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Megasphaera elsdenii (M.e.) NCIMB 41125 is a robust lactate utilizing strain of M.e. that is effective in minimizing the risk of ruminal acidosis in feedlot cattle. When dosed orally, cattle adapt smoothly to increasing concentrates in the diet, the incidence of digestive disturbances, morbidity and mortality is reduced, and carcass yield improves. One could therefore expect that the smooth transition should benefit overall performance. Dosing with the organism also provides the opportunity of a reduction in the time necessary for adaptation, rendering a further decrease in the cost of feeding. These two objectives were tested with 80 yearling crossbred steers blocked by weight before allotment to the respective treatments. The trial design was a randomized 2 x 2 factorial of two drench treatments (M.e. vs. placebo) and two adaptation periods (17 vs. 8 days). In the M.e. treatment, 40 steers were dosed orally on day 1 of the trial with 200 mL inoculum containing 1011 cells. In the placebo treatment, the other 40 steers were dosed orally with only the 200 mL inoculum. In the 17-day transition period, five diets (5-transition) were used, which increased progressively in concentrate percentage, whereas in the 8-day transition period only three of the five diets were fed (3-transition). The steers were fed individually for 63 days before being transferred to group pens and fed until day 95, when they were slaughtered. Dry matter intake was not affected by dose or transition treatment. Body weight at 28 days and 63 days did not differ between dose and transition treatments; neither did ADG and FCR. Hot carcass weight was higher in M.e. steers than in placebo steers. None of the parameters differed significantly between the 5-transition and the 3-transition treatments. It was concluded that dosing with M.e. NCIMB 41125 should provide a small benefit to performance of feedlot cattle, with a further benefit in cost savings as dosing with the organism should allow a shorter adaptation period. [ABSTRACT FROM AUTHOR]

The diversity of protozoan-associated methanogens in cattle was investigated using five universal archaeal small-subunit (SSU) rRNA gene primer sets. Methanobrevibacter spp. and rumen cluster C (distantly related to Thermoplasma spp.) were predominant. Significant differences in species composition among libraries indicate that some primers used previously to characterize rumen methanogens exhibit biased amplification. [ABSTRACT FROM AUTHOR]

Abstract: Bovine mastitis remains worldwide a major challenge for the dairy industry despite the widespread implementation of control strategies. The increasing number of coagulase-negative staphylococci (CNS) causing mastitis and of bacteria resistant to conventional antibiotics has become a serious problem in recent years. Marine sponges are a rich source of bioactive compounds, and many species can be useful for the development of new antimicrobial drugs. In the present study, 49 CNS strains were isolated from bovine mastitis cases from 21 different dairy herds kept at farms in Southeast Brazil. Strains were analyzed for antimicrobial susceptibility and mecA gene detection. Fifty-nine percent of the CNS strains were resistant to at least one of the drugs tested and 12.2% were classified as multiresistant. Three strains carried the mecA gene, confering resistance to the beta-lactamic antibiotics. In addition, the CNS strains were submitted to in vitro screening for antimicrobial activities of extracts from marine sponges. Extracts from the sponge species Cinachyrella sp., Haliclona sp. and Petromica citrina showed antibacterial activity against 61% of the CNS strains, including strains resistant to conventional antibiotics. Extracts from P. citrina showed the largest spectrum of inhibitory activity. The aqueous extract inhibited 51% of the CNS strains and presented a bactericidal effect over susceptible and multiresistant-bacteria at a minimal inhibitory concentration of 1.024μg/ml. This study shows the potential of marine sponges as new sources of antibiotics and disinfectants for the control of CNS involved in bovine mastitis. [Copyright &y& Elsevier]

Abstract: This study was undertaken to evaluate the acute phase responses via the assessment of the concentration of serum sialic acids (total, lipid bound and protein bound), inflammatory mediators (IFN-γ and TNF-α) and acute phase proteins (Hp and SAA) in 20 adult crossbred cattle naturally infected by Anaplasma marginale. The infected animals were divided into 2 subgroups on the basis of parasitemia rate (<20% and >20%). Also, as a control group, 10 clinically healthy cattle from the same farms were sampled. Our data revealed significant decreases in red blood cell count (RBC), hematocrite (PCV) and hemoglobine (Hb) in infected cattle compared to healthy ones. Conversely, the concentrations of Hp, SAA, ceruloplasmin, fibrinogen, serum sialic acids and the circulatory IFN-γ and TNF-α were increased in the diseased cattle (P <0.05). In addition, it was evident that the progression of parasitemia in infected cattle did not induce any significant alterations in the hematological indices (RBCs, PCV and Hb) and the concentrations of Hp, SAA, ceruloplasmin and fibrinogen. SAA was the most sensitive factor to change in the diseased cattle. Therefore, increase in SAA concentration may be a good indicator of inflammatory process in cattle naturally infected with Anaplasma marginale. [Copyright &y& Elsevier]

Volatile fatty acids (VFA) are the major products of microbial fermentation in the rumen. Besides serving as substrates for energy generation, VFA are known to stimulate rumen development, increase serum insulin and glucagon concentrations, and regulate gene expression in cattle and sheep. The mechanisms underlying these regulatory effects of VFA are unknown, but the recent discovery that VFA can bind to G protein-coupled receptor 43 (GPR43) and 41 (GPR41) suggests that the regulatory effects of VFA may be mediated by these receptors. As a step toward testing this possibility, we determined whether GPR43 was expressed in bovine rumen wall and the pancreatic islets of Langerhans. Polyclonal antibody against a bovine GPR43 peptide was generated. The specificity of the antibody for bovine GPR43 was confirmed by Western blot analysis of recombinant bovine GPR43 protein. Immunohistochemical analyses using this antibody revealed the presence of GPR43-immunoreactive cells in the epithelium, but not in the other layers of cattle rumen wall. The same immunohistochemical analyses did not reveal GPR43-immunoreactive cells in the islets of Langerhans or the surrounding exocrine tissue of cattle pancreas. These data support the possibility that the effect of VFA on rumen epithelial growth in cattle is directly mediated by GPR43 in the rumen epithelial cells and that the effect of VFA on pancreatic secretion of insulin and glucagon in cattle is unlikely to be directly mediated by GPR43. [ABSTRACT FROM AUTHOR]

