Comparative histopathology of the lymph nodes , spleen , liver and kidney in experimental ovine trypanosomosis

OMOTAINSE, S.O. & ANOSA, V.O. 2009. Comparative histopathology of the lymph nodes, spleen, liver and kidney in experimental ovine trypanosomosis. Onderstepoort­Journal­of­Veterinary­Research, 76:377–383 The infection of Yankassa rams with three important trypanosome species affecting livestock, namely, Trypanosoma­congolense,­T.­vivax­and T.­brucei produced both acute and chronic fatal conditions. Chronic infections were induced in the three infections by the application of subcurative doses of diaminazene aceturate (Berenil®). Pathological changes in the infected animals included splenomegaly and hepatomegaly which were more pronounced in acute than in chronic T.­congolense­ infection. However, these changes were more severe in chronic than in acute T.­vivax­infection. While splenomegaly was more pronounced in chronic T.­brucei­infection than in acute, hepatomegaly and lymphadenopathy were more severe in acute than in the chronic condition. The increases in size of the spleen, lymph nodes and liver were associated with congestion, increases in cell density related to increased immunological reactions in the spleen and lymph nodes as well as increase in numbers, size and activity of the phagocytic cells in these organs.


INTRODUCTION
Trypanosomosis is one of the most devastating and widespread diseases of African livestock.It is caused by the protozoan parasites of the genus Trypano so ma and is transmitted mainly by biting tsetse flies of the genusGlossina.It is a major constraint to livestock production due to its negative effects on milk production, mass gains, growth rates and reproduction.If left untreated it can lead to great economic losses in animals in terms of time and costs of treatment of animals, low productivity, infertility and mortality (Omamo & d'Ieteren 2003).
Several reports are available on the haemopoietic responses in trypanosomosis.In ovine trypanosomosis, there is emaciation, splenomegaly, hepa tomegaly, enlarged lymph nodes and generalized oedema.These lesions, which are characterized by a hyperactive mononuclear phagocytic system, are usually more prominent in the acute phase of the disease (Losos & Ikede 1972;Anosa & Isoun 1974;Anosa, Logan-Henfrey & Shaw 1992).
The mechanisms adduced to some of these observations include lysis of blood cells (Anosa & Kaneko 1983), haemodilution (Naylor 1971), haemorrhages (ILRAD 1984) and dyshaemopoiesis (Anosa et al. 1992;Omotainse & Anosa 1992).Production of toxins by the trypanosomes could have some lethal effects on the marrow cells (Kaaya, Valli, Maxie & Losos 1979;Lawson, Valli, Mills & Forsberg 1980) which can result in reduced production or maturation of the erythroid cells.Likewise, phagocytosis of immature cells as demonstrated in the spleen of T. brucei-infected mice (Anosa & Kaneko 1983, 1984) and in the bone marrow of T.vivax-infected calves (Anosa etal. 1992) can lead to the ineffective utilization of iron trapped in the macrophages in chronic trypanosomosis (Dargie, Murray, Grimshaw & McIntyre 1979).The capacity of monocytes to phagocytose red blood cells invitro in T.congolenseinfection of goats was increased (Witola & Lovelace 2001).These could contribute to the unsuccessful efforts of infected animals to increase the rate of erythropoiesis.To further understand the pathogenesis of anaemia in trypanosomosis, there is still the need to investigate the roles of some of the haemopoietic organs at various stages of infection involving different species of trypanosomes in different animal species.
The objective of this study, which was carried out using the Yankassa breed of sheep, was to investigate and compare the pathological changes in the lymph node, spleen, kidney and liver in acute and chronic ovine trypanosomosis, and to assess the possible roles of some of these organs in the pathogenesis of the anaemia in ovine trypanosomosis.

Experimental design
The study was designed to produce disease of varying duration, ranging from acute to chronic in Yankassa rams.Fifty-two rams were used.Three groups of 14 rams each were infected with T. brucei, T. vivax or Trypanosoma congolense, respectively.Ten uninfected rams were kept as a control group.

Parasites
The parasites used were T.brucei (CT 70 isolated from cattle at Gboko in 1988), T.vivax (CT 128, isolated from cattle at Kafanchan in 1988) and T.con golense (GT 12, isolated from a goat at Gboko in 1988), and obtained from the liquid nitrogen bank of the Nigerian Institute for Trypanosomosis Research, Vom, Nigeria.These parasites are resistant to most of the common trypanocides at usual therapeutic doses.

