Baseline analysis of Mycoplasma mycoides subsp. mycoides antigens as targets for a DIVA assay for use with a subunit vaccine for contagious bovine pleuropneumonia.

BACKGROUND: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia in cattle. A prototype subunit vaccine is being developed, however, there is currently no diagnostic test that can differentiate between infected cattle and those vaccinated with the prototype subunit vaccine. This study characterized Mmm proteins to identify potential antigens for use in differentiating infected from vaccinated animals. RESULTS: Ten Mmm antigens expressed as recombinant proteins were tested in an indirect ELISA using experimental sera from control groups, infected, and vaccinated animals. Data were imported into R software for analysis and drawing of the box and scatter plots while Cohen's Kappa assessed the level of agreement between the Mmm antigens. Two vaccine antigens (MSC_0499 and MSC_0776) were superior in detecting antibodies in sera of animals vaccinated with the subunit vaccines while two non-vaccine antigens (MSC_0636 and LppB) detected antibodies in sera of infected animals showing all clinical stages of the disease. Sensitivity and specificity of above 87.5% were achieved when the MSC_0499 and MSC_0636 antigens were tested on sera from vaccinated and infected animals. CONCLUSIONS: The MSC_0499 and MSC_0776 antigens were the most promising for detecting vaccinated animals, while MSC_0636 and LppB were the best targets to identify infected animals. Further testing of sera from vaccinated and infected animals collected at different time intervals in the field should help establish how useful a diagnostic test based on a cocktail of these proteins would be.

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

Morphological characterization and immunohistochemical detection of the proinflammatory cytokines IL-1ß, IL-17A, and TNF-α in lung lesions associated with contagious bovine pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease, is characterized by massive inflammation of the lung especially during the acute clinical stage of infection. Tissue samples from cattle, experimentally infected with Mycoplasma mycoides subsp. mycoides Afadé, were subjected to histopathological and immunohistochemical examination in order to provide insight into innate immune pathways that shape inflammatory host responses. Lung lesions were characterized by vasculitis, necrosis, and increased presence of macrophages and neutrophils, relative to uninfected animals. The presence of three cytokines associated with innate inflammatory immune responses, namely, IL-1ß, IL-17A, and TNF-α, were qualitatively investigated in situ. Higher cytokine levels were detected in lung tissue samples from CBPP-affected cattle compared to samples derived from an uninfected control group. We therefore conclude that the cytokines TNF-α and IL-1ß, which are prevalent in the acute phase of infections, play a role in the inflammatory response seen in the lung tissue in CBPP. IL-17A gets released by activated macrophages and attracts granulocytes that modulate the acute phase of the CBPP lesions.

Vaccination of cattle with the N terminus of LppQ of Mycoplasma mycoides subsp. mycoides results in type III immune complex disease upon experimental infection.

Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N') of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N' formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N' in a future subunit vaccine for CBPP.

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

BACKGROUND: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. RESULTS: There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. CONCLUSIONS: Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.

Role of the polymorphic immunodominant molecule in entry of Theileria parva sporozoites into bovine lymphocytes.

Theileria parva is a tick-transmitted apicomplexan parasite that infects cattle and African buffalo. In cattle, it causes a fatal lymphoproliferative disease called East Coast fever. The polymorphic immunodominant molecule (PIM) is expressed by two stages of the parasite: the sporozoite, which is inoculated by the tick to infect mammalian lymphocytes, and the schizont, the established intralymphocytic stage. Here, we demonstrate that monoclonal antibodies (MAb) to PIM can reduce the ability of sporozoites to infect bovine lymphocytes in vitro. This reduction appears to be due to blocking of sporozoite attachment by binding of the MAb to several regions of PIM. Interestingly, one MAb, which recognizes an epitope in the central variable region of PIM, did not inhibit sporozoite infectivity. We also demonstrate that PIM antigen, as a recombinant molecule, can also reduce sporozoite infectivity in vitro by blocking both attachment and internalization of sporozoites. Electron microscopic studies showed that PIM is present in microspheres below the sporozoite surface and is transported to the parasite surface soon after contact with bovine lymphocytes. The results suggest that at least two sporozoite molecules, PIM and the previously described p67, are involved in the entry of T. parva into mammalian lymphocytes.

