©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2009:47(4): 633(633-638)10.2478/v10042-008-0119-z

Introduction

The presence of polluting substances in the atmosphere is a phenomenon largely related to urban andindustrial areas. It is here that the emissions sourcesfrom heating plants, industrial activities and motor-vehicle waste gases are mostly concentrated. Suchsubstances can exert damaging effects to both humanand environmental health, according to the contami-nant's quantities being considered, the exposure time,and the harmful effects of the substance in question[1].Adecisive influence on the concentration of pollut-ing-agents is performed by the weather conditions,which can either encourage or inhibit the spreading of contaminants in the atmosphere. Among the earth'satmosphere contaminants, sulphur dioxide, carbondioxide, carbon monoxide, nitrogen oxides, andgaseous hydrocarbons are to be enumerated. Withrespect to gaseous hydrocarbons, which are emittedafter combustion as a particulate, the term hereemployed is generically referred to substances presentin the air in the form of solid or liquid particles. Inaddition to the aforesaid substances, several heavymetals, such as lead, mercury and zinc are normally present. Although in modest amounts, in the air, in thewater and in the soil. When in high concentration, suchmetals do embody threat to the ecosystems. The dan-gerousness of heavy metals is due both to their non- biodegradability and to the possibility of their accu-mulation effects within living organisms [1].The respiratory system is the main target of manychemicals compounds' toxic effect. In consideration of its anatomical location, and of the function its exerts,the respiratory system is constantly in contact withnoxious agents present in the atmosphere. It is alsowell known that most inhaled particles can build up inthe lung's tributary lymph nodes. Inhaling is the mainway through which target organs, lungs and lymph

FOLIAHISTOCHEMICAETCYTOBIOLOGICA

Vol. 47, No. 4, 2009 pp. 633-638

Lesions associated with mineral deposition in the lymphnodes and lungs of cattle: a case-control studyof environmental health hazard

A. Perillo

, O. Paciello

, A. Tinelli

, A. Morelli

, C. Losacco

, A. Troncone

Department of Animal Health and Welfare, Faculty of Veterinary Medicine, Bari, Italy

Department of Pathology and Animal Health, University of Naples Federico II, Naples, Italy

General practitioner Bari, Italy

Abstract:

This report focuses on the state of health of the cattle raised in the district of Taranto – city of Italy rated as envi-ronmentally at risk. Representative samples of lungs, bronchial and mediastinal lymph nodes of cattle from district of Taran-to's slaughterhouses were collected. After a macroscopic examination, samples with marked lesions were processed for lightmicroscopy. Samples were also observed with polarized light microscopy, scanning electron microscopy and with micro-analysis. The macroscopic examination revealed that 60 out of 183 samples showed marked lesions. Lung alterations werecharacterized by thickening of the alveolar septa and by the latter's modifying action on the alveolar spaces, foci of fibrosisand bronchopulmonary inflammation. For 51 out of the 60 samples observed, the histological examination confirmed the presence of pneumoconiosis and lymph nodal anthracosis. Energy-dispersive X-ray microanalysis of lung samples identi-fied a wide range of elements including silicon, aluminium, titanium, iron, carbon and small amount of the other metals. Inthe lymph-nodes the same kind of metals with a different levels of distribution was observed. Our survey on cattle farmedin areas at high risk of pollution may be helpful to the estimation of the exposure risk for man to environmental contami-nants and to the evaluation of the occurrence of the pathological manifestations as well.

Keywords:

pulmonary fibrosis, lymph-node, environmental pollution, cattle, mineral.

Correspondence:

O. Paciello, Dept. of Pathology and AnimalHealth, University of Naples Federico II, Via Delpino,1 - 80137, Naples, Italy; tel.: (+39) 0812536466,fax.: (+39) 0812536186, e-mail: paciello@unina.it

nodes chiefly, come into contact with the contaminantsdispersed in the air [2,3]. Absorption of toxic sub-stances depends on several factors, first of all the phys-ical, chemical, anatomical and functional properties of the cardio-respiratory system. The absorption mecha-nism can differ whether it interests a gas or a liquidsubstance. Nonetheless, in general the absorption-capacity in the lung increases as solubility in water increases. Highly hydro soluble gases, such as, sulphur dioxide (SO

), do not usually go beyond the nasophar-ynx. On the contrary, poorly hydro soluble gases, suchas nitrogen dioxide (NO

), or the hydro soluble gasesadsorbed by the particulate or in the form of aerosol,can reach the bronchial tree's peripheral areas thusinducing a toxic reaction. High concentrations of suchsubstances can provoke disorders and diseases [4-7].Among the most representative morphological alter-ations which interest the respiratory system, fibrosis,emphysema and neoplasias are to be mentioned [4-7].Contaminants of a factory origin which can most fre-quently cause such pathologies are: asbestos, cadmi-um, arsenic, sulphur dioxide, ozone, formaldehyde.Domestic as well as wild animals are exposed, justlike man, to contaminants present in the air, in the soil,in the water and in food. They can suffer from the potential acute and chronic effects of such exposure.Animal population exposed to contaminants in theenvironment are referred to as "animal sentinel sys-tems" (ASS) [8,9]. These are designed to the identifi-cation or the monitoring of a variety of environmentalcontaminants dangerous to the human, animal andecosystem health. Asentinel animal is defines like"one which points out or directs attention to some-thing", and more specifically, sentinel animals aredefines like "a group of animals whose presence actsas a sign of particular environmental conditions" [8,9].The present study focused on cattle farmed in thedistrict of Taranto, a city rated as environmentally atrisk. The aim of our research was to assess the effectsof the environmental pollution in lungs and tributarylymph nodes of cattle.

