Loa loa: immunological responses during experimental infections in mandrills (Mandrillus sphinx).

Six intact, adult mandrills (Mandrillus sphinx) were infected with human-derived, diurnal Loa loa infective larvae. Microfilaremia, hematological, and immunological parameters were followed for 2-4 years. A major aim was to investigate the relationship between specific humoral immunity to microfilariae and microfilaremia and also to assess whether infection led to generalized immune dysfunction. Microfilaremia was similar to previous studies for 4 mandrills, with a prepatent period of 153.5 +/- 10.1 days, a peak of 34-1,798 mf/ml around Day 200, followed by a decline to low, persistent microfilaremia. One mandrill (No. 20) had a longer prepatent period and very low, but persistent, levels of microfilaremia, and one (No. 19) had gradually increasing levels which remained > 10,000 mf/ml for 3 years. To assess generalized immune perturbations several parameters were studied. There was neither generalized leukocytosis nor relative or absolute eosinophila. Serum Ig concentrations were measured from 0-600 days postinfection by radial immunodiffusion using a rabbit anti-mandrill Ig serum, and these were remarkably stable. Proliferative responses of peripheral blood mononuclear cells from these infected mandrills and noninfected controls showed no significant differences in the magnitude of proliferation after stimulation with a range of doses of PHA, PWM, or Concanavalin A. Thus, no evidence of generalized immune dysfunction was found in the peripheral blood. Serum IgG levels to soluble mf antigens were estimated by an indirect ELISA and all animals had maximal levels around Week 22 postinfection, at the time of maximum microfilaremia, and these decreased over the next 2-3 years in all mandrills except No. 19, in which levels remained fairly constant. Serum IgG levels to adult worm antigens showed a similar pattern but were not, or were only slightly, diminished late in infection. Antibody to mf sheath antigens were detected by indirect immunofluorescence and agglutination of live mf. Antibody to sheath antigens were never detected in mandrill 19 but were present from Week 8 postinfection to 2-4 weeks before patency in all the others. Anti-sheath antibodies were not detected in serum at later time points, i.e., postpatency, in any mandrill, even at time points when microfilaremia was < 1 mf/ml. The anti-sheath antibody was IgM and no anti-sheath IgG was detected. Ig was detected on the surface of circulating mf in 1 mandrill (No. 20). The appearance of these antibodies prior to maturation of the adults indicates that certain L4 or immature adult antigens cross-react with the surface of mf.(ABSTRACT TRUNCATED AT 400 WORDS)

A highly defective HIV-1 strain isolated from a healthy Gabonese individual presenting an atypical western blot.

In central equatorial Africa the frequency of uninterpretable or atypical Western blots (WB)--ie. antibodies to gag proteins only--can represent up to 50% of enzyme-linked immunosorbent assay (ELISA)-positive samples. To date the significance of such serology remains unknown. Nevertheless, an unusual HIV-1 strain has been isolated from the blood of a healthy Gabonese individual who presented an atypical WB. This virus, identified as isolated HIV-1OYl, grew to low titres of reverse transcriptase activity (less than 50,000 cpm/ml) and was not obviously cytopathic. Radioimmunoprecipitation and peptide ELISA studies indicated that the lack of env-specific reactivity was probably due to the absence of antibodies to the viral glycoproteins, rather than the virus encoding a highly divergent envelope protein. Molecular cloning and sequencing of the provirus proved it to be a string of HIV-1 which was genetically closer to European and North American than to African strains. Furthermore the envelope protein sequence contained all the features of a typical HIV-1 env gene. However, the tat gene derived from the proviral clone was functionally defective. Site-directed mutagenesis of this gene showed that this was due to the substitution of an essential cysteine residue for a serine. Polymerase chain reaction amplification of the tat gene, as well as parts of the gag and env gene sequences of HIV-1OYl, showed that essentially all of the proviruses were defective. These data emphasize the need to view HIV isolates as populations of distinct genomes capable of complementing each other.

Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis.

