Antimicrobials in small-scale urban pig farming in a lower middle-income country - arbitrary use and high resistance levels.

Background: Administration of antimicrobials to food-producing animals is regarded as a major contributor to the overall emergence of resistance in bacteria worldwide. However, few data are available on global antimicrobial use and resistance (AMR) in livestock, especially from low- and middle-income countries. Methods: We conducted a structured survey of 91 small-scale pig farms in the urban and peri-urban areas of Phnom Penh, Cambodia, to assess the farmers' knowledge, attitudes and practices related to antimicrobial use in their pig production. Commensal Escherichia coli was isolated from three healthy pigs from each farm (n = 261) and susceptibility testing was performed against 14 antimicrobials, using broth microdilution. Univariable logistic regression and generalized linear mixed models were used to investigate potential associations between farm characteristics, management factors and resistance to different types of antimicrobials. Results: We found a widespread and arbitrary use of antimicrobials, often based on the farmer's own judgment. Around 66% of the farmers reported frequently self-adjusting treatment duration and dosage, and 45% had not heard about the term 'antimicrobial resistance'. The antimicrobials most commonly mentioned or kept by the farmers were amoxicillin, tylosin, gentamicin and colistin. Around 37% used a feed concentrate that contained antimicrobials, while antimicrobials for humans were used as a last-line treatment by 10% of the farmers. Commensal E. coli exhibited high prevalence of resistance to several antimicrobials considered to be of critical importance for human medicine, including ampicillin, ciprofloxacin and colistin, and multidrug-resistance was found in 79% of the samples. Higher prevalence of resistance was observed on farms that administered prophylactic antimicrobials and on farms that treated the entire group or herd in the event of disease. Conclusion: The widespread and arbitrary use of antimicrobials in pig farming in Cambodia is highly worrisome. Overall, farmers had a low awareness of the risks and consequences related to antimicrobial use and AMR. The results presented in this study confirm the hypothesis that non-rational use of antimicrobials results in higher prevalence of AMR and highlight the need for professional animal health systems that involve medically rational use of antimicrobials in emerging economies such as Cambodia.

Contamination of organic nutrient sources with potentially toxic elements, antibiotics and pathogen microorganisms in relation to P fertilizer potential and treatment options for the production of sustainable fertilizers: A review.

Organic nutrient sources such as farmyard manure, sewage sludge, their biogas digestates or other animal by-products can be valuable fertilizers delivering organic matter to the soil. Currently, especially phosphorus (P) is in the focus of research since it is an essential plant nutrient with finite resources, estimated to last only for some more decades. Efficient utilization of organic P sources in agriculture will help to preserve P resources and thereby has the potential to close nutrient cycles and prevent unwanted P-losses to the environment, one of the major causes for eutrophication of water bodies. Unfortunately, organic P sources usually contain also various detrimental substances, such as potentially toxic elements or organic contaminants like pharmaceuticals as well as pathogenic microorganisms. Additionally, the utilization of some of these substrates such as sewage sludge or animal by-products is legally limited in agriculture because of the potential risk to contaminate sites with potentially toxic elements and organic contaminants. Thus, to close nutrient cycles it is important to develop solutions for the responsible use of organic nutrient sources. The aim of this review is to give an overview of the contamination of the most important organic nutrient sources with potentially toxic elements, antibiotics (as one important organic contaminant) and pathogenic microorganisms. Changes in manure and sewage sludge management as well as the increasing trend to use such substrates in biogas plants will be discussed with respect to potential risks posed to soils and water bodies. Some examples for abatement options by which contamination can be reduced to produce P fertilizers with high amounts of plant available P forms are presented.

Treatment with Ca(OH)2 for inactivation of Salmonella Typhimurium and Enterococcus faecalis in soil contaminated with infected horse manure.

