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1.
This study investigated the effects of vehicles on penetration and retention of lidocaine applied to sheep skin in vitro. Thoracic skin from two sheep was clipped of wool and stored at −20 °C, until used. Skin samples were defrosted and mounted in Franz‐type diffusion cells, and then one of the following formulations, each saturated with lidocaine, was added: sodium lauryl sulphate (SLS) 0.5% in water, SLS 1% in water, dimethyl sulphoxide (DMSO) 50% in water (wt/wt), DMSO 100%, isopropyl myristate 100% (IPM), water alone, diethylene glycol monoethyl ether (DGME) 50% in water (wt/wt) and DGME 100%. The penetration of lidocaine in each skin sample was measured over 8 h. Significantly greater lidocaine skin concentrations and flux (JSS) were achieved with the nonaqueous vehicles, DMSO 100% (< 0.00001 and < 0.01, respectively), followed by DGME 100% and IPM (< 0.00001 and < 0.01, respectively). The lag time (tlag) for lidocaine penetration in the DMSO 100% vehicle was significantly shorter (< 0.01) compared with all other vehicles except water. Improved transdermal penetration of lidocaine in the DMSO 100% vehicle was likely due to skin barrier disruption, as determined by differences in pre‐ and post‐treatment transepidermal water loss (TEWL). This study has shown that nonaqueous vehicles enhanced penetration of lidocaine in sheep skin to a greater extent than aqueous vehicles, which has implications for topically applied local anaesthesia in sheep.  相似文献   

2.
The aim was to investigate diclofenac delivery into and across equine skin in vitro using Franz diffusion cells from a novel diclofenac epolamine (DIC‐EP; 1.3%) formulation and to compare the results to those of Surpass® (1% diclofenac sodium liposomal cream) and a 1% aqueous solution of diclofenac sodium. Skin was harvested from the lower legs of Freiberger geldings immediately after slaughter and sliced to a thickness of ~2 mm. Skin samples were divided into two groups [Group 1: 1 year old (n = 2) and Group 2: 6–8 years old (n = 3)]. Cumulative permeation of diclofenac in Groups 1 and 2 after 24 h using diclofenac sodium solution was 1.91 ± 0.27 and 1.76 ± 0.34 μg/cm2, respectively. The values for Surpass® and DIC‐EP were 3.2 ± 0.8/3.3 ± 0.7 μg/cm2 and 230 ± 59/89.2 ± 32.5 μg/cm2, respectively. Thus, diclofenac permeation from DIC‐EP was significantly greater and appeared to show an age‐dependent effect. Mathematical modelling showed that the DIC‐EP formulation significantly increased diclofenac partitioning into the skin and a linear correlation was observed between steady‐state flux and the partition parameter. Greater skin deposition of diclofenac was also observed with DIC‐EP. These preliminary results suggest that the DIC‐EP formulation may be effective in treating inflammatory conditions in horses.  相似文献   

3.
Phenylbutazone (PBZ) is a nonsteroidal anti‐inflammatory drug used in the treatment of chronic pain and arthritis. Topical and transdermal administration of PBZ would be beneficial in large animals in terms of minimizing gastro‐intestinal ulcerations and other side effects, easy administration to legs and joints and minimizing the dose to reduce systemic toxicity of the drug. A topical liposomal preparation with different concentrations of a mono‐substituted alkyl amide (MSA) and PBZ was formulated. The formulations were evaluated by in vitro skin‐permeation kinetics through deer skin using Franz diffusion cells. By increasing drug loading from 1% to 5% w/w, the steady‐state flux (μg/cm2/h) of PBZ was increased twofold (P < 0.001). Similarly, by increasing the MSA concentration from 0% to 4%, the steady‐state flux (μg/cm2/h) of PBZ was increased twofold (P < 0.001). Overall, by increasing the drug load and the use of an appropriate amount of the penetration enhancer, the steady‐state flux of PBZ through skin was increased fourfold (P < 0.001). MSA at both 2% and 4% w/w concentrations significantly increased the skin levels of PBZ as compared with control (P < 0.05). In conclusion, MSA served as an effective skin‐penetration enhancer in the liposomal gel of PBZ for deer.  相似文献   

