Immunohistochemistry for detection of avian infectious bronchitis virus strain M41 in the proventriculus and nervous system of experimentally infected chicken embryos
© Abdel-Moneim et al; licensee BioMed Central Ltd. 2009
Received: 01 December 2008
Accepted: 05 February 2009
Published: 05 February 2009
Infectious bronchitis virus primarily induces a disease of the respiratory system, different IBV strains may show variable tissue tropisms and also affect the oviduct and the kidneys. Proventriculitis was also associated with some new IBV strains. Aim of this study was to investigate by immunohistochemistry (IHC) the tissue tropism of avian infectious bronchitis virus (IBV) strain M41 in experimentally infected chicken embryos.
To this end chicken embryos were inoculated in the allantoic sac with 103 EID50 of IBV M41 at 10 days of age. At 48, 72, and 120 h postinoculation (PI), embryos and chorioallantoic membranes (CAM) were sampled, fixed, and paraffin-wax embedded. Allantoic fluid was also collected and titrated in chicken embryo kidney cells (CEK). The sensitivity of IHC in detecting IBV antigens in the CAM of inoculated eggs matched the virus reisolation and detection in CEK. Using IHC, antigens of IBV were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. These results were consistent with virus isolation and denote the wide tissue tropism of IBV M41 in the chicken embryo. Most importantly, we found infection of vasculature and smooth muscle of the proventriculus which has not seen before with IBV strain M41.
IHC can be an additional useful tool for diagnosis of IBV infection in chickens and allows further studies to foster a deeper understanding of the pathogenesis of infections with IBV strains of different virulence. Moreover, these results underline that embryonic tissues in addition to CAM could be also used as possible source to generate IBV antigens for diagnostic purposes.
Infectious bronchitis virus is the prototype species of the family Coronaviridae in the order Nidovirales. More than 25 genotypes are distributed worldwide. IBV causes an acute highly contagious viral respiratory disease of chickens which is characterized by respiratory rales, coughing and sneezing . Some IBV strains replicate in the gastrointestinal tract, oviduct, and kidney, and due to their nephropathogenic properties they have the potential to cause severe losses with up to 44% mortality [1, 2]. In other cases, infection of the proventriculus leads to 75% to 100% mortality in young birds . Most isolates of IBV replicate well in the developing chicken embryo following inoculation of the allantoic cavity, and high titers of virus can be isolated from the allantoic fluid . Replication of IBV strains M41 and Beaudette in vitro is restricted to primary chicken cells and depends on the expression of 2,3-linked sialic acids on the cell surface. Thus, these molecules are supposed to serve as receptor determinants for primary attachment of IBV to host cells [5, 6].
The conventional diagnosis of the IBV is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. Since two or three blind passages are often required for successful primary isolation of IBV, this procedure could be tedious and time consuming. Alternatively, IBV may be isolated by inoculation in chicken tracheal organ cultures. This method is sensitive  but highly laborious. Furthermore, IBV may be detected directly in tissues of infected birds by means of immunohistochemistry [8, 9] or in situ hybridization. The reverse transcription-polymerase chain reaction (RT-PCR) has proved useful in the detection of several RNA viruses . Aim of this study was first to evaluate the suitability of IHC for detection of IBV antigen in paraffin-wax embedded CAM and second to analyse the viral antigen distribution in different embryonic tissues between 48 and 120 h after experimental infection.
Detection of IBV in CAM and chicken embryo.
