Acta Veterinaria Hungarica 51 (1) (2003)

CONTENTS AND ABSTRACTS

Anatomy

Histological studies on embryonic development of the rabbit heart. Emese Balogh and P. Sótonyi 1-13

Multiple cardiac anomaly in sheep: A case study and review of the literature. Emese Balogh and P. Sótonyi 15-27

Bacteriology

Spontaneous antibiotic resistance mutation associated pleiotropic changes in Escherichia coli O157:H7. I. Tóth, Márta Csík and L. Emődy 29-44

Study of the role of Chlamydia, Mycoplasma, Ureaplasma and other microaerophilic and aerobic bacteria in uterine infections of mares with reproductive disorders. L. Szeredi, M. Tenk, I. Schiller and T. Révész 45-52

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials. I. Biksi, Andrea Major, L. Fodor, O. Szenci and F. Vetési 53-59

Clinical veterinary medicine

Immunological anomalies and thrombocytopenia in 117 dogs and cats diagnosed with Chronic Fatigue Syndrome (cfs). W. Tarello 61-72

Canine granulocytic ehrlichiosis (cge) in Italy. W. Tarello 73-90

Molecular genetics

N-ras mutation in a canine lymphoma: Short communication. B. Mayr, M. Holzheu, G. Schaffner and M. Reifinger 91-94

Reproduction

In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hcg injection. J. Rátky, D. Rath and K.-P. Brüssow 95-101

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187. Ö. Uçar and T. J. Parkinson 103-109

Involvement of high-density lipoprotein in stimulatory effect of hormones supporting function of the bovine corpus luteum. D. Skarżyński, J. Młynarczuk and J. Kotwica 111-120

Acta Veterinaria Hungarica 51 (1), pp. 1–13 (2003)

HISTOLOGICAL STUDIES ON EMBRYONIC DEVELOPMENT OF THE RABBIT HEART

Acta Veterinaria Hungarica 51 (1), pp. 15–27 (2003)

MULTIPLE CARDIAC ANOMALY IN SHEEP: A CASE STUDY AND REVIEW OF THE LITERATURE

Acta Veterinaria Hungarica 51 (1), pp. 29–44 (2003)

SPONTANEOUS ANTIBIOTIC RESISTANCE MUTATION ASSOCIATED PLEIOTROPIC CHANGES IN ESCHERICHIA COLI O157:H7

I. Tóth^1*, Márta Csík² and L. Emődy³

Acta Veterinaria Hungarica 51 (1), pp. 45–52 (2003)

STUDY OF THE ROLE OF CHLAMYDIA, MYCOPLASMA, UREAPLASMA AND OTHER MICROAEROPHILIC AND AEROBIC BACTERIA IN UTERINE INFECTIONS OF MARES WITH REPRODUCTIVE DISORDERS

L. Szeredi^1*, M. Tenk¹, I. Schiller² and T. Révész¹

1Central Veterinary Institute, H-1149 Budapest, Tábornok u. 2, Hungary; ²Institute for Veterinary Pathology, Veterinary Medical Faculty, University of Zurich, Zurich, Switzerland

(Received June 30, 2002; accepted September 11, 2002)

In six healthy mares and 24 mares showing reproductive disorders swab samples were taken from the fossa clitoridis to isolate Taylorella equigenitalis, and from the uterus to isolate mycoplasmas, ureaplasmas and other aerobic bacteria. Swab samples were also taken from the uterus for Chlamydia antigen ELISA and Chlamydia PCR studies. The uterus of 27 mares was examined cytologically, and biopsy samples were taken from the endometrium for histological examinations and for immunohistochemical examinations aimed at the detection of chlamydiae. T. equigenitalis, mycoplasmas, ureaplasmas and chlamydiae could not be detected from any of the mares examined. Aerobic facultative pathogenic bacteria were isolated from mares with endometritis in four cases. In 18 out of 22 mares with endometritis (82%) no infective agents could be demonstrated. Further studies are needed to elucidate the relative importance of non-infectious causes of endometritis and of anaerobic bacteria often detectable in the uterus in the aetiology of the reproductive disorders observed.

