Reduction of Trypanosoma equiperdum from equine semen by single layer centrifugation.

Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 102 to >5 × 106 trypanosomes, were added to semen samples. Subsequently, SLC was performed following standard procedures. The presence of the parasite in the purified semen was checked by wet smear examination, ITS1 PCR and in vivo inoculation in mice. Before SLC, all spiked semen samples, except the negative controls, were positive on PCR analysis. After SLC, all the pellets were found to be negative for T. equiperdum on microscopic examinations. Examination of the pellet by PCR could also not detect any parasite-DNA in the SLC-pellet of semen spiked with the lower number of parasites (102 to104 trypanosomes). However, in the SLC pellets spiked with 104 - 5 × 104 trypanosomes, only 1 out of the 4 replicates was negative for parasite DNA. All groups spiked with >5 × 104 trypanosomes were found to be positive on PCR. All mice in the positive controls exhibited parasitaemia (5/5). Mice inoculated with SLC-purified semen that was spiked with lower than 5 × 104 trypanosomes, remained free of parasitaemia, similar to the negative controls. However inoculation with SLC-pellets from samples with a higher number of trypanosomes (>5 × 104 - 5 × 106 and > 5 × 106), induced parasitaemia in 2 out of 5 and 3 out of 5 mice, respectively. This study indicates that single layer centrifugation can be used to clear T. equiperdum infected semen but that the success is dependent on the number of parasites.

Prevalence of Escherichia coli O157:H7 in beef cattle at slaughter and beef carcasses at retail shops in Ethiopia.

BACKGROUND: There is paucity of information regarding the epidemiology of Escherichia coli O157: H7 in developing countries. In this study, we investigated the occurrence of E. coli O157: H7 associated with beef cattle at processing plants and at retail shops in Ethiopia. METHODS: Various samples were collected from beef cattle at slaughter/processing plants, carcass at retail shops and humans at health centers. E. coli O157: H7 was isolated, identified and characterized for antimicrobial resistance, using standard microbiological methods. RESULTS: At the processing plants E. coli O157: H7 was detected in 1.89% of fecal, 0.81% of intestinal mucosal swab, 0.54% of skin swab and 0.54% of carcass internal swab samples. At retail shops it was detected in 0.8% of carcass and 0.8% of cutting board swab samples, while all samples from utensils, hands from workers, and fecal and stool samples were negative. All isolates were resistant to Amoxicillin, moderately resistant to Cefoxitine and Nitrofurantoins but susceptible to other antimicrobials tested. CONCLUSIONS: E. coli O157: H7 occurs at low prevalence in beef cattle, and the current sanitary dressing procedures in the processing plants and storage conditions in the retail shops are effective against E. coli O157: H7.

Prevalence and antimicrobial susceptibility of Escherichia coli O157 in beef at butcher shops and restaurants in central Ethiopia.

BACKGROUND: Ethiopia bears the largest burden of foodborne diseases in Africa, and diarrheal diseases are the second leading causes of premature deaths. Enterohemorrhagic Escherichia coli O157 causes an asymptomatic infection to severe diarrhea and/or hemolytic-uremic syndrome in humans. METHODS: A total of 440 beef carcass and in-contact surface swabs from 55 butcher shops and 85 minced beef samples from 40 restaurants in central Ethiopia were collected and examined for the presence of E. coli O157. Standard microbiological methods were used to isolate and identify E. coli O157 and to characterize the antimicrobial resistance of the isolates. RESULTS: E. coli O157 was detected in 4.5% carcass swabs (n = 5) and 3.6% cutting board swabs (n = 4) samples from butcher shops. E. coli O157 was not detected in any of the minced beef samples obtained from restaurants. All isolates (n = 9) were 100% susceptible to five drugs, but five isolates were resistant to amoxicillin, two isolates to streptomycin and three isolates to chloramphenicol. One isolate was resistant to two drugs and another to three drugs. CONCLUSIONS: The present study shows a low prevalence of E. coli O157 in beef sold at butcher shops. Nevertheless, given the low infective dose of this pathogen and the deep-rooted tradition of consuming raw or undercooked beef, the current prevalence should not be considered lightly from a public health perspective.

The Enrichment of Histomonas meleagridis and Its Pathogen-Specific Protein Analysis: A First Step to Shed Light on Its Virulence.

