The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

Aims: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracyclineresistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. Methods and Results: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l(-1) and a 10% increase in E. coli with an MIC > 50 mg l(-1) during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 mug g(-1) faeces during treatment. Conclusion: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. Significance and Impact of the Study: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.

Effect of a 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in the pig

Objectives: There are concerns that the use of enrofloxacin in livestock production may contribute to the development of fluoroquinolone resistance in zoonotic bacteria. The objective of our study was to investigate the effect of a single 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in a pig model. Results: Our results showed that a single treatment failed to eradicate S. Typhimurium DT104, which continued to be isolated up to 35 days after treatment. We also provide evidence that treatment positively selects for S. Typhimurium DT104 strains that are already nalidixic acid resistant (gyrA Asn-87) or cyclohexane resistant, the latter being indicative of an up-regulated efflux pump. Emergence of fluoroquinolone resistance was not detected during treatment or post-treatment in any of the Salmonella strains monitored. However, the effect of enrofloxacin on the nalidixic acid-resistant and cyclohexane-resistant S. Typhimurium DT104 outlasted the current withdrawal time of 10 days for Baytril (commercial veterinary formulation of enrofloxacin). Conclusions: In conclusion, our study has provided direct evidence that enrofloxacin-treated pigs could be entering abattoirs with higher numbers of quinolone-resistant zoonotic bacteria than untreated pigs, increasing the risk of these entering the food chain.

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig

Aim: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. Methods and Results: Pigs ( treated with avilamycin for 3 months and controls) were challenged with multiresistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter ( before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin- resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. Conclusion: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. Significance and Impact of the Study: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH2) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

Farmers’ attitudes to disease risk management in England: a comparative analysis of sheep and pig farmers

The UK Department for Environment, Food and Rural Affairs (Defra) identified practices to reduce the risk of animal disease outbreaks. We report on the response of sheep and pig farmers in England to promotion of these practices. A conceptual framework was established from research on factors influencing adoption of animal health practices, linking knowledge, attitudes, social influences and perceived constraints to the implementation of specific practices. Qualitative data were collected from nine sheep and six pig enterprises in 2011. Thematic analysis explored attitudes and responses to the proposed practices, and factors influencing the likelihood of implementation. Most feel they are doing all they can reasonably do to minimise disease risk and that practices not being implemented are either not relevant or ineffective. There is little awareness and concern about risk from unseen threats. Pig farmers place more emphasis than sheep farmers on controlling wildlife, staff and visitor management and staff training. The main factors that influence livestock farmers’ decision on whether or not to implement a specific disease risk measure are: attitudes to, and perceptions of, disease risk; attitudes towards the specific measure and its efficacy; characteristics of the enterprise which they perceive as making a measure impractical; previous experience of a disease or of the measure; and the credibility of information and advice. Great importance is placed on access to authoritative information with most seeing vets as the prime source to interpret generic advice from national bodies in the local context. Uptake of disease risk measures could be increased by: improved risk communication through the farming press and vets to encourage farmers to recognise hidden threats; dissemination of credible early warning information to sharpen farmers’ assessment of risk; and targeted information through training events, farming press, vets and other advisers, and farmer groups, tailored to the different categories of livestock farmer.

Weaning diet induces sustained metabolic phenotype shift in the pig and influences host response to Bifidobacterium lactis NCC2818

Background: The process of weaning causes a major shift in intestinal microbiota and is a critical period for developing appropriate immune responses in young mammals. Objective: To use a new systems approach to provide an overview of host metabolism and the developing immune system in response to nutritional intervention around the weaning period. Design: Piglets (n¼14) were weaned onto either an eggbased or soya-based diet at 3 weeks until 7 weeks, when all piglets were switched onto a fish-based diet. Half the animals on each weaning diet received Bifidobacterium lactis NCC2818 supplementation from weaning onwards. Immunoglobulin production from immunologically relevant intestinal sites was quantified and the urinary 1H NMR metabolic profile was obtained from each animal at post mortem (11 weeks). Results: Different weaning diets induced divergent and sustained shifts in the metabolic phenotype, which resulted in the alteration of urinary gut microbial co-metabolites, even after 4 weeks of dietary standardisation. B lactis NCC2818 supplementation affected the systemic metabolism of the different weaning diet groups over and above the effects of diet. Additionally, production of gut mucosa-associated IgA and IgM was found to depend upon the weaning diet and on B lactis NCC2818 supplementation. Conclusion: The correlation of urinary 1H NMR metabolic profile with mucosal immunoglobulin production was demonstrated, thus confirming the value of this multiplatform approach in uncovering non-invasive biomarkers of immunity. This has clear potential for translation into human healthcare with the development of urine testing as a means of assessing mucosal immune status. This might lead to early diagnosis of intestinal dysbiosis and with subsequent intervention, arrest disease development. This system enhances our overall understanding of pathologies under supra-organismal control.

