RPCV (2013) 108 (585-586) 69-91

XVIII Meeting of the Portuguese Society of Animal Pathology

- International Meeting of Leporidae Pathology

Editorial
The Portuguese Society of Animal Pathology (SPPA)
is a small non-profit organization and a daughter Society of the Portuguese Society of Veterinary Science
(SPCV), which normally holds an annual meeting in
the institutions from which its members belong (e.g.
universities or government laboratories with activities
in the field of Pathology).
In an effort to decentralize their meetings, a tradition
already started before, and both to aim at involving the
community of teachers and students of the research institutions that host the annual meeting and also to diversify the main subject of its meetings, SPPA targeted
new, younger and broader audiences, as well as new
sectors and knowledge fields, trying to cover the pathology of a wide animal species range. Hence, to the
present meeting we have chosen leporideae pathology
as the main subject and a necropsy workshop, open to
students and technicians - XVIII Meeting of the Portuguese Society of Animal Pathology. International
Meeting of Leporidae Pathology, 9 and 10th of May
2013.
Also, continuing the journey, already begun, of
aiming evermore towards internationalization, this
meeting will have reputable national and international speakers, to whom above all I acknowledge their
presence and immediate acceptance. Moreover, the
scientific committee with national and international
members, the three official languages, and the widest dissemination among technicians and students,
will certainly make this meeting more open, more involved and more attractive. So we hope, so we wish,
so we worked for!
Our special guests will include an international expert, who will share with us his scientific knowledge,
Prof. Dr. Daniel Fernandez de Luco Martinez, professor at the University of Zaragoza in the Department
of Animal Pathology, Faculty of Veterinary Science.
Graduated in Veterinary Medicine by the University
of Zaragoza in 1985 and obtained a PhD by the same
institution in 1991. He has developed his research in
pathology of wildlife and wildlife hunting health control in the region of Aragon in Spain. He has dozens of
publications in international peer-review journals and
was a guest speaker at dozens of conferences. He is
responsible for several research projects, having juried
several doctoral theses.

Also, not forgetting nationals with high value and

enriching practical experience in the field, an European expert, Dr. Jos Manuel Monteiro Azevedo. He has
earned his degree in Veterinary Medicine in the University of Tras-os-Montes and Alto Douro in 1999, and
is currently a Veterinarian for Coren, Spain, being the
Head of Industrial Rabbit Farming and the responsible
for the Industrial Cuniculture in the Iberian Peninsula.
His professional activity, since graduation, has been
dedicated to rabbits having taken several positions at
national and international companies. He collaborated
in teaching several courses at the University of Trsos-Montes and Alto Douro.
We will have the opportunity to share and exchange
experiences on animal pathology, results of research
projects, discuss pathological diagnosis of difficult or
rare cases, as well as the recent scientific advances in
this field in Europe, which is undoubtedly the largest
professional enrichment we could obtain. For the 18th
time this small scale but dynamic meeting, constitutes
an enrichment both scientifically, technically and professionally, and will allow us to grow a bit more professionally. Moreover, this will be also an excellent opportunity to meet again our colleagues and friends and
to discover Evora, one of the most interesting, beautiful and pleasant cities to visit in Portugal.
The success of this meeting is already notorious by
the number of members who decided to join us. Thank
you for believing in this project, thank you for being
here!
In the name of SPPA and the Organizing Committee, I want to acknowledge all those who contributed
to make this event possible, namely the University of
vora, in the person of its Rector Ex Sr. Prof. Dr.
Carlos Alberto dos Santos Braumann and of its ViceRector, Prof. Dr. Marta Silvrio, for immediately welcoming this endeavor, namely for all the logistical support provided and specially the generous availability in
providing their facilities. I would like to acknowledge
the Portuguese Society of Veterinary Sciences who
provided an important assistance in the financial management, and all those who helped us, teachers, students, staff and sponsors.

The President of SPPA

Helena Maria Vala Correia
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RPCV (2013) 108 (585-586) 69-91

Plenary sessions
Common gross lesions in farmed rabbits
Fernndez de Luco D. *
Departamento de Patologa Animal. Facultad de Veterinaria. Universidad de Zaragoza. C/ Miguel Servet
177. 50013 Zaragoza. Spain.
*

luco@unizar.es

Introduction: The identification of rabbit gross lesions by systems and organs in association with a disease, syndrome, aetiology or unknown cause is very
important for veterinary practitioners.
Material and methods: Necropsy has been made in
several farmed rabbits with mainly respiratory or digestive problems, as well as delayed growth.
Results: Dermatitis associated to myxomatosis,
ringworm, mange or staphylococci are common.
The respiratory system problems are localized in the
nasal cavity and lungs mainly associated to Pasteurella
multocida and Bordetella bronchiseptica. Young rabbits are especially sensitive to acute and chronic pneumonia. Haemorrhages can be seen in cases of rabbit
calicivirus disease.

Situaes de urgncia em cunicultura

industrial
Monteiro J.M.1*, Alegria N.2, Coelho A.C.2
2

Sumicor Portugal Lda; Coren S. C. G.

Dept. de Cincias Veterinrias, CECAV, Universidade
de Trs-os-Montes e Alto Douro (UTAD)

jos.monteiro@coren.es

Introduo: O mdico veterinrio durante a sua

atividade contribui para a reduo das perdas associadas morbilidade e mortalidade em cunicultura, para
a melhoria dos resultados produtivos, atuando como
consultor e orientando o produtor nos investimentos.
O presente trabalho teve como objetivo reportar a
atuao do mdico veterinrio na sua vertente clnica,
dando nfase s principais situaes de emergncia em
cunicultura.
Material e mtodos: Procedeu-se realizao de um
estudo observacional retrospetivo da actividade clnica
na Pennsula Ibrica, durante os anos de 2009 a 2012.
Foi efectuado o exame clnico dos animais provenientes de 76 exploraes, tendo-se procedido realizao de necrpsias, com recurso a exames laboratoriais e elaborao dos respetivos registos que serviram
de base ao presente estudo.

70

Probably, digestive processes are the most important

problems in growing rabbits. Epizootic rabbit enteropathy, with still unknown aetiology, is a relatively recent
process that has become the main cause of mortality
in farmed rabbit. Other enteritis processes are principally associated to coccidia, E. coli, Clostridium spp.,
C. spiroforme, C. piliforme (Tyzzers disease), Salmonella spp., as well as nematodes (Passalurus spp.). Inflammation and necrosis of the liver can be observed
as small white spots associated to Eimeria stiedae
(hepatic coccidiosis), Salmonella spp., C. piliforme,
Cysticercus pisiformis, etc. Necrosis with karyorrhexis
of the hepatocytes is a common finding in rabbit calicivirus disease.
Testis, uterus and mammary gland are the main organs affected in the reproductive system, as well as
dystocia in pregnant rabbits. Processes and agents
mainly involved are mixomatosis, syphilis (Treponema
cuniculi), P. multocida, Staphylococcus spp., etc
Finally, encephalitozoonosis by Encephalitozoon
cuniculi is present in many rabbitries. Nervous signs,
granulomatous encephalitis and interstitial nephritis
are present in the affected animals.

Resultados: Determinou-se que a maioria das chamadas de urgncia, se deveram s condies clnicas
de enteropatia mucide, colibacilose, pasteurelose, pododermatite, mixomatose e enterotoxemia cujas leses
anatomopatolgicas foram documentadas.
As exploraes com maternidade e engorda no mesmo pavilho, mau maneio, sem controlo ambiental e
sujeitas a variaes climticas, situaes potencialmente geradoras de maior stresse e consequente reduo
da imunidade, apresentaram aumento da morbilidade
e menor produtividade, enquanto as exploraes com
ambiente controlado e bom maneio, revelaram menos
situaes de emergncia clnica e boa produtividade.
Concluso: Foi possvel verificar que as exploraes
onde as medidas de bem-estar animal e de controlo ambiental rigoroso j se encontravam implementadas, apresentaram melhores resultados produtivos e menor morbilidade em todas as fases de produo. Os resultados
sugerem que a actuao do mdico veterinrio dever incidir tambm nas vertentes atrs referidas, pois s assim
se conseguir aumentar a rentabilidade das exploraes.

Emergency Situations in Industrial

Rabbit Farming
Introduction: The veterinary doctor in the course of
his activities contributes to the reduction of losses as-

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sociated with morbidity and mortality, by improving

the results of production, acting as a consultant, and
orienting the producer in his investments.
The present paper has as its objective to report on
the performance of the veterinary doctor in the clinical aspect of his work, giving emphasis to the principal emergency situations in rabbit farming.
Material and methods: A retrospective observational study was carried out of the clinical activity in
the Iberian Peninsula during the years 2009 to 2012.
The clinical examination of animals from 76 investigations was carried out, subsequent to the realization
of autopsies, with the use of laboratory tests whenever
necessary and the elaboration of the respective registers which served as a base for the present study.
Results: It was found that the majority of emergency
calls, resulted from the clinical conditions of mucoid
enteropathy, colibacillosis, pasteurellosis, foot pad dermatitis, myxomatosis and enterotoxaemia, of which the

RPCV (2013) 108 (585-586) 69-91

anatomopathological injuries were documented.

Farms with maternity and fattening stages in the same
building, poor handling, without environmental control
and subjected to climatic variations, all of which were
potential generators of major stress and consequently
reduction in immunity, showed an increase in morbidity
and lower productivity, while the farms with environmental control and good handling revealed less clinical
emergency situations and good productivity.
Conclusion: It was possible to verify that the farms
where the animal welfare measures being and rigorous environmental control had already been implemented showed better results in production and less
morbidity in all the phases of production. The results suggest that the activity of the veterinary doctor
should also occur in the aspects previously referred;
as it is only in this way that it will be possible to increase the profitability of the farms.

Oral communications
Controlo de pragas em parques Zoolgicos:
repercusses parasitrias, patolgicas e de
Sade Pblica
Crespo, A.P.1,2*, Lapo, N.1, Correia, J. 2, Gomes, L. 2,
Vaz, Y.2 Madeira de Carvalho, L.M.2
J ardim Zoolgico de Lisboa, Estrada de Benfica, 158160, 1549-004 Lisboa;
2
CIISA, Faculdade de Medicina Veterinria, Universidade Tcnica de Lisboa, Av. da Universidade Tcnica
de Lisboa, 1300-477 Lisboa, Portugal (madeiradecarvalho@fmv.utl.pt);
1

apamcrespo@gmail.com

Introduo: A ligao prxima entre animais,

Homem e pragas em zoos ou parques zoolgicos,
constitui um risco na transmisso de muitos agentes
de doena, alguns dos quais ocasionando doenas
zoonticas, podendo afectar no s os animais da
coleco zoolgica, como tambm trabalhadores do
zoo e visitantes.
Material e mtodos: Integrado num estudo sobre controlo de pragas no Jardim Zoolgico de Lisboa, efectuou-se um rastreio parasitolgico em 100
roedores (50 Mus musculus e 50 Rattus norvegicus).
Os endoparasitas adultos foram coligidos aps decantaes dos contedos gastrintestinais e esfregaos
por aposio do fgado (colorao pelo Giemsa) e de
fezes (colorao pelo Ziehl-Neelsen), efectuando-se
anlises coprolgicas por flutuao (mtodo de Willis) e de sedimentao espontnea. Os fgados foram
submetidos a anlise histolgica, fragmentos do f-

gado foram fixados durante 24 horas, procedeu-se

ao corte dos blocos de parafina e as amostras foram
fixadas e coradas pela colorao da Hematoxilina e
Eritrosina.
Resultados: Dos roedores observados, 82 apresentaram formas de endoparasitas, com prevalncias de
84,0% para R. norvegicus e de 80,0% para M. musculus. Das espcies de roedores, Rattus norvegicus representou maior importncia na transmisso dos parasitas assinalados. Identificaram-se um total de cinco
espcies de endoparasitas com potencial zoontico:
Hymenolepis diminuta (16,0%), Cysticercus fasciolaris (12,0%), Syphacia obvelata (37,0%), Calodium
hepaticum (32,0%) e Cryptosporidium sp. (5,0%).
Concluso Deste conjunto de parasitas, C. hepaticum, que j foi detectado aps a morte de primatas
no humanos com consequncias patolgicas no fgado daqueles hospedeiros. Alm da perda de animais, o risco zoontico de transmisso de parasitas,
bactrias e vrus aos trabalhadores e visitantes do
Jardim Zoolgico de Lisboa, deve ser sempre considerado como um problema de grande importncia
e impacto em Sade Pblica.

Pest control in Zoos: parasitic, pathological and Public Health repercussions

Introduction: The close connection between animals, humans and pests, poses a risk for the transmission of many agents, some of them causing zoonotic
diseases, affecting not only the animals in the zoological collection, as well as, zoo workers and visitors
themselves.

