Repeated disinfectant use in broiler houses and pig nursery units does not affect disinfectant and antibiotic susceptibility in Escherichia coli field isolates.

BACKGROUND: Disinfectants are frequently used in animal production to reduce or eliminate the load of infectious agents and parasites in buildings and equipment associated with the housing or transportation of animals. There are growing concerns that the use of disinfectants would select for resistance to antibiotics and disinfectants. The aim of this study was to determine the effect of repeated use of different disinfectants on the disinfectant and antibiotic susceptibility under practical conditions in a broiler and pig pilot farm. Therefore, the susceptibility of Escherichia coli (E. coli) to 14 antibiotics and 4 disinfectants was monitored over a one-year period. RESULTS: High (20-50%) to very high (> 50%) resistance levels for ampicillin, sulfamethoxazole, trimethoprim and tetracycline were observed in both animal production types. Disinfectant susceptibility did not change over time and did not depend on the used disinfection product. Compared to in-use concentrations of formaldehyde, benzalkoniumchloride and a peracetic acid - hydrogen peroxide formulation, all E. coli strains remained susceptible indicating that the use of disinfectants did not select for disinfectant resistance. Moreover, no association could be found between the use of disinfectants and antibiotic resistance. CONCLUSIONS: These findings suggest that repeated use of disinfectants in agricultural environments does not select for antibiotic resistance nor does it reduce disinfectant susceptibility.

Effect of a pre-milking teat foam and a liner disinfectant on the presence of mesophilic and (proteolytic) psychrotrophic bacteria prior to milking.

Contamination of raw milk by psychrotrophs can lead to the production of heat-resistant proteases and subsequent spoilage of UHT milk. Therefore, this research communication evaluated the effect of a pre-milking teat disinfectant (active components: L-(+)-lactic acid and salicylic acid) and a liner disinfectant (active components: peracetic acid and hydrogen peroxide) on the number of mesophilic and (proteolytic) psychrotrophic bacteria prior to milking. The teat orifices of 10 cows were sampled using a swabbing procedure before and after treatment with a pre-milking teat disinfectant on six subsequent days. On the teat orifices, there was a small but statistically significant decrease in the psychrotrophic bacterial counts between pre and post dipping. No differences were observed for the mesophilic bacterial counts and proteolytic active counts. Liners were also sampled using swabs pre and post disinfection. No statistically significant decrease in the bacterial counts was observed post liner disinfection, although there was a numerical decrease. Sixty-two percent of the proteolytic psychrotrophs were pseudomonads: 16.5% of which were P. fragi, 14.3% P. lundensis, 10.0% P. fluorescens and 2.9% P. putida. Trinitrobenzenesulfonic acid (TNBS) analysis revealed a wide variety in proteolytic activity (from 0 to 55 µmol glycine/ml milk) and the presence of high producers. It can be concluded that there was only a minor effect of teat and liner disinfection on the psychrotrophic bacterial counts indicating that the measures presented did not result in a reduction of the targeted bacteria on teat orifices and liners.

Exposure of ciprofloxacin-resistant Escherichia coli broiler isolates to subinhibitory concentrations of a quaternary ammonium compound does not increase antibiotic resistance gene transfer.

Resistance to antibiotics threatens to become a worldwide health problem. An important attributing phenomenon in this context is that pathogens can acquire antibiotic resistance genes through conjugative transfer of plasmids. To prevent bacterial infections in agricultural settings, the use of veterinary hygiene products, such as disinfectants, has gained popularity and questions have been raised about their contribution to such spreading of antibiotic resistance. Therefore, this study investigated the effect of subinhibitory concentrations of benzalkoniumchloride (BKC), a quaternary ammonium compound (QAC), on the conjugative transfer of antibiotic resistance genes. Five Escherichia coli field strains originating from broiler chickens and with known transferable plasmid-mediated ciprofloxacin resistance were exposed to subinhibitory BKC concentrations: 1/3, 1/10 and 1/30 of the minimum bactericidal concentration. Antibiotic resistance transfer was assessed by liquid mating for 4 h at 25°C using E. coli K12 MG1655 as recipient strain. The transfer ratio was calculated as the number of transconjugants divided by the number of recipients. Without exposure to BKC, the strains showed a ciprofloxacin resistance transfer ratio ranging from 10-4 to 10-7. No significant effect of exposure to subinhibitory concentrations of BKC was observed on this transfer ratio.

