The aetiology and significance of the phagocytosis of erythrocytes and leucocytes in sheep infected with Trypanosoma congolense (Broden, 1904).

The phagocytosis of erythrocytes and leucocytes in sheep infected with Trypanosoma congolense is shown to be due to the coating of the blood cells with trypanosomal antigen. The occurrence of the phagocytic activity is dependent on fluctuations of the parasitaemia and is significant in maintaining the anaemic state of the subject.     

Cellular immunity to canine mammary tumor cells demonstrated by the leucocyte migration technique.

Cellular immunity to canine mammary tumor cells was studied by means of the leucocyte migration technique (LMT). Intact tumor cells, separated either by enzymatical or mechanical disruption, were used as antigen, and efforts were made to cultivate tumor cells in vitro. Fifteen female tumorous dogs were studied, and 12 non-tumorous mainly male dags were used as controls. Leucocytes from tumor-bearing females were mixed with own autologous or foreign homologous tumor cells, and control leucocytes were presented with cells from the same source. In addition, leucocytes from tumorous animals and controls were mixed.Animal group A comprised 8 tumor-bearing females. In this group mixtures of different cell numbers and different tumor cell/leucocyte ratios were tried. Animal group B comprised 7 tumor-bearing females, and 40 × 10⁶ leucocytes from these were mixed with 2 × 10⁶ antigencells, antigen-cell/leucocyte ratio 0.05. A great number of tumor cells (tumor cell/leucocyte ratio > 0.05) caused strong non-specific inhibition of leucocyte migration, but in spite of marked inhibition (< 61%) in the homologous system in animal group A, inhibition in the autologous system was found to be stronger (72.2–92.3%). In animal group B, dogs presented with own tumor cells showed marked inhibition (23.7–90.1%), while the controls showed a migration inhibition below 20%. Mixtures of homologous leucocytes showed inhibition of the same order as mixtures of control leucocytes and tumor cells. Thus evidence of cellular immunity against own canine mammary tumor cells was obtained. It proved difficult to cultivate the tumor cells for more than 2–3 passages. Some evidence of antigenic cross reactivity was obtained between 2 adenocarcinomas. Enzymatical separation of tumor cells did not seem to alter antigenic characteristics of the cell surface. Mechanical separation, however, proved to be simpler, more rapid and yielded cell suspensions largely free of debris, and is therefore recommended for further work.     

Effect of trypanocides on jugular concentration of Trypanosoma congolense in the West African dwarf sheep

The effects of various trypanocides on parasitaemia was investigated in sheep experimentally-infected with Trypanosoma congolense strain 58/98. Intravenous injection of Berenil at the height of the first parasitaemic wave increased jugular parasite concentration by 12 and 16 times at the 9th and 20th minute post-treatment, respectively. With Pentamidine, maximum counts were 5.0–8.6 times zero-time concentration during the same periods. Peak effects of Samorin, Novidium and Ethidium were observed between the 60th and 90th minutes after drug administration and were 9.5, 6.3 and 3.5 times initial values, respectively. Injection of trypanocides resulted in double peaks of parasitaemia in which the second was usually higher than the first, except with Antrypol and Germani which had no significant effect on parasitaemia. The amplitude, but not the onset of the increase in parasitaemia in sheep, was found to be related to the therapeutic efficacy of the trypanocides in the treatment of Trypanosoma congolense infection in rats.Animals treated with the diamidines (Berenil and Pentamidine) exhibited apparent parasitologic cure of infection in sheep two to four days after treatment. However, administration of any of the drugs one week after the first treatment resulted in flushing of cryptic trypanosomes into the jugular vein and counts as high as 7.63 × 10³ μl^?1 were observed within ten minutes with Berenil. It is suggested that besides their therapeutic use, the diamidines may be of value in the parasitologic diagnosis of sub-patent trypanosomiasis due to Trypanosoma congolense.     

