Re: Babesiosis: persistence in the face of adversity

[Guest guest]

Nelly the cartoon doesn't open up.

tony

> I recently posted this on Lymenet, I'm sure some of you have seen

it before but as the link to the article is now defunct and as it is

a very very good article + animation on Babesiosis and how it

persists, I thought I would post it here.

>

> (Tony can look at the pretty cartoon, might keep him quiet for a

while!)

>

> Babesiosis: persistence in the face of adversity

>

> For animated version, go to:

http://archive.bmn.com/supp/part/allred.html.

>

> R. Allred allredd@m...

>

> Trends in Parasitology 2003, 19:51-55

>

> Dept of Pathobiology, College of Veterinary Medicine, University

of Florida, Gainesville, FL 32611-0880, USA

>

> Abstract

>

> Many babesial parasites establish infections of long duration in

immune hosts. Among different species, at least four mechanisms are

known that could facilitate evasion of the host immune response,

although no one species is (yet) known to use them all.

>

> This update strives to illustrate the ramifications of these

mechanisms and the interplay between them.

>

> Babesia spp. are a diverse group of tick-borne, obligate,

intraerythrocytic Apicomplexan parasites infecting a wide variety of

organisms.

>

> Infection of a vertebrate host is initiated by inoculation of

sporozoite stage parasites into the bloodstream during the taking of

a bloodmeal.

>

> Most babesial sporozoites directly invade circulating erythrocytes

without a tissue stage of development [1]. A few, notably Babesia

equi [2] and Babesia microti [3], first invade lymphocytes where

they form motile merozoites, which then invade erythrocytes.

Although this undoubtedly affects their interactions with the host,

any effects on immune evasion are at present unknown.

>

> Once erythrocyte invasion occurs, a seemingly perpetual cycle of

asexual reproduction is established, despite the rapid development

of a strong immune response [4].

>

> During the acute babesial infection, the host may become severely

ill. Typically, the infected host can suffer high fevers, severe

anemia, hemoglobinuria caused by intravascular hemolysis

considerably in excess of that correlated with parasitemia,

lethargy, inappetance, and sometimes hydrophobia [5]. Coagulatory

disturbances are also a frequent finding [69].

>

> Neurologic sequelae may occur with some babesial parasites, most

notably Babesia bovis. In the case of B. bovis, this is accompanied

by massive intravascular sequestration of infected red blood cells

(IRBC) carrying mature parasite stages [1013] .

>

> Despite the potential severity of the acute infection, individuals

who survive generally develop immunity against disease, but not

against infection per se, and could remain persistently infected

[1418] .

>

> In the case of B. bovis, infections can persist for years, perhaps

even the lifetime of the animal.

>

> Babesial parasites clearly have adapted well to survival in the

hostile environment that is the immune host. How do they do this?

>

> We understand very little about the mechanisms used by these

parasites to survive.

>

> However, at least five different phenomena are known that probably

contribute to parasite survival:

>

> (1) rapid antigenic variation ( Fig. 1);

>

> (2) cytoadhesion ( Fig. 2 and see animation on:

http://archive.bmn.com/supp/part/allred.html) and sequestration;

>

> (3) binding of host proteins to the IRBC surface;

>

> (4) the monoallelic expression of different members of multigene

families; and

>

> (5) establishment of a poorly understood transient

immunosuppression [1921] .

>

> The relative contributions of the individual phenomena are not

known, nor have definitive demonstrations yet been made that any of

these directly contributes to survival. Here, only brief discussions

of specific examples will be presented, along with an attempt to put

these phenomena into perspective relative to the establishment of

persistent infection.

>

> [graphic omitted]Fig. 1. Antigenic variation in Babesia bovis

appears to proceed via a segmental gene conversion mechanism. In

this mechanism, sequences are duplicated from donor gene copies [a

chromosomal segment containing donor genes (orange, purple and green

boxes) is shown in (a)] into an actively transcribed variant

erythrocyte surface antigen (ves) 1 gene (ves1 site of

transcription, yellow box) in (.

>

> With repeated instances of segmental gene conversion [presumably

only one per generation, e.g. (bi), (bii), (biii)], the actively

transcribed ves1 gene becomes progressively altered from the

original, whereas the donor gene copies appear to remain unaltered.

>

> The actual mechanism by which duplicated sequences are transferred

is not known. This site of transcription is shown in a hypothetical

placement near the telomeric end (shown as boxed red and black

repeat units). Hypothetical promoters are indicated by black flags

and hypothetical telomere terminal structures are represented by

pink boxes after the repeat units.

>

> [graphic omitted]Fig. 2. Cytoadhesion of Babesia bovis. Babesia

bovis-infected red blood cells (IRBC) can adhere to the capillary

endothelium of blood vessels of various tissues, a behavior that

could be abrogated by host antibodies recognizing parasite antigens

on the IRBC surface.

>

> (a) Babesia bovis IRBC carrying ring stages or mature stages

(trophozoites or meronts) that are expressing non-adhesive surface

molecules remain in the peripheral circulation, and will pass

through the spleen. Whereas ring-stage IRBCs will probably survive

splenic passage, IRBC carrying mature parasites will be removed and

destroyed as a result of antibody recognition of the antigens

expressed on IRBC surface.

>

> ( Babesia bovis IRBC carrying mature parasites that are

expressing adhesion-competent forms of proteins exported to the IRBC

surface (probably VESA1; shown as green triangles) will adhere to

the endothelium of the capillaries and post-capillary venous

circulation (cytoadhesion). These parasites will mature in the deep

microvasculature and release their merozoite stages there.

>

> © Babesia bovis IRBC expressing different isoforms of the

adhesive molecules on the IRBC surface (shown as green U-shaped

molecules) will bind to different receptors and endothelial cells.

This could account for apparent tissue tropism in this parasite,

including that occurring within the brain.

>

> (d) The development of antibodies (shown as Y-shaped molecules) by

the immune host is thought to be capable of preventing cytoadhesion

and of causing already-bound IRBCs to be released from the

endothelium. When this occurs, the IRBC are probably susceptible to

splenic removal.

>

> Note these antibodies recognize the ligands from (, but have no

effect on IRBC expressing the ligands from ©.

>

> For animated version, go to:

http://archive.bmn.com/supp/part/allred.html.

>

> Antigenic variation

>

> The first evidence for antigenic variation in babesial parasites

was obtained with Babesia rodhaini [22] and later with B. bovis

[23]. The results of these studies strongly suggested that

fundamental changes had occurred in the antigenicity of protective

antigen(s) in the surviving parasite populations, and demonstrated a

probable association between recognition of the variant antigen and

immune protection. Unfortunately, the parasite populations were not

clonal, precluding rigorous interpretation of these experiments, and

the identities of the variant antigen(s) in B. rodhaini remain

unknown.