AIM: To investigate the inactivation of Salmonella enterica serovar Typhimurium and the faecal indicator Enterococcus faecalis in horse manure:soil mixtures by application of hydrated lime (Ca(OH)(2)). METHODS AND RESULTS: In laboratory incubations, the inhibitory effect of different concentrations of Ca(OH)(2), as well as different application techniques, was tested. Other variables were horse manure:soil ratio, incubation temperature (6 and 14°C) and soil type (sand/clay). Bacterial enumeration by the plate count method in samples taken at increasing intervals revealed that Ca(OH)(2) effectively reduced Salmonella Typhimurium numbers. However, to achieve a sufficient reduction, the Ca(OH)(2) had to be applied at a sufficient rate, and the amount required varied because of manure:soil ratio and incubation temperature. The results showed that a pH above 11 was needed and that a high pH had to be maintained for up to 7 days. An appropriate application technique for the Ca(OH)(2) was also important, so that a high pH was obtained throughout the whole material to be treated. In addition, a high manure:soil ratio in combination with a higher incubation temperature was found to rapidly neutralize the pH and to increase the risk of Salmonella re-growth. CONCLUSIONS: Application of Ca(OH)(2) can be an efficient method for treating a Salmonella-contaminated horse paddock. A high pH is a key factor in Salmonella inactivation, and thus, monitoring the pH during the treatment period is necessary. To avoid re-growth excess manure should be removed for separate treatment elsewhere. SIGNIFICANCE AND IMPACT OF THE STUDY: Persistence of Salmonella in horse paddocks poses a risk of disease transmission to healthy animals and people who come into contact with these animals. An efficient method to de-contaminate a Salmonella-contaminated soil would be a valuable tool for animal welfare and for public health.

Growth potential of faecal bacteria in simulated psychrophilic/mesophilic zones during composting of organic waste.

AIM: This study investigated the growth potential of Salmonella serotype Typhimurium and faecal indicator organisms in compost materials and the correlation between bacterial growth potential and the physico-chemical composition of the compost substrate and temperature. METHODS AND RESULTS: Survival of Salm. Typhimurium, Enterococcus spp. and total coliforms at 14, 24 and 37 degrees C was determined in material of different degrees of maturity collected from composting plants for household waste and manure. All three micro-organisms showed the potential for growth in the material from active composts (Solvita index 4) but inactivation generally occurred over time in mature compost material (Solvita index 7-8). CONCLUSIONS: Salm. Typhimurium had the potential for growth in psychrophilic/mesophilic (P/M) zones of immature compost material and its growth potential correlated negatively with the maturity of the compost and the temperature within the simulated P/M zone. SIGNIFICANCE AND IMPACT OF THE STUDY: The risk of pathogen regrowth in P/M zones during organic waste composting further emphasizes the importance of good management practices and of avoiding P/M zones in combination with low compost maturity.

Verotoxinogenic Escherichia coli (VTEC) O157:H7--a nationwide Swedish survey of bovine faeces.

In the autumn of 1995 the first outbreaks of enterohemorrhagic Escherichia coli O157:H7 including ca 100 human cases were reported in Sweden. From outbreaks in other countries it is known that cattle may carry these bacteria and in many cases is the source of infection. Therefore, the present study was performed to survey the Swedish bovine population for the presence of verotoxin-producing E. coli (VTEC) of serotype O157:H7. Individual faecal samples were collected at the 16 main Swedish abattoirs from April 1996 to August 1997. Of 3071 faecal samples, VTEC O157 were found in 37 samples indicating a prevalence of 1.2% (CI95% 0.8-1.6). All 37 isolates carried genes encoding for verotoxin (VTI and/or VT2), intimin, EHEC-haemolysin and flagellin H7 as determined by PCR. Another 3 strains were of serotype O157:H7 but did not produce verotoxins. The 37 VTEC O157:H7 strains were further characterised by phage typing and pulsed-field gel electrophoresis. The results clearly show that VTEC O157:H7 is established in the Swedish bovine population and indicate that the prevalence of cattle carrying VTEC O157:H7 is correlated to the overall geographical distribution of cattle in Sweden. Results of this study have formed the basis for specific measures recommended to Swedish cattle farmers, and furthermore, a permanent monitoring programme was launched for VTEC O157:H7 in Swedish cattle at slaughter.

Production of capsular material by equine trophoblast transplanted into immunodeficient mice.

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

The potential for disinfection of separated faecal matter by urea and by peracetic acid for hygienic nutrient recycling.

