The present study was conducted with the aim of defining the morphology of lymphoid nodules and lymphoid aggregates in calves at different ages. A total of 9 calves, divided into 3 age groups (3-, 6- and 8-month-old) consisting of 3 calves in each group, was used in this study. Samples of the lungs were taken systematically and processed for histological examinations. The tissues were sectioned at 4, μm, stained with Haematoxylin and Eosin and examined under light microscope. The occurrence and structure of lymphoid nodules and lymphoid aggregates varied between the age groups. The number of lymphoid nodules and lymphoid aggregates in the lung of calves increased with age. The number of lymphoid nodules and lymphoid aggregates in the 8-month-old calves was significantly higher (P<0.05) than in the 3- and 6-month-old calves. Similarly, the size of lymphoid nodule and lymphoid aggregates increased with age and was significantly higher (P<0.05) in the 8-month-old calves as compared to the 3- and 6- month-old calves. The number of intraepithelial lymphocytes (IELs) within the lymphoid nodules and lymphoid aggregates was significantly higher (P<0.05) in the 8-month-old calves compared to the 3- and 6-month-old calves

Abu Bakar @ Zakaria, Md Zuki

Saad, Mohd Zamri

Saw, Po Po

Universiti Putra Malaysia Institutional Repository

J. Vet. Malaysia (2009) 21(2): 9 - 15MORPHOLOGY OF LYMPHOID NODULES AND AGGREGATESIN TIlE LUNGS OF CALVES AT DIFFERENT AGESSaw Po Po, A.B.Z. Zuki* and M. Zamri-SaadFaculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiaSUMMARYThe present study was conducted with the aim of defining the morphology of lymphoid nodules and lymphoidaggregates in calves at different ages. A total of 9 calves, divided into 3 age groups (3-, 6- and 8-month-old) consistingof 3 calves in each group, was used in this study. Samples of the lungs were taken systematically and processed forhistological examinations. The tissues were sectioned at 4 ,um, stained with Haematoxylin and Eosin and examinedunder light microscope. The occurrence and structure of lymphoid nodules and lymphoid aggregates varied betweenthe age groups. The number of lymphoid nodules and lymphoid aggregates in the lung of calves increased with age.The number oflymphoid nodules and lymphoid aggregates in the 8-month-old calves was significantly higher (P<0.05)than in the 3- and 6-month-old calves. Similarly, the size oflymphoid nodule and lymphoid aggregates increased withage and was significantly higher (P<0.05) in the 8-month-old calves as compared to the 3- and 6- month-old calves. Thenumber of intraepitheliallymphocytes (IELs) within the lymphoid nodules and lymphoid aggregates was significantlyhigher (P<0.05) in the 8-month-old calves compared to the 3- and 6-month-old calves.Keywords: Morphology, lymphoid nodules, aggregates, lungs, calves, ageINTRODUCTIONIntrapulmonary lymphoid tissue can be foundassociated with the bronchus of animals. It is known asbronchus-associated lymphoid tissue (BALT) and is amember of a family of mucosa-associated lymphoid tissue(Bienenstock, 1984). The BALT consists primarily of alymphoid cell compartment covered by an epithelium thatis structurally different from typical bronchial epithelium.The BALT is complex and consists of lymphoid nodulesand lymphoid aggregates (dense aggregated lymphoidtissues), scattered lymphoid tissues, intraepitheliallymphocytes (IEL) and interluminal lymphocytes. TheBALT typically contains lymphocytes and antigen-presenting cells as well as high endothelial venules(Anderson et al., 1986b). High endothelial venules arethought to be important in recirculation and homing ofmucosa specific lymphocyte (Woodrufff et al., 1987). Itsfunction is initiation of a local immune response forluminal antigen. The M or microfold cell characteristic ofBALT is a specialised epithelial cell engaged in activesampling of luminal content and capable of transfer ofsoluble and particulate matter bidirectional into or awayfrom the lumen (Bockman and Steven, 1977). The BALThas been described previously in cattle (Anderson et al.,1986a), mink (Jericho, 1970), rabbits, guinea pig, pig, rat,mice, dog, chicken and human (Bienenstock et al., 1973).However, the morphology of BALT in calves in relationto age in a single study has never been reported andcompared before. Thus, the present study reports themorphology of lymphoid nodules and aggregates atdifferent sites of lungs of calves at different ages. Inaddition, the intraepitheliallymphocytes (IELs) within thelymphoid nodule and aggregates were also determined.MATERIALSANDMETHODSAnimalsA total of nine local breed calves of different ages (3-, 6-and 8-month-old) were used in this study. The calves weredivided into three groups (n=3) according to age. The calveswere supplied by the University Research Park, UniversitiPutra Malaysia.Tissue sampling and processingThe lungs were removed immediately upon slaughterand washed with phosphate buffer saline solution (pH7.3). The right lung was infused with 10% buffered formalinvia the right principle bronchus and/allowed to fix for 24to 48 hours before sampling. The samples were takenfrom 4 different lobes of the right lung namely the anteriorcranial, posterior cranial, middle and caudal lobes. Thesamples were fixed in 10% buffered formalin andprocessed for histological examination. The tissues 'wereembedded in paraffin, sectioned at 4 ,um, stained withCorresponding author: Dr. A.B.Z. Zuki; Email: zuki@vet.upm.edu.my10 Saw Po Po, A.B.Z. Zuki and M. Zamri-SaadHaematoxylin and Eosin and examined under lightmicroscope of up to 400 magnification.Histological examinationThe size of lymphoid nodules and aggregates wasmeasured using the image analyser (Synoptic, FosterFinding Associated Ltd, Freepost NT 2042 NewcastleTechnopole, UK). Within the lymphoid nodules andlymphoid aggregates, the intraepithelial lymphocytes(lELs) of epithelium were counted based on theirmorphological structure. The IELs with well definednuclear were counted using the eyepiece gratitcule, andexpressed as per 50 epithelial cells nuclei in the lymphoidnodules and lymphoid aggregates.Statistical analysisThe data were analysed using the Statistical AnalysisSystem and Duncan's multiple range test.RESULTSThe lymphoid nodules were seen in the bronchi andbronchioles of the lung in all ages and were structurallymore complex than the lymphoid aggregates. Thelymphoid nodule consisted of dome area, follicle andcorona. Primary and secondary follicles of the lymphoidnodule extended from the submucosa to direct contactwith the basement membrane of the mucosal epithelium(Figure 1). The arterioles were present close to thelymphoid nodules. The lymphoid nodules were mainlycomposed of medium to large lymphocytes and a fewreticuloendothelial cells, plasma cells and macrophages.The basic structures of lymphoid nodules were similar inall parts of bronchi, bronchioles and respiratorybronchioles and could be divided into four areas: thefollicle, parafollicular area surrounding the follicle,lymphoepithelium and the dome area.The lymphoid aggregates were present beneath thetunica muscularis and mostly found in tunica submucosaand lamina propria. They were located randomly in thebronchi and bronchioles. In 3-month-old calves, lymphoidaggregates were frequently seen in bronchioles. In 6-and 8-month-old calves, they were mostly found at thebifurcation of bronchi, bronchioles and terminalbronchioles (Figure 2).The number of lymphoid nodules in lung increasedwith age (Figure 3). The number of lymphoid nodules indifferent lobes oflung was significantly different (P<0.05)between age groups. Within the group, the number oflymphoid nodules in different lobes of lung was alsodifferent. The number of lymphoid nodules in the craniallobes (anterior and posterior) was significantly higher(P<0.05) than in the middle and caudal lobes in the 6- and8-month-old calves. In 3-month-old calves, the lymphoidnodules were rarely present in the wall of bronchioles,and some part of the lobes had no evidence of lymphoidnodules. In 6-month-old calves, numerous well developednodules were present near the opening of secondarybronchi in the anterior cranial lobes. In the 8-month-oldcalves, several large complex nodules were seen in thebronchi, bronchioles and respiratory bronchioles.The length and width of lymphoid nodules indifferent lobes of lung of all age groups are shown inFigure 4. The size of lymphoid nodules increased withage. The length and width of lymphoid nodules of theanterior cranial lobe were significantly higher (P<0.05)than the other lobes of the lung. The length and width ofFigure 1: Histological section of lymphoid nodule at bronchial bifurcation in 8-month-old calf shows the nodule extendsfrom the submucosa [containing cartilage (C)] to the basement membrane of the mucosal epithelium (arrows).The lymphoid nodule consists of dome area, follicle and corona. H & E.Morphology of Lymphoid Nodules and Aggregates in the Lungs of Calves at Diffrent Ages 11Figure 2: Dense aggregated lymphoid tissue (DA) in terminal bronchiole of a 6-month-old calf. It is located in thesubmucosa of a terminal bronchiole and the epithelium (E) overlying the aggregate is unspecialised. MM=muscularis mucosae. H & E987No. of 6lymphoidnodules 54320ACAC = anterior cranial lobePC = posterior cranial lobeML = middle lobeCA = caudal lobe~3month-6month~8monthPC ML CAFigure 3: Number of lymphoid nodules in different parts of lung at different ages of calves450400350300Length (11m) 250200150100500AC = anterior cranial lobePC = posterior cranial lobeML = middle lobeCA = caudal lobe.................