Since the discovery of Histomonas meleagridis in 1893, the necessity of isolating pure H. meleagridis has been highlighted over the years in the battle against histomonosis. Insights into the molecular characteristics of this protozoon open possibilities to proper treatment. Axenization of H. meleagridis in vitro cultures cocultured with bacteria has been unsuccessful. Numerous unsuccessful attempts at culturing H. meleagridis axenically have reinforced the assumption that the protozoa had an obligate relationship with certain bacteria originating from the host ceca. Within these perspectives, we enriched H. meleagridis cells from a mono-eukaryotic culture copropagated with host cecal bacteria by flow cytometry. The enrichment of histomonads was confirmed through transmission electron microscopy and two-dimensional gel electrophoresis. For the first time several protein spots were successfully identified. The majority of spots were annotated as cytoskeletal proteins. Actin microfilaments are known to be a key player in cell spreading, cell adhesion, phagocytosis, signal transduction, and several other processes. Together with the identification of superoxide dismutase, the information generated from protein analysis of H. meleagridis may serve as a very first step toward understanding its pathogenesis and virulence.

New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels.

BACKGROUND: Trypanosoma (T.) evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia. METHODOLOGY/PRINCIPAL FINDINGS: T. evansi was isolated in mice by inoculation with the cryopreserved buffy coat of parasitologically confirmed animals. Fourteen stocks were thus isolated and subject to genotyping with PCRs targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, minisatellite markers and the F1-ATP synthase γ subunit gene. Nine stocks corresponded to type A, two stocks were type B and three stocks represented mixed infections between A and B, but not hybrids. One T. evansi type A stock was completely akinetoplastic. Five stocks were adapted to in vitro culture and subjected to a drug sensitivity assay with melarsomine dihydrochloride, diminazene diaceturate, isometamidium chloride and suramin. In vitro adaptation induced some loss of kinetoplasts within 60 days. No correlation between drug sensitivity and absence of the kinetoplast was observed. Sequencing the full coding sequence of the F1-ATP synthase γ subunit revealed new type-specific single nucleotide polymorphisms and deletions. CONCLUSIONS/SIGNIFICANCE: This study addresses some limitations of current molecular markers for T. evansi genotyping. Polymorphism within the F1-ATP synthase γ subunit gene may provide new markers to identify the T. evansi type that do not rely on variant surface glycoprotein genes or kinetoplast DNA.

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic.

Maternal colostral leukocytes appear to enhance cell-mediated recall response, but inhibit humoral recall response in prime-boost vaccinated calves.

Whether colostral leukocytes (CLs) of vaccinated dams influence the immune response of neonatal calves following vaccination against the same antigen as their respective dams remains unanswered. Therefore, we compared the induction of humoral and cellular immune responses after vaccination in calves that had received CL-free or maternal CL-enriched colostrum from a cell-free colostrum bank of nonvaccinated cows. Also, vaccinated calves that had received fresh maternal colostrum from their own dam were included in the study. Moreover, we analyzed whether the post-partum time of priming vaccination (day 2, 5 or 10) of the calves could influence the outcome of the immune responses. All calves received a booster vaccination 23 days after the priming vaccination. All calves showed only an increase in tetanus toxoid (TT)-specific antibodies and TT-induced proliferation after booster vaccination. Tetanus toxoid-specific antibody responses in calves increased immediately after booster vaccination, irrespective of whether or not their cell-free bank colostrum had been enriched with CLs from their own dam. Conversely, calves receiving their own plain dam colostrum displayed a later humoral response, due to colostral antibodies. After booster vaccination, calves of the CL-enriched colostrum group had a more pronounced antigen-specific proliferative response than the calves of the CL-free colostrum group. We propose that CLs might have a suppressive influence on the emergence of the TT-specific antibodies, but an enhancing effect on the TT-specific lymphocyte proliferation of newborn calves upon TT vaccination, which is dependent on the time point of the priming vaccination.

Trypanosome infection in dromedary camels in Eastern Ethiopia: Prevalence, relative performance of diagnostic tools and host related risk factors.

