Histopathological lesions in reproductive organs, distal spinal cord and peripheral nerves of horses naturally infected with Trypanosoma equiperdum.

BACKGROUND: Dourine, a venereal transmitted trypanosomosis caused by Trypanosoma equiperdum, has different clinical signs related to the reproductive and nervous system. Pathologic tissue changes associated with the disease are poorly described. The present study describes the histopathological lesions in naturally T. equiperdum-infected horses in the chronical stage of dourine. RESULTS: Four chronically dourine diseased horses underwent a post-mortem examination. They were Woo test negative, but CATT/T. evansi positive, had a low packed cell volume (PCV) and exhibited obvious clinical signs of dourine. Post-mortem examination did not reveal gross lesions in the organs assumed to be responsible for the symptomatology. On histopathology, genital organs were affected, with mononuclear cell infiltration and erosions and degeneration of seminiferous tubules and perivascular lymphoplasmacytic cuffing in the uterus. In the nervous system, mononuclear cell infiltration was located in peripheral nerves, ganglia and in the spinal cord, leading to axonal degeneration. Real-time PCR using ITS primer revealed the presence of trypanosomes in these organs and conventional PCRs using maxicircle and RoTat1.2 primers further confirmed the involvement of T. equiperdum since the DNAs from the vagina, testicle, distal spinal cord, sciatic and obturator nerves found to be positive for maxicircle and negative for RoTat 1.2. CONCLUSIONS: The histopathological lesions in the spinal cord and peripheral nerves explain the incoordination of the hind legs in T. equiperdum-infected horses, whilst its presence in the genital tract exemplifies the venereal transmission.

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 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.

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.

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.

Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae.

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA(+) B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA(+) B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.

Adaptive radiotherapy for locally advanced non-small cell lung cancer, can we predict when and for whom?

BACKGROUND: Adaptive radiotherapy (ART) could be a tool to reduce toxicity and to facilitate dose escalation in stage III NSCLC. Our aim was to identify the most appropriate time and potential benefit of ART. MATERIAL AND METHODS: We analyzed volume reduction and dosimetric consequences of 41 patients who were treated with concurrent (cCRT) (n = 21) or sequential (sCRT) chemoradiotherapy to a median dose of 70 Gy, 2 Gy/F. At every treatment fraction a cone-beam CT (CBCT) was performed. The gross tumor volume (GTV-T) was adapted (exclusion of lymph nodes) to create the GTV-T-F1. Every fifth fraction (F5-F30), the GTV-T-F1 was adapted on the CBCT to create a GTV-T-Fx. Dose volume histograms were recalculated for every GTV-T-Fx, enabling to create lookup tables to predict the theoretical dosimetric advantage on common lung dose constraints. RESULTS: The average GTV reduction was 42.1% (range 4.0-69.3%); 50.1% and 33.7% for the cCRT and sCRT patients, respectively. A linear relationship between GTV-T-F1 volume and absolute volume decrease was found for both groups. The mean V5, V20, V30 and mean lung dose increased by 0.8, 3.1, 5.2 and 3.4%, respectively. A larger increase (p < 0.05) was observed for peripheral tumors and cCRT. Lookup tables were generated. CONCLUSION: ART offers the most beneficial dosimetric effects when performed around fraction 15, especially for patients with a large initial GTV-T treated by cCRT.

Comparative clinico-pathological observations in young Zebu (Bos indicus) cattle experimentally infected with Trypanosoma vivax isolates from tsetse infested and non-tsetse areas of Northwest Ethiopia.

