Georg Wolf

Genomic (5′UTR) and serological differences among German BVDV field isolates

SummaryIsolates of bovine viral diarrhoea virus (BVDV) collected in Germany were examined for their genomic heterogeneity in sequences from the 5′untranslated region (UTR) of the viral genome. Polymerase chain reaction (PCR) tests based on the 5′UTR and the region coding for the NS2–3/4A polypeptide were used to differentiate between BVDV I and BVDV II genotypes. Eleven out of 96 BVDV-isolates were identified as BVDV II. Virus neutralization tests with BVDV I- or II-specific antisera raised in cattle were done. The mean titers were reduced by 7.2-fold (BVDV I-antiserum versus type II-isolates) or 35-fold (BVDV II-antiserum versus type I-isolates) when using the respective heterologous virus.

Associations between pathogens in healthy pigs and pigs with pneumonia

The aim of this study was to evaluate the associations between different pathogens in the development of pneumonia and bronchopneumonia in pigs. Samples of bronchoalveolar lavage fluid from 100 pigs showing no clinical signs and 239 pigs with clinical signs of respiratory disease were examined for Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, us-type porcine respiratory and reproductive syndrome virus (prrsv), eu-type prrsv, porcine circovirus type 2 (pcv-2), influenza virus type A, α-haemolytic Streptococcus species, β-haemolytic Streptococcus species, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Actinobacillus pleuropneumoniae. These potential pathogens were detected more frequently in the pigs with respiratory problems than in the pigs with no clinical signs. pcv-2 and α-haemolytic streptococci were the pathogens most frequently detected; A pleuropneumoniae was isolated in only two cases. There were more often associations between the organisms in the pigs with clinical signs than in the healthy pigs. In particular, α-haemolytic streptococci and M hyopneumoniae were both associated with the presence of M hyorhinis, eu-type prrsv, P multocida and B bronchiseptica, and α-haemolytic streptococci also occurred more often in pigs that were already infected with other pathogens. P multocida and B bronchiseptica were both significantly associated with M hyopneumoniae, α-haemolytic streptococci, eu-type prrsv and us-type prrsv.

Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus

A system for a reproducible in vitro restimulation of bovine viral diarrhea virus (BVDV)-specific cytotoxic T-cells (CTL) was developed. Lymphocyte cultures of BVDV-immunised cattle were stimulated with infectious BVDV isolate PT810 and recombinant bovine interleukin-2 for 12 to 25 days. A specific lysis of Concanavalin A-stimulated BVDV-infected autologous target cells was observed, whereas allogeneic BVDV-infected target cells were only marginally lysed as detected by flow cytometry. BVDV-specific lymphocyte transformation was further characterised by the expression of bovine lymphocyte activation antigens and bovine MHC class-II molecules. Secondary stimulation of CTL was influenced by in vitro production of BVDV-specific neutralising antibodies, which were secreted exclusively in BVDV-inoculated lymphocyte cultures of immunised cattle. These results demonstrate the presence of CTL in peripheral blood mononuclear cells (PBMC) of immunised cattle which can kill autologous BVDV-infected antigen-presenting cells after in vitro restimulation.

Endoscopically Visualized Lesions, Histologic Findings, and Bacterial Invasion in the Gastrointestinal Mucosa of Dogs with Acute Hemorrhagic Diarrhea Syndrome

Background Etiology of hemorrhagic gastroenteritis (HGE) syndrome in dogs is unknown and histopathologic and microbial investigations have only been performed post mortem. Objective To identify characteristic intra vitam endoscopic and histologic mucosal lesions, as well as bacterial species, within the mucosa of dogs with HGE. Animals Ten dogs diagnosed with HGE were included. Eleven dogs with gastroduodenoscopy and different intestinal diseases were used as controls for microbial changes. Dogs pretreated with antibiotics or diagnosed with any disease known to cause bloody diarrhea were excluded from the study. Methods In this prospective study, gastrointestinal biopsies were collected from 10 dogs with HGE. Endoscopic and histologic changes were assessed according to WSAVA guidelines. Biopsies from the stomach, duodenum, ileum, and colon were investigated by histology and by immunohistochemistry for the presence of Clostridium spp. and parvovirus. The first duodenal biopsy taken with a sterile forceps was submitted for bacterial culture. Results Acute mucosal lesions were only found in the intestines, not in the stomach. Clostridium spp., identified as Clostridium perfringens in 6/9 cases, were detected on the small intestinal mucosa in all dogs with HGE, either by culture or immunohistopathology. In the control group, C. perfringens could only be cultured in one of 11 dogs. Conclusions and Clinical Importance The results of this study demonstrate an apparent association between C. perfringens and the occurrence of acute hemorrhagic diarrhea. The term “HGE,” which implies the involvement of the stomach, should be renamed as “acute hemorrhagic diarrhea syndrome.”

