A.G. Luckins

Haematological changes in sheep experimentally infected with Trypanosoma evansi

Abstractu2002Sheep were infected with 2×106 Trypanosoma evansi TREU 2143 through the external jugular vein. The parasite kinetics as well as the effects on body temperature, packed cell volume (PCV), erythrocyte counts and total and differential white blood cell counts were monitored twice weekly for 3 months. The results showed that T. evansi produced a chronic form of the disease in sheep characterised by low-level and often cryptic parasitaemia, with self-cure occurring in two cases; mild anaemia as evidenced by decreases in PCV and erythrocyte counts; and significant (P<0.02) leucocytosis by day 22 post infection (p.i.). The leucocytosis was a result of marked lymphocytosis whose significant rises (P<0.02) parallelled the rises in total white blood cell (TWBC) counts. These changes were less obvious in the animals that underwent self-cure. We conclude that T. evansi produces pathological changes in the peripheral blood of sheep similar to those produced by its tsetse-transmitted counterparts. It would thus appear that the sheep/T. evansi model is suitable for long-term study of the immunopathology of pathogenic trypanosomes since the sheep is easily available, easy to handle and a natural host to all pathogenic trypanosomes.

Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine-biotin immunohistochemistry.

Twenty-four percent of hog deer (Cervus porcinus) that ranged free on a farm in Samut Prakarn province, Thailand, died showing nervous signs between September 1997 and February 1998. The nervous signs shown by most of them included ataxis, paresis of hind limbs, lateral recumbency, excitation and convulsion. Six animals and one carcass were submitted for diagnosis at the National Institute of Animal Health, Bangkok. Trypanosoma evansi was detected in blood and cerebrospinal fluid of four and five animals, respectively. Antibodies to T. evansi were found in all the hog deer by indirect enzyme-linked immunosorbent assay. Histopathological observation revealed a generalised non-suppurative meningoencephalitis affecting the white and grey matter at all levels of the brain. Typically, there were broad perivascular cuffs of mononuclear inflammatory cells, including lymphocytes, and some Mott cells. No trypanosomes were found in any tissue examined by conventional histopathology. However, numerous T. evansi were demonstrated by streptavidine-biotin immunohistochemistry in neuropil and Virchow-Robin spaces of brain in three animals.

Increase in CD5+ B cells and depression of immune responses in sheep infected with Trypanosoma evansi

The effects of Trypanosoma evansi on the cellular and humoral immune responses of sheep to Pasteurella haemolytica vaccine were studied. Peripheral blood lymphocytes (PBLs) from the sheep were analysed using single and double-colour indirect immunofluorescence staining and flow cytometry to monitor changes in circulating B and T cell subsets. Serum antibody responses were assayed using the enzyme-linked immunosorbent assay technique (ELISA), in addition to measuring local skin reactions at the site of vaccine administration. Results showed significant increases in circulating B cells in all sheep after the primary (p < 0.01) and secondary (p < 0.001) vaccinations although the increases were much more dramatic in the T. evansi-infected sheep. In addition, infection induced significant increases (p < 0.004) both in proportions and numbers of CD5+ B cells with more than 70% of circulating B cells expressing the CD5 antigen and showed significant differences (p < 0.01) from those of control sheep in which vaccination alone failed to induce similar increases. Also, infection resulted in significant decreases in CD5+ (p < 0.003), CD4+ (p < 0.03) and CD8+ (p < 0.03) T cell subsets in contrast to their increases in all control animals after vaccination. Moreover, there were significant suppression of both local skin reaction (p < 0.005) and serum Ig and IgG1 (p < 0.001) antibody responses to the vaccine antigen.

CELLULAR PHENOTYPES IN TRYPANOSOMA CONGOLENSE INFECTED SHEEP : THE LOCAL SKIN REACTION

Summary Mononuclear cell subpopulations in local skin reactions (chancres) in sheep infected with metacyclic forms of Trypanosoma congolense were studied by indirect immunoperoxidase staining using a panel of monoclonal antibodies (MoAbs) specific for ovine leucocyte subsets. Morphometric analysis revealed significant increases in numbers of cells expressing CD5, CD4, CD8, CD45R (mainly B cells), major histocompatibility complex (MHC) class II antigens, Fc receptors (FcR) on macrophages (VPM32) and FcR on B cells and macrophages (VPM33) from five days post‐infection. B cells which also expressed MHC class II were found mainly in dense aggregates. The CD4/CD8 ratios were raised over pre‐infection levels at 5–7 days post‐infection. In sheep which had been infected, treated with trypanocidal drugs and then challenged with an heterologous serodeme of T congolense, changes in cellular phenotype kinetics were similar to those seen in the skin in primary infections. Sheep superinfected with either an homologous or an heterologous, T. congolense serodeme showed only mild cellular infiltration and slight increases in various cellular phenotypes at the sites of inoculation.