>

> In contrast to the situation with B. rodhaini, a variant antigen

expressed by B. bovis has been identified. A size-polymorphic,

parasite-derived antigen was identified on the B. bovis IRBC

surface. This antigen was recognized in an isolate-specific manner

during live-cell immunofluorescence and surface-specific

immunoprecipitation assays [24]. The application of these assays to

parasites recovered at different times from a calf, infected once

with a clonal B. bovis line, revealed that the isolate-specific

antigen underwent rapid size and antigenic variation [16]. The

putative variant antigen was subsequently confirmed through the use

of monoclonal antibodies (mAb), and was named the variant

erythrocyte surface antigen (VESA) 1 [25]. This antigen migrates on

sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)

as a doublet, ranging in mass between 105 kDa and 135 kDa, among

different variants and isolates. Recently, the ves1 multigene family

encoding the larger subunit (VESA1a) was identified [26], and

preliminary estimates suggest a minimum of 50 gene copies. The

molecular mechanisms involved in expression of variation are only

beginning to be worked out, but all evidence to date is consistent

with progressive, segmental gene conversion playing a key role [26]

( Fig. 1). Some ves1 genes can donate sequences to the transcribed

gene copy, while apparently remaining unaltered themselves ( [26];

B. Al-Khedery et al., unpublished). It is not yet clear whether all

ves1 genes can participate in the phenomenon of gene conversion.

Coupled with a similarly polymorphic VESA1b subunit [16,24,25] , the

potential capacity of this parasite for presentation of unique VESA1

antigens is enormous.

>

> Cytoadhesion and sequestration

>

> The spleen is a remarkable organ in its ability to recognize and

remove damaged cells from circulation. It could be aided by the

presence of bound antibodies and, perhaps complement, on the damaged

cell's surface. Accordingly, passage through the spleen is a journey

to be avoided by parasitized erythrocytes. The ability to avoid

splenic passage has been observed in Babesia canis [27], the Babesia

Washington state isolate 1 (WA1) [28] and B. bovis [29,30] ,

although it is likely that different mechanisms are used to achieve

sequestration. It has been suggested that B. canis becomes trapped

in the deep vasculature through generalized vasodilation and

hypotensive pooling of blood. Under these conditions, IRBC might

fail to circulate, becoming trapped in local coagulatory masses

where they undergo proliferation [27]. Nothing is currently known of

the mechanism(s) used by the WA1 isolate to sequester, and the act

of sequestration itself could be host-specific [28,31] .

>

> By contrast to B. canis, B. bovis sequesters in the

microvasculature through specific binding of IRBC to the capillary

and post-capillary endothelium via knob-like protrusions of the IRBC

membrane ( Fig. 2 and http://archive.bmn.com/supp/part/allred.html)

[10,11,32] . This binding (cytoadhesion) and sequestration is

associated with the severe and frequently lethal cerebral form of

bovine babesiosis [1013,30] . Using an in vitro assay of

cytoadhesion to characterize this phenomenon, it was shown that

parasite-synthesized components must be present for binding to occur

[33]. Further, strong circumstantial evidence was found for

mediation of this phenomenon by the VESA1 antigen [34]. Clear

precedents exist, in the Plasmodium falciparum erythrocyte membrane

protein (PfEMP) 1 [35,36] , and the M proteins of Streptococcus

pyogenes [37], for mediation of pathogen adhesion to host cells by

highly variant components. The endothelial receptor for B. bovis

cytoadhesion is not known, but current data do not support a

significant role for compatibility determinant (CD)36 [33], the

major P. falciparum receptor.

>

> It has recently been hypothesized that sequestration results in an

enhanced parasite susceptibility to killing by reactive nitrogen

oxides due to unloading at reduced oxygen tension of erythrocytes

laden with nitric oxide (NO) [38]. It was previously shown that B.

bovis-stimulated macrophages produce NO, and that in vitro parasite

killing by NO can be achieved [39]. However, a diminished capacity

for macrophage NO production has been observed under reduced oxygen

tension [40], and other studies demonstrate NO consumption by

oxyhemoglobin, with the release only of nitrate and methemoglobin

[41]. Thus, sequestration might serve to enhance parasite survival

by preventing localized circulation of NO-carrying erythrocytes and

maintaining local hypoxia. Clearly, all the ramifications of

cytoadhesion are not yet understood, but the ability to sequester

and not suffer splenic removal is probably a very important

mechanism of long-term survival by this parasite.

>

> Binding of host proteins to the IRBC surface

>

> Not all babesial parasites appear to undergo antigenic variation,

and most do not cytoadhere or sequester. The bovine parasite,

Babesia bigemina, is a good example. Although B. bigemina expresses

parasite-derived antigens on the IRBC surface, the antigens appear

to be isolate-common and stable over the course of infection [42].

However, B. bigemina IRBC bind immunoglobulin (Ig) M on the IRBC

surface, in a non-immunospecific reaction [43]. Presentation of IgM

molecules, bound through the Fc region with the antigen recognition

domain facing the plasma, clearly represents a mechanism with the

potential to hide this parasite from immune recognition. It is

possible that the invariant IRBC surface antigens mediate IgM

binding, but such a connection has not been made. The number of IgM

bound on individual cells is also not known, but if sufficiently

dense could render these antigens inaccessible to specific

antibodies and leave IRBC 'invisible' to circulating immune effector

cells. How this would affect the interaction of IRBC with the spleen

is less clear, but might reduce susceptibility to splenic clearance

as well, and could help to explain the persistence (up to two years

[14]) of this parasite.

>

> Monoallelic expression of members of multigene familiesBabesia

microti is by far the most common cause of human babesiosis [44].

Similar to many babesial parasites, this species is capable of

persisting in the immune host, even after non-sterilizing

chemotherapy [18]. The mechanisms used to survive in individual

hosts have not yet been elucidated, but a recent clue to population

survival might have emerged. Using immune sera from individuals who

had recovered from babesiosis to screen complementary DNA (cDNA)

libraries, three immunodominant antigen gene families were

discovered [45]. In a limited study monitoring expression of the

Babesia microti MN1 strain (bmn1) gene family, it appeared that a

single member was transcribed. Further, it was revealed that

different alleles were expressed by parasites infecting patients

from different geographical areas. By contrast, the same allele was

expressed in several patients from a small area of transmission, and

this was stable upon transmission to hamsters [46]. These results

make it unlikely that the bmn1 gene family is involved in antigenic

variation as was suggested by the authors. However, the considerable

diversity within this gene family and its (apparently) subtelomeric

location would be consistent with frequent recombination events. If

bmn1 gene products are associated with immunoprotection, this

allelic diversity might provide the parasite with an opportunity to

establish mixed infections in immune hosts already primed by

exposure to other members of the family. In such a scenario, the

temporal nature might lie between the very rapid change of antigenic

variation and the relatively slow evolution associated with allelic

polymorphism. Despite the attractiveness of this possibility, the

association of allelic polymorphism per se with immune evasion is

not always clear. For example, in B. bigemina, four alleles of the

merozoite surface antigen (msa) 2 multigene family have been

described, the products of which are immunologically non-

crossreactive [47]. At least three of the four alleles are co-

expressed on individual merozoite- and sporozoite-stage parasites,

yet there is no additive effect on inhibition of invasion (or

binding) by simultaneous exposure to antibodies against all four

gene products [48].

