by coupling immunoblotting assays with alpha-C7Ag serum on the same immunoprecipitated material. During human babesiosis, the B divergens hsp70 appears as an early antigen during the acute phase. These results are in agreement with the use of the B divergens hsp70 as an essential valence antigen in an anti-babesiosis vaccine.     

The effects of sex,age and diet of mice and gerbils on susceptibility to Microphallus pygmaeus (Digenea: Microphallidae)

Molan A. L. and James B. L. 1984. The effects of sex, age and diet of mice and gerbils on susceptibility to Microphallus pygmaeus (Digenea: Microphallidae). International Journal for Parasitology14: 521–526. Mature male mice (40–100-day-old) and gerbils (60–150-day-old) harbour significantly more Microphallus pygmaeus than their female counterparts. Adult worms from male mice and gerbils consistently contain more eggs than those from females but the differences are not significant. No sex differences in worm recovery occur in immature or ageing mice and gerbils. Seventy-day-old mice and gerbils of both sexes harbour significantly more worms with a higher egg production than immature and ageing animals. The worm burden of sucklings, however, is higher than in weanlings and mature mice and gerbils of both sexes fed on cow's milk harbour significantly more worms than those fed on a normal pellet diet.     

Human babesiosis, an emerging zoonosis

Data of human babesiosis are shortly reviewed with particular emphasis to Europe. In Europe, most cases of human babesiosis are caused by Babesia divergens. Although both phenotypic and genotypic features suggest that zoonotic B. microti may occur in Europe, convincing medical evidence is lacking. Recently a non-Babesia divergens organism causing zoonotic infection has been found in Italy and Austria. Overall, the seroprevalence against both B. microti and B. divergens microrganisms in human ranges 1.5%-11.5% in Europe.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus)

A Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) is morphologically similar and genetically identical, based on SSU rRNA gene comparisons, to 2 agents responsible for human babesiosis in the United States. This zoonotic agent is closely related to the European parasite, Babesia divergens. The 2 organisms were characterized by in vitro comparisons. In vitro growth of the rabbit Babesia sp. was supported in human and cottontail rabbit erythrocytes, but not in bovine cells. Babesia divergens was supported in vitro in bovine and human erythrocytes, but not in cottontail rabbit cells. Morphometric analysis classifies B. divergens as a small babesia in bovine erythrocytes, but the parasite exceeds this size in human erythrocytes. The rabbit Babesia sp. is large, the same size in both human or rabbit erythrocytes, and is significantly larger than B. divergens. Eight or more rabbit Babesia sp. parasites may occur within a single erythrocyte, sometimes in a floret array, unlike B. divergens. The erythrocyte specificity and morphological differences reported in this study agree with previous in vivo results and validate the use of in vitro methods for characterization of Babesia species.     

Postnatal development of the testis in the Mongolian gerbil, Meriones unguiculatus

The testicular development in gerbils was studied over 16-week periods starting from birth. Testicular weight and seminiferous tubule diameter increased considerably between 1 and 11 weeks of age. At 16 weeks the testicular weight was about 540 mg. Spermatogenesis commenced at about 2 weeks when mitoses first appeared in spermatogonia. Spermatozoa appeared in a few of the seminiferous tubules by 7 weeks and consistently so in all of the tubules at 10 weeks. Epididymal spermatozoa appeared first in the cauda epididymis at 10 weeks and were consistently present at 12 weeks. Formation of mature Leydig cells with a grouped perivascular arrangement appeared by 3 weeks and continuously so thereafter. From these results, it is evident that the male Mongolian gerbil is almost sexually matured by 10 to 12 weeks of age.     

Subclinical infection and transmission of Tyzzer's disease in rats.

Two isolates of Bacillus piliformis originally obtained from rats from Japan and Indiana were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Protein and antigen profiles revealed heterogeneity between the two isolates, demonstrating that more than one isolate of B. piliformis is capable of infecting rats. Results of parallel infection and transmission studies with the two isolates were almost identical. Orally inoculated rats remained asymptomatic; however, enzyme-linked immunosorbent assay results revealed a significant increase in serum antibodies to B. piliformis. Formalin-killed B. piliformis elicited no serum antibody response among rats inoculated orally, indicating that viable organisms, capable of replicating within the host, are needed to induce a systemic humoral response. Naive rats and weanling gerbils were housed on soiled bedding from the experimentally infected, asymptomatic, seropositive rats. Although gerbils showed no clinical signs or histopathologic evidence of Tyzzer's disease, rats housed on bedding collected 1 or 2 weeks postinoculation seroconverted and remained seropositive but asymptomatic throughout the study. These results demonstrate that subclinically infected rats are capable of transmitting B. piliformis to naive rats and suggest that the histopathologic evaluation of sentinel gerbils may not be an effective method for detecting all strains of B. piliformis.     