Serum immunoglobulin levels and electrophoretic patterns of serum proteins in camels infected with Trypanosoma evansi

Abstract The relative proportions of serum proteins and serum immunoglobulins levels were determined for uninfected camels and for camels infected experimentally and naturally with Trypanosoma evansi in the Sudan. In experimentally infected camels, progressive changes in the relative proportions of serum proteins were observed leading to a decrease in albumin level and an overall increase in γ-globulin levels. No marked changes were observed in the levels of α- an β-globulins. Estimations on samples from camels exposed to natural infections showed higher serum protein levels in infected camels than in uninfected camels. The relative proportions of several serum protein components in infected camels differed markedly from those in uninfected camels. Infected camels had lower albumin levels, lower β-globulin levels and higher γ-globulin levels than uninfected camels. However, no significant difference was found between the levels of α-globulin in infected and uninfected animals. In experimentally infected animals, IgM levels increased to a maximum of five times preinfection values and remained high despite drug treatment. IgM levels were also increased significantly in naturally infected camels by comparison with control camels. Only minor fluctuations in IgG levels were observed in all groups.

The initial stage of infection with cyclically-transmitted Trypanosoma congolense in rabbits, calves and sheep.

Abstract Glossina morsitans infected with a stock of Trypanosoma congolense were used to infect rabbits, calves and a sheep. Local skin reactions developing after 6 to 10 days at the sites of infected tsetse bites were removed surgically or post mortem, and the tissues processed and examined for trypanosome distribution and pathological change by light, electron and fluorescence microscopy. Local reactions from rabbits were examined at intervals between 7 and 19 days after infection and from calves between 8 and 30 days after infection. A single reaction from a sheep was examined 10 days after infection. Observations were also made on the presence of trypanosomes in lymph nodes and blood from the infected animals. Substantial numbers of trypanosomes were located in the collagen of the deep dermis and near the bases of hair follicles following infection in all three host species. The parasites were numerous in reactions examined between days 7 and 12 and calf tissues were most heavily infected. Trypanosomes were also seen in lymphatic vessels of the skin and in superficial lymph nodes draining local reaction sites up to day 10. Although many trypanosomes in local reactions were destroyed by extracellular lysis, a few parasites were detectable in the dermal collagen until day 19 in rabbits and day 30 in calves. The multiplication and development of trypanosomes in each host was accompanied by focal disorganization and degeneration of infected areas of dermal collagen. Reactions examined 7 to 15 days after infection showed acute inflammatory and exudative changes with extensive mononuclear cell infiltration particularly in areas showing collagen necrosis and in the vicinity of dermal blood vessels. Macroscopically, reactions left in situ gradually disappeared by about day 16 in rabbits and day 21 in calves. Histological examinations confirmed that inflammatory changes subsided between days 14 and 21, leaving extensive areas of newly formed collagen and persistent perivascular and tissue foci of mononuclear cells.

Effects of Trypanosoma evansi on the output of cells from a lymph node draining the site of Pasteurella haemolytica vaccine administration.

The prefemoral efferent lymphatics of sheep infected with Trypanosoma evansi and inoculated with P. haemolytica vaccine and of those given only the vaccine, were surgically cannulated to study the effects of the infection on the total cellular output and output of blast cells from the node in response to the vaccine. T. evansi delayed and depressed the increases in total cell and lymphoblast outputs. In uninfected sheep, the total cellular output increased and peaked at more than twice the prevaccination values on days 4 and 5 after primary vaccination, but the increases were smaller and peaked on days 6 and 8 after primary vaccination in the infected sheep. The output of lymphoblasts mirrored the total cell output, though it was suppressed to a greater degree by T. evansi. The output of blasts peaked at more than 8 and 14 times the prevaccination values in the uninfected animals after primary and secondary (booster) vaccinations, respectively; but in infected animals, it peaked at twice the prevaccination values after the primary vaccination and showed no increase after booster vaccination until 11 days later. It is concluded that the inhibition of total and blast cell outputs by T. evansi may limit the early systemic dissemination of antigen-specific cells, thus playing a role in the induction of immunosuppression by the parasite.