A new inactivated BVDV genotype I and II vaccine - An immunisation and challenge study with BVDV genotype I

An inactivated vaccine containing BVDV I and II strains (PT810; BVDV I, and 890; BVDV II) and using different adjuvants and antigen dosages was tested in a cattle challenge model. Groups of six healthy, seronegative cattle were vaccinated twice with a low dose (10(6.6) TCID(50) PT810 and 10(7.2) TCID(50) 890) vaccine with the adjuvant Bay R1005 or a high dose (10(7.8) TCID(50) PT810 and 10(8. 2) TCID(50) 890) vaccine with two different adjuvants (Bay R1005 or Polygen). Thirty-eight days after the second vaccination, immunised animals (n=18) and non-vaccinated control animals (n=3) were challenged intranasally with 10(6) TCID(50) BVDV strain PT810. For a period of 16 days, virus was isolated from blood leukocytes and nasal swabs, and neutralising antibody titres were determined.The induction of antibodies following immunisation was strongly dependent on the antigen dosage in the vaccine. The high dose formulation induced high serum neutralising antibody titres against both genotypes of up to 32000 after the second immunisation. Animals with neutralising antibody titres >512 (n=14) did not show any marked leukopenia after challenge and only very little or no virus could be isolated from blood leukocytes and/or nasal swabs when compared to control cattle. Furthermore, some of these animals did not show any boost of neutralising or even NS3-specific antibodies, which renders viral replication unlikely and thus would prevent infection of the fetus. Both adjuvants (Bay R1005 or Polygen) were similarly efficient and induced nearly identical antibody responses. In contrast, four of the six low dosage vaccinates had a marked leukopenia and viraemia as well as detectable nasal virus shedding for several days. We conclude that the selected strains and the system of vaccine preparation with high BVDV antigen dosages and highly efficient new adjuvants provide an effective means of protection against BVDV I infections. Investigations to demonstrate the protection against BVDV II infections, the duration of immunity and the ability of fetal protection by using the high dose vaccine in a fetal challenge model will follow.

Transient elimination of circulating bovine viral diarrhoea virus by colostral antibodies in persistently infected calves: a pitfall for BVDV-eradication programs?

Infections with bovine viral diarrhoea virus (BVDV) cause substantial economic losses to cattle industries. Rapid detection of persistently BVDV infected (PI) calves is of utmost importance for the efficacy of BVDV control programs. Blood and ear skin biopsy samples are conveniently used for early mass screening of newborns. However, little is known about the impact of colostral antibodies on the outcome of relevant analyses. Here, we rigorously tested a series of samples obtained from five colostrum-fed PI calves from birth until they reached the status of seronegativity for NS3-specific antibodies. We comparatively quantified virus loads in blood samples and dried skin biopsies as detected with BVDV-NS3-, -Erns-capture ELISA and RT-qPCR. Monitoring of NS3-positive leukocytes was done with flow cytometry. Within seven days after colostrum intake, BVDV infected leukocytes disappeared for a three- to eight-week period. Immediately after colostrum ingestion, detectable Erns antigen levels dropped 10-100-fold in biopsy samples and in sera detection of Erns failed for one to two weeks. Virus demonstration in biopsy samples with a NS3-antigen-ELISA failed until days 90-158 after birth. Specific antibodies against BVDV also impaired the detection of viral RNA in leukocytes and blood. Mean RNA levels of the five calves were reduced in sera 2.500-fold and in leukocytes 400-fold, the lowest values were at week three of live. In contrast, levels of measurable viral RNA in biopsy samples remained constant during the observation period.

