ESLAV/ECLAM/LAVA/EVERI recommendations for the roles, responsibilities and training of the laboratory animal veterinarian and the designated veterinarian under Directive 2010/63/EU.

Directive 2010/63/EU was adopted in September 2010 by the European Parliament and Council, and became effective in January 2013. It replaces Directive 86/609/EEC and introduces new requirements for the protection of animals used for scientific purposes. In particular, it requires that establishments that breed, supply or use laboratory animals have a designated veterinarian (DV) with expertise in laboratory animal medicine, or a suitably qualified expert where more appropriate, charged with advisory duties in relation to the well-being and treatment of the animals. This paper is a report of an ESLAV/ECLAM/LAVA/EVERI working group that provides professional guidance on the role and postgraduate training of laboratory animal veterinarians (LAVs), who may be working as DVs under Directive 2010/63/EU. It is also aimed at advising employers, regulators and other persons working under the Directive on the role of the DV. The role and responsibilities of the DV include the development, implementation and continuing review of an adequate programme for veterinary care at establishments breeding and/or using animals for scientific purposes. The programme should be tailored to the needs of the establishment and based on the Directive's requirements, other legislations, and current guidelines in laboratory animal medicine. Postgraduate laboratory animal veterinary training should include a basic task-specific training module for DVs to complement veterinary competences from graduation, and continuing professional development on the basis of a gap analysis. A tiered approach to further training in laboratory animal veterinary medicine and science offers career development pathways that are mutually beneficial to LAVs and establishments.

Chlamydiae in free-ranging and captive frogs in Switzerland.

A total of 210 frog samples originating either from a mass mortality (1991/1992) or from routine postmortem investigations of the years 1990 to 2004 were examined retrospectively for a possible involvement of Chlamydiae. For a prevalence study of Chlamydia in a selected Swiss amphibian population, 403 samples from free-ranging Rana temporaria were examined. Histopathology, immunohistochemistry using a monoclonal antibody against chlamydial lipopolysaccharide, and a 16S rRNA polymerase chain reaction (PCR) followed by DNA sequencing were performed on the formalin-fixed and paraffin-embedded tissues. Using PCR, 8 of 54 (14.8%) frog samples from the mass mortality (1991/1992) were positive for Chlamydia suis S45. A control group of healthy Xenopus laevis had 3 of 38 positive samples, sequenced as C suis S45 (2/3) and an endosymbiont of Acanthamoeba species UWE1 (1/3). Chlamydophila pneumoniae TW-183 was detected from exotic frogs kept in a zoo. Of the frogs collected for the prevalence study, 6 of 238 (2.5%) tested positive, 1 each for C suis S45, Cp pneumoniae TW-183, and uncultured Chlamydiales CRG22, and the remaining 3 revealed Chlamydophila abortus S26/3. In immunohistochemistry, there were 2 positive labeling reactions, 1 in intestine and the other in the epithelium coating the body cavity, both testing positive for Cp pneumoniae TW-183 in PCR. Histologically there were no lesions recorded being characteristic for Chlamydia. Although there is a prevalence of Chlamydia in Swiss frogs, no connection to a mass mortality (1991/1992) could be established. For the first time, C suis S45 and Cp abortus S26/3 were detected in frog material.

Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks.