Changes in peripheral blood lymphocyte subpopulations and parasite-specific antibody responses in Trypanosoma evansi infection of sheep

Abstract This paper reports on changes in the lymphocyte composition of the peripheral blood in sheep infected with Trypanosoma evansi. In addition, parasite-specific IgG1 and IgM antibody responses were monitored using a double-sandwich enzyme-linked immunosorbent assay (ELISA) technique. Eight sheep were infected with 2u2009×u2009106T. evansi TREU 2143. The infection was characterised by chronicity and ended in self-cure in two of the sheep. These two sheep were designated group A, whereas the other six sheep, which remained parasitaemic until treated, were designated group B. Analysis of the peripheral blood lymphocytes (PBLs) by indirect immunofluorescence staining and flow cytometry revealed significant alterations in the numbers of T- and B-cell subsets detected in all infected sheep. In group A, whereas the numbers of CD8+ cells decreased, CD4+ cells showed marginal decreases, remaining at or above pre-infection figures and resulting in increase in the CD4:CD8 ratio. In group B, CD8+ cells showed few marginal decreases, being at or above pre-infection figures most of the time, whereas CD4+ cells decreased significantly from day 26 post infection (p.i.) such that the CD4:CD8 ratio decreased. Infection also resulted in significant increases (Pu2009<u20090.001) as of day 26 p.i. in circulating B-cells in group B as shown by the numbers of sIg+, CD45R+, CD1+ and major histocompatibility complex (MHC) II+ cells. The increases, however, were moderate and biphasic in group A.u2009T.u2009evansi-specific IgM and IgG1 antibody isotypes were detected in all infected sheep, but their levels were significantly higher in group A than in group B (IgM Pu2009<0.05; IgG1Pu2009<0.01). In addition, although an initially higher level of IgM response was subsequently replaced by a higher level of IgG1 response in group A, this was never the case in group B until after drug treatment.

Interference between different serodemes of Trypanosoma congolense in the establishment of superinfections in goats following transmission by tsetse

Summary When domestic ruminants cyclically infected with Trypanosoma congolense are superinfected with a different serodeme of the same species, an interference phenomenon occurs which delays the development of the second cyclical infection. Experiments were carried out to investigate the influence of the time interval between the two infections on the degree of interference and to follow the course of the superinfection clinically, serologically and histologically. Goats infected with tsetse‐transmitted T. congolense IL 1587 were either infected simultaneously or 7, 14, 18, 28 or 35 days later with a different serodeme, T. congolense IL 1180. Skin reactions due to superinfection were either absent or smaller in size and delayed in onset compared with control animals undergoing a primary infection with T. congolense IL 1180 which had been initiated by tsetse fly bites. All animals were treated with the trypanocidal drug Berenil 21 days after superinfection and 3 weeks later challenged with T. congolense IL 1180 using tsetse flies. The goats that had been infected simultaneously with the two serodemes were immune to the homologous challenge, but 11 (85%) of the animals that had been superinfected were not. The effect of interference on the host immune response to T. congolense IL 1180 was most marked in animals superinfected at day 7; thereafter there was evidence that the ability to respond immunologically against secondary infection was gradually recovered. Histological examination of skin removed 7 days after a simultaneous infection of goats with two serodemes, showed trypanosomes and a cellular reaction similar to that following infection with a single serodeme. In skin removed 7 days after superinfection of goats that had been infected for 7 days, the cellular response was less pronounced and trypanosomes were not seen, although by 14 to 16 days numerous trypanosomes were present and there was a marked cellular infiltrate. It is postulated that the presence or absence of a factor induced shortly after initiation of a trypanosome infection in the skin of goats might delay parasite development when metacyclic trypanosomes are deposited by tsetse following superinfection.

Interference with anti-trypanosome immune responses in rabbits infected with cyclically-transmitted Trypanosoma congolense

Summary Rabbits were infected with two clones of antigenically distinct stocks of Trypanosoma congolense transmitted through Glossina morsitans. Local skin reaction development and the appearance of neutralizing antibodies were followed in animals infected with one or other of the trypanosome stocks, with both stocks simultaneously or with both stocks consecutively. There was little difference in local skin reaction development on rabbits infected with a single stock or with both stocks simultaneously but, in rabbits exposed to a heterologous stock 14 or 21 days after a primary infection reactions were reduced in size or completely absent. Neutralizing antibodies against metacyclic‐derived trypano‐somes were detected 21 days after infection in animals infected with a single trypanosome stock and, in rabbits infected with both stocks simultaneously, antibodies against each stock were also detected 21 to 28 days after infection. In rabbits challenged 14 or 21 days after primary infection the appearance of trypanocidal antibodies against the stock used for challenge was delayed from 28 to 49 days.