Infection
The parasites were maintained in rams and harvested for the inoculation of the experimental animals.About 1 x 10 6 parasites were inoculated subcutaneously into each ram with the exception of those in the control group.Recommended curative but ineffective chemotherapy with a common trypano cide, diaminazene aceturate (Berenil ® , Hoechst, Frank furt, Germany), at the recommended curative dose of 3.5 mg/kg for T. congolense and T. vivax was administered intramuscularly at between 4 and 6 weeks of infection as packed cell volume of the infected animals decreased, to allow for a chronic course of infection in each infected group of animals.Trypanosomabrucei-infected rams were similarly given 7.0 mg/kg body mass.The periods of infection before treatment, 5, 6 and 5 weeks post infections (PI) with T. congolense, T. vivax and T. bru cei, respectively, were regarded as the acute phase while the periods post treatment up to 19 weeks PI in T.congolense and 24 weeks PI in both T. vivax and T. brucei infections were taken as chronic phase in this study.

Pathological examination
Post mortem examinations were carried out on the following numbers of animals in the different groups: the ten control animals, seven animals with acute T. congolense infection, six animals with chronic T. congolenseinfection, six animals with acute T.bru cei infection, eight animals with chronic T. brucei infection, six animals with acute T. vivax infection and eight animals with chronic T. vivax infection.Apart from rams with acute T.brucei-infection where sudden deaths were recorded, infected animals were sacrificed when recumbent at the points of death in order to obtain fresh samples for examination.Samples of lymph nodes, spleen, kidney and liver were collected in formalinized saline solution for histopathology, and were processed and routinely stained with haematoxylin and eosin (H&E) according to Drury & Wallington (1967).The lesions were scored on a scale of low (+), moderate (++) and high (+++).

Statistical analysis
The mass of some of the organs were measured and their means compared using Student t-test in the SPSS statistical package at significant level of P ≤ 0.05.

Gross pathological observation
Generally, in the three infections, there were cachexia and serous atrophy of fat, which were more pronounced in chronic infections.
There was enlargement of the prescapular and prefemoral lymph nodes, spleen, liver and kidneys (Table 1).Frequent urination was a prominent clini-cal sign observed in the three trypanosome infections from the second week of infection, particularly in the T.vivaxinfected animals.In both acute and chronic infections there were significant differences at various levels in the mass of the prefemoral and prescapular lymph nodes, spleens and livers (Table 1).

Histopathological changes
The lesions in the lymph nodes, spleen, kidney and liver of the rams in the three groups of infected rams are presented in Table 2.At the different phases of trypanosomal infections in this work, the lymph nodes and spleen demonstrated various degrees of severity of hyperplasia of the lymphoid follicles.The cortical and medullary regions of the lymph nodes showed congestion of the sinuses, proliferation of mononuclear cells, erythrophagocytosis and haemosiderosis.Similarly, the spleen of trypanosome-infected an imals showed depopulated red pulp, pro liferation of macrophages characterized by erythro phagocytosis and haemosiderosis as well as proliferation of plasma cells.There are differences in the extent of these reactions depending on the parasite species and stages of infection (Fig. 1 and 2).
The kidneys similarly demonstrated various degrees of congestion, perivascular and interstitial mononuclear cells infiltration, thickening of glomerular cap sules, desquamation of tubular cells and protein casts in the tubules (Fig. 3-5).
The livers showed various degrees of vascular congestion, perivascular cuffing of mononuclear cells, hepatocellular degeneration and erythrophagocyto- V sis according to the phase of the different trypanosomal infections (Table 2, Fig. 6 and 7).