Chlamydiae infect male genital organs of ruminants. However, little is known about their prevalence. Hence, we investigated fresh and cryopreserved semen (bulls: n=304; rams: n=78; bucks: n=44) by polymerase chain reaction (PCR), as well as genital organs (bulls: n=13; rams: n=10; bucks: n=6) by immunohistochemistry (IHC) and PCR. Sera from bulls (n=104) and small ruminants (n=61) were tested by LPS and rMOMP (recombinant major outer membrane protein) ELISA and competitive ELISA (cELISA), respectively. Three PCR assays were compared in this study for detection of chlamydial DNA in semen: 16S rRNA, IGS-S (intergenic spacer 16S/23S-short), and IGS-L (intergenic spacer 16S/23S-long) PCRs. PCR sensitivity and inhibitory effects were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. In bull semen, detection limits of the 16S, IGS-S and IGS-L PCRs were 10, 10, 100 templates, respectively. However, PCR sensitivity was reduced in ram and buck semen suggesting the presence of potential PCR inhibitors. Of 304 bull semen samples, the 16S PCR revealed DNA of chlamydiae in 20 samples (6.6%), including Cp. abortus (n=2), Cp. psittaci (n=1), Chlamydia suis (n=2), and Chlamydia-like organisms (n=15). In rams, one semen sample was positive for Chlamydia-like organism. All investigated male genital organs were negative for Chlamydia. Serology revealed 47.1% (49/104) positive bulls by LPS ELISA. Of these, 30 samples were positive by rMOMP ELISA, predominantly for Cp. pecorum. In small ruminants, cELISA displayed 34.8% (16/46) and 60% (9/15) positivity for Cp. abortus in rams and bucks, respectively. There was no correlation between serology and PCR of semen. The presence of chlamydiae in semen suggests the possibility of venereal transmission, although risk may be low in Switzerland.

Chlamydia-related abortions in cattle from Graubunden, Switzerland.

In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.

Detection of chlamydiae in boar semen and genital tracts.

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.

[A specific staining for the middle piece of sperm and its possible application]. / Eine spezifische Mittelstück-Färbung von Spermien und ihre mögliche Anwendung.

A special staining technique for the middle piece of sperms is described in different species (cow, horse, sheep, goat, pig). Staining was performed with frozen or fresh semen. The mitochondria in the middle piece could not always be stained with the same intensity. Using 600-fold magnification, subunits of the middle-piece could be observed, but only on fresh collected and immediately stained sperms. When kept in the refrigerator for more than 3 days, the observed subunits disappeared. A positive correlation between staining intensity and enzyme activity could be indicative for sperm motility and also fertilizing capacity.

Influence of sex and age on serum total immunoglobulin E concentration in Beagles.

OBJECTIVE: To establish an ELISA for detection of serum total IgE concentration in dogs and to analyze IgE values in a dog colony. ANIMALS: 147 healthy Beagles (31 males and 116 females). PROCEDURE: 2 canine IgE-specific polyclonal antibodies elicited by 2 recombinant fragments of the epsilon chain in hens were used to develop a capture ELISA specific for serum total IgE concentration. The IgE values were calculated by comparing serum dose-response curves (1:50 to 1:6,400) with a reference serum pool assigned 100 relative ELISA units (REU). Results-Mean IgE concentration in female Beagles was 51.2 REU (range, 0 to 337.8 REU; median, 31.4 REU), whereas mean IgE concentration in male dogs was only 7.5 REU (range, 0 to 32.6 REU; mean, 3.6 REU). Distribution of IgE values was skewed; approximately 80% of dogs had IgE values < 50 REU. Analysis of natural logarithmically transformed IgE values indicated that sex and age significantly (P < 0.05) influenced IgE values; mean serum IgE values increased until the age of 4 years. Heritability estimates of IgE concentration indicated a trend toward a genetic influence. CONCLUSION: A reliable capture ELISA specific for canine IgE was developed. Serum total IgE values vary with age and sex in the sample population. CLINICAL RELEVANCE: Serum total IgE concentration can now be evaluated in various dog breeds and, subsequently, in dogs with IgE-mediated diseases provided that these significant influences are accounted for. Serum total IgE values may then prove to be of diagnostic value, similar to their use in human beings.

Total serum IgE and IgA antibody levels in healthy dogs of different breeds and exposed to different environments.

Total serum immunoglobulin (Ig) E and A levels were analysed in 233 healthy dogs as basis for comparison with atopic dogs in future studies. They were measured by ELISA in a group of non- colonised dogs of various breeds (group A) and three groups of colonised dogs including one German Shepherd and two Beagle kennels (groups B-D). IgE levels from non-colonised dogs were significantly higher than the ones of German Shepherds and Beagles C (P<0.05). IgA levels were alike in all groups except for the German Shepherds which displayed the lowest levels. Age and sex were not identified as common significant cofactors for IgE and IgA levels in all groups and IgE levels correlated negatively with IgA only in non-colonised dogs. In conclusion, IgE and IgA levels seem to be mainly influenced by genetic background. Thus use of total serum IgE as a diagnostic tool in the atopic dogs required extensive family data and therefore appears most suitable for research purposes within specific, well defined dog populations.

