Biosecurity-based interventions and strategies to reduce Campylobacter spp. on poultry farms.

The prevention and control of Campylobacter colonization of poultry flocks are important public health strategies for the control of human campylobacteriosis. A critical review of the literature on interventions to control Campylobacter in poultry on farms was undertaken using a systematic approach. Although the focus of the review was on aspects appropriate to the United Kingdom poultry industry, the research reviewed was gathered from worldwide literature. Multiple electronic databases were employed to search the literature, in any language, from 1980 to September 2008. A primary set of 4,316 references was identified and scanned, using specific agreed-upon criteria, to select relevant references related to biosecurity-based interventions. The final library comprised 173 references. Identification of the sources of Campylobacter in poultry flocks was required to inform the development of targeted interventions to disrupt transmission routes. The approach used generally involved risk factor-based surveys related to culture-positive or -negative flocks, usually combined with a structured questionnaire. In addition, some studies, either in combination or independently, undertook intervention trials. Many of these studies were compromised by poor design, sampling, and statistical analysis. The evidence for each potential source and route of transmission on the poultry farm was reviewed critically, and the options for intervention were considered. The review concluded that, in most instances, biosecurity on conventional broiler farms can be enhanced and this should contribute to the reduction of flock colonization. However, complementary, non-biosecurity-based approaches will also be required in the future to maximize the reduction of Campylobacter-positive flocks at the farm level.

Influence of production system on the rate of onset of Campylobacter colonization in chicken flocks reared extensively in the United Kingdom.

1. Because thermophilic Campylobacter spp. are common in chicken flocks reared extensively, cross-sectional and longitudinal studies were carried out on organic and free-range farms to determine the onset of colonisation (lag phase) and likely sources of flock infection. 2. For 14 organic and 14 free range flocks, there was a difference in lag phases, with the former being colonized at a mean of 14·1 d in comparison with 31·6 d for the latter. Whereas most free-range flocks became colonized when released on to pasture, those reared organically were usually colonized at the housed brooding stage. 3. Further study of organic flocks on three farms over 7 successive crop cycles confirmed that colonisation was strongly influenced by the prevailing husbandry conditions and was not a consequence of the length of the rearing period. 4. Molecular epidemiological investigations on a farm showing the shortest lag phase, using PFGE typing with two different restriction enzymes (SmaI and KpnI) and flaA SVR sequence typing, revealed that potential sources of colonisation for organic chickens were already present on the farm at the time of chick placement. Such sources included the ante area of the brooding house, surrounding pasture and other livestock being kept on the farm. 5. Overall, the study demonstrated that, under UK conditions, the prevalence of colonisation was greater in extensive flocks (95-100%) than it was for conventional broilers (55%), similar to the situation in other countries, but all three management systems showed comparable levels of caecal carriage in positive birds (log(10)/g 6·2-6·7).

Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols.

AIMS: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. METHODS AND RESULTS: Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 10(7) -1 × 10(9 ) CFU g(-1) Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 µg ml(-1) ciprofloxacin, MICs of 16 µg ml(-1) and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. CONCLUSIONS: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.

Investigation of the presence and protective effects of maternal antibodies against Campylobacter jejuni in chickens.

The role of maternal antibodies in the lag phase of Campylobacter positivity, widely observed in commercial broiler flocks, was investigated. The results indicate that 3-wk-old birds derived from a commercial flock are more susceptible to colonization with Campylobacter jejuni than 1-to-2-wk-old birds. This increasing susceptibility parallels the loss of maternally derived, circulating, anti-Campylobacter, immunoglobulin Y antibodies as detected by enzyme-linked immunosorbent assay. The role of these antibodies in resistance to colonization was further investigated using progeny from breeder flocks of known Campylobacter status. These results confirmed that maternal antibodies confer partial protection against Campylobacter colonization on young chickens (1-2 wk old). This protection was directed against challenge with both homologous and heterologous strains of C. jejuni and even against strains with a high colonization potential. However, evidence presented indicates that newly hatched chicks, with the highest levels of maternal antibodies, were as susceptible to Campylobacter challenge as 3-wk-old birds. This conundrum was investigated further, and an increase in resistance was detected from 1 to 3 days of age. The reasons for this are, as yet, unknown, but the observation validates the use of newly hatched chicks in models of Campylobacter colonization. Moreover, this high susceptibility in the first few days of life may explain the occasional early flock colonization observed, especially when environmental exposure to Campylobacter is high, for example, in free-range birds.

Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine.

AIMS: A panel of pulsed field gel electrophoresis (PFGE) type variants of Campylobacter jejuni, previously identified as of clonal origin, were investigated to determine whether genomic instability could be observed during competitive growth. METHODS AND RESULTS: Upon recovery from frozen storage, some variants had undergone alterations in PFGE profiles, but subsequent culture produced constant genotypes. Individual variants did not display differences in colonization potential when tested in orally challenged 1-day-old chickens. However, competitive colonization using mixtures of two or three PFGE types generally resulted, by 4 weeks postchallenge, in one predominant PFGE type in all birds. For some variant mixtures, a minor population of novel PFGE types was detected in individual birds. The creation of new variants appeared to be dependent on the extent of competition and of the individual host. Genomic rearrangements most likely explain this increase in genetic diversity, apparently without the involvement of natural transformation or plasmid acquisition. In vitro cultivation of mixed inoculations were again selected for particular variants; but genetic diversity was not generated, suggesting that the selection pressures in vitro differed from those active in vivo. CONCLUSION: These observations support the hypothesis that by generating genetic diversity, C. jejuni can improve its phenotypic fitness to survive and colonize subsequent hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: The consequences of such observations for the development of campylobacter control strategies for poultry may be substantial.

Enterohaemorrhagic and other Shiga toxin-producing Escherichia coli (STEC): Where are we now regarding diagnostics and control strategies?

Escherichia coli comprises a highly diverse group of Gram-negative bacteria and is a common member of the intestinal microflora of humans and animals. Generally, such colonization is asymptomatic; however, some E. coli strains have evolved to become pathogenic and thus cause clinical disease in susceptible hosts. One pathotype, the Shiga toxigenic E. coli (STEC) comprising strains expressing a Shiga-like toxin is an important foodborne pathogen. A subset of STEC are the enterohaemorrhagic E. coli (EHEC), which can cause serious human disease, including haemolytic uraemic syndrome (HUS). The diagnosis of EHEC infections and the surveillance of STEC in the food chain and the environment require accurate, cost-effective and timely tests. In this review, we describe and evaluate tests now in routine use, as well as upcoming test technologies for pathogen detection, including loop-mediated isothermal amplification (LAMP) and whole-genome sequencing (WGS). We have considered the need for improved diagnostic tools in current strategies for the control and prevention of these pathogens in humans, the food chain and the environment. We conclude that although significant progress has been made, STEC still remains an important zoonotic issue worldwide. Substantial reductions in the public health burden due to this infection will require a multipronged approach, including ongoing surveillance with high-resolution diagnostic techniques currently being developed and integrated into the routine investigations of public health laboratories. However, additional research requirements may be needed before such high-resolution diagnostic tools can be used to enable the development of appropriate interventions, such as vaccines and decontamination strategies.

Crossing the boundaries.

In the past, research on zoonotic diseases has been fragmented. Teresa Belcher and Diane Newell discuss the formation of Med-Vet-Net, a "virtual institute", which aims to integrate activity between researchers in human and veterinary medicine across the European Union.

Serum anti-Helicobacter pylori IgG antibodies and pepsinogens A and C as serological markers of chronic atrophic gastritis.