>

> Interplay of these phenomena

>

> Currently, nothing is known of interplay between antigen masking

or monoallelic expression of invariant multigene families and

antigenic variation or cytoadhesion, if such interaction occurs.

Strong circumstantial evidence suggests that the rapidly variant

VESA1 antigen on the B. bovis IRBC surface serves as a parasite-

derived ligand mediating cytoadhesion [34]. One obvious potential

outcome of this is that an in vivo switch in an antigenic phenotype,

selected by immune recognition of the previously expressed VESA1a

isoform, probably results in alterations of the adhesive phenotype (

Fig. 2 and http://archive.bmn.com/supp/part/allred.html). This

effect was observed in vitro, using an assay of B. bovis

cytoadhesion to bovine brain endothelial cells [34]. Further, the

C9.1 line-derived mAb, 3F7.1H11, which also reacts with the

cytoadhesive CD7 line (as a result of selection for this trait), is

capable of both blockage and reversal of in vitro cytoadhesion, with

great efficacy [34]. In addition, the adhesion specificity of

different clonal B. bovis lines for different clonal bovine brain

endothelial cell lines varies (R.M. O'Connor and D.R. Allred,

unpublished), suggesting either the recognition of different

endothelial receptors, varied affinities for shared receptors, or

both. Thus, in vivo recognition and elimination of one parasite

population, with expansion of another, probably results in the

selection of parasites with differing adhesive phenotypes and

perhaps tissue tropism ( Fig. 2 and

http://archive.bmn.com/supp/part/allred.html). Given the tissue-

specific presentation of endothelial receptor motifs [49], a

protective immune response to one population could inadvertently

result in relocation of the infection focus, modifying the potential

pathology, and perhaps delaying the initiation of a response to

surviving parasites. Early studies on the histopathology of B. bovis

infection reported widely varying parasitemias for parasites

localized in different tissues, consistent with this possibility

[10,11,32] .

>

> Conclusions

>

> Babesia spp. are a seriously understudied group of parasites.

However, at least five mechanisms have been identified which might

contribute to their evasion of host immunity. Antigenic variation,

cytoadhesion/sequestration, host-protein binding, and induction of

immunosuppression probably facilitate persistence in the individual

immune host. Monoallelic expression of different members of a

multigene family might facilitate multiple infections of immune

hosts, and population dispersal in endemic areas.

>

> Acknowledgements

>

> D.R.A. gratefully acknowledges Basima Al-Khedery, Crabtree,

a O'Connor, Kristi Warren and Jervis for their

assistance. This work was supported by grants from the United States

Dept. of Agriculture (#20013520410144) and the American Heart

Association (#0051422B).

[Guest guest]

Try this (I think there was an extra . at the end of the previous link I gave)

