animals and direct fluorescent antibody test ( dfat ) : mandibular and 
 parotid gland samples were obtained from 22 rabid dogs , which had been submitted to the 
 research institute of tropical medicine ( ritm ) , philippines , for postmortem diagnosis of 
 rabies . small transverse sections ( 23 mm in thickness ) of ammon s horn and medulla
were 
 cut , and slide was touched against the cut surface of the section and then placed on cold 
 acetone overnight for fixing .
then , 450 l of fluorescence isothiocyanate conjugate anti - rabies 
 monoclonal antibody ( fujirebio , malvern , pa , u.s.a . ) was added .
the slides were 
 incubated for 30 min at 37c in a high humidity chamber .
slides were then dipped and rinsed 
 for 20 to 25 times in pbs twice followed by distilled water for further washing .
small 
 amounts of the mounting medium , 20% glycerol - tris buffered saline ph 9.0 , were placed on the 
 slides before covering with coverslips for examination .
the slides were examined under the 
 fluorescent microscope ( 80i , nikon , tokyo , japan ) .
histopathological examination : mandibular and parotid salivary glands of 
 rabid ( n=22 ) and control ( n=3 ) dogs were fixed in 10% neutral buffered formalin at room 
 temperature ( rt ) for more than 72 hr , embedded in paraffin , sectioned ( 3  m 
 thickness ) and mounted .
three rabies - vaccinated domestic japanese mixed dogs ( 8 to 10 years 
 old ) were used as a control group .
the sections were then subjected to hematoxylin and eosin 
 ( he ) , special staining ( alcian blue and reticulin silver impregnation ) and 
 immunohistochemistry as described below .
immunohistochemistry : for detection of rabies virus antigen in tissues , 
 sections were stained using the streptavidin - biotin - peroxidase complex ( lsab ) method with 
 anti - rabbit phosphoprotein ( p ) as described previously . for detection of cell type ,
the following primary antibodies were used : 
 anti - cd3 for t lymphocytes ( dako , kyoto , japan ) ; anti - cd20 ( spring bioscience , fremont , ca , 
 u.s.a . ) and cd79 ( dako ) ; anti - laminin for basement membranes ( thermo scientific , fremont , 
 ca , u.s.a . ) ; anti - neurofilament protein ( nf ) for nerve fibers ( dako ) ; anti - neuron specific 
 enolase ( nse ) for nerve cells ( dako ) ; anti - alpha smooth muscle actin 
 ( -sma ) for myoepithelial cells ( dako ) ; and anti - activated cysteine 
 aspartic acid specific protease ( cleaved caspase-3 ) for apoptotic cells ( cell signaling 
 technology , inc . , beverley , ma , u.s.a . ) .
briefly , tissue sections were treated for the 
 activation of antigens with 0.25% trypsin at rt for 30 min for anti - p antibodies , 
 microwaving at 750w for 5 min for anti cd-3 , cd20 and cd79 antibodies , heating in a water 
 bath at 95c for 15 min for anti - cleaved caspase-3 antibodies , proteinase k for 15 min for 
 anti - laminin antibodies or 30 min for anti - nf antibodies , and autoclaving at 121c for 15 
 min for anti - nse and anti--sma antibodies . to remove endogenous peroxidase ,
sections were 
 treated with 0.3% or 3% h2o2 in methanol . to block nonspecific 
 reactions ,
primary antibodies were diluted 
 1:1,000 ( anti - p and anti--sma ) , 1:100 ( anti - laminin , anti - nse and anti - nf ) or 1:50 
 ( anti - cleaved caspase-3 and anti - cd3 ) in pbs and incubated at rt for 30 min to 2 hr or at 
 4c overnight in a humidified chamber .
anti - rabbit igg ( nichirei , tokyo , japan ) was used as 
 a secondary antibody for anti - p . the envision + system labeled polymer - hrp anti - rabbit 
 antibody ( dako )
was used for detection of cd3 , cd20 , cd79 , laminin , cleaved caspase-3 and 
 nse .
the histofine simple stain max - po anti - mouse antibody was used for detection of 
 -sma and nf .
terminal deoxynucleotidyl transferase - mediated dutp - biotin nick end labeling 
 ( tunel ) assay : the presence of fragmented dna was evaluated using tunel assays as 
 described previously .
briefly , after 
 deparaffinizing the sections , endogenous peroxidase activity was removed with 0.3% 
 h2o2 in methanol for 30 min at rt .
