A simple cage-autonomous method for the maintenance of the barrier status of germ-free mice during experimentation.

The use of germ-free (GF) isolators for microbiome-related research is exponentially increasing, yet limited by its cost, isolator size and potential for trans-contamination. As such, current isolator technology is highly limiting to researchers engaged in short period experiments involving multiple mouse strains and employing a variety of mono-inoculated microorganisms. In this study, we evaluate the use of positive pressure Isocages as a solution for short period studies (days to 2-3 weeks) of experimentation with GF mice at multiple simultaneous conditions. We demonstrate that this new Isocage technology is cost-effective and room-sparing, and enables maintenance of multiple simultaneous groups of GF mice. Using this technology, transferring GF mice from isolators to Isocage racks for experimentation, where they are kept under fully germ-free conditions, enables parallel inoculation with different bacterial strains and simultaneous experimentation with multiple research conditions. Altogether, the new GF Isocage technology enables the expansion of GF capabilities in a safe and cost-effective manner that can facilitate the growth, elaboration and flexibility of microbiome research.

Biopsy of the mouse prostate.

Many transgenic and knockout mouse models of prostate cancer have become available over the past decade. In this paper we describe a simple biopsy technique of the murine prostate. This technique allows sequential follow-up of the prostate in an individual mouse. Its use could also reduce the number of mice used in studies of the prostate gland.

Vascular-targeted photodynamic therapy (VTP) of a canine-transmissible venereal tumour in a murine model with Pd-bacteriopheophorbide (WST09).

Abstract Treatment of canine-transmissible venereal tumour (CTVT) with local vascular-targeted photodynamic therapy (VTP) using Pd-bacteriopheophorbide (WST09) as a drug is suggested as an alternative to conventional chemotherapy. Male CD1 nude mice were subcutaneously grafted with the xenograft-transmissible canine venereal tumour (XTVT). The VTP protocol delivered once consisted of intravenous administration of WST09 (10 mg kg(-1)) followed by immediate local illumination with a diode laser (763 nm). Controls included animals treated with light or WST09 alone. Macroscopic and microscopic evaluations of tumour response were conducted 10, 24 and 48 h after treatment. Upon VTP, tumours underwent necrosis that lasted 8-10 days and exhibited complete healing by 25-35 days, reaching an overall long-term cure rate (83%) by 90 days after treatment. This study suggests that VTP with WST09 can efficiently treat CTVT in a single session, as compared with 4-6 sessions of chemotherapy and thus may be feasible for common veterinary practice, particularly under ambulatory conditions.

ErbB2 is a tumor associated antigen and a suitable therapeutic target in Wilms tumor.

PURPOSE: The modern multimodality therapeutic approach to Wilms tumor (WT), combining surgery with radiotherapy and chemotherapy results in high cure rates even for high stage disease. However, the combination of radiotherapy and chemotherapy is associated with severe early and late complications such as neutropenic sepsis, growth retardation and secondary malignancies. Therefore, novel therapeutic strategies, which would decrease the treatment burden, are required. We studied the expression of erbB2 growth factor receptor in WT cells as well as its role as a tumor therapeutic target in an in vivo xenograft model of Wilms tumor. MATERIALS AND METHODS: Paraffin embedded pathological samples from 14 different WT cases as well as xenografts derived thereof were immunostained with anti-erbB2 monoclonal antibody. The immunostaining was graded in comparison to a known positive control (breast cancer) and was scored by the intensity of staining (0 to +3) multiplied by the percentage of cells expressing the antigen. The expression of erbB2 in the human WT xenograft was verified also by fluorescence activated cell sorter analysis. In addition, nude mice bearing established subcutaneous human WT xenografts were treated with either 3 intraperitoneal injections of N29 anti-erbB2 monoclonal antibody or irrelevant antibody. RESULTS: All of the authentic human pathological samples, except 1 anaplastic WT as well the WT xenografts (at different stages), expressed erbB2. Expression was also observed in WT metastasis and in tumors which out grew chemotherapy. Systemic administration of anti-erbB2 monoclonal antibody inhibited and even prevented the growth of WT xenograft in vivo. CONCLUSIONS: ErbB2 is a tumor associated antigen in WT. Being expressed in almost all tumor stages, our in vivo model suggests that erbB2 may serve as a WT therapeutic target. Further work is needed to establish the role of erbB2 in the disease and its potential use in decreasing current treatment burden.

Overexpression of a set of genes, including WISP-1, common to pulmonary metastases of both mouse D122 Lewis lung carcinoma and B16-F10.9 melanoma cell lines.