Summary Twenty mammary lymph node samples were collected from cattle on a farm in the Republic of Korea. These cattle were serologically negative for Brucella by tube agglutination test (≤1 : 50) and serum agglutination test (≤1 : 50). Out of 20 lymph node samples, two samples were positive for Brucella growth on Brucella agar as well as blood agar. Tests for urease, hydrogen sulphide and reactions against monospecific sera A and M indicated that these two isolates (No. 15 and 16) belong to the genus Brucella. Genus specific, AMOS (abortus, melitensis, ovis, suis) and Bruce-ladder multiplex polymerase chain reaction (PCR) assays confirmed the Brucella isolates as either a B. abortus or a B. canis strain. This is the first report of the occurrence of a B. canis infection in cattle in Korea. More survey data are needed to determine whether B. canis is a significant aetiology in the cases of cattle brucellosis in Korea. [ABSTRACT FROM AUTHOR]

Abstract: The objective of the study was to investigate reservoirs and transmission of S. aureus in ewes and lambs in 3 meat sheep flocks. Repeated sampling of milk, teat skin, nasal- and vaginal mucous membranes was performed and samples were analysed for S. aureus. For comparison, samples were also collected from cows and young heifers in 3 dairy cattle herds. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE). S. aureus was detected in 8 (1.5%) of 520 milk samples from ewes and in 38 (6.4%) of 588 milk samples from cows. From body site swabs, S. aureus was found in 394 (32.6%) of 1208 samples from sheep and in 67 (16.0%) of 420 samples from cattle. The proportion of S. aureus-positive nasal swabs from ewes and cows were 56.7% and 13.9%, respectively. From lambs, 58.2% of the nasal swabs were S. aureus-positive. In each flock, one S. aureus pulsotype predominated. Identical S. aureus pulsotypes were found in milk and from body sites. Paired S. aureus isolates from the nasal cavity of (i) ewes and their lambs, (ii) twins and (iii) from repeated swabs of individual ewes were compared by PFGE, and in the majority of cases the two isolates were identical. The results contribute new knowledge indicating frequent transmission of S. aureus between the dam and her lambs and within animals in a flock. In contrast to cattle, S. aureus is frequently present in the nose of sheep which may represent the primary reservoir of S. aureus in sheep flocks. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from faeces and hide to dressed carcasses of Irish cattle as well as establishing the virulence potential of VTEC carried by these cattle. Individual cattle was tracked and faecal samples, hide and carcass (pre-evisceration and post-wash) swabs were analysed for verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay. Positive samples were screened for the five serogroups of interest by real-time PCR. Isolates were recovered from PCR positive samples using immunomagnetic separation and confirmed by latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR. Isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). Of the VTEC isolated, E. coli O157 was the most frequently recovered from hide (17.6%), faeces (2.3%) and pre-evisceration/post-wash carcass (0.7%) samples. VTEC O26 was isolated from 0.2% of hide swabs and 1.5% of faeces samples. VTEC O145 was isolated from 0.7% of faeces samples. VTEC O26 and VTEC O145 were not recovered from carcass swabs. Non-VTEC O103 was recovered from all sample types (27.1% hide, 8.5% faeces, 5.5% pre-evisceration carcass, 2.2% post-wash carcass), with 0.2% of hide swabs and 1.0% of faeces samples found to be positive for VTEC O103 isolates. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from hide to carcass was not observed. This study shows that while VTEC O157 are being carried by cattle presented for slaughter in Ireland, a number of other verotoxin producing strains are beginning to emerge. [Copyright &y& Elsevier]

Abstract: The aim of the study was to establish whether the visual cleanliness of cattle slaughtered was correlated to hide and carcass contamination as indicated by aerobic colony count (ACC), Enterobacteriaceae count (EC) and Escherichia coli count (ECC). Cattle in a slaughterhouse were visually inspected and assigned to a category from 1 (very clean) to 5 (very dirty) based on cleanliness. Fifteen animals for each category were randomly selected, hide and carcass sampled and analyzed for ACC, EC and ECC. Results showed that increasing dirt on cattle was associated with higher ACC, EC and ECC on hide and carcasses. Carcass ACC and ECC belonging to animals classified in cleanliness categories 3, 4 or 5 have a higher probability of exceeding the limits set by the Reg. EU 2073/2005. The study supports the conclusion that the pre-slaughter visual evaluation of animal cleanliness and application of corrective actions can be an effective aid to reduce carcass contamination. [Copyright &y& Elsevier]

Abstract: The aim of this study was to determine the ruminal cellulolytic bacterial population in swamp buffalo and beef cattle fed on rice straw by real-time PCR techniques. Four, rumen-fistulated swamp buffalo and four beef cattle (Native×Brahman) were randomly assigned to receive rice straw as a main feed with minimal amount of concentrate. All animals were allowed to adapt to feeds respective before samplings were conducted. It was found that diurnal ruminal pH and NH3–N concentration of buffalo were not different with those found in cattle, population of bacteria and fungal zoospores while the protozoal population were lower (P<0.05). In addition, the nutrient digestibilities of DM, OM, CP, NDF and ADF were found higher in buffalo than in cattle. Employing the real-time PCR techniques for the quantification of cellulolytic bacterial numbers (Ruminococcus albus) in the digesta of swamp buffalo, the results revealed significant higher in buffalo than those in cattle. However, at 4h post-feeding R. albus were significantly higher in buffalo than those in cattle measured in rumen fluid, while F. succinogenes resulted in higher number in cattle than those in buffalo. Interestingly, the values found in the digesta samples were higher in cellulolytic bacteria than those found in the rumen fluid. This finding indicates higher ability of swamp buffalo in digesting low-quality roughages, especially rice straw. [Copyright &y& Elsevier]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophage therapy is a potential alternative treatment for MRSA infections. The objective of this study was characterization of a novel virulent bacteriophage (MSA6) isolated from a cow with mastitis. Electron microscopy showed its resemblance to members of the family Myoviridae, with an isometric head (66 nm) and a long contractile tail (173 nm). The genome of phage MSA6 was tested by pulsed-field gel electrophoresis and estimated to be about 143 kb. It exhibited rapid adsorption (>82% in 5 min), a short latent period (15 min) and a relatively small burst size (23 PFU/cell). Isolated phage was capable of infecting a wide spectrum of staphylococcal strains of both human and bovine origin. The results of this investigation indicate that MSA6 is similar to other bacteriophages belonging to the family Myoviridae (Twort, K, G1, 812) that have been successfully used in bacteriophage therapy. [ABSTRACT FROM AUTHOR]