Nelly

http://archive.bmn.com/supp/part/allred.html

[infections] Re: Babesiosis: persistence in the face of adversity

Nelly the cartoon doesn't open up.tony> I recently posted this on Lymenet, I'm sure some of you have seen it before but as the link to the article is now defunct and as it is a very very good article + animation on Babesiosis and how it persists, I thought I would post it here.> > (Tony can look at the pretty cartoon, might keep him quiet for a while!)> > Babesiosis: persistence in the face of adversity> > For animated version, go to: http://archive.bmn.com/supp/part/allred.html. > > R. Allred allredd@m...> > Trends in Parasitology 2003, 19:51-55 > > Dept of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA> > Abstract > > Many babesial parasites establish infections of long duration in immune hosts. Among different species, at least four mechanisms are known that could facilitate evasion of the host immune response, although no one species is (yet) known to use them all. > > This update strives to illustrate the ramifications of these mechanisms and the interplay between them. > > Babesia spp. are a diverse group of tick-borne, obligate, intraerythrocytic Apicomplexan parasites infecting a wide variety of organisms. > > Infection of a vertebrate host is initiated by inoculation of sporozoite stage parasites into the bloodstream during the taking of a bloodmeal. > > Most babesial sporozoites directly invade circulating erythrocytes without a tissue stage of development [1]. A few, notably Babesia equi [2] and Babesia microti [3], first invade lymphocytes where they form motile merozoites, which then invade erythrocytes. Although this undoubtedly affects their interactions with the host, any effects on immune evasion are at present unknown. > > Once erythrocyte invasion occurs, a seemingly perpetual cycle of asexual reproduction is established, despite the rapid development of a strong immune response [4]. > > During the acute babesial infection, the host may become severely ill. Typically, the infected host can suffer high fevers, severe anemia, hemoglobinuria caused by intravascular hemolysis considerably in excess of that correlated with parasitemia, lethargy, inappetance, and sometimes hydrophobia [5]. Coagulatory disturbances are also a frequent finding [69]. > > Neurologic sequelae may occur with some babesial parasites, most notably Babesia bovis. In the case of B. bovis, this is accompanied by massive intravascular sequestration of infected red blood cells (IRBC) carrying mature parasite stages [1013] . > > Despite the potential severity of the acute infection, individuals who survive generally develop immunity against disease, but not against infection per se, and could remain persistently infected [1418] . > > In the case of B. bovis, infections can persist for years, perhaps even the lifetime of the animal. > > Babesial parasites clearly have adapted well to survival in the hostile environment that is the immune host. How do they do this? > > We understand very little about the mechanisms used by these parasites to survive. > > However, at least five different phenomena are known that probably contribute to parasite survival: > > (1) rapid antigenic variation ( Fig. 1); > > (2) cytoadhesion ( Fig. 2 and see animation on: http://archive.bmn.com/supp/part/allred.html) and sequestration; > > (3) binding of host proteins to the IRBC surface; > > (4) the monoallelic expression of different members of multigene families; and > > (5) establishment of a poorly understood transient immunosuppression [1921] . > > The relative contributions of the individual phenomena are not known, nor have definitive demonstrations yet been made that any of these directly contributes to survival. Here, only brief discussions of specific examples will be presented, along with an attempt to put these phenomena into perspective relative to the establishment of persistent infection. > > [graphic omitted]Fig. 1. Antigenic variation in Babesia bovis appears to proceed via a segmental gene conversion mechanism. In this mechanism, sequences are duplicated from donor gene copies [a chromosomal segment containing donor genes (orange, purple and green boxes) is shown in (a)] into an actively transcribed variant erythrocyte surface antigen (ves) 1 gene (ves1 site of transcription, yellow box) in (. > > With repeated instances of segmental gene conversion [presumably only one per generation, e.g. (bi), (bii), (biii)], the actively transcribed ves1 gene becomes progressively altered from the original, whereas the donor gene copies appear to remain unaltered. > > The actual mechanism by which duplicated sequences are transferred is not known. This site of transcription is shown in a hypothetical placement near the telomeric end (shown as boxed red and black repeat units). Hypothetical promoters are indicated by black flags and hypothetical telomere terminal structures are represented by pink boxes after the repeat units. > > [graphic omitted]Fig. 2. Cytoadhesion of Babesia bovis. Babesia bovis-infected red blood cells (IRBC) can adhere to the capillary endothelium of blood vessels of various tissues, a behavior that could be abrogated by host antibodies recognizing parasite antigens on the IRBC surface. > > (a) Babesia bovis IRBC carrying ring stages or mature stages (trophozoites or meronts) that are expressing non-adhesive surface molecules remain in the peripheral circulation, and will pass through the spleen. Whereas ring-stage IRBCs will probably survive splenic passage, IRBC carrying mature parasites will be removed and destroyed as a result of antibody recognition of the antigens expressed on IRBC surface. > > ( Babesia bovis IRBC carrying mature parasites that are expressing adhesion-competent forms of proteins exported to the IRBC surface (probably VESA1; shown as green triangles) will adhere to the endothelium of the capillaries and post-capillary venous circulation (cytoadhesion). These parasites will mature in the deep microvasculature and release their merozoite stages there. > > © Babesia bovis IRBC expressing different isoforms of the adhesive molecules on the IRBC surface (shown as green U-shaped molecules) will bind to different receptors and endothelial cells. This could account for apparent tissue tropism in this parasite, including that occurring within the brain.> > (d) The development of antibodies (shown as Y-shaped molecules) by the immune host is thought to be capable of preventing cytoadhesion and of causing already-bound IRBCs to be released from the endothelium. When this occurs, the IRBC are probably susceptible to splenic removal. > > Note these antibodies recognize the ligands from (, but have no effect on IRBC expressing the ligands from ©. > > For animated version, go to: http://archive.bmn.com/supp/part/allred.html. > > Antigenic variation > > The first evidence for antigenic variation in babesial parasites was obtained with Babesia rodhaini [22] and later with B. bovis [23]. The results of these studies strongly suggested that fundamental changes had occurred in the antigenicity of protective antigen(s) in the surviving parasite populations, and demonstrated a probable association between recognition of the variant antigen and immune protection. Unfortunately, the parasite populations were not clonal, precluding rigorous interpretation of these experiments, and the identities of the variant antigen(s) in B. rodhaini remain unknown. > > In contrast to the situation with B. rodhaini, a variant antigen expressed by B. bovis has been identified. A size-polymorphic, parasite-derived antigen was identified on the B. bovis IRBC surface. This antigen was recognized in an isolate-specific manner during live-cell immunofluorescence and surface-specific immunoprecipitation assays [24]. The application of these assays to parasites recovered at different times from a calf, infected once with a clonal B. bovis line, revealed that the isolate-specific antigen underwent rapid size and antigenic variation [16]. The putative variant antigen was subsequently confirmed through the use of monoclonal antibodies (mAb), and was named the variant erythrocyte surface antigen (VESA) 1 [25]. This antigen migrates on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a doublet, ranging in mass between 105 kDa and 135 kDa, among different variants and isolates. Recently, the ves1 multigene family encoding the larger subunit (VESA1a) was identified [26], and preliminary estimates suggest a minimum of 50 gene copies. The molecular mechanisms involved in expression of variation are only beginning to be worked out, but all evidence to date is consistent with progressive, segmental gene conversion playing a key role [26] ( Fig. 1). Some ves1 genes can donate sequences to the transcribed gene copy, while apparently remaining unaltered themselves ( [26]; B. Al-Khedery et al., unpublished). It is not yet clear whether all ves1 genes can participate in the phenomenon of gene conversion. Coupled with a similarly polymorphic VESA1b subunit [16,24,25] , the potential capacity of this parasite for presentation of unique VESA1 antigens is enormous. > > Cytoadhesion and sequestration > > The spleen is a remarkable organ in its ability to recognize and remove damaged cells from circulation. It could be aided by the presence of bound antibodies and, perhaps complement, on the damaged cell's surface. Accordingly, passage through the spleen is a journey to be avoided by parasitized erythrocytes. The ability to avoid splenic passage has been observed in Babesia canis [27], the Babesia Washington state isolate 1 (WA1) [28] and B. bovis [29,30] , although it is likely that different mechanisms are used to achieve sequestration. It has been suggested that B. canis becomes trapped in the deep vasculature through generalized vasodilation and hypotensive pooling of blood. Under these conditions, IRBC might fail to circulate, becoming trapped in local coagulatory masses where they undergo proliferation [27]. Nothing is currently known of the mechanism(s) used by the WA1 isolate to sequester, and the act of sequestration itself could be host-specific [28,31] . > > By contrast to B. canis, B. bovis sequesters in the microvasculature through specific binding of IRBC to the capillary and post-capillary endothelium via knob-like protrusions of the IRBC membrane ( Fig. 2 and http://archive.bmn.com/supp/part/allred.html) [10,11,32] . This binding (cytoadhesion) and sequestration is associated with the severe and frequently lethal cerebral form of bovine babesiosis [1013,30] . Using an in vitro assay of cytoadhesion to characterize this phenomenon, it was shown that parasite-synthesized components must be present for binding to occur [33]. Further, strong circumstantial evidence was found for mediation of this phenomenon by the VESA1 antigen [34]. Clear precedents exist, in the Plasmodium falciparum erythrocyte membrane protein (PfEMP) 1 [35,36] , and the M proteins of Streptococcus pyogenes [37], for mediation of pathogen adhesion to host cells by highly variant components. The endothelial receptor for B. bovis cytoadhesion is not known, but current data do not support a significant role for compatibility determinant (CD)36 [33], the major P. falciparum receptor. > > It has recently been hypothesized that sequestration results in an enhanced parasite susceptibility to killing by reactive nitrogen oxides due to unloading at reduced oxygen tension of erythrocytes laden with nitric oxide (NO) [38]. It was previously shown that B. bovis-stimulated macrophages produce NO, and that in vitro parasite killing by NO can be achieved [39]. However, a diminished capacity for macrophage NO production has been observed under reduced oxygen tension [40], and other studies demonstrate NO consumption by oxyhemoglobin, with the release only of nitrate and methemoglobin [41]. Thus, sequestration might serve to enhance parasite survival by preventing localized circulation of NO-carrying erythrocytes and maintaining local hypoxia. Clearly, all the ramifications of cytoadhesion are not yet understood, but the ability to sequester and not suffer splenic removal is probably a very important mechanism of long-term survival by this parasite. > > Binding of host proteins to the IRBC surface > > Not all babesial parasites appear to undergo antigenic variation, and most do not cytoadhere or sequester. The bovine parasite, Babesia bigemina, is a good example. Although B. bigemina expresses parasite-derived antigens on the IRBC surface, the antigens appear to be isolate-common and stable over the course of infection [42]. However, B. bigemina IRBC bind immunoglobulin (Ig) M on the IRBC surface, in a non-immunospecific reaction [43]. Presentation of IgM molecules, bound through the Fc region with the antigen recognition domain facing the plasma, clearly represents a mechanism with the potential to hide this parasite from immune recognition. It is possible that the invariant IRBC surface antigens mediate IgM binding, but such a connection has not been made. The number of IgM bound on individual cells is also not known, but if sufficiently dense could render these antigens inaccessible to specific antibodies and leave IRBC 'invisible' to circulating immune effector cells. How this would affect the interaction of IRBC with the spleen is less clear, but might reduce susceptibility to splenic clearance as well, and could help to explain the persistence (up to two years [14]) of this parasite. > > Monoallelic expression of members of multigene familiesBabesia microti is by far the most common cause of human babesiosis [44]. Similar to many babesial parasites, this species is capable of persisting in the immune host, even after non-sterilizing chemotherapy [18]. The mechanisms used to survive in individual hosts have not yet been elucidated, but a recent clue to population survival might have emerged. Using immune sera from individuals who had recovered from babesiosis to screen complementary DNA (cDNA) libraries, three immunodominant antigen gene families were discovered [45]. In a limited study monitoring expression of the Babesia microti MN1 strain (bmn1) gene family, it appeared that a single member was transcribed. Further, it was revealed that different alleles were expressed by parasites infecting patients from different geographical areas. By contrast, the same allele was expressed in several patients from a small area of transmission, and this was stable upon transmission to hamsters [46]. These results make it unlikely that the bmn1 gene family is involved in antigenic variation as was suggested by the authors. However, the considerable diversity within this gene family and its (apparently) subtelomeric location would be consistent with frequent recombination events. If bmn1 gene products are associated with immunoprotection, this allelic diversity might provide the parasite with an opportunity to establish mixed infections in immune hosts already primed by exposure to other members of the family. In such a scenario, the temporal nature might lie between the very rapid change of antigenic variation and the relatively slow evolution associated with allelic polymorphism. Despite the attractiveness of this possibility, the association of allelic polymorphism per se with immune evasion is not always clear. For example, in B. bigemina, four alleles of the merozoite surface antigen (msa) 2 multigene family have been described, the products of which are immunologically non-crossreactive [47]. At least three of the four alleles are co-expressed on individual merozoite- and sporozoite-stage parasites, yet there is no additive effect on inhibition of invasion (or binding) by simultaneous exposure to antibodies against all four gene products [48]. > > Interplay of these phenomena > > Currently, nothing is known of interplay between antigen masking or monoallelic expression of invariant multigene families and antigenic variation or cytoadhesion, if such interaction occurs. Strong circumstantial evidence suggests that the rapidly variant VESA1 antigen on the B. bovis IRBC surface serves as a parasite-derived ligand mediating cytoadhesion [34]. One obvious potential outcome of this is that an in vivo switch in an antigenic phenotype, selected by immune recognition of the previously expressed VESA1a isoform, probably results in alterations of the adhesive phenotype ( Fig. 2 and http://archive.bmn.com/supp/part/allred.html). This effect was observed in vitro, using an assay of B. bovis cytoadhesion to bovine brain endothelial cells [34]. Further, the C9.1 line-derived mAb, 3F7.1H11, which also reacts with the cytoadhesive CD7 line (as a result of selection for this trait), is capable of both blockage and reversal of in vitro cytoadhesion, with great efficacy [34]. In addition, the adhesion specificity of different clonal B. bovis lines for different clonal bovine brain endothelial cell lines varies (R.M. O'Connor and D.R. Allred, unpublished), suggesting either the recognition of different endothelial receptors, varied affinities for shared receptors, or both. Thus, in vivo recognition and elimination of one parasite population, with expansion of another, probably results in the selection of parasites with differing adhesive phenotypes and perhaps tissue tropism ( Fig. 2 and http://archive.bmn.com/supp/part/allred.html). Given the tissue-specific presentation of endothelial receptor motifs [49], a protective immune response to one population could inadvertently result in relocation of the infection focus, modifying the potential pathology, and perhaps delaying the initiation of a response to surviving parasites. Early studies on the histopathology of B. bovis infection reported widely varying parasitemias for parasites localized in different tissues, consistent with this possibility [10,11,32] . > > Conclusions > > Babesia spp. are a seriously understudied group of parasites. However, at least five mechanisms have been identified which might contribute to their evasion of host immunity. Antigenic variation, cytoadhesion/sequestration, host-protein binding, and induction of immunosuppression probably facilitate persistence in the individual immune host. Monoallelic expression of different members of a multigene family might facilitate multiple infections of immune hosts, and population dispersal in endemic areas. > > Acknowledgements > > D.R.A. gratefully acknowledges Basima Al-Khedery, Crabtree, a O'Connor, Kristi Warren and Jervis for their assistance. This work was supported by grants from the United States Dept. of Agriculture (#20013520410144) and the American Heart Association (#0051422B).

