SERUM IMMUNOGLOBULIN PROFILE OF Trypanosoma congolense - INFECTED SHEEP IN RELATION TO PARASITAEMIA AND CHANGES IN CIRCULATING LEUCOCYTE AND ERYTHROCYTE MASS

Trypanosomiasis is a major problem in developing countries most especially Africa and the incriminating agents are T. congolense, T.vivax. T.brucei in small ruminants. It has high morbity and mortality Rates of 70% and 20% respectively . The experimental animals were four Rams which were used as control and infected group, Parameters for this experiment were recorded before infection. Following inoculation with T.congolense, 2 mls of blood was collected from the sheep in vacutainers for serum biochemical assay. Enzyme linked immunosorbent assay was used to assay IGG and IGM concentrations and 1ml of blood was collected in vacutainers with anti coagulant to check leucocytic and erythrocytic index.This was done every day until day 6 and then every other day. Clinical signs observed post infection were , Anemia, Hematuria ,Rhinitis, dyspnea ,emaciation and paralysis of the fore and hind limbs and death .Statistical tables and charts were used for analyses. The role of Hypoprotenemia , IGG and IGM in experimental infection with Trypanosoma congolense were looked at. The anemia was Normocytic normochromic and later became Microcytic hypochromic, PCV Recorded on the first day prior to infection was 27% and on the last day of infection became 6% . Leucocytosis was Degenerative and Leucopenia was also observed Perhaps due to the antigenic nature of the parasite. On first day WBC count was 8.5 x10 9 /L and Prior to death Leucocytosis of 6.1x 10 9 /L. Hypoproteneimia was also seen with Blood urea Nitrogen and Alanine aminotransferase falling to values below normal 55U/L and 155mg/dl respectively. There were also fluctuations in the values of IGG and IGM with initial values of 1.4 and 0.71 µg/ML and terminal values of 0.34 and 0.21 µ𝑔 /L. The organs looked at Kidneys,Lungs,Heart and Liver were all in their initial stages of destruction , they were inflamed,edematous , congested and necrotic . Hypoprotenemia, , immune suppression,dyspnea ,Anemia and Leucopenia may be the cause of death in Trypanosomiasis.