Abstract: The prevalence of and factors associated with the protozoan enteropathogens Cryptosporidium and Giardia have been investigated in selected children and cattle populations from the province of Álava (Northern Spain). The presence of these organisms was detected in fecal samples using commercially available coproantigen-ELISA (CpAg-ELISA) and immunochromatographic (ICT) assays. A total of 327 caregivers of children participants were asked to answer questions on risk factors potentially associated to the prevalence of Cryptosporidium and Giardia, including water-use practices, water sports and contact with domestic or pet animals. Molecular analyses were conducted using a nested-PCR technique to amplify the small-subunit (SSU) rRNA gene of Cryptosporidium and the triosephosphate isomerase (tpi) gene of Giardia. Cryptosporidium oocysts and Giardia cysts were found in 3 and 16 samples using the CpAg-ELISA, and in 5 and 9 samples using the ICT test, respectively. Cryptosporidium and Giardia were also found in 7 and 17 samples by CpAg-ELISA, and 4 and 14 samples by ICT, respectively, of 227 cattle fecal samples. The overall Cryptosporidium and Giardia infection prevalences, based on a Bayesian approach accounting for the imperfect sensitivities and specificities of both diagnostic tests, were estimated to 1.0% (95% BCI: 0.2%–2.8%) and 3.1% (1.5%–5.3%) in children and 3.0% (0.5%–9.2%) and 1.4% (0.0%–6.4%) in cattle, respectively. In humans, a single Cryptosporidium isolate was characterized as C. hominis. Of seven Giardia isolates, four were identified as assemblage B, two as assemblage A-II and one was a mixed assemblage B+A-II infection. No Cryptosporidium or Giardia isolates could be obtained from cattle samples. Although limited, these results seem to suggest that cattle are unlikely to be an important reservoir of zoonotic Cryptosporidium and/or Giardia in the province of Álava. [Copyright &y& Elsevier]

Aims: European starlings ( Sturnus vulgaris) are an invasive species in the United States and are considered a nuisance pest to agriculture. The goal of this study was to determine the potential for these birds to be reservoirs and/or vectors for the human pathogen Escherichia coli O157:H7. Materials and Results: Under biosecurity confinement, starlings were challenged with various doses of E. coli O157:H7 to determine a minimum infectious dose, the magnitude and duration of pathogen shedding, and the potential of pathogen transmission among starlings and between starlings and cattle. Birds transiently excreted E. coli O157:H7 following low-dose inoculation; however, exposure to greater than 105.5 colony-forming units (CFUs) resulted in shedding for more than 3 days in 50% of the birds. Colonized birds typically excreted greater than 103 CFU g−1 of faeces, and the pathogen was detected for as long as 14 days postinoculation. Cohabitating E. coli O157:H7-positive starlings with culture-negative birds or 12-week-old calves resulted in intra- and interspecies pathogen transmission within 24 h. Likewise, E. coli O157:H7 was recovered from previously culture-negative starlings following 24-h cohabitation with calves shedding E. coli O157:H7. Conclusions: European starlings may be a suitable reservoir and vector of E. coli O157:H7. Significance and Impact of the Study: Given the duration and magnitude of E. coli O157:H7 shedding by European starlings, European starlings should be considered a public health hazard. Measures aimed at controlling environmental contamination with starling excrement, on the farm and in public venues, may decrease food-producing animal and human exposure to this pathogen. [ABSTRACT FROM AUTHOR]

The tegument of cattle may harbor a great diversity of filamentous fungi that can potentially cause skin diseases, including fungi belonging to the dermatophyte group. This study aimed to characterize the population of filamentous fungi from a healthy coat of cattle, checking for dermatophytes as part of the microbiota, as well as evaluating the presence of dermatophytes in the soil occupied by the animals. During the period of one year, 56 healthy cattle in four properties in the western boundary of the state of Rio Grande do Sul, Southern Brazil, were subjected to monthly samples of hair and skin scrapings. In the same period soil samples were obtained for the detection of dermatophytes. The results indicated a fungal biota classified into 30 different genera, among which the most frequent were (in order of frequency): Nigrospora, Fusarium, Curvularia, Alternaria, Epicoccum, Paecilomyces and Trichoderma. Isolates of Trichophyton mentagrophytes (0.4%), Microsporun gypseum (0.3%), and T. verrucosum (0.1%) were obtained in the samples of skin scrapings. M. gypseum was the dermatophyte most frequently isolated from soil. The results showed that the fungal biota of the external tegument of healthy cattle is transient. Moreover, it was shown that healthy cattle do not act as asymptomatic carriers of T. verrucosum, suggesting that this species of dermatophytes is not resident in the tegument of animals. [ABSTRACT FROM AUTHOR]

Aim: To develop a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis ( M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real-time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross-reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real-time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real-time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real-time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species. [ABSTRACT FROM AUTHOR]

Abstract: Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal. [Copyright &y& Elsevier]

Aims: To characterize class 1 integrons and resistance genes in tetracycline-resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). Methods and Results: Tetracycline-resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher ( P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 ( r = 0·44), aadA1 ( r = 0·61), cat ( r = 0·58) and intI1( r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. Conclusions: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline-resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline-resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. Significance and Impact of the Study: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline-resistant E. coli isolated from feedlot cattle. [ABSTRACT FROM AUTHOR]

Objectives of this study were to compare fatty acid (FA) composition of ruminal bacterial (B) and protozoal (P) cells, and to investigate effect of protozoa on FA profile in the rumen of cattle. Three cows were used to prepare ruminal B and P cells. Four faunated and three defaunated cattle (half-siblings) were used to study effect of protozoa on ruminal FA profile. Proportions of C16:0 and C18:0 in total fatty acids in B cells were 20.7% and 37.4%, whereas those in P cells were 33.4% and 11.6%, respectively. Proportions of trans-vaccenic acid (VA) and cis-9, trans-11 conjugated linoleic acid (CLA) in B cells were 3.9% and 1.0%, and those in P cells were 5.5% and 1.6%, respectively, being higher in P cells. Proportions of C18:1, C18:2 and C18:3 in P cells were two to three times higher than in B cells. Proportions of unsaturated fatty acids, VA and CLA in B cells of faunated cattle were higher than those of defaunated. VA and CLA in the ruminal fluid of faunated were also 1.6 to 2.5 times higher than those of defaunated. This tendency was similar for cell-free fraction of ruminal fluid. These results indicate that protozoa contribute greatly in VA and CLA production in the rumen. [ABSTRACT FROM AUTHOR]