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RPCV (2013) 108 (585-586) 69-91

Material and methods: Following a pest control

study at the Lisbon Zoo, a survey on endo and ectoparasites was performed on 100 commensal rodents (50
Mus musculus and 50 Rattus norvegicus). Endoparasites were collected from the gastrointestinal contents
by a decanting process (adult specimens). Smears by
liver apposition (Giemsa staining) and by extension
feces (Ziehl-Neelsen staining), together with coprological exams by Willis flotation and spontaneous sedimentation techniques, were performed. The livers subjected to histological analysis of liver fragments were
fixed for 24 hours, proceeded to cut the paraffin blocks
and the samples were fixed and stained by Hematoxylin staining and Erythrosine.
Results: From the total number of studied rodents,
82 were positive for endoparasites, with prevalence
rates of 84.0% for R. norvegicus and 80.0% for M.

musculus. Concerning the role of rodent species in

parasite transmission, Rattus norvegicus represented
the greater importance. Five species of endoparasites
with zoonotic potential were identified: Hymenolepis
diminuta (16.0%), Cysticercus fasciolaris (12.0%),
Syphacia obvelata (37.0%), Calodium hepaticum
(32.0%) and Cryptosporidium sp. (5.0%).
Conclusion: Of this set of parasites, C. hepaticum
constitutes an extreme example, since its presence was
already detected after death in non-human primates,
with severe consequences for these hosts liver. Beside
the loss of animals, the risk of transmission of zoonotic
parasites, bacteria and viruses to workers and visitors
of the Lisbon Zoo should always be considered as a
problem of great importance and impact on Public
Health.

Um caso de intoxico por cebola (Allium

cepa) em bovinos

que se trata dum caso de intoxicao por cebolas;

acresce-se ainda que, aps ter sido retirado o referido
suplemento e administrada uma teraputica de suporte, os animais que estavam doentes recuperaram e no
houve mais mortalidade.

Monteiro M. 1*, Mendona P. 1, Carvalho P.1, Mariano I.2

1
2
*

Laboratrio de Patologia/INIAV;
Laboratrio Veterinrio de Montemor-o-Novo
madalena.monteiro@iniav.pt

Introduo: A anemia hemoltica resultante do

consumo prolongado de cebolas, Allium cepa, tem
sido descrita em vrias espcies animais nomeadamente em ces, bovinos e caprinos; o princpio txico
presente na cebola o n-propil-dissulfito responsvel
por alteraes da hemoglobina.
Os Autores descrevem vrios casos de mortes em
bovinos adultos que eram alimentados com pastagem
natural e recebiam um suplemento de silagem de desperdcio de tomate misturada com cebolas e alhos.
Caso clnico: Os animais apresentavam anemia,
hemoglobinria e em alguns casos, ictercia.
Foram realizadas as necrpsias de dois animais; a
abertura do cadver revelou um intenso cheiro a cebolas; observaram-se alteraes hepticas e renais.
Discusso: Microscopicamente havia extensas zonas de necrose heptica centrolobular e necrose dos
tubos renais simultaneamente com acumulao dum
material compatvel com hemoglobina.
Os exames efectuados em animais coabitantes, para
diagnstico diferencial com doenas que podem apresentar sinais clnicos idnticos, resultaram negativos
para leptospirose sendo detectada, em apenas um animal, uma pequena infeco de Theileria annulata.
Concluso: As leses microscpicas so sugestivas
de uma anemia hemoltica tendo a sua associao ao
suplemento alimentar em causa, permitido concluir

72

A case of onion (Allium cepa) intoxication

in cattle
Introduction: In domestic animals the prolonged
ingestion of onion (Allium cepa) has long been associated with the emergence of hemolytic anemia. This
toxicity is due to the presence of n-propil- disulphide.
In this presentation the authors describe the occurrence of several cases of toxicity in a grazing bovine
herd due to ingestion of silage containing tomato, onion and garlic by-products.
Clinical case: The affected animals showed anemia, haemoglobinuria and, occasionally, jaundice
leading to death in some severe cases.
Three animals were submitted to necropsy and the
most prominent feature was a marked onion odor. The
kidneys were dark brown and the liver slightly orange.
Microscopically extensive areas of centrolobular liver
necrosis as well as renal tubular necrosis with accumulation of hemoglobin-like material were seen.
Differential diagnostic testing excluded leptopirosis and revealed one Theileria annulata infected animal.
Conclusion: The symptoms and the lesions observed were strongly suggestive of a haemolytic anemia. Taken together with the feed supplementation
these facts enabled us to conclude for onion toxicity.
Furthermore, after the discontinuation of the referred
silage supplement and upon implementation of supportive care, the affected animals began to recover
and no further deaths occurred.

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Um caso raro de neoplasia retrobulbar

Vala H1,2*, Mega A1,2, Santos C1,2, Cruz R1,2, Nbrega
C1,2, Mesquita JR1,2, Esteves F1,2, Vieira F3, Ferraz A4
Escola Superior Agrria de Viseu, Instituto Politcnico de Viseu, Portugal;
2
Centro de Estudos em Educao, Tecnologias e Sade,
Instituto Politcnico de Viseu, Portugal;
3
Histocit, Portugal;
4
Clnica Veterinria Planeta Animal, Aveiro, Portugal.
1

* hvala@esav.ipv.pt
Introduo: Neoplasias envolvendo o nervo tico
so raras. O meningioma intraorbitrio, a neoplasia
mais comum sediada no nervo tico, constitui apenas
3% de todos os meningiomas em ces, razo pela qual
existem poucos estudos para permitir prever os seus
sintomas e comportamento biolgico.
Objetivos: Os autores descrevem e submetem discusso, um caso raro de meningioma retrobulbar, em
particular para apreciao da graduao histolgica e
o prognstico.
Histria Clnica: Um Labrador Retriever de 10
anos de idade, foi apresentado com queixas de edema
e descarga purulenta do olho direito. Apresentava exoftalmia, protuso da terceira plpebra, quemose, lcera
profunda da crnea e ausncia de reflexo pupilar luz,
no mesmo olho. O exame fsico, oftlmico e ecogrfico permitiu diagnosticar exoftalmia devido a massa
retrobulbar.
Material e Mtodos: A amostra foi fixada em formol tamponado a 10 %, processada de acordo com o
protocolo de rotina, tendo igualmente sido realizados
estudos imunohistoqumicos, usando o mtodo do
Complexo Estreptavidina-biotina-peroxidase.
Resultados: O exame macroscopico revelou uma
massa cnica, localizada na poro retrobulbar do nervo tico, adjacente poro posterior do globo ocular,
apresentando colorao esbranquiada, aspeto homogneo, lobulado e consistncia firme.
O exame microscopico revelou clulas fusiformes,
com ncleo redondo a oval, cromatina em padro pontilhado, citoplasma eosinoflico, moderado, organizadas
em ninhos, em torno de capilares, separados por feixes
interlaados, com estroma muito vascularizado e escasso.

RPCV (2013) 108 (585-586) 69-91

Concluso: Os autores sugerem o diagnstico de

meningiomado tipo transicional, tendo em conta as caratersticas histopatolgicas e os resultados do estudo
de imunohistoqumica.
Agradecimentos: FCT e CI&DETS (PEst-OE/
CED/UI4016/2011)

A rare case of retrobulbar neoplasia

Introduction: Neoplasms involving the optic nerve
are rare. Intraorbital meningiomas, the most common
tumour of the optic nerve, represent only 3% of all
meningiomas in dogs, so few data exists to predict its
symptoms and biologic behaviour.
Objectives: The authors describe and submit for
discussion, a rare case of retrobulbar meningioma,
in particular for assessment of histological grade and
prognosis.
Medical History: A 10 year old Labrador Retriever
male dog was presented for complaints of swelling and
purulent discharge of the right eye. The animal had exophthalmos, protrusion of the third eyelid, chemosis,
deep corneal ulceration and no pupillary light reflex.
Exophthalmos due to a retrobulbar mass was diagnosed
after physical, ophthalmic and ultrasound examination.
Material and Methods: The sample was fixed in
10 % buffered formalin solution, processed for routine
histopathological diagnosis and a standard Streptavidin-Biotin Complex technique for immunohistochemical evaluation was also performed.
Results: Macroscopic examination revealed a conic
mass, located in the retrobulbar portion of the optic
nerve, adjacent to the posterior aspect of the globe with
homogeneous, lobulated and whitish appearance and
firm consistency.
Microscopic examination revealed spindled cells,
with round-to-oval, finely stippled nuclei, small or
indistinct nucleoli, moderate amount of eosinophilic
cytoplasm, arranged in concentric whorls around capillaries, separated by interlacing fascicles, with small
amount of well vascularized, collagenous stroma.
Conclusion: The authors suggested the diagnosis of
transitional meningioma, based on the histopathological diagnosis and immunohistochemistry results.
Acknowledgments: FCT and CI&DETS (PEst-OE/
CED/UI4016/2011)

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Ocorrncia de parasitismo externo em

coelhos de companhia
Almeida, C.1*, Henriques C. 1, Bernardino, M. 1,2, Madeira de Carvalho, L.M. 2
 xoclinic, R. Dom Antnio Ribeiro n1 loja B, 1495E
145 Algs, Portugal
2
CIISA, Fac Med Vet, Univ Tecn Lisboa, Av. da Universidade Tcnica de Lisboa, 1300-477 Lisboa, Portugal
1

exoclinic@mail.com

Introduo: Em Portugal, tal como sucedeu anteriormente noutros pases como Inglaterra, Estados
Unidos e Alemanha, o coelho domstico tem revelado
crescente popularidade como animal de companhia,
devido sua grande inteligncia e interessante interao com a famlia.
Durante o perodo de doze meses (Jan-Dez 2012)
foram consultados 98 coelhos na Exoclinic sobretudo por problemas de sobrecrescimento dentrio,
problemas gastrointestinais e manifestaes dermatolgicas.
Material e mtodos: Dos 25 casos de manifestaes
cutneas, para alm do exame fsico foram submetidos a raspagem cutnea e citologia direta das leses.
Foram identificados 16 coelhos positivos a parasitismo
externo: Psoroptes cuniculi (69%) e Scarcoptes scabiei (31%). Dos animais afetados, 81% apresentavam
menos de seis meses de idade, verificando-se casos positivos em machos e fmeas e nas raas Fuzzy-Lop,
Lion Head e Mini Holands.
O tratamento sugerido foi de injees subcutneas
de ivermetina (200g/kg) repetindo ao fim de 14 dias,
28 dias, sugerindo uma quarta injeo e quinta injeo
at desaparecimento total de sinais clnicos. Em alternativa, utilizou-se em metade dos casos, selametina soluo tpica cutnea 15mg, repetindo-se ao fim de 28
dias, sugerindo uma terceira aplicao se necessrio.
Resultados: Em termos de desaparecimento das leses cutneas e crescimento do plo nas zonas de alopcia os resultados obtidos foram bem sucedidos ao
fim de 28 dias de tratamento e com eficcia equivalente
com ambos os protocolos.
Em simultneo foram aplicadas medidas de melhor
maneio e higienizao das instalaes do animal, bem
como o alerta do potencial zoontico de alguns tipos
de sarna. Ainda assim, verificou-se a presena de dois
episdios de manifestaes cutneas de sarna no proprietrio, j instaladas aquando do incio tratamento do
animal.
Concluso: De acordo com a nossa experincia,
o clnico deve estar apto para a correta identificao
dos agentes causais de problemas dermatolgicos em
coelhos. O aumento da casustica destes pacientes representa tambm novas perspetivas de diagnstico e

74

RPCV (2013) 108 (585-586) 69-91

novos desafios teraputicos. na nossa opinio uma

oportunidade para instituir boas prticas veterinrias
de preveno e profilaxia destas doenas, pois o bom
estado geral de sade est condicionado pela correta
informao e educao do proprietrio.

Occurrence of external parasitism

in pet rabbits
Introduction: In Portugal, as happened previously
in other countries like England, Germany and the
United States, the domestic rabbit has shown increasing popularity as a pet, due to its great intelligence
and interesting interaction with the family.
During a twelve month period (Jan-Dec 2012) 98
rabbits were consulted in Exoclinic, mainly due to
dental problems, gastrointestinal diseases and dermatological manifestations.
Material and methods: Of 25 cases of cutaneous
manifestations, in addition to the physical examination, they were subjected to scrape and direct cytology of cutaneous lesions. We identified 16 rabbits
positive for external parasitism: Psoroptes cuniculi
(69%) and Scarcoptes scabiei (31%). Of the affected
animals, 81% were less than six months old, with positive cases in both males and females and in FuzzyLop, Lion Head and Mini Dutch breeds.
The suggested treatment was subcutaneous injections of ivermectin (200g/kg) repeating after 14 days
and 28 days, suggesting a fourth and a fifth injection
until total disappearance of clinical signs. Alternatively, in half the cases was used a topicalolution of
selamectin 15mg, repeating the treatment 28 days after the first one, suggesting a third application if necessary.
Results: In terms of disappearance of skin lesions
and hair growth in areas of alopecia, results were successful after 28 days of treatment, with equivalent efficacy for both protocols.
Simultaneously were applied measures for a better
handling and hygiene of the animal facilities, as well
as the alert for the zoonotic potential of some types of
mange. Still, there was the presence of two episodes
of cutaneous manifestations of scabies in the owners,
already onset before initiating the animal treatment.
Conclusion: According to our experience the clinician must be able to correctly identify the causative
agents of skin problems in rabbits. The increase of
these patients casuistic as pets also opens new prospects for diagnosis and new therapeutic challenges.
It is in our opinion, an opportunity to establish good
veterinary practice prevention and prophylaxis of
these diseases, since the good general health is conditioned by proper education and information of the
owner.