Statement of research problem
African trypanosomosis continues to decimate human and livestock populations unabated and constitute an impediment to human and livestock production in the disease endemic countries of Tropical Africa (Dubois et al.,2005).Anaemia which is evidenced by low values of erythrocyte count, packed cell volume(PCV) and haemoglobin concentration ,had for long been identified as one of the principal features and major causes of death in acute phase of trypanosomosis in livestock,as well as been an aggravating factor in the development of most of the reported disorders in affected animals (Esievo and Saror,1991) The recognition of the importance of this clinical feature of trypanosomosis stimulated diverse forms of research interest aimed at elucidating the mechanism of it,s development in affected animals.These efforts resulted in the emergence of numerous reports that vary in concept (Igbokwe,1989).The varying nature of the reports not withstanding, the general consensus is that the anemia in Animal trypanosomosis is predominantly the result of hemolytic crisis in which the red blood cells are being destroyed by mononuclear phagocytic system (Igbokwe and mohammed,1991).
Trypanosomosis (trypanosomiasis), a globally occurring parasitic disease, poses as a major obstacle to livestock production in tropical and subtropical regions consequent to huge economic losses it causes in affected livestock (Boada-Sucre et al., 2016). The principal trypanosomes which cause disease in ruminants in sub-Saharan Africa are Trypanosoma congolense (T. congolense), T. vivax, T. brucei and T. evansi and a resultant decrease in serum sialic acid and erythropoietin Adamu et al., 2009;Boada-Sucre et al., 2016;Ode et al., 2017). Animal trypanosomosis is characterized by periodic fevers, parasitemia, and anemia, changes in blood chemistry, body mass loss, and often death. The anemia developed by animals is considered the most characteristic symptom (Boada-Sucre et al., 2016). It seems that the anemia is hemolytic in nature, occurring intravascularly in the early stages of the disease, and extravascularly in the chronic phase. There are many reported possible mechanisms of erythrocyte destruction that lead to the anaemia in affected animals (Boada-Sucre et al., 2016). Animals infected with trypanosomes develop a number of immunological disorders of varying severity. First, circulating immune complexes may be generated as a result of the persistent antigenemia and resulting immune stimulation. The deposition of these biologically active complexes on erythrocytes may contribute to the anemia often associated with T. congolense (Fadiga et al., 2013). Serum immunoglobulin levels in infected animals show significant differences from normal. For example, serum immunoglobulin M (IgM) levels are greatly elevated (Iizard et al.,1978; rising on occasion to 20 times normal values. In contrast to the rise in IgM levels, serum IgG tends to remain relatively constant, at values close to normal (Nagele et al., 2013;Iizard et al.,1978). Catabolic studies indicate that the half-life of all serum immunoglobulin classes is shortened (Iizard et al.,1978).
Parasite antigenic variation and the induction of alterations in the hosts defense system, such as excessive activation of the complement system leading to persistent hypocomplementemia, down regulation of nitric oxide production, polyclonal B-lymphocyte activation and marked immunosuppression are the main mechanisms to survive in the chronically infected host . The production of antibodies to VSG of T. congolense is the major early immune response. The first antibody to the VSG is of immunoglobulin M (IgM) class and is produced independently of T cells. It was reported that at peak of parasitaemia a type 2 helper T cell (TH2) like cytokines were prevalent in the typanosusceptible Boran cattle (Ayele et al .,2017). Hemolytic material from T. congolense has been shown to consist of a mixture of free fatty acids and phospholipase A. Hemolytic factors have been found in the sera of trypanosome-infected cattle (Mc crorie et al.,1980). A number of immunological mechanisms have been proposed to be involved in the hemolysis. Trypanosome antigen has been demonstrated on the surface of erythrocytes. Antiglobulin tests showed that the erythrocytes of calves infected with T. congolense had absorbed immunoglobulins. Preformed antigen-antibody complexes may be absorbed onto erythrocytes, a phenomenon found in immune complex diseases , this would account for the detection of both antigen and immunoglobulin. Such immune complexes may fix complement on the erythrocyte surface, resulting in intravascular hemolysis or erythrophagocytosis or both. Complement has been detected on the surfaces of erythrocytes of patients infected with African trypanosomes (Ayele et al ., 2017; Baral et al., 2007 ;Vali et al.,1978). These observations would be consistent with antibody or antibody complement coating of erythrocytes and host parasite interactions (Nicholas et al., 2009;Babatunde et al., 1982). African trypanosomiasis is one of the major disease constraints to livestock production in sub-Saharan Africa. While the pathology of the disease has been well documented, surprisingly little is known about the mechanisms that cause disease. An unusual feature associated with the immune response during trypanosome infections in man and animals is that a large fraction of the antibodies produced are IgM. (Noyes et al.,2009 ). Although increases in serum IgM have been consistently observed in many trypanosome infections of cattle,the appearance of trypanosome non-specific antibodies was not a consistent finding. Recently, following the observation that numbers of CD5+B cells increased in blood and spleen concomitantly with an increase in serum IgM during T. congolense-infection of cattle (Naessens et al., 1992) CD5+ B cells form a distinct B-cell lineage (B-1), and spontaneously produce IgM with the features of natural antibodies in normal individuals/animals. (Buzza et al., 1997) 1.2 Significance of the study In spite of the rapid development of medicine in recent years trypanosomosis continues to be a major constraint to Human health and increased livestock production,and broadly,to agricultural development in most countries of Tropical Africa (Kristjanson et al.,1999;FAO,2002).The disease still remains one of the most important diseases of livestock and Human especially in Sub-saharan Africa (Odoya et al.,2003).Verily,the prevalence of trypanosome infections in Nigeria and perhaps most African countries appears to be on the increase (Odoya et al.,2003).More so, it is estimated that 46 million cattle are exposed to the hazard of contacting the disease just as are millions of other species of domestic animals in Africa (Kristjanson et al.,1999)Like-wise curative and preventive treatments which have remained the only means of curbing the ravaging effects of the desease cost livestock owners and Government colossal amounts.Efforts to use vaccines as alternatives to prevent the desease have been rendered futile due to the antigenic variation of the organisms in question (Teale,1991). For example trypanosomes have multiple genes that code for different surface coat glycoproteins that are not vulnerable to immune response and this causes the persistence of the organism in blood. The major source of concern is the fact that Human and Animal trypanosomes are re-emerging (Waiswa et al.,2003)while development of resistance to trypanocidal drugs by the organism is on the increase.Also, with the enormous losses incurred and the disturbing epidemiologic trends of this desease further studies need to be done so as to ascertain ways of prevention and cure. It has recently been demonstrated that the Boran breed produces anti-trypanosome antibodies mainly of the IgM isotype, while the IgGl isotype antibodies are detectable in N'Dama cattle. Since immunoglobulin class switching is dependent upon the release of cytokines from activated helper T cells, it is possible that differences in T-cell function between trypanotolerant and trypanosusceptible cattle may contribute to the differences observed in their antibody production (Gachohi et al.,2012). This may be obtainable in different breeds of sheep where the trypanotolerant ones may produce mostly IgG and the tyrpanosusceptible ones will produce mostly the IgM isotype (Flynn et al., 1992;Buza et al., 1997). Another reported mechanism of erythrocyte destruction is through the action of phospholipases, possibly released in trypanosome infections, which can destroy cell membrane phospholipids leading to the erythrocyte destruction.
Since trypanosomosis continues to ravage Nigerian indigenous livestock, knowledge of immunologic responses and activities of phospholipases may give an insight and a better understanding of these mechanisms of trypanosome infection-induced erythrocyte destruction in these species of animals.