A cross-sectional study was conducted in Chifra and Dewe districts of Afar region, Eastern Ethiopia, to determine the prevalence, agreement between diagnostic tests and host related risk factors of trypanosome infection in camel. An overall prevalence of 2%, 24.1%, 21.3%, 9.5% and 7.8% was recorded with respectively Giemsa stained thin blood smear, CATT/T. evansi, RoTat1.2 PCR, 18S PCR and ITS-1PCR in a cohort of 399 animals. Only one T. vivax infection was confirmed by TvPRAC PCR indicating T. evansi as the predominant species affecting camels in the study area. No single animal was positive when tested with T. evansi type B specific EVAB PCR. There was slight agreement between the CATT/T. evansi and the molecular tests. Among the PCR methods, RoTat 1.2 PCR yielded a significantly higher positivity rate compared to 18S PCR and ITS-1 PCR. There was no significant difference in the positivity rate observed in each gender of camels (p>0.05). The positivity rate was significantly higher in camels with poor body condition and in older animals when tested using the CATT/T.evansi or RoTat 1.2 PCR (p>0.05). Camels that tested positive with all tests had significantly lower PCV's (p<0.05). This study provides further evidence that T. evansi is endemic in the Afar region of Ethiopia. The latent class analysis indicated an estimate overall prevalence of 19% (95% CI: 13-28). Moreover, the model indicated low sensitivity of CATT/T. evansi (43%) and the PCR tests (39-53%) but higher specificity of the PCR tests (86-99%) and low specificity of CATT/T. evansi (80%). This study suggests that improved sensitivity and reliability of the tests would help diagnosis of trypanosomosis. Further studies are required to determine the prevalence of clinical disease and losses due to trypanosomosis.

Surra Sero K-SeT, a new immunochromatographic test for serodiagnosis of Trypanosoma evansi infection in domestic animals.

Trypanosoma evansi, the causative agent of surra, infects different domestic and wild animals and has a wide geographical distribution. It is mechanically transmitted mainly by haematophagous flies. Parasitological techniques are commonly used for the diagnosis of surra but have limited sensitivity. Therefore, serodiagnosis based on the detection of T. evansi specific antibodies is recommended by the World Organisation for Animal Health (OIE). Recently, we developed a new antibody detection test for the serodiagnosis of T. evansi infection, the Surra Sero K-SeT. Surra Sero K-SeT is an immunochromatographic test (ICT) that makes use of recombinant variant surface glycoprotein rVSG RoTat 1.2, produced in the yeast Pichia pastoris. In this study, we compared the diagnostic accuracy of the Surra Sero K-SeT and the Card Agglutination Test for T. evansi Trypanosomososis (CATT/T. evansi) with immune trypanolysis (TL) as reference test on a total of 806 sera from camels, water buffaloes, horses, bovines, sheep, dogs and alpacas. Test agreement was highest between Surra Sero K-SeT and TL (κ=0.91, 95% CI 0.841-0.979) and somewhat lower between CATT/T. evansi and TL (κ=0.85, 95% CI 0.785-0.922) and Surra Sero K-SeT and CATT/T. evansi (κ=0.81, 95% CI 0.742-0.878). The Surra Sero K-SeT displayed a somewhat lower overall specificity than CATT/T. evansi (94.8% versus 98.3%, χ(2)=13.37, p<0.001) but a considerably higher sensitivity (98.1% versus 84.4%, χ(2)=33.39, p<0.001). We conclude that the Surra Sero K-SeT may become an alternative for the CATT/T. evansi for sensitive detection of antibodies against T. evansi in domestic animals.

Epidemiology of Trypanosoma evansi and Trypanosoma vivax in domestic animals from selected districts of Tigray and Afar regions, Northern Ethiopia.

