No evidence of PEDV infection in French artificial insemination centers in 2015.

Pigs infected by porcine epidemic diarrhea virus (PEDV) are affected by severe diarrhea, vomiting and dehydration. The severity of clinical signs depends on the virus strain. Two genetically different PEDV strains are known to infect pigs, the PEDV S-InDel strains which circulate on all continents and the highly virulent PEDV S-non-InDel strains found in Asia and in America. We have previously demonstrated the presence of PEDV RNA in semen from boars experimentally infected with an S-non-InDel PEDV strain. If naturally infected boars may shed PEDV in semen, this would have important consequences for the breeding sector. Thus we sought to determine whether PEDV has been circulating in populations of breeding boars from French artificial insemination (AI) centers. The current study reports on a serological survey conducted on one hundred and twenty boars from six AI centers, representing 18.6% of the total population of breeding boars in French AI centers in 2015. All of them were found negative for PEDV antibodies, showing no evidence of PEDV circulation in French AI centers at that time.

Effect of an endurance-like exercise on the disposition and detection time of phenylbutazone and dexamethasone in the horse: application to medication control.

REASONS FOR PERFORMING STUDY: Equine antidoping rules were established to prevent a horse's performance being altered after the administration of prohibited substances, including approved drugs used for legitimate treatment. Veterinarians have to advise owners or trainers on appropriate withholding times to guarantee that their horses may safely compete after drug administration. In order to propose tailored withdrawal times, several horse organisations released detection time (DT) values, for the main veterinary drugs used in horses. One of the possible limits to the information provided by published DTs in horses is the fact that they are determined from classic pharmacokinetic studies performed at rest under laboratory conditions. In field conditions, training and exercise programmes may have an influence on drug elimination. METHODS: Dexamethasone (DMX) and phenylbutazone (PBZ) have been quantified in plasma and urine after solid phase extraction. The kinetic disposition of DXM (8 microg/kg) and PBZ (8 mg/kg) administered by i.v. route in 8 horses, was investigated in rest conditions and during a standardised 3 h test exercise according to a cross-over design. OBJECTIVES: The aim of the present study was to compare the kinetic disposition of 2 test drugs, DMX and PBZ in rest vs. exercising conditions. RESULTS: It was shown in 8 horses that a sustained 3 h of mild exercise slightly decreased the plasma clearance of both drugs (about 25% for DXM and 37% for PBZ) and this is mainly explained by the significant decrease of the corresponding hepatic clearance. In addition, as the volume of distribution was correlatively decreased, the plasma terminal half-life, which is a hybrid parameter of plasma clearance and volume of distribution, remains unchanged overall. CONCLUSION AND POTENTIAL RELEVANCE: Establishing DTs or withdrawal times (WTs) are relevant as plasma and urine half-lives, but not clearance, are the main determinants of DT length. Veterinarians may realistically decide upon a WT for a legitimate drug based on the corresponding DT obtained under resting conditions providing this drug has a low hepatic extraction ratio and a safety margin is added to allow for all possible sources of variability.

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.

Purification and characterisation of a trypsin-like serine oligopeptidase from Trypanosoma congolense.

Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. Here a similar enzyme from Trypanosoma congolense is described. This oligopeptidase, called OP-Tc, was purified using three-phase partitioning, and ion-exchange and affinity chromatography. OP-Tc is inhibited by alkylating agents, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumarin, 4-(2-aminoethyl)benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidyl chloromethyl ketones. Reducing agents such as dithiothreitol enhanced activity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino acid residues (Arg or Lys) in the P1 position. Potential substrates without a basic residue in P1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P2 and P3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower k(cat)/Km values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC (Km = 0.72 microM, k(cat) = 96 s(-1)). OP-Tc preference for amino acids in the P2 position was (Gly,Lys,Arg) > Phe > Leu > Pro. The results also suggest that the P3-binding site has hydrophobic characteristics. OP-Tc may not be a naturally immunodominant molecule because neither IgG nor IgM anti- OP-Tc antibodies were detected in the blood of experimentally infected cattle.

