leishmaniasis caused by the intracellular protozoan leishmania is the second most important parasitic disease after malaria , affecting humans and animals in wide areas of the americas , asia , africa and europe .
domestic dogs are considered the main reservoir of leishmania infantum , and a risk factor for anthroponotic human visceral infection .
canine leishmaniasis ( canl ) is endemic and affects millions of dogs in asia , europe , north africa and south america , and is currently an emergent disease in north america .
it has been estimated that at least 2.5 million dogs are infected in southwestern europe ( moreno and alvar , 2002 , athanasiou et  al . , 2012 ) .
the infection is spreading to non - endemic areas and cases of canl are reported with increasing frequency in northern european countries and in the united kingdom , even in dogs that have never visited endemic zones ( shaw et  al . , 2009 ) .
the introduction and spread of the disease to regions where infections had not previously been observed may create new epidemiological scenarios , further complicating the zoonotic potential and control of the disease . in the absence of effective human and canine vaccines , the only feasible way to treat and control leishmaniasis is through the use of affordable chemotherapy .
meglumine antimoniate is one of the most common drugs used in europe to treat canl ( solano - gallego et  al . , 2011 ) .
the combination of meglumine antimoniate with allopurinol is considered to be the most effective therapy and constitutes the first line protocol against canl ( mir et  al .
anti - leishmania therapy can usually decrease the parasite load , although it is extremely difficult to achieve a parasitological cure in dogs .
for this reason , the majority of short - term therapeutic interventions are usually followed by a relapse within 1 year of discontinuing treatment ( baneth and shaw , 2002 , ikeda - garcia et  al .
as infected dogs never achieve parasitological cure and are frequently treated several times with the same compounds , selection and transmission of antimony - resistant parasites could appear ( campino and maia , 2012 , sereno et  al . , 2012 ) .
therapeutic failure and relapse in leishmaniasis is known to have a multifactorial origin , involving features related to the host ( immunity , genetic and nutritional factors , among others ) , the drug ( quality , pharmacokinetics ) and the parasite ( drug resistance , increased infectivity , coinfection with other pathogens ) ( vanaerschot et  al . , 2014 ) . while the occurrence of leishmania resistance to pentavalent antimonials is well known in human medicine ( croft et  al . , 2006 ,
l.  infantum isolates taken from dogs which had received several treatment courses have demonstrated contradictory results , with no differences in susceptibility in isolates from treated and untreated dogs ( carri and ports , 2002 ) , or a decreased susceptibility to antimony after several therapeutic interventions ( gramiccia et  al . , 1992 ) . in the present study
we investigate if a l. infantum isolate from a dog with canl relapse after a therapeutic intervention has developed drug resistance as a factor responsible of therapeutic failure , and the mechanism of resistance developed .
the singularity of this study was based on the use of leishmania isolates at the time of diagnosis and after therapeutic interventions , facilitating the comparative studies .
it is important to detect resistant parasites in dogs with therapeutic failure in order to avoid and reduce the emergence and spread of resistant parasites throughout the canine populations ; a factor that requires special consideration when dogs are treated with the same anti - leishmania drugs that are available for human visceral leishmaniasis .
trivalent antimony ( sb ) , amphotericin b ( amb ) , paromomycin , triton x-100 , paraformaldehyde , 4,6-diamidino-2-phenylindole dilactate ( dapi ) , n - dodecyl--d - maltoside ( ddm ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) and phorbol 12-myristate 13-acetate ( pma ) were obtained from sigma - aldrich ( st . louis , usa ) .
a five - years - old male boxer dog was positively diagnosed for leishmaniasis through detection of anti - leishmania specific antibodies by indirect immunofluorescence ( igg titer    1280 ) and by detection of l. infantum parasites with qpcr ( 1.06 parasites/l ) using specific primers for kdna as described ( corpas - lpez et  al . , 2015 ) .
the animal had many clinical signs compatible with canl , including onychogryphosis , adenopathy , skin lesions and uveitis , among others .
