Anthelmintic resistance: is a solution possible?

More than 50 years after anthelmintic resistance was first identified, its prevalence and impact on the animal production industry continues to increase across the world. The term "anthelmintic resistance" (AR) can be briefly defined as the reduction in efficacy of a certain dose of anthelmintic drugs (AH) in eliminating the presence of a parasite population that was previously susceptible. The main aim of this study is to examine anthelmintic resistance in domestic herbivores. There are numerous factors playing a role in the development of AR, but the most important is livestock management. The price of AH and the need to treat a high number of animals mean that farmers face significant costs in this regard, yet, since 1981, little progress has been made in the discovery of new molecules and the time and cost required to bring a new AH to market has increased dramatically in recent decades. Furthermore, resistance has also emerged for new AH, such as monepantel or derquantel. Consequently, ruminant parasitism cannot be controlled solely by using synthetic chemicals. A change in approach is needed, using a range of preventive measures in order to achieve a sustainable control programme. The use of nematophagous fungi or of plant extracts rich in compounds with anthelmintic properties, such as terpenes, condensed tannins, or flavonoids, represent potential alternatives. Nevertheless, although new approaches are showing promising results, there is still much to do. More research focused on the control of AR is needed.

Bacteria and antibiotic resistance detection in fractures of wild birds from wildlife rehabilitation centres in Spain.

Anatomic adaptations make birds more prone to open fractures with exposed bone parts losing vascularization. As a result of this exposure, fractures are colonized by different microorganisms, including different types of bacteria, both aerobic and anaerobic, causing osteomyelitis in many cases. For this reason, antibiotic treatment is common. However, carrying out antibiotic treatment without carrying out a previous antibiogram may contribute to increased resistance against antibiotics, especially in migratory wild birds. In this paper, bacterial counts regarding fracture type, bacterial identification and antibiotic resistance have been analysed in wild birds from wildlife rehabilitation centres in Spain. The results obtained showed that open fractures had higher bacterial counts (CFU/mL) than closed ones. Bacteria in family Enterobacteriaceae, identified were Escherichia spp., Enterobacter spp., Shigella spp., Hafnia alvei, Proteus mirabilis, Leclercia adecarboxylata and Pantoea agglomerans. Other bacteria present in wild birds' fractures were Aeromonas spp., Enterococcus spp. Bacillus wiedmannii and Staphylococcus sciuri. All species found presented resistance to at least one of the antibiotics used. Wild birds can be implicated in the introduction, maintenance and global spreading of antibiotic resistant bacteria and represent an emerging public health concern. Results obtained in this paper support the idea that it is necessary to take this fact into account before antibiotic administration to wild animals, since it could increase the number of bacteria resistant to antibiotics.

Dogs as a source of Salmonella spp. in apparently healthy dogs in the Valencia Region. Could it be related with intestinal lactic acid bacteria?

BACKGROUND: Although salmonellosis is considered one of the most important food-borne zoonotic diseases in Europe, close contact between dogs and their owners can also be a potential source of Salmonella spp. for humans. This study assessed the prevalence and antimicrobial resistance of Salmonella spp. in apparently healthy dogs in the Valencian Region, eastern Spain. Moreover, a macroscopic comparison of lactic acid bacteria in both Salmonella-positive and Salmonella-negative dogs was carried out. RESULTS: Of a total of 325 dogs sampled, 6 (1.85%) were positive for Salmonella spp. with 3 different serotypes, Havana (3), Mikawasima (2) and monophasic Typhimurium (1). All isolates were susceptible to all antimicrobials tested except monophasic S. Typhimurium, which was resistant to ampicillin. Finally, macroscopic results revealed that lactic acid bacteria had higher heterogeneity in the Salmonella-negative dogs than in the Salmonella-positive dogs. Although the results in our study showed a low prevalence of Salmonella spp., raw food has been suggested as a risk factor for bacteria in dog faeces. CONCLUSIONS: Public awareness campaigns on good hygiene practices, especially after handling canine faeces or raw food, are necessary. Furthermore, to reduce the potential transmission of bacteria, dogs should be fed food that has been properly cooked, as raw or undercooked food can be a source of zoonotic pathogens. Moreover, further studies must be performed to determine the relationship between lactic acid bacteria and Salmonella spp. in dog faeces.

effect of embryonic genotype on reference gene selection for RT-qPCR normalization.

To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. In rabbits, there are classic stable reference genes that have been identified for normalization in oocytes and pre-implantation stage embryos. However, effects of embryonic genotype on reference gene selection have not been elucidated. The aim of this study was to test (i) the stability of mRNA transcription level for histone (H2afz) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in rabbit blastocysts from two lines selected by different criteria (litter size and post-weaning daily weight gain) and (ii) its influence on biological significance examined by means of a set of embryonic transcripts, such as POU5F1 (Oct-4), epidermal growth factor receptor (erbB3), transforming growth factor-beta2, vascular endothelial growth factor and gamma interferon (Ifn-gamma). The geNorm, NormFinder and BestKeeper programs showed similar results, pointing out that H2afz and GAPDH were the most stable reference genes in rabbits selected on litter size at weaning. Moreover, our study revealed that embryonic genotype affected target gene expression when a single reference gene was used to analyse mRNA expression in blastocysts. Results showed that GAPDH gene is better than H2afz for gene expression studies of both embryo genotypes. A normalization factor derived from H2afz and GAPDH is likely to be appropriate when RT-qPCR was performed in rabbit embryos with different genotypes.

mRNA expression in rabbit blastocyst and endometrial tissue of candidate gene involved in gestational losses.