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Um caso de localizao exclusivamente

gstrica de Spirocerca lupi numa raposa
(Vulpes vulpes) em Portugal
Silva J.F.*, Madeira de Carvalho L., Moreira A.S.
CIISA - Faculdade de Medicina Veterinria (Universidade Tcnica de Lisboa), Av. da Universidade Tcnica
(Polo Universitrio Alto da Ajuda), 1300-477 Lisboa
*

jfsilva@fmv.utl.pt

Introduo: O Spirocerca lupi localiza-se caracteristicamente na parede esofgica de Candeos. Ferrantelli et al.(2010) e Diakou et al.(2012), assinalaram a
presena deste Nemtode na parede gstrica de raposas.
Material e mtodos: O cadver de uma raposa adulta, sexo masculino, pesando 5,85 kg foi necropsiado e
colhido material, que foi processado segundo a tcnica
histolgica de rotina. A identificao dos Nemtodes
em cortes histolgicos foi feita de acordo com o artigo
de Chitwood e Lichtenfels (1972) e tendo em conta aspectos bioecolgicos e epidemiolgicos.
Resultados: Na parede do estmago observaram-se
dois ndulos coalescentes, com dimetros respectivamente de 1,2 cm e 1,8 cm. Estes apresentavam superfcie de corte branca, uma cavidade contendo material
necrtico e um Nemtode enrolado sobre si prprio, de
cor avermelhada, com cerca de 1 mm de espessura. O
exame microscpico dos ndulos revelou uma cpsula de tecido conjuntivo fibroso envolvendo Nemtodes
adultos. Estes apresentavam musculatura celomiria e
polimiria, cordes laterais proeminentes, tero encerrando ovos no operculados de casca fina, que continham uma larva. O intestino apresentava um epitlio
simples cbico com microvilosidades alongadas.
Concluso: As caractersticas histolgicas, bioecolgicas e epidemiolgicas do Nemtode permitem

Identificao de Parasitas de Peixes

em Cortes Histolgicos
Ramos, P.1*, Carvalho, R.1, Rosa, F.2
Laboratrio de Patologia dos Animais Aquticos. Instituto Portugus do Mar e da Atmosfera, IPMA, I.P.
Avenida de Braslia, 1449-006 Lisboa, Portugal
2
Instituto de Investigao Cientfica Tropical, IICT.
Rua da Junqueira n 14, 1300-343 Lisboa, Portugal
1
J no se encontra a trabalhar na Instituio
*
Autor correspondente. Tel.: + 00351 213027000 Fax:
+ 00351 962467682
1

pramos@ipma.pt

Introduo: A identificao de parasitas de peixes

em cortes histolgicos poder apresentar alguma dificuldade, sobretudo quando as caractersticas morfol-

RPCV (2013) 108 (585-586) 69-91

classific-lo como Spirocerca lupi, pelo que este um

caso de localizao gstrica deste Nemtode em raposa
em Portugal.

A case of location exclusively in the gastric

wall of Spirocerca lupi in a fox (Vulpes vulpes)
in Portugal
Introduction: One of the Spirocerca lupi current locations is the oesophagic wall in Canidae. Ferrantelli et
al. (201) and Diakou et al. (2012) reported the gastric
wall location of this Nematode in foxes.
Material and methods: An adult male fox weighting 5.85 kg was necropsied and samples were collected, which were processed according to the routine
histological technique. The identification of Nematode
specimens in histological sections was based in the paper by Chitwood and Lichtenfels (1972) and considering bioecological and epidemiological aspects.
Results: In the gastric wall were observed two coalescent nodules, which diameters were, respectively;
1.2 cm and 1.8 cm. Their cut surface was white, with
a cavity containing necrotic material and a red Nematode coiled over itself, with 1 mm thick. The microscopic exam of the nodules revealed a capsule of dense
connective tissue surrounding adult Nematoda. These
worms possessed muscle cells of the coelomyarian and
polymyarian types, prominent lateral chords, and an
uterus containing thin-shelled, non-operculated ova,
with a larva inside. The intestinal wall consisted of a
simple cuboidal epithelium with long microvilli.
Conclusion: The histological, bioecological, and
epidemiological characteristics of this Nematode allowed us to classify it as Spirocerca lupi. The present
case relates to an exclusively gastric location of this
Nematode in a Portuguese fox.

gicas usadas na sua identificao no so observveis.

Material e Mtodos: Apresentam-se trs casos de
infeco parasitria: peritoneu de safio, Conger conger, com aspecto nodular; msculo de faneca, Trisopterus luscus, com cor negra e corao de perca sol, Lepomis gibbosus, envolvido por quistos de cor branca.
Os tecidos foram fixados em formol salgado a 10% e
processados histologicamente.
Resultados: A estrutura nodular de peritoneu correspondia a um aglomerado de larvas enquistadas. A
ausncia de progltides indicou tratar-se de espcimes
imaturos. Nuns cortes, o parnquima larvar apresentava aspecto de tecido laxo, sem estruturas morfolgicas e noutros, observou-se o esclex com botrdeas
e quatro tentculos retrcteis armados com ganchos,
bem como corpsculos calcreos correspondendo a
larvas plerocercides de Trypanorhyncha. No mesmo
tecido foram observadas larvas filiformes, com corpo
75

SUPLEMENTO

circular em corte transversal, esfago de aspecto simtrico radial e lmen triradial, correspondendo a larvas
de nematodos.
No msculo de faneca identificaram-se ovos bioperculados, morulados ou castanhos embrionados, bem
como pores de larvas ou adultos com corpo cilndrico localizados entre as fibras musculares e dentro
delas, resultando numa infeco parasitria por nematodos Trichosomoididae, Huffmanella spp.
No corao da perca sol, os quistos continham uma
larva no segmentada, com dois cecos, uma ventosa
oral e outra ventral, do trematode digentico, Posthodiplostomum minimum centrarchi.
Concluso: Na identificao dos parasitas foi importante observar os caracteres morfolgicos, mas o
conhecimento do peixe parasitado, o seu habitat e os
tecidos afectados, auxiliaram o diagnstico.

Identification of Fish Parasites in Stained

Tissue Samples
Introduction: In stained tissue sections prepared
for light microscopy, taxonomic features may not be
present, thus making parasite identification difficult.
Three cases of parasite infection were described: European conger, Conger conger peritoneum with nodular
appearance; pouting, Trisopterus luscus with abnormal
dark colour muscle and pumpkinseed sunfish, Lepomis
gibbosus heart, surrounded by white colour cysts.

Caracterizao de um surto da nova variante

da Doena Hemorrgica Viral (DHV) em
coelhos bravos (Oryctolagus cuniculus algirus), Barrancos, Sudeste de Portugal
Melo P.1*, Abrantes J.2,3, Ramada M. 1, Lopes A.M. 2,3,4,
Alves P.C. 2,4, Esteves P.J. 2,5, Correia J.J. 6
VETNATURA, Lisboa, Portugal *vetnatura@gmail.com
CIBIO/UP, Centro de Investigao em Biodiversidade
e Recursos Genticos/Universidade do Porto, InBio,
Laboratrio Associado, Campus Agrrio de Vairo,
Vairo, Portugal
3
INSERM, U892, Universit de Nantes, Nantes, France
4
Departamento de Zoologia e Antropologia, Faculdade
de Cincias da Universidade do Porto, Porto, Portugal
5
CITS, Centro de Investigao em Tecnologias da Sade, IPSN, CESPU, Gandra, Portugal
6
CIISA, Faculdade de Medicina Veterinria - Universidade Tcnica de Lisboa, Portugal

1

2

Introduo: A doena hemorrgica viral (DHV)

do coelho tem como agente etiolgico um calicivrus
e causa elevada mortalidade em coelhos domsticos e
76

RPCV (2013) 108 (585-586) 69-91

Material and Methods: Tissues were fixed in 10%

buffered neutral formalin and processed using standard
methods.
Results: The nodular peritoneum consists of multitude of encysted and clustered larvae. The absence of
complete segmentation into proglottis identifies this
specimen as immature. In sections, the body larvae
vary from loosely parenchyma to prominent scolex
features with bothridia and four tentacles armed with
hooks. The larvae were identified as Trypanorhynchean
plerocercoid. In this nodular peritoneum, filiform larvae were also present showing a round body surface
in cross-section and the histological features of the
oesophagus with its radial symmetry and triradiate lumen which allowed classifying them as nematodes.
The histopathological findings of the pouting muscle, revealed bioperculated eggs, immature and embryonated brown eggs. The eggs and portions of larvae or
adult nematodes Trichosomoididae of the genus Huffmanella were observed in either intracellular or extracellular locations.
The heart of pumpkinseed sunfish was found infected
by Posthodiplostomum minimum centrarchi larvae. The
larvae were differentiated by the absence of the body
segmentation and by an addition sucker, the ventral one.
Conclusion: The morphological characters are important in parasites identification but the knowledge on
infected fish species, their habitat and infected tissues
also provide additional information to the parasites
identification.

bravos. Na Pennsula Ibrica, a DHV foi descrita em

1989 e todas as estirpes virais isoladas pertenciam ao
genogrupo 1. Em 2011 detectou-se em Espanha uma
nova variante deste vrus, descrita anteriormente em
Frana, diferenciando-se por originar mortalidade em
juvenis (<2 meses) e adultos vacinados.
Caso clnico: Entre Dezembro de 2012 e Janeiro de
2013, ocorreu um surto atpico de DHV em Barrancos, afectando coelhos bravos jovens no vacinados e
adultos vacinados, registando uma taxa de mortalidade
de 42%.
Efectuou-se a necrpsia a sete cadveres, com recolha de pulmo, fgado, timo, traqueia, rim e bao. A
deteco de RNA viral foi efectuada por RT-PCR e os
casos positivos foram sequenciados. Para a anlise filogentica foram includas estirpes de cada genogrupo
e estirpes da nova variante.
Resultados: As leses macroscpicas foram maioritariamente epistaxis, hemorragia do pulmo e traqueia,
congesto do rim, bao, timo. Os fgados apresentavam
predominantemente colorao plida e em alguns casos congesto. Os animais vacinados apresentavam leses menos severas. As leses histopatolgicas foram
hemorragias do pulmo e traqueia e a nvel heptico,
congesto e hepatite necrosante.

SUPLEMENTO

A anlise filogentica demonstrou que as estirpes

isoladas agrupam com a nova variante com elevado
suporte estatstico.
Concluses: Este estudo confirma a presena da
nova variante em Portugal e levanta questes quanto
conservao e sustentabilidade das populaes naturais de coelho bravo. Recomendam-se medidas adicionais de gesto nas unidades de criao.
Characterization of an outbreak of the

newly identified variant of rabbit haemorrhagic disease virus (RHDV) in wild rabbits
(Oryctolagus cuniculus algirus), Barrancos,
Southeast of Portugal
Introduction: Rabbit haemorrhagic disease virus
(RHDV) is a calicivirus that causes a highly fatal disease in wild and domestic adult European rabbits. In
Iberian Peninsula, the first description of RHDV dates
from 1989 and all isolated strains clustered within genogroup 1. In 2011 a new variant of RHDV was detected in Spain, already described in France, that differs by
causing mortalities in young rabbits (<2 months old)
and in vaccinated adult rabbits.

Principais dificuldades encontradas

na aplicao das tcnicas de deteo
de apoptose em tecidos animais
Garcia C1*, Cruz R1,2, Mega A1,2, Oliveira AL3, Teixeira
de Lemos E1,2,5, Ferreira D4, Reis F5, Vala H1,2
Escola Superior Agrria de Viseu, Instituto Politcnico de Viseu, Portugal;
2
Centro de Estudos em Educao, Tecnologias e Sade, Instituto Politcnico de Viseu, Portugal
3 
Universidade de Lon, Lon, Espanha
4
Laboratrio de Farmacologia e Teraputica Experimental, IBILI, Faculdade de Medicina da Universidade de Coimbra, Coimbra, Portugal
5
CICV- Centro de Investigao em Cincias Veterinrias, Universidade Lusfona de Humanidades e Tecnologia, Lisboa, Portugal
1

cspgarcia@gmail.com

Introduo: A aplicao de metodologias capazes

de identificar clulas apoptticas constitui uma valiosa
ferramenta em vrios estudos biomdicos. Muito do
conhecimento adquirido acerca dos mecanismos reguladores da apoptose deriva de observaes em clulas
isoladas. No entanto, as propriedades demonstradas
por estes ensaios nem sempre se aplicam ao estudo
de amostras tecidulares. Para deteo da apoptose em
tecidos, muitos investigadores recomendam o mtodo

RPCV (2013) 108 (585-586) 69-91

Clinical case: Between December of 2012 and January of 2013, an atypical RHDV outbreak was detected
in Barrancos, where unvaccinated young and vaccinated adult wild rabbits were fatally susceptible, with a
mortality rate of 42%.
Necropsies of seven cadavers were performed and
samples of lungs, liver, thymus, trachea, kidneys and
spleen were collected. Detection of viral RNA was performed by RT-PCR and when positive, products were
sequenced. For the phylogenetic analysis, strains from
each genogroup and of the new variant were included.
Macroscopic lesions were mostly epistaxis, hemorrhage in the lungs and trachea, congestion in the kidney, spleen and thymus. Livers were predominantly
pale and in some congested. Vaccinated animals had
less severe lesions. Histopathological lesions were hemorrhages in the lungs and trachea and congestion and
necrotizing hepatitis in the liver.
Phylogenetic analysis clustered the isolates with the
newly described variant with high bootstrap support.
Conclusions: This study confirms that the new variant of RHDV is circulating in Portugal and raises concerns regarding the conservation of the natural populations of European rabbit. Additional management
measures are recommended in breeding facilities.

