121-130
A novel method, based on lithium sulfate precipitation for purification of chicken egg yolk immunoglobulin Y, applied to immunospecific antibodies against Sendai virus
by G. Bizhanov1, I. Jonauskiene & J. Hau

Egg-laying hens were immunized with Sendai virus (SV) that had been grown in chicken embryos. The titres of immunospecific SV antibodies varied from log212 to log216 during the 5-month immunization period and total immunoglobulin Y (IgY) concentrations varied from 1.6 to 4.0 mg per ml of egg yolk. Two IgY purification methods based on salt precipitation using lithium sulfate or sodium citrate were developed. These methods were compared with two other purification methods based on polyethylene glycol (PEG) precipitation and chloroform extraction, respectively in terms of yield, total protein content, IgY concentration and immunospecific anti Sendai IgY activity. The total protein and IgY contents when purified by chloroform were 1.4-2.8 times and 1.3-2.3 times higher, respectively than in corresponding preparations purified by the other methods. However, the proportion of nonsense proteins was approximately 10% higher in the IgY preparation purified by chloroform than in those purified by salt precipitation. The immunospecific IgY activity recorded in the preparations from the new salting out methods was lower compared with the PEG and chloroform purification methods. However, the purity analysis of IgY by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that IgY purified with lithium sulfate contained only two major components with molecular weights of 40 kDa and 66 kDa and one minor protein component of 45 kDa. By contrast, IgY extracted with ammonium sulfate, which is a classical method used to obtain purified IgY, contained two major protein of 40 kDa and 66 kDa and at least three less intense protein bands corresponding to proteins of molecular weights 31.4 kDa, 33.5 kDa and 45 kDa. The results indicate that the purification of IgY by lithium sulfate results in very pure IgY in high quantities (94% +/- 5% of total egg yolk protein).

131-141
Twenty six-week exposure to 2 amino-3 methylimidazo [4,5-ƒ]quinoline (IQ) does not significantly increase the incidence of tumours in HMGCR/mts1 tg579 transgenic mice
by Alicja Mortensen, Eugene Lukanidin, Noona S. Ambartsumian& Ilona K. Sørensen

HMGCR/mts1 tg579 transgenic mice were designed to direct the expression of metastasis-promoting mts1 (S100A4) gene to all the tissues. In order to test the usefulness of this mouse model for carcinogenicity tests shorter than that recommended by OECD guideline nr. 451, HMGCR/mts1 tg579 transgenic and C57BL/6ByA (wild type) mice (15 males and 15 females of each genotype per group) received either a control diet for 53 weeks or a control diet plus 0.03% 2-amino-3 methylimidazo[4,5-ƒ]quinoline (IQ) for 26 weeks and a control diet for the remaining 27 weeks. IQ is a food mutagen with a carcinogenic effect in non-human primates and rodents. IQ is a liver carcinogen and also causes lung tumours and tumours of the forestomach in mice. Body weight gain and feed intake were decreased (p<0.05) during IQ feeding in IQ-dosed mice of both genotypes and sexes. The daily dose of IQ, as calculated based on the feed intake, was 43 and 46 mg/kg bw in HMGCR/mts1 tg579 transgenic males and females, and 43 and 45 mg/kg bw in C57BL/6ByA males and females. The survival rates of HMGCR/mts1 tg579 transgenic mice were 100% for males and 93% for females in the control group, and 93% for both sexes in the IQtreated group. The survival rates of C57BL/6ByA mice of both sexes were 100% in the control group and 93% in the IQ-treated group. Non-neoplastic lesions were found in all groups, except for HMGCR/mts1 tg579 transgenic control males, primary in the liver and were of various type but of single incidence with not statistically significant difference between controls and IQ-treated groups of both genotypes and sexes. Pre-neoplastic lesions were observed preferentially in the liver in IQ-treated HMGCR/mts1 tg579 transgenic animals of both sexes. The total number of animals with tumours was: in HMGCR/mts1 tg579 transgenic mice: males - 0/15 and 3/15, females - 2/14 and 1/15 and in C57BL/6ByA: males - 0/15 and 1/15, females - 1/15 and 4/15, in control and IQ-treated animals, respectively. The primary tumours recorded in HMGCR/mts1 tg579 transgenic mice were: one pleomorphic malignant lymphoma and one histiosarcoma in the female control group, one liver hemangiosarcoma, one colon adenocarcinoma, and one malignant lymphoma/lympholytic in IQ-treated males, and one colon adenoma in IQ-treated females. The primary tumours recorded in C57BL/6ByA mice were: one histiocytic sarcoma in control females, one colon adenoma in IQ-treated males, one colon adenocarcinoma, one pleomorphic malignant lymphoma, one malignant lymphoma/lymphocytic, one thymic lymphoma, and one histiocytic sarcoma in IQ-treated females. In conclusion, IQ feeding did not statistically significantly increase the incidence of tumours in HMGCR/mts1 tg579 transgenic and C57BL/6ByA mice in this limited bioassay. The results in HMGCR/mts1 tg579 transgenic mice obtained under current experimental conditions suggest that 53 weeks may be not a sufficient time span to demonstrate a carcinogenic potential of a test compound in this mouse model.

