human lagochilascariosis is caused by lagochilascaris minor , an infection that has been reported in individuals of both sexes .
lagochilascariosis is considered an emerging helminthosis described in the american continents and its diagnosis is underestimated .
the main clinical symptoms in humans are chronic lesions with abscess formation , usually affecting the neck and head tissues [ 2 , 3 ] .
sometimes the parasite invades the pulmonary tissue and central nervous system , leading to death [ 4 , 5 ] .
frequently , l. minor lesions contain multiple stages of the parasite ( eggs , larvae , and adult worm ) , indicating autoinfection and favoring the development of chronic disease .
the natural life cycle of l. minor and its infection mechanism remain unknown . an experimental heteroxenous life cycle for the parasite has been described in mice and domestic cats [ 5 , 6 ] as well as in wild rodents and cats .
the capacity of l. minor to migrate across different human tissues is also observed in animal models of the disease . in mice orally inoculated with l. minor infective eggs
, hatched larvae can be observed migrating in the intestinal tract ( 612  h ) . after hatching , 3rd stage larvae ( l3 )
migrate through intestinal mucosa into vessels and hepatic parenchyma ( 12  h ) and disseminate to other tissues .
granulomatous lesions containing encysted l3 larvae have been found 30 days after infection in lungs , skeletal muscles , subcutaneous tissues , and lymph nodes . in cats that eat carcasses of infected mice ,
l3 migrate from the stomach ( 6  h ) to the upper portions of the digestive tract , where they develop into 4th - stage larvae ( l4 ) , reaching maturity after 12 days of infection .
adult worms located in the esophagus , pharynx and trachea , and rhino - oropharynx and cervical lymph nodes , can expel eggs that will be found in feces of infected cats .
we recently showed that balb / c mice are more resistant to l. minor infection than c57bl/6 mice , having less severe lesions in the lungs , lower numbers of nodules with encysted larvae , fewer adult worms , and higher serum levels of ifn- .
the availability of isogenic strains of mice with different genetic backgrounds and the same h-2 haplotype has enabled the study of this host - parasite relationship , which is crucial to the establishment of susceptibility or resistance to infection .
the manipulation of the host immune response system by l. minor antigens may be a key determinant of survival within the mammalian host .
therefore , the aims of this study were to evaluate infection in b10.a and a / j mice ( both h-2 ) , compare the survival rate and tissue lesions , and analyze the proliferative response and production of ifn- and il-10 in cultures of spleen cells from normal and immunized mice stimulated with the crude extract of the parasite ( ce ) or concanavalin a ( cona ) .
six- to eight - week - old a / j and b10.a male mice were purchased from the university of so paulo animal facility .
eggs from the parasite were collected from feces of felis domesticus experimentally infected with a human isolate of l. minor .
feces from infected animals were subjected to hoffman 's method   and kept in culture in formalin solution ( 1% ) at room temperature for 30 days .
after the development of infective eggs containing third - stage ( l3 ) larvae , cultures were submitted to faust 's method   for optimal recovery of eggs free from fecal debris .
egg suspensions were washed five times ( 20 minutes/4.000  rpm ) with phosphate - buffered saline ( pbs , ph 7.4 ) and transferred to a graduated centrifuge tube .
the final concentration was adjusted to 2  10  eggs / ml and then used to infect the mice . in total
, 60  a / j and 66  b10.a mice were orally inoculated with a suspension of 2  10  200  l. minor eggs per animal . in total , 20 
a / j and 26  b10.a animals were followed for one year to determine survival rates .
forty animals were sacrificed ( 5 per day ) at different time points ( 30 , 45 , 60 , 90 , 120 , 150 , 180 , and 210 days postinfection ) and submitted to necropsy for collection of organs for histopathology .
additionally , 40  a / j and 40  b10.a uninfected mice received saline orally and were used as follows : 20 animals served as controls for mortality ( followed for one year ) , and 20 were necropsied and served as controls for histopathology ( 5 animals sacrificed at each time point : 30 , 90 , 150 and 210 days after infection ) .
sections of spleen , lung , lymph node , liver , muscle , and subcutaneous nodules derived from groups of 5 uninfected and 5 infected b10.a and a / j mice were collected 30 to 210 days after infection , fixed in 10% neutral buffered formalin , embedded in paraffin and subsequently stained with hematoxylin and eosin ( h & e ) , masson 's trichrome for detection of fibrosis   and lunas ' staining   for characterization of eosinophils . the granulomas were classified as primary or exudative granuloma , secondary or exudative - productive granuloma and tertiary or productive - fibrotic granuloma .
