F. Seixas

Grade is an independent prognostic factor for feline mammary carcinomas: A clinicopathological and survival analysis

Feline mammary carcinomas (FMC) are highly infiltrative tumours which show a strong tendency for local recurrence and metastasis. Histological type assessment of these tumours is not sufficiently discriminatory in predicting prognosis and in this study the prognostic significance of the Elston and Ellis method of histological grading was evaluated. Ninety-two feline mammary carcinomas from 84 cats were graded and 64 queens were included in a follow-up study. Grade was significantly related to tumour size (P=0.006), clinical stage (P=0.005), lymphovascular invasion (P<0.0001), mitotic index (P<0.0001), Ki67 index (P=0.001), overall survival (P=0.0001) and disease-free survival (P<0.0001). Cox regression analysis identified grade as an independent prognostic factor. Multivariable analysis also showed regional lymph node metastasis and lymphovascular emboli as independent prognostic factors related to overall survival and to disease-free-survival, respectively. The study demonstrated that histological grading can be used as a prognostic factor to evaluate the biological behaviour of FMC.

Immunohistochemical characterization of 13 canine renal cell carcinomas.

Canine renal cell carcinomas (RCCs) are uncommon aggressive tumors that occur mainly in middle-aged male dogs. Their histologic classification bears no relationship with prognosis, and little information is available concerning their immunohistochemical properties. In this retrospective study, formalin-fixed, paraffin-embedded tissues from 13 canine RCCs were retrieved from the archives, classified histologically, and evaluated immunohistochemically. The dogs were 7 males and 6 females (1 spayed) of 10 different breeds, averaging 8 years in age. The tumors were classified as papillary, tubulopapillary, papillary–cystic, solid, or sarcomatoid. All 13 tumors were immunohistochemically positive for uromodulin, 12 for c-KIT, 11 for vimentin, 9 for wide-spectrum-screening cytokeratins, 7 for cytokeratins AE1/AE3 and carcinoembryonic antigen, 4 for cytokeratins CAM 5.2, and 3 for CD10. All 3 solid RCCs expressed vimentin, c-KIT, and carcinoembryonic antigen and were negative for cytokeratins. All 7 papillary and tubulopapillary tumors expressed vimentin; 6 (86%), cytokeratins; and 6 (86%), c-KIT. Both papillary–cystic RCCs were positive for cytokeratins and c-KIT and negative for vimentin. These results indicate that the different histologic types of RCC have characteristic immunohistochemical profiles and that c-KIT may be involved in the pathogenesis of canine RCC.

Mammary Invasive Micropapillary Carcinoma in Cats: Clinicopathologic Features and Nuclear DNA Content

Invasive micropapillary carcinoma (IMC) is a variant of infiltrating ductal carcinoma of the breast associated with poor outcome. In this study, we report 16 carcinomas of the feline mammary gland displaying histologic features that correspond to IMC of the breast in women. The clinicopathologic findings, overall survival time, disease-free survival time, and nuclear DNA content of these cats were compared with 65 more common invasive mammary carcinomas (other feline mammary carcinoma [FMC]) of nonspecified type. IMC was associated with larger tumor size, higher histologic grade (P < .0001), deeper muscle invasion (P = .004), and more frequent lymphovascular invasion and nodal metastases (P = .009 and P = .001, respectively) than other FMCs. The aneuploid pattern was more frequent in IMC lesions. IMCs were also associated with lower survival rates. In summary, all cases of feline IMC were associated with clinicopathologic features of high biologic aggressiveness and should be classified as independent histologic types of FMC.

The effects of repeated oral gavage on the health of male CD-1 mice

Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.

Differential mortality of birds killed at wind farms in Northern Portugal

Capsule The Skylark Alauda arvensis had the highest overall mortality in ten Northern Portuguese wind farms surveyed between 2006 and 2011. Analysis from the integration of conventional and molecular techniques suggest a sex and age biased mortality affecting mainly adult males (90.9%), which may be related to their characteristic breeding male song-flights making them highly vulnerable to collision with wind turbines. The results highlight the added value of more complete population impact assessments that go beyond simple carcass identification at wind farms.

High-resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples.