The evolutionary basis for differences between the immune systems of man, mouse, pig and ruminants

Studying the pathogenesis of an infectious disease like colibacillosis requires an understanding of the responses of target hosts to the organism both as a pathogen and as a commensal. The mucosal immune system constitutes the primary line of defence against luminal micro-organisms. The immunoglobulin-superfamily-based adaptive immune system evolved in the earliest jawed vertebrates, and the adaptive and innate immune system of humans, mice, pigs and ruminants co-evolved in common ancestors for approximately 300 million years. The divergence occurred only 100 mya and, as a consequence, most of the fundamental immunological mechanisms are very similar. However, since pressure on the immune system comes from rapidly evolving pathogens, immune systems must also evolve rapidly to maintain the ability of the host to survive and reproduce. As a consequence, there are a number of areas of detail where mammalian immune systems have diverged markedly from each other, such that results obtained in one species are not always immediately transferable to another. Thus, animal models of specific diseases need to be selected carefully, and the results interpreted with caution. Selection is made simpler where specific host species like cattle and pigs can be both target species and reservoirs for human disease, as in infections with Escherichia coli.

A metabolic system-wide characterisation of the pig: a model for human physiology

The pig is a single-stomached omnivorous mammal and is an important model of human disease and nutrition. As such, it is necessary to establish a metabolic framework from which pathology-based variation can be compared. Here, a combination of one and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy (NMR) and high-resolution magic angle spinning (HR-MAS) NMR was used to provide a systems overview of porcine metabolism via characterisation of the urine, serum, liver and kidney metabolomes. The metabolites observed in each of these biological compartments were found to be qualitatively comparable to the metabolic signature of the same biological matrices in humans and rodents. The data were modelled using a combination of principal components analysis and Venn diagram mapping. Urine represented the most metabolically distinct biological compartment studied, with a relatively greater number of NMR detectable metabolites present, many of which are implicated in gut-microbial co-metabolic processes. The major interspecies differences observed were in the phase II conjugation of extra-genomic metabolites; the pig was observed to conjugate p-cresol, a gut microbial metabolite of tyrosine, with glucuronide rather than sulfate as seen in man. These observations are important to note when considering the translatability of experimental data derived from porcine models.

Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

Sequence analysis of a CTX-M-1 IncI1 plasmid found in Salmonella enterica 4,5,12,i:-, Escherichia coli and Klebsiella pneumoniae on a single UK pig farm

OBJECTIVES: In 2009, CTX-M Enterobacteriaceae and Salmonella isolates were recovered from a UK pig farm, prompting studies into the dissemination of the resistance and to establish any relationships between the isolates. METHODS: PFGE was used to elucidate clonal relationships between isolates whilst plasmid profiling, restriction analysis, sequencing and PCR were used to characterize the CTX-M-harbouring plasmids. RESULTS: Escherichia coli, Klebsiella pneumoniae and Salmonella 4,5,12:i:- and Bovismorbificans resistant to cefotaxime (n = 65) were recovered and 63 were shown by PCR to harbour a group 1 CTX-M gene. The harbouring hosts were diverse, but the group 1 CTX-M plasmids were common. Three sequenced CTX-M plasmids from E. coli, K. pneumoniae and Salmonella enterica serotype 4,5,12:i:- were identical except for seven mutations and highly similar to IncI1 plasmid ColIb-P9. Two antimicrobial resistance regions were identified: one inserted upstream of yacABC harbouring ISCR2 transposases, sul2 and floR; and the other inserted within shfB of the pilV shufflon harbouring the ISEcp1 transposase followed by blaCTX-M-1. CONCLUSIONS: These data suggest that an ST108 IncI1 plasmid encoding a blaCTX-M-1 gene had disseminated across multiple genera on this farm, an example of horizontal gene transfer of the blaCTX-M-1 gene.