4.
Topical application of ectoparasiticides for flea and tick control is a major focus for product development in animal health. The objective of this work was to develop a quantitative structure permeability relationship (QSPeR) model sensitive to formulation effects for predicting absorption and skin deposition of five topically applied drugs administered in six vehicle combinations to porcine and canine skin in vitro. Saturated solutions (20 μL) of 14C‐labeled demiditraz, fipronil, permethrin, imidacloprid, or sisapronil were administered in single or binary (50:50 v/v) combinations of water, ethanol, and transcutol (6 formulations, n = 4–5 replicates per treatment) nonoccluded to 0.64 cm2 disks of dermatomed pig or dog skin mounted in flow‐through diffusion cells. Perfusate flux over 24 h and skin deposition at termination were determined. Permeability (logKp), absorption, and penetration endpoints were modeled using a four‐term Abrahams and Martin (hydrogen‐bond donor acidity and basicity, dipolarity/polarizability, and excess molar refractivity) linear free energy QSPeR equation with a mixture factor added to compensate for formulation ingredient interactions. Goodness of fit was judged by r2, cross‐validation coefficient, coefficients (q2s), and Williams Plot to visualize the applicability domain. Formulation composition was the primary determinant of permeation. Compounds generally penetrated dog skin better than porcine skin. The vast majority of permeated penetrant was deposited within the dosed skin relative to transdermal flux, an attribute for ectoparasiticides. The best QSPeR logKp model for pig skin permeation (r2 = 0.86, q2s = 0.85) included log octanol/water partition coefficient as the mixture factor, while for dogs (r2 = 0.91, q2s = 0.90), it was log water solubility. These studies clearly showed that the permeation of topical ectoparasiticides could be well predicted using QSPeR models that account for both the physical–chemical properties of the penetrant and formulation components.  相似文献   

5.
Commercial formulations of non-steroidal anti-inflammatory drugs (NSAIDs) are developed for human use but the extent to which they will pass through equine skin is unknown. Skin was harvested from five Thoroughbred geldings from the thorax, groin and leg (dorsal metacarpal) regions and frozen (-20 degrees C) until required. Two grams of methylsalicylate (MeSa) gel was applied to defrosted full-thickness samples in diffusion cells and the penetration of MeSa and its active metabolite, salicylate (Sa), through skin samples were measured over 24 h. Significantly higher (P < or = 0.02) total salicylate (AUC; MeSa + Sa) penetrated through skin from the leg region (5491.3 h mg/L), compared to thorax (3710.7 h mg/L) and groin (3571.5 h mg/L). In addition, there was a significantly higher (P0.01) rate of penetration of total Sa through leg skin in the first 6h after application. It was concluded that the commercial formulation of MeSa would achieve therapeutic levels of total salicylate beneath sites of topical application, with a faster and more pronounced response through the leg region, compared to the upper body.  相似文献   

6.
An increasing number of formulations are applied to equine skin, yet variable penetration can affect efficacy, or the incidence of adverse effects, or both. To investigate the effects of common methods of skin preparation on transdermal drug penetration in vitro, we clipped, harvested, and froze skin samples from 5 Thoroughbred geldings. Thawed samples were prepared as follows: control (no preparation); cleaned with aqueous chlorhexidine (Aq-C, 0.1% w/v); cleaned with alcoholic chlorhexidine (Al-C, 0.5% w/v); shaved (Sh); or tape-stripped (Ta) with the use of adhesive tape. The samples were then placed in diffusion cells, and 2 g of methylsalicylate (MeSa) gel (Dencorub) was applied to the stratum corneum side. The penetration of MeSa and its analyte, salicylate (Sa), through the skin samples was measured over 10 h. Compared with control skin, significantly more MeSa penetrated through skin prepared with Al-C or Sh (P < 0.01) or with Aq-C or Ta (P < 0.05), and significantly more Sa was recovered in the receptor phase from skin prepared with Aq-C, Al-C, or Sh (P < 0.05) or with Ta (P < 0.01). A significantly higher rate of penetration and shorter lag time were also noted for MeSa with all the prepared skin samples, compared with the control samples. The results show that clinical techniques routinely used to clean or prepare skin can significantly affect the rate and extent of penetration of a topically applied drug. This may result in greater systemic availability of active drug, which could lead to enhanced efficacy and, possibly, a higher incidence of adverse effects.  相似文献   