Objectives
Objectives of the study are to determine changes in-1. serum concentration of immunoglobulins (IgM and IgG) in relation to the level of parasitaemia in Trypanosoma congolense-infected sheep 2. haematologic and eucocytic parameters of Trypanosoma congolense-infected sheep in relation to parasitaemia and free fatty acid and aminoacid concentrations .
3. some vital organ functions (kidney and liver) in relation to parasitemia in Trypanosoma congolense-infection. Trypanosomes are protozoa belonging to the order Kinetoplastida,family Trypanosomatidae and the genus Trypanosoma (Kirti Dua,2012).The family ,Trypanosomatidae consist of eight genera.Four of these are monoxenous(single host)parasites of invertebrates,mainly insects.The genera include Leptomonas, Herpetomas, Crithidia and Blastocrithidia. (Kirti Dua,2012).The other four genera are dixenous(digenetic)parasites that require two successive hosts in their life cycles. These host constitute the intermediate host or vectors, which are usually Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021 invertebrates, and the other ,the definitive host, which are vertebrates or plants.Members of these genera include Phytomonas ,Leishmania, Trypanosoma and Endotrypanum.

Trypanosomatidae
Members of these group are characteristically leaf-like in shape ,have a single flagellum that is attached to the body by an undulating membrane.The blood stream forms of mammalian trypanosomes(Trypomastigote)are basically lanceolate in shape (Hoare,1972).The flagellum of trypanosomes represents the locomotor apparatus and is a single structure under light microscope. The kinetoplast represents the mitochondrion.

General characteristics of Trypanosomes
Flagellated protozoan parasites that live in blood and other body fluids of vertebrate hosts ,the flagellum is a contractile fibre and when it beats the cell membrane is pulled to form an undulating membrane .It is also with the help of the flagellum that the protozoa swims and penetrates the host cell. It posseses a Kinetoplast which it uses to undergo cyclic development in arthropod host. (Kirti Dua,2012).

General life cycle of the family Trypanosomatidae
Most Trypanosomes of this group undergo a cyclical development (Kirti Dua,2012 The course of development of trypanosomes in the mammalian host begins when the metacyclic trypanosomes are introduced directly by inoculating through bite, as seen in tsetse borne salivarian species,or passively by contamination of the mucous membrane or skin which occurs in stercorarian species.

Developmental stages
The different stages involved are; (A) Trypomastigote stage; formerly referred to as trypanosome stage, mainly seen in the vertebrate host but can occur in arthropod vectors as infective stage for the vertebrate. (B) Epimastigote stage; formerly called crithidial stage, found principally in arthropods but can be found in vertebrate host as part of the vertebrate developmental life cycle. (C) Promastigote stage; formerly referred to as leptomonal stage and found in arthropods or plants. (D) Amastigote stage ;also known as Leishmania stage and these occurs in vertebrates and arthropods.