BACKGROUND: The Northwest region of Ethiopia is affected by both tsetse and non-tsetse transmitted trypanosomosis with a huge impact on livestock productivity. The objective of this experimental study was to determine clinical and pathological findings in young Zebu cattle experimentally infected with Trypanosoma vivax isolates from tsetse infested and non-tsetse infested areas of Northwest Ethiopia. A total of 18 cattle (Bos indicus) aged between 6 and 12 months, purchased from a trypanosome-free and confirmed to be trypanosome negative divided into three groups of six animals were used. Animals in the first two groups (Group TT: tsetse infested isolate infected and Group NT: non-tsetse infested isolate infected) received 2 mL of infected blood from donor animals at 10(6) trypanosomes/mL, and the remaining group was non-infected control (NIC). Each group was observed for a period of eight consecutive weeks, daily for clinical signs and once per week for parasitaemia. Postmortem examinations were done on euthanized animals, and tissue samples were taken for histopathological analysis. RESULTS: The prepatent period of the disease was earlier in the NT group 6 days post infection (dpi) than TT group 12 dpi. The infection was characterized by reduced feed intake, intermittent pyrexia and parasitaemia, enlarged lymph nodes, lacrimation, reduced feed intake and emaciation. Less frequently diarrhea, oedema and nervous signs were observed in both groups of infected animals. At necropsy, infected animals showed enlarged spleen, enlarged lymph nodes, pneumonic and emphysematous lung, enlarged liver, and haemorrhages on the brain and intestine. Histopathological analysis revealed lymphoid hyperplasia of the spleen, necrosis of the liver, encephalitis and hyperplasia of lymph nodes. CONCLUSION: Trpanosoma vivax isolates from both tsetse infested and non-tsetse areas showed a variety of virulence factors leading to the development of acute clinical signs, gross and histopathological lesions. However, the parasitaemia and clinical signs appeared earlier in the NT compared to TT infected groups.

Susceptibility of avian pathogenic Escherichia coli from laying hens in Belgium to antibiotics and disinfectants and integron prevalence.

Avian pathogenic Escherichia coli (APEC) causes huge annual losses in the poultry industry worldwide. Multiresistance against antibiotics of APEC strains is increasingly seen in broilers, although much is still unknown about strains from laying hens where use of antibiotics is limited. Disinfection can reduce the infection burden. However, little is known about the presence of resistance against these products. Ninety-seven APEC strains were isolated from Belgian laying hens. The resistance to different classes of antibiotics was determined as well as the minimum inhibitory concentrations (MIC; agar and broth dilution) and minimum bactericidal concentrations (MBC) of five disinfectants most often used in the poultry industry (formaldehyde, glutaraldehyde, glyoxal, hydrogen peroxide, and a quaternary ammonium compound). The presence of integrons was determined by PCR Resistance to ampicillin (35.1%), nalidixic acid (38.1%), sulfonamides (SULFA, 41.2%), and tetracycline (TET, 53.6%) was high but resistance to other tested antibiotics was low. Nevertheless, two extended spectrum beta-lactamase producers were found. The MIC of the disinfectants for the APEC strains showed a Gaussian distribution, indicating that there was no acquired resistance. MBCs were similar to MICs via the broth dilution method, showing the bactericidal effect of the disinfectants. Twenty-one strains (21.6%) were found positive for class 1 integrons and a positive association between integron presence and resistance to trimethoprim, SULFA, and TET was found. No association could be found between integron presence and phylogenetic group affiliation. Susceptibility of APEC strains from laying hens to antibiotics is, in general, very high. Phenotypic resistance to commonly used disinfectants could not be found, indicating that the current use of disinfectants in the laying hen industry did not select for resistance.

Analysis for prevalence and physical linkages amongst integrons, ISEcp1, ISCR1, Tn21 and Tn7 encountered in Escherichia coli strains from hospitalized and non-hospitalized patients in Kenya during a 19-year period (1992-2011).

BACKGROUND: We determined the prevalence and evidence for physical linkage amongst integrons, insertion sequences, Tn21 and Tn7 transposons in a collection of 1327 E. coli obtained over a 19-year period from patients in Kenya. RESULTS: The prevalence of class 1 integrons was 35%, class 2 integrons were detected in 3 isolates but no isolate contained a class 3 integron. Integron lacking the 3'-CS or those linked to sul3 gene or IS26 or those containing the ISCR1 were only detected in multidrug resistant (MDR) strains. The dfrAs were the most common cassettes and their prevalence was: - dfrA1(28%), dfrA12(20%), dfA17(9%), dfrA7(9%), and dfrA16(5%). The aadA were the second most abundant cassettes and their prevalence was: - aadA1(25%), aadA2(21%), and aadA5(14%). Other cassettes occurred in lower prevalence of below 5%. Prevalence of Tn21, ISEcp1, ISCR1 and IS26 was 22%, 10%, 15%, and 7% respectively. Majority of Tn21 containing integrons carried a complete set of transposition genes while class 2 integrons were borne on Tn7 transposon. The qnrA genes were detected in 34(3%) isolates while 19(1%) carried qnrB. All qnr genes were in MDR strains carrying integrons containing the ISCR1. Close to 88% of bla(TEM-52) were linked to IS26 while ≥ 80% of bla(CTX-Ms) and bla(CMYs) were linked to ISEcp1. Only a few studies have identified a bla(CTX-M-9) containing an ISEcp1 element as reported in this study. Multiple genetic elements, especially those borne on incIl, incFII, and incL/M plasmids, and their associated resistance genes were transferrable en bloc to E. coli strain J53 in mating experiments. CONCLUSIONS: This is the first detailed study on the prevalence of selected elements implicated in evolution of resistance determinants in a large collection of clinical E. coli in Africa. Proliferation of such strains carrying multiple resistance elements is likely to compromise the use of affordable and available treatment options for majority of poor patients in Africa. There is therefore a need to monitor the spread of these highly resistant strains in developing countries through proper infection control and appropriate use of antimicrobials.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens.