High‐resolution melting (HRM) analysis is a very attractive and flexible advanced post‐PCR method with high sensitivity/specificity for simple, fast and cost‐effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real‐time PCR systems, along with improved saturating DNA‐binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex‐specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed‐tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real‐time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.

Histopathologic and immunohistochemical exam in one case of canine endometrial adenocarcinoma

Canine endometrial carcinomas are rare, and mostly occur in geriatric bitches. In this work, the uterus of a 10-year-old female Boxer evidencing an endometrial carcinoma on the body of the uterus was used to describe the histopathological features of the tumour and to study its immunophenotype. In this work, a panel of immunomarkers (cytokeratins AE1/AE3 and 14, vimentin, CD10 and Ki-67) was applied to the endometrial carcinoma to establish the staining patterns indicative of the tumour agressiveness and cellular differentiation. Additionally DNA ploidy was also performed. In this case, the tumour showed papillar pattern, with large pleomorphic, anaplastic cells and also some aberrant multinucleated and giant cells. In some areas of the tumour, it was also observed cytotrophoblastic-like cells outlining the papillae. Cytokeratin AE1/AE3 expression was detected in the luminal neoplasic cells. Cytokeratin 14 positivity was sporadic and irregular, and was observed mainly in the luminal epithelium. Only stromal and aberrant cells showed a positive staining to vimentin. Positive membranous staining to CD10 was evidenced by clear epithelial, cytotrophoblastic-like cells at the tumour surface but not by the stromal cells. The mitotic and Ki-67 indices were low, suggestive of a weak aggressiveness of the tumour. The multinucleated and giant cells evidenced a positive immunostaining to CK AE1/AE3, and CD 10; its positivity to vimentin was sporadic. This study aims to contribute to the advancement of the knowledge in canine endometrial carcinoma immunophenotype.

A case of pulmonary cryptococcosis in a free-living toad (Bufo bufo).

Pulmonary cryptococcosis was observed in a free-living adult female common toad (Bufo bufo) that was killed by a vehicle. Both lungs had various eosinophilic, monomorphic, and spherical to elliptical organisms identified as Cryptoccocus spp. The yeasts were demonstrated by Grocotts silver method and the periodic acid-Schiff reaction and the capsule was positive for mucin with a mucicarmine stain. The agent was confirmed by immunohistochemistry, using the monoclonal antibody anti-Cryptococcus neoformans, and by a polymerase chain reaction-based method using a C. neoformans-specific primer. This report, to the best of our knowledge, represents the first case of cryptococcosis in a common toad.

Pulmonary adiaspiromycosis in a European hedgehog (Erinaceus europaeus) in Portugal