Development of an improved vaccine for contagious bovine pleuropneumonia: an African perspective on challenges and proposed actions.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides (Mmm) is an economically very important cattle disease in sub-Saharan Africa. CBPP impacts animal health and poverty of livestock-dependent people through decreased animal productivity, reduced food supply, and the cost of control measures. CBPP is a barrier to trade in many African countries and this reduces the value of livestock and the income of many value chain stakeholders. The presence of CBPP also poses a constant threat to CBPP-free countries and creates costs in terms of the measures necessary to ensure the exclusion of disease. This opinion focuses on the biomedical research needed to foster the development of better control measures for CBPP. We suggest that different vaccine development approaches are followed in parallel. Basic immunology studies and systematic OMICs studies will be necessary in order to identify the protective arms of immunity and to shed more light on the pathogenicity mechanisms in CBPP. Moreover a robust challenge model and a close collaboration with African research units will be crucial to foster and implement a new vaccine for the progressive control of this cattle plague.

A Theileria parva isolate of low virulence infects a subpopulation of lymphocytes.

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1(+) γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4(+), CD8(+), and WC1(+) T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8(+) T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+), or WC1(+) T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8(+) lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.

Plasma levels of TNF-α, IFN-γ, IL-4 and IL-10 during a course of experimental contagious bovine pleuropneumonia.

BACKGROUND: Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4+ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4+ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated. RESULTS: Plasma cytokine concentrations varied between animals in each group. Depletion of CD4+ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4+ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4+ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection. CONCLUSIONS: Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.

A minor role of CD4+ T lymphocytes in the control of a primary infection of cattle with Mycoplasma mycoides subsp. mycoides.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known.We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.

Clinical chemistry of congenic mice with quantitative trait loci for predicted responses to Trypanosoma congolense infection.

Trypanosoma congolense is a protozoan parasite that causes severe diseases in livestock. Three major quantative trait loci (QTL), Tir1, Tir2, and Tir3, control the survival time of mice after infection with T. congolense. Congenic mice carrying the C57BL/6 resistance alleles on the A/J background were developed for each of these loci. The congenic mice were used to physically map the regions containing the QTL gene(s) and to investigate the physiological effect of each locus. Clinical chemistry data for infected A/J, C57BL/6, and BALB/c mice were obtained for 15 analytes at five time points. Congenic mice were assessed for survival, parasitemia, and anemia as well as seven clinical-chemical analytes. The survival times were significantly increased in the Tir1 and Tir2 mice but not Tir3 congenic mice. The survival time of the parental inbred mice correlated negatively with parasitemia but positively with alanine aminotransferase activities in serum, suggesting that inflammatory reactions in the liver had a beneficial effect possibly associated with reduced parasitemia. However, there was no difference in parasitemia or liver enzyme activities of Tir1 and Tir2 congenic mice relative to their controls, showing that survival, parasitemia, and degree of liver damage are not associated with each other, despite the correlation in the parental lines. These data suggest that the congenic loci affect survival but do not affect control of parasite number. They may therefore act by limiting the pathological consequences of T. congolense infection.

Transcriptional profiling of cattle infected with Trypanosoma congolense highlights gene expression signatures underlying trypanotolerance and trypanosusceptibility.