Clinical signs and pathogenesis
The incubation period for T.congolense infection is usually 4 to 24 days while that for T.vivax is 4 to 40 days and T.brucei 5 to 10 days. Infected biting flies inoculate metacyclic trypanosomes into the skin of animal during feeding, trypanosomes grow at the site and cause localized swelling called chancres. They then migrate to the lymph node and then into the blood stream where they divide rapidly and initiate parasitemias (Kirti Dua, 2012).The anemia has complex pathogenesis that involves erythrophagocytosis ,some hemolysis and dyshemopoiesis.The clinical signs are usually stress associated for example, transportation, inclement weather, malnutrition or concurrent infection which may cause acute or per acute form of the desease where nervous symptoms are present.In the acute form there is high fever, emaciation, lacrimation, reduced milk yield ,circling movements. anemia and death within 24 hours.In chronic cases there is dullness, anorexia ,intermittent fever and cachexia though the cardinal sign of this desease is anaemia (Kirti Dua,2012)Within a week of infection there is usually a decrease in packed cell volume(PCV),hemoglobin, red blood cell and white blood cell levels which may within two months drop up to 50% their preinfection values. Mucous membrane become pale and some animals may exhibit edema of the throat and underline.The disease often always result in immunosuppresion and mortality and morbidity depends on the virulence of the trypanosome ,age and breed of the animal.(Kirti Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021 56 Dua,2012).Necropsy findings vary and are not specific but major anomalies include, anemia, serous atrophy of fat, edema(subcutaneous edema)and excessive fluid in the body cavities, emaciation, petechial hemorrhages and an enlarged liver,the spleen may be enlarged,normal or atrophied. (Huan,1975)

Trypanosoma congolense
This organism is mostly seen in Tropical Africa and they have a sub-terminal kinetoplast and short undulating membrane,may or may not posses flagellum,and may be pleomorphic or monomorphic in shape. and causes the disease known as African trypanosomosis,also referred to as Nagana,Tsetse disease,Trypanosomosis,the vector is tsetse flies or Glossina and species of animals infected include cattle,pig ,sheep and goats and horses.Several reports or observations were made on the clinical desease caused by T.congolense in experimentally infected livestock (Sekoni et al.,1990).T.congolense infection are often highly pathogenic with fatal infections.The acute phase of T.congolense infection in sheep was reported to last for 4-6 weeks(Logan-Henfrey et al.,1992)but their susceptibility to this infection varies,a breed of sheep known as Djalonke are more trypanotolerant than other species (Leak et al .,2002).Host infected with T.congolense may die within 6-10weeks post infection.In chronic cases T.congolense may last for months or years in blood during which anemia or extreme emaciation(cachexia) may develop (Tizard and Holmes,1976).

Immunoglobulin
Animals infected with trypanosomes develop a number of immunological disorders of varying severity. First, circulating immune complexes may be generated as a result of the persistent antigenemia and resulting immune stimulation. The deposition of these biologically active complexes on erythrocytes may contribute to the anemia often associated with T. congolense (Boada-Sucre et al , Itard et al.,1978). Serum immunoglobulin levels in infected animals show significant differences from normal. For example, serum immunoglobulin M (IgM) levels are greatly elevated (Iizard et al.,1978; rising on occasion to 20 times normal values. In contrast to the rise in IgM levels, serum IgG tends to remain relatively constant, at values close to normal (Nagele et al., 2013;Iizard et al.,1978). Catabolic studies indicate that the half-life of all serum immunoglobulin classes is shortened (Iizard et al.,1978). Parasite antigenic variation and the induction of alterations in the hosts defense system, such as excessive activation of the complement system leading to persistent hypocomplementemia, down regulation of nitric oxide production, polyclonal B-lymphocyte activation and marked immunosuppression are the main mechanisms to survive in the chronically infected host . The production of antibodies to VSG of T. congolense is the major early immune response. The first antibody to the VSG is of immunoglobulin M (IgM) class and is produced independently of T cells. It was reported that at peak of parasitaemia a type 2 helper T cell (TH2) like cytokines were prevalent in the typanosusceptible Boran cattle (Ayele et al .,2017). Hemolytic material from T. congolense has been shown to consist of a mixture of free fatty acids and phospholipase A (Herbert and Miller, 1980). Hemolytic factors have been found in the sera of trypanosome-infected cattle (Mc crorie et al.,1980). A number of immunological mechanisms have been proposed to be involved in the hemolysis. Trypanosome antigen has been demonstrated on the surface of erythrocytes. Antiglobulin tests showed that the erythrocytes of calves infected with T. congolense had absorbed immunoglobulins. Preformed antigen-antibody complexes may be absorbed onto erythrocytes, a phenomenon found in immune complex diseases , this would account for the detection of both antigen and immunoglobulin. Such immune complexes may fix complement on the erythrocyte surface, resulting in intravascular hemolysis or erythrophagocytosis or both. Complement has been detected on the surfaces of erythrocytes of patients infected with African trypanosomes (Ayele et al ., 2017; Baral et al., 2007 ;Vali et al.,1978). These observations would be consistent with antibody or antibody complement coating of erythrocytes and host parasite interactions (Nicholas et al., 2009;Babatunde et al., 1982). African trypanosomiasis is one of the major disease constraints to livestock production in sub-Saharan Africa. While the pathology of the disease has been well documented, surprisingly little is known about the mechanisms that cause disease. An unusual feature associated with the immune response during trypanosome infections in man and animals is that a large fraction of the antibodies produced are IgM. (Tacket et al., 1989). Although increases in serum IgM have been consistently observed in many trypanosome infections of cattle,the appearance of trypanosome non-specific antibodies was not a consistent finding. Recently, following the observation that numbers of CD5+B cells increased in blood and spleen concomitantly with an increase in serum IgM during T. congolense-infection of cattle (Naessens et al., 1992)  IgM is one of several isotypes of antibody (also known as immunoglobulin) that are produced by vertebrates. IgM is the largest antibody and it has a molecular weight of 500-990 Daltons, it is the first antibody to appear in the response to initial exposure to an antigen. [1][2] In the case of humans and other mammals that have been studied, the spleen, where plasmablasts responsible for antibody production reside, is the major site of specific IgM production.(Baron 2019)