the secondary granuloma was characterized by a necrotic zone of variable extension , a cellular exudation zone , and presence of epithelioid cells .
the tertiary granuloma was characterized by less necrotic cells and diminished cellular exudation with predominance of conjunctive tissue [ 14 , 15 ] . 
 l.
minor - infected mice were euthanized 6090 days postinfection . encysted l3 larvae were collected from nodules under sterile conditions upon puncturing to allow spontaneous release of the parasite . after 20 washes in pbs
, live l3 larvae were resuspended in pbs and sonicated in an ika - tk8 disruptor . the crude extract ( ce ) of l3 larvae
was then centrifuged at 50000    g for 1  h at 4c .
the supernatant was collected and the protein content was estimated by the micro bca method ( pierce , usa ) .
seven naive mice of each strain were immunized with 10  g of ce of l. minor in a volume of 0.1  ml .
mice were immunized subcutaneously four times at one - week intervals , and their spleens were collected one week after the final immunization for evaluation of lymphocyte proliferation and cytokine production .
spleens from a / j and b10.a mice were collected in rpmi medium supplemented with 2  mm l - glutamine , 1  mm sodium pyruvate , 100  u / ml penicillin , 100  g / ml streptomycin ,   2  10  m 2-mercaptoethanol , 1% nonessential amino acids and 5% fetal calf serum ( fcs ) and were   passed through nylon mesh to obtain spleen cells .
after erythrocyte lyses , the cell suspension was washed twice in supplemented rpmi medium and plated in triplicate at 5  10 cells / well of 96-well round - bottomed plates in a total volume of 200  l medium .
spleen cells were stimulated with 5  g / ml of cona ( sigma ) or 5  g / ml of ce of l. minor .
this concentration of cona and ce produced optimum responses in immunized animals when tested in within a range of 0.5 and 50  g / ml . mitogen- and antigen - stimulated cultures were maintained for 5 days at 37c and 5% co2 in a humidified atmosphere .
h - thymidine was added ( 0.5  ci per well ) for the last 18 hours of culture .
cells were then harvested on glass fiber filters , and the incorporated radioactivity was measured in a liquid scintillation counter .
data are indicated as counts per minute ( cpm ) . for analysis of secreted cytokines ,
5  10 spleen cells / well ( as above ) were plated in 96-well u - bottom tissue culture plates ( costar ) and stimulated with cona or ce ( as above ) . after 48 hours , cell - free culture supernatants were collected and stored at 80c until use . il-10 and
ifn were measured by a sandwich elisa using the opeia kit ( bd bioscience , usa ) according to the manufacturer 's instructions .
cytokine analysis was performed based on a standard cytokine concentration curve with a detection limit of 15  pg / ml for both il-10 and ifn. results are expressed in pg / ml .
the survival curve was analyzed using the kaplan and meier   method , and the differences between groups were tested using the log - rank test .
b10.a mice began to die on day 13 postinfection , reaching only 26% survival on day 340 , with a median survival time of 246.5 days .
in contrast , a / j mice began to die later , on day 252 of infection , reaching 90% survival on day 340 ( figure 1 ) .
a / j mice displayed significantly greater survival rates than b10.a throughout the entire period of infection ( p = .0003 ) . in the control group , only two b10.a mice and one a / j mouse died by day 340 postinfection . in the initial infection phase ( 0 to 30 days postinfection ) , a / j mice presented a primary granuloma in the lungs containing preserved larvae , perivascular and peribronchial moderate inflammatory infiltration of macrophages and discrete infiltration of neutrophils ( figure 2(a ) ) . in the intermediate infection phase ( 45 to 90 days postinfection ) , a secondary granuloma was observed with severe and diffuse inflammatory infiltration of foaming macrophages , multinuclear giant cells and a small number of eosinophils and fibroblasts in the lungs ( figure 2(c ) ) . in late infection phase ( 120 days postinfection ) , a tertiary granuloma was observed in the lungs , containing disrupted third stage larvae with moderate necrosis and destruction of the tissue , concentric fibrosis , and inflammatory infiltration foci composed of macrophages .  
in contrast , b10a mice in the initial phase of the infection ( 0 to 30 days postinfection ) , presented severe and diffuse inflammatory infiltration in the lungs , composed of macrophages , neutrophils and small number of eosinophils ( figure 2(b ) ) . in the intermediate phase ( 45 to 90 days postinfection )
, a secondary granuloma with severe and diffuse inflammatory infiltration was observed in the lungs with a predominance of macrophages and a small number of neutrophils ( figure 2(d ) ) . in late - phase infection
( 120 days postinfection ) tertiary granuloma with concentric fibrosis intercalated by foaming macrophages and multinuclear giant cells was observed in the lungs .