Time after inocul ation (h PI)
Results of Immunohistochemistry
Virological findings‡ (TCID50/ml)
Gizzard (smooth muscle, vasculature) ++
Heart (vasculature) ++
Kidney (glomerular tufts) ++
Liver (Kupffer cells) ++
Lung (vasculature) ++
Proventriculus (smooth muscle) ++
Spleen (macrophages) +
Heart (vasculature) ++
Kidney (glomerular tufts) ++
Liver (Kupffer cells) +
Spleen (Macrophages) +++
Nasal mucosa (epithelium) ++
Skin (epidermis) +
Nasal mucosa (epithelium) ++
Skin (epidermis) +
Brain (neurons) +
Eye (sclera) +
Spinal cord (neurons) +
Spleen (Macrophages) ++
Gizzard (mucosa) ++
Kidney (tubuli) +
Nasal cavity (mucosa) +++
Skin (epidermis) ++
Spinal cord (neurons) +
Spleen (macrophages) ++
Kidney (glomeruli) +
Liver (Kupffer cells) +
Lungs (epithelium, vasculature) +
Nasal cavity (mucosa)++
Proventriculus (smooth muscle, mucosa) ++
Skin (epidermis) ++
Spleen (macrophages) +++
Brain (neurons) +
Nasal cavity (mucosa) ++
Proventriculus (mucosa) +
Skin (epidermis) ++
Trachea (mucosa) ++
Classic methods for IBV diagnosis include serological tests for analysis of antibody titers against IBV  and virus isolation in embryonated chicken eggs since IBV grows well in the developing chicken embryo. These methods are inherently slow and time consuming. Currently, detection and serotype analysis of IBV is performed by RT-PCR and restriction fragment length polymorphism analysis  or by sequencing RT-PCR products of S1 gene . To establish IHC on paraffin-wax embedded tissues using a polyclonal rabbit anti-IBV serum, embryonated eggs were inoculated with 103 EID50 IBV M41. CAM and embryos were collected till 120 h PI, since the presence of IBV antigen in inoculated eggs by an antigen detection method is preferably performed 2 to 3 days after inoculation . This also confirmed in the current study where IBV titers declined sharply at 120 h PI. It is well known that maximum IBV virus titers reached 1 to 2 days post-inoculation [16–18] but interestingly, allantoic fluid showed high virus titers 24–87 h PI. Immunostaining of the CAM was positive in all inoculated eggs from which infectious virus was recovered. It is worth to note that also samples of infected CAM with respective allantoic fluid virus titers of only 102 or 103 TCID50 were obtained, stained positively with the IBV antiserum which equals approximately 104 or 105 EID50 . This indicates a relatively high sensitivity of the immunohistochemistry applied in this study. Using a monoclonal antibody for immunostaining, the detection limit for IBV antigen was 106.2 EID50 as described by . These differences in sensitivity may be due to larger number of antigenic epitopes recognized by the polyclonal serum. This finding prompted us to apply IHC for screening of antigen distribution in different tissues in the IBV inoculated embryos. Antigens were detected in the nasal epithelium, trachea, lung, spleen, myocardium, liver, gizzard, proventriculus, kidney, skin, sclera of the eye, spinal cord, as well as in neurons of the central nervous system in infected embryos. However, isolation or detection of virus by virological methods may indicate only that virus is present in the tissue due to viraemia and thus does not necessarily prove productive replication in the respective organs. Hence, tissue tropism can not be determined by virus isolation or detection only  but requires morphologically based techniques. Although Chong & Apostolov , failed to detect virus by immunofluorescence in the intestine and cecal tonsils of chickens experimentally infected with M41 of IBV, Lucio & Fabricant  found that M41 can infect a variety of tissues and that some isolates may be recovered frequently from the digestive tract. IBV infection of the proventriculus was firstly recorded in China  then detected with UNAM-97 IBV Mexican variant that produced decrease in the proventricular gland papillary branching and electrodense material scattered in proventriculus with a structure consistent with coronaviruses . To the best of our knowledge, this is the first time that the prototype IBV strain M41 was also detected within the muscle layer of the proventriculus. Because IBV causes an upper respiratory tract disease, viral antigens in nasal mucosa, trachea and lung were expected. IBV M41 viral antigen was found in the renal tubules and glomeruli. This observation is consistent with the finding that IBV M41 has also been isolated and/or detected in kidneys of naturally or experimentally inoculated