Despite advances in the management of solid tumours, the development of metastases continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastases, we have applied oligonucleotide microarrays to established murine models of highly metastatic D122 Lewis lung carcinoma and B16-F10.9 melanoma cell lines. These models are characterised by primary subcutaneous growth in C57BL/6J mice, a period of minimal residual disease and spontaneous pulmonary metastases. Microarray analysis defined seven genes, namely - arginase, brain natriuretic peptide (BNP), interleukin-1 alpha (IL-1 alpha), plasminogen activator inhibitor-2 (PAI-2), surfactant protein C (SP-C), uteroglobin (UG) and wnt-1-induced secreted protein-1 (WISP-1), which were consistently elevated in pulmonary metastases compared to the primary tumour of both D122 and B16-F10.9 models. Previous studies demonstrated that two of these seven genes, IL-1 alpha and PAI-2, are involved in the metastatic process. The results obtained by the microarrays were confirmed by real-time quantitative PCR, for three chosen genes - PAI-2, WISP-1 and UG. Our approach aimed to identify genes essential for the metastatic process in general and for pulmonary metastases specifically. Further research should address the precise role of these genes in the metastasising process to the lungs and test if they could be used as targets for future therapies.

Experimental Corynebacterium pseudotuberculosis mastitis in dairy cows.

The inoculation of 2000 colony-forming units of Corynebacterium pseudotuberculosis into one teat canal of each of three cows resulted in severe, chronic, pyogranulomatous mastitis. Within three days the cows had a reduced haematocrit, haemoglobin concentration and red cell count. The anaemia was initially normocytic, normochromic and non-regenerative, and was associated with a brief peak of neutrophilia; a regenerative response became evident two to three weeks later. Clinical signs of mastitis appeared seven to 14 days after the inoculation, with a peak of high fever, more severe anaemia, a second peak of neutrophilia and the complete cessation of milk production from all quarters; extensive and severe pyogranulomatous mastitis developed in the inoculated quarters. No other lesions were detected postmortem, and C pseudotuberculosis was cultured from the affected quarters but not from the supramammary lymph nodes and viscera.

Lack of MHC expression and retention of ultrastructural characteristics by xenograft transmissible venereal tumor cells in SCID mice.

Canine transmissible venereal tumor (CTVT) is primarily a tumor of adult dogs with a high incidence of spontaneous regression. We recently reported a xenograft model of CTVT (XTVT) in NOD/SCID mice. XTVT cells retain cytological and histological features of CTVT as well as characteristic rearranged LINE/c-MYC junction [Am. J. Vet. Res. 62 (2001) 907]. In this paper, we demonstrate that XTVT cells maintain ultrastructural characteristics of CTVT and do not express MHC classes I and II molecules.

Use of a murine xenograft model for canine transmissible venereal tumor.

OBJECTIVE: To develop a murine model for canine transmissible venereal tumor (CTVT). ANIMALS: Thirty-three 6-week-old NOD/LtSz-scid (NOD/SCID) mice and seven 6-week-old C57BL/6J mice. PROCEDURE: Samples of CTVT were excised from a 3-year-old dog and inoculated SC into ten 6-week-old NOD/SCID mice to induce growth of xenograft transmissible venereal tumor (XTVT). To establish mouse-to-mouse transmission, samples of XTVT were removed and inoculated SC into 4 groups of 6-week-old NOD/SCID mice and into a control group. Samples of CTVT were also inoculated into immunocompetent C57BL/6J mice for a mouse antibody production (MAP) test. The canine and xenografted tumors were evaluated cytologically and histologically, and polymerase chain reaction was performed for detection of the rearranged LINE/c-MYC junction. RESULTS: 8 of 10 NOD/SCID mice that were inoculated with CTVT developed tumors 3 to 10 weeks after inoculation. In the second-generation xenograft, all mice developed tumors by postinoculation day 47; 1 X 10(6) of XTVT cells were enough to create a xenograft. Metastases developed in 4 of 20 mice. Xenografted and metastatic tumors retained cytologic, histologic, and molecular characteristics of CTVT. Results of the MAP test were negative for all pathogens. CONCLUSION: We established an NOD/SCID murine model for XTVT and metastasis of CTVT. This model should facilitate study of tumor transplantation, progression, and metastasis and should decrease or eliminate the need for maintaining allogenic transfer in dogs.

WISH-PC2: a unique xenograft model of human prostatic small cell carcinoma.