7.
Photobiomodulation therapy (PBMT) effects depend on the energy settings and laser penetration. We investigated the penetration time profiles of two different light therapy devices, at the dark and light skin regions in horses. Six light skin and six dark skin adult clinically healthy Arab and Quarter horses were used. A cutometer was used to measure the width of the skin fold from both sides of the cervical area, followed by three measurements of the thickness of the same skin fold by transversal and longitudinal ultrasonography (US). The depth of light penetration was compared based on the percentage of penetration versus power, between a portable PBMT device versus a class IV laser device. The laser mean power output was measured with an optical power meter system for 120 seconds after penetrating the skin. Skin width and laser penetration were compared among equipment by paired “t” test. There was no difference in the width of the skin fold between measurements acquired by the cutometer against either longitudinal or transversal US or between the US measurements at cervical versus metacarpus area. Light penetration was greater in both kinds of skins in the PBMT (0.01303 ± 0.00778) versus class IV laser (0.00122 ± SD 0.00070) (P < .001). The PBMT device provided a greater energy penetration than the class IV laser in unclipped light and dark skin, suggesting that the former may produce a better therapeutic effect. The color of the skin changes penetration profiles of PBMT.  相似文献   

8.
The effects of three vehicles, phosphate-buffered saline (PBS), ethanol (50% in PBS w/w) and propylene glycol (50% in PBS w/w) on in vitro transdermal penetration of testosterone was investigated in the horse. Skin was harvested from the thorax of five Thoroughbred horses after euthanasia and stored at −20°C until required. The skin was then defrosted and placed into Franz-type diffusion cells, which were maintained at approximately 32°C by a water bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled [14C]testosterone, in each vehicle were applied to the outer (stratum corneum) surface of each skin sample and aliquots of receptor fluid were collected at 0, 2, 4, 8, 16, 20, 22 and 24 h and analysed for testosterone by scintillation counting. The maximum flux (J max) of testosterone was significantly higher for all sites when testosterone was dissolved in a vehicle containing 50% ethanol or 50% propylene glycol, compared to PBS. In contrast, higher residues of testosterone were found remaining within the skin when PBS was used as a vehicle. This study shows that variability in clinical response to testosterone could be expected with formulation design.  相似文献   

9.
Abstract

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis in postmetamorphic amphibians, has been linked to amphibian population declines. Different amphibian species, however, exhibit different susceptibility to Bd pathogenicity. At the same time, agricultural pesticides commonly found contaminating aquatic habitats have been reported to increase the susceptibility of amphibians to pathogens. To investigate whether certain pesticides are able to alter the pathogenicity of Bd to larval amphibians, we exposed larval American bullfrogs Lithobates catesbeianus to end-use formulations of the herbicides atrazine or glyphosate, and then exposed them to Bd. Following the experimental exposures, a preexisting infection of the tadpoles by the monogenean ectoparasite Gyrodactylus jennyae was detected in all experimental and control tadpoles. Gyrodactylus jennyae infection intensity varied, and individuals with heavy G. jennyae infections suffered more skin erosion due to grazing by the parasite. Tadpoles experimentally exposed to Bd, or to Bd and either herbicide, had significantly reduced survival rates compared with untreated tadpoles that were only infected by G. jennyae. Increased mortality was also correlated with degree of skin erosion; survival of tadpoles with severe skin erosion was significantly reduced compared with that of tadpoles with no, or mild, skin erosion. While infected with G. jennyae, the group of tadpoles with the lowest survival rate (exposed only to Bd) included significantly more individuals exhibiting severe skin erosion and significantly fewer individuals without skin erosion, compared with the control group. These results emphasize the potential pathogenicity of gyrodactylid infections in larval amphibian hosts and suggest that concomitant exposures to Bd may enhance infections and effects of G. jennyae in bullfrog tadpoles.

Received February 3, 2012; accepted August 10, 2012  相似文献   

10.
A case of Listeria monocytogenes skin infection in a man is presented. A 54‐year‐old male veterinary practitioner developed pustular changes on the skin of arms and hands after assisting with the delivery of a stillborn calf. Listeria monocytogenes was isolated from the skin lesions on the arms and from the bovine placenta. Listeria monocytogenes isolates were serotyped and genotyped with pulsed‐field gel electrophoresis (PFGE) to confirm the suspected transmission of the pathogen from animal to human. All isolates were of serotype 4b with identical pulsotype. To the best of our knowledge, this is the first case of cutaneous listeriosis in which the evidence for zoonotic transmission of L. monocytogenes is supported by genotyping methods.  相似文献   

11.
The paper analyses the results of a survey of 37 Russian biosphere reserves using questionnaires concerning the presence of alien species of mammals, their pathways of penetration, and their impacts on protected ecosystems. The penetration of alien mammals into terrestrial ecosystems of Russia is extensive, both in places with maximum human environmental impact (inhabited areas and agricultural lands) and in biosphere reserves with minimal human impact. There are 62 mammal species registered as alien in Russian ecosystems and they account for 22% of the terrestrial mammal fauna of Russia. The percentage of alien species in biosphere reserves is 32.6% at most. In most regions, Castor fiber, Ondatra zibethicus, Nyctereutes procyonoides, Canis familiaris, Neovison vison and Sus scrofa are very dangerous, and both Castor fiber and Sus scrofa can have environment‐forming impacts.  相似文献   