TUNEL. Contudo, outros mtodos esto disponveis

para avaliao da apoptose, devendo a sua escolha ser
criteriosamente avaliada, tendo em conta as alteraes
morfolgicas que se pretendem detetar. O presente trabalho pretende, como principal objetivo, partilhar as
dificuldades encontradas na implementao de dois
diferentes mtodos para deteo de clulas apoptticas
em tecidos animais.
Material e mtodos: Foram utilizados tecidos de diferentes animais (rato, porco e co), fixados em formalina a
10% e includos em parafina, os quais foram submetidos
a tcnicas imunohistoqumicas com recurso ao mtodo
TUNEL (Kit In situ cell death, Roche) e aos anticorpos
policlonais Bax e Bcl-2 (Santa Cruz Biotechnology).
Resultados: Aps um longo perodo de testes e de
custos, em que se obtiveram reaes inespecficas, bem
como vrios artefactos - obstculos que, para serem
superados, requerem persistncia, todos os mtodos
provaram ser eficazes na deteo de apoptose, tendo
sido as diferenas de marcao observadas relacionadas com o tipo de tecidos testados.
Concluso: Os mtodos imunohistoqumicos constituem uma abordagem a considerar no que respeita
deteo da apoptose em tecidos animais, sendo que as
dificuldades encontradas representam um desafio supervel. Partilhar esta esperincia poder, certamente,
evitar testes e custos desnecessrios sua implementao noutras unidades laboratoriais.
Agradecimentos: FCT e CI&DETS (PEst-OE/
CED/UI4016)
77

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Main difficulties encountered in implementation of techniques for apoptosis detection

in animal tissues
Introduction: The implementation of methodologies capable of identifying apoptotic cells is a valuable tool in many biomedical studies. Much of the
knowledge acquired about the mechanisms regulating apoptosis comes from observations in isolated
cells; however, the properties shown by these assays
do not always apply to the study of tissue samples.
For detection of apoptosis in tissues, many investigators recommend the TUNEL method, nonetheless,
other methodologies are available, and their choice
must be carefully evaluated, taking into account the
morphologic alterations which they intend to detect.
The main goal of this work is to share difficulties
encountered in the implementation of two different
methods for the detection of apoptotic cells in animal
tissue.

Otimizao da imunodeteo do recetor

HER2 em tumores mamrios de cadela
Beselga, A. 1, Soares, M. 1, Carvalho, S. 1, Matos, J. 2,
Cabeadas, J. 2, Correia, J. 1, Peleteiro, M. 1, Ferreira,
F. 1*
 IISA, Faculdade de Medicina Veterinria, UniverC
sidade Tcnica de Lisboa. Alto da Ajuda, 1300-477
Lisboa, Portugal
2
IPOFG-EPE, Servio de Anatomia Patolgica, 1099023 Lisboa, Portugal
1

* fernandof@fmv.utl.pt
Introduo: O recetor para o fator de crescimento
epidrmico de tipo 2 (HER2) apresenta-se sobrexpresso em 15-30% dos tumores mamrios na mulher,
mostrando um elevado valor prognstico e teraputico. Na cadela, o papel oncognico do HER2 permanece duvidoso devido, em parte, falta de padronizao da tcnica de imunohistoqumica.
Material e mtodos: Vinte e uma amostras de
tumores mamrios caninos (TMC) foram colhidas
e fixadas de acordo com as indicaes da American
Society of Clinical Oncology (ASCO). Aps classificao histopatolgica, as amostras foram sujeitas a
diferentes protocolos imunohistoqumicos, tendo-se
feito variar o mtodo de recuperao antignica e o
anticorpo primrio usado: TAB250, diluio 1:250
(Zymed); SP3, 1:100 (Zytomed); CB11, 1:60 (Invitrogen); 4B5, pronto a usar (Ventana). A interpretao dos resultados foi realizada de acordo com os
critrios da ASCO.
Resultados: Os melhores resultados foram obtidos
78

RPCV (2013) 108 (585-586) 69-91

Material and methods: Tissues from different animals (rat, pig and dog) tissues, fixed in 10% neutralbuffered formalin and paraffin embedded were submitted to immunohistochemical techniques using the
TUNEL method (In situ Cell Death Kit, Roche) and
polyclonal Bax and Bcl-2 antibodies (Santa Cruz Biotechnology).
Results: After a long period of performed tests and
expenses made, in which unspecific reactions, as well
as several artifacts were obtained - obstacles that required persistence to be overcome, all methods have
proven to be effective in the detection of apoptosis,
with differences probably related to the tissues tested.
Conclusion: Immunohistochemical methods are a
good approach to consider in the detection of apoptosis
in animal tissues. Sharing the challenging difficulties
found in our work, might obviate many tests and expenses in its implementation in other laboratory facilities.
Acknowledgments: FCT and CI&DETS (PEst-OE/
CED/UI4016)

com o anticorpo CB11 quando associado ao mtodo de recuperao antignica por panela de presso.
Com este protocolo, 42,9% dos tumores analisados
mostraram sobrexpresso do recetor HER2. Todos os
anticorpos testados mostraram marcao membranar
especfica no tecido mamrio de cadela, exceo do
clone TAB250.
Concluso: A variabilidade dos resultados obtidos
demonstra a necessidade de padronizao da tcnica de imunohistoqumica para deteo do HER2 em
Medicina Veterinria. Neste estudo, 42,9% dos TMC
analisados mostraram sobreexpresso do HER2, um
percentagem superior descrita na Mulher, reforando a possvel importncia deste biomarcador em Oncologia Veterinria.

Optimization of HER2 receptor immunodetection in canine mammary tumours

Introduction: The epidermal growth factor type2 receptor (HER2) is overexpressed in 15-30% of
breast tumors in women, presenting a prognostic and
high therapeutic value. In the bitch, the role of the
HER2 oncogene remains doubtful due to the lack of
immunohistochemistry technique standardization.
Material and methods: Twenty-one canine mammary tumors (CMT) were collected and fixed in
agreement to the American Society of Clinical Oncology (ASCO) guidelines. After histopathological
classification, the samples were subjected to different
immunohistochemical protocols, varying the recovery antigen method and the primary antibody used:
TAB250, dilution 1:250 (Zymed); SP3, 1:100 (Zytomed); CB11, 1:60 (Invitrogen); 4B5, ready-to-use

SUPLEMENTO

RPCV (2013) 108 (585-586) 69-91

(Ventana). Results interpretation was performed according to the ASCO criteria.

Results: The best results were obtained when the
antibody CB11 was coupled with the pressure cooker
as antigen retrieval method. With this protocol, 42.9%
of the analyzed tumors showed HER2 overexpression.
All antibodies tested show specific immunostaing of
the cytoplasmic membrane, except the clone TAB250.

Conclusion: The variability of the results obtained

demonstrates the need of immunohistochemistry
standardization for HER2 evaluation in Veterinary
Medicine. In this study, 42.9% of TMC analyzed
showed HER2 overexpression, a higher percentage
than the one described in women, reinforcing the
possible importance of this biomarker in Veterinary
Oncology.

Linfoma intestinal do tipo B com diferenciao em clulas de Mott num gato.

celular superior a 30% com o anticorpo Ki-67 (Dako

M7240 1/50).
Concluso: Com base nos achados morfolgicos,
histoqumicos e imunohistoqumicos, o diagnstico
histolgico compatvel com linfoma intestinal de tipo
B, com diferenciao em clulas de Mott.

Pedro Fasca1,2*; Maria Joo Costa3, Rita Pontes3, Dulce Carujo3, Matilde Gonalves4, Marli Angua1, Ins
Carvalho2
Faculdade de Medicina Veterinria da ULHT
DNATech
3
Clnica veterinria de Telheiras
4
Servio de Anatomia Patolgica Centro Hospitalar
de Setbal
1
2

* pedrofaisca@ulusofona.pt
Introduo: Gato, macho, 11 anos de idade, que se
apresentou consulta para vacinao anual. No exame fsico detetou-se massa abdominal, no dolorosa
e firme.
Materiais e mtodos: Realizou-se ecografia abdominal que confirmou massa intestinal.
Posteriormente foi realizada a sua remoo por enterectomia.
Resultados: Macroscopicamente, ao nvel do jejuno, identificou-se neoformao ulcerada, no estenosante, com 9x5x4,5 cm; em seco era branca amarelada, brilhante, homognea, envolvendo toda a espessura
da parede intestinal.
Histologicamente observou-se proliferao difusa
de clulas linfoides desde a mucosa at serosa. Associado aos linfcitos identificam-se inmeras clulas
grandes e redondas, entre 20 a 30 m, com ncleo empurrado para a periferia e citoplasma preenchido por
glbulos eosinoflicos e hialinos, positivos com cido
peridico de Schiff, compatveis com clulas de Mott.
Os linfcitos apresentaram imunomarcao positiva
para Pax5 (Leica NCL-L-PAX-5 1/80), CD79alphacy
(Dako M7051, 1/50) e imunomarcao negativa para
CD3 (Dako A0452, 1/50); as clulas de Mott demonstraram positividade para CD79alphacy, cadeias leves
lambda (Ventana 760-2515) e negatividade para Pax5,
CD3 e cadeias leves kappa (Ventana 760-2514). Demonstrou-se ndice mittico baixo (0-2 mitoses por
campo com objetiva de 40x) e ndice de proliferao

Feline intestinal B-cell lymphoma with

Mott cell differentiation.
Introduction: An 11year-old, male cat, was present
for the annual vaccination booster without any abnormal clinical signs. On clinical examination, a firm,
non-painful abdominal mass was palpated.
Materials and methods: An abdominal ecography
confirmed the presence of an intestinal mass, which
was posteriorly extirpated by enterectomy.
Results: The jejunum presented a 9x5x4,5 cm, homogenous, bright whitish-yellow mass surrounding the
intestinal lumen and invading all intestinal layers.
Histologically, there was a diffuse non-capsulated
proliferation of round cells, disposed in dense sheets
through all intestinal layers. The neoplastic cells were
of lymphoid type associated with large (20-30um)
round cells with cytoplasmatic eosinophilic, periodic
acidSchiff positive, globules compatible with Mott
cells.
The lymphoid cells presented positive immunostaining for CD79alphacy (Dako M7051, 1/50), and Pax5
(Leica NCL-L-PAX-5 1/80), and negative immunostaining for CD3 (Dako A0452, 1/50); the Mott cells
presented positive immunostaining for CD79alphacy
and lambda light-chain (Ventana 760-2515 ready to
use), and were negative for Pax5, CD3 and kappa lightchain (Ventana 760-2514 ready to use). The mitotic
figures were rare (02 per 40x field of view), and the
Ki 67 proliferation index was higher than 30% (Dako
M7240 1/50).
Conclusion: On the basis of histopathology, histochemistry and immunohistochemistry, a B-cell lymphoma with Mott cell differentiation was diagnosed.

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Adenocarcinoma do endomtrio em gatas

com menos de um ano de idade: descrio
de quatro casos
Pires M.A.1*, Saraiva A.L. 1,2, Santos T.3, Vilhena H.4,
Sousa A.5, Santos C.1, Payan-Carreira R.1
 ECAV, University of Trs-os-Montes and Alto DouC
ro, P.O. Box 1013, 5001-801 Vila Real, Portugal.
2
EUVG Escola Universitria Vasco da Gama, 3040714 Castelo Viegas, Coimbra, Portugal.
3
Veterinary Teaching Hospital (HVUTAD), University
of Trs-os-Montes and Alto Douro, P.O. Box 1013,
5001-801 Vila Real, Portugal.
4
Hospital Veterinrio do Baixo Vouga, 3750-742
gueda, Portugal
5
Policlnica Veterinria de Aveiro - Vetsanus, 3800118 Aveiro, Portugal.
1

apires@utad.pt

Introduo: As neoplasias uterinas de origem epitelial so raras em todas as espcies animais, exceto
em vacas e coelhas. Nas gatas, o adenocarcinoma do
endomtrio (AEG) , geralmente descrito em fmeas
com idade superior a 8 anos, ainda que, esporadicamente, possa ser observado em idades compreendidas
entre os 2 e os 4 anos.
Material e mtodos: foram enviados ao Laboratrio
de Histologia e Anatomia Patolgica da UTAD 4 peas
de ovariohisterectomia (OVH) de gata, provenientes
de diferentes Hospitais Veterinrios. Os rgos, fixados em formol tamponado a 10% foram processados
por rotina para parafina e corados com hematoxilina
e eosina.
Resultados: A sintomatologia observada foi varivel, desde a ausncia at presena de corrimento hemorrgico acastanhado,, facilmente confundidos com
pimetra ou aborto.
Macroscopicamente os teros apresentavam-se distendidos, firmes, com parede espessada, dilataes e
irregularidade dos cornos uterinos em diferentes graus.
Microscopicamente observou-se proliferao papilar
do endomtrio, com invaso do miomtrio em dois dos
casos. O diagnstico foi de AEG de tipo papilar seroso.
Concluso: Embora as neoplasias de origem epitelial do tero sejam raras nos animais domsticos, o
nosso grupo nos ltimos anos tem registado mais AEG

80

RPCV (2013) 108 (585-586) 69-91

do que o documentado. Embora esta neoplasia seja

mais frequente em animais velhos, nos ltimos 2 anos
tivemos 4 casos de AEG em gatas de idade inferior a
um ano no momento da cirurgia.
Esta situao clnica pode ser confundida com aborto ou pimetra, pelo que se recomenda a anlise sistemtica de todas as peas de OVH, seja qual for a idade
ou diagnstico clnico.