Prostatic small cell carcinoma is an aggressive subtype of prostate cancer that usually appears as a progression of the original adenocarcinoma. We describe here the WISH-PC2, a novel neuroendocrine xenograft of small cell carcinoma of the prostate. This xenograft was established from a poorly differentiated prostate adenocarcinoma and is serially transplanted in immune-compromised mice where it grows within the prostate, liver, and bone, inducing osteolytic lesions with foci of osteoblastic activity. It secretes to the mouse Chromogranin A and expresses prostate plasma carcinoma tumor antigen-1, six-transmembrane epithelial antigen of the prostate, and members of the Erb-B receptor family. It does not express prostate-specific antigen, prostate stem cell antigen, prostate-specific membrane antigen, and androgen receptor, and it grows independently of androgen. Altogether, WISH-PC2 provides an unlimited source in which to study the involvement of neuroendocrine cells in the progression of prostatic adenocarcinoma and can serve as a novel model for the testing of new therapeutic strategies for prostatic small cell carcinoma.

Systemic lupus erythematosus in mice, spontaneous and induced, is associated with autoimmunity to the C-terminal domain of p53 that recognizes damaged DNA.

The tumor suppressor molecule p53 features a regulatory domain at the C terminus that recognizes damaged DNA. Since damaged DNA might be involved in activating anti-DNA autoantibodies, we tested whether autoimmunity to the C terminus of p53 might mark murine systemic lupus erythematosus (SLE). We now report that MRL / MpJ-Fas(lpr) mice, which spontaneously develop SLE, produce antibodies both to the C terminus of p53 and to a monoclonal antibody (PAb-421) that binds the p53 C terminus. Anti-idiotypic antibodies to PAb-421 (sampled as monoclonal antibodies) could also bind DNA. Thus, the PAb-421 antibody mimics DNA, and the anti-idiotypic antibody to PAb-421 mimics the p53 DNA-binding site. This mimicry was functional; immunization of BALB / c mice to PAb-421 induced anti-DNA antibodies and antibodies to the C terminus of p53, and most of the mice developed an SLE-like disease. Immunization of C57BL / 6 mice to PAb-421 induced antibodies to p53, but not to its C-terminal domain. The C57BL / 6 mice also did not develop anti-DNA antibodies or the SLE-like disease. Thus, network autoimmunity to the domain of p53 that recognizes damaged DNA can be a pathogenic feature in SLE in genetically susceptible strains of mice.

Dirofilaria repens infection in a dog in Israel.

Only one case of canine infection with Dirofilaria repens has been reported in Israel to date; this occurred in 1934. This publication, 65 years later, represents the second case of D. repens infection in a dog in Israel. This dog was infected locally since it was born in Israel and was never taken abroad, suggesting that the life cycle of the filaria was completed in Israel. Since dogs, cats, and foxes serve as a reservoir for the filaria and these are abundant in Israel, and mosquitoes of the genera Culex and Aedes (both of which occur in Israel and the Middle East) are vectors, the conditions for establishment of the filaria in Israel exist, and warrant regional epidemiologic investigation.

Epithelial cells of different organs exhibit distinct patterns of p53-dependent and p53-independent apoptosis following DNA insult.

The present study shows that DNA damage induces different patterns of p53-dependent and p53-independent apoptosis in epithelial cells of various organs of adult mice. Genotoxic stress induced a biphasic apoptotic response in the small intestine and tongue. While the first immediate apoptotic wave was p53-dependent, the second was slower in rate and was p53-independent. Under the same experimental conditions a single rapid, but a more extended, p53-independent response was evident in the skin of the tail. Indeed, exposure of p53+/+ mice to 400 R induced in epithelium of the small intestine and tongue an immediate rapid response that was followed by a second delayed p53-independent apoptotic wave. p53-/- mice exhibited in these organs the second wave only. However, epithelium of the tail derived from the same mice showed a single rapid apoptotic response that lasted much longer than the p53-dependent response and was similar in the p53-/- and the p53+/+ mice. Variations in apoptotic patterns observed in epithelial cells derived of the different tissues may point to differences in the physiological pathways expressed.

Immunoreactivity of canine mammary neoplasms with monoclonal antibody CC49.

Monoclonal antibody (MAb) CC49 binds to human tumour-associated glycoprotein termed TAG-72. CC49 is a second-generation MAb with higher affinity to TAG-72 than the original MAb B72.3. CC49 was applied to 42 samples from different canine mammary tumours, belonging to seven different histopathological types. Immunoreactivity was detected by the use of an avidin-biotin complex immunoperoxidase method. Most sections from all types of mammary neoplasm reacted with this MAb. Normal tissue did not stain or stained only weakly. The results of this study suggest CC49 has selective immunoreactivity for a variety of canine mammary tumours, which seems superior to that reported with MAb 72.3. These results support the proposal for further study of diagnostic and therapeutic uses of CC49 in the management of canine mammary tumours.

Acute maduramicin toxicity in calves.