Polymerase chain reaction (PCR) detection of porcine Chlamydia trachomatis and ruminant Chlamydia psittaci serovar 1 DNA in formalin-fixed intestinal specimens from swine.

In previous studies chlamydiae were detected immunohistologically in the gut of 66 out of 311 pigs. The aim of the present investigation was the classification of these intestinal porcine chlamydiae. For the study, DNA extracted from 52 paraffin-embedded intestinal tissues was amplified in nested polymerase chain reactions (PCRs) with Chlamydia omp1 genus- and species-specific primers. Some of the amplification products were cloned and sequenced. In 45 cases DNA could be amplified with genus-specific primers. Species-specific PCR and sequencing showed that in 42 cases the chlamydial omp1 genotype was Chlamydia trachomatis. Sequenced DNA fragments were 95-99% identical with the porcine strain S45. In three further cases sequencing analysis provided DNA sequences which were 100% identical with Chlamydia psittaci B577 (serovar 1) omp1 genotype. So far as the authors are aware this is the first report on the occurrence of C. psittaci serovar 1 in pigs.

Mixed infections with porcine Chlamydia trachomatis/pecorum and infections with ruminant Chlamydia psittaci serovar 1 associated with abortions in swine.

In a previous immunohistological study, chlamydiae were detected in 5 out of 139 cases of swine abortion, and a possible implication of C. psittaci serovar 1 was suggested. The present study sought to classify the chlamydiae found in the fetal organs of these abortions. DNA extracted from 15 paraffin-embedded tissue specimens (10 livers and 5 lungs, obtained from 10 fetuses from 9 cases of abortion) was amplified in a nested PCR with Chlamydia omp1 genus-specific primers. Chlamydia DNA was amplified in 9 liver and 2 lung specimens. Eight of the amplification products were cloned, and 5 clones of each amplification were sequenced. Sequence analysis demonstrated in 7 specimens the simultaneous presence of porcine C. trachomatis S45 and C. pecorum 1710S omp1 genotypes. All DNA fragments of 1 amplification were identical to the ruminant C. psittaci B577 omp1 genotype (serovar 1). The results suggest that mixed infections with porcine C. trachomatis and C. pecorum dominate chlamydial infections associated with abortion in swine, but ruminant abortigenic C. psittaci are also found.

[Short anesthesia in rats: an injection or an inhalation anesthesia]. / Kurznarkose bei Ratten: Eine Injektions- bzw. Inhalationsnarkose.

First we describe the application of an injection anaesthesia in a specific strain of rats and test its usefulness under practical conditions. Application (i.p.) of the described dose according to the animals body-weight resulted in an acceptable anaesthesia, which lasted approximately 50 minutes in male rats and 80 minutes in female rats. Next we compare the common ether anaesthesia with an other inhalation anaesthesia (Methoxyflurane) which, by using a self-made mask, could be prolonged without any problem for at least 15 minutes ("prolonging-time"). A correlation was found between the duration of the prolonging-time and the awakening time. Inhalation anaesthesia using Methoxyflurane obviously affected the animals health and well-being less than the conventional ether anaesthesia.

[Estradiol and progesterone concentrations in the plasma of nonpregnant bitches during the sexual cycle]. / Ostradiol- und Progesteronkonzentrationen im Plasma von nicht trächtigen Hündinnen während des Sexualzyklus.

Measurement of the plasma concentrations of oestradiol and progesterone in six bitches on 180 consecutive days showed large fluctuations in the levels of both hormones. The concentrations of oestradiol began to increase 10-12 days before the peak levels of 38.1-89.7 pg/ml were reached, then declined again over the next 3-7 days to or below the minimum measurable value of 9 pg/ml. On the day after the maximum concentrations of oestradiol were recorded, plasma progesterone began to rise rapidly, reaching a plateau after approximately two weeks, then declining gradually after a further two weeks. At the height of the luteal phase, peak levels of 12.6-70.1 ng/ml were measured, although on some days values of less than 1 ng/ml were recorded. The time of occurrence of the initial rise in the progesterone concentration during oestrus presumably indicates that preovulatory luteinization had taken place. During anoestrus the basal concentration of progesterone was generally less than 1 ng/ml and that of oestradiol less than 9 pg/ml. The normal values derived from these observations are discussed with regard to the interpretation of oestradiol and progesterone concentrations in domestic pets under treatment in veterinary practice.