Functions
-Opsonization and cytotoxic destruction of antigens -Found on mucosal surfaces and serve as precursor for light chain Immunoglobulins -Able to bind with light chain Immunoglobulin to effect desired response.
-Aids in diagnosis of infections in early stage.

Immunoglobulin G (IgG)
IgG is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. Hypothyroidism can cause both decreased GFR and hypovolemia, but BUN-to-creatinine ratio has been found to be lowered in hypothyroidism and raised in hyperthyroidism.(Myoclinic.org 2013) The main causes of a decrease in BUN are severe liver disease, anabolic state, and syndrome of inappropriate antidiuretic hormone. [5] 2.2.2 Alanine transaminase (ALT) Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021 .It is also called alanine aminotransferase (ALAT) and was formerly called serum glutamate-pyruvate transaminase (SGPT) or serum glutamic-pyruvic transaminase (SGPT) and was first characterized in the mid-1950s by Arthur Karmen and colleagues.
[1] ALT is found in plasma and in various body tissues but is most common in the liver. It catalyzes the two parts of the alanine cycle. Serum ALT level, serum AST (aspartate transaminase) level, and their ratio (AST/ALT ratio) are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels(Analyte monogragh 2013)

Function
ALT catalyses transfer of an amino group from L-alanine to Alpha ketoglutarate to produce pyruvate and Lglutamate(Transamination) Signifacant levels of ALT often suggest medical problems such as Hepatitis,Congestive heart failure.Liver damage or Myopathy.
ALT is commonly used as a tool for screening for Liver problems.

Free Fatty Acid
Free fatty acid is carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 28.
[1] Fatty acids are usually exist as three main classes of esters: triglycerides, phospholipids, and cholesteryl esters. In any of these forms, fatty acids are both important dietary sources of fuel for animals and they are important structural components for cells. When circulating in the plasma (plasma fatty acids), not in their ester, fatty acids are known as non-esterified fatty acids (NEFAs) or free fatty acids (FFAs). FFAs are always bound to a transport protein, such as albumin Carbohydrates are converted into pyruvate by glycolysis as the first important step in the conversion of carbohydrates into fatty acids.
[23] Pyruvate is then decarboxylated to form acetyl-CoA in the mitochondrion. However, this acetyl CoA needs to be transported into cytosol where the synthesis of fatty acids occurs. This cannot occur directly. To obtain cytosolic acetyl-CoA, citrate (produced by the condensation of acetyl-CoA with oxaloacetate) is removed from the citric acid cycle and carried across the inner mitochondrial membrane into the cytosol.
[23] There it is cleaved by ATP citrate lyase into acetyl-CoA and oxaloacetate. The oxaloacetate is returned to the mitochondrion as malate.
[24] The cytosolic acetyl-CoA is carboxylated by acetyl CoA carboxylase into malonyl-CoA, the first committed step in the synthesis of fatty acids The "uncombined fatty acids" or "free fatty acids" found in the circulation of animals come from the breakdown (or lipolysis) of stored triglycerides.[23