Possible endocrinological repercussions of infection with Loa loa and Mansonella perstans filariae were studied in Gabonese subjects. Microfilaremic males were compared with amicrofilaremic controls. In the infected group 13/105 subjects (12%) presented only abnormally low serum levels of testosterone (less than 4 ng/ml), 25/105 (24%) only abnormally high serum levels of gonadotrophins, FSH (greater than 15 mIU/ml) and LH (greater than 20 mIU/ml), and 22/105 (21%) presented anomalies in both testosterone and gonadotrophin levels. One out of 68 control subjects had 3.6 ng/ml seric testosterone and all had normal levels of gonadotrophins. Ecdysteroids were detected (greater than 0.025 ng/ml) in the serum of 87/97 (90%) microfilaremic subjects (GM 0.123 ng/ml) compared to 12/64 (19%) controls (GM 0.030 ng/ml). Ecdysteroids were detected in the urine of all subjects, infected (GM 8.468 ng/ml) as well as control (GM 1.245 ng/ml). The hormonal perturbations were correlated with the levels of Loa loa microfilaremia but not with those of serum and urinary ecdysteroids. These results demonstrate that microfilaremic subjects often show endocrinal signs of hypogonadism and present appreciable levels of ecdysteroids in serum and urine. A direct role for parasitic ecdysteroids in hypogonadism remains to be demonstrated.

Isolation and characterization of simian immunodeficiency virus from mandrills in Africa and its relationship to other human and simian immunodeficiency viruses.

Two isolates of simian retrovirus related to the human immunodeficiency virus (HIV) were obtained from apparently healthy mandrills, Papio (Mandrillus) sphinx, in western equatorial Africa. This virus, designated SIVMND (simian immunodeficiency virus from mandrills), appeared morphologically similar to HIV by electron microscopy, showed Mg2+-dependent reverse transcriptase activity, and induced cytopathic effect in human CD4-positive cells. Western blotting (immunoblotting) analyses revealed that the gag and pol products of SIVMND showed cross-reactivity with those of known HIVs and SIVs. Molecular clones covering full-length viral DNA were obtained from closed circular extrachromosomal DNA of SIVMND-infected cells. By clone-on-clone hybridization with known retroviruses of the HIV and SIV groups, SIVMND showed similar cross-hybridization with HIV-1, HIV-2, SIVAGM (African green monkey-derived SIV), and SIVMAC (rhesus macaque-derived SIV) in the gag and pol regions only at low stringency but not at high stringency, a result indicating that SIVMND is a new member of the HIV-SIV group. The existence of distinct SIVs in different monkey species suggest that recent interspecies transfer of HIV-SIV is unlikely in nature.

Trypanosoma brucei: polyamine oxidase mediated trypanolytic activity in the serum of naturally resistant cattle.

Trypanosoma brucei brucei are lysed when incubated in vitro in a mixture of bovine serum and polyamine. Normal bovine serum alone or polyamine alone does not show any trypanocidal activity. The bovine serum in the mixture can be replaced by purified polyamine oxidase, and addition of polyamine oxidase inhibitors blocks trypanolysis. Using this in vitro lysis test, it is shown that West African cattle which are resistant naturally to trypanosomiasis have a higher trypanolytic activity in their serum than do trypanosensitive cattle (P less than 10(-5]. Seric trypanolytic activity of individual animals remains stable when tested over a period of 18 months; moreover, it is not modified by trypanosome infection. Higher levels of seric polyamine oxidase in resistant cattle were demonstrated also by enzymatic analysis. The factors responsible for trypanolysis have been analyzed. Oxidation of spermidine by polyamine oxidase leads to the production of unstable aldehydes, acrolein, ammonia, O2-, HO, and H2O2. Acrolein and H2O2 show strong trypanolytic activity while the other products do not appear to be toxic for trypanosomes. The physiological importance of polyamine oxidase mediated trypanolysis is unclear; even at peak parasitemia in cattle (10(7) organisms/ml) it can be calculated that trypanosomes would not release enough spermidine for the generation of sufficient quantities of toxic degradation products. Additional polyamines could be released in serum from tissues damaged as a result of the infection.

Trypanosoma congolense: lack of correlation between the resistance of cattle subjected to experimental cyclic infection or to field challenge.

Twelve male cattle of the Baoulé breed were exposed to natural trypanosome challenge in an area of high Glossina density, to characterize them as trypanoresistant or trypanosensitive. Weekly blood samples were taken for the determination of parasitemia and packed cell volume, as a measure of anemia. Seven Zebu cattle were also exposed to challenge at the same time. The Zebu proved to be trypanosensitive with high parasitemia, pronounced anemia and died or were drug treated in extremis. Five Baoulé were as sensitive as the Zebu while 7 others were trypanoresistant since they showed little or no patent parasitemia, only mild transient anemia and survived in good condition. The 12 Baoulé were allowed to recover from challenge in the field and along with 7 Zebu were subjected to experimental fly challenge in fly-proof accommodation. Glossina morsitans submorsitans infected with a clone of Trypanosoma congolense derived from the stock Serengeti/71/STIB/212 were allowed to engorge on the shaven flanks of tranquilized animals. All animals showed persistent parasitemia for at least 7 weeks, including all the Baoulé resistant to natural challenge. Two Baoulé, one resistant and one sensitive to natural challenge, and 4/7 Zebu appeared unable to control parasitemia, had severe anemia, and were drug treated in extremis. The remaining Baoulé, 6 resistant and 4 sensitive, appeared to be undergoing spontaneous cure by Week 9-10, as did 3/7 Zebu. In Zebu, anemia was as pronounced as under natural challenge. Three resistant Baoulé maintained packed cell volume above 30 as under field challenge but the others showed marked anemia. On the contrary, 4 sensitive Baoulé showed only slight anemia after artificial fly challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of antibody in natural resistance to African trypanosomiasis.