12.
The use of transdermal medications in cats has become popular in veterinary medicine due to the ease of administration compared to oral medication. However, the research to support systemic absorption of drugs applied to the pinna after transdermal administration in cats is limited. The aim of this study was to characterize the percutaneous absorption pharmacokinetics of methimazole in a lipophilic vehicle compared to methimazole in Pluronic® lecithin organogel (PLO) using a finite dose applied to feline ear skin in an in vitro Franz cell model. The two formulations of methimazole (10 mg) were applied to the inner stratum corneum of six pairs of feline ears. The receptor medium was sampled up to 30 h post–administration, and methimazole concentrations were measured using high‐performance liquid chromatography (HPLC). Histological examination of all ears was undertaken as small differences in the thickness of ear skin may have contributed to inter‐individual differences in methimazole absorption between six cats. Methimazole was absorbed more completely across the pinnal skin when administered in the lipophilic vehicle compared to administration in the PLO gel (P < 0.001).  相似文献   

13.
Little is known about the transdermal penetration of hydrocortisone in the horse and, although commercial formulations containing hydrocortisone are registered for topical use in the horse, there have been no studies investigating the movement of this glucocorticoid through different regions of equine skin. Skin was harvested from the thorax, groin and leg (dorsal metacarpal) regions of five Thoroughbred geldings and frozen (-20 degrees C) until required. Defrosted skin was placed in Franz-type diffusion cells and the amount of radiolabelled ((3)H) hydrocortisone, in a saturated solution of unlabelled hydrocortisone in 50% ethanol (w/w), which penetrated through and remained within skin samples was measured over 24 h. Significantly higher (P < 0.001) maximum flux (J(max); mol/cm(2)/h) was measured when hydrocortisone was applied to skin from the leg, compared to thorax and groin, although significantly less hydrocortisone (P < 0.001) was retained within skin from the leg at 24 h. Topical application of hydrocortisone in a vehicle containing ethanol would penetrate faster through leg skin from the lower leg when compared with the thorax or groin, which depending on cutaneous blood flow, may result in higher systemic drug concentrations or greater efficiency in treating local inflamed tissue.  相似文献   

14.
Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD. Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT‐PCR. Real‐time quantitative RT‐PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full‐length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll‐like receptor ligands lipopolysaccharide and poly I:C. Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.  相似文献   

15.
Diclofenac has been responsible for the deaths of millions of vultures on the Asian subcontinent. While the pathology of toxicity is well described, the mechanism of toxicity remains elusive. However, it was postulated that toxicity could be related to the unique avian renal vascular structure known as the renal portal valve and that that diclofenac altered valve functionality with subsequent renal ischaemia. While plausible, the valva renalis portalis has only been described in a small number of other bird species such as the chicken (Gallus domesticus), the domestic duck (Anas platyrhynchos domesticus) and ostrich (Struthio camelus) but not a raptor. The aim of this study was to evaluate the renal anatomy and related vasculature of the Cape griffon vulture (Gyps coprotheres) (CGV), a species sensitive to the toxic effects of diclofenac, using gross anatomy, histology and vascular casting. The vasculature of the vulture was found to be almost identical to that of the domestic chicken with the valva renalis portalis present in the v. iliaca externa between the v. renalis renalis cranialis and the v. renalis caudalus. The valve was ring-shaped with finger-like processes and histologically was composed of smooth muscle. The valve was also well vascularized and was associated with a nerve plexus. Based on the findings of this study, the proposed mechanism of toxicity is anatomically possible.  相似文献   

