End Point Multiplex PCR for Diagnosis of Haemoprotozoan Diseases in Cattle.

BACKGROUND: Theileriosis, trypanosomosis and babesiosis are the three major haemoprotozoan diseases causing huge economic losses worldwide. Difficulty in diagnosis of these diseases lies with the detection of carrier state with low parasitemia and concurrent infection. PURPOSE: The present study was conducted to standardize and evaluate multiplex PCR assay for specific, fast and simultaneous detection of Theileria annulata, Trypanosoma evansi and Babesia bovis in bovines. METHODS: Positive parasitic DNA was obtained from microscopically positive samples. Simplex PCR assay was developed targeting repetitive nucleotide sequences for Trypanosoma evansi and gene coding enzyme carbamoyl phosphate synthetase II for Babesia bovis. For theileriosis conditions already standardized targeting cytochrome b gene was used. Gradient PCR assay was used to determine common amplification conditions and develop multiplex PCR assay. Limit of detection was determined using tenfold serial dilution of parasitic DNA. Blood samples collected from 117 bovines suspected for haemoparasite infection was tested by simplex and multiplex PCR assay. RESULTS: Simplex PCR assay was able to detect Theileria annulata, Trypanosoma evansi and Babesia bovis at dilution 10-9, 10-8 and 10-8 which corresponds to copy number 1, 10 and 10, respectively, whereas of multiplex PCR assay was found to be 10-7 dilution corresponding to 100 copy number. PCR products bands obtained in multiplex PCR assay at 257, 312 and 446 bp were easily distinguishable. Results of simplex PCR assay for detection of individual parasites revealed 48 (41.02%), 27 (23.07%) and 5 (4.27%) samples positive for T. annulata, T. evansi and B. bovis, respectively. Sixty-three (53.8%) samples were found positive by multiplex PCR assay with 15 samples (23.8%) showing mixed infection. CONCLUSION: Multiplex PCR assay was found to be highly specific and can be used for easy, early, sensitive, specific and simultaneous diagnosis of haemoprotozoan diseases in epidemiological survey as a robust tool.

Comparative evaluation of polymerase chain reaction assay with microscopy for detection of asymptomatic carrier state of theileriosis in a herd of crossbred cattle.

AIM: This study aims to develop and to standardize a polymerase chain reaction (PCR) assay that will diagnose clinical as well as carrier state of the disease and to compare the results with conventional microscopy technique. MATERIALS AND METHODS: A herd of crossbred cattle with the previous history of theileriosis in village Lahli, district Rohtak, Haryana, was selected for this study. A total of 29 blood samples were collected randomly from cows including five clinically ill cattle. Blood smears from all animals and lymph node biopsy smears from animal with swollen lymph nodes were examined microscopically after conventional Giemsa staining. Phenol chloroform isoamyl alcohol method was used for extracting DNA from blood. Previously published primers targeting cytochrome b gene sequence of Theileria annulata were used in the PCR assay that was standardized to use in the laboratory. RESULTS: Out of 29 samples tested,18 (62.06%) were found positive for theileriosis by PCR assay, whereas only 10 (34.48%) samples were detected positive by conventional microscopic technique using Giemsa staining method. CONCLUSIONS: On the basis results of comparative studies, it can be concluded that PCR assay is a more sensitive than microscopic examination for detection of theileriosis. This can be attributed to the ability of PCR assay to detect small amounts of genomic DNA of T. annulata or low parasitemia in cows. Therefore, PCR assay can serve as a more sensitive tool to detect Theileria for detection of theileriosis even in asymptomatic carrier cattle which is important for the implementation of successful control programs.

Modification of accessory activity of sheep monocytes in vitro by a coenurus antigen from Taenia multiceps.