Inhibition of trypanosomal cysteine proteinases by their propeptides.

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

Trypanotolerance, an option for sustainable livestock production in areas at risk from trypanosomosis.

Trypanosomosis is one of the major constraints on animal production in areas of Africa which have the greatest potential for significant increases in domestic livestock populations and livestock productivity. While the eradication of trypanosomosis from the entire continent is an unrealistic goal, considerable effort has been invested in the control of this disease through the use of trypanocidal drugs, management of the vector and exploitation of the genetic resistance exhibited by indigenous breeds. There is little hope that a conventional, anti-infection vaccine will be produced in the near future. Drug resistance is developing faster than generally thought. The control of the tsetse fly has been attempted over many decades. The decreasing efficacy of available trypanocidal drugs and the difficulties of sustaining tsetse control increase the imperative need to enhance trypanotolerance through selective breeding, either within breeds or through cross-breeding. Trypanotolerance has been defined as the relative capacity of an animal to control the development of the parasites and to limit their pathological effects, the most prominent of which is anaemia. A major constraint on selection for trypanotolerance in cattle, for both within-breed and cross-breeding programmes, has been the absence of practical reliable markers of resistance or susceptibility. Distinct humoral immune response to trypanosome infection is the major feature of bovine trypanotolerance. The role that these responses play in the control of infection or disease is being addressed by ongoing research, but remains a matter of speculation at present. Results in recent years have shown that packed cell volume (PCV) in particular and parasitaemia, the two principal indicators of trypanotolerance, are strongly correlated to animal performance. However, although direct effects of trypanosome infections on PCV and growth are obvious, more sensitive diagnostic methods for reflecting parasite control are required so that individual animals can be categorised reliably for their parasite control capability. One key finding is the major contribution made by each of the indicators evaluated to the overall trypanotolerance variance. Preliminary genetic parameters for PCV provide evidence that trypanotolerance is not only a breed characteristic but is also a heritable trait within the N'Dama population; this brings new opportunities for improved productivity through selection for trypanotolerance. More reliable estimation of genetic parameters of the indicators may well show that these parameters must be handled simultaneously for optimal progress. This would require diagnostics for assessing parasite control capability that identify trypanosome species more accurately, especially in mixed infections. A major advantage of trypanotolerant livestock, particularly N'Dama cattle, is the resistance or adaptation of this breed to many of the important pathogenes which prevail in the sub-humid and humid tropics. Research on practical indicators of resistance to these conditions will be required to establish relevant integrated strategies based on disease-resistant livestock. Selective breeding will require the integration of the traits that farmers hold important for their production systems.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi.

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Trypanosoma congolense: a comparison of T-cell-mediated responses in lymph nodes of trypanotolerant and trypanosusceptible cattle during primary infection.

A comparison of T-cell-mediated immune responses in trypanotolerant N'Dama and susceptible Boran cattle during primary infection with tsetse-transmitted Trypanosoma congolense was conducted to assess whether different patterns of T-cell activation occurred during trypanosome infection. Proliferation and IFN-gamma synthesis in response to trypanosome antigens and to the mitogen Con A were measured in LNC before infection and 10 and 35 days postinfection. Phenotypic analysis of LNC was also carried out. No significant differences in the in vitro proliferation of LNC to VSG, to hsp70/BiP, or to Con A were detected between the breeds. In contrast, IFN-gamma production in response to Con A was higher in Boran cattle at 35 days p.i. A reduction in the number of CD2+ and CD4+ T-cells and an increase in the percentage of B-cells, CD8+ T-cells, and gamma delta T-cells during infection in both N'Dama and Boran was revealed by cytofluorimetric analysis of lymph node cells.

Effect of tsetse control on trypanosome prevalence in livestock: problems of experimental design and statistical interpretation--a case study in northern Côte d'Ivoire.