[Guest guest]

>Nelly the cartoon doesn't open up.>tony

Tony, Isn't past your bedtime? Just one cartoon and off to bed you go, nightynight!

Nelly

[Guest guest]

D. Allred DVM has been studing and publishing about b.bovis and b.

divergens (european strains of Babs) and comparing it to Malaria for

over 20 years in Vet journals.

Seach on Pubmed on ALLRED D R and all his papers will come up.

Barb

> > I recently posted this on Lymenet, I'm sure some of you have seen

> it before but as the link to the article is now defunct and as it

is

> a very very good article + animation on Babesiosis and how it

> persists, I thought I would post it here.

> >

> > (Tony can look at the pretty cartoon, might keep him quiet for a

> while!)

> >

> > Babesiosis: persistence in the face of adversity

> >

> > For animated version, go to:

> http://archive.bmn.com/supp/part/allred.html.

> >

> > R. Allred allredd@m...

> >

> > Trends in Parasitology 2003, 19:51-55

> >

> > Dept of Pathobiology, College of Veterinary Medicine, University

> of Florida, Gainesville, FL 32611-0880, USA

> >

> > Abstract

> >

> > Many babesial parasites establish infections of long duration in

> immune hosts. Among different species, at least four mechanisms are

> known that could facilitate evasion of the host immune response,

> although no one species is (yet) known to use them all.

> >

> > This update strives to illustrate the ramifications of these

> mechanisms and the interplay between them.

> >

> > Babesia spp. are a diverse group of tick-borne, obligate,

> intraerythrocytic Apicomplexan parasites infecting a wide variety

of

> organisms.

> >

> > Infection of a vertebrate host is initiated by inoculation of

> sporozoite stage parasites into the bloodstream during the taking

of

> a bloodmeal.