A herd of 277 beef-breed calves in three age groups was mistakenly given the poultry coccidiostat maduramicin in a total mixed ration. It caused an acute toxicosis in which sudden death was the sole clinical finding in most cases. One group of 212 calves aged five to eight months suffered a mortality of 51 per cent in eight days and a total mortality of 56 per cent during the 40 days in which mortality was recorded. Mortality of only 3 per cent was recorded in two other groups of calves aged nine to 16 months in eight days and a total mortality of 11 per cent over the 40-day period.

Meningoencephalitis and brain abscessation due to Escherichia coli in a 2 week old alpaca cria.

An alpaca cria which received insufficient colostrum, presented with lethargy, anorexia, no passage of faeces and neurological signs. Differential diagnosis included neonatal septicaemia, meningitis and gastrointestinal obstruction. The cria died despite treatment. Necropsy revealed brain abscesses with Escherichia Coli in pure culture. The importance of the amount and timing of colostrum intake is discussed.

Spirocerca lupi infection in the dog: aberrant migration.

The nematode Spirocerca lupi (S. lupi), a parasite of dogs and other carnivores, affects mainly the esophagus and the aorta leading to gastrointestinal, respiratory, and circulatory signs. Aberrant migration of the worm to unusual anatomical structures, especially the thoracic cavity, resulting in atypical clinical signs is being reported more frequently. Aberrant migration of S. lupi is reviewed, and two such cases (i.e., migration to the heart, causing an aortico-pulmonary "window-like" opening, and to a subcutaneous abscess in the caudal thoracic region) are presented.

Hepatozoon canis infection in two dogs.

Infection of 60 to 90% of neutrophils with the protozoa, Hepatozoon canis, was detected in 2 dogs. Clinical signs included lethargy, anorexia, and weight loss. Both dogs had severe anemia, leukocytosis, and thrombocytopenia as well as hypoalbuminemia, hyperglobulinemia, and high activities of serum alkaline phosphatase and creatine kinase. Both dogs were treated with imidocarb dipropionate and doxycycline. One dog recovered clinically, with disappearance of parasites from WBC. The other dog died, despite treatment. Necropsy revealed widespread dispersion of schizonts in the parenchymal tissues, but no involvement of skeletal muscle tissues. The disease syndrome that has been identified in the Texas Gulf region is characterized by gait abnormalities associated with multifocal pyogranulomatous myositis, thus, it is distinct clinicopathologically from the syndrome observed in these 2 dogs.

Correlation of Ag-NOR protein measurements with prognosis in canine transmissible venereal tumour.

Ten canine transmissible venereal tumour (TVT) cases were studied by digital image analysis quantification on sections stained with silver to demonstrate nucleolar organizer regions (Ag-NORs). In each animal, 100 neoplastic cells were randomly selected for evaluation. The following parameters were measured or calculated: area of nucleus, area of Ag-NOR dot, the mean number of Ag-NOR dots per nucleus, the mean area of Ag-NOR dots per nucleus and the ratio of mean nuclear dot area to nuclear area. All 10 cases were treated with vincristine at a dose of 0.6 mg/m2 intravenously once a week. Two, which showed malignant characteristics (i.e. uncontrolled growth, local invasion or metastasis), did not respond to multiple (12) treatments and had a fatal outcome. Of the remaining eight cases, seven responded to two to six treatments and one required 12 treatments. The average number of Ag-NORs per nucleus and the area of Ag-NORs per nucleus were lower in the seven cases that responded to two to six treatments than in the other three cases, but the difference was not significant. However, there was a significant difference in the ratios of Ag-NOR area to nuclear area between the two groups of cases. Thus, poor prognosis was (1) possibly correlated with an increase in the mean number of Ag-NORs per nucleus and an increase in the mean area of Ag-NORs per nucleus, and (2) definitely correlated with an increase in the mean ratio of Ag-NOR area to nuclear area.

Intracranial gangliocytoma in a dog.

The clinical, pathologic, immunohistochemical, and electron microscopic characteristics of a pure intracranial gangliocytoma in a 6-year-old spayed female dog are presented. The tumor is very rare in humans, and has not been previously reported in the dog. The most remarkable feature of this tumor was that it was composed of a single neuronal cell type without the presence of glial elements, as demonstrated by the negative immunohistochemical reaction to glial fibrillary acidic protein.

Trypanosoma congolense infection in two dogs.

Trypanosomiasis, caused by Trypanosoma congolense, was diagnosed for the first time in Israel in two boxer dogs imported from Kenya. The dogs developed clinical signs two days after arrival and succumbed to the disease within four days. The major clinical and clinicopathological findings included anaemia, haemorrhages, lymphadenomegaly, hepatosplenomegaly and neurological signs. Histopathology showed lymphocytic-plasmacytic infiltration in the skin, brain, meninges, kidney and liver.