BACKGROUND: African animal trypanosomiasis (AAT) caused by tsetse fly-transmitted protozoa of the genus Trypanosoma is a major constraint on livestock and agricultural production in Africa and is among the top ten global cattle diseases impacting on the poor. Here we show that a functional genomics approach can be used to identify temporal changes in host peripheral blood mononuclear cell (PBMC) gene expression due to disease progression. We also show that major gene expression differences exist between cattle from trypanotolerant and trypanosusceptible breeds. Using bovine long oligonucleotide microarrays and real time quantitative reverse transcription PCR (qRT-PCR) validation we analysed PBMC gene expression in naïve trypanotolerant and trypanosusceptible cattle experimentally challenged with Trypanosoma congolense across a 34-day infection time course. RESULTS: Trypanotolerant N'Dama cattle displayed a rapid and distinct transcriptional response to infection, with a ten-fold higher number of genes differentially expressed at day 14 post-infection compared to trypanosusceptible Boran cattle. These analyses identified coordinated temporal gene expression changes for both breeds in response to trypanosome infection. In addition, a panel of genes were identified that showed pronounced differences in gene expression between the two breeds, which may underlie the phenomena of trypanotolerance and trypanosusceptibility. Gene ontology (GO) analysis demonstrate that the products of these genes may contribute to increased mitochondrial mRNA translational efficiency, a more pronounced B cell response, an elevated activation status and a heightened response to stress in trypanotolerant cattle. CONCLUSION: This study has revealed an extensive and diverse range of cellular processes that are altered temporally in response to trypanosome infection in African cattle. Results indicate that the trypanotolerant N'Dama cattle respond more rapidly and with a greater magnitude to infection compared to the trypanosusceptible Boran cattle. Specifically, a subset of the genes analyzed by real time qRT-PCR, which display significant breed differences, could collectively contribute to the trypanotolerance trait in N'Dama.

Synthetic nonamer peptides derived from insect defensin mediate the killing of African trypanosomes in axenic culture.

Synthetic antimicrobial 9-mer peptides (designated as peptides A and B) designed on the basis of insect defensins and their effects on the growth of African trypanosomes were examined using two isolates of Trypanosoma congolense, IL1180 and IL3338, and two isolates of Trypanosoma brucei brucei, ILTat1.1and GUTat 3.1, under axenic culture conditions. Both peptides inhibited the growth of all bloodstream form (BSF) trypanosomes at 200-400 microg/mL in the complete growth medium, with peptide A being more potent than peptide B. In addition, these peptides exhibited efficient killing at 5-20 microg/mL on BSF trypanosomes suspended in phosphate-buffered saline, whereas procyclic insect forms in the same medium were more refractory to the killing. Electron microscopy revealed that the peptides induced severe defects in the cell membrane integrity of the parasites. The insect defensin-based peptides up to either 200 or 400 microg/mL showed no cell killing or growth inhibition on NIH3T3 murine fibroblasts. The results suggest that the design of suitable synthetic insect defensin-based 9-mer peptides might provide potential novel trypanocidal drugs.

Identification of targets of monoclonal antibodies that inhibit adhesion and growth in Mycoplasma mycoides subspecies mycoides.

Mycoplasma mycoides subspecies mycoides (Mmm) adhesion is tissue and host specific. Inhibition of adhesion will prevent Mmm from binding to lung cells and hence prevent colonization and disease. The aim of this study was to develop a panel of Mmm monoclonal antibodies against Mmm and use these antibodies to investigate their inhibitory effect on the adherence of Mmm to bovine lung epithelial cells (BoLEC), and to further identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoides subsp. mycoides (AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that the antibodies bound to proteins of variable sizes. AMMY 10, a previously characterized Mmm- capsular polysaccharide (CPS) specific antibody, inhibited growth of Mmm in vitro and also caused agglutination of Mmm total cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion of Mmm to BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune response in vivo.

Gene-knockout mice in malaria research: useful or misleading?