Effects of the timing of antigen stimulation on parasitaemia profile and subsequent immunodepression in an experimentally induced Trypanosoma brucei infection

The influence of administering sheep red cells (SRC) as antigens, either after or before a trypanosome challenge, on parasitaemia profile and antibody response was assessed in albino Wistar rats. High levels of parasitaemia associated with significantly depressed antibody response and packed cell volume (PCV) values were observed when trypanosome challenge preceded antigen stimulation. In contrast, a clear delay in the onset and development of parasitaemia occurred when antigen priming preceded trypanosome challenge. At the beginning, PCV values and antibody response to the antigen were in the range of levels found in control rats. However, as infection progressed, parasitaemia rose and significant immunological hyporesponsiveness developed which at least reached levels found in rats that had received trypanosome challenge prior to antigen stimulation. These findings should be taken into consideration when evaluating serological tests used for assessing responses to specific vaccinations, or for the diagnosis of infections based on rising antibody titres in the host.     

Studies on the role of humoral and cell-mediated immunity in pigs after vaccination with Aujeszky's disease virus

Humoral and cellular immunity in pigs vaccinated twice with Aujeszky's disease virus (ADV) was studied by seroneutralizing test and direct leucocyte migration inhibition technique. Significant migration inhibition of leucocytes (LMI) was found on the fifth day, whereas specific antibodies began to appear at that time only in very low titers. Anamnestic reaction due to the second injection of ADV did not bring about a significant increase of migration inhibition of leucocytes, instead the level of antibodies elevated markedly.     

Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

The pathophysiology of ovine trypanosomosis: haematological and blood biochemical changes.

The course of Trypanosoma congolense infection in sheep was followed for 96 days. Infected animals developed fluctuating parasitaemia, macrocytic normochromic anaemia and leucocytosis which was principally a lymphocytosis. Following treatment with the trypanocidal drug, diminazene aceturate at 84 days after infection, the haematological values returned to normal within 12 days. Infected sheep developed hypocholesterolaemia and hypophospholipidaemia leading to a reduction in total serum lipids. This study has shown that sheep infected with T. congolense develop anaemia, the onset of which follows the first wave of parasitaemia. The changes in blood lipids observed in infected sheep appeared to be related to the intensity and duration of parasitaemia.     

Activation of small ruminant aortic endothelial cells after in vitro infection by caprine arthritis encephalitis virus

Small ruminants infected by the lentiviruses caprine arthritis-encephalitis virus (CAEV), originally isolated from a goat, or maedi-visna virus, originally from sheep, typically develop an organising lymphoid infiltration of affected tissues. This could reflect modulation of the migration pattern of lymphocytes in infected animals. Possible active contribution by vascular endothelial cells was investigated using an in vitro model. Low-passage cultured ovine aortic endothelium proved susceptible to productive infection by CAEV without significant cytotoxicity. Infected endothelial cells maintained expression of endothelial markers, increased MHC class I antigen expression and initiated expression of the adhesion molecule VCAM -1 and, at a late stage, MHC class II antigens. Infected endothelial cells showed a two-fold increase in binding capacity for sheep peripheral blood leucocytes over uninfected controls. Such events could contribute to the tissue distribution of lymphoid cells and local immune responses in lentiviral infections of small ruminants.     

Phagocytosis of Bacteroides nodosus by ovine peripheral blood leucocytes

Phagocytosis of Bacteroides nodosus by ovine peripheral blood leucocytes (PBL) was examined after organisms had been opsonized in sera from normal sheep, or from animals immune to, or infected with ovine footrot. Ingestion of bacteria, as assessed microscopically or by counting isotopically-labelled organisms spectrometrically was effected in suspensions by polymorphonuclear leucocytes (PMN). Opsonization of bacteria in immune serum, particularly its IgG₂ isotype, enhanced the rate of phagocytosis by PMN compared with that promoted by normal serum or medium alone. Whereas IgG₂ from immune serum also increased the rate of ingestion of B. nodosus by adherent PMN, IgM and IgG₁ from immune serum also initiated phagocytosis of bacteria by adherent ovine monocytes. Leucocytes from normal, immune or infected sheep of different breeds ingested B. nodosus with equal facility.     

Parasitaemia and clinical manifestations in Trypanosoma brucei infected dogs.