~.-__.. ""_3 rronth....... 6rronth-"-8 rronth+-----~------~----~----~ACL M...LFigure 4A: Length of lymphoid nodules in different lobes of lung at different ages of calves12 Saw Po Po, A.B.Z. Zuki and M. Zamri-Saad~IJO250200Width (11m)150100500ADNACL = anterior cranial lobePCL = posterior cranial lobeMLL = middle lobeCAL = caudal lobe.,. • • 3rronth-6rronth-8rronthFigure 4B: Width of lymphoid nodules in different lobes of lung at different ages of calvesM..W"'C9Cl) 8-C'GCl 7~ Cl)Cl ~ 6Cl II)C'G II)Cl):;= 5II) 3:!s:: 0 4Cl) ..s::"'C c.. 3- E0~ ~ 2Cl).c 1E~ 0ZAC = anterior cranial lobePC = posterior cranial lobeML = middle lobeCA = caudal lobeACA PGADifferent lobes-+-3 rronth---- 6 rronth_"_8 rronthMLA CAAFigure 5: Mean number of lymphoid aggregates in different lobes of lung at different ages of calveslymphoid nodules in 8-month-old calves was significantlyhigher (P<O.05) than in the 3- and 6- month-old calves.The distribution of the lymphoid aggregates indifferent lobes of lung varied individually and betweenage groups. Figures 5, 6 and 7 show the number and sizeof lymphoid aggregates in different lobes of lung of allage groups. The number and size of the lymphoidaggregates increased with age. The number of lymphoidaggregates in the cranial lobe was higher than in otherlobes (Figure 5). In the 3- and 6-month-old calves, thewidth of the lymphoid aggregates was not significantlydifferent (P>O.05) within the same age (Figure 7). Thewidth and length oflymphoid aggregates in 8-month-oldcalves were significantly higher (P<O.05) than in the 3-and 6-month-old calves.Figure 8 shows the number of IELs in lymphoidnodules in different lobes of the lung in 3-, 6- and 8-month-old calves. The number of IELs in the cranial lobewas significantly higher (P<O.05) than in the other lobes;also the number of IELs increased with age. The numberof IELs in lymphoid nodules in 8-month-old calves wassignificantly higher (P<O.05) than those of the 3- and 6-month-old calves. The number of IELs in lymphoidaggregates in the different lobes of the lung in the 3-, 6-and 8-month-old calves is shown in Figure 9. The numberof IELs in lymphoid aggregates of the posterior craniallobe was higher than in other lobes in 6 and 8 month-oldcalves. The number of IELs in lymphoid aggregates in 8-month-old calves was significantly higher (P<O.05) thanin that of the 3- and 6-month-old calves.DISCUSSIONThe lymphoid tissues at different levels of therespiratory tract identified by our method are basicallysimilar and histologically resemble those examined in otherMorphology of Lymphoid Nodules and Aggregates in the Lungs of Calves at Diffrent Agesr13300 AC = anterior cranial lobe"0 PC = posterior cranial lobeQ)- 250 ML = middle loberaC'Ie Q) CA = caudal lobeC'I :::III) 200C'I II)ra :;:;Q)"'C •II) ':::::::::r:::: ·0 150Q) ..r::"0 c..- E 100 . --+-- 3 month0 ~..r::C,50r:::: ---6 monthQ)...J ___._ 8 month0ACAL PCAL MLAL CAALDifferent lobesFigure 6: Length of lymphoid aggregates in different lobes of lung at different ages of calves160AC = anterior cranial lobe"C 140~PC = posterior cranial lobeQ)- ML = middle lobeIIICl Cl) 120 CA = caudal lobee ::JCl In 100Cl InIII ;;Cl) "C 80 ~'~;~'''i,___",,0In ·0 ' ."___ .-,.cCl) .c"C c. 60- E -<>-- 3 rronth0 ~ 40.c .-- -_.6 rronth-"C 20~ __._ 8 rronth0ACAW PCAW WLAW CAAWDifferent lobesFigure 7: Width of lymphoid aggregates in different lobes of lung at different ages of calvesAC = anterior cranial lobePC = posterior cranial lobeML = middle lobeCA = caudal lobe353025In..J!!:! 20-0...Cl) 15 ~",.,..""",,-~''''-''''.cE::JZ 1050AC3month-6month-8monthPC fvL CAFigure 8: Number of IELs of lymphoid nodules in different lobes of lung at different ages of calvesSaw Po Po, A.B.Z. Zuki and M. Zamri-Saad1418161412NO.ofIELs 10 •86420ACAC = anterior cranial lobePC = posterior cranial lobeML = middle lobeCA = caudal lobe• __..,_ 3 month-6month_"_8 monthPC CAMLFigure 9: Number of IELs of lymphoid aggregates in different lobes of lung at different ages of calvesmammals (Bienenstock et al., 1973; McDermott et al., 1982;Anderson et al., 1986a; Mair et al., 1987; Huang et al.,1990). The frequency of occurrence of BALT ranges from100% in rabbit and rats, to 50% in guinea pigs, 33% in pigand 0% in cats (Pabst and Gehrke, 1990). In horse, BALTwas absent immediately after birth, common in foals andless common in