The nature and usefulness of trypanoresistance in West African Baoule cattle was studied by exposing animals in areas of high Glossina density. Under such conditions, Zebu and some Baoule died soon with high parasitaemia while resistant Baoule showed little patent parasitemia, almost no anaemia and thrived. Subsequently the role that specific antibody may play in such trypanoresistance was analyzed. In the first instance we examined protective antibody titres during T. congolense infection of inbred mice strains, comparing results obtained in trypanosome-mouse combinations where the host controls parasitemia and survives with those obtained when the host fails to control parasitemia and dies. We then attempted to extend these observations to cattle by following the disease course and appearance of neutralizing antibodies in animals of known sensitivity to natural Glossina challenges, following artificial challenge with T. congolense infected Glossina.

The virulence of Trypanosoma congolense can be determined by the antibody response of inbred strains of mice.

Three inbred strains of mice, BALB/c, C57Bl/6 and CBA/J were infected with three clones of Trypanosoma congolense, DIND/3.1, SAM/28.1 and KAR/57.1, which were obtained from three different stocks. DIND/3.1 was of high virulence for BALB/c and CBA/J but of negligible virulence for C57Bl/6. SAM/28.1 was of high virulence and KAR/57.1 of negligible virulence for the three strains of mice. In each case, high virulence was correlated with a late, transient and low titre protective antibody response measured by complement mediated lysis of live organisms. Negligible virulence was correlated with an early, high titre protective antibody response. Suppression of the antibody response by sub-lethal irradiation or cyclophosphamide treatment of the host turned a trypanosome infection of negligible virulence into one of high virulence. In mice with mixed infections it was shown that highly virulent trypanosomes did not influence the course of infection and antibody response to trypanosomes of negligible virulence and vice-versa. The relationship of total antigen mass to the kinetics of the antibody response suggests that 1000- to 10,000-fold less antigen is required in good responder than in bad responder mice to trigger the immune response. Thus the virulence of T. congolense can be determined by the antibody response of inbred strains of mice. The specificity and dose dependency of this antibody response seem to implicate the involvement of Ir genes.

Identification and selection of cattle naturally resistant to African trypanosomiasis.

Cattle were exposed to natural trypanosome challenge in an area of high Glossina density (Samandéni, Burkina Faso) for various periods of time during 1982, 1983, 1984 and 1985. All of 30 Zebu proved to be sensitive to trypanosomiasis i.e. they died or were treated in extremis in 10 +/- 4 weeks. Twenty-one (31%) Baoulé were as sensitive as the Zebu while 47 (69%) were resistant i.e. they survived in good condition. Twenty Ndama/Baoulé crosses, indigenous to Samandéni were all resistant. Weekly blood samples were taken (2,317 in total) for the determination of parasitaemia and packed cell volume (PCV) as a measure of anaemia, the most important pathological feature of cattle trypanosomiasis. In both Zebu and sensitive Baoulé 59% of the blood samples showed positive parasitaemia, of which 38% and 52% respectively were T. congolense the major cattle pathogen in the area considered. In resistant Baoulé and Ndama/Baoulé 11% and 10% of the samples were positive for trypanosomes of which only 4% and 2% were T. congolense respectively. PCV decreased from 35 to 20 in Zebu, 39 to 20 in sensitive Baoulé and 40 to 34 in resistant Baoulé, there was no change in the indigenous Ndama/Baoulé. Six Ndama/Baoulé indigenous to Samandéni remained resistant to trypanosomiasis when moved to another area of high Glossina challenge. Seven Ndama/Baoulé calves, conceived in Samandéni but born and kept for 2 1/2 years in a Glossina-free area, also proved to be resistant to challenge. Twelve Baoulé calves, born from cattle selected under natural field challenge, and which had not come in contact with trypanosomes for the first 10 months of their life, proved to be resistant when exposed in the field. These observations show that some, but not all, cattle from the Baoulé breed are naturally resistant to African trypanosomiasis, that this resistance does not need repeated exposure to trypanosomes early in life but appears to be inherited and functional against many types of antigenically different trypanosomes. Thus, selective breeding of trypanoresistant animals and their successful introduction, without trypanocidal drug protection, into areas of high Glossina density appears feasible.