Four dogs were infected with Trypanosoma brucei (Mkar strain) while another four were used as uninfected controls. Two of the dogs showed acute disease and died in the first wave of parasitaemia on days 7 and 8 post infection (PI) while the other two died from the sub-acute disease on days 24 and 28 PI corresponding to the second wave of parasitaemia. In the first wave of parasitaemia there was a sharp decrease in the packed cell volume, red blood cell, haemoglobin, total leucocytes, eosinophil, neutrophil and lymphocyte values, but during the period of low parasitaemia there was a slight recovery of the values of total leucocytes and lymphocytes although these and the other values showed a continuous decrease during the second wave of parasitaemia. In contrast, there was a consistent monocytosis in both acute and sub-acute diseases. The general picture was that of loss of condition, anaemia, leucopenia, monocytosis, ocular impairment, elevated temperature, pulse and respiratory rates, the difference between the acute and sub-acute diseases being in the degree of intensity. The degree of anaemia noted and the circulatory disturbances associated with the infection could have caused the death of all the infected dogs.     

The effects of Eperythrozoon ovis in sheep

Infection of adult sheep with a single strain of Eperythrozoon ovis led to three different situations. First, the animal resisted the organism and no haematological changes occurred. Second, the host developed a controllable parasitaemia in which erythrocyte values fell shortly after peak parasitaemia and then returned to normal. Third, the host failed to control the parasitaemia and chronic low grade anaemia developed. Dexamethasone sometimes caused a resurgence of parasitaemia in sheep.     

Lymphocyte Stimulation and Leucocyte Migration Inhibition as Diagnostic Tools for the Detection of Mycobacterium Avium Infection in Cattle

The tuberculin skin test is the conventional method of detecting infections with mycobacteria in animals. A positive reaction is considered to reflect cell-mediated immunity (CMI). CMI against mycobacteria can be studied by in vitro systems using suspensions of blood lymphocytes or leucocytes. The reactivity of these cells to different antigens can be measured in the lymphocyte stimulation (LS) (Muscoplat et al 1975, Bergman 1976, Johnson & Morein 1976), or leucocyte migration inhibition (LMI) (Aalund 1970, Clausen 1973) tests.     

Failure to induce homologous immunity to Fasciola hepatica in sheep vaccinated with irradiated metacercariae

Vaccination of sheep with either 100 or 1000 γ-irradiated (2.5 krad) metacercariae of Fasciola hepatica, on two occasions six weeks apart, did not generate significant protection against intraruminal challenge with F. hepatica six weeks after the second vaccinating dose as measured by recovery of flukes from liver and bile ducts, twenty weeks after challenge. There was, however, a significant increase in the proportion of flukes retarded in the parenchyma of both vaccinated groups. The percentage of retarded flukes was positively correlated with the degree of liver damage and increased weight of the hepatic lymph nodes. It was not possible to determine if the retarded flukes were derived from the vaccine or challenge infections or both.Challenge infection of both vaccinated and unvaccinated sheep significantly increased the numbers of eosinophils and globule leucocytes in the parenchymal bile duct and the numbers of mast cells and globule leucocytes in the abdominal bile duct. In addition the numbers of eosinophils and globule leucocytes in the parenchymal bile duct were significantly correlated with the percentage of retarded flukes in both vaccinated groups. In the abdominal bile duct, only the numbers of eosinophils in the low level vaccination group were significantly correlated with fluke retardation.Vaccination did not protect against the pathogenic effects of challenge infection as measured by reduced packed cell volumes and weight gain.     

Humoral immune response of sheep to infection with Eperythrozoon ovis

Circulating antibody was detected by an indirect fluorescent antibody test (IFAT) in the serum of sheep infected experimentally with Eperythrozoon ovis. Antibodies were first detected 15 to 32 days after infection with E ovis and titres peaked at 41 days. This antibody may be associated, at least in part, with protection against infection with E ovis since the initial increase in antibody titre coincided with a fall in the primary parasitaemia. A role for antibody is suggested further by the fact that the prepatent period of infection was prolonged by one day and the parasitaemia initially remained at low levels in infected sheep protected by passively transferred hyperimmune serum. Moreover, following primary infection, acquired immunity was manifest by a lack of parasitaemia following challenge infections while increased IFA titres were observed. No evidence of opsonic activity was observed in an in vitro erythrophagocytosis test in that neither mouse macrophages nor sheep monocytes phagocytosed E ovis infected or uninfected erythrocytes sensitised with hyperimmune serum.     

Cell-mediated immunity in mouse experimental coxsackie A9 virus infection

Using a human A₉ Coxsackievirus strain as antigen and different-aged A₂G inbred mice as receptors an in vitro secondary cell-mediated immune response was noticed, revealed by means of two markers, i.e. leukocyte migration inhibition and T spleen lymphocyte blastogenesis, in the presence of viral antigen.     