> >

> > Most babesial sporozoites directly invade circulating

erythrocytes

> without a tissue stage of development [1]. A few, notably Babesia

> equi [2] and Babesia microti [3], first invade lymphocytes where

> they form motile merozoites, which then invade erythrocytes.

> Although this undoubtedly affects their interactions with the host,

> any effects on immune evasion are at present unknown.

> >

> > Once erythrocyte invasion occurs, a seemingly perpetual cycle of

> asexual reproduction is established, despite the rapid development

> of a strong immune response [4].

> >

> > During the acute babesial infection, the host may become severely

> ill. Typically, the infected host can suffer high fevers, severe

> anemia, hemoglobinuria caused by intravascular hemolysis

> considerably in excess of that correlated with parasitemia,

> lethargy, inappetance, and sometimes hydrophobia [5]. Coagulatory

> disturbances are also a frequent finding [69].

> >

> > Neurologic sequelae may occur with some babesial parasites, most

> notably Babesia bovis. In the case of B. bovis, this is accompanied

> by massive intravascular sequestration of infected red blood cells

> (IRBC) carrying mature parasite stages [1013] .

> >

> > Despite the potential severity of the acute infection,

individuals

> who survive generally develop immunity against disease, but not

> against infection per se, and could remain persistently infected

> [1418] .

> >

> > In the case of B. bovis, infections can persist for years,

perhaps

> even the lifetime of the animal.

> >

> > Babesial parasites clearly have adapted well to survival in the

> hostile environment that is the immune host. How do they do this?

> >

> > We understand very little about the mechanisms used by these

> parasites to survive.

> >

> > However, at least five different phenomena are known that

probably

> contribute to parasite survival:

> >

> > (1) rapid antigenic variation ( Fig. 1);

> >

> > (2) cytoadhesion ( Fig. 2 and see animation on:

> http://archive.bmn.com/supp/part/allred.html) and sequestration;

> >

> > (3) binding of host proteins to the IRBC surface;

> >

> > (4) the monoallelic expression of different members of multigene

> families; and

> >

> > (5) establishment of a poorly understood transient

> immunosuppression [1921] .

> >

> > The relative contributions of the individual phenomena are not

> known, nor have definitive demonstrations yet been made that any of

> these directly contributes to survival. Here, only brief

discussions

> of specific examples will be presented, along with an attempt to

put

> these phenomena into perspective relative to the establishment of

> persistent infection.

> >

> > [graphic omitted]Fig. 1. Antigenic variation in Babesia bovis

> appears to proceed via a segmental gene conversion mechanism. In

> this mechanism, sequences are duplicated from donor gene copies [a

> chromosomal segment containing donor genes (orange, purple and

green

> boxes) is shown in (a)] into an actively transcribed variant

> erythrocyte surface antigen (ves) 1 gene (ves1 site of

> transcription, yellow box) in (.

> >

> > With repeated instances of segmental gene conversion [presumably

> only one per generation, e.g. (bi), (bii), (biii)], the actively

> transcribed ves1 gene becomes progressively altered from the

> original, whereas the donor gene copies appear to remain unaltered.

> >

> > The actual mechanism by which duplicated sequences are

transferred

> is not known. This site of transcription is shown in a hypothetical

> placement near the telomeric end (shown as boxed red and black

> repeat units). Hypothetical promoters are indicated by black flags

> and hypothetical telomere terminal structures are represented by

> pink boxes after the repeat units.

> >

> > [graphic omitted]Fig. 2. Cytoadhesion of Babesia bovis. Babesia

> bovis-infected red blood cells (IRBC) can adhere to the capillary

> endothelium of blood vessels of various tissues, a behavior that

> could be abrogated by host antibodies recognizing parasite antigens

> on the IRBC surface.

> >

> > (a) Babesia bovis IRBC carrying ring stages or mature stages

> (trophozoites or meronts) that are expressing non-adhesive surface

> molecules remain in the peripheral circulation, and will pass

> through the spleen. Whereas ring-stage IRBCs will probably survive

> splenic passage, IRBC carrying mature parasites will be removed and

> destroyed as a result of antibody recognition of the antigens

> expressed on IRBC surface.

> >

> > ( Babesia bovis IRBC carrying mature parasites that are

> expressing adhesion-competent forms of proteins exported to the

IRBC

> surface (probably VESA1; shown as green triangles) will adhere to

> the endothelium of the capillaries and post-capillary venous

> circulation (cytoadhesion). These parasites will mature in the deep

> microvasculature and release their merozoite stages there.

> >

> > © Babesia bovis IRBC expressing different isoforms of the

> adhesive molecules on the IRBC surface (shown as green U-shaped

> molecules) will bind to different receptors and endothelial cells.

> This could account for apparent tissue tropism in this parasite,

> including that occurring within the brain.

> >

> > (d) The development of antibodies (shown as Y-shaped molecules)

by

> the immune host is thought to be capable of preventing cytoadhesion

> and of causing already-bound IRBCs to be released from the

> endothelium. When this occurs, the IRBC are probably susceptible to

> splenic removal.

> >

> > Note these antibodies recognize the ligands from (, but have no

> effect on IRBC expressing the ligands from ©.

> >

> > For animated version, go to:

> http://archive.bmn.com/supp/part/allred.html.

> >

> > Antigenic variation

> >

> > The first evidence for antigenic variation in babesial parasites

> was obtained with Babesia rodhaini [22] and later with B. bovis

> [23]. The results of these studies strongly suggested that

> fundamental changes had occurred in the antigenicity of protective

> antigen(s) in the surviving parasite populations, and demonstrated

a

> probable association between recognition of the variant antigen and

> immune protection. Unfortunately, the parasite populations were not

> clonal, precluding rigorous interpretation of these experiments,

and

> the identities of the variant antigen(s) in B. rodhaini remain

> unknown.

> >

> > In contrast to the situation with B. rodhaini, a variant antigen

> expressed by B. bovis has been identified. A size-polymorphic,

> parasite-derived antigen was identified on the B. bovis IRBC

> surface. This antigen was recognized in an isolate-specific manner

> during live-cell immunofluorescence and surface-specific

> immunoprecipitation assays [24]. The application of these assays to

> parasites recovered at different times from a calf, infected once

> with a clonal B. bovis line, revealed that the isolate-specific

> antigen underwent rapid size and antigenic variation [16]. The

> putative variant antigen was subsequently confirmed through the use

> of monoclonal antibodies (mAb), and was named the variant

> erythrocyte surface antigen (VESA) 1 [25]. This antigen migrates on

> sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)

> as a doublet, ranging in mass between 105 kDa and 135 kDa, among

> different variants and isolates. Recently, the ves1 multigene

family

> encoding the larger subunit (VESA1a) was identified [26], and

> preliminary estimates suggest a minimum of 50 gene copies. The

> molecular mechanisms involved in expression of variation are only

> beginning to be worked out, but all evidence to date is consistent

> with progressive, segmental gene conversion playing a key role [26]

> ( Fig. 1). Some ves1 genes can donate sequences to the transcribed

> gene copy, while apparently remaining unaltered themselves ( [26];

> B. Al-Khedery et al., unpublished). It is not yet clear whether all

> ves1 genes can participate in the phenomenon of gene conversion.