briefly , each symptom was assigned a value according to the severity of it ( 0  =  absence ; 1  =  mild ; 2  =  moderate ; 3  =  severe ) and the cs was the sum of the values for every symptom . the initial clinical scores ( cs  =  18 ) rises after the first treatment series ( cs  =  24 ) and declined to the initial cs value after the second one ( cs  =  18 ) . in the follow - up period without drug pressure , the dog showed a higher cs value ( cs  =  20 ) , due to the aggravation of eye symptoms ( uveitis , keratitis ) besides increased creatinine .
after the trial , the dog was returned to its owner with full information on its clinical and parasitological state , who decided to treat the animal with other drugs .
the dog was housed in the facilities of an animal shelter , with access to water and food ad libitum .
the building where the dog was housed was protected with adequate mosquito nets to prevent sandflies from entering .
animal experiments were approved by the ethics committee for animal experimentation of the university of granada ( protocol : 450 - 26113 ) and all procedures were carried out according to the international guidelines and the european union directive ( 86/609/eec ) .
the dog was treated with meglumine antimoniate ( glucantime ) at 100  mg / kg / day administered subcutaneously for two 28-day periods , separated by a 30-day interval .
the parasites were isolated from bone marrow and ganglion aspirates in evans modified tobie s medium ( emtm ) .
after their isolation , parasites were cultured at 28  c in rpmi-1640 medium supplemented with 10% heat - inactivated fetal bovine serum ( hifbs , invitrogen ) .
after 6 passages ( 2 months ) , parasite strains were labeled , mixed with dmso and stored in liquid nitrogen . to determine parasite susceptibility to sb and other anti - leishmania drugs such as amb , paromomycin and miltefosine ,
2    10 promastigotes were incubated in 96-well plates ( 100  l ) at 28  c for 72  h in the presence of increasing drug concentrations before determining cell proliferation by the mtt colorimetric assay as described previously ( gmez - prez et  al . , 2015 ) .
the 50% effective concentration ( ec50 ) was defined as the drug concentration required for half maximal inhibition of cellular growth rate .
the ec50 for each line was calculated by nonlinear regression analysis using sigmaplot 2000 software for windows ( spss inc . ,
resistance index ( ri ) was calculated by dividing the ec50 obtained for the resistant line by the ec50 obtained for the parental susceptible line .
thp-1  cells were grown at 37  c and 5% co2 in rpmi-1640 supplemented with 10% hifbs , 2  mm glutamate , 100  u / ml penicillin and 100  g / ml streptomycin .
3    10  cells / well , in 96-well plates , were differentiated to macrophages with 20  ng / ml of pma treatment for 48  h followed by 24  h of culture in fresh medium ( gmez - prez et  al . , 2014 ) . to determine the susceptibility of intracellular leishmania amastigotes to sb , sb in the form of meglumine antimoniate ( glucantime ) , amb , paromomycin and miltefosine ,
late stationary - phase promastigotes were used to infect macrophage differentiated - thp-1  cells at a macrophage / parasite ratio of 1:10 .
infected cell cultures were then incubated at different compound concentrations for 72  h , as described previously ( gmez - prez et  al . , 2014 ) .
samples were then fixed for 30  min  at 4  c with 2% paraformaldehyde in pbs , followed by permeabilization with 0.1% triton x-100 in pbs for 30  min .
the percentage of infection and the mean number of amastigotes from infected macrophages were determined in 200 macrophages / well .
promastigotes ( 1    10 per ml ) were incubated with 100  m sb for 60  min  at 28  c , then centrifuged and the resultant pellet stored at 80  c until antimony accumulation was measured as described previously ( manzano et  al . , 2013 ) .
to determine antimony efflux , the different isolates were incubated with compensated sb concentrations ( 50  m for line 576 - 1 and 500  m for line 576 - 3 ) in culture medium at 28  c for 1  h to allow a similar labeling in the leishmania lines .
the parasites were then washed with pbs , resuspended in culture medium at 28  c and pelleted at different times ( 0 , 30 , 60 and 120  min ) . the cell pellet was dissolved in 200  l of concentrated nitric acid at room temperature for 24  h. the sample was diluted to 3  ml with distilled water and then injected into an inductively coupled plasma mass spectrometer ( icp - ms ; perkinelmer ) for quantitation .
antimony was measured at its m / z ratios of 121 and 123 with rhodium as an internal standard .