16.
A dog being treated with immunosuppressive doses of prednisone and azathioprine for pancytopenia of unknown origin, developed, over a 2‐week period, multiple erythematous nodular lesions in the skin including footpads. Skin samples revealed lesions identical to those of human bacillary angiomatosis (BA). The nodules were composed of multifocal proliferations of capillaries, each lined by protuberant endothelial cells. The capillary clusters were separated by an oedematous connective tissue, lightly infiltrated with degenerate inflammatory cells, including neutrophils and macrophages. Tissue sections stained with Warthin–Starry silver stain revealed large numbers of positively stained bacilli in the stromal tissue, most heavily concentrated around the proliferating capillaries. Lesions of vascular degeneration and inflammation were evident. Bartonella vinsonii subsp. berkhoffii genotype 1 was independently amplified and sequenced from the blood and the skin tissue. The pathognomonic nature of the histological lesions, demonstration of compatible silver‐stained bacilli in the tissue, and identification of B. vinsonii subsp. berkhoffii in the blood and tissue indicates that this is most likely the aetiologic agent responsible for the lesions. Antibiotic therapy was successful in resolving the nodules. It would appear that B. vinsonii subsp berkhoffii, like Bartonella henselae and Bartonella quintana, has the rare ability to induce angioproliferative lesions, most likely in association with immunosuppression. The demonstration of lesions identical to those of human BA in this dog is further evidence that the full range of clinical manifestations of human Bartonella infection occurs also in canines.  相似文献   

17.
This study investigated the effects of freezing canine skin on the penetration kinetics of hydrocortisone. Skin samples from three dogs were used for in vitro penetration studies commencing on the day of skin collection (fresh skin) and again after freezing at -20 degrees C for 1, 4, 8 and 12 months. When the data from the dogs was averaged, the pseudo-steady-state flux (Jss) of hydrocortisone through skin frozen for any duration was significantly (P < 0.023) greater than through fresh skin and there was a positive relationship (P < 0.007) between the length of freezing and DeltaJss. For all dogs, the lag times (tlag) calculated for hydrocortisone penetration were significantly (P < 0.029) shorter through skin that had been frozen, compared with fresh skin. However, the shapes of the permeation profiles of hydrocortisone appeared similar through the fresh and frozen dog skins and no differences were detected between the groups on histological examination. The results of this study have shown that freezing dog skin at -20 degrees C can significantly increase the transdermal penetration of hydrocortisone in vitro, and that the extent of this enhancement can increase with duration of freezing.  相似文献   

18.
The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17β‐estradiol, 17α‐estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism‐based inhibitions were examined. 7‐benzyloxyresorufin (BR) and 7‐benzyloxy‐4‐trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7‐benzyloxyresorufin O‐dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism‐based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17β‐oestradiol. Mechanism‐based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α‐oestradiol through the mechanism‐based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.  相似文献   

19.
The bioavailability of three formulations of ivermectin was determined following oral administration to dogs. The average peak plasma level (C max) of ivermectin administered in the standard tablet formulation at 6 and 100 µg/kg of body weight was 2.97 and 44.31 ng/g, respectively. This suggest dose-dependent pharmacokinetics.C max and total ivermectin bioavailability, as assessed from the area under the plasma curve (AUC), were similar between two tablet formulations of ivermectin administered at 100 µg/kg. Furthermore,C max was similar following administration of radiolabelled ivermectin at 6 µg/kg in either a beef-based chewable formulation or in the standard tablet formulation.  相似文献   

20.
Swimmer's itch is caused by the penetration of free‐swimming larvae of trematodes of the family Schistosomatidae in human skin. It is usually reported in people engaged in recreational water activities in freshwater bodies and in most of cases, it is provoked by bird schistosomes of the genus Trichobilharzia. In the summer 2017, many cases of dermatitis were recorded in people bathing in the waters of the Albano Lake (Rome, Italy) and a parasitological investigation was carried out in order to ascertain the causative agent of these cases. Snails of the family Lymnaeidae, natural intermediate hosts of bird schistosomes, were collected from lake shallow waters to detect the presence of trematodes of the genus Trichobilharzia. Pools of maximum 10 snails were placed in Petri dishes, and cercarial emergence was stimulated exposing snails to strong artificial light intensity at 25°C. Three hundred and thirty‐seven snails were collected and screened for the shedding of cercariae. Furcocercariae of the family Schistosomatidae, with a morphology overlapping that of the genus Trichobilharzia, were detected in seven Petri dishes. Assuming that in each positive Petri dish just one snail was shedding furcocercariae, the minimum infectious rate was 2.1%. Molecular analysis of furcocercariae allowed ascribing them to the species Trichobilharzia franki. Snails of the species Radix auricularia were identified as intermediate hosts of the parasite. This is the second record of T. franki causing cercarial dermatitis in Central Italy, the third in Italy. The 2017 was in Italy exceptionally warm and dry. Trematodes are sensitive to changes in temperature, being cercarial production and emission rates temperature dependent. Small increases in water temperature would speed up parasite development and transmission, leading to a manifold increase in cercarial emergence. Moreover, high temperatures raise chances to acquire the infection, due to increased time spent in water by people.  相似文献   

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