A factor in Taenia multiceps coenurus fluid (TMCF) has previously been shown to modify the accessory activity of murine macrophages in vivo and in vitro. The factor (TMCF-F24) has been purified by ion exchange in a fast protein liquid chromatography (FPLC) system. This study was conducted to determine whether TMCF-F24 is an antigen in naturally occurring cerebral coenuriasis, and whether it can also modify normal sheep blood monocytes. Specific IgG antibodies to TMCF-F24 were detected, using ELISA, in serum and cerebrospinal fluid of sheep with clinical coenuriasis. Alterations in monocyte accessory activity were detected by an assay which measured the rate of increase in mitogen-induced lymphocyte transformation caused by addition of increasing numbers of the monocytes. Normal monocytes caused a positive increase in lymphocyte transformation. Monocytes incubated with TMCF-F24 caused progressive inhibition of transformation. This factor may therefore modify monocyte-T cell interaction in natural infection.

Comparison of cellular schizont, soluble schizont and soluble piroplasm antigens in ELISA for detecting antibodies against Theileria annulata.

The efficacy and suitability of cellular schizont, soluble schizont and soluble piroplasm antigens was compared for detecting antibodies against Theileria annulata. Fifty bovine sera of known identity were evaluated in ELISA using the above mentioned antigens. Antibody titres of 1:100 to 1:51,200 were detected while using soluble piroplasm and cellular schizont antigen in ELISA. The titres ranged between 1:100 to 1:25,600 with the soluble schizont antigen. Soluble piroplasm antigen exhibited the highest antibody titres followed by cellular schizont and soluble schizont antigens. Cellular schizont antigen proved to be better than soluble schizont antigen for detecting anti-schizontal antibodies. Antibody titres obtained by the three antigens exhibited a good linear correlation amongst each other. The study showed that soluble piroplasm and cellular schizont antigens can be used successfully for detecting antibodies against piroplasm and schizont stages of T. annulata, respectively in bovine sera.

Immunological activities of a lymphocyte mitogen isolated from coenurus fluid of Taenia multiceps (Cestoda).

The purification of a mitogen from Taenia multiceps coenurus fluid has been previously reported. In the present study, this activity, which was independent of endotoxin, stimulated the expression of lymphocyte IL-2 and Fc receptors, enhanced mitotic response to phylohaemogglutinin and concanavalin A and antagonised the previously described suppressive effects of the macrophage modifying fraction of coenurus fluid. The mitogen also increased peritoneal macrophage count and viability, Fc receptor expression and Fc receptor-mediated phagocytosis. The mitogenic activity could be destroyed by a combination of protease and amylase, but not by either enzyme alone. It is suggested that the mitogen forms part of a homeostatic mechanism for the preservation of a balanced host-parasite relationship.

Taenia multiceps (Cestoda): Ia antigen expression and prostaglandin secretion by parasite-modified, murine peritoneal macrophages.

Taenia multiceps secretions modify accessory cell activity in macrophages. The present experiments were designed to elucidate the cellular mechanisms involved. While normal, murine peritoneal macrophages amplified mitogen-activated T-cell proliferation, macrophages modified by exposure to parasite secretions inhibited this proliferation. The modified behaviour was shown by glutaraldehyde-fixed as well as living macrophages, and modification was inducible by FPLC fraction 24 of coenurus fluid and was associated with an expanded population of 1a- macrophages. Secretory products of parasite-activated macrophages also inhibited T-cell proliferation, and secretion was prevented by indomethacin. The measurement of modified accessory activity was not influenced by the concentration of tritiated thymidine in lymphocyte proliferation assays. Consequently there is no evidence that the reported events are affected by macrophage-derived, cold thymidine secretion. It is concluded that T. multiceps si able to manipulate macrophage accessory function by mechanisms which involve altered histocompatibility antigen expression and the secretion of prostaglandin.

An immunisation trial with in vitro produced Babesia bigemina exoantigens.

Bovine babesiosis, caused by Babesia bigemina, is an important tick-transmitted haemoprotozoan disease in the tropics. This study evaluated the immunoprotective efficacy of in vitro produced B. bigemina exoantigens in bovine calves. The calves inoculated with B. bigemina exoantigens did not show any clinical, parasitological or hypersensitivity reactions after inoculation. They withstood challenge without showing any clinical symptoms except a transient thermal reaction. In contrast, two out of four control calves exhibited clinical symptoms of babesiosis and one died. On challenge, there was a significant reduction in the haematological values of both groups. However, this was more pronounced in the control animals. Challenge resulted into a normocytic hypochromic anaemia. The vaccinated animals revealed a significant rise in antibody titres after vaccination as well as after challenge as detected by a single dilution ELISA. The rise in antibody titres of control animals was only moderate. Inoculation of B. bigemina exoantigens induced a protective immune response in the vaccinated animals which could protect them from infected blood challenge.