Abstract: Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is the most prevalent EHEC serotype that has been recovered from patients with haemolytic uremic syndrome (HUS) worldwide. Vaccination of cattle, the main reservoir of EHEC O157:H7, could be a logical strategy to fight infection in humans. This study evaluated a vaccine based on the carboxyl-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and EspB, two key colonization factors of E. coli O157:H7. Intramuscular immunization elicited significantly high levels of serum IgG antibodies against both proteins. Antigen-specific IgA and IgG were also induced in saliva, but only the IgA response was significant. Following experimental challenge with E. coli O157:H7, a significant reduction in bacterial shedding was observed in vaccinated calves, compared to control group. These promising results suggest that systemic immunization of cattle with intimin and EspB could be a feasible strategy to reduce EHEC O157:H7 faecal shedding in cattle. [Copyright &y& Elsevier]

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT1O4) or U302. The cluster! isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VI! isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfon- amides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. !n the 19 isolates representative of cluster VH, the bIaTEM1 gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the blaTEM1-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan. [ABSTRACT FROM AUTHOR]

Abstract: This study was conducted to investigate the toxicity of 12 heavy metals (i.e., HM), namely Hg, Cd, Co, Ni, Cr, Cu, Ag, Mo, Zn, Pb, Li and Na on bacterial isolates from the rumen of sheep, cattle and buffaloes. Rumen samples were collected immediately after slaughter during the dry season. Ruminal bacterial isolates, 59, were obtained from two sheep, five cows and nine buffaloes. Sensitivity of ruminal bacterial isolates to each HM was determined by the clearance zone (CZ) using the Kirby-Bauer disc diffusion susceptibility test. Bacterial populations isolated from the rumens of sheep and buffalo had a lower resistance (P<0.001) to HM toxicity than did cattle. Regardless of ruminant species, bacterial isolates revealed a higher tolerance (P<0.001) to Li and Na, whereas Hg and Cd were the most toxic HM for all isolates. We conclude that inhibition of these HM to the isolated microbial population of sheep, cattle and buffalo is ranked: Hg (most toxic)>Cd>Co∼Ag∼Cu∼Cr∼Ni>Mo∼Zn∼Pb>Li∼Na. Further research is required to explain the mechanism of toxicity of these HM, and also to explore the variation among ruminant species. [ABSTRACT FROM AUTHOR]

Shiga toxin producing Escherichia coli (STEC) O26:H11 is an enteric pathogen capable of causing severe hemorrhagic colitis that can lead to hemolytic uremic syndrome. This organism is able to colonize cattle and human intestinal epithelial cells by secreting effectors via a type III secretion system (T3SS). In this investigation, we examined the role of 2 effectors, Tir and NleB, and the structural translocator component EspA in the adherence of STEC to epithelial cells and in the colonization of cattle. Isogenic deletion mutants were constructed and using microscopy and flow cytometry compared to the wild-type strain in their ability to adhere to HEp-2 cells. A competitive assay was also used to measure the capacity of the mutants to colonize the intestinal tract of cattle, where both the mutant and the parental strains were introduced orally at the same time. Genomic DNA was extracted from enriched fecal samples collected at various time points, and quantitative real-time PCR was used to quantify bacteria. A significant reduction in fecal shedding was observed, and adherence to HEp-2 cells was decreased for the tir and espA mutants. Deletion of the nleB gene did not have a significant effect on the adherence of HEp-2 cells; however, in an in vivo model, it strongly reduced the ability of STEC O26:H11 to colonize the bovine intestinal tract. La souche d'Escherichia coli productrice de la toxine Shiga (STEC) O26:H11 est un pathogène entérique qui cause des colites hémorragiques sévères pouvant conduire à un syndrome urémique hémolytique. Cet organisme est capable de coloniser les cellules épithéliales intestinales bovines et humaines par l'intermédiaire du système de sécrétion de type III (T3SS). Dans cette recherche, nous avons examiné le rôle de 2 effecteurs, Tir et NleB, ainsi que de la composante structurelle EspA du translocateur, sur l'adhérence aux cellules épithéliales et la colonisation chez le bovin. Des mutants de délétion isogéniques ont été construits et leur propriété d'adhérence aux cellules HEp-2 a été comparée à celle de la souche sauvage par microscopie et cytométrie de flux. Un essai de compétition a aussi été réalisé pour mesurer la capacité des mutants à coloniser le tractus intestinal du bovin, alors que les souches parentale et mutantes ont été introduites oralement simultanément. L'ADN génomique a été extrait d'échantillons enrichis de fèces récoltés à différentes périodes de temps et une PCR quantitative en temps réel a été utilisée pour quantifier les bactéries. Une réduction significative de libération dans les fèces a été observée chez les mutants tir et espA, et leur adhérence aux cellules HEp-2 était aussi diminuée. La délétion du gène nleB n'a pas eu d'effet significatif sur l'adhérence aux cellules HEp-2; cependant, dans un modèle in vivo, elle réduisait fortement la capacité des cellules STEC O26:H11 à coloniser le tractus intestinal du bovin. [ABSTRACT FROM AUTHOR]

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance. Cette étude visait à examiner la présence de bactéries commensales résistantes aux antibiotiques dans une ferme bovine, représentant les zones fortement touchées par l'agriculture, dans des localités urbaines, représentant des zones touchées par les activités urbaines et indirectement par l'agriculture, et enfin, dans un parc national, représentant une zone non touchée par l'agriculture. Un total de 288 échantillons de sol, de fèces et d'eau a été récolté dans la ferme bovine, dans la ville de Fort Collins, et dans le parc national Rocky Mountain National Park, au Colorado. En plus, 42 nouveaux échantillons de nourriture, de litière non utilisée, de compost et de fumier ont été récoltés dans la ferme bovine. Les populations bactériennes totale, résistante à la tétracycline et résistante au ceftiofur ont été dénombrées par culture standard sur plats de Pétris et par PCR en temps réel. Seuls les échantillons d'eaux usées de la ferme bovine montraient un nombre plus élevé de bactéries résistantes à la tétracycline (dénombré par la culture sur plats) et de copies de gènes de résistance à la tétracycline (quantifié par PCR en temps réel), comparativement aux échantillons d'eau récoltés d'environnements non agricoles. Le gène de résistance au ceftiofur, blaCMY-2 était indétectable dans tous les échantillons, alors que les gènes de résistance à la tétracycline examinés dans cette étude, tet(B), tet(C), tet(W) et tet(O), ont été détectés dans tous les échantillons, exception faite des échantillons de sols du parc national. Les pools de gènes de résistance à la tétracycline quantifiés à partir des gènes tet(O) et tet(W) étaient plus grands que ceux des gènes tet(B) et tet(C) dans les échantillons de fèces et d'eau. Même si, pour la quantification par PCR en temps réel, un nombre limité de gènes de résistance ont été choisis dans cette étude plutôt que la totalité de ces gènes, nos résultats suggèrent que des études plus poussées sont nécessaires pour évaluer les impacts des milieux naturels et urbains sur la résistance aux antibiotiques. [ABSTRACT FROM AUTHOR]