No efficient, reliable, and scale independent disinfection methods for toilet waste are available today for safe recycling of plant nutrients. Therefore, two chemical treatment methods, addition of urea or of PAA (a quaternary mixture of 15% peracetic acid, 15% hydrogen peroxide and 30% acetic acid), were evaluated for disinfection of faecal matter.Degradation of the added urea resulted in 30 g of ammonia nitrogen per kilogram of treated matter and a pH increase to approximately 9.3. This produced an efficient disinfection of E. coli, Enterococcus spp., and Salmonella spp. within 3 weeks (>6log(10) reduction) and a reduction of the chemical resistant Salmonella typhimurium 28b phage, corresponding to a decimal reduction within 7.5 days. No viable Ascaris suum eggs were found after 50 days of treatment. No reduction of spore forming Clostridia spp. was observed. Urea treatment proved to be efficient for disinfection of source separated faecal matter in a scale independent method used for safe recycling of nutrients found in the faecal matter.PAA reduced all of the above indicator organisms within 12 h after application. For this faecal material, with a dry matter content of approximately 10%, an addition of 0.5-1% of PAA (active substance, corresponding to 3.3-6.7% of the Proxitane 15 used) was required before no viable organisms were found in the material. However, this was not tested for the A. suum. No viable spore-forming bacteria or phages were detected. A high rate of bacteria regrowth occurred at 0.15% dosage and 5 days of treatment. PAA is an efficient alternative for disinfection of separated faeces if a rapid treatment is needed.

Uterine microbiology and antimicrobial susceptibility in isolated bacteria from mares with fertility problems.

Uterine microbiology and antimicrobial susceptibility was investigated in 239 mares with fertility problems in a prospective study in Sweden. Uterine swab samples were collected with double guarded swabs and transported overnight before being cultured. The Minimum Inhibitory Concentrations (MIC) was determined for a panel of antimicrobials. From 152 of the 239 mares at least one bacterial species was isolated, most frequently E. coli (104 isolates), beta-haemolytic streptococci (31) and fungi (16). beta-haemolytic streptococci were more frequently (p < 0.01) associated with clinical endometritis than with repeat breeding. The opposite was true for E. coli (p < 0.01). Among beta-haemolytic streptococcal isolates some resistance was noted for 4 of 11 tested antibiotics, however, all isolates were susceptible to the widely used penicillin G. Among E. coli isolates enrofloxacin was the only of the 10 tested antibiotics for which no resistance was noted. Resistance was most commonly noted to cephalothin (39% of the isolates), streptomycin (22%), trimethoprim/sulphamethoxazole (15%) and ampicillin (11%). In conclusion, we show that both E.coli and beta-haemolytic streptococci are frequently associated with fertility problems in mares and that antimicrobial resistance is a common feature of E. coli but also recognised for beta-haemolytic streptococcal uterine isolates.

Recycling biowaste--human and animal health problems.

Biowaste from the food chain is of potential benefit to use in agriculture. Agriculture in general and organic farming in particular needs alternative plant nutrients. However, the quality concerning hygiene and soil contaminants must be assured. This recycling has to be regulated in a way that harmful effects on soil, vegetation, animals and man are prevented. The problems with heavy metals and organic contaminants have been focused on. Still, maximum threshold values are continuously discussed to avoid an increase of soil concentrations. The effect on the ecosystems of residues from use of medicines needs further attention. There is also a risk for a spread of antibiotic resistant micro-organisms in the environment and then to animals and man. Infectious diseases may be spread from biowaste and new routes of disease transmission between animals and humans can be created. Zoonotic diseases in this context play a central role. Pathogens recently introduced to a country may be further spread when biowaste is recycled. The very good health status of domestic animals in the Nordic countries may then quickly change. The quality of biowaste is of enormous importance if biowaste is to gain general acceptance for agricultural use, especially for organic production. A balance needs to be maintained between risk and advantage for its use.

Inhibition of cell growth and apoptosis by inducible expression of the transcriptional repressor Mad1.