Evaluation of the hygienogram scores and related data obtained after cleaning and disinfection of poultry houses in Flanders during the period 2007 to 2014.

Cleaning and disinfection (C&D) of poultry houses is an essential aspect in farm hygiene management. Adequate performance of the different steps of a C&D protocol and the use of suitable products are key to prevent and control zoonoses and animal diseases. Hygiene monitoring on total aerobic flora through sampling with agar contact plates at different locations of the poultry house results in a hygienogram score that is used to evaluate the proper execution of C&D.This study analyzed the hygienogram scores of 19,739 poultry flocks in Flanders after C&D. Data relating to the C&D protocol, i.e., year, season, husbandry system, production type, cleaning product, sampler, active components of the disinfectant, disinfection time, disinfection temperature, and disinfection responsible, were collected.The average hygienogram score decreased significantly over time, suggesting a general improvement between 2007 and 2014. Differences in scores were found among the husbandry systems, with the barn/aviary system having a significantly better hygienogram score compared to the floor house, furnished cage, and battery. Significantly better scores also were found when a cleaning product was used in the C&D protocol. Disinfection with a peracetic acid and hydrogen peroxide combination or formaldehyde gave the best scores. In addition, C&D protocols using ≥2 different disinfectants showed improved results compared to the use of one single disinfectant. Finally, disinfection applied by a specialist contractor resulted in a better score compared to disinfection by the farmer.In conclusion, analysis of the hygienogram scores and related data allowed identifying several factors, resulting in an improvement, which may reduce the total bacterial load in poultry stables and, consequently, the number of zoonotic and pathogenic micro-organisms.

Farmers' preferences for automatic lameness-detection systems in dairy cattle.

As lameness is a major health problem in dairy herds, a lot of attention goes to the development of automated lameness-detection systems. Few systems have made it to the market, as most are currently still in development. To get these systems ready for practice, developers need to define which system characteristics are important for the farmers as end users. In this study, farmers' preferences for the different characteristics of proposed lameness-detection systems were investigated. In addition, the influence of sociodemographic and farm characteristics on farmers' preferences was assessed. The third aim was to find out if preferences change after the farmer receives extra information on lameness and its consequences. Therefore, a discrete choice experiment was designed with 3 alternative lameness-detection systems: a system attached to the cow, a walkover system, and a camera system. Each system was defined by 4 characteristics: the percentage missed lame cows, the percentage false alarms, the system cost, and the ability to indicate which leg is lame. The choice experiment was embedded in an online survey. After answering general questions and choosing their preferred option in 4 choice sets, extra information on lameness was provided. Consecutively, farmers were shown a second block of 4 choice sets. Results from 135 responses showed that farmers' preferences were influenced by the 4 system characteristics. The importance a farmer attaches to lameness, the interval between calving and first insemination, and the presence of an estrus-detection system contributed significantly to the value a farmer attaches to lameness-detection systems. Farmers who already use an estrus detection system were more willing to use automatic detection systems instead of visual lameness detection. Similarly, farmers who achieve shorter intervals between calving and first insemination and farmers who find lameness highly important had a higher tendency to choose for automatic lameness detection. A sensor attached to the cow was preferred, followed by a walkover system and a camera system. In general, visual lameness detection was preferred over automatic detection systems, but this preference changed after informing farmers about the consequences of lameness. To conclude, the system cost and performance were important features, but dairy farmers should be sensitized on the consequences of lameness and its effect on farm profitability.