Key words: Mares, endometritis, infections, bacterium

*Corresponding author; E-mail: szeredil@oai.hu; Fax: +36 (1) 222 6071

Acta Veterinaria Hungarica 51 (1), pp. 53–59 (2003)

IN VITRO SENSITIVITY OF HUNGARIAN ACTINOBACULUM SUIS STRAINS TO SELECTED ANTIMICROBIALS

I. Biksi^1*, Andrea Major², L. Fodor², O. Szenci¹ and F. Vetési³

1Large Animal Clinic, Faculty of Veterinary Science, Szent István University, H-2225 Üllő, Dóra major, Hungary; ²Department of Microbiology and Infectious Diseases and ³Department of Pathology and Forensic Veterinary Medicine, Faculty of Veterinary Science, Szent István University, Budapest, Hungary

(Received June 14, 2002, accepted September 11, 2002)

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC₅₀ values in the range of 0.05–3.125 µg/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC₅₀ value of oxytetracycline and spectinomycin was 6.25 and 12.5 µg/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC₅₀ values were in the range of 25–100 µg/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman’s rho 0.789, p < 0001). MIC values of the type strain and MIC₅₀ values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.

Key words: Actinobaculum suis, antimicrobial sensitivity, novel antimicrobials, MIC

*Corresponding author; E-mail: ibiksi@univet.hu; Fax: +36 (29) 521 303

Acta Veterinaria Hungarica 51 (1), pp. 61–72 (2003)

IMMUNOLOGICAL ANOMALIES AND THROMBOCYTOPENIA IN 117 DOGS AND CATS DIAGNOSED WITH CHRONIC FATIGUE SYNDROME (CFS)⁺

W. Tarello^*

(Received May 5, 2002; accepted September 11, 2002)

Retrospective analysis of immune dysfunctions found in 55 dogs and 62 cats diagnosed with Chronic Fatigue Syndrome (CFS), revealed leukopenia in 11% of dogs (n = 6) and 22.5% of cats (n = 14), lymphopenia in 14.5% of dogs (n = 8) and 10% of cats (n = 6), hypogammaglobulinaemia in 9% of dogs (n = 5) and 13% of cats (n = 8) and thrombocytopenia in 20% of dogs (n = 11) and 68% of cats (n = 42). All patients had creatine kinase enzyme levels above the normal range (CK = 5–100 IU/L) and carried micrococcus-like organisms on erythrocytes. Blood cultures proved positive for Staphylococcus spp. in 16 cases. After low-dosage arsenic-based therapy (thiacetarsamide sodium) all animals experienced complete clinical remission. Subsequent controls demonstrated immune restoration in 4 representative FIV-FeLV negative cats, previously diagnosed with CFS associated with leukopenia, lymphopenia, hypogammaglobulinaemia and thrombocytopenia. The main conclusion is that a CFS-like disease in dogs and cats, characterised by the common hallmarks of high CK levels, absence of known causes of chronic fatigue in animals and presence of micrococcus-like organisms in the blood, can be associated with humoral and/or cellular immune deficiencies in 9–22.5% of cases and with thrombocytopenia in 20–68% of cases. Considerations are made on the possible role of micrococci in the aetiology of the condition and on the similarities with CFS in humans.

Key words: Chronic fatigue syndrome, CFS, dog, cat, lymphopenia, leukopenia, hypogammaglobulinaemia, thrombocytopenia

+This work has been carried out in the ‘Clinica Veterinaria Airone’ of Nus, Aosta (Italy).