[Estrus induction in bitches by the administration of PMSG and HCG]. / Brunstinduktion bei Hündinnen durch Verabreichung von PMSG und HCG.

Oestrus was induced in 14 anoestrous beagle bitches by intramuscular injection of PMSG in a dose of 20 I.U./kg once daily on five consecutive days, followed by an additional single i.m. injection of 500 I.U. of hCG on the fifth day. The day on which the first injection was given was counted as Day 1 of the experiment. Between the fourth and sixth day, the bitches began to attract the attention of male dogs and between Days 9 and 15 all bitches came in heat. Matings occurred on two to ten occasions, and six of the bitches conceived. The maximum concentrations of oestradiol in the plasma were in most cases reached on Days 10 or 12 and ranged from 42 to 195 pg/ml. In all cases progesterone concentrations rose sharply between Days 10 and 20. The incretion phase of the corpora lutea was noticeably brief in the non-gravid bitches; in five of these eight bitches, anoestrous values below 2 ng/ml were already obtained before the 65th day of the experiment. These results indicate that the administration of PMSG on five consecutive days supplemented by a final single injection of hCG stimulates the ovaries adequately to afford good prospects of conception. The concentrations of ovarian hormones are discussed.

Specific diagnosis of parvovirus enteritis in dogs and cats by in situ hybridization.

In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesions characteristic of parvovirus infection were assayed for the presence of parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissue in all 10 dogs and in nine of the 10 cats, whereas antigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistochemistry. We conclude that an aetiological diagnosis can be established with a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissue sections.

Retrospective study of myocardial canine parvovirus infection by in situ hybridization.

The diagnosis of myocardial canine parvovirus (CPV) infection used to depend on the presence of pathognomonic intranuclear inclusion bodies. The in situ hybridization technique, however, allowed to detect CPV specific nucleic acid in myocardial tissue where no inclusion bodies were found. Hence, we applied this technique to check formalin-fixed paraffin-embedded myocardial tissue from puppies with heart lesions for the presence of CPV. The tissues had been collected between 1977 and 1989. A biotinylated probe was used for in situ hybridization. This way CPV specific nucleic acid was detected in 3 dogs where CPV myocarditis had not been diagnosed on routinely stained slides because of the lack of intranuclear inclusion bodies. However, in spite of the application of the in situ hybridization technique no further myocardial CPV infection was detected in puppies with heart lesions from after 1979, confirming that the number of puppies with myocardial CPV infection declined after that year.

Effect of duration of PMSG treatment on induction of oestrus, pregnancy rates and the complications of hyper-oestrogenism in dogs.

In anoestrous bitches, the use of 5 or 10 days of daily PMSG treatments (20 i.u./kg/day) before a single injection of hCG (500 i.u./dog) did not affect the timing of induced pro-oestrus (4-6 days) but did result in a greater incidence of conception (3 of 6 vs 6 of 17), a high rate of successful pregnancy (50% vs 0%), lower preovulatory oestrogen concentrations, and reduced incidence of complications due to hyper-oestrogenism such as thrombocytopenia (0% vs 29%). These results suggest an advantage of 5 over 10 days of priming with PMSG, at the doses used before hCG injection, for induction of fertile oestrus in bitches.

Demonstration of rotavirus antigen in trypsin-digested paraffin tissue sections by immunofluorescence.

Trypsin-digested paraffin tissue sections were used to demonstrate rotavirus particles in small intestinal epithelial cells of dirrheic neonatal calves, using direct fluorescent-antibody assay. The results were compared with other techniques to demonstrate rotavirus particles in tissue sections by immunofluorescence. Enzyme treatment of deparaffinized tissue sections gave excellent results with distinct specific fluorescence and minimal background. Prior staining of the tissue section with Mayer's hematoxylin made simultaneous observation of the same tissue section by light and ultraviolet microscopies possible.