Feline Endometrial Adenocarcinoma in females under one year-old: A description of

four cases
Introduction: Uterine neoplasms of epithelial origin
are rare in all domestic animals, except for cows and
rabbits. In female cats, endometrial adenocarcinoma
(FEA) is usually described in females over 8 years-old.
Yet, sporadically it may be found in younger females
of age of 2-4 years.
Material and methods: 4 ovariohysterectomy surgical specimens from queens aged under one year old,
at the time of surgery, were submitted from different
practices to the Laboratory of Histology and Anatomical Pathology, at UTAD. The organs were fixed in 10%
buffered formalin, processed to paraffin embed tissue,
and stained with haematoxylin and eosin.
Results: Different clinical symptoms were observed
in these animals, ranged from absent to a haemorrhagic
or brownish discharge, easily confused with pyometra
or abortion.
Macroscopic outcomes revealed a distended and firm
uterus, with wall thickness, dilatations and irregularity
along both uterine horns, at different grades. Microscopic findings revealed a papillary proliferation of the
endometrium with myometrium invasion in two cases.
The final diagnosis was FEA of the papillary type.
Conclusion: Although uterine neoplasms of epithelial origin are rare in domestic animals, in the last years
our group has recorded more FEA than documented in
the literature. Despite this neoplasia to be related with
older animals, in less than two years, we untypically
diagnosed 4 FEA in queens aged under one year-old
at time of surgery that could be clinically mistaken for
pyometra or abortion. For this reason, we strongly recommend histopathological analysis of all ovariohysterectomy surgical specimens, despite animals age or
clinical diagnose.

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RPCV (2013) 108 (585-586) 69-91

Poster presentations
Abate de Lagomorfos nos Matadouros
Portugueses: Estudo Descritivo (2011-2012)

para possibilitar a implementao de medidas preventivas.

Jesus J.1, Santos S.1*, Lamela M.2

Slaughtering of Lagomorphs in the Portuguese Slaughterhouses: Descriptive Study

(2011-2012)

Centro de Investigao em Cincias Veterinrias

(CICV) - FMV- ULHT, Lisboa, Portugal
2
Chefe de Diviso na Direco de Servios de Segurana Alimentar da Direco Geral de Alimentao e
Veterinria (DGAV)
1

sarasantos.med.vet@gmail.com

Introduo: Este trabalho d a conhecer os volumes

de abate actuais de lagomorfos e as principais causas
de reprovao sanitria nos matadouros portugueses.
Material e mtodos: Analisou-se a totalidade de
abates de lagomorfos entre 1 de Janeiro de 2011 e 31
de Dezembro de 2012, registados no Sistema de Informao do Plano de Aprovao e Controlo dos Estabelecimentos (SIPACE), plataforma informtica da Direo Geral de Alimentao e Veterinria, utilizada por
todos os inspetores sanitrios desde Outubro de 2010.
Resultados: Foram apresentados para abate regular
10 901 526 lagomorfos. Desses, 321 (0,03) foram
rejeitados ante-mortem e 93 665 (8,6) post-mortem.
De entre os principais tipos de processos patolgicos
observaram-se leses fibrino-purulentas, abcessos e
caquexia. Registaram-se 20 497 mortes no transporte.
Concluso: No perodo em anlise, o volume de
abate manteve-se estvel e no apresentou sazonalidade, sugerindo assim um consumo regular desta carne ao longo do ano. As leses fibrino-purulentas e os
abcessos foram as principais causas de reprovao. A
identificao das causas de reprovao fundamental

Caracterizao da populao linfocitria

em leses granulomatosas de javali (Sus
scrofa) com diagnstico presuntivo de tuberculose
Matos A.C1., Andrade S.2, Pires M.A.2*, Coelho A.C.2,
Pinto M.L.2
Instituto Politcnico de Castelo Branco, Castelo
Branco, Portugal
2
CECAV - Centro de Cincia Animal e Veterinria;
ECAV- Escola de Cincias Agrrias e Veterinrias,
Vila Real, Portugal
1

lpinto@utad.pt

Introduo: A tuberculose doena infeciosa que

ameaa a vida de milhes de pessoas, quer em pases

Introduction: The present study was conducted to

evaluate lagomorphss slaughtering data such as the
current number of slaughtered animals and the major
causes to condemnation in Portugal.
Material and methods: This study includes the
total number of lagomorphs slaughtered between 1st
January 2011 and 31st December 2012. The data were
obtained from Informatiom System of Establishments
Approval and Control Plan (SIPACE) software, belonging to Directorate General of Food and Veterinary
(DGAV), where all veterinary inspectors insert the
records since October 2010.
Results: Out of 10 901 526 slaughtered lagomorphs,
321 (0,03) were rejected ante-mortem and 93 665
(8,6) were rejected post-mortem. The majority of
injuries were fibrinopurulent lesions, abscesses and
cachexia. Deaths in the transport accounted 20 497
animals.
Conclusion: In the past two years the number of
slaughtered lagomorphs remained stable and no seasonality in the number of slaughtered animals was
noted, meaning that this type of meat is consumed
regularly over the year. Fibrinopurulent lesions and
abscesses were the most frequent causes of condemnation. Preventive actions can only be taken after identification of the prevalent injuries, proving that studies
like this are useful.

em desenvolvimento, quer em pases desenvolvidos,

sendo tambm responsvel por elevadas perdas econmicas em animais de espcies pecurias. A patognese
da tuberculose bovina, causada por Mycobacterium bovis e da tuberculose humana, cujo agente principal o
microrganismo da espcie Mycobacterium tuberculosis, so as que se encontram melhor caracterizadas. No
entanto, a tuberculose tambm afeta outras espcies de
animais, sendo estes muitas vezes os responsveis pelo
contgio s espcies pecurias e ao Homem. Em particular, o estudo da tuberculose nas espcies selvagens
pode elucidar a forma como esta doena se mantm e
se transmite.
Material e mtodos: Este trabalho foi realizado com
o objetivo de avaliar a resposta inflamatria crnica
em animais selvagens com diagnstico presuntivo de
tuberculose, em que se procurou investigar a expresso dos anticorpos anti-CD3 e anti-CD79 em leses

81

SUPLEMENTO

granulomatosas dos gnglios mesentricos de javalis

(n=30), pela tcnica de imuno-histoqumica.
Resultados: Pela anlise imuno-histoqumica observou-se marcao citoplasmtica das clulas linfocitrias em 11 gnglios mesentricos (36,6%) para
o anticorpo anti-CD79, e em 15 gnglios mesentricos (50%) para o anticorpo anti-CD3. A quantificao dessas mesmas clulas permitiu agrupar as leses
consoante a percentagem de clulas linfocitrias que
exibiam.
Concluso: As leses em estudo apresentam percentagens relativamente semelhantes de linfcitos T e B,
sugerindo assim uma resposta celular e humoral com
igual participao na resposta imunitria, desencadeada pelo organismo face entrada do agente patognico.
Lymphocyte

population in the granulomatous lesions of wild-boars (Sus scrofa) suspected of tuberculosis.

Introduction: Tuberculosis is an infectious disease
that threatens the lives of millions of people both in
developing and developed countries and is responsible
for important economical losses in farm animals. The
pathogenesis of bovine tuberculosis due to Mycobacterium bovis and human tuberculosis mainly caused
by the microorganism of the species Mycobacterium

Estudo Preliminar sobre a Descalcificao

de Tecido sseo em Micro-ondas
Angua M.*, Fasca P.
Centro de Investigao em Cincias Veterinrias- Faculdade de Medicina Veterinria - Universidade Lusfona de Humanidades e Tecnologias
* marli.anagua@ulusofona.pt
Introduo: A anlise histopatolgica de tecido sseo exige uma etapa de descalcificao, que possibilite a
microtomia. O mtodo mais comum consiste na imerso
das amostras em solues cidas, mas para alm de provocarem danos tecidulares, o processo muito prolongado, o que atrasa significativamente o diagnstico. A
utilizao de micro-ondas acelera a descalcificao, mas
no deve comprometer a imagem microscpica.
O objetivo deste trabalho consistiu na diminuio da
durao da descalcificao, mantendo a qualidade da
imagem microscpica.
Material e mtodos: Foram testadas amostras de
osso compacto e esponjoso, provenientes de caprinos,
com dimenses que pretenderam simular casos de bipsias e casos de peas cirrgicas ou necrpsias. Realizou-se a descalcificao pelo mtodo convencional

82

RPCV (2013) 108 (585-586) 69-91

tuberculosis are more extensively characterized. However, tuberculosis also affects other animal species
and these are often responsible for the contamination
of cattle and humans. In particular, an overview of the
wildlife hosts will provide insight into how these reservoirs maintain and spread the disease.
Material and methods: This study was conducted
to evaluate the chronic inflammatory response in wild
animals with a presumptive diagnosis of tuberculosis.
In order to better understand the pathogenesis of the
disease and further characterize its lesions, it was our
aim to investigate the expression of antibodies antiCD3 and anti-CD79 in the granulomatous lesions of
wild boars mesenteric lymph nodes (n=30) through
the immunohistochemistry technique.
Results: Immunohistochemical analysis allowed us
to observe the lymphocyte cells population in 11 mesenteric nodes (36.6%) for the anti-CD79 antibody,
and in 15 mesenteric nodes (50%) for the anti-CD3 antibody. The quantification of these same cells allowed
grouping of lesions depending on the percentage of
lymphocyte cells that they exhibit.
Conclusion: The lesions of this study showed relatively similar percentages of B and T lymphocytes,
thus suggesting a cellular and humoral response and
a similar immune response triggered by the organism
against the entry of the pathogen.

e pelo mtodo em micro-ondas, atravs da adaptao

de um protocolo conhecido. Utilizou-se cido ntrico
a 5% e 10%.
Resultados: Nos fragmentos de maiores dimenses,
o protocolo foi interrompido aps 4 horas com cido
ntrico a 10% em micro-ondas, por no se conseguir
uma descalcificao completa e estar a ocorrer destruio do osso compacto. Nos fragmentos de osso esponjoso, verificou-se uma reduo de cerca de 25 horas
relativamente ao mtodo convencional. Nas bipsias,
houve uma reduo de aproximadamente 10 horas, utilizando cido ntrico a 5%. Com cido ntrico a 10%
houve destruio tecidular. Nos casos em que se obteve
uma descalcificao completa, a imagem microscpica
apresenta fraca qualidade.
Concluso: A utilizao de micro-ondas na descalcificao com cido ntrico a 5% ou 10%, aplicando o
protocolo deste estudo em amostras de caprinos, reduz
a durao do processo, mas compromete significativamente a imagem microscpica, o que se torna invivel
para diagnstico.

Preliminary Study about Microwave Decalcification of Bone Tissue

Introduction: Histological analysis of bone tissue

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RPCV (2013) 108 (585-586) 69-91

requires a decalcification process. The most common

method consists on samples immersion in acidic solutions. However, it causes tissue damage and is time
consuming, delaying the diagnosis. The use of microwaves accelerates the decalcification process, but
shouldnt compromise the histological image.
The aim of this work was to reduce the decalcification
time, maintaining the quality of the histological image.
Material and methods: Goat compact and spongy
bone, with dimensions that intended to simulate biopsies and surgical or necropsies specimens, were
used. Decalcification was performed by conventional
method and microwave method with an adaptation on a
known protocol, using 5% and 10% nitric acid.
Results: For larger fragments, the protocol was dis-

continued after 4 hours of microwave cycles** with

10% nitric acid, because the decalcification was incomplete, in spite of evident compact bone destruction. In the fragments of spongy bone, there was a
reduction of approximately 25 hours, comparing with
conventional method. In biopsies, there was a reduction of approximately 10 hours using 5% nitric acid.
With 10% nitric acid, there was tissue destruction. The
cases with complete decalcification had a poor quality
of the histological image.
Conclusion: The use of microwave decalcification
with 5% or 10% nitric acid, applying the protocol of
this study on goats samples, reduces the process time
but compromises the histological image, which is impractical for diagnosis purpose.

Necrpsia em equinos. Descrio de um

caso de doena granulomatosa disseminada

O diagnstico de doena granulomatosa disseminada compatvel com sarcoidose equina baseou-se nas leses identificadas na necrpsia e na anlise histopatolgica de leses granulomatosas presentes em diversos
rgos, ricas em clulas gigantes de Langhans e negativas para a presena de bacilos lcool-cido resistentes.
Concluso: Como concluso, pode referir-se que realizao de necrpsia permite, na grande maioria dos
casos, identificar a causa da morte em equinos, preferencialmente com recurso a outros exames complementares de diagnstico.

Miranda I.*, Tilley P., Pomba C., Pissarra H., Correia

J.J., Peleteiro M.C.
Centro de Investigao Interdisciplinar em Sanidade
Animal, Faculdade de Medicina Veterinria, Universidade Tcnica de Lisboa. Alto da Ajuda, 1300-477
Lisboa, Portugal
*

ileanamiranda@gmail.com

Introduo: Tendo em conta a importncia de avaliar a prevalncia das situaes ocorridas e a capacidade de emisso de diagnsticos definitivos, procedeu-se
identificao das causas de morte dos casos de necrpsia de cavalos registados no Laboratrio de Anatomia Patolgica da Faculdade de Medicina Veterinria,
da Universidade Tcnica de Lisboa. De entre os casos
identificados foi dado particular relevo a um de doena
granulomatosa disseminada (DGD), atendendo raridade do processo, complexidade da evoluo clnica
e aos diagnsticos diferenciais envolvidos.
Material e mtodos: Foram analisados os registos
compreendidos entre Janeiro de 2009 e Janeiro de
2013, num total de 38 necrpsias. No caso da DGD, o
diagnstico baseou-se na anlise histopatolgica de rgos fixados em formol e submetidos a processamento
histolgico de rotina. As coloraes efetuadas foram
H&E e Ziehl Neelsen. Foi ainda realizada anlise bacteriolgica.
Resultados: Dos 39 casos de necrpsia analisados,
apenas um foi inconclusivo. Os restantes foram divididos em seis grupos: Doena Infeciosa Sistmica (5),
Doena Cardiorrespiratria (5), Doena Renal (1), Doena Gastrointestinal (21), Doena Neurolgica (3),
Doena Ortopdica/Trauma (3). Os exames complementares realizados foram anlise histopatolgica (24)
e anlise bacteriolgica (10).