the moderate inflammatory infiltration of the lungs was diffuse and composed of macrophages and few neutrophils . in both mouse lineages ,
the liver developed large hepatocytes with bilobular nuclei and congestion with occasional perivascular inflammatory infiltrate .
spleen cells from a / j mice immunized with the crude extract of the parasite showed higher levels of proliferation in the presence of ce ( p = .01 ) when compared to nonimmunized controls ( figures 3(a ) and 3(b ) ) .
a / j immunized mice also exhibited a greater proliferative response against ce antigen compared to b10.a immunized mice ( p = .008 ) .
in addition , spleen cells of a / j and b10.a mice immunized with the crude extract ( ce ) of the parasite showed higher levels of proliferation in the presence of cona when compared to nonimmunized mice , although the difference was not statistically significant .
spleen cells from a / j immunized mice produced higher levels of il-10 induced by ce than did nonimmunized mice , but in the b10.a strain , the difference in il-10 production was not statistically significant ( figure 4(a ) ) . additionally , a / j immunized mice produced more il-10 against ce antigen compared to b10.a immunized mice ( p = .04 ) .   in a / j immunized mice ,
the il-10 production by spleen cells stimulated with cona was higher than that of nonimmunized animals , although the results were not statistically significant ( figure 4(b ) ) .
in contrast , b10.a immunized mice produced lower amounts of il-10 when compared to nonimmunized animals , but these results were also not statistically significant .
however , il-10 production induced by cona stimulation of spleen cells from a / j immunized mice was significantly higher than the amounts produced by b10.a mice ( p = .02 ) .  
levels of ifn- induced by ce in spleen cells of a / j immunized mice were higher than nonimmunized mice , although not statistically different .
ifn- production induced by ce stimulation of spleen cells from b10.a immunized mice was similar to from nonimmunized animals .
moreover , the ifn- production induced by ce stimulation of spleen cells from a / j immunized mice was higher compared to b10.a mice ( p = .049 ) ( figure 5(a ) ) . in addition , levels of ifn- produced by spleen cells of immunized a / j mice stimulated with cona were higher than those presented by nonimmunized mice ( p = .014 ) .
in contrast , spleen cells of b10.a immunized mice produced lower levels of ifn- induced by cona than did nonimmunized control animals ( p = .02 ) .
the ifn-   production induced by cona stimulation of spleen cells from a / j immunized mice was also higher than that of b10.a mice ( p = .005 ) ( figure 5(b ) ) .
this paper provides evidence that the mhc complex ( h-2 haplotype ) of mice infected with l. minor does not influence survival during experimental infection .
the survival rates of infected a / j mice were significantly higher than those of infected b10.a mice , although these isogenic mouse strains have different genetic backgrounds and the same h-2 haplotype ( h-2 ) .
h-2 is an important factor , but we must consider that susceptibility to parasitic infection is polygenic .
in fact , host genetic factors have a major influence on the susceptibility of mammals to infection by a variety of microorganism .
the genetically determined differences between individuals in a population affect the efficiency of the immune response , and thus the host phenotype of susceptibility .
in experimental paracoccidioidomycosis , b10.a mice behave as susceptible and a / j as resistant strain to fungal infection , that correlates with differences in the immune response , since resistant mice showed prevalent type 1 immunity whereas susceptible b10.a presented a progressive form of infection with impaired ifn- secretion [ 18 , 19 ] . acquired immunity against leishmania donovani
has been shown to be under the control of genes within the h-2 locus : in a b10 genetic background , mice carrying the h-2 haplotype present a cure phenotype ; whereas , the h-2 haplotype strains developed a noncure disease , in a manner dependent of the cytokines produced .  
the influence of the h-2 linked as wells as non - h-2 linked genes on the immune response was also shown by the infection of mice with trichinella spirallis , which affects the expulsion of the nematode from inbred and congenic mice [ 21 , 22 ] .  
the lesions detected in infected b10.a mice were similar to those described by semerene et al .   in c57bl/6 mice .
severe perivasculitis , vasculitis , and interstitial inflammation were visualized in the lungs , in the early phase of infection .