Experimental animals
Three apparently healthy sheep of Yankasa breed aged between 1.5 and 2 years will be purchased from sheep markets in an apparently tsetse fly-free area. The ages of these animals will be confirmed using the dental eruption pattern described by Wilson and Durkin (I984). The animals will be kept in a fly-proof experimental house of the Department of Veterinary Medicine and Surgery, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, and screened of haemoparasites. They will be fed adequately (with concentrates, cotton seed cake, groundnut and cowpea hays, corn bran and salt licks). They will also be provided with water ad libitum.
The animals will be dewormed using a broad spectrum dewormer ( albendazole at a dose of 5 mg/kg and with a broad spectrum antibiotic , oxytetracyclive LA at a dose rate of 20 mg/kg). The animals will also be sprayed against external parasites using diazintol at a concentration of 2 ml/litre of water. Each of the animals will be ear tagged for proper identification. The sheep will be allowed to acclimatize for a period of three weeks and they will be Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021 routinely examined and subjected to usual handlings to determine temperature,puse and respiratory rates. Blood collection twice weekly for parasite screening and establishment of baseline haematological and biochemical analyses.
Just before commencement of the experiment,the Rams blood will be examined for the preinfection blood parameters . 1 ml of blood would be taken to parasitology to check for parasitemia ,1 ml to clinical pathology laboratory for hematologic blood analysis and 2 mls to public health laboratory to check the immunoglobulin level.

Trypanosome stock
The parasite, T .congolense, will be sourced from Nigerian Institute for trypanosomiasis research in Kaduna. The stabilate of the parasite will be inoculated into two Wistar rats intraperitoneally and subcutaneously. The infected rats will be immediately transported to the department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria. The rats will be kept separately and fed with commercial pelleted feeds. Blood samples will be collected from each of the rats everyday for parasitaemia detection using the method of Woo (1969).

Infection of sheep
When parasitaemia reaches an estimated level of 10 6 trypanosomes/ml, the rat will be put on chloroform anaesthesia, and its jugular vein severed to obtain sufficient blood for infecting the Rams intravenously.

Infection and Detection of Parasitaemia in the experimental sheep
Following the infection of the Rams through the jugular vein, blood sample (3 ml) will be collected, everyday until parasitaemia is detected and then every other day, from each of the sheep , beginning from day 1 postinfection. One millilitre of the blood will be dispensed into a vacutainer containing 2 mg ethylenediamine tetraacetic acid(EDTA) to determine parasitaemia and its level, and also used in for haematologic and eucocytic analyses. The other 2 ml will be used to obtain serum for use in biochemical analyses (Woo,1969). Parasitaemia will be detected in the T. congolense-infected sheep as described earlier. Estimation of the level of parasitaemia will be carried out according to the modified method of Mutayoba et al.( 1994).

Haematologic and eucocytic evaluations
Packed cell volume (PCV), red blood cell count, and Leucocyte(WBC) count and differencia eucocyte count will be estimated as described by Coles (1986) .

PCV And Red bood ce count
Boob is coected in capiary tubes and then seaed at one end and centrifuged at 2000rpm for 5mins and haematocrit reader is used to determine packed red bood ce in percentage.The Red bood ce wi be counted after diuting 0.5ms of norma saine with 0.5ms of bood and then haemocytometer is used to determine the number of Red ces. 3.5.2 WBC and differentia count WBC count woud be determined by diuting 0.5ms of bood in 0.5ms of 2% acetic acid and haemocytometer woud be used to determine the number of ces. Thin bood smear woud be made on a gass side air dried and then fixed in methano for 5mins thereafter paced in giemsa for 25mins. The side is the viewed under oi immersion for Differencia WBC count .

Determination of serum immunoglobulin levels( IgM and IgG quantitation)
Abcam's Immunogobuin Sheep ELISA Kit (ab190546) is a two-site enzyme linked immunoassay (ELISA) for the measurement of IgG and IgM in sheep biological samples.
In this assay the IgG and IgM present in samples reacts with the anti-IgG or anti IgM antibodies which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound proteins by washing, anti-IgG antibodies or anti IgM antibodies conjugated with horseradish peroxidase (HRP), are added. These enzymelabeled antibodies form complexes with the previously bound IgG or IgM. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3',5,5'tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of Immunoglobulins in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of IgG Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021 60 or IgM in the test sample. The quantity of IgG or IgM in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for sample dilution.