Necropsy in horses. A case of disseminated

granulomatous disease
Introduction: With the objective of estimating the
prevalence of the occurrences and the capability to issue definitive diagnosis, an evaluation of the various
causes of death in horses was performed based on the
necropsy cases registered in the Laboratory of Pathology, Faculty of Veterinary Medicine, Technical University of Lisbon. Among the cases identified, particular
emphasis was given to a case of disseminated granulomatous disease (DGD), due to the rarity of the pathology, complexity of the clinical evolution and the
differential diagnosis involved.
Material and methods: The reports from January
2009 to January 2013 were analyzed, and a total of 39
necropsies recovered. In the case of DGD the conclusion was based on the histopathology of the organs
fixed in 10% formalin and subject to regular histological processing. H&E and Ziehl Neelsen stains were
performed. Bacteriology was also performed.
Results: Out of 39 cases of necropsy analyzed, only
one was inconclusive. The remainders were divided
into six groups: Systemic Infectious Disease (5), Cardiopulmonary Disease (5), Kidney Disease (1), Gastrointestinal Disease (21), Neurological Disease (3)
and Trauma/Orthopedic Disease (3). Complementary

83

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RPCV (2013) 108 (585-586) 69-91

exams were histopathological examination (24) and

bacteriological analysis (10).
The diagnosis of disseminated granulomatous disease consistent with equine sarcoidosis was based on
the gross morphology at necropsy and histopathology
of granulomatous lesions with Langhans giant cells

present in different organs that were negative for the

presence of acid-fast bacilli.
Conclusion: It can be concluded that, in most cases,
necropsy allows for the identification of the cause of
death in horses, preferentially using other complementary exams to achieve a correct and complete diagnosis.

Fibromatosis in Iberian hare Lepus granatensis. Preliminary data.

similar disease has been reported in mountain hare Lepus timidus in Finland, where gross and microscopic
lesions resembled to be associated to a Poxvirus infection.
Material and methods: We describe here two natural cases in wild Iberian hares Lepus granatensis in
Aragon region (Spain). Both hares were adults female
and male hunted in the autumn period in 1998 and
2006, respectively.
Results: Noticeable swelling eyelids were observed
in both animals and the male also showed small nodular lesions in nose and lips, swelling prepuce and an
irregular swelling of scrotum skin. The dermis histopathology of skin gross findings consisted in fibroblast
cells proliferation and oedema, whereas the epidermis
showed swelling and vacuolated cells with intracytoplasmic eosinophilic inclusion bodies. Infiltration of
plasma cells and lymphocytes were observed in the
dermis. Neutrophils were present in the dermis and
epidermis, probably by secondary bacterial infections.
Conclusion: The observed lesions could be associated to hare fibromatosis in Iberian hare Lepus granatensis, in Spain.

Arnal MC, Martnez-Durn D, Revilla M, Fernndez

de Luco D *
Departamento de Patologa Animal. Facultad de Veterinaria. Universidad de Zaragoza. C/ Miguel Servet
177. 50013 Zaragoza. Spain.
* luco@unizar.es
Introduction: Hare fibromatosis is a rare and transmissible viral disease, caused by a Leporipoxvirus,
antigenically more related to the Shope fibroma virus
than myxoma virus. The disease is characterized by
single or multiple skin tumours on ears, head and legs.
The affected animals appear in late summer and autumn with high morbidity and low mortality. Fibromatosis has been described in wild and farmed brown
hare Lepus europaeus in Europe (France, Germany,
Italy, U.K.). African hare fibromatosis is a very similar
disease caused by an unclassified Poxvirus affecting
Cape hare Lepus capensis in Kenya. Recently, another

Processos patolgicos prevalentes em

carnvoros silvticos mortos em acidentes
rodovirios
Matos A.C1., Figueira L1., Martins M.1, Matos M.2, lvares S.1, Mendes A.1, Pires M.A.3, Coelho A.C.3, Pinto
M.L.3*
I nstituto Politcnico de Castelo Branco, Castelo
Branco, Portugal
2
Centro de Gentica e Biotecnologia, Instituto de Biotecnologia e Bioengenharia, Universidade de Trs-osMontes e Alto Douro, Vila Real, Portugal
3
CECAV - Centro de Cincia Animal e Veterinria;
ECAV- Escola de Cincias Agrrias e Veterinrias,
Universidade de Trs-os-Montes e Alto Douro, Vila
Real, Portugal
1

* lpinto@utad.pt
Introduo: As doenas que afetam os animais
selvagens tm sido alvo de um interesse crescente,

84

uma vez que se demonstrou que, para alm das implicaes relacionadas com a conservao das espcies,
podem ter um impacto significativo na sade animal
e humana. Devido ao seu comportamento furtivo, os
carnvoros selvagens constituem uma populao difcil
de monitorizar e a investigao de doena nestas espcies resulta geralmente do exame de animais que so
encontrados mortos.
Material e mtodos: Este estudo foi realizado em
carnvoros selvagens vtimas de acidentes de viao,
em Portugal, entre 2009 e 2012, e inclui 3 famlias e 6
espcies diferentes (n=72). Os animais foram submetidos a exame post-mortem e foram efetuadas colheitas,
sendo os tecidos fixados em formol tamponado a 10%,
processados para incluso em parafina e corados com
hematoxilina-eosina.
Resultados: As leses pulmonares foram as mais
prevalentes, (15/72; 20,8%), especialmente em raposas (Vulpes vulpes, 8/49; 16,3%) e saca-rabos
(Herpestes ichneumon, 7/15; 46,6%). As pneumonias
parasitrias, sugestivas de infestao por Crenosoma
vulpis ou Angiostrongylus vasorum foram observadas

SUPLEMENTO

em 6 raposas. A adiaspiromicose, provocada por Emmonsia crescens, foi a leso pulmonar mais comum em
saca-rabos (4/15, 26,6%). A nefrite intersticial crnica
e a linfadenite macrofgica afetaram, respetivamente,
10/72 (13,8%) e 6/72 (8,3%) animais das famlias Canidae e Mustelidae.
Concluso: De acordo com este estudo, as pneumonias parasitrias e micticas encontram-se entre
as leses mais prevalentes em carnvoros selvagens e
podem ter um impacto significativo na sade destas espcies, quer devido magnitude das leses observadas,
quer devido ao aumento da susceptibilidade a outros
agentes infeciosos.

Disease processes identified in road killed

wild carnivores
Introduction: Wildlife diseases have been considered
increasingly important due to their potential effect upon
human health, veterinary medicine, wildlife, and conservation biology. Since wild carnivores are elusive animals, the occurrence of disease in these species has been
typically investigated by identification of pathological
processes in carcasses that are found incidentally.
Material and methods: This study was conducted
in wild carnivores found death on the roads in Portu-

Doena vrica hemorrgica (DVH)

uma nova variante?
Vala H1,2*, Coelho C1,2, Nbrega C1,2, Mesquita JR1,2,
Oliveira J1,2
Escola Superior Agrria de Viseu, Instituto Politcnico de Viseu, Portugal;
2
Centro de Estudos em Educao, Tecnologias e Sade, Instituto Politcnico de Viseu, Portugal
1

* hvala@esav.ipv.pt
Introduo: A doena vrica hemorrgica (DVH),
causada por um RNA vrus (gnero Lagovrus, famlia
Caliciviridae), uma doena fatal que afeta coelhos domsticos e selvagens. Estudos anteriores a 1984, demonstraram a existncia de anticorpos especficos para DVH
em coelhos saudveis de vrias regies europeias e ainda
a possibilidade de estes animais apresentarem alguma resistncia contra a DVH conferida por uma imunidade cruzada com calicivrus benignos, fazendo com que a doena
exibisse um quadro clnico menos severo. A doena foi
controlada em cuniculturas graas vacinao, mas ainda uma ameaa nas populaes selvagens no vacinadas.
Uma nova variante desta doena, descrita em 2010
na Pennsula Ibrica, afeta principalmente animais jovens, que exibem hemorragias no corao, traqueia,
timo, pulmes, fgado, rins, intestino, assim como

RPCV (2013) 108 (585-586) 69-91

gal, from 2009 to 2012 (n=72), representing 3 families

and 6 different species. Post mortem examination was
performed and multiple tissues were fixed in 10% neutral buffered formalin, processed for routine paraffin
embedding and stained with hematoxylin and eosin for
histopathological examination.
Results: Lung lesions were the most prevalent finding (15/72, 20.8%), particularly in fox (Vulpes vulpes,
8/49, 16.3%) and Egyptian mongooses (Herpestes
ichneumon, 7/15, 46.6%). Six foxes showed lesions
of parasitic pneumonia suggestive of Crenosoma vulpis or Angiostrongylus vasorum infestation. In Egyptian mongooses adiaspiromycosis due to Emmonsia
crescens were the most common lung lesions (4/15,
26.6%). Chronic interstitial nephritis was registered in
10/72 (13.8%) animals, while in 6/72 (8.3%) macrophage lymphadenitis was observed, both conditions affecting members of the Canidae and Mustelidae.
Conclusion: According to our study, parasitic and
mycotic lung pneumonias are amongst the most prevalent lesions in wild carnivores. Although parasites and
fungus may often be regarded as a burden that has little
or no impact on the animals, our findings suggest that
they can severely affect animal health by the magnitude of their lesions and by making turning the host
more susceptible to other infectious agents.

CID, ictercia, esplenomeglia e enterite catarral.

Objetivos: Descrever um surto de elevada mortalidade numa cunicultura, principalmente em animais
jovens, com leses tpicas de DVH.
Material e mtodos: Foram colhidas amostras de coelhos vacinados e no vacinados, alvo de morte sbita.
As amostras foram fixadas em formalina a 10% (48 horas), embebidas em parafina e processadas seguindo os
procedimentos de rotina para avaliao histopatolgica
Resultados: Principais leses observadas: opisttonus; congesto e edema pulmonar; ictercia severa;
esplenomeglia; lceras gstricas punctiformes; congesto renal bilateral, petquias (timo).
Concluso: Embora esta infeo seja mais comum
em animais adultos, as leses observadas so consistentes com DVH. A presena concomitante de leses
associadas nova variante de DVH (CID, ictercia,
esplenomeglia, enterite catarral) leva-nos a suspeitar
da sua presena em Portugal, ressalvando o facto da
necessidade de estudos mais aprofundados.
Agradecimentos: FCT e CI&DETS (PEst-OE/
CED/UI4016/2011)

Rabbit haemorrhagic disease (RHD)

a new variant?
Introduction: Rabbit haemorrhagic disease (RHD)
is an acute fatal disease of domestic and wild Euro85

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RPCV (2013) 108 (585-586) 69-91

pean rabbits caused by a RNA virus (genus Lagovirus,

family Caliciviridae). Prior to 1984, healthy rabbits in
European regions had RHDV-specific antibodies and
their infection with benign rabbit caliciviruses conferred them a low degree of cross-protective immunity
against RHD, determining less severe clinical courses.
Vaccination allowed to control the disease in the rabbit industry, but RHD still threatens non-vaccinated
wild populations. A new variant of this disease is being described since 2010 in Iberian Peninsula. It affects mainly young rabbits that exhibit haemorrhages
in heart, trachea, thymus, lungs, liver, kidneys and gut,
as well as DIC, jaundice, splenomegaly and catarrhal
enteritis.
Objectives: To describe a case of high mortality rate
on a rabbitry, mostly in young rabbits, with typical lesions of RHD.
Material and methods: Samples from domestic

vaccinated and non vaccinated rabbits which died suddenly were taken and fixed in 10% neutral-buffered
formalin for 48 hours and embedded in paraffin. 3m
sections were stained for routine histopathological diagnosis (H&E) and examined by light microscopy.
Results: Main macroscopic lesions observed:
opisthotonos, oedema and pulmonary congestion, severe jaundice, splenomegaly, gastric punctiform ulcers, bilateral renal congestion and thymic petechiae,
Conclusion: Macroscopic lesions in infected
youngs, consistent with RHD infection usually observed only in adult rabbits, and the presence of lesions mostly associated to the new variant of RHD
(DIC, jaundice, splenomegaly, catarrhal enteritis), led
us to consider the presence of the new variant of RHD
in Portugal, although further studies are needed.
Acknowledgments: FCT and CI&DETS (PEst-OE/
CED/UI4016/2011)

Timo de Coelho um potencial marcador

para tcnicas de deteo de apoptose

cinoma in situ revelou reao positiva moderada com

os anticorpos pro-apoptticos e mais intensa com
o anticorpo anti-apopttico. O timo de co revelou
reao positiva moderada nos timcitos do crtex com
os anticorpos pr-apoptticos e dbil nos da medular.
Com o anticorpo anti-apopttico revelou reao dbil
na medular e negativa no crtex. Idnticos resultados
foram obtidos no timo de coelho, tendo sido, contudo,
mais forte a intensidade das respetivas reaes.
Concluso: O timo de coelho revelou a reao mais
especfica dos tecidos estudados, sendo assim recomendado como um bom controlo positivo para estudos
de apoptose.
Agradecimentos: FCT e CI&DETS (PEst-OE/
CED/UI4016/2011)