Susceptibility and immune responses of zebu and taurine cattle of West Africa to infection with Trypanosoma congolense transmitted by Glossina morsitans centralis.

Following tsetse-transmitted infection with Trypanosoma congolense, major differences in development of localised skin reactions, the ability to control parasitaemia, the degree of anaemia and in antibody response to trypanosomes were found between the reputedly trypanotolerant breeds of cattle (N'Dama, N'Dama/Baoule crosses, Baoule) and the trypanosusceptible West African Zebu. The local skin reactions that developed in the Zebu were large and severe while those that occurred in the other breeds were smaller and less severe or mild. The timing of appearance of parasitaemia and the height of the first peaks were similar in all the animals, but the Zebu were less able to control subsequent waves of parasitaemia. Possibly reflecting these events, it was only in the Zebu that significant anaemia developed. Neutralizing antibody against homologous metacyclic trypanosomes developed between 14 to 18 days after infection in all breeds of cattle; however, marked differences were found when antibody to trypanosomes derived from first peak parasitaemias were tested in the Zebu and Baoule. Neutralizing antibody against these parasites appeared in the Baoule on day 24 but were not detected in Zebu until day 51. Furthermore, the antibody titres were 3 log2 higher in the Baoule. It was concluded that the trypanotolerance exhibited by the West African taurine cattle might be related to a) their ability to control trypanosome numbers in the skin and in the bloodstream, an outcome that was possibly brought about by the earlier and superior immune response and b) failure to develop anaemia which might be associated with their capacity to control parasitaemia.

Natural resistance to African trypanosomiasis.

Glossina infected with African trypanosomes infest 10(7) Km2 of intertropical Africa. Ten thousand cases of human sleeping sickness are officially recorded each year and 35 X 10(6) human beings are at risk. Animal trypanosomiasis impedes the use of 7 X 10(6) Km2 of land adequate for cattle raising and constitutes a major constraint to increasing protein production in Africa. Several approaches are used to combat trypanosomiasis (a) vector eradication has been successful in certain defined situations but cannot be realistically extended to the whole area at risk, (b) prophylactic drugs, which are too toxic for humans, are widely used to protect cattle. This practice has led to field resistance to many drugs used in the past (reviewed in Holmes & Scott 1982) and the appearance of resistance to the last available prophylactic drug, isometamidium, is being reported (Bourn & Scott 1978, Küpper & Wolters 1983, Pinder & Authié 1984), (c) the hope for a vaccine has been largely abandoned, in the present state of knowledge, due to the considerable extent of antigenic diversity in trypanosomes (reviewed in Doyle 1977, Turner 1982, Roelants & Pinder 1984), (d) consequently, the possible use of certain West African breeds of cattle, which appear resistant to trypanosomiasis, has been emphasized as a solution to this problem in domestic animals. The analysis of this natural resistance is the subject of the present essay.

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle.

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.

Correlation of serum zinc levels with resistance of cattle to trypanosomiasis.

Zinc, copper and magnesium levels were determined by atomic absorption spectrophotometry in the serum of 32 cattle (Zebus and Baoulés) which were proven to be sensitive to African trypanosomiasis under field challenge and 45 cattle (Baoulés and Ndamas/Baoulés) which were proven to be resistant. Copper and magnesium levels were similar in all animals but zinc levels were higher in sensitive animals (1.50 ppm) than in resistant ones (1.10 ppm) (p less than 10(-5)); the reported normal levels of serum zinc is 1.00 ppm. These differences persisted on repeated measurements and whether individuals were infected with trypanosomes or not. Elevated levels of zinc depressed the stimulation of bovine T cells by trypanosomes in vitro and is reported to inhibit antigen presentation by macrophages. Zinc levels may be an influencial factor determining susceptibility or resistance of West African cattle to trypanosomiasis.

High and low responsiveness of bovine lymphocytes to Trypanosoma brucei in vitro: lack of correlation with resistance to trypanosomiasis.