sections were then treated with 
 20 mg / ml proteinase - k in 0.1 m pbs for 15 min at rt to activate antigens . 
 after washing in pbs
, the slides were prepared according to the manufacturer s guidance and 
 were counterstained with hematoxylin .
double immunofluorescence staining and immunostaining with a special 
 stain : double staining of a single tissue section was used for identification of 
 cell type and the co - expression of antigens .
after deparaffinization , sections were 
 rehydrated in a graded alcohol series , incubated with 0.25% trypsin for 30 min to allow 
 enzymatic - induced antigen retrieval and incubated with 10% normal goat serum to block the 
 non - specific binding .
sections were incubated with a mixture of the primary antibodies , 
 rabbit anti - p together with anti--sma overnight at 4c .
dilution for anti - p and anti- -sma 
 antibodies was performed as described above .
after washing , sections were incubated with a 
 mixture of fluorochrome - conjugated regents , alexa fluor 546-goat anti - rabbit igg ( molecular 
 probes , eugene , or , u.s.a . ) together with 488-goat anti - mouse igg ( molecular probes ) , for 30 
 min at rt , rinsed in pbs and mounted with prolong gold antifade mountant with 
 dapi ( molecular probes ) . for immunostaining combination with special stain ,
anti - p antibody 
 staining was performed as described above , and slides were incubated with dab until color 
 developed .
the reaction was stopped by washing the slides in distilled water , and the slides 
 were then incubated in 3% acetic acid for 5 min , followed by staining with alcian blue 
 solution for 30 min .
slides were then washed in distilled water for 5 min , stained with 
 nuclear - fast red for 1 min , washed in distilled water for 5 min and mounted for light 
 microscopy .
clinical characteristics of the dogs : eighteen dogs were discovered after 
 having died , while four dogs were subjected to euthanasia .
the 22 dogs ( 11 males , 8 females 
 and 3 of unknown sex ) ranged in age from 1 to more than 24 months , with six dogs having 
 unknown ages .
sixteen of the 22 dogs had no history of rabies vaccination , one dog had a 
 history of rabies vaccination , and no information on rabies vaccination status was available 
 for five dogs .
sixteen dogs were free - roaming dogs with owners , three were strays , two were 
 confined with household contact , and one had unknown living conditions .
the primary clinical 
 symptoms of canine rabies infection , such as unprovoked aggressiveness , mad biting of 
 inanimate objects , aimless running and excessive salivation , were observed in 18 of the 22 
 dogs .
all brain specimens were diagnosed as positive for rabies virus antigen through the 
 direct immunofluorescence antibody test ( dfat ) .
the clinical information and results of dfat 
 are summarized in table 1table 1.clinical information and laboratory findings of rabid dogs ( samples obtained 
 postmortem)no.age ( months)sexownedmanner of deathvaccinationanimal conditionsneurological symptomsdfat148maleyesfound deadnofree - roaming and ownedunprovoked aggressiveness , excessive salivation+2unknownmaleyeseuthanasiaunknownstrayunprovoked aggressiveness , mad biting of inanimate objects+324femaleyesfound deadyesfree - roaming and ownedexcessive salivation , mad biting of inanimate objects , 
 apprehensive , watchful look , paralysis+41maleyesfound deadnofree - roaming and ownedunknown+56maleyesfound deadnofree - roaming and ownedunprovoked aggressiveness , mad biting of inanimate objects+66maleyesfound deadnofree - roaming and ownedunknown+71maleyesfound deadnofree - roaming and ownedunprovoked aggressiveness+860maleyesfound deadnoconfined with household contactunprovoked aggressiveness , excessive salivation , apprehension , 
 watchful look , mad biting of inanimate objects+92femaleyesfound deadnofree - roaming and ownedunprovoked aggressiveness ,
mad biting of inanimate objects , 
 aimless running+1012femaleyesfound deadnofree - roaming and ownedunknown+111femaleyesfound deadnounknownunknown+1236maleyesfound deadnofree - roaming and ownedunprovoked aggressiveness , mad biting of inanimate objects , 
 paralysis of jaw and tongue+1316femaleunknownfound deadnofree - roaming and ownedunprovoked aggressiveness+14unknownunknownunknowneuthanasiaunknownstrayunprovoked aggressiveness+152maleunknownfound deadunknownfree - roaming and ownedapprehensive , watchful look , paralysis+16unknownunknownunknownfound deadnofree - roaming and ownedunprovoked aggressiveness+172femaleunknownfound deadnofree - roaming and ownedunprovoked aggressiveness+188femaleunknownfound deadnofree - roaming and ownedunprovoked aggressiveness , mad biting of inanimate objects+19unknownmaleunknowneuthanasiaunknownfree - roaming and ownedunprovoked aggressiveness+20unknownmaleunknownfound deadnofree - roaming and ownedunprovoked aggressiveness , aimless running+21unknownunknownunknowneuthanasiaunknownstrayunprovoked aggressiveness+223femaleunknownfound deadnoconfined with household contactunprovoked aggressiveness , mad biting of inanimate objects , 
 aimless running++ : positive , dfat : direct fluorescent antibody test .. + : positive , dfat : direct fluorescent antibody test .