ADIASPIROMYCOSIS (haplomycosis) is a self-limiting pulmonary mycosis caused by the dimorphic fungi Chrysosporium parvum var crescens and C parvum var parvum (Burek 2001, Carter 2002) that occurs worldwide in small mammals (Turner and others 1999, Travis and others 2002). The disease has been reported in 124 mammal species and subspecies (Acha and Szyfres 1989) of the Carnivora, Rodentia and Insectivora (Burek 2001). The infection is widespread, especially among rodents of the Arvicolidae and Muridae families, but it rarely occurs in the Sciuridae and Cricetidae (Fischer 2001); the Mustelidae appear to be particularly susceptible to the infection (Simpson and GavierWiden 2000, Burek 2001). C parvum has also been found in dogs (Jones and others 1997, Carter 2002) and goats, and several cases of adiaspiromycosis have been reported in human beings since 1964 (Jones and others 1997). Human infection is uncommon (England and Hochholzer 1993, Turner and others 1999, Travis and others 2002), but cases have been reported in the USA, Europe, the former Soviet Union, and Central and South America (Chandler and Watts 1988). Most human infections are attributed to C parvum var crescens (Chandler and Watts 1988, Travis and others 2002), but two reports documented disseminated disease caused by C parvum var parvum in patients with AIDS (Echevarria and others 1993, Turner and others 1999). This short communication describes a case of adiaspiromycosis due to C parvum var crescens in a European hedgehog (Erinaceus europaeus). In May 2001, a young male European hedgehog was found dead in a road near Vila Real in northern Portugal. The animal was collected and submitted for postmortem examination at the Histopathology Laboratory of Tras-os-Montes e Alto Douro University. The animal was in normal body condition, weighing 638 g. Postmortem examination revealed multiple traumatic lesions in the mandible and ribs, and rupture of the abdominal wall with evisceration. The pericardial sac was ruptured. The lungs were detached from their normal anatomical position and free in the thoracic cavity. They presented multiple sites of damage, oedema, congestion and extensive haemorrhagic areas, and various grey-white, nodular, focal lesions, measuring 0·5 to 1·0 mm in diameter, were disseminated on the surfaces of both lungs. The lesions appeared to have no predilection for particular areas or lobes of the lungs. No significant abnormalities were observed in the thymus. The liver was pale and friable, and had multiple ruptures, and the spleen was also damaged. The small intestine was ruptured at four points, and its contents had spilled into the peritoneal cavity. Samples of the organs were fixed in 10 per cent buffered formalin and processed for light microscopy using standard methods. Paraffin sections (2 μm) for histological examination were stained with haematoxylin and eosin and by Grocott’s technique. Microscopically, the lung presented atelectasis, emphysema and numerous granulomatous lesions in the interstitium (Fig 1), each containing one or two spherical adiaconidia surrounded by epithelioid cells, giant multinucleated cells in a palisade arrangement, and fibrous tissue. The diameter of the fungi ranged from 119 to 285 μm. The double contour, refractile wall had a thickness of 38 to 59 μm. The outer third of the trilaminar wall was eosinophilic and refractile, and there was a non-staining layer between the outer and inner walls that appeared black with Grocott’s technique (Fig 2). The interior of the adiaconidia was either empty or contained small eosinophilic globules. The structures were consistent with descriptions of the morphology of the adiaspores of C parvum var crescens (Chandler and Watts 1988, Jones and others 1997, Burek 2001, Travis and others 2002). The hedgehog also had a verminous bronchitis. No adiaspores were observed in the regional lymph nodes. The liver showed lipidosis, and the kidney presented pigmentary nephrosis and interstitial nephritis. No significant lesions were observed in the remaining organs. Adiaspiromycotic infection is believed to occur as a result of the inhalation of dustborne spores (conidia) found in soil, which accumulate in the bronchioles and alveoli (England and Hochholzer 1993, Burek 2001, Travis and others 2002). The genus Chrysosporium consists of ubiquitous fungi, which produce 2 μm to 4 μm conidia in the saprophytic form (Simpson and Gavier-Widen 2000, Travis and others 2002). When inhaled by a mammalian host, the conidia progressively enlarge to form thick-walled spherules, designated as adiaconidia or adiaspores (Acha and Szyfres 1989, Simpson and Gavier-Widen 2000, Fischer 2001). The adiaspores of C parvum var parvum grow to up to 20 to 40 μm (Burek 2001), whereas the C parvum var crescens adiaspores have a diameter ranging from 200 to 700 μm (Chandler and Watts 1988, Simpson and Gavier-Widen 2000, Travis and others 2002). C parvum var crescens has also a broader host range and geographical distribution than C parvum var parvum (Peterson and Sigler 1998, Burek 2001). Adiaconidia seem to be incapable of replication or dissemination in the host (Chandler and Watts 1988, Travis and others 2002), yet massive infections have been observed in small mammals (Burek 2001). The disease is not contagious (Jones and others 1997), thus each adiaspore constitutes a separate infection (Simpson and Gavier-Widen 2000). The infection resembles foreign-body inhalation pneumonia, with a granulomatous reaction due to the inhaled spores (Chandler and Watts 1988, Travis and others 2002). The severity of the disease depends on the number of spores inhaled (Acha and Szyfres 1989, England and Hochholzer 1993) and the immune response of the host (Chandler and Watts 1988, Jones and others 1997). When clinical signs are present, they can be attributed to mechanical displacement of the tissues and the inflammatory reaction caused by the proVeterinary Record (2006) 158, 274-275