BACKGROUND: African animal trypanosomosis, transmitted cyclically by tsetse flies or mechanically by other biting flies, causes serious inflictions to livestock health. This study investigates the extent of non-tsetse transmitted animal trypanosomosis (NTTAT) by Trypanosoma (T.) evansi and T. vivax in domestic animals in the tsetse-free regions of Northern Ethiopia, Afar and Tigray. METHODS: A cross sectional study was conducted on 754 dromedary camels, 493 cattle, 264 goats, 181 sheep, 84 donkeys, 25 horses and 10 mules. The microhaematocrit centrifugation technique was used as parasitological test. Plasma was collected for serodiagnosis with CATT/T.evansi and RoTat 1.2 immune trypanolysis (ITL) while buffy coat specimens were collected for molecular diagnosis with T. evansi type A specific RoTat 1.2 PCR, T. evansi type B specific EVAB PCR and T. vivax specific TvPRAC PCR. RESULTS: The parasitological prevalence was 4.7% in Tigray and 2.7% in Afar and significantly higher (z = 2.53, p = 0.011) in cattle (7.3%) than in the other hosts. Seroprevalence in CATT/T.evansi was 24.6% in Tigray and 13.9% in Afar and was significantly higher (z = 9.39, p < 0.001) in cattle (37.3%) than in the other hosts. On the other hand, seroprevalence assessed by ITL was only 1.9% suggesting cross reaction of CATT/T.evansi with T. vivax or other trypanosome infections. Molecular prevalence of T. evansi type A was 8.0% in Tigray and in Afar and varied from 28.0% in horses to 2.2% in sheep. It was also significantly higher (p < 0.001) in camel (11.7%) than in cattle (6.1%), donkey (6%), goat (3.8%), and sheep (2.2%). Four camels were positive for T. evansi type B. Molecular prevalence of T. vivax was 3.0% and was similar in Tigray and Afar. It didn't differ significantly among the host species except that it was not detected in horses and mules. CONCLUSIONS: NTTAT caused by T. vivax and T. evansi, is an important threat to animal health in Tigray and Afar. For the first time, we confirm the presence of T. evansi type B in Ethiopian camels. Unexplained results obtained with the current diagnostic tests in bovines warrant particular efforts to isolate and characterise trypanosome strains that circulate in Northern Ethiopia.

A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.

Serological evidence of Ostertagia ostertagi infection in dairy cows does not impact the efficacy of rabies vaccination during the housing period.

Nematode infections modulate the immune reaction of humans and livestock and may impair immune responses to non-parasitic antigens such as those present in vaccines. In this study, the relationship between antibodies directed against Ostertagia ostertagi, the economically most important nematode infection of cattle in temperate regions, and the magnitude and the kinetics of the antibody response to rabies vaccination was investigated in a commercial dairy herd of 46 cows. During the stabling period, all animals received a single intramuscular administration with a commercial inactivated rabies vaccine (Rabisin®, Merial). The serum antibody levels against O. ostertagi on day 0 were compared with anti-rabies IgM, IgA, IgG1, IgG2 and virus-neutralizing antibodies on days 0, 7, 14 and 21 after vaccination. In addition, to explore the potential effect of newly acquired O. ostertagi infections, the kinetics of the O. ostertagi antibody levels during the first 2 months after turnout on pasture were compared with concurrent changes in the rabies antibodies. During the stabling period the O. ostertagi antibody level tended to be positively associated with the magnitude, rate of increase and rate of decrease of the rabies antibodies. However, none of these associations were significant (P>0.05). Over the first 2 months at pasture, an increase in O. ostertagi antibody level tended to be associated with a decrease in rabies IgG2 and IgM, but again these associations lacked statistical significance (P>0.20). We conclude that the O. ostertagi antibody level in adult cattle over the housing period has no significant association with the antibody response to rabies vaccination. We recommend that future studies aiming to assess the relationship of nematode infections with humoral immune responses to vaccines are conducted on a larger scale and focus on the summer period when cattle are exposed continuously to nematode challenge from the pasture and hence are actively responding immunologically to nematode antigen exposure.

Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection.

Oral feed-based passive immunization can be a promising strategy to prolong maternal lactogenic immunity against postweaning infections. Enterotoxigenic Escherichia coli (ETEC)-caused postweaning diarrhea in piglets is one such infection that may be prevented by oral passive immunization and might avert recurrent economic losses to the pig farming industry. As a proof of principle, we designed anti-ETEC antibodies by fusing variable domains of llama heavy chain-only antibodies (VHHs) against ETEC to the Fc part of a porcine immunoglobulin (IgG or IgA) and expressed them in Arabidopsis thaliana seeds. In this way, four VHH-IgG and four VHH-IgA antibodies were produced to levels of about 3% and 0.2% of seed weight, respectively. Cotransformation of VHH-IgA with the porcine joining chain and secretory component led to the production of light-chain devoid, assembled multivalent dimeric, and secretory IgA-like antibodies. In vitro analysis of all of the antibody-producing seed extracts showed inhibition of bacterial binding to porcine gut villous enterocytes. However, in the piglet feed-challenge experiment, only the piglets receiving feed containing the VHH-IgA-based antibodies (dose 20 mg/d per pig) were protected. Piglets receiving the VHH-IgA-based antibodies in the feed showed a progressive decline in shedding of bacteria, significantly lower immune responses corroborating reduced exposure to the ETEC pathogen, and a significantly higher weight gain compared with the piglets receiving VHH-IgG producing (dose 80 mg/d per pig) or wild-type seeds. These results stress the importance of the antibody format in oral passive immunization and encourage future expression of these antibodies in crop seeds.