*Correspondence: Walter Tarello DVM, C.P. 1644, 06129 Perugia 5, Italy; E-mail: wtarello@yahoo.it

Acta Veterinaria Hungarica 51 (1), pp. 73–90 (2003)

CANINE GRANULOCYTIC EHRLICHIOSIS (CGE) IN ITALY⁺

W. Tarello^*

(Received May 5, 2002; accepted September 11, 2002)

Medical records of thirty-five consecutive cases of canine granulocytic ehrlichiosis (CGE) diagnosed cytologically in Central Italy in 1995–2000 were analysed retrospectively. Tick exposure was reported in 16 dogs (45.7%) and concurrent babesiosis in 19 dogs (54.3%). Ehrlichia-like inclusion bodies were found in neutrophils in a percentage varying from 0.5% to 11%. Frequently recorded clinical signs included anorexia (71.4%), lethargy (45.7%), conjunctivitis (31.4%), fever (25.7%), lameness (20%) and ataxia (20%). Among the 16 representative dogs in which protein electrophoresis was performed, 10 (62.5%) showed high globulin levels and 6 (37.5%) had concurrent high total protein levels. During treatment with doxycycline, all associated symptoms, including those unusually described, such as pyoderma intertrigo, erythema, apparent blindness and oral papillomatosis, progressively disappeared in 31 (89%) out of 35 dogs. The efficacy of treatment was marked in dogs simultaneously treated twice with imidocarb dipropionate: among the 14 dogs in which a fast recovery was noted, 11 (80%) were concurrently affected by babesiosis and consequently treated with the specific medicament leading to excellent outcomes. The main conclusion is that CGE is present among dogs from Central Italy and should be included in the differential diagnosis of possible zoonotic agents affecting the canine population.

Key words: Canine granulocytic ehrlichiosis, CGE, Anaplasma phagocytophila, dog, zoonosis, doxycycline, babesiosis

+This work was carried out at the ‘Airone Veterinary Clinic’ of Castiglione del Lago (Perugia) and in a private veterinary practice of Fermo (Ascoli Piceno), Italy

*Correspondence: Walter Tarello DVM, C.P. 1644, 06129 Perugia 5, Italy; E-mail: wtarello@yahoo.it

Acta Veterinaria Hungarica 51 (1), pp. 91–94 (2003)

N-ras MUTATION IN A CANINE LYMPHOMA: SHORT COMMUNICATION

B. Mayr^1*, M. Holzheu¹, G. Schaffner² and M. Reifinger³

1Institute for Animal Breeding and Genetics, ³Institute for Pathology and Forensic Veterinary Research, Veterinary University, Veterinärplatz 1, A-1210 Vienna, Austria; ²Research Institute of Molecular Pathology, Vienna, Austria

(Received May 27, 2002; accepted September 11, 2002)

Lymphomas of dogs were investigated by molecular genetic methods. Regions of exon 1 and 2 of the N-ras gene, which harbours the mutation hot spots (codons 12, 13 and 61) were screened. A GGT –> GAT (glycine –> aspartic acid) mutation in codon 13 was present in a multicentric-type lymphoma of a 1-year-old male dog.

Key words: Lymphoma, dog, mutation, N-ras

*Corresponding author; E-mail: Burkhard.Mayr@vu-wien.ac.at; Fax: +43 (1) 25077-5693

Acta Veterinaria Hungarica 51 (1), pp. 95–101 (2003)

IN VITRO FERTILISATION OF IN VIVO MATURED PORCINE OOCYTES OBTAINED FROM PREPUBERAL GILTS AT DIFFERENT TIME INTERVALS AFTER HCG INJECTION

J. Rátky¹, D. Rath² and K.-P. Brüssow^3*

1Research Institute for Animal Breeding and Nutrition, Herceghalom, Hungary; ²Institute for Animal Breeding and Animal Behaviour, Mariensee, 31535 Neustadt, Germany; ³Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany

(Received May 7, 2002; accepted September 11, 2002)

The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.