histopathological findings of salivary glands of control and rabid dogs : 
 no gross findings were observed in the parotid glands , mandibular glands and mandibular 
 lymph nodes in control dogs ( fig .
1.cutting position of the right external ear ( e ) , parotid gland ( pg ) , mandibular gland 
 ( mg ) and mandibular lymph node ( ln ) after formalin fixation in a control dog .
the parotid gland was composed exclusively of serous acinar epithelium in 
 association with striated ducts and interlobular excretory ducts , but interlobular ganglion 
 cells were not found ( fig .
the parotid gland is composed of the serous acinar gland ( a ) and 
 interlobular stroma .
the positions of the interlobular duct ( d ) , peripheral nerve 
 bundles ( arrows ) and vessels ( v ) are indicated in the interlobular stroma . he 
 staining .
the mandibular glands were divided into several lobules by dense connective tissue 
 septa and were composed of serous and mucous acinar epithelia .
many ganglion cells and 
 peripheral nerve fibers were observed in the interlobular septa ( fig . 3fig .
the mandibular gland is composed of mixed components of mucous and 
 serous acinar cells ( a ) .
the positions of the interlobular duct ( d ) , ganglion cells 
 ( arrows ) and vessels ( v ) are indicated in the stroma . he staining .
cutting position of the right external ear ( e ) , parotid gland ( pg ) , mandibular gland 
 ( mg ) and mandibular lymph node ( ln ) after formalin fixation in a control dog .
the parotid gland is composed of the serous acinar gland ( a ) and 
 interlobular stroma .
the positions of the interlobular duct ( d ) , peripheral nerve 
 bundles ( arrows ) and vessels ( v ) are indicated in the interlobular stroma . he 
 staining .
the mandibular gland is composed of mixed components of mucous and 
 serous acinar cells ( a ) .
the positions of the interlobular duct ( d ) , ganglion cells 
 ( arrows ) and vessels ( v ) are indicated in the stroma . he staining .
bar=100 
 m . in the mandibular glands , 19 out of 22 rabid dogs exhibited mild to moderate 
 non - suppurative sialadenitis characterized by fragmentation and cytolysis of the acinar 
 epithelium with infiltration of moderate to marked lymphocytes and plasma cells ( fig .
4.mandibular gland . non - suppurative sialadenitis characterized by cytolysis of acinar 
 epithelium and infiltration of lymphoplasmacytic cells ( asterisks ) .
infiltration of inflammatory cells was not found in three of the 
 four euthanized dogs , but small foci of necrotic acinar cells were scattered throughout the 
 samples ( fig . 5fig .
necrotic foci of the acinar epithelium ( arrowheads ) without 
 inflammatory cells were observed in the euthanasia case . he staining .
no histopathological changes in striated and interlobular ductal 
 epithelial cells and interlobular ganglion cells were found . in the parotid glands , no 
 morphological evidence of acinar , duct units and interlobular stroma
no pathological findings were observed in the serous acinar epithelium 
 or striated ducts ( arrows ) . he staining .
mandibular gland . non - suppurative sialadenitis characterized by cytolysis of acinar 
 epithelium and infiltration of lymphoplasmacytic cells ( asterisks ) .
necrotic foci of the acinar epithelium ( arrowheads ) without 
 inflammatory cells were observed in the euthanasia case . he staining .
no pathological findings were observed in the serous acinar epithelium 
 or striated ducts ( arrows ) . he staining .
immunohistochemical examination of the mandibular glands and parotid 
 glands : on immunohistochemistry using anti - rabies p antibody , no viral antigen 
 was detected in control dogs ( data not shown ) . in all rabid dogs ,
viral antigens were 
 detected in the cytoplasm of the mucous acinar epithelium and interlobular ganglion cells in 
 the mandibular gland ( figs .