A 10-day vacancy period after cleaning and disinfection has no effect on the bacterial load in pig nursery units.

BACKGROUND: Biosecurity measures such as cleaning, disinfection and a vacancy period between production cycles on pig farms are essential to prevent disease outbreaks. No studies have tested the effect of a longer vacancy period on bacterial load in nursery units. METHODS: The present study evaluated the effect of a 10-day vacancy period in pig nursery units on total aerobic flora, Enterococcus spp., Escherichia coli, faecal coliforms and methicillin resistant Staphylococcus aureus (MRSA). Three vacancy periods of 10 days were monitored, each time applied in 3 units. The microbiological load was measured before disinfection and at 1, 4, 7 and 10 days after disinfection. RESULTS: No significant decrease or increase in E. coli, faecal coliforms, MRSA and Enterococcus spp. was noticed. Total aerobic flora counts were the lowest on day 4 after disinfection (i.e. 4.07 log CFU/625 cm2) (P < 0.05), but the difference with other sampling moments was limited (i.e. 0.6 log CFU/625 cm2) and therefore negligible. Furthermore, this observation on day 4 was not confirmed for the other microbiological parameters. After disinfection, drinking nipples were still mostly contaminated with total aerobic flora (i.e. 5.32 log CFU/625 cm2) and Enterococcus spp. (i.e. 95 % of the samples were positive) (P < 0.01); the feeding troughs were the cleanest location (total aerobic flora: 3.53 log CFU/625 cm2 and Enterococcus spp.: 50 % positive samples) (P < 0.01). CONCLUSIONS: This study indicates that prolonging the vacancy period in nursery units to 10 days after disinfection with no extra biosecurity measures has no impact on the environmental load of total aerobic flora, E. coli, faecal coliforms, MRSA and Enterococcus spp..

Comparison of competitive exclusion with classical cleaning and disinfection on bacterial load in pig nursery units.

BACKGROUND: Colonisation of the environment of nursery units by pathogenic micro-organisms is an important factor in the persistence and spread of endemic diseases in pigs and zoonotic pathogens. These pathogens are generally controlled by the use of antibiotics and disinfectants. Since an increasing resistance against these measures has been reported in recent years, methods such as competitive exclusion (CE) are promoted as promising alternatives. RESULTS: This study showed that the infection pressure in CE units after microbial cleaning was not reduced to the same degree as in control units. Despite sufficient administration of probiotic-type spores, the analysed bacteria did not decrease in number after 3 production rounds in CE units, indicating no competitive exclusion. In addition, no differences in feed conversion were found between piglets raised in CE and control units in our study. Also, no differences in faecal consistency (indicator for enteric diseases) was noticed. CONCLUSION: These results indicate that the CE protocol is not a valuable alternative for classical C&D.

Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, ß-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in food.

Environmental and cow-related factors affect cow locomotion and can cause misclassification in lameness detection systems.

To tackle the high prevalence of lameness, techniques to monitor cow locomotion are being developed in order to detect changes in cows' locomotion due to lameness. Obviously, in such lameness detection systems, alerts should only respond to locomotion changes that are related to lameness. However, other environmental or cow factors can contribute to locomotion changes not related to lameness and hence, might cause false alerts. In this study the effects of wet surfaces, dark environment, age, production level, lactation and gestation stage on cow locomotion were investigated. Data was collected at Institute for Agricultural and Fisheries Research research farm (Melle, Belgium) during a 5-month period. The gait variables of 30 non-lame and healthy Holstein cows were automatically measured every day. In dark environments and on wet walking surfaces cows took shorter, more asymmetrical strides with less step overlap. In general, older cows had a more asymmetrical gait and they walked slower with more abduction. Lactation stage or gestation stage also showed significant association with asymmetrical and shorter gait and less step overlap probably due to the heavy calf in the uterus. Next, two lameness detection algorithms were developed to investigate the added value of environmental and cow data into detection models. One algorithm solely used locomotion variables and a second algorithm used the same locomotion variables and additional environmental and cow data. In the latter algorithm only age and lactation stage together with the locomotion variables were withheld during model building. When comparing the sensitivity for the detection of non-lame cows, sensitivity increased by 10% when the cow data was added in the algorithm (sensitivity was 70% and 80% for the first and second algorithm, respectively). Hence, the number of false alerts for lame cows that were actually non-lame, decreased. This pilot study shows that using knowledge on influencing factors on cow locomotion will help in reducing the number of false alerts for lameness detection systems under development. However, further research is necessary in order to better understand these and many other possible influencing factors (e.g. trimming, conformation) of non-lame and hence 'normal' locomotion in cows.