Association between sequence polymorphism in an epitope of Babesia divergens Bd37 exoantigen and protection induced by passive transfer

In Europe, Babesia divergens is the major agent responsible for babesiosis in cattle and can occasionally infect splenectomised humans. Recently, we reported the characterisation of a 37 kDa exoantigen (Bd37) anchored in the merozoite membrane of B. divergens by a glycosylphosphatidyl-inositol. After phospholipase hydrolyse of the glycosylphosphatidyl-inositol anchor, the Bd37 antigen could be isolated in the plasma of the infected host and from the in vitro culture supernatants. Immunisation of mice with a gel-filtration protective fraction of B. divergens exoantigens, produced a monoclonal antibody (MAb), called F4.2F8-INT, directed against Bd37. In the present study, we report data on passive protection using MAb F4.2F8-INT. This MAb was able to completely protect against virulent challenges with B. divergens isolates Rouen 1987 (Rouen87) and Weybridge 8843 (W8843) but had no protective effect against another French isolate from Massif Central (6303E). Physical characterisation of the epitope recognised by F4.2F8-INT allowed us to explain the differences observed between these isolates by western blotting and passive protection. These results suggest that the antigen carrying this epitope could be used as a target in the development of a recombinant vaccine against B. divergens babesiosis.     

Morphology and genetics of a Babesia isolate from Capreolus capreolus

A Babesia isolate that was morphologically distinct from Babesia capreoli and very similar to B. divergens was found in the blood of a roe deer (Capreolus capreolus) found dead in central Italy. Sequences corresponding to the full coding region of the 18S ribosomal RNA (rRNA) gene were identical to a sequence reported for Babesia divergens from a reindeer (Rangifer tarandus) and 99.9% and 99.8% similar to those reported for B. capreoli and bovine origin B. divergens, respectively.     

Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1 x 10(9) red blood cell (RBC)/ml, 1-2.5 x 10(5) infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [(3)H]hypoxanthine incorporation. An excellent correlation was demonstrated between A(405) of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A(405) and [(3)H]hypoxanthine incorporation for [(3)H]hypoxanthine concentrations lower than 4 microCi/ml. Our assays also highlighted the inhibitory effect of [(3)H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 microCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A(405) and parasitemia counting. In conclusion, A(405) measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.     

Antibody prevalence and molecular identification of Babesia spp. In roe deer in France

In a region-wide serologic study carried out in 2004 on free-ranging hunted roe deer in various landscapes, we found that 58% of the animals (237 out of 406) were antibody positive for Babesia divergens antigen. Serologic and infection status was also analyzed for 327 roe deer live-trapped in two fenced forest areas over 5 yr (2004-08). For two consecutive years during this period, 92 and 94% of the deer in these closed populations were antibody-positive for B. divergens. Babesia spp. were isolated in autologous red blood cell culture for 131 of the trapped animals (40%). Molecular typing was done on 76 isolates with polymerase chain reaction (PCR)-restriction fragment length polymorphism methods targeted at the 18S ribosomal subunit gene (18 isolates) and the Bd37 gene coding for a merozo?te surface antigen implicated in a protective response (60 isolates). Results indicated continuous cocirculation of B. capreoli and B. venatorum in both forests and possible coinfection of animals with both species. No infection with B. divergens was detected. Fifteen isolates were confirmed to be B. capreoli by sequencing part of the 18S rRNA gene. Using PCR detection of the Bd37 gene, all nine isolates of B. venatorum in this study were negative, whereas the 15 confirmed and 50 putative B. capreoli isolates showed very variable restriction profiles, distinct from those known for Bd37 in B. divergens. Two isolates showed conflicting results, suggestive of mixed infection.     

North-east Atlantic and Mediterranean species of the genus Buffonellaria (Bryozoa, Cheilostomata): implications for biodiversity and biogeography

In this study we revise the cheilostome bryozoan genus Buffonellaria Canu & Bassler, 1927 and its Mediterranean and north-east Atlantic species, thereby addressing several existing problems. First, a lectotype for the type species, Buffonellaria divergens (Smitt, 1873) from Florida, is chosen, which proves to be distinct from the European species. Second, the two hitherto established north-east Atlantic species [ Buffonellaria nebulosa ( Jullien & Calvet, 1903 ) and Buffonellaria porcellanum Arístegui Ruiz, 1987], are redescribed, which were poorly documented until now. Third, close inspection of material, collected from Spitsbergen to tropical West Africa, using scanning electron microscopy reveals that the actual number of species, all previously referred to either B. divergens or Stephanosella biaperta (Michelin, 1848), is distinctly greater in the north-east Atlantic than has been previously acknowledged. As a result, seven new species are introduced ( Buffonellaria acorensis sp. nov. , Buffonellaria antoniettae sp. nov. , Buffonellaria arctica sp. nov. , Buffonellaria harmelini sp. nov. , Buffonellaria jensi sp. nov. , Buffonellaria muriella sp. nov. , and Buffonellaria ritae sp. nov. ), whereas two are left in open nomenclature. With the increase in number of species, the extremely broad geographical range of distribution assumed for B. divergens breaks down to numerous restricted areas. However, although most species have only been reported from a single location, B. arctica sp. nov. seems to have a fairly wide distribution in the Arctic region.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 537–566.