yearlings and 2 -year-old animals (Blundenand Gower, 1999). In cattle, the mean number of BALTfoci increases with age to the maximum at 18 months andthen declines in adults (Anderson et al., 1986a). The majorspecies and age differences in BALT might be the reasonfor the marked differences in pulmonary immuneresponses between species and between individual ofdifferent ages (Pabst and Gehrke, 1990). The present studyhad found that the distribution of lymphoid nodules andlymphoid aggregates in the lung of calves varies betweendifferent ages and between different lobes of the sameage. The number and size of lymphoid nodules andlymphoid aggregates increased with age. The findingsare similar to the previous studies in cattle (Anderson etal., 1986a), chicken and turkey (Fagerland andArp, 1993),human (Tscherning et al., 1995) and horse (Mair et al.,1987; Horohov, 2004). However, the number of BALTdeclines in adult cattle (Anderson et al., 1986a). Basedon the current findings, it is suggested that the occurrenceof BALT is influenced by age and location. Results ofthis study show that the lymphoid nodules and lymphoidaggregates are significantly higher (P<0.05) in the craniallobes than other lobes.Few aggregated lymphoid tissues and lymphoidnodules were found in the 3-month-old calves. Comparedwith most species (Bienenstock et al., 1973a; Chen et al.,1989; Mair et al., 1987; Huang et al., 1990), the intra-pulmonary tract of 6- and 8- month-old calves possessedrelatively more aggregated lymphoid tissues andlymphoid nodules and they were seen in bronchi,bronchioles and respiratory bronchioles. However, thisfinding is similar to the finding in equine and ovine (Mairet al., 1987; Chen et al., 1989). The number of IELsgradually increased with age and a regional variation inthe number of IELs was found. Thus, the IELs maycorrelate with the region which firstly contacted withenvironmental air. This finding supports the observationby Horohov (2004). The number ofIELs was considerablyless in 3-month-old calves compared to that of 6- and 8-month-old calves. This finding suggests that the exposureto airborne antigens or formites stimulate the IELs.Boismenu and Havran (1994) and Goto et al. (2000) reportthat bronchial IELs may be stimulated by bronchialepithelial cell growth and may play important roles inairway mucosal immunity. According to Erie and Pabst(2000), IELs maintain non pathogenic allergy in theairway. Thus, an increase in the number of IELs mayincrease the mucosal immunity of the lung. According tomorphometric analysis, it is concluded that the highestcellular organisations of BALT was observed in 8-month-old calves.ACKNOWLEDGEMENTThis study was supported by the Ministry of Science,Technology and Innovation Malaysia IRPA ResearchGrant No 5484200. The authors wish to thank Mr. Mohd.Jamail Samad from the Histopathology Laboratory,Faculty of Veterinary Medicine, Universiti Putra Malaysiafor technical support.REFERENCFSAnderson, M.L., Moore, P.F., Hyde, D.M. andDungworth, D.L. (1986a). Bronchus associatedlymphoid tissue in the lung of cattle. Relationship toage. Res. Vet. Sci. 41: 211-220.Morphology of Lymphoid Nodules and Aggregates in the Lungs of Calves at Diffrent AgesAnderson, M.L., Moore, P.F., Hyde, D.M. andDungworth, D.L. (1986b). Immunoglobulincontaining cells in the tracheobronchial tree of cattle:Relationship to age. Res. Vet. Sci. 41: 221-227.Bienenstock, J. (1984). Bronchus-associated lymphoidtissue. In: Immunology of the Lung and UpperRespiratory Tract. J. Bienenstock (Ed.) McGraw- Hill.New York. pp.96-118.Bienenstock, J., Johnston, N. and Perey, D.Y.E. (1973).Bronchial lymphoid tissue. Morphologicalcharacteristic. Lab. Investi. 28: 686-692.Blunden, AS and Gower, S.M. (1999). 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Exper. Path. 23: 47-112.Pabst, R and Gehrke, I. (1990). la the bronchus-associatedlymphoid tissue (BALT) all integral structure of thelung in normal mammals, including humans? Amer.J. Resp. Cel. Mole. Bio. 3: 131-135.Tscherning, T., Kleemann, WJ. and Pabst, R. (1995).Bronchus-associated lymphoid tissue (BALT) in thelung of children who had died from sudden infantdeath syndrome and other causes. Thrax. 50: 658-660.Woodruff, JJ., Clarke, L.M. and Chin, Y.H. (1987). Specificcell-adhesion mechanisms determining migrationpathways of recirculation lymphocytes. Ann. Revi.Immun. 5: 201-222.

Morphology of lymphoid nodules and aggregates in the lungs of calves at different ages

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Morphology of lymphoid nodules and aggregates in the lungs of calves at different ages

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