On-farm comparisons of different cleaning protocols in broiler houses.

The present study evaluated the effectiveness of 4 cleaning protocols designed to reduce the bacteriological infection pressure on broiler farms and prevent food-borne zoonoses. Additionally, difficult to clean locations and possible sources of infection were identified. Cleaning and disinfection rounds were evaluated in 12 broiler houses on 5 farms through microbiological analyses and adenosine triphosphate hygiene monitoring. Samples were taken at 3 different times: before cleaning, after cleaning, and after disinfection. At each sampling time, swabs were taken from various locations for enumeration of the total aerobic flora and Enterococcus species pluralis ( SPP:). In addition, before cleaning and after disinfection, testing for Escherichia coli and Salmonella was carried out. Finally, adenosine triphosphate swabs and agar contact plates for total aerobic flora counts were taken after cleaning and disinfection, respectively. Total aerobic flora and Enterococcus spp. counts on the swab samples showed that cleaning protocols which were preceded by an overnight soaking with water caused a higher bacterial reduction compared to protocols without a preceding soaking step. Moreover, soaking of broiler houses leads to less water consumption and reduced working time during high pressure cleaning. No differences were found between protocols using cold or warm water during cleaning. Drinking cups, drain holes, and floor cracks were identified as critical locations for cleaning and disinfection in broiler houses.

Comparison of sampling procedures and microbiological and non-microbiological parameters to evaluate cleaning and disinfection in broiler houses.

Cleaning and disinfection of the broiler stable environment is an essential part of farm hygiene management. Adequate cleaning and disinfection is essential for prevention and control of animal diseases and zoonoses. The goal of this study was to shed light on the dynamics of microbiological and non-microbiological parameters during the successive steps of cleaning and disinfection and to select the most suitable sampling methods and parameters to evaluate cleaning and disinfection in broiler houses. The effectiveness of cleaning and disinfection protocols was measured in six broiler houses on two farms through visual inspection, adenosine triphosphate hygiene monitoring and microbiological analyses. Samples were taken at three time points: 1) before cleaning, 2) after cleaning, and 3) after disinfection. Before cleaning and after disinfection, air samples were taken in addition to agar contact plates and swab samples taken from various sampling points for enumeration of total aerobic flora, Enterococcus spp., and Escherichia coli and the detection of E. coli and Salmonella. After cleaning, air samples, swab samples, and adenosine triphosphate swabs were taken and a visual score was also assigned for each sampling point. The mean total aerobic flora determined by swab samples decreased from 7.7±1.4 to 5.7±1.2 log CFU/625 cm2 after cleaning and to 4.2±1.6 log CFU/625 cm2 after disinfection. Agar contact plates were used as the standard for evaluating cleaning and disinfection, but in this study they were found to be less suitable than swabs for enumeration. In addition to measuring total aerobic flora, Enterococcus spp. seemed to be a better hygiene indicator to evaluate cleaning and disinfection protocols than E. coli. All stables were Salmonella negative, but the detection of its indicator organism E. coli provided additional information for evaluating cleaning and disinfection protocols. Adenosine triphosphate analyses gave additional information about the hygiene level of the different sampling points.

Phage and MLVA typing of Salmonella enteritidis isolated from layers and humans in Belgium from 2000-2010, a period in which vaccination of laying hens was introduced.