Mad1 is a Myc antagonist that heterodimerizes with Max and functions as a transcriptional repressor. We have studied the effects of Mad1 on cell growth, cell cycle distribution, and apoptosis using Mad1-inducible cell lines. Expression of Mad1 inhibited cell proliferation, S-phase entry, and colony formation, changes that were accompanied by a reduction in CDK2 activity. The inhibition of Mad1 on cell proliferation was potentiated by serum starvation and was paralleled by accumulation of cells in the G0/G1 and the G2 phases of the cell cycle. Mad1 also reduced apoptosis induced by serum withdrawal and by the cytostatic drug cisplatinum. The effects on both cell growth and apoptosis were dependent on the mSin3 interaction domain of Mad1, which is necessary for recruitment of histone deacetylases and corepressors, suggesting that transcriptional repression is mediating these functions. Taken together with the expression pattern of Mad1, these results suggest that Mad1 plays an important role during initiation of differentiation by inhibiting cell proliferation and blocking apoptosis.

Aerobic bacterial flora of semen and stallion reproductive tract and its relation to fertility under field conditions.

This study was initiated in order to investigate the bacterial flora of the stallion genital tract by taking consecutive samples from normal stallions in regular use. The objective was to determine whether any growth of potential pathogens, particularly P. aeruginosa and K. pneumoniae, in fresh semen and urethra was associated with the presence of inflammatory cells in the semen and whether bacterial growth had any effect on sperm morphology and pregnancy results. Sixteen stallions, only used for A.I., housed at 3 different commercial stud farms, were used. A wide variety of microorganisms was found in almost all samples from fresh semen (total 115 samples). P. aeruginosa was isolated from 46/115 (40%) of the samples and from 12 of the 16 stallions. K. pneumoniae was isolated from the semen of one stallion. Samples taken from the distal urethra after ejaculation contained fewer microorganisms than samples from fresh semen. No bacteria were found in 51% of the extended semen samples. Most of the stallions had an acceptable sperm morphology, and very few of the ejaculates contained inflammatory cells. Pregnancy results among the stallions varied, but were acceptable for most of them. There was no correlation between the frequency of samples testing positive for P. aeruginosa in raw semen and pregnancy results.

Ultrastructure of the secretory endometrium during oestrus in young maiden and foaled mares.

Cyclical accumulation of uterine fluid occurs during oestrus and is often seen in excessive volumes in mares considered susceptible to endometritis. Since the mechanisms behind the formation of free uterine fluid remain to be clarified, the fine structure of the secretory equine endometrium was studied in biopsies collect during videoendoscopy from 14 endometritis-free, 4-9-year-old mares during oestrus. A distinct oedema of the tunica mucosa was evident. The surface epithelium had both ciliated and nonciliated cells and, particularly at the uterine body, often presented intra-epithelial macrophages. The epithelial cells of the gland duct were similar to the surface epithelium, except that the nonciliated cells lacked secretory vesicles in the non ciliated cells. This glandular epithelium presented clear signs of secretory activity with conspicuous secretory vesicles holding electron-dense granula in the adluminal cytoplasm and a well developed supranuclear Golgi apparatus. Secretory products as well as cell debris were commonly found in the lumen of the glands. No clear signs of apocrine secretion were found and it seemed therefore, that the mechanism of secretion is merocrine, i.e. by exocytosis. The endometrial oedema and intense secretory activity, both under oestrogenic influence, contribute to the building up of the uterine fluid during oestrus. No differences in morphology of the secretory endometrium could be noticed between nulliparous mares and mares that had had 1 or 2 foals.

The contribution of complement to opsonic activity in the uterine secretions of mares free of endometritis.

The aim of the present study was to investigate if complement contributes to opsonic activity in the uterine secretions of mares with normal reproductive functions. Five mares with a mean age of 9 years were used in the study. The mares were considered to be free of endometritis based upon clinical history, palpation per rectum and ultrasonogaraphy of the genital tract, videoendoscopic inspection of the uterus, electronmicroscopy of endometrial biopsies, and bacteriological and cytological examination of swabs from the endometrium. The hormonal status of the mares was also determined. Uterine secretions were collected by a tampon inserted into the uterus during estrus. Secretion and serum samples were divided in half, one half of which was subjected to heat treatment (56 degrees C in 30 minutes) in order to inactivate the complement. Opsonic activity in the samples was then determined in a chemiluminescence assay using zymosan as the target particle. Opsonic activity was expressed as peak chemiluminescence and the time to peak chemiluminescence. Heat treatment of the secretions reduced peak luminescence significantly (P<0.01), and it prolonged the time to peak luminescence significantly (P<0.05). These data show that complement contributes a considerably to opsonic activity in uterine secretions prior to an inflammatory response in the equine uterus.