chickens [23, 25, 26]. As earlier described for other IBV genotypes, antigens of M41 were present not only in renal tubules, but also in the glomerular tuft epithelium . The detection of IBV antigen in the spleen, is discussed controversially in the literature. In some reports, IBV antigens or mRNA were not found within the spleen [28–30], in others, splenic infection was observed [27, 31, 32]. Studies to determine virus distribution in embryonic tissues were conducted previously [29, 30] indicating that IBV antigen or nucleic acid was present in trachea, bursa, kidney, intestine, lung, heart, esophagus, mesentery, shell gland, and air sac, but not in spleen or thymus. The different virus distribution between the present paper and other papers may be due to the difference of embryo age at inoculation; 10 days old embryos (present paper) and 17 or 18 days old embryos [29, 30]. It is probable that more immature tissues are more susceptible for IBV. The susceptibility for IBV infection is associated with the expression of 2,3-linked sialic acid which is used by the virus for primary attachment to the cell surface [5, 6]. For tight binding and subsequent fusion with the cellular membrane interaction with a second receptor appears to be required . The initial target of avian influenza viruses and IBV in chicken is the respiratory epithelium. Presence of 2,3-linked sialic acid is the prerequisite for avian influenza viruses to initiate respiratory infection. This molecule may also be used by IBV for infection of the respiratory tract. IBV, like avian influenza viruses, infects many non-respiratory tissues, including alimentary tract, oviduct and kidney [33, 34]. The broad distribution of 2,3-linked sialic acid in different organs and species contradicts the view that this type of sugar is a major determinant of the narrow host tropism of IBV. In this study IBV antigen was found in musculature of both gizzard and proventriculus of inoculated embryos which is consistent with the known presence of 2,3-linked sialic acid receptors in the intestinal tract of chicken . Thus it raises the question whether IBV infection of the proventriculus is a classic feature of IBV and occurs in association with an old IBV strain. A recently isolated M41 strain  resulted in an increased proventriculus index 7–28 days after experimental infection of 1-day-old chickens [Mohamed AA: Studies on infectious proventricultis in broiler chicken. Master thesis, In progress].
For our knowledge, IBV was neither isolated from nor detected in the brain of young or adult chickens . Our finding that IBV antigens are present in the nervous system of embryonic chicken, may be explained by the presence of polysialylated N-CAM (neural cell adhesion molecules) in chicken embryos which might mediate virus entry into the neurons. The abundance of polysialylated N-CAM declines gradually during the embryonic development and the synthesis dramatically decreases right before birth [36, 37].
Our results show that the classic IBV strain M41 exhibits a wide tissue tropism including the nervous system and the proventriculus in chicken embryos and demonstrate that IHC as described here is a very sensitive tool for detection of productive virus replication in situ and therefore allows further studies to improve the understanding of the pathogenesis of the IBV infection.
Cells and viruses
The IBV strain M41 used in the current study was kindly provided by M. Hess, Intervet, Boxmeer, NL. Chicken embryo kidney cells (CEK) were prepared from specific pathogen free (SPF) embryonated chicken eggs (ECE) as described elsewhere . Briefly, kidneys from 18-day-old SPF ECE were isolated, washed with Hanks' balanced salts solution, minced and disaggregated in trypsin solution. After centrifugation the cells were resuspended in Dulbeccos' modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and grown at 37°C in a 5% CO2 incubator to confluent monolayers.
Inoculation of embryonated eggs
Twelve ten days old SPF ECE were inoculated in the allantoic sac with 200 μl of IBV strain M41(103 EID50). Inoculated ECE were candled twice daily. At 48, 72, or 120 h dead and/or living whole embryos as well as allantoic fluid and CAM were collected. Embryos died at any time after inoculation, were also sampled accordingly. The allantoic fluid of inoculated eggs was harvested and clarified by centrifugation for 10 min at 925 g. The obtained supernatants were used for titration in CEK.