Abstract: Clostridium chauvoei is the causative agent of blackleg in cattle and sheep. The clinical symptoms of this severe disease are very similar to that of malignant edema (Clostridium septicum), infections of other Clostridium species belonging to the gas edema complex, and anthrax (Bacillus anthracis). C. chauvoei and C. septicum are closely related taxa and share many phenotypic properties hampering diagnosis by using traditional microbiological methods. Thus, there is a need for a fast and reliable identification method for specific detection of both species in clinical samples. The multiplex real-time PCR assay presented here is based on the detection of the spo0A gene and enables the simultaneous identification of C. chauvoei and C. septicum. The assay design includes an amplification control DNA template for the recognition of PCR-inhibitors. Assay validation was performed using a collection of 29 C. chauvoei, 38 C. septicum strains and 26 strains of other Clostridium species. Furthermore, the real-time PCR assay was successfully tested on tissue samples from 19 clinical blackleg cases. The assay allowed the reliable detection of one picogram DNA which represents approximate 239 genome equivalents. [Copyright &y& Elsevier]

Shiga-toxigenic Escherichia coli (STEC) 0157 occurrence was determined along the entire gastrointestinal tract (GIT) of each of four naturally shedding cattle and at three sites in 61 slaughter cattle. STEC 0157 was distributed along the entire GIT, though interanimal distribution was variable. Neither feces nor rectoanaljunction samples accurately predicted the STEC 0157-negative status of any particular animal. [ABSTRACT FROM AUTHOR]

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified. [ABSTRACT FROM AUTHOR]

Reducing Escherichia coli O157:H7 in cattle and their manure is critical for reducing the risk for human foodborne and waterborne illness. The objective of this study was to evaluate the effects of soil and pond ash surfaces for feedlot pens on the prevalence, levels, and/or persistence of naturally occurring E. coli O157:H7 and total E. coli in cattle (feces and hides) and manure. Cattle (128 beef heifers) were sorted among 16 pens: 8 surfaced with soil and 8 surfaced with pond ash. The prevalence of E. coli O157:H7 in feces decreased (P < 0.0001) during the study from 57.0% on day 0 to 3.9% on day 84 but did not differ (P ≥ 0.05) between cattle on soil and on pond ash pens at any sampling period. The prevalence of the pathogen on hides and in feedlot surface material (FSM) also decreased (P < 0.0001), with no effect of soil or pond ash surface (P ≥ 0.05). Similarly, levels of E. coli in FSM did not differ (P ≥ 0.05) at any sampling period, and there were no clear trends for survival differences of E. coli O157:H7 or E. coli in FSM between pond ash and soil surfaces, although E. coli populations survived at 5.0 log CFU/g of FSM on the pen surfaces 6 weeks after the cattle were removed. These results indicate that housing cattle on pens surfaced with pond ash versus pens surfaced with soil does not affect E. coli O157:H7 in cattle or their manure. [ABSTRACT FROM AUTHOR]

Thirty-five serum samples and six hygroma fluid samples were collected from sexually mature cattle in one herd with clinical signs of brucellosis (abortion and hygromas) in the Western Region of the Gambia in order to isolate and characterise Brucella species. Information on the sex, age, number of calvings, number of abortions, presence of hygromas, and presence of orchitis was also collected for each animal sampled. Twenty-six (74 per cent) of the serum samples were positive in the rose bengal test and 29 (83 per cent) were positive by indirect ELISA. Three isolates of Brucella, biotyped as Brucella abortus biovar 3, were cultured from six hygroma fluid samples. The multiple locus variable number tandem repeat analysis assay clustered the isolates as B abortus with the same profile for the three isolates, suggesting a common origin of contamination. [ABSTRACT FROM AUTHOR]

Feces from 2,255 cattle (calves, young beef cattle, and culled cows) were collected at slaughter from nine departments across France. Campylobacter was recovered from 16.5% of the 2,255 samples (C. jejuni from 12.8% and C. coli from 3.7%), predominantly from calves. Antimicrobial resistance to six antibiotics of medical and/or veterinary interest was tested with the E-test. Resistance to tetracycline was found in most isolates (52.8% of C. jejuni isolates and 88.1% of C. coli isolates) in contrast to low but consistent resistance to ampicillin and erythromycin. Only two C. coli isolates were resistant to gentamicin. Multiple resistance was frequently detected in C. jejuni and C. coli isolates, and 0.8% (3 of 372) of the isolates were resistant to five of the six antimicrobials. An upward trend in the resistance to quinolones and fluoroquinolones in C. jejuni from calves was found; resistance to nalidixic acid reached 70.4% in 2006 and fluoroquinolone resistance increased from 29.7 to 70.4% during 2002 through 2006. All data were analyzed in parallel using clinical breakpoints or epidemiological cutoff values, and the results overlapped largely, except those for gentamicin. This 5-year survey (2002 through 2006) gives the first overview of the prevalence and antimicrobial resistance of C. jejuni and C. coli in cattle in France and documents to what extent cattle may contribute to the environmental reservoir of Campylobacter in France in the context of recurrent reports on links between human campylobacterioses and livestock. The results underline a notable increase in the resistance to fluoroquinolones in C. jejuni from cattle that may be of significant importance for public health. [ABSTRACT FROM AUTHOR]