Intracloacally passaged low-virulent Histomonas meleagridis protects turkeys from histomonosis.

Histomonosis (blackhead disease or infectious enterohepatitis) caused by the extracellular protozoon parasite Histomonas meleagridis is an important disease of turkeys and a threat to the poultry industry. Due to recent legislation on drug restrictions, research to find new alternatives is an urgent matter in the battle against histomonosis. In the present study, intracloacal inoculation of a low-virulent H. meleagridis strain isolated after serial passages in turkeys clearly demonstrated a reduction of virulence and hence its effectiveness as a vaccine against histomonosis. The low-virulent isolate has been evaluated in a comparative experimental infection study. No mortality nor predominant caecal or liver lesions could be observed in the groups inoculated with 10(3), 10(4) or 10(5) histomonads per bird. Only dilated caeca with a yellow and foamy content could be noticed. Groups inoculated with similar doses of a virulent strain displayed a dose-related pathology and mortality up to 100%. The protective capacity of the strain with reduced virulence could be demonstrated as none of the birds cloacally inoculated with 10(3), 10(4) or 10(5) histomonads died upon challenge with 10(5)H. meleagridis of the virulent strain. Hereby, 71% of the challenge control group died. Interestingly, no or only very minor pathological lesions in the caeca and liver could be detected after challenge of the birds inoculated with the passaged histomonads. In conclusion, cloacal inoculation of the low-virulent strain obtained after serial backpassages was able to induce protection of turkeys against challenge with a virulent H. meleagridis strain.

In silico analysis of tkt1 from avian pathogenic Escherichia coli and its virulence evaluation in chickens.

Extraintestinal pathogenic Escherichia coli (ExPEC) contain tktA and tktB which code for transketolases involved in the pentose phosphate pathway. Recent studies demonstrated that a third gene coding for transketolase 1 (tkt1) was located in a pathogenicity island of avian and human ExPEC belonging to phylogenetic group B2. In the present study, in silico analysis of tkt1 revealed 68% and 69% identity with tktA and tktB, respectively, of ExPEC and 68% identity with tktA and tktB of E. coli MG1655. The translated tkt1 shared 69% and 68% identity with TktA and TktB proteins, respectively, of ExPEC and E. coli MG1655. Phylogenetically, it is shown that the three genes (tktA, tktB and tkt1) cluster in three different clades. Further analysis suggests that tkt1 has been acquired though horizontal gene transfer from plant-associated bacteria within the family Enterobacteriaceae. Virulence studies were performed in order to evaluate whether tkt1 played a role in avian pathogenic E. coli CH2 virulence in chickens. The evaluation revealed that mutant virulence was slightly lower based on LD50 when compared to the wild type during infection of chickens, but there were no significant differences when the two strains were compared based on the number of deaths and lesion scores.

Several enteropathogens are circulating in suckling and newly weaned piglets suffering from diarrhea in the province of Villa Clara, Cuba.

Intestinal contents of suckling (n = 45) and newly weaned (n = 45) piglets, suffering from diarrhea in the province of Villa Clara in Cuba, were tested for viral, bacterial, and parasitic enteropathogens from May to June 2008. At least one enteropathogen was identified in 53.3 % of piglets and enterotoxigenic Escherichia coli (ETEC; 25.6 %) was the major pathogen; mostly STa(+)/STb(+) or F4(+)/STa(+)/STb(+) ETEC were isolated. The overall occurrence of the rest of pathogens was 10 % for transmissible gastroenteritis virus (TGEV) and Cryptosporidium parvum, 6.7 % for rotavirus A and Isospora suis, 5.6 % for α-toxigenic Clostridium perfringens, 3.3 % for verotoxigenic E. coli (VTEC), and 2.2 % for Salmonella enterica subspecies enterica serovar Newport. TGEV and α-toxigenic C. perfringens were only identified in suckling piglets, while Salmonella Newport and VTEC were only detected in weaned pigs. Porcine epidemic diarrhea virus (PEDV), ß-toxigenic C. perfringens, Eimeria spp., and helminths were not identified. Eight kinds of mixed infections were detected in 25 % of enteropathogen positive piglets. ETEC was present in 10 of 12 mixed infections, and TGEV infections were never combined. This survey demonstrates that several enteropathogens are circulating in piggeries located in the province of Villa Clara in Cuba, and that is necessary to improve surveillance, prevention, and control of enteric infections in order to increase production efficiency.