genomic dna and total rna were extracted from the leishmania isolates using the rneasy plus mini kit ( qiagen ) and dneasy blood and tissue kit ( qiagen ) , respectively .
isolated rna was transcribed into cdna using the qscript cdna synthesis kit ( quanta biosciences , inc ) following the manufacturer s instructions .
the synthetized cdnas were diluted 1:10 and 1:50 and specific fragments amplified with sense and antisense primers ( 5-acgatggcgtccaagtacag ) and ( 5-gagtatgcgaggtgacaagtc ) for aquaglyceroporin-1 ( aqp1 ) , ( 5-gctgtacatgcagaacgtctc ) and ( 5-acgtactcgtgctccacttc ) for mrpa , ( 5-cttcaactgcatcctcttcg ) and ( 5-gcagtccatcgaatcgcagg ) for ornithine decarboxylase  ( odc ) , ( 5-cctcgtgtgccgctatctag ) and ( 5-gcatgcgctgcacaaggtca ) for -glutamylcysteine synthetase ( -gcs ) , and ( 5-gaagtacacggtggaggctg ) and ( 5-cgctgatcacgaccttcttc ) for gapdh using 35 amplification cycles at an annealing temperature of 54  c .
pcr products were electrophoresed on 1% agarose gel , stained with ethidium bromide , viewed under a uv illuminator and the relative intensity against gapdh was measured as an internal control .
the full - length coding sequence of aqp1 from l. infantum ( genedb l. infantum , accession code linj.31.0030 ) was isolated from the genomic dna of the clinical isolates by pcr using sense and antisense primers ( 5-caacagcacgcgcacttc ) and ( 5-cagtcagca cgagctgtg ) .
a single - band 945  bp pcr product was obtained , cloned in the pgem - t vector system ( promega ) and sequenced .
intracellular non - protein thiol levels were measured by flow cytometry using celltracker , as described previously ( garca - hernndez et  al . , 2012 ) .
promastigotes ( 1    10 per ml ) were washed twice with pbs , incubated with 2  m celltracker for 15  min  at 37  c , washed again with pbs and analyzed by flow cytometry using a facscan flow cytometer ( becton - dickinson , san jose , ca ) equipped with an argon laser operating at 488  nm .
fluorescence emission between 515 and 545  nm was quantified using cell quest software . for preparation of total protein lysate , l. infantum promastigotes ( 3    10 per ml ) were harvested , washed in pbs , lysed by the addition of lysis buffer ( 2% ddm , 150  mm nacl , 50  mm tris - hcl ph 7.4 plus a protease inhibitor cocktail from sigma ) and incubated at 4  c for 1  h. the clarified lysate was mixed with 2    laemmli sample buffer and resolved by sds - polyacrylamide gel electrophoresis ( sds - page ) . the samples ( 25  g protein per lane ) were resolved by 10% sds - page and transferred to pvdf membranes .
membranes were subsequently blocked at room temperature for 1  h in 5% skimmed milk in pbs with 0.05% tween 20 ( pbs - t ) and washed three times with pbs - t .
immunodetection was performed by incubating the blocked membranes at room temperature for 1  h in the presence of antibodies against cytosolic tryparedoxin ( ctxn ) , mitochondrial tryparedoxin ( mtxn ) , cytosolic tryparedoxin peroxidase ( ctxnpx ) , mitochondrial tryparedoxin peroxidase ( m - txnpx ) , trypanothione synthetase ( trys ) and trypanothione reductase ( tr ) : anti - ctxn ( dilution 1:3000 ) ( castro et  al . , 2004 ) , anti - mtxn ( 1:3000 ) ( castro et  al . , 2004 ) , anti - ctxnpx ( 1:3000 ) ( castro et  al . , 2002 ) , anti - mtxnpx ( 1:3000 ) ( castro et  al . , 2002 ) , anti - trys ( 1:1000 ) ( sousa et  al . , 2014 ) or anti - tr ( 1:1000 ) ( h. castro and a. toms , personal communication ) .
the membranes were washed and incubated with hrp anti - mouse igg ( 1:5000 ) or hrp anti - rabbit igg ( 1:5000 ) ( promega ) .