Bovine peripheral blood lymphocytes (PBL) were stimulated to proliferate in vitro by live, irradiated or freeze-thawed Trypanosoma brucei, but not by the isolated variant surface glycoprotein. The optimal dose was 10(5) trypanosomes per 5 X 10(5) lymphocytes in 0.2 ml. Maximal proliferation was at day 5. Of the 98 cattle tested, 36 were high-responders (stimulation indexes 20-104), 49 were low or non-responders (SI 1-10) and 13 were intermediate. The responder status of individual animals did not change over a period of 1 year, nor did it alter following deliberate trypanosome infection. The stimulation was dependent on macrophage/monocyte type accessory cells, and this co-operation did not seem to be MHC restricted. Lack of stimulation of non-responder PBL did not appear to be due to the activation of suppressor cells. Accessory cells from non-responder animals could complement PBL from responders, but accessory cells from responders could not complement non-responder PBL. Responsiveness is therefore a characteristic of lymphocytes. Analysis of the surface markers of these lymphocytes or the blast cells generated in culture showed that they were a subpopulation of T cells, possibly TH cells. Analysis of PBL from 98 animals, which had been selected for trypanoresistance or trypanosensitivity under natural tsetse fly challenge, failed to establish a correlation between resistance and level of lymphocyte stimulation by trypanosomes in vitro.

Alpha-helical coiled-coil structures of Trypanosoma brucei variable surface glycoproteins.

We have used electron microscopy to examine purified intact variable surface glycoproteins (VSGs) from clones derived from two distinct stocks of Trypanosoma brucei. The VSG molecule from MITat 1.2 has a large elongated domain consistent with the shape of the dimeric N-terminal domain determined by X-ray analysis (see preceding paper), and a heretofore unseen short, thin fibrous tail presumed to be the C-terminal domain. Electron microscopy on DiTat 1.3, however, indicates a morphology quite distinct from that of MITat 1.2. Analysis of four VSG amino acid sequences reveals 7-fold periodicities (heptad repeats) which indicate that alpha-helical coiled-coil secondary structure elements occur in all of these VSGs, consistent with the observation of helical bundles in one VSG. These results suggest the possibility that VSG antigenic diversity may be related to a diversity in length and disposition of alpha-helical bundles and coiled-coil domains.

Anti-trypanosome specific immune responses in bovids of differing susceptibility to African trypanosomiasis.

A clone of Trypanosoma brucei brucei (DiTat 1.1) was injected into 32 bovids of various breeds, 11 animals being kept as controls. Five animals, Simmental-Ndama F1 crosses, were extremely sensitive. They showed overt symptoms and one died on day 18 of infection despite treatment with a trypanocidal drug. Seven other animals became ill but recovered progressively and cleared the infection. Twenty animals, of breeds generally considered to be trypanosensitive as well as ones from trypanotolerant breeds, did not show symptoms apart from anaemia and cleared the infection. Putative protective antibody, i.e. directed against exposed determinants on the coat variant-specific glycoprotein, was detected by agglutination, complement-mediated lysis and inhibition of infectivity. All animals showed a primary immune response consisting of IgM whose kinetics and amplitude were indistinguishable between animals of differing sensitivity. The response was long-lasting, whether the animals had been treated or not with a trypanocidal drug 3 weeks after infection, and antibody of IgG1 and IgG2 types were detected in certain sensitive as well as resistant animals after 2 months. Some animals were rechallenged with DiTat 1.1 either 1 year after the primary infection or 6 months after inoculation of irradiated trypanosomes. Peak titres of antibody were lower than was the case following primary infection but higher levels of mercaptoenthanol-resistant antibodies were seen. In no case was there any difference in the response of sensitive or tolerant animals. Our results do not support the idea that resistance of certain bovids to African trypanosomiasis is due to a better protective antibody response.

Trypanotolerance. an individual not a breed character.

Ten animals from a reputedly trypanosensitive bovine breed, the Zebu, and ten from a reputedly trypanotolerant breed, the Baoulé, were exposed to natural trypanosome challenge in an area of high Glossina density. The ten Zebus and five of the Baoulés died within 6 to 11 weeks with heavy parasitaemia and severe anaemia. The five other Baoulés showed little or no parasitaemia, no anaemia and were all in good condition after weeks of exposure: Seven animals of a herd of Baoulé/Ndama crosses indigenous to the same area were followed concurrently and showed very rare or no patent parasitaemia. Clearly, only some individuals within a "trypanotolerant breed" are able to withstand natural fly challenge. Selection of trypanotolerant animals has to be done on an individual basis and not on the biometrical characteristics of the breed.