The aim of the study was to characterize isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from humans and layer farms in Belgium collected during 2000-2010. Three periods were compared, namely (i) before implementation of vaccination (2000-2004), (ii) during voluntary vaccination (2005-2006) and (iii) during implementation of the national control program (NCP) for Salmonella including mandatory vaccination against S. Enteritidis (2007-2010). The characteristics compared across time periods were distributions of phage type and multiple-locus variable number tandem-repeat assay (MLVA). While PT4 and PT21 were predominantly isolated in Belgium in layers and humans before 2007, a significant reduction of those PTs was observed in both populations in the period 2007-2010. The relative proportion of PT4b, PT21c and PT6c was found to have increased considerably in the layer population since 2007. In the human population, PT8, PT1 and the group of 'other' PTs were more frequently isolated compared to the previous periods. When comparing the proportion of the predominant MLVA types Q2 and U2, no significant difference was found between the layer and human population in the three periods and between periods within each category (layer and human). A significant difference in isolate distribution among MLVA clusters I and II was found between human and layer isolates recovered during Period 3 and in the human population between Period 1 and 3. Results suggest that the association between S. Enteritidis in layers and the occurrence of the pathogen in humans changed since implementation of the NCP in 2007.

Prevalence of lameness and claw lesions during different stages in the reproductive cycle of sows and the impact on reproduction results.

Lameness in sows is an emerging disease condition with major effects on animal welfare and economics. Yet the direct impact on reproduction results remains unclear. The present field study investigated the impact of lameness and claw lesions throughout the reproductive cycle on (re)production results of sows. In five farms, a total of 491 group-housed sows were followed up for a period of one reproductive cycle. Sows were assessed for lameness every time they were moved to another area in the farm. Claw lesions were scored at the beginning and at the end of the cycle. Reproduction results included the number of live-born piglets, stillborn piglets, mummified fetuses and crushed piglets, weaning-to-oestrus interval and the presence of sows not showing oestrus post weaning, returning to service and aborting. Sows that left the group were recorded and the reason was noted. A mean prevalence of lameness of 5.9% was found, although it depended on the time in the productive cycle. The highest percentage of lame sows (8.1%) was found when sows were moved from the post-weaning to the gestation stable. No significant associations were found between lameness and reproduction parameters with the exception of the effect on mummified foetuses. Wall cracks, white line lesions, heel lesions and skin lesions did have an effect on farrowing performance. Of all sows, 22% left the group throughout the study, and almost half of these sows were removed from the farm. Lameness was the second most important reason for culling. Sows culled because of lameness were significantly younger compared with sows culled for other reasons (parity: 2.6 ± 1.3 v. 4.0 ± 1.8). In conclusion, the present results indicate that lameness mainly affects farm productivity indirectly through its effect on sow longevity, whereas claw lesions directly affect some reproductive parameters. The high percentage of lame sows in the insemination stable indicate that risk factor studies should not only focus on the gestation stable, but also on housing conditions in the insemination stable.

Genetic characteristics of Shiga toxin-producing E. coli O157, O26, O103, O111 and O145 isolates from humans, food, and cattle in Belgium.

In this study, we characterized 272 Shiga toxin-producing Escherichia coli (STEC) isolates from humans, food, and cattle in Belgium [O157 (n = 205), O26 (n = 31), O103 (n = 15), O111 (n = 10), O145 (n = 11)] for their virulence profile, whole genome variations and relationships on different genetic levels. Isolates of O157 displayed a wide variation of stx genotypes, heterogeneously distributed among pulsogroups (80% similarity), but with a concordance at the pulsosubgroup level (90% similarity). Of all serogroups evaluated, the presence of eae was conserved, whereas genes encoded on the large plasmid (ehx, espP, katP) occurred in variable combinations in O26, O103, and O145. The odds of having haemolytic uraemic syndrome was less for all genotypes stx2a, stx2c, stx1/stx2c, and stx1 compared to genotype stx2a/stx2c; and for patients aged >5 years compared to patients aged ≤ 5 years. Based on the genetic typing and by using epidemiological data, we could confirm outbreak isolates and suggest epidemiological relationships between some sporadic cases. Undistinguishable pulsotypes or clones with minor genotypic variations were found in humans, food, and cattle in different years, which demonstrated the important role of cattle as a reservoir of STEC O157, and the circulation and persistence of pathogenic clones.