immunoreactive proteins were viewed using a chemiluminescence detection kit ( pierce ecl western blotting substrate ) according to the manufacturer s instructions .
in this study , we have analyzed parasites isolated from ganglion aspirate at the time of diagnosis ( mcan / es/2014/dp576 - 1 , abbreviated as line 576 - 1 ) and 30 days after two therapeutic interventions with glucantime ( 100  mg / kg / day , subcutaneously for 28 days ) , separated by a 30-days interval ( mcan / es/2014/dp576 - 3 , abbreviated as line 576 - 3 ) .
several factors could have contributed to the absence of a therapeutic response to glucantime in this dog , including factors related to the parasite , the drug or the host .
we evaluated whether line 576 - 3 presents modifications in drug susceptibility that could be associated with the development of resistance and , in positive case , we analyzed the mechanism of resistance . the susceptibility profile to sb of the different isolates of l. infantum
was analyzed in promastigotes and intracellular amastigotes ( table  1 ) , the ec50 values for line 576 - 3 were significantly higher than for line 576 - 1 , in both promastigotes and intracellular amastigotes ( table  1 ) .
these data suggest that parasites developed resistance to sb after the second therapeutic intervention , presenting a ri of around 6 for promastigotes and greater than 3 for intracellular amastigotes ( table  1 ) .
considering glucantime was the drug used to treat this infected dog , we analyzed the susceptibility profile to this drug in intracellular amastigotes from line 576 - 3 , obtaining an ec50 of 159.3  m , 3.3-fold higher than line 576 - 1 ( table  1 ) . our data clearly substantiate that relapse after the second therapeutic intervention was due to the acquisition of drug resistance in line 576 - 3 .
we also studied the cross - resistance profile to other anti - leishmania drugs , including amb , miltefosine and paromomycin ( table  1 ) .
the results showed that intracellular amastigotes from line 576 - 3 present a significant cross - resistance to paromomycin ( ri around 2.8 ) .
however , we did not observe any cross - resistance to amb and miltefosine in promastigotes and intracellular amastigotes .
as previously described , promastigotes of l.  donovani resistant to antimony present a significant cross - resistance to paromomycin and not to amb and miltefosine ( garca - hernndez et  al . ,
2012 ) ; however , other studies using antimony resistant and sensitive field l.  donovani isolates shown equal susceptibility to paromomycin ( kulshrestha et  al . ,
these results support the variability in the response of leishmania to drugs . additionally , drug pressure could induce genomic changes in parasites that could be responsible of the resistance to paromomycin in intracellular amastigotes . furthermore , lack of correlation between the promastigote and intracellular amastigote susceptibilities to paromomycin observed in the present study , supports the intracellular amastigote model as more appropriate approach for susceptibility studies , as has been previously reported ( vermeersch et  al .
we then tried to elucidate the mechanism of antimony resistance in line 576 - 3 by analyzing antimony uptake and the expression profiles of known genes involved in transport and thiol based redox metabolism .
a decrease in drug concentration within the parasite , either by reducing drug uptake or by increasing efflux / sequestration of the drug , represents the primary mechanism of antimonial resistance in leishmania ( brochu et  al . , 2003 ) .
to determine whether a reduction in sb uptake was one of the mechanisms of resistance developed in line 576 - 3 , the intracellular accumulation of this metal ion was measured by icp - ms after incubation with 100  m sb for 1  h. sb accumulation was found to be significantly lower ( 90% ) in the resistant line 576 - 3 compared to line 576 - 1 ( fig . 
the lower sb accumulation in line 576 - 3 could explain the resistance to antimonials . to determine whether the reduced level of accumulation was due to an increase in antimony efflux
, both l. infantum lines were loaded under conditions that yielded similar amounts of intracellular antimony and the portion of antimony retained in the parasites was measured at different times .
the efflux of sb was found to be time - dependent and similar in both leishmania lines ( fig . 