Abstract: The bovine rumen undergoes substantial changes in environmental conditions during the animal''s feeding cycle, but the effects of these changes on microbial populations have not been examined systematically. Two dairy cows fed a mixed forage/concentrate ration at 12 h intervals over 4 feeding cycles displayed substantial changes in ruminal pH and volatile fatty acid (VFA) concentrations. Automated ribosomal intergenic spacer analysis (ARISA) of solid- and liquid-associated bacterial populations in samples collected at 2, 4, 6, 9, and 12 h after feeding revealed a high degree of bacterial diversity. A total of 155 different amplicon lengths (ALs) were detected across all 83 samples, and 11–74 detected per sample. A substantial proportion (11%) of the ALs was detected in one cow but not in the other. The proportions of ALs that were detected only in the liquid phase or the solid phase were 13.5% and 1.9%, respectively. Correspondence analysis indicated that bacterial community composition differed between cows and between solid or liquid phases, but overall the solid-associated population displayed less change in composition within and across feeding cycles. The data support the notion that cows fed the same diets can have substantial differences in bacterial community composition, and that the solids-associated (biofilm) communities display greater stability than do associated planktonic communities. [Copyright &y& Elsevier]

We report the complete genome sequences of a buffalo coronavirus (BufCoV HKU26) detected from the faecal samples of two domestic water buffaloes ( Bubalus bubalis ) in Bangladesh. They possessed 98–99% nucleotide identities to bovine coronavirus (BCoV) genomes, supporting BufCoV HKU26 as a member of Betacoronavirus 1. Nevertheless, BufCoV HKU26 possessed distinct accessory proteins between spike and envelope compared to BCoV. Sugar-binding residues in the N-terminal domain of S protein in BCoV are conserved in BufCoV HKU26. [ABSTRACT FROM AUTHOR]

Cattle with high feed efficiencies (designated "efficient") produce less methane gas than those with low feed efficiencies (designated "inefficient"); however, the role of the methane producers in such difference is unknown. This study investigated whether the structures and populations of methanogens in the rumen were associated with differences in cattle feed efficiencies by using culture-independent methods. Two 16S rRNA libraries were constructed using ∼800-bp amplicons generated from pooled total DNA isolated from efficient (n = 29) and inefficient (n = 29) animals. Sequence analysis of up to 490 randomly selected clones from each library showed that the methanogenic composition was variable: less species variation (22 operational taxonomic units [OTUs]) was detected in the rumens of efficient animals, compared to 27 OTUs in inefficient animals. The methanogenic communities in inefficient animals were more diverse than those in efficient ones, as revealed by the diversity indices of 0.84 and 0.42, respectively. Differences at the strain and genotype levels were also observed and found to be associated with feed efficiency in the host. No difference was detected in the total population of methanogens, but the prevalences of Methanosphaera stadtmanae and Methanobrevibacter sp. strain AbM4 were 1.92 (P < 0.05) and 2.26 (P < 0.05) times higher in inefficient animals, while Methanobrevibacter sp. strain AbM4 was reported for the first time to occur in the bovine rumen. Our data indicate that the methanogenic ecology at the species, strain, and/or genotype level in the rumen may play important roles in contributing to the difference in methane gas production between cattle with different feed efficiencies. [ABSTRACT FROM AUTHOR]

The possibility to detect mastitis-causing pathogens based on their volatile metabolites was previously studied. In that study, the mastitis samples were incubated overnight. To minimize the total analysis time, no incubation, or a short incubation, would be preferred. We therefore investigated the effect of the incubation time on the formation of volatile metabolites in mastitis samples. A selection of 6 volatile metabolites with the highest impact on the prediction model for identifying the mastitis-causing pathogen, was compared at different incubation times between 0 and 24 h. Identification of the pathogens was not possible without incubation. The minimum incubation time for detection of most of the 6 metabolites was 4 to 8 h. Although a longer incubation time increased the differences between pathogens, after 8 h all metabolites could be detected and the pathogens could be differentiated. Eight hours was therefore selected as the optimal incubation time. This optimal incubation time was evaluated with a set of 25 mastitis samples, of which 88% were correctly classified after 8 h of incubation. The total analysis time for this method is therefore considerably shorter than current microbiological culturing. [ABSTRACT FROM AUTHOR]

Abstract: Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis. [Copyright &y& Elsevier]

Abstract: A triplex real-time (TRT-PCR) assay was developed to ensure a rapid and reliable detection of Mycobacterium avium subsp. paratuberculosis (Map) in faecal samples and to allow routine detection of Map in farmed livestock and wildlife species. The TRT-PCR assay was designed using IS900, ISMAP02 and f57 molecular targets. Specificity of TRT-PCR was first confirmed on a panel of control mycobacterial Map and non-Map strains and on faecal samples from Map-negative cows (n =35) and from Map-positive cows (n =20). The TRT-PCR assay was compared to direct examination after Ziehl-Neelsen (ZN) staining and to culture on 197 faecal samples collected serially from five calves experimentally exposed to Map over a 3-year period during the sub-clinical phase of the disease. The data showed a good agreement between culture and TRT-PCR (kappa score=0.63), with the TRT-PCR limit of detection of 2.5×102 microorganisms/g of faeces spiked with Map. ZN agreement with TRT-PCR was not good (kappa=0.02). Sequence analysis of IS900 amplicons from three single IS900 positive samples confirmed the true Map positivity of the samples. Highly specific IS900 amplification suggests therefore that each single IS900 positive sample from experimentally exposed animals was a true Map-positive specimen. In this controlled experimental setting, the TRT-PCT was rapid, specific and displayed a very high sensitivity for Map detection in faecal samples compared to conventional methods. [Copyright &y& Elsevier]

Three commercially available Shiga toxin (Stx) enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their ability to detect Stx in pure cultures of Stx-producing Escherichia coli (specificity). The detection limits (sensitivity) of each ELISA kit were also evaluated. Seventy-eight Stx-producing E. coli (STEC) isolates that produced Stx1, Stx2, or Stx1 and Stx2 variants were examined in this study. The specificities of the tests were comparable, and the sensitivities of two of the tests (Premier EHEC and rBiopharm Ridascreen Verotoxin Enzyme Immunoassay) were within the same order of magnitude. The ProSpecT Shiga Toxin E. coli Microplate Assay was approximately 10-fold less sensitive. The inability of all three tests to detect the Stx2d and Stx2e variants indicated that some STEC strains may not be detected by Stx ELISA. The ability of the Premier EHEC ELISA to detect toxin in artificially inoculated bovine fecal samples (following enrichment) indicated that this kit may be used to screen cattle for the presence of Stx as an indicator of the presence of STEC. In particular, such a screening method could be useful during the summer, when the number of STEC-positive animals and the number of STEC that they shed increase. [ABSTRACT FROM AUTHOR]