Enzyme-linked immunosorbent assay (ELISA) for Sheep antibodies
The method of Buza et al., 1997 will be adapted. Serum will be prepared from blood samples collected sequentially over a 5-week period during trypanosome infections and immediately stored at -70° until use. Each well of a 96well Dynatech ELISA plate (Dynatech,Plochingen, Germany) will be coated with 100 ug of a 40 ug/ml solution of trypanosome lysate in coating buffer (0 05 M bicarbonatebuffer, pH 9 6) and the plates will be incubated overnight at 4°C. The plates will then be washed three times with washing buffer (phosphate-buffered saline pH 74, containing 0 1%Tween 20). Thereafter, 100 ul of serum diluted 1/100 in washing buffer will be added in duplicate wells and incubated for 2 hr at 37°. The plates will then be washed and 100 gl of horseradish peroxidase-conjugated monoclonal antibodies (mAb) IL-A2, specific for sheep IgG,` or mAb IL-A30,specific for IgM,` were added for 1 hr at room temperature.After washing, 100 ul of substrate K-blue ELISA)Technologies, Lexington, KY) will be added to each well and the optical density determined at 450 nm on a Titertek Multiskan MCC/340 ELISA plate reader (Flow, Oy, Finland) following a 30-min incubation. Mix,absorbance of sample will be read against the reagent blank after 5minutes. Activity of ALT and AST will then be calculated. Reagent 1 =( 100mmol/I,pH 7.4 of phosphate buffer , 200mmol//lof L-alanine 2.0mmol/l of α-oxoglutarate). Reagent 2= 2.0mmol of 2,4 dinitrophenylhydrazine (Reitman, 1957;Schmidt 1963;Gometi et al.,2014)

Measurement of Blood urea Nitrogen
Creatinine gives a red colour with alkaline solution of picric acid.The red colour is measured in the presence of a high background yellow colour,the solutions will appear orange at low creatinine concentration.

Statistical analysis
Values of individual parameters in both the infected and contro group will be subjected to simple descriptive statistics using tables and charts . Anaysis of variants wi be used to check Significant changes in mean and standard deviation in the infected and contro groups through comparison of data obtained.
9. Results and Discussion 9.1 Parasitaemia, Leucocytic index and variable surface glycoprotein(VSG) Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021 61 The parasitaemia in T.congolense infection in the infected Rams were as presented in Table I .The organisms were detected in the blood of the infected Rams simultaneously on day 6 post infection.Hence the incubation period for this experiment was six days.Mean parasitemia rose from 8x10 6 trypanosomes per milliliter(tryps/ml) of blood(2tryps/field of view) from the 6day and reached peak values of 16x10 6 Tryps/ml of blood on day 22 post infection (5tryps/field). On day 15 post infection two of the Rams died and parasitaemia at that stage was 8x10 6 (2tryps/field)prior to death they showed clinical signs which were Anorexia, Rhinitis, dyspnea ,hemoglobinuria,Hematuria,paper white ocular mucous membranes,paralysis of the fore and hind limbs,diarrhoae and the Rams died after this signs were observed. .Blood was taken from the Rams prior to infection so as to compare the parameters required pre and post infection. White blood cell count was normal pre infection with a mean value of 8.5x10 9 /L. Post infection on day 7, leukopenia was observed and the value was 3.4 x 10 9 /L and on day 14 leukocytosis was seen again and the value was 7.9 x 10 9 /L but on day 22 there was a relapse back to leucopenia and the value was 4.8 x 10 9 /L but on day 28 leukocytosis was seen again and the value was 6.1 x 10 9 /L. On the 29 day the animal died.The fluctuation in Leukocytosis and Leukopenia might be as a result of the parasite varying it's surface glycoprotein at several intervals( Antigenic variation). Table 1 shows the relationship between WBC and VSG . The control Mean Leukocytic index was 8.5x10 9 L at the beginning of the experiment but this gradually started to decrease until day 7 probably due to the response of the body to the parasite but on day 14 there was a build up of Leukocytes to a value of 7.9 x10 9 L. This maybe due to the parasite varying its surface glycoprotein and the immune system recognizes the parasite as new. This pattern was sustained with increasing parasitaemia until the animals died.