Widespread occurrence of Trypanosoma vivax in bovines of tsetse- as well as non-tsetse-infested regions of Ethiopia: a reason for concern?

A cross-sectional study was undertaken to assess the prevalence of bovine trypanosomosis in some tsetse-infested and tsetse-free areas of Ethiopia. From August 2010 till April 2011, a total of 1524 animals were parasitologically examined and compared by the haematocrit centrifugation technique (Woo test) and polymerase chain reaction (ITS-1 PCR). The ITS-1 PCR was more sensitive and more accurate in species identification than the Woo test. In ITS-1 PCR, an overall trypanosome prevalence of 31.0% was observed that is significantly (P<0.001) higher than in the Woo test (5.3%). Trypanosoma vivax was the predominant taxon (24.9%), followed by T. theileri (6.0%), T. congolense (2.9%) and Trypanozoon (1.6%). Mixed infections were quite common (14% of all infections). The overall prevalence of trypanosome infections in tsetse area (32.4%) was not different from non-tsetse area (30.5%) neither were the prevalences of T. vivax in both areas (respectively 22.6% and 25.7%). With these high prevalences, bovine trypanosomosis continues to hinder animal production and productivity in Ethiopia, both in tsetse-infested and non-infested parts of the country. Attempts to control African trypanosomosis should also pay attention to mechanically transmitted pathogenic trypanosomes and should adopt the most advanced molecular tests for species identification.

Escherichia coli ghosts or live E. coli expressing the ferri-siderophore receptors FepA, FhuE, IroN and IutA do not protect broiler chickens against avian pathogenic E. coli (APEC).

The aim of this study was to investigate if immunization with the ferri-siderophore receptors FepA, FhuE, IroN and IutA could protect chickens against avian pathogenic Escherichia coli (APEC) infection. The antigens were administered as recombinant proteins in the outer membrane (OM) of E. coli strain BL21 Star DE3. In a first immunization experiment, live E. coli expressing all 4 recombinant ferri-siderophore receptors (BL21(L)) were given intranasally. In a second immunization experiment, a mixture of E. coli ghosts containing recombinant FepA and IutA and ghosts containing recombinant FhuE and IroN was evaluated. For both experiments non-recombinant counterparts of the tentative vaccines were administered as placebo. At the time of challenge, the IgG antibody response for BL21(L) and a mixture of E. coli ghosts containing recombinant FepA and IutA and ghosts containing recombinant FhuE and IroN was significantly higher in all immunized groups as compared to the negative control groups (LB or PBS) confirming successful immunization. Although neither of the tentative vaccines could prevent lesions and mortality upon APEC infection, immunization with bacterial ghosts resulted in a decrease in mortality from 50% (PBS) to 31% (non-recombinant ghosts) or 20% (recombinant ghosts) and these differences were not found to be significant.

Identification of Avian pathogenic Escherichia coli genes that are induced in vivo during infection in chickens.

Avian pathogenic Escherichia coli (APEC) is associated with extraintestinal infections in poultry causing a variety of diseases collectively known as colibacillosis. The host and bacterial factors influencing and/or responsible for carriage and systemic translocation of APEC inside the host are poorly understood. Identification of such factors could help in the understanding of its pathogenesis and in the subsequent development of control strategies. Recombination-based in vivo expression technology (RIVET) was used to identify APEC genes specifically expressed during infection in chickens. A total of 21 clones with in vivo-induced promoters were isolated from chicken livers and spleens, indicative of systemic infection. DNA sequencing of the cloned fragments revealed that 12 of the genes were conserved E. coli genes (metH, lysA, pntA, purL, serS, ybjE, ycdK [rutC], wcaJ, gspL, sdsR, ylbE, and yjiY), 6 of the genes were phage related/associated, and 3 genes were pathogen specific (tkt1, irp2, and eitD). These genes are involved in various cellular functions, such as metabolism, cell envelope and integrity, transport systems, and virulence. Others were phage related or have yet-unknown functions.