Acyl-homoserine-lactone autoinducer (AHL) produced by nonenterohemorrhagic Escherichia coli species in cattle appears to be required for enterohemorrhagic E. coli (EHEC) colonization of the gastrointestinal tract (GIT). The current research aimed to examine the effect of season, diet, EHEC shedding, and location within the GIT on AHL prevalence in the ruminant. Luminal content samples were collected from the rumen and rectum of feedlot cattle at slaughter in the spring, summer, fall, and winter for culture of E. coli O157:H7 and AHL determination. During the spring collection, samples were additionally collected from the cecum and small intestine, but these samples all were AHL negative and therefore not examined again. To assess the influence of diet on AHL prevalence, 14 lambs were fed either 100% forage or 80% concentrate diets and experimentally inoculated with EHEC. At 8 days after infection, all the lambs were killed, and necropsies were taken, with luminal contents collected from the GIT. The collections from the feedlot cattle had AHL in 100% of the rumen content samples from the spring, summer, and fall, but not in any of the winter samples. No other GIT samples from feedlot cattle were AHL positive, and all the samples from the sheep study were AHL negative. The cattle seemed to show a weak correlation between ruminal AHL and EHEC prevalence. This research found AHL only in the rumen and not in the lower GIT of feedlot cattle. However, it is unclear whether this is because the pH of the lower gut destroys the AHL or because a lack of certain bacteria in the lower gut producing AHL. [ABSTRACT FROM AUTHOR]

Abstract: Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis. [Copyright &y& Elsevier]

Transportation of cattle to the slaughter plant could influence hide contamination with Salmonella enterica. Fecal and hide samples were obtained from 40 lots of cattle at the feedlot and again at the slaughter plant. Potential risk factors for hide contamination were evaluated. A multilevel Poisson regression model was used to determine whether transportation and lairage were associated with hide contamination by Salmonella. Cattle with hide samples positive for Salmonella at the feedlot had twice the risk of having positive slaughter hide samples compared with cattle without positive feedlot hide samples (relative risk [RR], 1.9). Cattle transported in trailers from which samples positive for Salmonella were collected had twice the risk of having positive slaughter hide samples compared with cattle transported in culture-negative trailers (RR, 2.3). Cattle transported for long distances had twice the risk of having positive hide samples at slaughter compared with cattle transported shorter distances (RR, 2.3). Cattle held in lairage pens contaminated with feces had twice the risk of having positive slaughter hide samples compared with cattle held in clean pens (RR, 1.8). Cattle held off feed longer than 18 h before loading had twice the risk of having positive slaughter hide samples compared with cattle held off feed for shorter times (RR, 1.7). Cattle that were agitated during loading had twice the risk of having positive slaughter hide samples compared with cattle that were calm (RR, 2.2). These findings suggest that variables associated with transportation and lairage can impact the presence of Salmonella on the hides of cattle at slaughter. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7 outbreaks associated with produce consumption have brought attention to livestock manures and manure-based soil amendments as potential sources of pathogens for the contamination of these crops. Procedures for enumeration of E. coli O157:H7 are needed to assess the risks of transmission from these manures and their by-products. A direct plating method employing spiral plating onto CHROMagar O157 was investigated for enumeration of E. coli O157:H7 in feedlot surface material, aged bovine manure, bovine manure compost, and manure-amended soil. In studies utilizing samples spiked with a five-strain cocktail of E. coli O157:H7 at levels ranging from 10² to 105 CFU/g of sample, there were strong correlations between the observed and predicted levels of this pathogen. Although the addition of 2.5 mg/liter potassium tellurite and 5 mg/liter novobiocin made the medium more restrictive, these amendments enhanced the ability to identify and enumerate E. coli O157:H7 in feedlot surface material, which contained a higher proportion of fresh feces than did the other three sample types and therefore higher levels of interfering bacterial microflora. The spiral plating method was further assessed to determine its ability to enumerate E. coli O157:H7 in naturally contaminated feedlot surface material. Comparison of E. coli O157:H7 counts in feedlot surface material obtained by the spiral plating method and a most probable number technique were well correlated. We conclude that direct spiral plating onto CHROMagar O157 is effective for estimating E. coli O157: H7 levels in a variety of manures and manure-containing sample types to a lower detection limit of 200 CFU/g. The method has application for determining E. coli O157:H7 concentrations in manures and composts before their sale and use as soil amendments and for measuring the effectiveness of manure treatment processes to reduce or inactivate this pathogen. [ABSTRACT FROM AUTHOR]

This study was conducted to compare fecal grab (FEC) and rectoanal mucosal swab (RAMS) techniques as sampling methods for surveillance of Escherichia coli O157:H7 in conjunction with administration of a mitigation therapy. The study was nested within a larger experiment that investigated bacteriophage as a preharvest strategy for controlling E. coli O157: H7 in feedlot steers. Samples (FEC and RAMS) were collected from 16 of the 32 feedlot steers (control and oral bacteriophage treatment; n = 8) involved in the mitigation study. All steers had been inoculated on day 0 with 1010 CFU of nalidixic acid-resistant E. coli O157:H7, and samples were collected on 16 occasions over the next 83 days. FEC samples were assessed by direct plating of serial dilutions in PBS, plus a 6-h enrichment and immunomagnetic separation when E. coli O157:H7 concentrations were below limits detectable by direct plating (i.e., <1 log CFU/g). All RAMS samples were assessed by enrichment and immunomagnetic separation. E. coli O157:H7 was detected more frequently (P < 0.01) by FEC than by RAMS. Overall, 213 of 256 samples were positive either by FEC or RAMS. Discrepancies between sampling techniques were observed in 63 of the 213 positive samples; FEC missed 11 samples that were positive by RAMS, and RAMS missed 52 of those positive by FEC (miss rates of 5.16 and 24.41%, respectively). Kappa values (0.36 to 0.45) indicated only fair to moderate agreement between FEC and RAMS results, but this agreement was higher at lower levels of E. coli O157:H7 shedding (later in the experimental period). Selection of sampling procedure could significantly influence the assessed merit during testing of potential strategies for controlling E. coli O157:H7 on the farm. [ABSTRACT FROM AUTHOR]

Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion. [ABSTRACT FROM AUTHOR]