Packed cell volume(PCV)and parasitaemia
The Mean PCVs of the control Rams pre infection was 27% which was within the Normal Range. Following infection with T.congolense there was a gradual and progressive drop in mean PCV value. On day 6 when parasitaemia was noticed the packed cell volume had dropped considerably to 21% and parasitaemia at this stage was 8x10 6 (2tryps/field).This parasitaemia was maintained for fourteen days post infection while the PCV at this stage was 13% and at this stage, two of the infected Rams had died(day 15).PCV continued to drop as parasitaemia increased and when parasitaemia was 16x10 6 (5tryps/field) on day 28 ,PCV was 6% and on day 29 the last Ram was found dead. It is therefore evident that increasing parasitaemia causes intravascular and Extravascular hemolysis because tissues observed had petechial hemorrhages,and a severe decline in PCV was observed. More so there was Hemorrhage by diapedesis and rhexis as whole blood was lost in urine(Hematuria) probably due the fact that the kidney was gradually failing to filter blood as a result of the infection with the organism in question. There was a mean PCV decline rate of ±2.. Table II showing relationship between PCV and parasitaemia. 9.3 Changes seen in Vital Organs associated with Trypanasoma congolense infection The Liver,Kidney,Heart and the Lungs were observed for Gross Pathogic lesions associated with this infection,but prior to that the Trachea was seen to contain froathy exudates and petechial hemorrhages and,the thoracic muscles were severely emaciated. Thoracic cavity contained copious amount of Sero-fibrinous exudates which were blood tinged. But the area of interest were the changes seen in the vital organs.

Heart
The Muscles of the heart were inflamed especially the Pericardium and Myocardium and there was serous atrophy of fat(yellowish-red discoloration) which is an indication of starvation and inflammation. There was Edema ,slight enlargement and congestion of the heart on cut surface .

Liver
The liver was also inflamed and the parietal Surface had areas of Focal Necrosis which were grey , round and unraised .It was slightly enlarged and edematous because the sharp edges had been lost and congestion was seen on cut surface.

Lungs
The Lungs were congested and inflamed slightly edematous and the left lung had Rib impression. It showed no sign of consolidation because it did not sink when a small portion was immersed in water ,there were areas of petechial hemorrhages.

Kidney
The Kidney was inflamed because the Kidney capsule had croupous exudates and it detatched easily form the Cortex .It was congested and slightly enlarge

Changes in serum IGG and IGM Concentration
The Mean serum immunoglobulin IGG and IGM were as presented in Table V .The mean values for the preinfected groups were 1.40µg/ML and 0.71µg/ML for IGG and IGM respectively .These values are comparable and the concentration of IGG was higher than that of IGM in both groups(infected and control).On Day 7 post infection a gradual decrease of 0.30µg/ML and 0.50µg/ML was observed for both IGG and IGM respectively and on Day 14 peak values of 0.75µg/ML and1.3µg/ML was observed for IGG and IGM respectively until day 16 when both values started to decrease to 0.35µg/ML and 0.25µg/ML respectively. Prior to the death of the Rams on Day 22 the values of IGG and IGM had decreased to 0.21µg/ML and 0.34µg/ML respectively. These fluctuations may be associated with the massive invasion of blood by the parasites and the inability of the host immune system to produce enough humoral immunity. More so the WBC,s had been compromised with a remarkable decrease in their number. Memory cells produced could not be sustained for long due to parasite varying its surface glycoprotein at irregular intervals.

Conclusion
Anemia found in this experiment was hemorrhagic and hemolytic and the red blood cell morphological appearance was at first Normocytic normochromic but towards the terminal stages of the experiment became Microcytic hypochromic. The Leucocytes where degenerative with the appearance of many band cells( immature cells with fragmented and spherical nucleus with weak cell membrane) in their formative stages and these band cells where more numerous than the mature normal neutrophils. Leucocytosis was relative because the leucocytes observed in the infected group were not up to the value seen in the control. Cellular and humoral immunity were not conferred due to this reasons, more so macrophages in tissues were also overwhelmed by the parasites because most of the organs observed such as the liver, lungs, kidney and heart were in their early stages of destruction. In this experiment it is therefore evident that Anemia, leucopenia ,immune suppression , hypoprotenemia and hypoglycemia may be the cause of death in Trypanosoma congolense infection in sheep more so the anemia was both hemorrhagic and hemolytic. This experiment has also shown that erythrocyte and leucocyte(Plasmids) proteins ,lipids and carbohydrates covering their cell membranes might be a source of nourishment for the parasite in concern and despite the VSG of Trypanosoma ,infected animals still respond to treatment so in my opinion suppressed immunity might be the major problem. I wish to acknowledge the following professors namely S. 11. Recommendations i. Funds should be granted to researchers carrying out experiments on Trypanosomiasis because much work still need to be carried out as it is still endemic in Africa. ii. Laboratories should be well equipped with standard laboratory equipments so as to be able to compete favorably with developed countries especially in areas of emerging diseases. iii .Training and re-training of staff to help facilitate research experiments and also in areas of emerging diseases. iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11 No.6, 2021