This study was designed to test the sensitivity and specificity of serum anti-Helicobacter pylori IgG antibodies and the ratio of serum pepsinogen A to pepsinogen C (PGA:PGC) in detecting chronic atrophic gastritis (CAG) and intestinal metaplasia. Parallel gastric biopsies and a serum sample were collected from a series of 87 patients aged 20-69 years attending a routine upper endoscopy clinic. The seroprevalence (> 10 micrograms IgG/ml) of anti-H. pylori antibodies was 42.7%, and of a low PGA:PGC ratio (< 1.5) was 17.7%. A positive H. pylori IgG antibody level was more sensitive than the level of PGA:PGC in diagnosing CAG (71.4% and 25.0%, respectively), moderate CAG (86.7% and 26.7%, respectively), and intestinal metaplasia (90.9% and 50.0%, respectively). Anti-H. pylori IgG antibody levels were less specific than PGA:PGC levels in diagnosing CAG (90.9% and 93.9%, respectively), moderate CAG (78.3% and 89.1%, respectively), and intestinal metaplasia (72.6% and 92.2%, respectively). A combination of anti-H. pylori antibodies and a low PGA:PGC ratio for the detection of CAG resulted in a specificity of 100%, but the sensitivity was 21.4%.

Production and characterization of monoclonal antibodies directed against guinea pig IgG subclasses.

Monoclonal antibodies directed against guinea pig IgG subclass-specific epitopes have been generated and characterized. Three monoclonal antibodies designated GP1, GP2 and GP3 have been compared to rabbit polyclonal reagents for specificity against purified IgG subclass antigens and for the detection of herpes simplex-specific IgG subclasses, from infected guinea pig, by an indirect ELISA. The monoclonal antibodies were shown to have greater specificity than the corresponding polyclonal reagents.

The intracellular localisation of immunoglobulin in human lymphoid cells and haematopoietic cell lines by immunoperoxidase electron microscopy.

A technique is described for the ultrastructural localisation of intracellular immunoglobulin (Ig) in human lymphoid cell suspensions by the immunoperoxidase method. The technique involves restricted saponin digestion of glutaraldehyde prefixed cells to enhance conjugate penetration. With this method of staining Ig was located in the rough endoplasmic reticulum, perinuclear space and Golgi apparatus in human lymphoid cells from a variety of sources, which is consistent with previously published observations using other ultrastructural techniques. In contrast, diffuse intracytoplasmic staining was predominant in cells prefixed with glutaraldehyde but not treated with saponin. These differences in patterns are discussed in terms of membrane permeability. Although saponin treatment was necessary for consistent localisation of intracellular Ig it resulted in unavoidable loss of Ig from the surface of the cells.

Estimation of prevalence of Helicobacter pylori infection in an asymptomatic elderly population comparing [14C] urea breath test and serology.

A non-invasive serological assay devised in this laboratory had a sensitivity and specificity of 100% as determined by culture and confirmed by histology in a group of 47 patients who had undergone endoscopy. The correlation between serology and the non-invasive [14C] breath test was very good. Only one of 24 culture positive patients was, while all 23 culture negative patients were, breath test negative. In a group of 46 healthy elderly persons, however, significant anomalies between serology and breath test were observed. Only 83% of the breath test negative persons were seronegative, while only 68% of the breath test positive persons were seropositive. These results can be explained in terms of age related atrophic gastritis and immune incompetence, causing reduced colonisation and decreased antibody production, respectively. These investigations suggest that non-invasive tests for H pylori infection may not be reliable in the elderly.

The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy.

Fast protein liquid chromatography and SDS-PAGE have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH.

Antigenic conservation of the ureases of spiral- and helical-shaped bacteria colonising the stomachs of man and animals.