As part of a study on livestock productivity under trypanosomosis risk in the region of Boundiali, northern Côte d'Ivoire, 21 herds of cattle (N'Dama, Baoulé and Zebu crosses) and 20 flocks of Djallonké and Djallonké x Sahel sheep were monitored monthly for body weight, packed red cell volume and trypanosomal parasitaemia over various periods between January 1984 and December 1992. A tsetse control campaign using biconical traps impregnated with alpha-cypermethrin started in December 1987. Tsetse control reduced the relative tsetse density by over 95% between 1988 and 1992, and this was associated with reductions in the prevalence of Trypanosoma congolense over the same period of over 90% both in sheep and cattle. Average reductions in the prevalence of T. vivax were lower, on average 68% in adults and 85% in young animals. Attempts were made in the design of the study to allow comparisons between controlled and uncontrolled areas; however, there were too many confounding and uncontrollable factors to allow such comparisons to be made. It was necessary, therefore, to compare data collected from all herds and flocks before and after the intervention, with the consequential difficulties in accounting for uncontrollable year-to-year variations in factors affecting trypanosome prevalence in livestock.

Trypanosoma congolense: B-lymphocyte responses differ between trypanotolerant and trypanosusceptible cattle.

Trypanosomiasis is a serious constraint to livestock production in sub-Saharan Africa. Some breeds of cattle are genetically more resistant to the pathogenic effects of trypanosome infection. We measured B-cell activation and the quantity and isotype of antibody produced at the cellular level in six trypanotolerant N'Dama and five trypanosusceptible Boran cattle. The frequencies of spleen cells secreting total and parasite-specific IgM and IgG were measured prior to and 16, 28, and 35 days after a primary challenge with Trypanosoma congolense. Boran cattle had higher frequencies of splenic cells secreting IgM specific for trypanosome-derived variable surface glycoprotein (VSG), cysteine protease (congopain, CP), and heat shock protein (hsp 70/BiP) and the nonparasite antigen, ovalbumin, than did N'Dama cattle. In contrast, the number of VSG-specific IgG-secreting cells was significantly greater in N'Dama than in Boran cattle. During infection, low titers of anti-VSG IgM were detected transiently in the serum of all animals. However, N'Dama had significantly more VSG-specific IgG in blood than Boran during infection. The peripheral blood mononuclear cell population of N'Dama cattle contained a higher percentage of surface IgM-positive B-cells prior to and throughout infection than were found in the blood of Boran. In addition, during infection N'Dama cattle had more circulating lymphocytes that could be activated in vitro to undergo differentiation into IgM- and IgG-secreting cells. These findings demonstrate differences in the frequency of trypanosome-specific antibody-secreting cells in the spleen and in the activation state of B-cells in the blood between N'Dama and Boran cattle during a primary infection with T. congolense.

Trypanosoma congolense: tissue distribution of long-term T- and B-cell responses in cattle.

Memory T- and B-cell responses to trypanosome antigens were measured in peripheral blood mononuclear cells, spleen and lymph node cells obtained from four trypanotolerant N'Dama cattle which had been exposed to six experimental infections with Trypanosoma congolense. These cattle were treated with trypanocidal drugs following each infection and had remained aparasitemic for 3 years prior to this study. The antigens used were whole trypanosome lysate, variable surface glycoprotein, a 33-kDa cysteine protease (congopain) and a 70-kDa heat-shock protein. As parameters of T-cell-mediated immunity, we measured T-cell proliferation and IFN-gamma production. Lymph node cells, spleen cells and peripheral blood mononuclear cells all proliferated to a mitogenic stimulus (concanavalin A) but only lymph node cells responded to trypanosome antigens. Similarly, IFN-gamma was produced by both lymph node and spleen cells stimulated with concanavalin A but only by lymph node cells stimulated with variable surface glycoprotein and whole trypanosome lysate. T. congolense-specific antibodies were detected in sera and in supernatants of cultured lymph node and spleen cells after in vitro stimulation with lipopolysaccharide and recombinant bovine interleukin-2. In conclusion, we have demonstrated that memory T- and B-cell responses are detectable in various lymphoid organs in cattle 3 years following infection and treatment with T. congolense.

Trypanosoma congolense: proliferative responses and interleukin production in lymph node cells of infected cattle.