viral antigens were detected by immunohistochemistry with anti - p 
 antibody in the acinar epithelium ( a ) and ganglion cells ( arrow ) , but the interlobular 
 duct ( d ) and blood vessels ( v ) were negative .
viral antigens were detected by immunohistochemistry with anti - p 
 antibodies in the cytoplasm of mucous acinar cells ( a ) .
viral antigens were detected by immunohistochemistry with anti - p 
 antibodies in the ganglion cells and peripheral nerves .
some viral antigens were observed in the myoepithelium and peripheral nerves , but 
 viral antigens were not found in the striated and interlobular ductal epithelia . in parotid 
 gland ,
these 
 immunohistochemistry results are summarized in table 
 2table 2.summary of inflammatory features and distribution of viral antigen in the 
 mandibular glands of 22 rabid dogsno.inflammatory cellsvirus antigen distributionmucous epitheliumserous epitheliumductal units ( striated and interlobular 
 ducts)interlobular ganglion1moderate++2mild++3mild++4moderate++5mild++6mild++7moderate++8moderate++9moderate++10mild++11none+++12mild++13moderate++14none++15mild++16moderate++17mild++18moderate++19moderate++20mild++21none++22moderate+++ : positive ,  : negative .. mandibular gland .
viral antigens were detected by immunohistochemistry with anti - p 
 antibody in the acinar epithelium ( a ) and ganglion cells ( arrow ) , but the interlobular 
 duct ( d ) and blood vessels ( v ) were negative .
viral antigens were detected by immunohistochemistry with anti - p 
 antibodies in the cytoplasm of mucous acinar cells ( a ) .
viral antigens were detected by immunohistochemistry with anti - p 
 antibodies in the ganglion cells and peripheral nerves .
for identification of the virus infected cells , double staining was performed by 
 combination of anti - p antibody and either anti--sma or alcian blue staining . in the 
 mandibular gland of rabid dogs , anti--sma - positive myoepithelial cells 
 showed co - expression with anti - p antibody reactivity ( fig .
myoepithelial cells were double positive ( arrows , yellow ) for 
 anti--sma ( green ) and anti - p antibodies ( red ) .
in addition , the mucous acinar epithelium showed co - stained with anti - p antibody 
 reactivity and alcian blue staining ( fig .
the mucous acinar epithelium showed co - expression with anti - p 
 antibody reactivity ( arrows ) and alcian blue staining .
in contrast , -sma - positive myoepithelial cells of the parotid glands were negative 
 for anti - p antibody reactivity .
myoepithelial cells were double positive ( arrows , yellow ) for 
 anti--sma ( green ) and anti - p antibodies ( red ) .
the mucous acinar epithelium showed co - expression with anti - p 
 antibody reactivity ( arrows ) and alcian blue staining .
myoepithelial cells surrounding acinar epithelium 
 showed strong reactivity for anti--sma ( 12a ) , whereas the intensity 
 of immunostaining decreased in rabid dogs ( 12b ) .
bar=50 
 m . ) and intercalated duct epithelium of the mandibular and parotid glands .
however , in 
 rabid dogs , the intensity of immunostaining of anti--sma - positive cells was decreased in 
 glands that showed severe inflammation ( fig . 

myoepithelial cells surrounding acinar epithelium 
 showed strong reactivity for anti--sma ( 12a ) , whereas the intensity 
 of immunostaining decreased in rabid dogs ( 12b ) .
there are moderate numbers of anti - cd3 positive cells 
 appeared in the interstitial connective tissue and around fragmented acinar epithelium , 
 while anti - cd20 and anti - cd79 ( fig .
many of anti - cd79 positive cells were observed in the interstitial 
 connective tissue .
immunohistochemistry . bar=20 m . ) positive cells were mainly detected in the interstitial connective tissue and 
 periductal areas .
many of anti - cd79 positive cells were observed in the interstitial 
 connective tissue .
interlobular ganglion cells and 
 peripheral nerve fibers were positive for anti - nse antibodies in the mandibular glands . 

anti - nf immunoreactivity 
 was observed as a fine network in the nerve fibers distributed throughout the stroma and 
 around acini in the mandibular glands .
the staining patterns were the same between control 
 and rabid dogs . in control dogs ,
laminin immunoreactivity and reticulin silver impregnation staining 
 appeared as linear , continuous staining around individual acini and ducts of the mandibular 
 and parotid glands . in the mandibular glands of rabid dogs ,
staining was irregular and weak , 
 because acinar epithelium and basement membranes were disrupted ( fig .
reticulin silver impregnation staining revealed irregular positive 
 reactions , with disruption of acinar epithelium ( asterisk ) and basement membranes . 