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.

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.

Protection of piglets against Edema disease by maternal immunization with Stx2e toxoid.

Edema disease (ED) in piglets is caused by Shiga toxin Stx2e-producing Escherichia coli. We show that a genetically disarmed Stx2e toxoid is a safe antigen that generates antiserum protecting piglets against the Stx2e toxin. Immunization of suckling piglets with the Stx2e toxoid was safe, had no adverse effects on growth of the piglets, and resulted in effective prevention of edema disease clinical symptoms after challenge with the Stx2e toxin. Our data showed that maternal immunity against the Stx2e toxoid can be transmitted from the vaccinated sows to the piglets via the colostrum. Very high levels of Stx2e-specific serum antibodies persisted in these piglets until 1 month postweaning, bridging the critical period in which the weaned piglets are most susceptible to edema infection. Challenge with Stx2e toxin resulted in clinical signs of edema disease and death of all control piglets from nonimmunized sows, whereas none of the piglets from immunized sows developed clinical signs of ED.

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.

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.

Analysis of ß-lactamase phenotypes and carriage of selected ß-lactamase genes among Escherichia coli strains obtained from Kenyan patients during an 18-year period.

BACKGROUND: Although ß-lactam antibiotics are heavily used in many developing countries, the diversity of ß-lactamase genes (bla) is poorly understood. We screened for major ß-lactamase phenotypes and diversity of bla genes among 912 E. coli strains isolated from clinical samples obtained between 1992 and 2010 from hospitalized and non-hospitalized patients. RESULTS: None of the isolates was resistant to carbapenems but 30% of all isolates were susceptible to cefepime, cephamycins and piperacillin-tazobactam. Narrow spectrum ß-lactamase (NSBL) phenotype was observed in 278 (30%) isolates that contained bla(TEM-1) (54%) or bla(SHV-1) (35%) or both (11%). Extended Spectrum ß-lactamase (ESBL) phenotype was detected in 247 (27%) isolates which carried blaCTX-M-14 (29%), bla(CTX-M-15) (24%), bla(CTX-M-9) (2%), bla(CTX-M-8) (4%), bla(CTX-M-3) (11%), bla(CTX-M-1) (6%), blaSHV-5 (3%), bla(SHV-12) (5%), and bla(TEM-52) (16%). Complex Mutant TEM-like (CMT) phenotype was detected in 220 (24%) isolates which carried bla(TEM-125) (29%), while bla(TEM-50), bla(TEM-78), bla(TEM-109), bla(TEM -152) and bla(TEM-158) were detected in lower frequencies of between 7% and 11%. Majority of isolates producing a combination of CTX-M-15 + OXA-1 + TEM-1 exhibited resistance phenotypes barely indistinguishable from those of CMT-producers. Although 73 (8%) isolates exhibited Inhibitor Resistant TEM-like (IRT) phenotype, bla(TEM-103) was the only true IRT-encoding gene identified in 18 (25%) of strains with this phenotype while the rest produced a combination of TEM-1 + OXA-1. The pAmpCs-like phenotype was observed in 94 (10%) isolates of which 77 (82%) carried bla(CMY-2) while 18% contained blaCMY-1.Isolates from urine accounted for 53%, 53%, 74% and 72% of strains exhibiting complex phenotypes such as IRT, ESBL, CMT or pAmpC respectively. On the contrary, 55% isolates from stool exhibited the relatively more susceptible NSBL-like phenotype. All the phenotypes, and majority of the bla genes, were detected both in isolates from hospitalized and non-hospitalized patients but complex phenotypes were particularly common among strains obtained between 2000 and 2010 from urine of hospitalized patients. CONCLUSIONS: The phenotypes and diversity of bla genes in E. coli strains implicated in clinical infections in non-hospitalized and hospitalized patients in Kenya is worryingly high. In order to preserve the efficacy of ß-lactam antibiotics, culture and susceptibility data should guide therapy and surveillance studies for ß-lactamase-producers in developing countries should be launched.