A monoclonal antibody, CP11, has been produced which is directed against the ureas of Campylobacter pylori. This antibody has been used to look for antigenic cross-reactivity, in other ureolytic and non-ureolytic campylobacters, by immunohistological techniques. It has also been used to investigate the helical-shaped organisms found in the stomach of the human, monkey and cat (CS1) and the ileum of the rat (ST1). Interestingly the antibody cross-reacted with the gastric helical organisms from the human, monkey and cat but not with the rat helical organism. No cross-reactivity was observed with C. mustelae or the other ureolytic campylobacters, C. nitrofigilis and the urease positive thermophilic campylobacters. These results are discussed in relation to the phylotaxonomy of these organisms.

Isolation of a gastric campylobacter-like organism from the stomach of four rhesus monkeys, and identification as Campylobacter pylori.

Campylobacter-like organisms, isolated from the gastric antrum of Rhesus monkeys, were compared with Campylobacter jejuni and C. pylori. They were similar to C. pylori by light microscopy, in ultrastructural morphology, in enzymic, fatty-acid-methyl-ester, and protein-profile analysis, and in antigenic reactivity with rabbit antisera to C. jejuni and C. pylori and with C. pylori-specific monoclonal antibody. Because this natural infection of the Rhesus monkey is associated with chronic gastritis, resembling the disease in humans colonised with C. pylori, we recommend the animal as a model for the investigation of human gastritis.

Helicobacter pylori transmission: evidence from a comparison with hepatitis A virus.

OBJECTIVE: To assess the possibility that faecal-oral contact might play a role in the transmission of Helicobacter pylori. DESIGN: A cross-sectional comparison of the patterns of hepatitis A and H. pylori seropositivity. METHODS: At interview, blood samples and questionnaire data were collected from a group of 467 male volunteers, aged 18-65, from Stoke-on-Trent, UK. Serum samples from each subject were then analysed for anti-H. pylori and anti-hepatitis A antibodies. RESULTS: Overall, 100 of 175 H. pylori seropositive subjects (57.1%) and 113 of 292 H. pylori seronegative subjects (38.7%) were hepatitis A seropositive (chi 2 = 15.0, P < 0.001). This difference was not statistically significant after adjustment for age group and father's occupation, as a surrogate for socioeconomic status in childhood (P = 0.15). The seroprevalence of hepatitis A increased with age at a rate of 2.3% per year, compared to only 1.0% per year for H. pylori (P = 0.015). CONCLUSION: These data suggest that the case for faecal-oral transmission of H. pylori, in a manner similar to the spread of hepatitis A, is not proven and that other modes of transmission, for instance through oral-oral contact, should also be considered.

Production and characterization of a monoclonal badger anti-immunoglobulin G and its use in defining the specificity of Mycobacterium bovis infection in badgers by western blot.

A mouse monoclonal anti-badger IgG antibody was produced to investigate the specificity of the antibody response of badgers infected with Mycobacterium bovis. The monoclonal antibody generated was directed against badger IgG heavy chain and appeared to be species restricted, reacting only with badger and dog IgGs but not cat, rabbit, mouse, guinea pig, bovine or ferret IgGs. This monoclonal antibody detection system functioned well in both ELISA and Western blot analyses and was successfully used to investigate the humoral response of the badger to M. bovis infection. Sera from infected badgers detected a 25 kDa antigen which was not detected by sera from M. bovis culture-negative animals. This antigen was conserved in all field strains of M. bovis tested and seroconversion to it was detected during experimental infection. The immunodominance of this antigen in the badger during infection with M. bovis suggests that this 25 kDa polypeptide is a suitable candidate on which to base an antibody detection test for M. bovis infection.

Genotyping Campylobacter jejuni strains isolated from the gut and oviduct of laying hens.