Observations on the pathology of Eperythrozoon ovis infection in sheep

The main pathological features of experimental Eperythrozoon ovis infection were an increase of spleen weight by up to 250% at the peak of parasitaemia and an increase in liver weight by 36%. Haemosiderin was present in the kidneys, livers and spleens of all infected sheep at the peak and late stages of parasitaemia. Control sheep had haemosiderin in the spleen only. On the basis of these findings, intravascular haemolysis appears to be the predominant mode of red cell removal. Although not observed histologically, some erythrophagocytosis by the spleen and liver probably occurs in the course of the infection.     

Effects of vasopressor drugs on number of Trypanosoma congolense in ruminant blood

The effects of intravenous administration of vasopressor drugs on parasite concentration was studied in cows and sheep infected with Trypanosoma congolense. Ephedrine (USP), adrenaline (BP) and insulin increased jugular concentration of the parasite. Maximum effects were reached between 1 1/2 and 2 h with ephedrine and at 2 1/2-3 h with adrenaline. Increases in parasitaemia resulting from epinephrine were sustained only in sheep and adrenaline gave similar effects in both ruminants. Only epinephrine significantly increased parasitaemia within 45 min of administration. Ephedrine, hydralazine and guanethidine had no significant effects on parasitaemia. It is suggested that one of the mechanisms by which trypanocides increase jugular concentration of T. congolense may be by vasoconstriction of the capillary endothelium.     

Immune response to haemophilus parahaemolyticus (HP) infection in the pig I. In vitro tests to detect sensitized cells in the blood of infected and vaccinated animals

The in vitro immune response of pigs in herds infected or non-infected with H. parahaemolyticus (HP) was investigated by lymphocyte stimulation and leucocyte migration inhibition tests. The stimulation of pig lymphocytes with HP bacteria has been measured by ³H-thymidine incorporation. The response of the cells seems to be specific, lymphocytes from chronically HP-infected animals showed positive reactions with high frequency, while on the contrary, cells from SPF (specific pathogen free) animals reacted only occasionally to the HP antigens. However, in the leucocyte migration inhibition test the reactions were less specific; the widely occurring H. parasuis bacteria seem to possess antigen(s) which causes cross reactions with HP in this test.Vaccination with HP bacteria induced sensitive lymphocytes in the blood of SPF pigs and an increase of the in vitro lymphocyte reactions in already positive animals.     

Prevalence of peste des petits ruminants among sheep and goats in India

This study measured the clinical prevalence of peste des petits ruminants (PPR) among sheep and goats in India between 2003 and 2009 by analyzing clinical samples from suspected cases of PPR that were submitted to the Rinderpest and Allied Disease Laboratory, Division of Virology, IVRI, Mukteswar for PPR diagnosis. PPR outbreaks were confirmed by detecting PPR virus (PPRV)-specific antigen in the clinical samples. Clinical samples (blood, nasal swabs, spleen, lymph node, kidney, liver, intestine, and pooled tissue materials) were taken from a total of 592 sheep and 912 goats in different states of India and screened for the presence of PPRV antigen using a monoclonal antibody-based sandwich ELISA kit. A total of 20, 38, and 11 laboratory-confirmed PPR outbreaks occurred among sheep, goat, and combined sheep and goat populations, respectively. Our findings provide evidence of widespread PPR endemicity in India. The underlying reasons could be variations in husbandry practices in different geographical regions, agro-climatic conditions, and livestock migration. Furthermore, decrease in the number of PPR outbreaks over time might be due to the effectiveness of current live PPR vaccines and timely vaccination of target species. Vaccination against PPR has been practiced in India since 2002 to control this disease.     

Serological investigation of Corynebacterium pseudotuberculosis infection in sheep--correlation between the hemolysis inhibition test and the ELISA test

Corynebacterium pseudotuberculosis causes caseous lymphadenitis in sheep and goats. The animals may be infected without showing clinical symptoms. Several serotests have therefore been employed to detect infected animals. Shen et al (1982) performed an enzyme linked immunosorbent assay (ELISA) to detect antibodies against the organism using cell wall antigens. Maki et al (1985) found that the toxin of the bacterium was a better antigen for assessing infection in the ELISA test. They reported that the antitoxin ELISA appeared to be as sensitive as the antihemolysin inhibition test (Zaki 1968).