> Coupled with a similarly polymorphic VESA1b subunit [16,24,25] ,

the

> potential capacity of this parasite for presentation of unique

VESA1

> antigens is enormous.

> >

> > Cytoadhesion and sequestration

> >

> > The spleen is a remarkable organ in its ability to recognize and

> remove damaged cells from circulation. It could be aided by the

> presence of bound antibodies and, perhaps complement, on the

damaged

> cell's surface. Accordingly, passage through the spleen is a

journey

> to be avoided by parasitized erythrocytes. The ability to avoid

> splenic passage has been observed in Babesia canis [27], the

Babesia

> Washington state isolate 1 (WA1) [28] and B. bovis [29,30] ,

> although it is likely that different mechanisms are used to achieve

> sequestration. It has been suggested that B. canis becomes trapped

> in the deep vasculature through generalized vasodilation and

> hypotensive pooling of blood. Under these conditions, IRBC might

> fail to circulate, becoming trapped in local coagulatory masses

> where they undergo proliferation [27]. Nothing is currently known

of

> the mechanism(s) used by the WA1 isolate to sequester, and the act

> of sequestration itself could be host-specific [28,31] .

> >

> > By contrast to B. canis, B. bovis sequesters in the

> microvasculature through specific binding of IRBC to the capillary

> and post-capillary endothelium via knob-like protrusions of the

IRBC

> membrane ( Fig. 2 and http://archive.bmn.com/supp/part/allred.html)

> [10,11,32] . This binding (cytoadhesion) and sequestration is

> associated with the severe and frequently lethal cerebral form of

> bovine babesiosis [1013,30] . Using an in vitro assay of

> cytoadhesion to characterize this phenomenon, it was shown that

> parasite-synthesized components must be present for binding to

occur

> [33]. Further, strong circumstantial evidence was found for

> mediation of this phenomenon by the VESA1 antigen [34]. Clear

> precedents exist, in the Plasmodium falciparum erythrocyte membrane

> protein (PfEMP) 1 [35,36] , and the M proteins of Streptococcus

> pyogenes [37], for mediation of pathogen adhesion to host cells by

> highly variant components. The endothelial receptor for B. bovis

> cytoadhesion is not known, but current data do not support a

> significant role for compatibility determinant (CD)36 [33], the

> major P. falciparum receptor.

> >

> > It has recently been hypothesized that sequestration results in

an

> enhanced parasite susceptibility to killing by reactive nitrogen

> oxides due to unloading at reduced oxygen tension of erythrocytes

> laden with nitric oxide (NO) [38]. It was previously shown that B.

> bovis-stimulated macrophages produce NO, and that in vitro parasite

> killing by NO can be achieved [39]. However, a diminished capacity

> for macrophage NO production has been observed under reduced oxygen

> tension [40], and other studies demonstrate NO consumption by

> oxyhemoglobin, with the release only of nitrate and methemoglobin

> [41]. Thus, sequestration might serve to enhance parasite survival

> by preventing localized circulation of NO-carrying erythrocytes and

> maintaining local hypoxia. Clearly, all the ramifications of

> cytoadhesion are not yet understood, but the ability to sequester

> and not suffer splenic removal is probably a very important

> mechanism of long-term survival by this parasite.

> >

> > Binding of host proteins to the IRBC surface

> >

> > Not all babesial parasites appear to undergo antigenic variation,

> and most do not cytoadhere or sequester. The bovine parasite,

> Babesia bigemina, is a good example. Although B. bigemina expresses

> parasite-derived antigens on the IRBC surface, the antigens appear

> to be isolate-common and stable over the course of infection [42].

> However, B. bigemina IRBC bind immunoglobulin (Ig) M on the IRBC

> surface, in a non-immunospecific reaction [43]. Presentation of IgM

> molecules, bound through the Fc region with the antigen recognition

> domain facing the plasma, clearly represents a mechanism with the

> potential to hide this parasite from immune recognition. It is

> possible that the invariant IRBC surface antigens mediate IgM

> binding, but such a connection has not been made. The number of IgM

> bound on individual cells is also not known, but if sufficiently

> dense could render these antigens inaccessible to specific

> antibodies and leave IRBC 'invisible' to circulating immune

effector

> cells. How this would affect the interaction of IRBC with the

spleen

> is less clear, but might reduce susceptibility to splenic clearance

> as well, and could help to explain the persistence (up to two years

> [14]) of this parasite.

> >

> > Monoallelic expression of members of multigene familiesBabesia

> microti is by far the most common cause of human babesiosis [44].

> Similar to many babesial parasites, this species is capable of

> persisting in the immune host, even after non-sterilizing

> chemotherapy [18]. The mechanisms used to survive in individual

> hosts have not yet been elucidated, but a recent clue to population

> survival might have emerged. Using immune sera from individuals who

> had recovered from babesiosis to screen complementary DNA (cDNA)

> libraries, three immunodominant antigen gene families were

> discovered [45]. In a limited study monitoring expression of the

> Babesia microti MN1 strain (bmn1) gene family, it appeared that a

> single member was transcribed. Further, it was revealed that

> different alleles were expressed by parasites infecting patients

> from different geographical areas. By contrast, the same allele was

> expressed in several patients from a small area of transmission,

and

> this was stable upon transmission to hamsters [46]. These results

> make it unlikely that the bmn1 gene family is involved in antigenic

> variation as was suggested by the authors. However, the

considerable

> diversity within this gene family and its (apparently) subtelomeric

> location would be consistent with frequent recombination events. If

> bmn1 gene products are associated with immunoprotection, this

> allelic diversity might provide the parasite with an opportunity to

> establish mixed infections in immune hosts already primed by

> exposure to other members of the family. In such a scenario, the

> temporal nature might lie between the very rapid change of

antigenic

> variation and the relatively slow evolution associated with allelic

> polymorphism. Despite the attractiveness of this possibility, the

> association of allelic polymorphism per se with immune evasion is

> not always clear. For example, in B. bigemina, four alleles of the

> merozoite surface antigen (msa) 2 multigene family have been

> described, the products of which are immunologically non-

> crossreactive [47]. At least three of the four alleles are co-

> expressed on individual merozoite- and sporozoite-stage parasites,

> yet there is no additive effect on inhibition of invasion (or

> binding) by simultaneous exposure to antibodies against all four

> gene products [48].

> >

> > Interplay of these phenomena

> >

> > Currently, nothing is known of interplay between antigen masking

> or monoallelic expression of invariant multigene families and

> antigenic variation or cytoadhesion, if such interaction occurs.