In 2003 in the Netherlands, a new methicillin-resistant Staphylococcus aureus (MRSA) strain emerged that could not be typed with Sma1 pulsed-field gel electrophoresis (NT-MRSA). The association of NT-MRSA in humans with a reservoir in animals was investigated. The frequency of NT-MRSA increased from 0% in 2002 to >21% after intensified surveillance was implemented in July 2006. Geographically, NT-MRSA clustered with pig farming. A case-control study showed that carriers of NT-MRSA were more often pig or cattle farmers (pig farmers odds ratio [OR] 12.2, 95% confidence interval [CI] 3.1-48.6; cattle farmers OR 19.7, 95% CI 2.3-169.5). Molecular typing showed that the NT-MRSA strains belonged to a new clonal complex, ST 398. This study shows that MRSA from an animal reservoir has recently entered the human population and is now responsible for >20% of all MRSA in the Netherlands. [ABSTRACT FROM AUTHOR]

Host association of Campylobacter jejuni was analyzed by using multilocus sequence typing data for 713 isolates from chickens and bovids (cattle and sheep). Commonly used summary measures of genotypes (sequence type and clonal complex) showed poor accuracy, but a method using the full allelic profile showed 80% accuracy in distinguishing isolates from these 2 host groups. We explored the biologic basis of more accurate results with allelic profiles. Strains isolated from specific hosts have imported a substantial number of alleles while circulating in those host species. These results imply that 1) although Campylobacter moves frequently between hosts, most transmission is within species, and 2) lineages can acquire a host signature and potentially adapt to the host through recombination. Assignment using this signature enables improved prediction of source for pathogens that undergo frequent genetic recombination. [ABSTRACT FROM AUTHOR]

The objective of this study was to document temporal trends in bulk tank somatic cell count (SCC) and total bacterial counts (TBC) in Irish dairy herds during the years 1994 to 2004. Three milk processors participated in the study, providing data on 2,754,270 individual bulk tank SCC and 2,056,992 individual bulk tank TBC records from 9,113 herds. Somatic cell counts decreased during the years 1994 to 2000, followed by an annual increase thereafter of more than 2,000 cells/mL. A tendency existed for TBC to decrease over time. Across all years, bulk tank SCC were the lowest in April and highest in November; TBC were the lowest in May and highest in December. The significant seasonal pattern observed in herd SCC and TBC was an artifact of seasonal calving in Ireland. In general, herds selling more milk had lower bulk tank SCC and TBC. Herds having the highest SCC (i.e., >450,000 cells/mL) and the lowest SCC (i.e., ≤150,000 cells/mL) both contributed substantially to the mean SCC of the milk pool collected by the milk processors. Derived transition matrices showed that between adjacent years, herds had the greatest probability of remaining in the same annual mean SCC or TBC category. [ABSTRACT FROM AUTHOR]

Aims: Bovine meat is the principal source of human contamination of attaching and effacing Escherichia coli, including enterohaemorrhagic E. coli O157. The aim was to study the prevalence of these strains on bovine carcasses in Algeria. Methods and Results: Two-hundred and thirty carcasses were swabbed and analysed by classical microbiological methods for total E. coli counts and for the presence of pathogenic E. coli. The E. coli counts were high, with a 75th percentile of 444·75 CFUs cm−2. For pathogenic E. coli, more than 7% of the tested carcasses were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by multiplex PCR. The main isolated pathotype (78%) was eae+ stx2+ ehxA+. In addition to E. coli O157, other attaching and effacing E. coli (AEEC) were also detected from carcasses by colony hybridization after pre-enrichment and plating on sorbitol MacConkey agar using eae, stx1 and stx2 probes. Thirty carcasses (13%) on the 230 analysed harboured at least one colony positive for one of the tested probes. These positive carcasses were different from those positive for E. coli O157. Sixty-six colonies (2·9%) positive by colony hybridization were isolated. The majority (60·6%) of the positive strains harboured an enteropathogenic E. coli-like pathotype ( eae+ stx−). Only three enterohaemorrhagic E. coli (EHEC)-like ( eae+ stx1+) colonies were isolated from the same carcass. These strains did not belong to classical EHEC serotypes. Conclusions: In this study, the global hygiene of the slaughterhouse was low, as indicated by the high level of E. coli count. The prevalence of both E. coli O157 and other AEEC was also high, representing a real hazard for consumers. Significance and Impact of the Study: This is the first study of this type in Algeria, which indicates that the general hygiene of the slaughterhouse must be improved. [ABSTRACT FROM AUTHOR]

The spread of microbial contamination on the hides of beef was investigated at two stages in the meat chain: (i) in a simulated livestock market ("the market") using 33 animals, and (ii) in the unloading-to-skinning area of a commercial abattoir using 18 animals. At both stages, harmless bacterial markers (nalidixic acid—resistant Escherichia coli K-12; rifampicin- and nalidixic acid-resistant Pseudomonas fluorescens; and a tetracycline-resistant E. coli) were inoculated on the hides of a small number of selected animals, and their transfer to other animals and the environment was examined. At the market, the initial prevalence of animals positive for the hide markers (9.1% in each phase) introduced in the presale pen, sale ring, and postsale pen changed to 39.4, 15.1, and 54.5%, respectively, by the end of the market process. In addition, widespread contamination of the market environment with the hide markers was observed. At the abattoir, the initial prevalence of animals positive for the hide marker (11.1%) inoculated at unloading increased to 100% (hide before skinning) and 88.8% (skinned carcass). In addition, another marker inoculated on environmental surfaces in lairage pens, races, and stunning box was detected on 83.3% (hide before skinning) and 88.8% (skinned carcass). These results, although obtained with a relatively small number of animals, demonstrate that both the livestock market process and the unloading-to-skinning process at abattoirs can facilitate the extensive spread of microbial contamination on hides not just within, but also between, batches of animals. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae (group B streptococcus [GBS]) causes invasive human infections and bovine mastitis. This study examined the genetic relationship between bovine and human serotype III GBS by using molecular techniques that classify human serotype III GBS into four distinct phylogenetic lineages. Bovine serotype III GBS were largely contained in two lineages, which are distinct from the two major lineages (restriction digest types III-2 and III-3) that infect human neonates. One of the bovine lineages closely resembles the human III-1 lineage, whose members occasionally cause human neonatal infections. The bovine strains in the other lineage characteristically have an initiation factor IF2 gene (infB) H allele and multilocus sequence types that are not found in human GBS strains. Evidence suggests that this "H allele" lineage is related to the human III-3 lineage. These results support the assertion that human and bovine GBS are largely unrelated and provide further insight into the genetic relation between human and bovine GBS. [ABSTRACT FROM AUTHOR]