Vala H1,2*, Santos C1,2, Cruz R1,2, Nbrega C1,2, Ortiz

AL3, Mesquita JR1,2, Esteves F1,2, Mega A1,2,
Escola Superior Agrria de Viseu, Instituto Politcnico de Viseu, Portugal;
2
Centro de Estudos em Educao, Tecnologias e Sade,
Instituto Politcnico de Viseu, Portugal
3
Len University, Len, Spain
1

* hvala@esav.ipv.pt
Introduo: As tcnicas de determinao de apoptose tm sido preconizadas para fins de diagnstico,
designadamente para determinao das caratersticas
biolgicas das neoplasias e, indiretamente, determinao do prognstico.
A implementao de uma nova tcnica num laboratrio pressupe o uso de um controlo positivo eficaz para aferio da mesma. Muitas das publicaes
da especialidade sobre o tema no indicam, de modo
claro, os controlos positivos usados e no divulgam os
resultados dos mesmos.
O objetivo deste trabalho consistiu na implementao de trs mtodos de deteo de apoptose, utilizando trs amostras de tecidos de co: neoplasia mamria
- carcinoma in situ, timo e plasmocitoma; e uma amostra de tecidos de coelho timo.
Material e mtodos: Os mtodos de imunohistoqumica incluram a deteo de protenas pro- e antiapoptticas, respetivamente, Bax e Bcl-2 (1:100; Santa
Cruz, Biotechnology) e o mtodo de TUNEL, com o
kit de deteo de apoptose in situ (1:2,5; Roche).
Resultados: O plasmacitoma revelou reao positiva moderada com todos os mtodos testados. O car-

86

Rabbit thymic tissue a potential marker

for apoptosis techniques
Introduction: Apoptosis methods have been proposed for diagnostic purposes, in particular for determining the biological characteristics of tumours and,
indirectly, their prognosis.
Implementation of a new technique requires the use
of an effective positive for correct assessment.
Most specialty publications on the subject, do not
indicate the positive controls used and do not disclose
the obtained results therein.
Objective: The aim of this work was to implement
three different methods for the detection of apoptosis,
using three canine tissue samples, consisting of breast
cancer - carcinoma in situ, thymus and plasmacytoma;
and a sample of rabbit tissues - thymus.
Material and methods: Immunohistochemistry
was performed, using pro- and antiapoptotic proteins,
respectively, Bax and Bcl2 (1: 100; Santa Cruz, Bio-

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RPCV (2013) 108 (585-586) 69-91

technology) and the TUNEL technique (1: 2,5; Roche),

using the in situ cell death detection kit.
Results: Canine plasmacytoma revealed moderate
positive reaction with all methods. Canine carcinoma
in situ showed moderate positive reaction with pro-apoptotic antibodies and more intense with anti-apoptotic
antibody. Dogs thymus showed moderate positive reaction in cortical thymocytes with pro-apoptotic antibodies and weak in the medullary thymocytes. With
the anti-apoptotic antibody, medullary thymocytes

showed weak reaction and negativity in the cortex.

Identical results were obtained in rabbits thymus, but
with stronger intensity.
Conclusion: The rabbit thymus showed the most
specific reaction of the tissues studied, by which the
authors recommended it as a viable positive control for
apoptosis studies.
Acknowledgments: FCT and CI&DETS (PEst-OE/
CED/UI4016)

Um caso de hemangiosarcoma cardaco

em co

rentes aos vasos sanguneos. Todo o tecido adiposo adjacente ao corao apresentava proliferao de clulas
neoplsicas.
Concluso: O diagnstico foi de hamangiossarcoma
da aurcula direita com metstases pulmonares, sem
envolvimento esplnico.

Pires M.A.1,2*, Pinheiro D. 1, Silva N.3, Montenegro L.3

 ECAV, University of Trs-os-Montes and Alto DouC
ro, P.O. Box 1013, 5001-801 Vila Real, Portugal.
2
ECAV, University of Trs-os-Montes and Alto Douro,
P.O. Box 1013, 5001-801 Vila Real, Portugal.
3
Veterinary Montenegro Hospital (HVM), 4200 - 447
Porto, Portugal.
1

* apires@utad.pt
Introduo: Segundo a literatura, o hemangiossarcoma cardaco uma situao comum nos ces Pastor
Alemo, comumente associado a leso esplnica.
Os autores descrevem um caso de hemangiossarcoma cardaco sem envolvimento esplnico, com parmetros clnicos e imagens macroscpicas, associado a
metstases pulmonares.
Material e mtodos: Foram remetidos ao Laboratrio de Histologia e Anatomia Patolgica da UTAD, o
corao e pulmo de um Pastor Alemo, macho, de 11
anos de idade. Os rgos fixados em formol tamponado a 10%, foram processados por rotina e corados com
hematoxilina e eosina.
Resultados: O historial do animal indicava ausncia
de vacinao e desparasitao, fraqueza extrema, dispneia e choque. Ao RX a silhueta cardaca apresentavase arredondada e com imagens de efuso torcica e
pericrdica, confirmada por ecografia.
Foi solicitada a eutansia, tendo a necrpsia revelado
hemotrax e hemopericrdio, leso papilar exofitica na
aurcula direita, vilosa, de 3 cm de dimetro que invadia o miocrdio, at ao pericrdio com 6x3x4,5cm. O
pulmo apresentava mltiplas pequenas leses redondas
e negras, a maior com 0,9 cm de dimetro. No se observaram outras leses macroscpicas dignas de registo.
O corao apresentava proliferao de clulas fusocelulares, com elevado pleomorfismo e clulas atpicas,
com 4 a 5 mitoses/40x, com disposio estoriforme e
fendas com eritrcitos. O pulmo apresentava mltiplas metstases com clulas e disposio semelhantes
ao observado no corao, algumas intimamente ade-

A case of heart hemangiosarcoma in a dog

Introduction: The consulted literature, referred that
hear hemangiosarcoma, a common condition in German Shepherd dog, generally associated with spleen
hemangiosarcoma.
The authors report a case of heart hemangiosarcoma
without spleen implication, with clinical parameters and
macroscopic images, associated with lung metastasis.
Material and methods: an heart and lung from an
eleven years old male German Shepherd dog was to the
Laboratory of Histology and Anatomical Pathology, at
UTAD. The organs were fixed in 10% buffered formalin,
and processed and stained with haematoxylin and eosin.
Results: The clinical history indicated absence of
vaccination or dewormed medication, generalized
weakness, dyspnoea and shock. At x-ray the cardiac
silhouette is rounded with pleural and pericardial effusion confirmed by ultrasonography.
Euthanasia was requested and at necropsy an abundant hemothorax, and hemopericardium was recorded.
The right auricle presents a papillary villous exofitic
proliferation with 3cm of diameter, that invaded the
myocardium until the pericardial wall with overall
dimensions 6x3x4.5cm. The lung presented multiple
black round nodules, the larger with 0.9 cm diameter.
There were no other gross lesions noteworthy.
The histology of the heart lesion revealed fusocellular proliferation with high pleomorphic and atipic
cells, with 4-5 mitosis for HPF. The neoplasia had a
storiform pattern with clefts with erythrocytes. The
lung had multiple metastases with similar cells, some
of them within vascular structures. All adipose tissue
around the heart had neoplasic cells.
Conclusion: The final diagnosis was hemangiosarcoma of the right auricle with lung metastasis, without
spleen implication.
87

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RPCV (2013) 108 (585-586) 69-91

Prolapso da cloaca associado a um adenocarcinoma intestinal em cobra-falsa-coral

(Lampropeltis triangulum)

diagnstico de um adenocarcinoma intestinal mucinoso. Os autores consideram que se deveria incluir nas
etiologias de prolapsos da cloaca em cobras, os adenocarcinomas intestinais.

Martinho F.1, Fasca P.1, Santos S.1, Carvalho I.2, Angua M.1

Cloacal prolapse associated with an intestinal

adenocarcinoma in a milk snake (Lampropeltis triangulum)

 ICV- Faculdade de Medicina Veterinria - ULHT,

C
Lisboa, Portugal7
2
DNATech
1

Caso clnico: Uma cobra-falsa-coral fmea, com 4

anos de idade, apresentou-se com anorexia e prolapso
da cloaca recorrente. Com ecografia celmica visualizou-se massa na parede do tero final do clon, qual
se seguiu uma enterectomia da zona afetada.
Macroscopia: Parede intestinal com 2 x 1 x 1 cm,
que apresenta ndulo ulcerado, branco homogneo,
com limites bem definidos.
Microscopia: Histologicamente, corresponde a um
fragmento de intestino grosso, ulcerado que apresenta
uma proliferao celular no capsulada com origem na
mucosa e que invade as restantes camadas intestinais
at serosa. As clulas so colunares altas intercaladas por clulas caliciformes dispostas em quistos, cinos e lbulos num estroma desmoplsico. As clulas
apresentam citoplasma acidfilo e um ncleo no polo
basal redondo com a cromatina finamente ponteada e
um nuclolo evidente. No lmen das estruturas epiteliais e no estroma envolvente observa-se a deposio
de mucinas. O ndice mittico baixo.
As clulas epiteliais apresentaram imunomarcao
positiva para CK AE1/AE3 (Dako M3515).
Concluso: Com base nos achados histolgicos, o

Hrnia de Bexiga associada a Angioleiomiossarcoma Cutneo em Coelho

Santos S.1*, Patrcio. R.1,2
CICV - FMV- ULHT, Lisboa, Portugal
AlcabidecheVet Clnica Veterinria, Cascais, Portugal
1
2

sarasantos.med.vet@gmail.com

Introduo: Em Agosto de 2012 um coelho de estimao com 6 anos de idade, macho esterilizado foi
submetido a cirurgia para extirpao de uma massa
subcutnea ulcerada, localizada na regio inguinal e
associada a uma hrnia de bexiga.
Material e mtodos: Aps a cirurgia a massa foi
enviada para anlise histopatolgica, fixando-se o tecido em formol tamponado a 10% para colorao com
hematoxilina & eosina (H&E). Realizou-se imunohistoqumica com -actina (clone, 1A4 DAKO; 1\180)
88

Clinical case: A 4 years old, female, milk snake, was

presented with anorexia and recurrent rectal prolapse.
By ultrasound, an intestinal mass in the final third of
the colon was identified, and an enterectomy was performed.
Macroscopically: Intestinal wall with 2 x 1 x 1 cm,
presenting an ulcerated, well demarcated, white, homogenous nodule.
Microscopically: A section of colon, focally ulcerated, presented epithelial cell proliferation originating in
the mucosa and invading the adjacent intestinal layers.
The neoplastic cells were tall, columnar, interspersed
with goblet cells, and were disposed in cysts, acini and
lobules in a desmoplastic stroma. The cells presented
acidophilic cytoplasm, and a round, basally located,
nuclei with stippled chromatin and distinct nucleoli.
The lumen of the epithelial structures was filled with
mucin. Mitotic index was low.
Epithelial cells presented positive immunostaining
for CK AE1/AE3 (Dako M3515).
Conclusion: In the basis of the histological findings,
the diagnosis is mucinous adenocarcinoma of the colon. In the authors opinion, colon adenocarcinomas
should be included in the differential diagnosis of cloacal prolapses in snakes.

e desmina (clone DE-R-11, DAKO; 1\50) utilizando

intestino e estmago de co como controlo positivo.
Resultados: A H&E revelou uma neoplasia subcutnea, no capsulada e infiltrativa constituda por clulas
fusiformes anaplsicas organizadas em feixes compactos e ninhos. As clulas neoplsicas apresentavam-se
intimamente relacionadas com as estruturas vasculares
parcendo proliferar da camada mdia dos vasos sanguneos drmicos os quais apresentavam a camada mdia esclorosada. Foi observado pleomorfismo intenso,
ndice mittico elevado (0-4 mitoses por campo 40x) e
focos de necrose.
Os anticorpos foram fortemente positivos em aproximadamente 90% das clulas neoplsicas.
Discusso: Trata-se de uma neoplasia maligna, rara
em coelhos, sobre a qual existe pouca informao referente aos estudos imunohistoqumicos e comportamento biolgico esperado. Enfatiza-se a eficcia dos
anticorpos utilizados e a no reincidncia.
Concluso: Angioleiomiossarcoma cutneo.