Oestrogen receptors in a case of hydrometra in a bitch

HYDROMETRA – the accumulation of fluid in the uterine lumen – alone is uncommonly observed in bitches. McAfee and McAfee (1976) reported a case of hydrometra in a young bitch that was presented for elective ovariohysterectomy. Other reports of hydrometra have associated the condition with cystic endometrial hyperplasia (CEH) which evolves with a high degree of mucous hydration (Kennedy and Miller 1992). In other species, hydrometra has been associated with chronic stimulation of oestrogen receptors by exogenous substances (Biolatti and others 1994, Re and others 1995). Granulosa cell tumours are one possible source of endogenous oestrogens. However, they do not always induce uterine disease. These tumours are common in bitches, but their endocrine secretion is not constant and may vary between tumours and with the stage of tumour development (Cotchin and others 1961, Norris and others 1970, McEntee 1990). Several studies (De Cock and others 1997, Dhaliwal and others 1997, Vermeirsch and others 1999) have demonstrated that the number and distribution of oestrogen receptors in the uterus varies within the oestrous cycle, and have correlated the expression of oestrogen receptors with the concentrations of oestrogen and progesterone in the blood. This short communication describes a case of hydrometra in a bitch in which the macroscopic and microscopic features differed from those associated with the CEH/pyometra complex. A 10-year-old Yorkshire terrier cross bitch was presented for examination with a history of depression, anorexia and vomiting, dark faeces with an abnormal consistency, polyuria and polydipsia; the owner also described trembling and muscular weakness that rendered the dog incapable of climbing up stairs. According to the owner, its last heat had been one month previously. On admission, the bitch was conscious but lethargic, and the rectal temperature was within normal values. The conjunctival mucosa was slightly pale, but the capillary refill time was normal. The respiratory rate was normal; cardiac auscultation revealed a heart murmur. Signs of pain were evident on abdominal palpation. The results of a complete blood count indicated mild anaemia, but the leucogram was normal (Table 1). Serum biochemical analysis revealed that biochemical variables were within normal range limits, with the exception of albumin, which was very close to the lower end of the range (Table 1). No haematological signs of disseminated intravascular coagulation were present. Ultrasonographic examination revealed enlargement of the uterus, atrophy of the uterine wall and hypoechogenic contents, which led to a presumptive diagnosis of CEH/pyometra complex. The dog received supportive therapy, including lactated Ringer’s solution supplemented with glucose and potassium chloride, enrofloxacin, ranitidine and metaclopramide. Once the dog’s condition had been stabilised it underwent ovariohysterectomy. The dog was premedicated with 0·2 mg/kg diazepan (Bialzepan; Bial) and 0·4 mg/kg butorphanol (Turbogesic; Fort Dodge) intravenously, and then anaesthetised by intravenous administration of 2 mg/kg propofol (Diprivan; AstraZeneca). After intubation, the anaesthesia was maintained with 2 per cent isoflurane (Abbott Laboratories). Within 10 days after surgery, the bitch recovered from its clinical signs and showed normal PCV values and also normal serum albumin and calcium levels. The uterus presented a bilateral increase in the dimensions of the horns (11·5 cm in length and 2 cm in diameter) and its wall was very thin but had no ulcers or cysts (Fig 1), and had normal transverse folding. The fluid content of the uterus was watery and slightly yellow, and no particles in suspension were observed. The ovaries had a smooth surface; one ovary measured 1·5 x 1 x 0·5 cm, and had an apparently normal consistency, the other measured 1 x 0·8 x 0·5 cm and had a soft consistency, and a massive, white structure was present in one of the poles of the ovary. The ovaries and uterus were fixed in 10 per cent buffered formalin, embedded in paraffin wax, sectioned at 2 μm and stained with haematoxylin and eosin. Uterine sections were also stained with periodic acid-Schiff and alcian blue for staining mucopolysaccharides. For immunohistochemical characterisation, a mouse monoclonal antibody that binds to human oestrogen receptors (Clone CC4-5; Novocastra Laboratories) was used. The tissues were sectioned, placed on a slide, deparaffinised and hydrated. Antigen retrieval was performed in a pressure cooker at above 100°C for two minutes in a citrate buffer at pH 6, and then hydrogen peroxide inactivation and immunohistochemistry were performed. The primary antibody was applied at a dilution of 1:40, overnight at 4°C. The expression of oestrogen receptors in the uterine tissue was Veterinary Record (2006) 158, 487-489