Key words: Oocyte, in vivo maturation, in vitro fertilisation, ovum pick up, swine

*Corresponding author; E-mail: bruessow@fbn-dummerstorf.de; Fax: +49-38208-68752

Acta Veterinaria Hungarica 51 (1), pp. 103–109 (2003)

IN VITRO INDUCTION OF THE ACROSOME REACTION IN OVINE SPERMATOZOA BY CALCIUM IONOPHORE A23187

Ö. Uçar^* and T. J. Parkinson^**

Department of Clinical Veterinary Sciences, Bristol University, Bristol, United Kingdom

(Received May 31, 2002; accepted September 11, 2002)

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8–36 months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60 min in modified Tyrode’s medium (TALP) which contained either zero, 0.1 or 1.0 µM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3 ± 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P < 0.001) increased after incubation with A23187. After incubation with 0.1 µM/l A23187 for 45 and 60 min there were 22.4 ± 3.0% and 31.7 ± 4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0 µM/l A23187 for 45 and 60 min there were 46.2 ± 6.5% and 53.8 ± 5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0 ± 2.7% and 22.3 ± 4.2%. There was also a significant (P < 0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.

Key words: Ram, semen, acrosome reaction, calcium ionophore, A23187

*Corresponding author: Dr. Ömer Uçar (DVM, PhD), Present address: Department of Reproduction and AI, Faculty of Veterinary Medicine, Kafkas University, 36040 Kars, Turkey; E-mail: oucar6975@hotmail.com; Fax: +90 (474) 242 6853

**Present address: Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand

Acta Veterinaria Hungarica 51 (1), pp. 111–120 (2003)

INVOLVEMENT OF HIGH-DENSITY LIPOPROTEIN IN STIMULATORY EFFECT OF HORMONES SUPPORTING FUNCTION OF THE BOVINE CORPUS LUTEUM

D. Skarżyński, J. Młynarczuk and J. Kotwica^*

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-718 Olsztyn-Kortowo, Poland

(Received April 8, 2002; accepted September 11, 2002)

The hypothesis that epinephrine (noradrenaline, NA) enhances utilisation of high-density lipoproteins (HDL) by bovine luteal cells and that this process involves phospholipase (PL) C and protein kinase (PK) C intracellular pathway was tested. Luteal cells from days 2–4, 5–10 or 11–17 of the oestrous cycle were pre-incubated for 20 h. Subsequently DMEM/Ham’s F-12 medium was replaced by fresh medium and the cells were treated for 6 h as follows: In Experiment I with HDL (5–75 µg cholesterol per ml), NA, isoprenaline (ISO) or luteinising hormone (LH). In Experiment II cells were incubated for further 24 h in deficient medium (without FCS) and next treated as in Experiment I. In Experiment III cells were stimulated with NA, ISO or LH alone and together with HDL. In Experiment IV cells were treated with PLC inhibitor (U-73122) or with PKC inhibitor (staurosporine) or stimulator (phorbol 12-myristrate 13-acetate) and with either NA, insulin or LH. Only luteal cells from days 5–10 of the cycle responded on HDL and ß-mimetics (P < 0.05). LH stimulated progesterone secretion from the luteal cells during all stages of the cycle (P < 0.001). Cells incubated in deficient medium and supplemented with HDL secreted as much progesterone as those stimulated by LH in all stages of the cycle. Beta-mimetics were unable to enhance the stimulatory effect of HDL. Blockade of PLC had no influence on progesterone secretion from cells treated with either NA or LH, but this did impair the stimulatory effect of insulin (P < 0.05). Similarly, blockade of PKC by staurosporine impaired (P < 0.05) the effect of insulin only but not that observed after LH or NA treatment. We suggest that: (a) noradrenergic stimulation does not enhance utilisation of cholesterol from HDL for progesterone secretion; (b) the fasting of luteal cells seems to activate enzymes responsible for the progesterone synthesis; (c) effect of NA on progesterone secretion from luteal cells does not involve the PLC-PKC pathway.

Key words: Cattle, corpus luteum, HDL, progesterone, PKC

*Corresponding author; E-mail: janko@pan.olsztyn.pl; Fax: +48 (89) 524 0347