Regulation of macrophage-mediated larvicidal activity in Echinococcus granulosus and Mesocestoides corti (Cestoda) infection in mice.

Killing of metacestodes by normal or post-infection macrophages and the regulation of this activity by cytokines were studied in vitro. The protoscolecidal activity of normal macrophages against Echinococcus granulosus was inhibited by a product of naive T-enriched lymphocytes co-cultured with protoscoleces (PSC). By contrast, supernates from co-cultures of Mesocestoides corti tetrathyridia (MCT) and T-enriched or B-enriched normal lymphocytes increased killing of MCT by normal macrophages. Larvicidal activity (against both PSC and MCT) was enhanced by high concentrations of macrophage-activating factors produced by Con A-stimulated rat lymphocytes (Con A-LK), but was reduced by low concentrations of these factors. Activation by synergism between Con A-LK and recombinant interferon-gamma(r. IFN-gamma) was demonstrated in macrophage-mediated killing of MCT at high effector to target ratio. Cytokine-activation of normal or post-MCT infection macrophages was compared. Macrophages from both 8 and 20 week post-infection mice were refractory to lymphokines from lymphocyte-MCT cultures and displayed greatly reduced killing of MCT. Macrophage activation by Con A-LK and r.IFN-gamma was also impaired, implying a general defect in the ability of these post-infection macrophages to respond to macrophage activating signals. The data indicate that two different mechanisms may exist by which metacestodes regulate potentially larvicidal effector mechanisms. E. granulosus can elicit the production of lymphokines suppressive for PSC killing, whereas M. corti appears directly to induce a refractory state in effector macrophages.

Differential regulation of murine Mesocestoides corti infection by bacterial lipopolysaccharide and interferon-gamma.

Many liver-invasive parasites cause extensive liver damage which may result in an impaired ability to catabolize endotoxin. The influence of endogenous endotoxin on the progress of liver-invasive parasitic diseases has been investigated in murine Mesocestoides corti infection. Invasion of liver tissue by tetrathyridia resulted in extensive parenchymal destruction with fibrosis. In association with this, undetoxified endotoxin, in potentially biologically active concentration, was found on peritoneal macrophages, 5 months post-M, corti infection. Host susceptibility was influenced by the Lps gene for responsiveness to lipopolysaccharide (LPS). The parasite burden of LPS-responsive (C3H/HeN) mice was significantly increased in the livers of these mice when compared to LPS-resistant (C3H/HeJ) mice. LPS reduced the ability of normal peritoneal macrophages to kill tetrathyridia, when co-cultured in vitro. LPS also abrogated the ability of recombinant interferon-gamma (r.IFN-gamma) to enhance macrophage larvicidal activity. These in vitro findings were confirmed in vivo. Daily intraperitoneal administration of LPS, at low concentration, caused a 4-fold increase in parasite burden in the liver, while r.IFN-gamma at optimal concentration reduced parasite burden by 57%. Post-infection macrophages have previously been shown to be refractory to cytokine-activation for larval killing. In this report, we conclude that (1) this refractoriness may be due to the presence of undetoxified endotoxin on post-infection macrophages and (2) endotoxin may reduce host resistance by abrogating effector macrophage response to IFN-gamma.

Responses in animals vaccinated with the Theileria annulata (Hisar) cell culture vaccine.