Gene-knockout mice have been extensively used in the study of several malaria-induced pathologies. Some investigators believe that the deficient, infected mice mimic disease aspects produced in the absence of the target gene, but others believe that the deficient mice models mainly explain the effects of compensatory, related molecules. Comparison of some of the most relevant knockout mouse studies for understanding cerebral malaria and parasitemia and their related human reports shows that gene-knockout mice are useful tools that support conclusions from human genetic studies. These mice have helped to indicate new resistance genes against human malaria and have provided valuable information about mechanisms of malaria resistance in mice.

Cytokine mRNA profiles in bovine macrophages stimulated with Trypanosoma congolense.

It is known that different breeds of cattle display differential susceptibilities to Trypanosome congolense infections, and that N'Dama cattle remain more productive after infection than Boran cattle which are more susceptible to T. congolense. Macrophages from both breeds were cultured in vitro and the expressions of a number of cytokines and iNOS mRNA were analyzed using real time RT-PCR after stimulation with antibody-opsonized trypanosomes. No significant difference was seen between the responses of the two breeds. However, RNA levels of TNF-alpha in the IFN-gamma-primed macrophages were about 100-fold higher than those in the non-primed macrophages. A significant ten-fold decrease was seen for the anti-inflammatory cytokine IL-10. These results indicate that priming of the cells with IFN-gamma cause a serious shift toward an inflammatory response.

Antimycoplasmal Activities of Compounds from Solanum aculeastrum and Piliostigma thonningii against Strains from the Mycoplasma mycoides Cluster.

Infections caused by Mycoplasma species belonging to the 'mycoides cluster' negatively affect the agricultural sector through losses in livestock productivity. These Mycoplasma strains are resistant to many conventional antibiotics due to the total lack of cell wall. Therefore, there is an urgent need to develop new antimicrobial agents from alternative sources such as medicinal plants to curb the resistance threat. Recent studies on extracts from Solanum aculeastrum and Piliostigma thonningii revealed interesting antimycoplasmal activities hence the motivation to investigate the antimycoplasmal activities of constituent compounds. The CH2Cl2/MeOH extracts from the berries of S. aculeastrum yielded a new ß-sitosterol derivative (1) along with six known ones including; lupeol (2), two long-chain fatty alcohols namely undecyl alcohol (3) and lauryl alcohol (4); two long-chain fatty acids namely; myristic acid (5) and nervonic acid (6) as well as a glycosidic steroidal alkaloid; (25R)-3ß-O-α-L-rhamnopyranosyl-(1→2)-O-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyloxy-22α-N-spirosol-5-ene (7) from the MeOH extracts. A new furan diglycoside, (2,5-D-diglucopyranosyloxy-furan) (8) was also characterized from the CH2Cl2/MeOH extract of stem bark of P. thonningii. The structures of the compounds were determined on the basis of spectroscopic evidence and comparison with literature data. Compounds 1, 3, 4, 7, and 8 isolated in sufficient yields were tested against the growth of two Mycoplasma mycoides subsp. mycoides (Mmm), two M. mycoides. capri (Mmc), and one M. capricolum capricolum (Mcc) using broth dilution methods, while the minimum inhibitory concentration (MIC) was determined by serial dilution. The inhibition of Mycoplasma in vitro growth was determined by the use of both flow cytometry (FCM) and color change units (CCU) methods. Compounds 4 and 7 showed moderate activity against the growth of Mmm and Mmc but were inactive against the growth of Mcc. The lowest MIC value was 50 µg/ml for compound 7 against Mmm. The rest of the compounds showed minimal or no activity against the strains of Mycoplasma mycoides tested. This is the first report on the use of combined FCM and CCU to determine inhibition of in vitro growth of Mycoplasma mycoides. The activity of these compounds against other bacterial strains should be tested and their safety profiles determined.

Cytokine mRNA profiling of peripheral blood mononuclear cells from trypanotolerant and trypanosusceptible cattle infected with Trypanosoma congolense.