The presence of IBV antigens was investigated in CAM and embryos using the avidin-biotin complex (ABC) method for immunohistochemistry . To this end, sampled CAM and individual whole embryos, containing all organs were fixed for 4 h in Carnoy solution (absolute ethyl alcohol:chloroform:acetic acid; 6:3:1) followed by dehydration in absolute ethanol, and paraffin wax-embedding. Sections were cut at 2 μm, and mounted on electrostatically charged glass slides. Tissue sections (CAM and whole embryos cut in transversal or sagittal orientation) were dewaxed in xylene, rehydrated, treated with 3% hydrogen peroxide and then subjected to antigen retrieval by microwaving in 10 mM citric buffer (pH 6.0) for 10 min. Treated slides were cooled at room temperature for 20 min. Non specific background staining was blocked by incubating the sections for 20 min. with goat serum. The sections were then incubated for 1 h with 1:3000 rabbit anti-IBV polyclonal serum (prepared previously in the laboratory of G. Keil, FLI, using purified IBV M41) in Tris buffered saline (TBS), 1% bovine serum albumin (BSA), followed by 30 min. incubation with each of 1:200 biotinylated goat anti-rabbit immunoglobulin and subsequent extra avidin peroxidase conjugate. To visualize bound antibodies, the sections were then incubated in amino-ethyl-carbazole substrate chromogen (DakoCytomation, Hamburg, Germany) for 10 min. Finally, the sections were counter-stained with Mayer's haematoxylin and mounted with aqueous mounting medium. The staining intensities observed microscopically were divided into four grades: - (negative = no positive cells), + (weak = small number of positive cells per high power field [HPF, approx. 400×]), ++ (moderate = moderate number of positive cells per HPF), +++ (strong = accentuated staining pattern with large numbers of positive cells per HPF). In each tissue 10 randomly selected areas of each compartment were evaluated at high power by light microscopy. The judgments were made semiquantitatively via side-by-side comparisons of one section to another and the purpose was to evaluate antigen distribution in relation to labelling intensity in different embryonic tissues.
100 μl of tenfold serial dilutions in MEM were added to 105 CEK cells per well. After 48 h of incubation, cells were fixed with acetone-methanol, and virus titers expressed as tissue culture infective dose fifty (TCID50) were determined by indirect immunofluorescence using a polyclonal serum raised in rabbits against purified IBV M41 virions.
IBV infected CEK cells were fixed with acetone-methanol for 15 min. After washing, cells were incubated with the polyclonal rabbit anti-IBV serum (1:2000) for 1 h and subsequently with FITC-conjugated goat anti-rabbit antibodies (Sigma) (1:2000) for 1 h. Both primary and secondary antibodies were diluted in PBS. Plates were rinsed three times with PBS after each step.
We thank Katrin Giesow and Gabriele Czerwinski for excellent technical assistance.
- Cavanagh D, Naqi S: Infectious bronchitis. In Diseases of Poultry. 11th edition. Edited by: Saif YM, Barnes HJ, Glisson JR, Fadly AM, McDougald LR, Swayne DE. Ames: Iowa State University Press; 2003:101-119.Google Scholar
- Cavanagh D: Coronaviruses in poultry and other birds. Avian Pathol 2005, 34: 439-448. 10.1080/03079450500367682View ArticlePubMedGoogle Scholar
- Yu L, Jiang Y, Low S, Wang Z, Nam SJ, Liu W, Kwang J: Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens. Avian Dis 2001, 45: 416-424. 10.2307/1592981View ArticlePubMedGoogle Scholar
- McMartin DA: Infectious bronchitis. In Virus Infections of Vertebrates. Virus Infections of birds. Edited by: McFerran JB, McNulty MS. Amsterdam: Elsevier Science Publishers; 1993:249-275.Google Scholar