Bovine tropical theileriosis caused by Theileria annulata is an economically important disease of cattle in India. The disease has assumed paramount importance with the intensification of cross-breeding programmes aimed at enhancing milk production in the country. To control this disease, a cell culture vaccine was developed in this department by continuous passaging of T. annulata (Hisar) schizonts in vitro. Current work in this department has concentrated on the epidemiology of theileriosis: development of the cell culture vaccine for very young calves and pregnant cows; evaluation of serological responses using immunofluorescent antibody (IFA) tests and Enzyme-linked immunosorbent antibody assays (ELISA); studies on the duration of immunity stimulated by the cell culture vaccine; the immune/susceptible status of calves born to vaccinated dams. Results have shown the following. Clinical cases of theileriosis were mainly observed in young calves below two months of age followed by adults in exotic and cross-bred animals. Amongst indigenous animals, only young calves below two months of age suffered from clinical disease. Clinical cases of theileriosis mainly occurred between the months of April to October. The T. annulata schizont cell culture vaccine developed in the department was extensively used in the susceptible calves and pregnant/lactating cows in the field. Sufficiently high antibody titres were detected by both schizont as well as piroplasm antigen using both ELISA and IFAT. The results indicated that the vaccine was safe, potent and effective for all breeds and age groups of cattle under field conditions. ELISA was standardised for T. annulata using three antigens, viz.: soluble piroplasm, soluble schizont and cellular schizont antigens. Comparison of results with IFAT showed that ELISA is more sensitive, objective, reliable and specific as well as less cumbersome than IFAT. Piroplasm, cellular schizont and soluble schizont antigens were found to be suitable for the detection of antitheilerial antibodies as per their order in ELISA. Studies on the duration of immunity stimulated by the T. annulata schizont cell culture vaccine indicated that immunity started waning after six months. Calves born of dams immunised against T. annulata with the cell culture vaccine were found to be fully susceptible to theileriosis soon after birth. This indicated that there was no passive transfer of immunity from dams to their offspring through colostrum.

Echinococcus multilocularis antigens modify accessory cell function of macrophages.

Peritoneal macrophages and splenic lymphocytes were collected from BALB/c mice, normal or previously infected with Echinococcus multilocularis. In an accessory cell function assay, peritoneal macrophages, in increasing numbers, were added to cultures of splenic lymphocytes. Cultures were stimulated by concanavalin A (Con A) or E. multilocularis culture supernatant (EMSN). Post-infection macrophages, unlike normal macrophages, suppressed Con A- and EMSN-driven lymphocyte transformation. Modification of accessory cells could also be repeatedly induced in vivo by EMSN or a single FPLC fraction of EMSN. Lymphocytes were made more sensitive to accessory cell signals following incubation with EMSN.

Modification of cellular immunity by Taenia multiceps (Cestoda): accessory macrophages and CD4+ lymphocytes are affected by two different coenurus factors.

Taenia multiceps coenurus fluid was analysed by fast protein liquid chromatography in order to separate the factors responsible for previously reported modification of immunological activity in macrophages and T-cells. One factor, F7, was found to be mitogenic for murine L3T4+ T-cells, to be macrophage dependent, to require macrophage compatibility at the I region of the H2 complex, to increase the sensitivity of T-cells to regulatory signals from macrophages and to increase the rate of generation of splenic rosette-forming cells (RFC) against sheep red cells. A second factor, F24, was found to alter macrophages so as to render them suppressive, rather than stimulatory, for parasite-activated and Con A-activated lymphocyte transformation, to depress the rate of generation of RFC and to antagonize the mitogenic effect of F7. The combined actions of these two factors are, therefore, sufficient to explain the known immunomodulatory effects of the metacestode.

Lymphoreticular responses to metacestodes: Taenia multiceps (Cestoda) can modify interaction between accessory cells and responder cells during lymphocyte activation.

This study was designed to test the accessory function of macrophages after activation with products of Taenia multiceps coenuri. Activation was carried out by intraperitoneal injection of mice with coenurus fluid or protoscolex culture supernatant, and function was assessed by adding these macrophages in progressively increasing numbers to macrophage-depleted lymphocyte cultures transforming under the influence of plant mitogens or coenurus-fluid mitogen. In contrast to normal macrophages, which have a progressively enhancing action on the above reactions, parasite-activated macrophages at similar concentrations were progressively inhibitory. However, low concentrations of the activated macrophages enhanced mitosis as well as, or better than, normal. Lymph node cells from injected mice showed abnormal response to macrophage-derived signals. In particular there was subnormal reaction to macrophages in the presence of coenurus mitogen. These results suggest that T. multiceps coenuri may survive in the host because of their ability to reduce effective interaction between lymphocytes and accessory cells.