Materials and methods

183 samples from lungs and bronchial as well as mediastinallymph nodes of cattle from district of Taranto's slaughterhouseswere collected from September 2004 through November 2005. Theanimals were end-of-career milk cows, Bruna, Italian Frisona,Podolica and cross breeds. By consulting the daily slaughter – list-tracing animals' serial number, age and company code – it was pos-sible to infer that all animal were from pollutant districts.After a careful macroscopic examination, samples with markedlesions were processed for light microscopy. Lung and bronchiallymph node samples were fixed in buffered formalin 10% and processed for paraffin embedding. Histological diagnosis wasassessed on 6-nm-thick hematoxylin-eosin (HE) and Van-Gieson-stained sections. In addition, some sections were stained withZiehl-Neelsen method and with Fite's new fuchsine formaldehydemethod for the acid-fast bacilli.For scanning electron microscopy (SEM), lung and bronchiallymph-node samples were fixed in paraformaldehyde 4% in phos- phate buffer, dehydrated in a graded ethanol series and critical point dried. Specimens were mounted on aluminium stubs andcoated with a layer of gold in a polaron E 5400 sputter-coater andexamined with a Cambridge Stereoscan 240 scanning electronmicroscope. Some samples were mounted on carbon stubs andcoasted with a layer of carbon and analysed by means of an AN10/25 S Link X-ray analytical system with an energy dispersivespectrometer (EDS).

Results

Lungs and lymph-nodes with pathologic changes asso-ciated to some infectious agent (other than smallamounts of terminal pneumonia) were excluded fromthese studies. The macroscopic examination revealedthat 60 out of 183 samples showed noticeable lesionsrelated to air pollution.Lung alterations were marked by thickening of thealveolar septa and by the latter are modifying actionson the alveolar spaces, initial foci of fibrosis and bron-chopulmonary inflammation (Fig. 1-2). In some cases,the presence of small, barely visible blackish dots,identified as carbon particles within macrophages,would stood out in the subpleuric interstice principal-ly, giving the lung a blackish colour (Fig. 1). Micro-scopically the medullary sinus macrophages had smallneed-like intracellular crystals and large stranslucentestracellular crystals observed with the polarized lightmicroscopy (Fig. 3 and 4). In the subpleuric interstice,carbuncular particles present in macrophages' cyto- plasm were accountable for a responsiveness of afibrous kind, with a tendency towards interstice andalveolus invasion. Further histological investigationevidenced, moreover, a peribronchial fibrosis as wellas lymph-plasmocyte and granulocyte infiltrates (Figs5 and 6).The accumulation-effect of given contaminantschanges in a linear way as age increases, althoughlesions in animals no older than 4 or 5 months from adistant zone from the most-at-risk industrial pole weredetected.Ablackish, diffused pigmentation marked the lunglymph nodes' surface once cut (Fig. 2). Carbon-dustaccumulation in many samples would look as anorganization of minute, dark spots arranged as a crownalong the edge between cortex and medulla. Involvedlymph nodes would appear normal or increased in vol-ume, with a reactive hyperplasia area of variable enti-ty (Fig. 2). Under both macroscopic and microscopicexamination, a distinction between follicular hyperpla-sia phenomena, circulatory disorders, exudation imbi- bition and fibrosis proliferation was practicable.The histological examination revealed that lymphnode displayed a severe lymphoid depletion, anintense connective reaction which could alter the organarchitecture completely. Sparse or aggregated

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Fig. 1.

Cattle, lung: pleural thickness and severe interstitial emphysema.

Fig. 2.

Cattle, lymph node: Mineral associated lymphadenopa-thy with follicular hyperplasia and little dots of blackish pigments in the parenchyma.

Fig. 3.

Cattle, lung: Presence of carboniotic exsoge-nous particles within macrophages (H+E original magnification ×40).

Fig. 4.

Cattle, lung: macrophage with a large translucent intracel-lular crystals (H+E, polarized light. original magnification ×40).

Fig. 5.

Cattle, lung: Severe peribronchial fibrosis associated with mus-cular hypertrophy and broncospasm (E.E. original magnification ×20).

Fig. 6.

Cattle, lung: Marked proliferation of peribronchial con-nective tissue associated with the presence of macrophages full of carbon particles (Van-Gieson original magnification ×10).