1b ) , thus confirming that the lower sb accumulation was not due to increased efflux activity , but rather a decrease in drug uptake .
in leishmania , antimony uptake is mediated by the transporter aqp1 ( gourbal et  al .
previous studies indicated aqp1 rna levels were down - regulated in several leishmania promastigote species ( l. tarentolae , l. major and l. infantum ) that were experimentally resistant to antimonials ( marquis et  al . , 2005 ) , as well as in different antimony - resistant leishmania donovani clinical isolates ( decuypere et  al . , 2005 ,
in general , the level of aqp1 transcript correlated well with the accumulation of sb and resistance levels in leishmania parasites ( marquis et  al . , 2005 ) .
aqp1 gene expression in lines 576 - 1 and 576 - 3 was analyzed by rt - pcr to ascertain whether there was a correlation between aqp1 gene expression and antimony susceptibility and accumulation .
the results showed that aqp1 expression in the resistant line 576 - 3 was around 5-fold lower than the expression in the susceptible line 576 - 1 ( fig . 
gene deletion and point mutations in aqp1 have been reported in antimony - resistant leishmania parasites ( monte - neto et  al . ,
the full - length coding sequence of the l. infantum aqp1 gene was cloned and sequenced from the 576 - 1 and 576 - 3 isolates .
only one of five clones sequenced presented a point mutation that lead to a premature stop codon , while the rest of the clones did not show any point mutations or deletions that could be associated with a reduction in aqp1 activity .
our data therefore support the suggestion that down regulation of aqp1 is the main resistance mechanism in l. infantum isolate from a case of canl with therapeutic failure .
abc transporter mdr - related proteins ( mrpa / pgpa ) are known to be involved in antimonial - resistance in leishmania through sequestration of the metal - thiol conjugates in an intracellular organelle located close to the flagellar pocket ( lgar et  al . , 2001 ) .
the role of mrpa in conferring antimony resistance by sequestration of metal - thiol conjugates has also been reported in clinical isolates of leishmania ( mukherjee et  al . , 2007 ) .
mrpa gene expression analysis in antimony susceptible and resistant l. infantum lines was determined by rt - pcr using specific primers .
the results showed that there were no differences in expression levels between susceptible and resistant lines ( fig . 
1d ) , suggesting that mrpa is probably not responsible for the defect in antimonial accumulation in resistant l. infantum line 576 - 3 .
an increase in thiol levels is considered to be one detoxification mechanism described in antimony - resistant l.  donovani isolates ( mandal et  al .
, 2011 , rai et  al . , 2013 ) . in order to study the involvement of thiols in the antimony resistant line
the results , quantified in terms of relative fluorescence units ( rfu ) , demonstrate that the resistant line 576 - 3 showed 1.5-fold higher intracellular non - protein thiol levels ( 239.17    38.43 rfu ) than the susceptible line 576 - 1 ( 152.57    23.38 rfu ) , and the difference showed statistical significance ( p  <  0.005 ) .
trypanothione ( t[sh]2 ) , a conjugate of glutathione ( gsh ) and spermidine , is one of the major reduced thiols in leishmania parasites ( fairlamb and cerami , 1992 ) . increased levels of t[sh]2 are achieved through overexpression of -gcs , the rate limiting enzyme of gsh biosynthesis , and odc , an enzyme involved in spermidine biosynthesis ( grondin et  al . , 1997 ,
2003 ) . mechanisms of antimony resistance based on t[sh]2 overproduction through overexpression of -gcs / odc have previously been described in both experimental leishmania antimony - resistant lines and in some clinical isolates of antimony - resistant l.  donovani ( haimeur et  al . , 1999 , decuypere et  al . , 2005 ,
, 2006 , mukherjee et  al . , 2007 , goyeneche - patino et  al . , 2008 ,
this led us to probe the possible relationship between intracellular thiol content and the gene expression status of -gcs , odc and t[sh]2 synthetase ( trys ) .
the results show that odc exhibited increased rna levels ( around 2-fold ) in the antimony - resistant line 576 - 3 compared to the susceptible line 576 - 1 ( fig . 
2a ) , whereas -gcs and trys expression levels were unaffected ( fig .  2 ) .
therefore , overexpression of odc may contribute to increased levels of t[sh]2 in the 576 - 3 antimony - resistant parasites .
the above results concur with previous reports that revealed an increased expression of odc in antimony - resistant isolates of l.  donovani ( mukherjee et  al . , 2007 ) . in trypanosomatids
tr is a flavoenzyme that catalyzes the nadph - dependent reduction of trypanothione disulfide ( ts2 ) to t[sh]2 .
previous reports have described the amplification of the tr gene in antimony - resistant l.  donovani field isolates ( mittal et  al . , 2007 ) .
using western blot analysis we observed a 4-fold increase  in the levels of tr protein in the resistant line versus the susceptible one ( fig . 