reticulin silver impregnation staining revealed irregular positive 
 reactions , with disruption of acinar epithelium ( asterisk ) and basement membranes . 

detection of apoptotic cells by tunel assays and anti - cleaved caspase-3 
 antibodies : in mandibular glands of rabid dogs , the acinar epithelium and 
 lymphocytes exhibited apoptotic features , such as nuclear fragmentation and cytolysis , and 
 were positive in tunel assays ( fig .
necrotic foci ( asterisk ) in the acinar epithelium were tunel 
 positive ( arrows ) .
the numbers of tunel - positive cells were higher in glands that showed severe 
 inflammation .
however , tunel - positive signals were not detected in the ductal epithelium , 
 interlobular ganglion cells and serous demilune . in parotid glands ,
the patterns of anti - cleaved 
 caspase-3 immunostaining were similar to those of tunel staining in the mandibular glands , 
 but with fewer positive cells .
necrotic foci ( asterisk ) in the acinar epithelium were tunel 
 positive ( arrows ) .
centrifugal spread from the cns to peripheral sites along neuronal routes is essential for 
 transmission of rabies virus to its natural hosts .
salivary gland infection is necessary for 
 the transfer of infectious oral fluids by rabid vectors . in this study , non - suppurative 
 sialadenitis , characterized by fragmentation
, cytolysis of the acinar epithelium , basement 
 membrane disruption and lymphoplasmacytic infiltration , was observed in the mandibular 
 glands of most rabid dogs .
no inflammatory cells were found in three of four euthanized 
 dogs ; however , small foci of necrotic acinar cells were scattered throughout the samples , 
 and these cells were positive for anti - caspase-3 and tunel staining .
these pathological 
 findings suggested that early sialadenitis was induced by direct disruption of the acinar 
 epithelium by rabies virus infection . in our study ,
the viral antigen was mainly detected in the mucous acinar epithelium and 
 myoepithelium in the mandibular glands .
these findings are consistent with previous reports 
 of skunks and foxes infected with the street rabies virus . in addition , the viral antigen was present in the myoepithelium between the 
 basal lamina and acinar epithelial cells .
thus , the rabies virus may propagate in the 
 myoepithelium and affect nerve terminal innervation .
previous studies have described the 
 morphological structure of the neuro - effector that innervates the acinar epithelium and 
 myoepithelium of the salivary glands of carnivores [ 11 , 20 , 27 ] .
these neuro - effectors may be the hypolemmal type ( i.e. , non - myelinated axons 
 that penetrate below the acinar basement membrane and adjacent to the myoepithelium ) or the 
 epilemmal type ( i.e. , non - myelinated axons found outside the acinar epithelium and 
 myoepithelium ) .
furthermore , the intra - acinar nerve endings in the mandibular glands are 
 restrictively localized only in the intercellular space between myoepithelial cells and 
 mucous acinar cells , whereas no intra - acinar nerve endings occur among the serous cells 
 .
therefore , it was suggested the rabies virus 
 may directly enter mucous acinar epithelial cells and myoepithelial cells via both 
 innervation
. however , several mammalian species lack apparent hypolemmal innervation in the 
 striated ducts and interlobular excretory ducts [ 31 , 
 32 ] . in the present study
, we did not observe 
 degeneration , inflammation or viral antigen in the duct unit of striated and interlobular 
 ducts in the mandibular gland , suggesting that hypolemmal and epilemmal innervations were 
 not present in the duct system of dogs .
thus , viral proliferation and cytotoxicity could not 
 occur there , ensuring that secretions containing the virus were efficiently excreted into 
 the oral cavity .
interlobular ganglions were observed in the interlobular septa of the mandibular glands , 
 similar to findings in humans   and rats .
these ganglions included a number of cholinergic 
 ganglion neurons that received motor impulses from pre- and postganglionic parasympathetic 
 fibers carried by the facial nerve .
ganglion 
 neurons are responsible for innervation of the salivary parenchyma and for regulation of 
 saliva secretion [ 24 , 26 ] . in the present study ,
after the rabies virus replicates within the facial nerve nuclei , 
 located in the ventral part of the rostral medulla oblongata , the virus then descends along 
 the facial nerve to reach the submandibular ganglion and the interlobular ganglions [ 3 , 30 , 35 ] . in this study
, we did not observe interlobular 
 ganglia in the parotid gland , and also no viral antigens were detected here .
therefore , it 
 was suggested that these ganglia are important for viral replication and serve as a main 
 source of virus to acinar epithelium of the mandibular glands . in the present study ,
virus - infected ganglion neurons were negative for tunel staining and 
 cleaved caspase-3 , indicating that virus - infected neurons did not undergo apoptosis , similar 
 to results reported in natural infection of dogs and humans by a street rabies virus strain 
 [ 15 , 30 ] . 