we also observed organized granulomas around intact l3/l4 larvae with concentric fibrosis , macrophages , neutrophils , and small number of eosinophils in the lungs of these mice , in the intermediate phase of the infection ( 4590 days after infection ) .
in contrast , the lungs of infected a / j mice had moderate perivasculitis , vasculitis , and interstitial inflammation , in the early phase of the infection , while during the intermediate phase of infection we detected less severe organized granulomas around intact or disrupted l3/l4 larvae with discrete fibrosis and presence of macrophages , plasma cells , few neutrophils and rare eosinophils , these results indicate that pulmonary lesions are of greater severity in the b10.a strain and correlate with mortality .
the differences noted in mouse susceptibility to l. minor infection with distinct survival rates and tissue lesions may also be influenced by individual host immune response to the parasite .
different cell populations are involved in the immune response to the parasite : t and b cells , and mononuclear phagocytes .
th2 cells produce il-4 , il-5 , and il-13 , which stimulate humoral responses and are known to be involved in resistance to extracellular helminths such as nipostrongylus brasiliensis .
antibodies to l. minor ( igg , igm and iga ) were detected in higher levels in a / j infected mice , when compared to b10.a infected mice , and it may reflect its greater resistance to infection .
the recently described th17 cells that produce il-17 play a key role in inflammation and the activation of neutrophils and immunity to microorganisms , particularly at mucosal surfaces . regulatory t cells ( tr ) of different subtypes produce il-10 and/or tgf- , and can regulate th1 , th2 and th17 cells , b cells , and macrophages [ 2628 ] .  
ifn- is the signature cytokine produced by th1 cells , but can be also derived from tc , nk cells , and b cells in small proportion .
il-10 is the major cytokine produced by tr cells , but can be also derived from b cells and monocytes [ 29 , 30 ] .  
considering that the participation of these different cell populations and the manipulation of the host immune response system by l. minor antigens may be a key determinant to their survival within a mammalian host , we immunized mice with the ce parasite antigen and analyzed the immune response . in this paper , we investigated the proliferative response and cytokine production of spleen cells from mice immunized with the ce of l. minor l3 larvae .
spleen lymphocytes from a / j mice immunized with ce and stimulated with cona ( positive control ) or ce of the parasite showed better proliferative response when compared to b10.a mice , particularly against the ce of the parasite .
ce is composed of intracellular and membrane components of the parasite that may stimulate specific spleen cells from a / j mice better than b10.a mice .   immunized a / j mice showed significantly increased production of il-10 against cona and ce . on the other hand , spleen cells of immunized
spleen cells from immunized a / j mice produced more ifn- when stimulated with cona or ce than did spleen cells from b10.a immunized mice .
moreover , immunized b10.a mice produced less ifn- when stimulated with cona when compared to nonimmunized controls .   in conclusion
, the a / j mice , which are more resistant to infection , displayed a better proliferative response and greater production of il-10 and ifn- against ce antigen and cona when immunized with l. minor antigens , compared to the susceptible b10.a mice which presented a lower proliferative response and less il-10 and ifn-. this intense inflammatory response may be a consequence of an uncontrolled reaction caused by the small amount of il-10 produced in b10.a mice . in a / j mice ,
the concomitant production of il-10 and ifn may control the tissue lesions and the number of parasites , limiting collateral tissue damage caused by a robust antiparasitic immune response .
in fact , when we compared experimental lagochilascariosis in c57bl/6 and balb / c mice , the latter were more resistant to infection and produced more ifn and il-10 .  
the high mortality observed in b10.a infected mice may be a consequence of the intense inflammatory reaction without control by regulatory cytokines .
we are currently investigating the kinetics of cytokine production in a / j and b10.a mice infected with l. minor to better understand the immunological mechanisms involved in the resistance to the infection .
 recently , we demonstrated that c57bl/6 mice are more susceptible to experimental lagochilascariosis than balb / c mice . to investigate the pattern of infection and the role of the genetic background on susceptibility to infection 
 , we studied experimental lagochilascariosis in h-2a identical b10.a and a / j mice . infected b10.a mice had a lower survival ratio and more severe lesions in the lungs than did a / j mice . 
 splenocytes of a / j mice immunized with the crude extract of the parasite showed increased proliferation and produced a higher level of interleukin 10 and interferon- in the presence of ce or concanavalin a when compared to b10.a mice . 
 this suggests that resistance of a / j mice may be due to less severe lesions in lungs and other organs and a better immune response to parasite antigens . 
 this paper provides evidence that major histocompatibility complex haplotype does not influence the survival to experimental infection with l. minor . 