2b ) . increased levels of tr will help to maintain increased levels of t[sh]2 , which is required for conjugation with antimony and to overcome the inhibitory effect of sb . in leishmania the tryparedoxin / tryparedoxin peroxidase ( txn / txnpx ) system
is crucial to defend parasites against the oxidative stress that can be found in different compartments ( floh et  al . , 1999 ) . increased levels of both proteins
have been detected in antimony - resistant l.  donovani field isolates ( wyllie et  al . , 2010 ) .
leishmania have two txnpx , one localized in the cytoplasm ( ctxnpx ) and the other in the mitochondrion ( mtxnpx ) , which protect the cell from peroxide - induced damage ( castro et  al . , 2002 ) .
proteomic analysis of experimental antimony - resistant lines from l.  braziliensis and l. infantum showed an overexpression of ctxnpx ( matrangolo et  al .
additionally , overexpression of ctxnpx confers resistance to antimony in l. braziliensis and l.  donovani ; however , overexpression of ctxnpx in l. infantum does not seem to be directly associated with resistance to sb ( lyer et  al .
therefore , we studied whether txn / txnpx proteins were upregulated in the antimony - resistant line 576 - 3 .
the results showed an overexpression of mtxn ( by around 12-fold ) and mtxnpx ( around 4-fold ) , while no differences were observed in the expression of ctxn and ctxnpx ( fig . 
the overexpression of the mitochondrial pathway could induce an increase in t[sh]2 levels which forms a complex with sb , thus inactivating the toxic effects of antimony and enhancing the parasite s antioxidant defense .
the present study has characterized the mechanisms of antimony resistance for the first time in a clinical isolate of l. infantum from a dog with naturally acquired canl .
we confirmed that l. infantum line 576 - 3 confers resistance against sb by significantly decreasing expression of the aqp1 transporter which leads to a reduction in intracellular accumulation of sb .
other factors including an increase in thiols levels and overexpression of enzymes involved in thiol metabolism could also contribute to the sb detoxification mechanism .
a major contribution of this study in a canine l. infantum isolate is to find an antimony - resistant mechanism similar to that previously described in other human clinical isolates .
therefore , more studies are required in l. infantum isolates from dogs that relapse after treatment to better understand the therapeutic failure in canl .
the use of naturally infected dogs and a controlled therapeutic intervention represent an excellent experimental strategy to study therapeutic failure / resistance and search for more effective therapeutic strategies following relapse , to avoid the spread of drug - resistant lines in endemic areas in attempts to control the disease . however , the use of naturally infected dogs has limitations mainly due to the economic costs and the requirements and controls to prevent new reinfections and follow - up analyses for drug efficacy .
 visceral leishmaniasis ( vl ) caused by the protozoan parasite leishmania infantum , is one of the most important zoonotic diseases affecting dogs and humans in the mediterranean area . 
 the presence of infected dogs as the main reservoir host of l. infantum is regarded as the most significant risk for potential human infection . 
 we have studied the susceptibility profile to antimony and other anti - leishmania drugs ( amphotericin b , miltefosine , paromomycin ) in leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate ( subcutaneous glucantime , 100  mg / kg / day for 28 days ) . after the therapeutic intervention , these parasites were significantly less susceptible to antimony than pretreatment isolate , presenting a resistance index of 6-fold to sbiii for promastigotes and > 3-fold to sbiii and 3-fold to sbv for intracellular amastigotes . 
 the susceptibility profile of this resistant l. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels . 
 additionally , other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism , such as ornithine decarboxylase , trypanothione reductase , mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase , could contribute to the resistance as antimony detoxification mechanisms . 
 a major contribution of this study in a canine l. infantum isolate is to find an antimony - resistant mechanism similar to that previously described in other human clinical isolates . 
Introduction
Materials and methods
Results and discussion
Conclusions