Concentrations of nuclear progesterone receptors in endometrium of virgin and repeat breeder heifers after embryo transfer.

The present study aimed to correlate the repeat breeder syndrome in the bovine with an impaired or suboptimal uterine progestational response. Concentrations of nuclear (i.e. transformed) receptors for progesterone (PRn) were determined with a binding and exchange method in endometrial samples from virgin (VH) and repeat breeder (RBH) heifers 15 days post oestrus. The heifers were recipients of Day 7 demi-embryos collected from donors with normal fertility and transferred 8 days prior to tissue sampling. Results were compared with both the type of heifer, the condition of the embryo present within the uterus and the temporal relationship to the hormone plasma levels. The binding data for PRn indicated that a single class of high-affinity, low-capacity sites existed. The amount of PRn in VH endometria holding embryonic structures was significantly greater than in RBH, but no statistical differences were found in their plasma progesterone levels. PRn concentrations were also higher in the uterine horn in which an elongated (greater than 15 mm), morphologically normal embryo was present, when compared to cornua with small (less than 5 mm) embryos, regardless of recipient group. Furthermore, in endometria with an elongated embryo in the lumen, the relative amount of PRn was significantly greater in VH than in RBH. The present results indicate that the RBH recipients of Day 7 demi-embryos by Day 15 have fewer numbers of specific receptors for maternal progesterone, which could explain in part the poorer development of the transferred embryos compared with that in virgin heifers.

Standing oestrus, ovarian function and early pregnancy in virgin and repeat breeder heifers.

Seventeen virgin heifers (VH) and 14 repeat breeder heifers (RBH) were inseminated in spontaneous oestrus during a total of 86 oestrous cycles. Pregnancy was checked either by non-surgical embryo collections 14 or 17 days after ovulation or by real time ultrasonography 20-26 days after ovulation. The two categories of heifers were compared with respect to duration of standing oestrus, ovarian function and plasma progesterone levels after insemination and during early pregnancy. The results indicate that the duration of standing oestrus was longer in RBHs than in VHs. Ovulation disturbances occurred and were more common among the RBHs than among the VHs. Disturbances in the development of the corpus luteum were noted in both categories of heifers, the RBHs having less luteal tissue volume than the VHs. In general, plasma progesterone levels did not differ significantly between the two categories of heifers. Despite the strict control of oestrus and ovulation as well as additional AI when necessary in this study, a poorer pregnancy result for the RBHs than for the VHs was seen.

Plasma levels of prostaglandin F2 alpha metabolite and progesterone in repeat breeder heifers.

A detailed clinical-endocrine investigation was performed in 6 repeat breeder heifers (RBH) with the aim being to ascertain whether endocrine asynchronism exists at luteal regression and during early pregnancy. The heifers were first studied during an open cycle and then after insemination when 3 heifers became pregnant. Circulating plasma levels of PGF2 alpha metabolite were measured every 2nd h, while progesterone (P4) levels were measured every 6th h. The oestrous period and intervals between the onset of oestrus and ovulation were relatively longer, compared with what is normally seen in heifers. Plasma levels of P4 at the onset of oestrus were higher than normal, but it was concluded that the plasma levels of PGF2 alpha metabolite and P4 in RBH at luteal regression and early pregnancy were normal.

Morphology of day 7 bovine demi-embryos during in vitro reorganization.

Non-surgically collected day 7 bovine embryos were microsurgically cleaved and reinserted into surrogate zonae pellucidae. The demi-embryos were fixed immediately after splitting and at various intervals of in vitro incubation at 35 degrees C in modified Dulbecco's medium. At the light-microscopical level, the demi-embryos were found to have restored their prebisection morphology within 30 min after splitting. The electron microscopy confirmed these findings, by the presence of mitosis, cell polarization, neoformation and reaggregation of mitochondria, increased pinocytosis and cell-to-cell contact between trophoblastic and/or inner cell mass cells. The present observations therefore suggest that day 7 bovine demi-embryos intended for transfer should be cultured for 30 min before being morphologically evaluated.