> Strong circumstantial evidence suggests that the rapidly variant

> VESA1 antigen on the B. bovis IRBC surface serves as a parasite-

> derived ligand mediating cytoadhesion [34]. One obvious potential

> outcome of this is that an in vivo switch in an antigenic

phenotype,

> selected by immune recognition of the previously expressed VESA1a

> isoform, probably results in alterations of the adhesive phenotype

(

> Fig. 2 and http://archive.bmn.com/supp/part/allred.html). This

> effect was observed in vitro, using an assay of B. bovis

> cytoadhesion to bovine brain endothelial cells [34]. Further, the

> C9.1 line-derived mAb, 3F7.1H11, which also reacts with the

> cytoadhesive CD7 line (as a result of selection for this trait), is

> capable of both blockage and reversal of in vitro cytoadhesion,

with

> great efficacy [34]. In addition, the adhesion specificity of

> different clonal B. bovis lines for different clonal bovine brain

> endothelial cell lines varies (R.M. O'Connor and D.R. Allred,

> unpublished), suggesting either the recognition of different

> endothelial receptors, varied affinities for shared receptors, or

> both. Thus, in vivo recognition and elimination of one parasite

> population, with expansion of another, probably results in the

> selection of parasites with differing adhesive phenotypes and

> perhaps tissue tropism ( Fig. 2 and

> http://archive.bmn.com/supp/part/allred.html). Given the tissue-

> specific presentation of endothelial receptor motifs [49], a

> protective immune response to one population could inadvertently

> result in relocation of the infection focus, modifying the

potential

> pathology, and perhaps delaying the initiation of a response to

> surviving parasites. Early studies on the histopathology of B.

bovis

> infection reported widely varying parasitemias for parasites

> localized in different tissues, consistent with this possibility

> [10,11,32] .

> >

> > Conclusions

> >

> > Babesia spp. are a seriously understudied group of parasites.

> However, at least five mechanisms have been identified which might

> contribute to their evasion of host immunity. Antigenic variation,

> cytoadhesion/sequestration, host-protein binding, and induction of

> immunosuppression probably facilitate persistence in the individual

> immune host. Monoallelic expression of different members of a

> multigene family might facilitate multiple infections of immune

> hosts, and population dispersal in endemic areas.

> >

> > Acknowledgements

> >

> > D.R.A. gratefully acknowledges Basima Al-Khedery, Crabtree,

> a O'Connor, Kristi Warren and Jervis for their

> assistance. This work was supported by grants from the United

States

> Dept. of Agriculture (#20013520410144) and the American Heart

> Association (#0051422B).

[Guest guest]

D. Allred DVM has been studing and publishing about b.bovis and b. divergens (european strains of Babs) and comparing it to Malaria for over 20 years in Vet journals.Seach on Pubmed on ALLRED D R and all his papers will come up.

Barb,

Thanks, you know I am sure I have read his abstracts a zillion times and thought yeah OK cytoadherence, microvasculature, antigenic variability etc but for some reason this full-text article with great pictures as well as the article with the animation is making it all a lot clearer for me at this point in time.

Now I feel I have a bit more of a grasp on the mechanisms behind my improve/relapse when on then off mefloquine.

I know that in the area where I was bitten in France (well one of the areas) I know that Babesia bovis is rampant (I was in contact with a University vet researching it in Nantes).

Nelly

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve & db=pubmed & dopt=Abstract & list_uids=10417157 & query_hl=1

[infections] Re: Babesiosis: persistence in the face of adversity

D. Allred DVM has been studing and publishing about b.bovis and b. divergens (european strains of Babs) and comparing it to Malaria for over 20 years in Vet journals.Seach on Pubmed on ALLRED D R and all his papers will come up.Barb> > > Nelly the cartoon doesn't open up.> tony

[Guest guest]

<dumbaussie2000@y...> wrote:

> Nelly the cartoon doesn't open up.

> tony

The cartoon crashed my internet. :-(

Hey Tony, you really should go back to school. With the right

credentials, you'd be a formidable force to reckon with. Not only

would people finally respect you at least a little, (no matter how

begrudgingly), EVERYBODY would get to hate you then!

Hey, you live in Oz! You should go see the Wizard! Get yourself a

diploma. :-)

penny

p.s. pick me up a brain while you're at it. I can always use a

spare. (Although a few people don't think I have one now. :-)

--- In infections , " dumbaussie2000 "

[Guest guest]

>The cartoon crashed my internet. :-(

Penny, what do you mean?

Did you use this link:

http://archive.bmn.com/supp/part/allred.html

it works fine for me.

Has anyone else been able to open it?

Nelly

[Guest guest]

Yeah, I don't know. Can't open it. The last link isn't even

clickable for some reason, although it looks like it should be, so I

cut and paste it, and it still won't open. Everything just freezes

up, even my back button. Don't know why.

penny

> >The cartoon crashed my internet. :-(

>

> Penny, what do you mean?

>

> Did you use this link:

>

> http://archive.bmn.com/supp/part/allred.html

>

> it works fine for me.

>

> Has anyone else been able to open it?

>

> Nelly

[Guest guest]

Worked like a charm for me, Nelly. And I agree with you, the

animation is actually quite helpful. I read the text version first,

and was amazed at how much of it was condensed in that flash piece.

Penny, I've had that happen before, usually installing the latest

flash player from Macromedia takes care of it (even if you have the

current version).

Cheers,

S.

> >The cartoon crashed my internet. :-(

>

> Penny, what do you mean?

>

> Did you use this link:

>

> http://archive.bmn.com/supp/part/allred.html

>

> it works fine for me.

>

> Has anyone else been able to open it?

>

> Nelly

[Guest guest]

I was able to play it. Cool

Barb

> >The cartoon crashed my internet. :-(

>

> Penny, what do you mean?

>

> Did you use this link:

>

> http://archive.bmn.com/supp/part/allred.html

>

> it works fine for me.

>

> Has anyone else been able to open it?

>

> Nelly

[Guest guest]

Penny

I opened it and found it very informative how those little red cells

get parasitized.My problem is why isn't it being discovered clearly

and cleanly.If you have a red cell parasite- it has to rear it's

ugly head with a correct staining procedure.

Jill what is the fish test, is it as clear as the cartoon or is it

checking for an event occured type thingy.

tony

> > Nelly the cartoon doesn't open up.

> > tony

>

>

> The cartoon crashed my internet. :-(

>

> Hey Tony, you really should go back to school. With the right

> credentials, you'd be a formidable force to reckon with. Not only

> would people finally respect you at least a little, (no matter how

> begrudgingly), EVERYBODY would get to hate you then!

>

> Hey, you live in Oz! You should go see the Wizard! Get yourself a

> diploma. :-)

>

> penny

>

> p.s. pick me up a brain while you're at it. I can always use a

> spare. (Although a few people don't think I have one now. :-)

>

>

>

> --- In infections , " dumbaussie2000 "