therefore , we concluded that lack of neuronal apoptosis in rabies in dogs infected by street 
 virus may promote prolonged infection within ganglion neurons and continually supply virus 
 to the salivary acinar epithelium . on the other hand , virus - infected mucous acinar 
 epithelial cells exhibited fragmentation and cytolysis , and migrating t lymphocytes were 
 positive for tunel and cleaved caspase-3 , suggesting that rabies virus may trigger apoptotic 
 cascades through the fas / fas ligand and caspase - dependent apoptotic pathways . in the present study ,
the inflammatory cells were composed mainly of cd3 , cd20 and 
 cd79-positive lymphocytes and were present in the virus - infected mandibular gland .
these 
 findings suggested that a combination of cell - mediated and humoral immune responses was 
 important for the clearance of rabies virus from the salivary gland and that the 
 inflammatory cytokines released by t lymphocytes activated plasma cell infiltration .
b lymphocytes play an important role 
 in producing virus - neutralizing antibodies and are critical for control of virus replication 
 and essential in the clearance rabies virus from the cns . in addition ,
high titers of tissue - neutralizing antibody suppress viral 
 spread to salivary epithelial cells .
the plasma 
 cells in the salivary tissue are the source of locally produced antibodies , such as 
 immunoglobulin a ( iga ) , which provides mucosal surface immunity against various antigens and 
 neutralizes viruses [ 5 , 22 ] .
furthermore , iga - producing plasma cells infiltrate the salivary 
 gland via regulation of t lymphocytes and various inflammatory cytokines , such as 
 interleukin ( il)-5 and interferon ( ifn)- . in 
 the present study
, however , the direct roles of lymphocytes in the salivary gland of rabid 
 dogs remain unresolved , because most of present cases were discovered after died , and 
 therefore , further studies are required . in summary ,
our results confirmed the path through which the rabies virus descends along 
 the facial nerve after proliferation in the brain to reach the ganglion neurons of the 
 mandibular gland , subsequently traveling to the acinar epithelium and salivary gland 
 myoepithelium .
furthermore , viral proliferation and cytotoxicity did not occur in the duct 
 system , ensuring that secretions containing the virus were efficiently excreted into the 
 oral cavity .
 rabies is a zoonotic disease caused by the rabies virus . while the salivary glands are 
 important as exit and propagation sites for the rabies virus , the mechanisms of rabies 
 excretion remain unclear . here , we investigated the histopathology of the salivary glands 
 of rabid dogs and analyzed the mechanism of excretion into the oral cavity . 
 mandibular and 
 parotid glands of 22 rabid dogs and three control dogs were used . 
 mild to moderate 
 non - suppurative sialadenitis was observed in the mandibular glands of 19 of the 22 dogs , 
 characterized by loss of acinar epithelium and infiltration by lymphoplasmacytic cells . 
 
 viral antigens were detected in the mucous acinar epithelium , ganglion neurons and 
 myoepithelium . 
 acinar epithelium and lymphocytes were positive for anti - caspase-3 
 antibodies and tunel staining . 
 in contrast , no notable findings were observed in the 
 ductal epithelial cells and serous demilune . in the parotid gland , 
 the acinar cells , 
 myoepithelium and ductal epithelium all tested negative . 
 these findings confirmed the path 
 through which the rabies virus descends along the facial nerve after proliferation in the 
 brain to reach the ganglion neurons of the mandibular gland , subsequently traveling to the 
 acinar epithelium via the salivary gland myoepithelium . 
 furthermore , the observation that 
 nerve endings passing through the myoepithelium were absent from the ductal system 
 suggested that viral proliferation and cytotoxicity could not occur there , ensuring that 
 secretions containing the virus are efficiently excreted into the oral cavity . 
MATERIALS AND METHODS
RESULTS
DISCUSSION