T-cell-mediated immune responses to defined antigens of Trypanosoma congolense were measured in cattle undergoing primary infection. The antigens used were the variable surface glycoprotein and two invariant antigens, a 33-kDa cysteine protease (congopain) and a recombinant form of a 69-kDa heat-shock protein. Proliferative responses were highest during the second week postinfection and were detected in cells obtained from the lymph node draining the site of infection but not in peripheral blood mononuclear cells. Production of IL-2 and IFN-gamma was measured in supernatants from antigen-stimulated lymph node cell cultures. Expression of IL-2, IL-4, and IFN-gamma mRNA was detected in antigen-stimulated lymph node cells by reverse transcription-polymerase chain amplification.

A 69 kDa immunodominant antigen of Trypanosoma (Nannomonas) congolense is homologous to immunoglobulin heavy chain binding protein (BiP).

An immunodominant antigen in Trypanosoma congolense-infected cattle is a 69 kDa protein which is conserved among species and developmental stages of African trypanosomes. Immunoscreening of a cDNA expression library identified a 2.35 kbp clone which contains a complete open reading frame encoding a protein of 653 amino acids with a predicted molecular mass of 71 kDa. Protein sequence analyses revealed 45-65% identity with hsp70s from a broad range of organisms, the highest homology being with the mammalian BiP (immunoglobulin heavy chain binding protein). The 69 kDa trypanosome protein shares with other BiP-related molecules two characteristics that are associated with their localization in the endoplasmic reticulum and their function as chaperonins, i.e., a hydrophobic N-terminal signal sequence and a conserved C-terminal tetrapeptide (X)DEL. Divergence between the 69 kDa antigen and other BiP-homologues occurs in the C-terminal region. This may be responsible for the high immunogenicity of the trypanosome protein. The gene for the 69 kDa antigen appears to be present as a cluster of several copies which are not organized in tandem repeats. It is expressed in all developmental stages of T. congolense, but the specific mRNA levels are higher in metacyclics than in other stages.

Trypanosomiasis and trypanotolerance in cattle: a role for congopain?

A Trypanosoma congolense cysteine protease (congopain) elicits a high IgG response in trypanotolerant but not in trypanosusceptible cattle during primary infections. As discussed here by Edith Authié, this observation suggests that congopain, like other parasite cysteine proteases, may play a role in pathogenicity and that more efficient immune responses to congopain may contribute to trypanotolerance.

Antibody responses to a 33 kDa cysteine protease of Trypanosoma congolense: relationship to 'trypanotolerance' in cattle.

A cysteine protease of Trypanosoma congolense (congopain) elicited IgG1 antibodies in those cattle which exhibited a degree of resistance to disease during experimental infections (Authié et al. 1992, 1993). The aim of the present study was to investigate further the association between anti-congopain antibodies and resistance to trypanosomiasis, and to provide a lead into the mechanisms responsible for the differential responses to congopain in cattle. Isotype characteristics and kinetics of the antibody response to congopain were studied in three N'Dama (trypanoresistant) and three Boran (susceptible) cattle during primary infection with T. congolense ILNat 3.1. In both groups an IgM response to congopain was elicited, thus demonstrating that congopain is antigenic in both types of cattle. Most of the IgM appeared to be incorporated into immune complexes. IgG was detected as free antibody; IgG1 but not IgG2 was detected. All three N'Dama, but none of the three Boran cattle, mounted a significant IgG response to congopain. Sera from 70 primary-infected cattle belonging to five breeds of differing susceptibility were tested for their reactivity to congopain. High levels of IgG to congopain were observed in the two trypanotolerant breeds, whereas the three susceptible breeds had lower levels of these antibodies. Crosses between N'Dama and Boran cattle, which exhibit an intermediate susceptibility, had intermediate levels of antibodies. Thus, the results from experimental infections confirmed our initial observations. However, under natural tsetse challenge, repeated infections and trypanocidal treatments in Zebu cattle stimulated as high anti-congopain antibody levels as in non-treated trypanotolerant taurine cattle.

Epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia. 1. Tsetse challenge and its relationship to trypanosome prevalence in cattle.

In this, the first of a series of papers on the epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia, the tsetse populations and their relationships to the prevalence of trypanosome infections in cattle are described. The tsetse challenge to cattle at two sites sites in the area was estimated as the product of tsetse relative density and the trypanosome infection rate in flies. The proportion of feeds taken by tsetse from cattle was also considered. Three tsetse species were detected in the area, Glossina pallidipes, G. fuscipes and G. morsitans submorsitans. A significant correlation (r = 0.60, P < 0.001) was observed between the mean monthly estimates of tsetse challenge due to G. pallidipes and the prevalence of trypanosome infections in cattle the following month at one site, whilst at the other, no significant relationship was observed (P = 0.08). The tsetse density at both sites showed seasonal changes which were related to the monthly rainfall. Finally, variations in tsetse density appeared to be the main factor responsible for variation in tsetse challenge and thus trypanosome prevalence in cattle.

Epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia. 2. Factors associated with variations in trypanosome prevalence, incidence of new infections and prevalence of recurrent infections.

An average of 840 East African Zebu cattle from nine herds in the Ghibe valley, southwest Ethiopia were monitored from January 1986 to April 1990. Each month blood samples were collected for analysis of packed red cell volume (PCV) and detection of trypanosomes. Animals found to be parasitaemic and with a PCV less than 26% were treated with diminazene aceturate at a dose of 3.5 mg/kg body weight. The majority of infections were associated with Trypanosoma congolense (84% of infections in adult cattle and 71% in cattle less than 24 months of age), and the mean percentage of adult animals detected parasitaemic 1 month after treatment of an infection with T. congolense was 27%. In order to assess possible existence of drug resistance, a model was applied which allowed monthly incidences of new infections to be distinguished from recurrent infections. This model showed that the monthly incidence of new infections of T. congolense in adult cattle increased significantly from 11% in 1986 to 24% in 1989 following a concomitant increase in the tsetse challenge. The corresponding increase in overall prevalence of T. congolense was from 17% to 38% and the mean prevalence of recurrent infections increased significantly from 6% to 14%. These findings ruled out the possibility that the high prevalence of trypanosome infections in cattle was due only to a high tsetse challenge and pointed to the existence of T. congolense populations which expressed resistance to diminazene. There were variations associated with season, herd, age and sex in the incidence of new infections, prevalence of recurrent infections and relapse to treatment.

Epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia. 3. Occurrence of populations of Trypanosoma congolense resistant to diminazene, isometamidium and homidium.

In July 1989, blood samples were collected from parasitaemic cattle in the Ghibe valley, Ethiopia, frozen in liquid nitrogen and transported to Nairobi, Kenya. Twelve of the stabilates were inoculated into individual Boran (Bos indicus) calves and characterised for their sensitivity, in turn, to diminazene aceturate (Berenil), isometamidium chloride (Samorin) and homidium chloride (Novidium). All 12 stabilates produced infections which were shown to be Trypanosoma congolense and resistant to treatment with diminazene aceturate at a dose of 7.0 mg kg-1 body weight (b.w.). Eleven of the infections were also resistant to isometamidium chloride at a dose of 0.5 mg kg-1 b.w. and homidium chloride at a dose of 1.0 mg kg-1 b.w. The drug-sensitivity phenotypes of three of the same isolates were also determined in goats which were each treated with only one of the three trypanocides: all expressed the same phenotypes as the populations expressed in the aforementioned Boran calves. Five clones were derived from one of the isolates which expressed a high level of resistance to all three trypanocides; each clone expressed high levels of resistance to all three trypanocides when characterised in mice. Thus, the multi-resistance phenotype of the parental isolate was associated with expression of mutli-resistance by individual trypanosomes. Finally, molecular karyotypes and electrophoretic variants of six enzymes were determined for seven and eight of the isolates, respectively. Six different karyotypes were observed and all eight of the latter isolates belonged to different zymodemes, indicating that the multi-resistance phenotype at Ghibe was associated with many genetically distinct populations.