To examine differences in cytokine profiles that may confer tolerance/susceptibility to bovine African trypanosomiasis, N'Dama (trypanotolerant, n = 8) and Boran (trypanosusceptible, n = 8) cattle were experimentally challenged with Trypanosoma congolense. Blood samples were collected over a 34-day period, and RNA was extracted from peripheral blood mononuclear cells. The expression levels of a panel of 14 cytokines were profiled over the time course of infection and between breeds. Messenger RNA (mRNA) transcript levels for the IL2, IL8, and IL1RN genes were significantly downregulated across the time course of infection in both breeds. There was an early increase in transcripts for genes encoding proinflammatory mediators (IFNG, IL1A, TNF, and IL12) in N'Dama by 14 days postinfection (dpi) compared with preinfection levels that was not detected in the susceptible Boran breed. By the time of peak parasitemia, a type 2 helper T cells (T(H)2)-like cytokine environment was prevalent that was particularly evident in the Boran. Increases in transcripts for the IL6 (29 and 34 dpi) and IL10 (21, 25, and 29 dpi) genes were detected that were higher in the Boran compared with N'Dama. These findings highlight the implications for using murine models to study the bovine immune response to trypanosomiasis, where in some cases cytokine expression patterns differ. Overall, these data suggest that the trypanotolerant N'Dama are more capable of responding very early in infection with proinflammatory and T(H)1 type cytokines than the trypanosusceptible Boran and may explain why N'Dama control parasitemia more efficiently than Boran during the early stages of infection.

Selected ethno-medicinal plants from Kenya with in vitro activity against major African livestock pathogens belonging to the "Mycoplasma mycoides cluster".

ETHNOPHARMOCOLOGICAL RELEVANCE: Members of 'Mycoplasma mycoides cluster' are important ruminant pathogens in Africa. Diseases caused by these Mycoplasma negatively affect the agricultural sector especially in developing countries through losses in livestock productivity, mortality and international trade restrictions. There is therefore urgent need to develop antimicrobials from alternative sources such as medicinal plants to curb these diseases. In Kenya, smallholder farmers belonging to the Maasai, Kuria and Luo rely on traditional Kenyan herbals to treat respiratory symptoms in ruminants. In the current study extracts from some of these plants were tested against the growth of members of Mycoplasma mycoides cluster. AIM: This study aimed at identifying plants that exhibit antimycoplasmal activities using an ethnobotanical approach. MATERIALS AND METHODS: Kenyan farmers of Maasai, Luo and Kuria ethnic groups were interviewed for plant remedies given to livestock with respiratory syndromes. The plant materials were thereafter collected and crude extracts prepared using a mixture of 50% of methanol (MeOH) in dichloromethane (CH2Cl2), neat methanol (MeOH), ethanol (EtOH) and water to yield four crude extracts per plant part. The extracts were tested in vitro against five strains of Mycoplasma mycoides subsp. capri, five strains of Mycoplasma mycoides subsp. mycoides and one strain of Mycoplasma capricolum subsp capricolum using broth micro-dilution assays with an initial concentration of 1mg/ml. Minimum inhibitory concentration (MIC) of the most active extracts were determined by serial dilution. RESULTS: Extracts from five plants namely: Solanum aculeastrum, Albizia coriaria, Ekebergia capensis, Piliostigma thonningii and Euclea divinorum exhibited the highest activities against the Mycoplasma strains tested. Mycoplasma mycoides subsp. mycoides were more susceptible to these extracts than Mycoplasma mycoides subsp. capri and Mycoplasma capricolum susp. capricolum. The activities of the crude extracts varied with the solvent used for extraction. The MICs mean values of the active extracts varied from 0.02 to 0.6mg/ml. CONCLUSIONS: The results suggested that these plants could potentially contain antimicrobial compounds that might be useful for the treatment of respiratory diseases in ruminants. Future work should focus on the isolation and identification of the active compounds from the plant extracts that showed interesting activities and evaluation of their antimicrobial and cytotoxic potential.