- Winter C, Schwegmann-Weßels C, Cavanagh D, Neumann U, Herrler G: Sialic acid is a receptor determinant for infection of cells by avian Infectious bronchitis virus. J Gen Virol 2006, 87: 1209-1216. 10.1099/vir.0.81651-0View ArticlePubMedGoogle Scholar
- Winter C, Herrler G, Neumann U: Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent. Microbes Infect 2008,10(4):367-373. 10.1016/j.micinf.2007.12.009View ArticlePubMedGoogle Scholar
- Cook JKH, Darbyshire JH, Peters RW: The use of chicken tracheal organ cultures for isolation and assay of avian bronchitis. Arch Virol 1976, 50: 109-118. 10.1007/BF01318005View ArticlePubMedGoogle Scholar
- Nakamura K, Cook JKA, Otsuki K, Huggins MB, Frazier JA: Comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohistochemistry. Avian Pathol 1991, 20: 241-257. 10.1080/03079459108418761View ArticlePubMedGoogle Scholar
- Chen BY, Hosi S, Nunoya T, Itakura C: Histopathology and immunohistochemistry of renal lesions due to infectious bronchitis virus in chicks. Avian Pathol 1996, 25: 269-283. 10.1080/03079459608419141View ArticlePubMedGoogle Scholar
- Collisson EW, Li J, Sneed LW, Peters ML, Wang L: Detection of avian infectious bronchitis using in situ hybridization and recombinant DNA. Vet Microbiol 1990, 24: 261-271. 10.1016/0378-1135(90)90176-VView ArticlePubMedGoogle Scholar
- Cavanagh D, (Ed): Advances in avian diagnostic technology. Xth Wld Vet Poult Assoc Congress. Sydney 1993, 57-70.Google Scholar
- Lukert PD: Infectious bronchitis. In Isolation and identification of avian pathogens. 2nd edition. Edited by: Hitchner SB, Domermuth CH, Purchase HG, Williams JE. American Association of Avian Pathologists, Kennett Square, PA; 1980:70-72.Google Scholar
- Kwon HM, Jackwood MW, Gelb J Jr: Differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction-fragment-length polymorphism analysis. Avian Dis 1993, 37: 194-202. 10.2307/1591474View ArticlePubMedGoogle Scholar
- Kingham BF, Keeler CL Jr, Nix WA, Ladman BS, Gelb J Jr: Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Dis 2000, 44: 325-335. 10.2307/1592547View ArticlePubMedGoogle Scholar
- De Wit JJ: Detection of infectious bronchitis virus. Avian Pathol 2000, 29: 71-93. 10.1080/03079450094108View ArticlePubMedGoogle Scholar
- Purchase HG, Cunningham CH, Burmester BR: Genetic differences among chicken embryos in response to inoculation with an isolate of infectious bronchitis virus. Avian Dis 1966, 10: 162-172. 10.2307/1588346View ArticlePubMedGoogle Scholar
- Clarke JK, McFerran JB, Gay FW: Use of allantoic cells for the detection of avian infectious bronchitis virus. Archiv fur die Gesamte Virusforschung 1972, 36: 62-70. 10.1007/BF01250296View ArticlePubMedGoogle Scholar
- Darbyshire JH, Cook JKA, Peters RW: Comparative growth kinetic studies on avian infectious bronchitis virus in different systems. J Comp Pathol 1975, 85: 623-630. 10.1016/0021-9975(75)90128-0View ArticlePubMedGoogle Scholar
- Ferreira HL, Pilz D, Mesquita LG, Cardoso T: Infectious bronchitis virus replication in the chicken embryo related cell line. Avian Pathol 2003, 32: 413-417. 10.1080/0307945031000121167View ArticlePubMedGoogle Scholar
- Naqi SA: A monoclonal antibody-based immunoperoxidase procedure for rapid detection of infectious bronchitis virus in infected tissues. Avian Dis 1990, 34: 893-898. 10.2307/1591379View ArticlePubMedGoogle Scholar
- Hofstad MS, Yoder HW Jr: Avian infectious bronchitis virus distribution in tissues of chicks. Avian Dis 1966, 10: 230-239. 10.2307/1588355View ArticlePubMedGoogle Scholar
- Chong KT, Apostolov K: The pathogenesis of nephritis in chicks induced by infectious bronchitis virus. J Comp Pathol 1982, 92: 199-211. 10.1016/0021-9975(82)90078-0View ArticlePubMedGoogle Scholar