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Bladder Hernia associated to a Cutaneous

Angioleiomyosarcoma in a Rabbit
Introduction: A 6 year-old neutered male pet rabbit
presented on August 2012 with a bladder hernia associated to an ulcerated subcutaneous neoplasm in the
inguinal area.
Material and methods: After the surgery the mass
was submitted for histopathology. Tissue samples were
fixed in 10% neutral buffered formalin and stained
with hematoxylin and eosin (H&E). We used antibodies against -smooth-muscle actin (clone, 1A4 DAKO;
1\180) and desmin (clone DE-R-11, DAKO; 1\50) for
immunohistochemical studies. Dogs intestine and
stomach samples were used as a positive control.
Results: The H&E revealed an unencapsulated and
infiltrative neoplasm in the subcutaneous tissue, com-

Caracterizao do ndice apopttico em

mastocitomas caninos
Paiva D1*, Cruz R1,2, Mega A1,2, Vala H1,2
Escola Superior Agrria de Viseu, Instituto Politcnico de Viseu, Portugal;
2
Centro de Estudos em Educao, Tecnologias e Sade, Instituto Politcnico de Viseu, Portugal
1

RPCV (2013) 108 (585-586) 69-91

posed of anaplastic spindle cells arranged in compact

bundles and whorls. The neoplasm cells were closely
connected with vascular structures and seemed to
proliferate from the tunica media of the subcutaneous blood vessels which presented sclerosis of tunica
adventitia. It was observed an intense pleomorphism,
higher mitotic index (0-4 mitotic figures by fields at
40x) and foci of necrosis.
Around 90% of the neoplastic cells were strongly
positive for antibodies.
Discussion: This case presents a rare malignant neoplasm in rabbits and there is little information about
immunohistochemical studies and neoplastic behaviour. It is important to share the effectiveness of the
used antibodies and the not recurrence of this neoplasm.
Conclusion: Cutaneous angioleiomyosarcoma.

gamento da vida e do nmero das clulas tumorais e,

consequentemente, progresso tumoral.
Os mastocitomas caninos revelam sensibilidade
com os marcadores Bax e Bcl-2, podendo estes ser
aplicveis em estudos de apoptose nestas neoplasias
de modo a consolidar o prognstico em relao sua
graduao histolgica.
Agradecimentos: FCT e CI&DETS (PEst-OE/
CED/UI4016/2011)

* dany_fullmoon@hotmail.com
Introduo: Encontram-se descritos estudos de
apoptose em vrias neoplasias. A apoptose um processo biolgico de morte celular programada que, devido ao seu importante papel na oncognese, poder
elucidar alguns aspetos.
Objetivo: Deteo da apoptose em mastocitoma
canino, procurando determinar a sua relao com a
classificao histolgica, ndice apopttico, e prognstico.
Material e mtodos: 12 casos de mastocitoma canino, do ficheiro de anlises do Laboratrio de Anatomia Patolgica da ESAV. Os mtodos de imunohistoqumica foram implementados, utilizando protenas
pro- e anti-apoptticas Bax e Bcl (Santa Cruz, Biotechnology).
Resultados: Todos os mastocitomas includos no
estudo revelaram reao positiva aos anticorpos testados, verificando-se uma reao mais intensa com o
anticorpo pr-apopttico Bax em relao ao anticorpo anti-apopttico Bcl-2. Enquanto a expresso de
Bax foi mais intensa nos mastocitomas de grau I, o
Bcl-2 evidenciou maior expresso nos mastocitomas
de grau III, apresentando ambos uma reao mais fraca no grau II.
Concluso: A diminuio de Bax e aumento de
Bcl-2, segundo o grau histolgico, sugere um prolon-

Characterization of the apoptotic index in

canine mastocytomas
Introdution: Studies on apoptosis have been described in several neoplasias. Apoptosis is a biological process of programmed cell death that, due to its
important role in oncogenesis, may elucidate some
aspects.
Objective: Detection of apoptosis in canine mastocytoma to determine its relationship with histologic
grading, apoptotic index and prognosis.
Material and methods: 12 cases of canine mastocytoma, from the analytic files of the Laboratory of
Anatomical Pathology of ESAV. Immunohistochemical methods were implemented, targeting pro- and
anti-apoptotic proteins, Bax and Bcl (Santa Cruz, Biotechnology).
Results: All mastocytomas included in this study
revealed a positive reaction to the tested antibodies, a
more intense reaction was found to pro-apoptotic antibody Bax in comparison to the anti-apoptotic antibody Bcl-2. While Bax expression was more intense in
grade I mastocytomas, Bcl-2 had a higher expression
in grade III mastocytomas, and both, had a weaker expression in grade II.
Conclusion: The decrease of Bax and increase of
Bcl-2 expression in accordance with increasing histo89

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RPCV (2013) 108 (585-586) 69-91

logic grade, suggests an extension in the life of tumor

cells, an increase their number and, consequently, tumoral progression.
Canine mastocytomas revealed sensitivity with Bax
and Bcl-2 markers and may be applied to apoptotic

studies in such tumors to consolidate prognosis in relationship to histologic grading.

Acknowledgments: FCT and CI&DETS (PEst-OE/
CED/UI4016/2011)

Fibrosarcoma Cutneo em Robalo, Dicentrarchus labrax de Tanque de Terra

reaco positiva ao Van Gieson, diagnosticou-se um

fibrosarcoma.

Ramos, P.1*, Grade, A.1, Branco, S.2

Laboratrio de Patologia dos Animais Aquticos. Instituto Portugus do Mar e da Atmosfera, IPMA, I.P.
Avenida de Braslia, 1449-006 Lisboa, Portugal
2
Instituto de Cincias Agrrias e Ambientais Mediterrnicas, Universidade de vora. Apartado 94, 7002554 vora, Portugal
1

* pramos@ipma.pt
Introduo: Os tumores espontneos em peixes so
morfologicamente semelhantes e em muitos casos,
idnticos aos que ocorrem em vrias espcies animais,
incluindo o homem. Assim, o seu estudo importante
no s do ponto de vista da patologia comparada mas
tambm pela escassez de registos.
Material e mtodos: Num robalo proveniente de um
tanque de terra foi observada a presena de duas formaes nodulares localizadas na face lateral esquerda.
Amostras do ndulo maior foram fixadas em formol
salgado a 10%. Obtiveram-se cortes com 3 que foram
corados com hematoxilina & eosina e Van Gieson.
Resultados: O ndulo maior encontrava-se na face
lateral esquerda, prximo da base da barbatana dorsal
e tinha aproximadamente 2,7 cm de dimetro. Este ndulo, de cor amarela, apresentava superfcie ulcerada e
ligeira congesto. Ao corte, tinha consistncia branda.
O segundo ndulo com cerca de 1 cm de dimetro foi
observado abaixo da linha mdia. A imagem histolgica do ndulo permitiu observar a presena de clulas
com morfologia fibroblstica, fusiformes, com disposio em feixes, num estroma bem vascularizado e rico
em colagnio, entrecruzando-se com feixes de clulas
com ncleo redondo em reas com aspecto mixomatoso. A neoplasia invadia o tecido muscular adjacente.
Na derme, observou-se reaco inflamatria com infiltrao de macrfagos e formao de granulomas. Foram identificados melancitos ao longo da membrana
basal, dispersos na derme ou em centros melanomacrofgicos. O estroma abundante em colagnio corou
positivamente com o Van Gieson.
Concluso: Com base nas imagens histolgicas e na

90

Cutaneous Fibrosarcoma in Earth Pond

Reared European Seabass, Dicentrarchus
labrax
Introduction: The spontaneous neoplasms in fishes
are morphologically similar and, in many cases identical, to those found in several animal species, including man. Thus their study is valuable not only from the
standpoint of comparative pathology but also by the
scarcity of the records.
Material and methods: A european bass in a earth
pond culture was discarded because of the presence of
two nodules on the left side of the body surface. Portion of the largest tumour was collected, fixed in 10%
neutral buffered formalin and sections 3 thick were
stained with both haematoxilyn & eosin and Van Gieson.
Results: The largest nodule was located on the left
flank, near the dorsal fin and measures approximately
2,7 cm in diameter. In appearance, its surface was yellowish, with superficial congested ulcerations and soft
consistency. The second one was a little round nodule
with 1,2 cm diameter, ventral and bellow the medial
line. The histological appearance of the tumour was
a well vascularized, no encapsulate dermal mass of
spindle-shaped cells, forming dense whorls in a collagen rich stroma. Adjacent less cellular areas containing stellate cells with criss-crossed fibrils and loosely
organized, suggested a myxomatous area. These spindle-shaped cells were seen infiltrating the subcutaneous tissue and invading the adjacent muscle tissue. Infiltration of inflammatory cells and granulomas could
be observed within the dermis. Melanophores were
scattered along the basement membrane and dermis or
forming melanomacrophage centres. The histochemical reactions of the stroma were positive with the specific coloration of Van Gieson.
Conclusion: The diagnosis of fibrosarcoma was
based on the examination of elongated fibroblasts and
dense collagenous fibres that stains positively with Van
Gieson.

SUPLEMENTO

Imortalizao de linhas celulares caninas

primrias de tumores mamrios: optimizao de protocolo
Lus R Raposo1,2, Susana Santos1,2,3,4, Joaquim Henriques5, Pedro Faisca5, Margarida Alves5, Andr Beselga6, Jorge Correia6, Alexandra R Fernandes1,2, 4*
Centro de Qumica Estrutural, Complexo I, Instituto
Superior Tcnico, Universidade Tcnica de Lisboa,
Lisbon, Portugal,
2
Escola de Engenharia, Universidade Lusfona de Humanidades e Tecnologias, Lisbon, Portugal,
3
Department of Biochemistry and Human Biology,
Faculty of Pharmacy, University of Lisbon, Lisbon,
Portugal,
4
Departamento de Cincias da Vida, Faculdade de Cincias e Tecnologias, Universidade Nova de Lisboa,
Caparica, Portugal,
5
Centro de Investigao em Cincias Veterinrias, Faculdade de Medicina Veterinria - Faculdade de Medicina Veterinria- Universidade Lusfona de Humanidades e Tecnologias, Lisbon, Portugal,
6
Faculdade de Medicina Veterinria, Universidade
Tcnica de Lisboa, Lisbon, Portugal * alexandrancrfernandes@gmail.com
1

Introduo: Os tumores mamrios caninos (CMTs)

so o tipo de tumor mais frequente nas cadelas, correspondendo a 52% de todas as suas neoplasias. Para as variantes malignas de CMT, a cirurgia a nica opo de
sucesso, uma vez que a quimioterapia, no aumenta significativamente o tempo de sobrevivncia. Por esta razo, crucial desenvolver modelos celulares por forma
a testar novos compostos com actividade anti-tumoral
para os CMTs. As linhas celulares imortalizadas derivadas de CMTs podero, ainda, constituir uma ferramenta
importante na investigao dos mecanismos celulares e
moleculares subjacentes tumorignese dos CMTs.
Material e mtodos: Para a imortalizao das linhas
celulares derivadas de CMT, as amostras das bipsias so
lavadas, maceradas e incubadas com tripsina durante uma
hora a 37C em vez da incubao em gelo descrita na bibliografia. As clulas individuais so recolhidas e semeadas em meio DMEM suplementado. As clulas crescem
at atingir uma monocamada confluente sendo, de seguida, tripsinizadas e colocadas em frascos maiores (5x105
clulas so colocadas a crescer em frascos de 75cm2).
Resultados: A nossa metodologia permitiu-nos obter
monocamadas de clulas confluentes para todas as amostras das bipsias. Microscopicamente, a morfologia das
clulas compatvel com uma origem epitelial. Actualmente, temos trs linhas celulares que foram tratadas com
tripsina 10 vezes, sugerindo uma possvel capacidade
para originarem linhas derivadas de CMT imortalizadas.
Uma das linhas celulares derivada de um carcinoma mamrio simples (tipo mucinoso) de grau II e as restantes de
carcinomas mamrios complexos de grau II.

RPCV (2013) 108 (585-586) 69-91

Concluso: O presente trabalho descreve um protocolo optimizado para a imortalizao de linhas celulares desenvolvido pela nossa equipa. Foi possvel obter
consistentemente monocamadas confluentes de clulas
para todas as amostras processadas at data. Estas
monocamadas de clulas so compostas por clulas
com morfologias diferentes (algumas mais fusiformes,
outras mais cuboidais) e tamanhos diferentes independentemente do grau de malignidade e do tipo de tumor
que lhe deu origem. Com as tripsinizaes, as clulas
tendem a tornar-se mais homogneas, com uma forma
mais cuboidal e mais pequenas.

Immortalization of primary canine cell

lines from mammary tumors: a protocol
optimization
Introduction: Canine mammary tumors (CMTs) are
the most common tumor type in female dogs, comprising 52% of all neoplasms. For malignant CMT variants, surgery is the only successful treatment option
since chemotherapy has not radically increased overall
survival time. In this regard it is a key issue to develop
cellular models to validate new therapeutic compounds
for CMT treatment. Immortalized CMT cell lines may
also be a valuable tool that will allow achieving a better
knowledge on the cellular and molecular mechanisms
underlying CMT tumorigenesis.
Material and methods: For CMT cell line immortalization, intra operative tumour samples were obtained, washed, minced and incubated with trypsin for
one hour at 37C instead of cooling as described in the
bibliography. Individual cells were then collected and
seeded in supplemented DMEM medium. The cells are
allowed to grow until a confluent primary cell monolayer is formed followed by trypsinization and seeded
in larger flasks (5x105 cells are grown in 75cm2 flasks)
than in the previous protocol.
Results: Our changes in the original methodology
allowed us to growth primary cell monolayers from all
biopsy samples. Microscopically, the cell morphology
is compatible with their epithelial origin. Currently we
achieved three CMT cell lines that have been trypsinised
for 10 times, suggesting a potential capacity to originate
immortalized primary CMT cell lines. One of the cells
lines was established from a grade II mammary simple
carcinoma (mucinous type) and the remaining from
grade II mammary complex carcinomas.
Conclusion: An optimized protocol for cell lines
immortalization was developed by our team. Until
now, we were able to establish a primary cell monolayer for all processed samples. These cell monolayers are
composed of cells with different morphologies (some
more fusiform other more cuboidal) and different sizes
regardless the tumour type or grade that originated the
monolayer. With trypsinizations the cell populations
tend to become more homogenous, with a more cuboidal shape and smaller size.
91