The reactions of specific neuron types to intestinal ischemia in the guinea pig enteric nervous system

Damage following ischemia and reperfusion (I/R) is common in the intestine and can be caused during abdominal surgery, in several disease states and following intestinal transplantation. Most studies have concentrated on damage to the mucosa, although published evidence also points to effects on neurons. Moreover, alterations of neuronally controlled functions of the intestine persist after I/R. The present study was designed to investigate the time course of damage to neurons and the selectivity of the effect of I/R damage for specific types of enteric neurons. A branch of the superior mesenteric artery supplying the distal ileum of anesthetised guinea pigs was occluded for 1 h and the animals were allowed to recover for 2 h to 4 weeks before tissue was taken for the immunohistochemical localization of markers of specific neuron types in tissues from sham and I/R animals. The dendrites of neurons with nitric oxide synthase (NOS) immunoreactivity, which are inhibitory motor neurons and interneurons, were distorted and swollen by 24 h after I/R and remained enlarged up to 28 days. The total neuron profile areas (cell body plus dendrites) increased by 25%, but the sizes of cell bodies did not change significantly. Neurons of type II morphology (intrinsic primary afferent neurons), revealed by NeuN immunoreactivity, were transiently reduced in cell size, at 24 h and 7 days. These neurons also showed signs of minor cell surface blebbing. Calretinin neurons, many of which are excitatory motor neurons, were unaffected. Thus, this study revealed a selective damage to NOS neurons that was observed at 24 h and persisted up to 4 weeks, without a significant change in the relative numbers of NOS neurons.

Fatores que contribuem para a manutenção da dor em pacientes com síndrome miofascial : um estudo observacional

A Sindrome Miofascial constitui, dentre os quadros de dor crônica, o que leva um número expressivo de pacientes a buscar atendimento médico nos Serviços de Tratamento de Dor. Caracteriza-se pela presença de pontos dolorosos localizados na musculatura, chamados de “pontos de gatilho”, e de espasmo muscular, podendo ocorrer limitação funcional e disfunção do sistema vegetativo (Sola & Bonica, 2001). A dor e a limitação funcional decorrentes da Sindrome Miofascial constituem atualmente uma das grandes causas de falta ou afastamento do trabalho, o que acarreta graves problemas econômicos e sociais (Roth et al, 1998; Sola & Bonica, 2001). Seu tratamento pode ser demorado e requer participação ativa por parte do paciente. O índice de melhora dos pacientes portadores de Síndrome Miofascial parece sofrer influência de diversos fatores tais como sintomas depressivos, ansiedade, ganho com os sintomas, etc. Os objetivos deste trabalho foram estabelecer a incidência de sucesso e insucesso da terapêutica proposta e verificar qual a relação daqueles fatores com o desfecho clínico estabelecido. Efetuou-se um estudo observacional. O delineamento experimental realizado foi um estudo de incidência. Estudaram-se 62 pacientes adultos (mais de 18 anos), de ambos os sexos, com diagnóstico de Síndrome Miofascial, que procuraram atendimento no Serviço de Tratamento de Dor e Medicina Paliativa do HCPA. Os instrumentos de aferição utilizados foram a Escala Análoga Visual de Dor (VAS), Escala de Ansiedade Traço- Estado (IDATE), Escala para Depressão de Montegomery-Äsberg, Self-Reporting Questionnaire (OMS), Questionário sobre Expectativa de Futuro e questionário estruturado. Os testes psicológicos e de avaliação da dor foram aplicados em dois momentos, na primeira consulta e ao final do tratamento proposto. Foi considerado como desfecho clínico o insucesso terapêutico, avaliado ao final do estudo. Observaram-se incidência de 71% e 29%, respectivamente, para sucesso e insucesso terapêutico. Os pacientes que estavam afastados do trabalho apresentaram aproximadamente 9 vezes mais chances de insucesso terapêutico. Aqueles que obtiveram ganho com os sintomas apresentaram em torno de 7 vezes mais chances de manutenção da dor ao final do tratamento. Maior ansiedade-estado (razão de chances ou RC = 3,4), expectativa negativa de futuro (RC = 22), sintomas depressivos moderados a intensos (RC =4,5) e presença de distúrbios psiquiátricos menores (RC = 3,6) associaram-se com maiores chances de insucesso terapêutico. Características demográficas, familiares, de ocupação e clínicas não se associaram ao desfecho clínico avaliado. Dos pacientes analisados, 29% permaneceram sem alívio da dor, com dificuldades para dormir e afastados de suas atividades, caracterizando o insucesso terapêutico. Observou-se a associação entre insucesso e presença de distúrbios psiquiátricos menores, sintomas depressivos moderados a intensos, maior estado de ansiedade, ganho com os sintomas e afastamento do trabalho. A abordagem multidisciplinar destes pacientes se faz necessária para elevar os índices de sucesso terapêutico no tratamento da SMF.