Gestation is a complex process that involves different growth factors, cytokines and adhesion proteins related with embryo development, cellular differentiation and proliferation, embryo-endometrium interaction, angiogenesis, maternal-embryonic recognition and growth development of placenta and embryos. In this study, we examine pre-implantational (at 6 days of gestation) and gestational (at 12 days and total from ovulation to birth) losses in two rabbit lines selected by different criteria (post-weaning daily gain and litter size) and the pattern of a set of candidate transcripts, at 6 days of gestation, related with embryo development and implantation process, such as Oct-4, epidermal growth factor receptor 3 (erbB3), Transforming Growth Factor ß2, Vascular Endothelial Growth Factor and Interferon γ and related with insulin-like growth factors signalling as insulin growth factors I and II and their receptors in rabbit blastocysts and endometrial tissue. Similar pre-implantational losses were obtained in both lines. However, the gestational losses of the line selected by post-weaning daily gain clearly mirrored an increase in losses by 50% at 12 days and at birth (22.4 vs 9.5 and 50.2 vs 25.4, respectively, between line selected by post-weaning daily gain and line selected by litter size). In blastocysts and endometrial tissue at 6 days of gestation qRT-PCR assays indicated that the mean insulin-like growth factor (IGF)-IIR mRNA expression was down-regulated in line selected by post-weaning daily gain. Dysregulation of the IGF-IIR could be potential reasons for induced gestational losses. We conclude that IGF-IIR gene expression in blastocyst and endometrial tissue at 6th day of gestation tends to decline in line selected by post-weaning daily gain. The functional significance related with gestational losses is uncertain.

Gestational losses in a rabbit line selected for growth rate.

Prenatal death can occur due to several genetic and environmental factors which alter normal embryo development, maternal environment to support normal fertilisation, development of embryos, placenta and foetus, or affect the necessary relationship between embryo and endometrium. The aim of this work was to study gestational losses and progesterone, 17 ß-estradiol and IGF I serum levels in a rabbit line selected for growth rate (paternal line). In this study, a maternal line well characterised in previous studies was used as a reference line. A total of 211 laparoscopies were carried out, and the number of corpora lutea and implanted embryos at 12th days, total born and live born were recorded per female. To analyse the endocrine levels, blood serum was collected from 54 females with implanted embryos at 12th and 24th day of gestation (27 from each line). The paternal line showed the lowest ovulation frequency, number of implanted embryos, total born and live born (0.70, 11.3, 7.4, and 6.4 vs 0.86, 12.8, 11.1 and 10.6 for maternal line, respectively) and consequently, the highest implantation, gestational, foetal and perinatal losses (0.31, 0.60, 0.40, and 0.15, respectively). Progesterone serum levels at 12th days of gestation were similar between lines; however, progesterone serum level at 24th day of gestation was significantly lower in the paternal line (4.8 vs 8.2 ng/mL). Serum levels of 17ß-estradiol and IGF-I at 12th days of gestation were different between lines (14.6 vs 26.5 pg/mL, 237 vs 149 ng/mL for paternal and maternal lines respectively). These higher gestational losses of the paternal line could be explained by differences in 17 ß-estradiol level at 12th days of gestation and the possible effect on low progesterone serum levels at 24th days of gestation. Further studies in steroid production and bioavailability have to be done during oestrus and pregnancy related with metabolic activity of this line.

Differential mRNA expression in rabbit in vivo pre-implantatory embryos.

To study genes involved in embryo developmental competence and implantation in rabbits, the expression of a panel of genes related to pluripotency, angiogenesis, proliferation, apoptosis and differentiation were evaluated in late rabbit blastocysts. Thirty nulliparous does were used to obtain a total of 184 in vivo-derived blastocysts on days 4, 5 and 6 of development. The relative transcript abundance of vascular endothelial growth factor (VEGF), epidermal growth factor receptor 3 (erbB3), transforming growth factor ß2 (TGF ß2) and transcription factor OCT-4 were analysed from eight pools of each stage of development, using quantitative real-time reverse transcriptase-polymerase chain reaction (qrtRT-PCR). mRNA expression was detected for all genes in 4-, 5- and 6-day-old blastocysts, according to blastocyst growth and implantation proximity. Significant differences in OCT-4, VEGF and TGF ß2 expression were observed between days of development. Results show a down-regulation of OCT-4 from the 4th day, contrasting with the up-regulation of VEGF and TGF ß2 at 6-day-old blastocyst. These findings corroborate the importance of VEGF and TGF ß2 in rabbit embryo development and implantation and suggest a possible regulator role of OCT-4 in embryonic angiogenetic factors. On the other hand, no differences were found for erbB3 expression. Therefore, the study of specific gene transcripts in rabbit blastocyst could provide novel embryo developmental competence markers and might be used as a new tool for further studies of embryo quality and in vitro development.