Campylobacter jejuni frequently colonizes the avian intestine. Recent evidence suggests that this organism can also colonize the oviduct of laying hens. However, the source and role of this colonization are unknown. Isolates from the ceca, cloacae, and oviducts of 11 laying hens in three intensive egg-producing flocks were genotyped by Fla typing with the restriction fragment length polymorphism of the polymerase chain reaction product of the flaA and flaB genes (fla typing) and pulsed-field gel electrophoresis (PFGE). A diversity in fla types and PFGE types was observed within and between flocks. Individual birds could be colonized by different genotypes at various intestinal and oviduct sites. However, the oviduct of individual birds appeared to be colonized by only one genotype at the time of sampling. In two birds, matching isolates investigated from the intestinal and reproductive tracts were genotypically identical but different from those oviduct isolates found in other birds in the same flock. Interestingly, not all cecal isolates appeared to be equally able to colonize the oviduct. These results suggest that oviduct colonization may result from ascending infection via the cloaca and that some strains of C. jejuni may be better adapted than others to oviduct colonization.

Sequential spread of Campylobacter infection in a multipen broiler house.

Generally, colonization with Campylobacter jejuni is first detected in broilers 2-3 wk after hatching. Once introduced into a flock, this infection spreads very rapidly. The sources and routes of transmission of C. jejuni in broilers remain debatable. In this study, the spread of infection was monitored in a commercial multipen broiler house in which birds were contained in discrete groups and sampled sequentially. Colonization was monitored in two broiler flocks up to slaughter. Serotyping and fla typing methods were applied to differentiate all the C. jejuni strains isolated. In flock 1, colonization was first detected at 32 days of age in birds located at the rear of the house. By 40 days, nearly all the birds were infected with the same strain (fla type 1.9). However, at 46 days of age, a second strain (fla type 3.7) was detected in some of the birds. These birds were also located toward the rear of the house. In flock 2, infection was detected at 5 wk of age. This infection was once again first detected in birds located at the rear of the house. In this flock, only a single fla type (1.1) was isolated throughout. A survey of the broiler house relative to the location of first point of infection indicated the use of an entrance door unprotected by boot dips. However, securing this door during the second flock study did not prevent infection.

Isotype, specificity, and kinetics of systemic and mucosal antibodies to Campylobacter jejuni antigens, including flagellin, during experimental oral infections of chickens.

The immune response of chickens to Campylobacter jejuni infection was studied as a step in the search for vaccine candidates. One-day-old chicks orally challenged with C. jejuni strain 81116 showed significant increases in specific IgG, IgA, and IgM circulating antibodies, as detected by enzyme-linked immunosorbent assay (ELISA). These levels peaked at 9, 5, and 7 weeks postinfection, respectively. Maternal IgG antibodies were also detected over the first 2 weeks. Specific mucosal IgG and IgA antibody levels also increased significantly. All of the birds demonstrated a major response to the 62-kDa flagellin protein by Western blotting techniques. The immunodominance of flagellin was confirmed by ELISA using an antigen preparation from an aflagellate mutant. When overlapping recombinant polypeptide fragments of flagellin were used, epitopes detected by chicken antibodies were observed in region IV, between residues 95-340 of the protein. Thus flagellin may be suitable candidate for a vaccine, although its role in protection must first be established.

The carry-over of Campylobacter isolates between sequential poultry flocks.

The carry-over of Campylobacter strains from one flock to a subsequent flock in the same broiler house has been studied using molecular epidemiological techniques. In all, 524 Campylobacter strains, isolated from two sequential broiler flocks from 60 broiler houses, were typed by restriction fragment polymorphism of the polymerase chain reaction (PCR) product of the flaA and flaB genes (fla typing). Selected strains were also typed using pulsed field gel electrophoresis (PFGE). By fla typing, 15 (21%) of the 60 houses with Campylobacter-positive sequential flocks had identical genotypes. In 10 (16% overall) of these houses the strains were also identical by PFGE. The difference in PFGE patterns in the strains from the three remaining houses may be indicative of genetic instability. Overall, these results suggest that carry-over from one flock to a subsequent flock in the same house is a relatively infrequent event and, therefore, that routine broiler house cleansing and/or disinfection is largely adequate to eliminate Campylobacter contamination. An alternative explanation of the low level carry-over is a persistent source or reservoir, external to the environment of the broiler houses.