Short communication: Intraoperator repeatability and interoperator reproducibility of devices measuring teat dimensions in dairy cows.

Various methods have been applied to measure teat dimensions. However, the accuracy and precision needed to obtain reliable results are often poor or have not yet been investigated. To determine the precision of the ruler, the caliper, and a recently developed 2-dimensional (2D) vision-based measuring device under field conditions, for respectively teat length, teat diameter, and both teat length and diameter, 2 experiments were conducted in which the consistency of measurements within operators (repeatability) and between operators (reproducibility) was tested. In addition, the agreement of the 2D device with the ruler and the caliper was studied. Although the ruler and the 2D device poorly agreed, both methods were precise in measuring teat length when the operators had experience in working with cows. The caliper was repeatable in measuring teat diameter, but was not reproducible. The 2D device was also repeatable in measuring teat diameter, and reproducible when the operators had experience with the device. The methods had poor agreement, most likely due to the operator-dependent pressure applied by the caliper. Because the 2D device has the advantage of measuring both teat length and teat diameters in a single measurement and is accurate and practical, this method allows efficient and fast collection of data on a large scale for various applications.

Voluntary feed intake and leptin sensitivity in ad libitum fed obese ponies following a period of restricted feeding: a pilot study.

The relation between plasma leptin and daily ad libitum roughage intake was evaluated during a 14-day period in eight obese Shetland ponies. When the feeding strategy was changed from maintenance feeding to ad libitum feeding, feed intake increased the first day, decreased the second day and increased again during the following days to reach a constant level after 8 days. Plasma leptin concentration increased during the first 2 days, but remained constant afterwards. Although the same pattern was found in all ponies, the magnitude of the increase in leptin on day 1 and the resulting decrease in feed intake on day 2 differed between ponies. A lower anorectic effect was seen in ponies with higher initial leptin concentration, suggesting the presence of different degrees in leptin sensitivity in obese ponies. High leptin production in a attempt to compensate for the decrease in leptin sensitivity might explain large variations in plasma leptin among obese ponies with similar body condition score. Further research is necessary to clarify whether the reduced leptin sensitivity precedes obesity in equines or vice versa.

Short communication: Associations between teat dimensions and milking-induced changes in teat dimensions and quarter milk somatic cell counts in dairy cows.

Although many studies have examined the relation between a wide range of factors and quarter milk somatic cell count (qSCC), including physical characteristics of the teat and changes in teat tissue due to milking, the effect of short-term, milking-induced changes in teat dimensions on somatic cell count has not yet been investigated. To identify teat dimensions and milking-induced changes in teat dimensions associated with qSCC, we conducted a longitudinal study (n(herds)=6, n(cows)=72, n(measurements)=12). Parity, stage of lactation, teat barrel diameter, and changes in teat barrel diameter during milking were identified as factors associated with qSCC. Teats with wider barrels had higher qSCC. Negative changes in the diameter of the teat barrel during milking (i.e., thinner teats postmilking compared with premilking) were associated with lower qSCC, whereas positive changes (i.e., thicker teats postmilking compared with premilking) were associated with higher qSCC. Selection toward more optimal teat characteristics may therefore result in improved milk quality and udder health. However, a threshold might exist for the maximum reduction in teat barrel diameter below which udder health is negatively influenced. If so, changes in teat barrel diameter might serve as an indicator for suboptimal milking and incorrect choice of teatcup liner or milking machine settings and thus help improve management of the herd.