Genome wide analysis of the bovine mucin genes and their gastrointestinal transcription profile.

BACKGROUND: Mucins are large glycoproteins implicated in protection of all mucosal surfaces. In humans and rodents, the mucin gene family has been well described and previous studies have investigated the distribution and function of mucins in the gastrointestinal (GI) tract. In contrast, little data is available on the mucin gene family in polygastric species, such as cattle. The aim of the current study was to identify all members of the bovine mucin family by genome mining and subsequently investigate the transcription pattern of these mucins in the GI tract. RESULTS: Nine bovine membrane-associated mucins (MUC1, MUC3A, MUC4, MUC12, MUC13, MUC15, MUC16, MUC20 and MUC21) and six secreted mucins (MUC2, MUC5AC, MUC5B, MUC6, MUC7 and MUC19) were identified in the bovine genome. No homologues could be identified for MUC3B, MUC8 and MUC17. In general, domain architecture of the membrane-associated mucins was found to be similar between humans and cattle, while the protein architecture of the gel-forming mucins appeared to be less conserved. Further analysis of the genomic organization indicated that the previously reported bovine submaxillary mucin (BSM) may be part of a larger gene encoding for MUC19. Analysis of the transcription profile showed that the secreted mucins were transcribed from the abomasum onwards, whereas the membrane associated mucins MUC1 and MUC20 were transcribed throughout the whole GI tract. In contrast to humans, MUC5B transcript was found in both the small and large intestine, but was absent in oesophageal tissue. CONCLUSIONS: This study provides the first characterization of the mucin gene family in cattle and their transcriptional regulation in the GI tract. The data presented in this paper will allow further studies of these proteins in the physiology of the GI tract in ruminants and their interactions with pathogens.

Infection with the gastrointestinal nematode Ostertagia ostertagi in cattle affects mucus biosynthesis in the abomasum.

The mucus layer in the gastrointestinal (GI) tract is considered to be the first line of defense to the external environment. Alteration in mucus components has been reported to occur during intestinal nematode infection in ruminants, but the role of mucus in response to abomasal parasites remains largely unclear. The aim of the current study was to analyze the effects of an Ostertagia ostertagi infection on the abomasal mucus biosynthesis in cattle. Increased gene expression of MUC1, MUC6 and MUC20 was observed, while MUC5AC did not change during infection. Qualitative changes of mucins, related to sugar composition, were also observed. AB-PAS and HID-AB stainings highlighted a decrease in neutral and an increase in acidic mucins, throughout the infection. Several genes involved in mucin core structure synthesis, branching and oligomerization, such as GCNT3, GCNT4, A4GNT and protein disulphide isomerases were found to be upregulated. Increase in mucin fucosylation was observed using the lectin UEA-I and through the evaluation of fucosyltransferases gene expression levels. Finally, transcription levels of 2 trefoil factors, TFF1 and TFF3, which are co-expressed with mucins in the GI tract, were also found to be significantly upregulated in infected animals. Although the alterations in mucus biosynthesis started early during infection, the biggest effects were found when adult worms were present on the surface of the abomasal mucosa and are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells.

High prevalence of F4+ and F18+ Escherichia coli in Cuban piggeries as determined by serological survey.

Little information is available on the prevalence of swine enteropathogens in Cuba where diarrheic diseases are responsible for 31% and 37% of the total mortality during the neonatal and postweaning periods. F4+ and F18+ enterotoxigenic Escherichia coli and F18+ verotoxigenic E. coli induce diarrhea and edematous disease in pigs, but their distribution has never been thoroughly studied in the Cuban swine population. Therefore, the present study estimated the prevalence of F4- and F18-specific antibodies in sera of 1,044 6-month-old gilts distributed in 34 piggeries spread over the Cuban territory. For the data analysis, which included the optical density of individual samples tested by ELISA, random-effects models and a mixture model in R (package "mixAK"; Komárek, Computational Statistics and Data Analysis 53:3932-3947, 2009) were fitted. Low, moderate, and high levels of F4-specific antibodies were found in 67.6%, 26.8%, and 5.6% of the gilts, while 66.4% and 33.6% of gilts showed low and high levels of F18-specific antibodies. Hereby, we show that F4+ and F18+ E. coli are highly prevalent as potential enteropathogens in Cuban piggeries.