143-148
Nephrocalcinosis in rabbits – a case study
by Merel Ritskes-Hoitinga, Ole Skott, Torben R. Uhrenholt, Inger Nissen, Inez Lemmens, Anton Beynen

Varying degrees of kidney calcification have been found in New Zealand White rabbits used for in vitro physiological studies in our animal unit over the past 4 years. Histologically, the lesion is characterised by the deposition of calcified deposits inside tubular structures in the cortical and (cortico)medullary region of the rabbit kidney. The kidney calcifications could be so severe, that the isolation of renal resistance vessels, dissected for intended in vitro studies, was no longer possible. Because 40% of the rabbit kidneys had to be discarded at the start of the year 2000, since then, a routine histological check of all rabbit kidneys used until 1 January 2004, was performed. The yearly incidence of numbers of animals having kidneys with calcified deposits in cortex and/or medulla as compared to the total number of rabbits used, ranged from 16 % to 63%. Kidney calcifications were seen in both sexes. A large interindividual variation in the degree of nephrocalcinosis was found. Of the dietary factors involved in the ethiopathogenesis in rats, phosphorus (P) concentration is an important determinant for kidney calcification. Also in rabbits it has been proven that dietary phosphate supplements and increased dietary P-levels in semipurified diets will lead to kidney calcifications in higher frequencies and degrees of severity. The recommended dietary Plevel for growing rabbits is 0.22%, according to the National Research Council guidelines of 1977. Commercial, natural-ingredient rabbit diets always have dietary P levels that exceed 0.22%. It is therefore considered recommendable to lower dietary P concentration in marketed rabbit diets, in order to reduce or prevent nephrocalcinosis in rabbits.

149-154
Reduction in the spread of rodent urinary allergens during cage changing by Laminar Air Flow cabins. Reduction in rodent allergens by LAF cabins
by Thomas C. Krohn* & Axel Kornerup Hansen

As spread of allergens, especially during handling and cage changes, is a problem in an animal facility, initiatives are taken to reduce this spread. The aim of the present study was to evaluate the effectiveness of a LAF-cabin (Laminar Air Flow cabin), and its ability to protect the staff against inhaling allergens during cage changing of rats, and mice. The allergens were sampled from the handler’s breathing zone and from different places inside the cabin. Subsequently the amount of allergens was quantified by an ELISA testing for MUS m1 and RAT n1. The use of the LAF-cabin reduced the amount of rat and mouse allergens in the breathing zone by at least 90-95%. The study shows that by using a LAF-cabin for cage changing it is possible both to achieve an essential reduction in allergen exposure for the staff and at the same time keep the allergens inside the ventilated area of the cabin, thereby preventing allergen contamination of the surrounding facilities. The potential advantages compared with other techniques are outlined.

177-183
The use of the API 20 NE bacteria classification procedure to identify Pasteurellaceae strains in rodents and rabbits
by R. Boot, M. Van den Brink, P. Handgraaf& R. Timmermans

Forty growth-factor independent Pasteurellaceae strains representing most known taxa from rodents and rabbits were API 20 NE profiled by four laboratory animal diagnostic laboratories using their routine methodology. Significant differences were found in the number of bacterial strains classified with the Pasteurellaceae. The results of the four laboratories taken together showed that 136 (85 %) of the 160 tests carried out with the 40 strains led to classification with the family. The 23 Pasteurellaceae strains from species (Pasteurella aerogenes, P. multocida, P. pneumotropica) present in the API 20 NE data base (API taxa strains) and the 17 strains from taxa not included (non - API taxa strains) were classified with the family with similar frequency. Pasteurella species designation differed significantly between the laboratories and full agreement in speciation was found with only 9 (22.5 %) of the 40 strains. Of the tests carried out with P. multocida and P. pneumotropica, 42 and 52 % respectively led to misclassification. Conversely 38 % of the profiles obtained with non- API taxa strains led to identification as P. multocida or P. pneumotropica. We conclude that identification of Pasteurellaceae to the species level by the API 20 NE system is unreliable, but the system is useful to classify various Pasteurellaceae taxa from rodents and rabbits with the bacterial family.

185-187
IAT & AALAS
by Bruce W Kennedy, MS RLATG

The Institute of Animal Technology's meeting was ultimately held in Athens, Greece at the end of April. Let me review. I am the 2002 recipient of the B&K Universal Group Limited International Travel Fellowship Award which sends an AALAS member to the comparable IAT meeting held in Europe. Typically the venue is in the UK but because of ARE terrorist-type activities it has been necessary to hold it elsewhere. I was supposed to go to Dublin, Ireland in the spring of 2003 but due to animal rights activity at the hotel it was re-scheduled for Cyprus in early June and because of a prior commitment I could not attend. Fortunately I was able to attend this year.