DISCUSSION
The lesions in the lymph nodes of trypanosomeinfected animals included increased presence of macro phages with erythrophagocytosis, congestion, enlarged lymphoid nodules as well as increased plasma and Mott's cell populations.Apart from congestion, these lesions were more pronounced in the chronic than in the acute phase.Trypanosomavivax and T. brucei infections showed more intense lesions than those in the rams infected with T.congo lense.Perivascular mononuclear cell infiltration, mainly of lymphocytes, plasma cells and macrophages, in the liver was also a common feature in ovine trypanosomosis.These changes conform with those described earlier reports of T.vivax infection (ILRAD 1984;Anosa etal. 1992) and in T.rhodesi ense infection of humans (Robins-Browne, Schneider & Metz 1975), and similarly were more intense in the chronic phase of the disease.
In this study, hyperplasia of the red pulp, congestion of the sinuses, enlargement of the lymphoid nodules, increased erythrophagocytosis, haemosiderosis and proliferation of plasma cells were the lesions seen in the spleen in acute T.congolense,T.vivax and T.brucei infections.The lesions were generally more pronounced in T.brucei infections than in T. congolense and T. vivax infections.The enlarged germinal centres of lymphoid nodules, especially during the acute phase of ovine trypanosomosis, are immunological reactions, presumably associated with the need for increased production of immunoglobulins.The presence of increased haemosiderosis is an indication of the major role the spleen plays in the destruction of red blood cells during try- panosomosis.This observation conforms to that of Anosa & Isoun (1980) and Taylor & Authie (2004).The presence of greater level of lesions such as erythrophagocytosis in T. brucei infections especially those in the acute phase of infection could contribute to the rapid development of anaemia despite the increased erythroid hyperplasia usually present in this phase of infection (Anosa etal.1992).The mild enlargement of the lymphoid nodules of the spleen in the chronic T.congolenseand T.bru cei infections could be an indication of the declining role of humoral immunity in chronic trypanosomosis (Moulton 1986).This probably indicates exhaustion of the immune apparatus which would most probably lead to immunosuppression, thereby giving rise to the secondary bacterial infections reported by Kramer (1966) and Anosa & Isoun (1983).The depopulation of lymphoid nodules was more pronounced in T. brucei infection than in the T. vivax and T.congolense infections.The immunosupression could be partly responsible for the sudden and higher mortality experienced with T.brucei infection as compared to others in this study.
Vascular congestion, disorganization of the hepatic cords, perivascular mononuclear cell infiltration, hepat ocyte degeneration, hyperplasia of Kupffer cells and erythrophagocytosis were common lesions observed in the liver in acute form of ovine trypanosomosis in this study.These lesions were more pronounced in T.brucei infection than in T.congolense and T.vivax infections.Apart from the vascular congestion, these pathological changes were more pronounced in chronic T.bruceiand T.vivaxinfections.The lesions associated with the various species of trypanosomes in this study were in good agreement with earlier reports on animal trypanosomosis (Losos & Ikede 1972;Anosa & Kaneko 1984;Taylor & Authie 2004).This might have played a significant role in the inability of the animals to successfully recover from the anaemia experienced in trypanosomosis.
In summary, the lesions seen in the spleen, lymph nodes and the liver included the presence of activated macrophages, erythrophagocytosis and haemosiderosis indicating massive destruction of red blood cells in these organs, but these were more prominent in the spleen and lymph nodes.These changes confirm that these organs are the sites of red blood cell destruction in trypanosomosis.
The diffuse oedema, thickening of the glomerular capsule, tubular cell desquamation as well as accumulation of mononuclear cells in the intertubular septae and around the glomeruli of the kidneys in acute infections were more severe with chronicity.These lesions were more pronounced in T. vivax than in T.brucei and T.congolenseinfections, and might have been responsible for the constant urination which was most observed particularly in T. vivax-infected animals.Glomerulonephritis, characterized by thickened glomerular capsules, thickened intertubular and perivascular spaces by lymphocytes, plasma cells and macrophages, have been reported in T.brucei infection of rodents (Anosa & Kaneko 1984), in T. gambiense infection of mice and in T.rhodesiense infection of monkeys (Nagle, Ward, Lindsey, Sadun, Johnson, Berkaw & Hilderbrandt 1974).Damage to renal tissues, especially the juxta-glomerular cells (Jubb, Kennedy & Perma 1985) can affect the production of erythropoietin, a hormone produced by the kidney to stimulate the bone marrow for erythropoiesis, thereby leading to inadequate stimulation of the bone marrow for the required increased production of red blood cells well above normal to reverse anaemia.Tubular damage can also lead to proteinuria, which may have been responsible for the constant urination in this study and can lead to death (Goodwin 1970).Therefore the lack of adequate erythrocytic response to the anaemia in ovine trypanosomosis may be attributed in part to the renal damage.

CONCLUSION
Trypanosoma congolense, T. vivax and T. brucei infections in Yankassa rams produced clinical disease with prominent lesions in several organs, with variations in severity depending on the parasite species and duration of infection.Pathological lesions including erythrophagocytosis, haemosiderosis and vascular congestion in the lymph nodes, spleen and liver as well as glomerular degeneration and necrosis of the kidneys, may have contributed to the clinical signs particularly the anaemia which is a consistent sign in animal trypanosomosis.Folicular hyperplasia of the lymphoid organs, vascular damage and tubular cell desquamation of the kidneys as well as cellular and vascular disruptions of the liver, coupled with the presence of erythrophagocytosis in these organs, further explain the possible cause of anaemia and other clinical signs and lesions in ovine trypanosomosis.ence, as co-sponsors of this work.We deeply appreciate the technical assistance of Mr Christopher A. Ibitola for processing the tissues for histology.

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FIG. 1 Medullar region of a lymph node of a ram with acute T. brucei infection showing congested blood vessels (S).H & E, x630

FIG. 7 VFIG. 4
FIG. 7 Micrograph of the liver of a ram with chronic T. brucei infection showing congestion of a periportal vein (V) and perivascular accumulation of lymphocytes and macrophages (M).H & E, x1200

TABLE 1
Mean mass (gm) of some organs of control and trypanosome infected Yankassa rams

TABLE 2
Summary of lesions in lymph nodes, spleen, liver and kidney in ovine trypanosomosis