- Lucio B, Fabricant J: Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus. Avian Dis 1990, 34: 865-870. 10.2307/1591375View ArticlePubMedGoogle Scholar
- Escorcia M, Fortoul TI, Petrone VM, Galindo F, López C, Téllez G: Gastric gross and microscopic lesions caused by the UNAM-97 variant strain of infectious bronchitis virus after the eighth passage in specific pathogen-free chicken embryos. Poult Sci 2002, 81: 1647-1652.View ArticlePubMedGoogle Scholar
- Jones RC: Nephrosis in laying chickens caused by Massachusetts-type infectious bronchitis virus. Vet Rec 1974, 5: 319.View ArticleGoogle Scholar
- Abdel-Moneim AS, El-Kady MF, Ladman BS, Gelb J Jr: S1 gene sequence analysis of a nephro-pathogenic strain of avian infectious bronchitis virus in Egypt. Virol J 2006, 3: 78. 10.1186/1743-422X-3-78PubMed CentralView ArticlePubMedGoogle Scholar
- Abdel-Moneim AS, Madbouly HM, El-Kady MF: In vitro characterization and pathogenesis of Egypt/Beni-Suef/01; a novel genotype of infectious bronchitis virus. Beni-Suef Vet Med J Egypt 2005, 15: 127-133.Google Scholar
- Ambali AG, Jones RC: Early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus. Avian Dis 1990, 34: 809-817. 10.2307/1591367View ArticlePubMedGoogle Scholar
- Kapczynski DR, Sellers HS, Rowl GN, Jackwood MW: Detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa. Avian Dis 2002, 46: 679-685. 10.1637/0005-2086(2002)046[0679:DOIOII]2.0.CO;2View ArticlePubMedGoogle Scholar
- Lee C-W, Brown C, Jackwood MW: Tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization with antisense digoxigenin-labeled universal riboprobe. J Vet Diag Invest 2002, 14: 377-381.View ArticleGoogle Scholar
- Cumming RB: The control of avian infectious bronchitis/nephrosis in Australia. Aust Vet J 1969, 45: 200-203. 10.1111/j.1751-0813.1969.tb01930.xView ArticlePubMedGoogle Scholar
- Naqi S, Gay K, Patalla P, Mondal S, Liu R: Establishment of persistent avian infectious bronchitis virus infection in antibody-free and antibody-positive chickens. Avian Dis 2003, 47: 594-601. 10.1637/6087View ArticlePubMedGoogle Scholar
- Casais R, Dove B, Cavanagh D, Britton P: Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J Virol 2003, 77: 9084-9089. 10.1128/JVI.77.16.9084-9089.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Liu S, Kong X: A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in China. Avian Pathol 2004, 33: 321-327. 10.1080/0307945042000220697View ArticlePubMedGoogle Scholar
- Guo C-T, Takahashi N, Yagi H, Kato K, Takahashi T, Yi S-Q, Chen Y, Ito T, Otsuki K, Kida H, Kawaoka Y, Hidari KI, Miyamoto D, Suzuki T, Suzuki Y: The quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza A viruses to human type receptors. Glycobiology 2007,17(7):713-724. 10.1093/glycob/cwm038View ArticlePubMedGoogle Scholar
- Oka S, Bruss JL, Nelson RW, Rutishauser U: Properties and developmental regulation of polysialyltransferase activity in the chicken embryo brain. J Biol Chem 1995, 270: 19357-19363. 10.1074/jbc.270.33.19357View ArticlePubMedGoogle Scholar
- Ong E, Nakayama J, Angata K, Reyes L, Katsuyama T, Arai Y, Fukuda M: Developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, PST and STX. Glycobiology 1998,8(4):415-424. 10.1093/glycob/8.4.415View ArticlePubMedGoogle Scholar
- Schat KA, Purchase HG: Cell culture methods. In A laboratory manual for isolation and identification of avian pathogens. 4th edition. Edited by: Swayne DE, Glisson JR, Jackwood MW, Pearson JE, Reed WM. American Association of Avian Pathologists, Kennett Square, PA; 1998:223-234.Google Scholar
- Luppi PH, Fort P, Jouvet M: Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res 1990, 534: 209-224. 10.1016/0006-8993(90)90131-TView ArticlePubMedGoogle Scholar
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