A retrospective study of the clinical presentation of 140 dogs and 39 cats with bacteraemia

OBJECTIVES To evaluate retrospective data from 140 dogs and 39 cats with positive blood cultures that were presented to the Clinic for Small Animal Medicine in Munich from 1995 to 2004. METHODS The identity of bacteria isolated from blood cultures of dogs and cats with bacteraemia was determined, and clinical and laboratory findings and outcome of animals with Gram-negative versus Gram-positive bacteraemia were compared. RESULTS Sepsis was diagnosed in 81.7 per cent of dogs and 59.5 per cent of cats with bacteraemia. Escherichia coli was isolated in one third of the animals. Dogs with bacteraemia more often showed monocytosis and increased alkaline phosphatase activity, while in cats, hyperglycaemia was found more commonly. Dogs with Gram-negative bacteraemia had hypoalbuminaemia significantly more often than dogs with Gram-positive bacteraemia, while among the remaining parameters, there were no statistically significant differences. CLINICAL SIGNIFICANCE Not all dogs and cats with a positive blood culture met the criteria for sepsis. Bacteraemia caused by Gram-positive versus Gram-negative bacteria cannot be distinguished based on clinical or laboratory parameters, and bacterial culture and susceptibility testing have to be performed for the right choice of antibiotic treatment.

Bacteriological and antibiotic sensitivity test results in 271 cats with respiratory tract infections.

INFECTIONS of the respiratory tract are still a common cause of morbidity and mortality in cats, despite improved options for their prevention and treatment. Disease associated with bacterial respiratory tract infection (RTI) can occur following infection with certain bacterial pathogens or following proliferation of the normal bacterial inhabitants of the respiratory tract if the natural defence mechanisms are impaired. Predisposing factors include virus infection, toxoplasmosis, lungworm infection, trauma, aspiration pneumonia, neoplasia, tooth root infection, nasopharyngeal polyps, congenital anomalies, otitis media or interna, metabolic dysfunctions and immunosuppressive therapy (Van Pelt and Lappin 1994). Few data are available on the prevalence of the bacterial species involved in these infections. This short communication describes a study to evaluate the distribution of bacteria isolated from the upper and lower airways of cats presenting with signs consistent with infectious respiratory tract disease, and to determine their antibiotic susceptibility. Data were collected from the medical records of 271 cats from private households that had been presented to the University of Munich Veterinary Teaching Hospital since 1989 showing clinical signs consistent with RTI. The criteria for selection of cases were the presence of signs such as sneezing, fever, nasal discharge, coughing and dyspnoea, and no obvious signs of underlying disease such as neoplasia, trauma, or heart failure. Information about the cats’ age, sex and breed was available for 265 cats. The median age of the cats was five years (mean 6·2 years), and 112 of them (42·3 per cent) were female and 153 (57·7 per cent) were male. There were 238 domestic shorthair cats (89·8 per cent), 12 Persian or Persian cross (4·5 per cent), nine Siamese or Siamese cross (3·4 per cent), two Birmans (0·8 per cent), two angoras (0·8 per cent) and two British shorthair cats (0·8 per cent). One hundred and eighty samples were obtained from nasal discharge or from lesions inside the nasal cavity using cotton-tipped swabs, and 118 samples were taken from the lower respiratory tract by tracheal lavage or bronchoalveolar lavage via a sterile endotracheal catheter in cats under anaesthesia. The samples were inoculated on to agar, sheep blood agar, colistin/nalidixic acid agar and Gassner’s water blue metachrome yellow lactose agar. The plates were incubated at 37°C under aerobic conditions and examined after 24 and 48 hours. The results of bacterial culture are shown in Table 1. The antibiotic susceptibility of the isolates was tested by the agar disc diffusion method; the antibiotics tested are listed in Table 2. Sixteen different bacterial species were cultured from the nasal samples. The bacteria isolated most frequently were Pasteurella, Streptococcus and Staphylococcus species and Escherichia coli. These results are similar to the bacterial distribution in cats reported by Dossin and others (1998). All the bacterial species isolated from the nasal samples have also been cultured from the airways of healthy cats (Greene 1998); this suggests that bacterial cultures from the nose of cats with RTI may not always indicate the underlying problem. A significantly higher proportion of the lower respiratory tract samples than the nasal samples yielded negative culture results. The numbers of bacteria in the trachea and bronchi are considered to be lower than those in the upper respiratory tract because the filtration of air in the nose and the mucociliary clearance mechanisms prevent bacteria from entering the lower airways (Greene 1998). Thirteen different bacterial species were isolated from the lower airway samples. The most frequently isolated bacteria were Pasteurella species, E coli, Streptococcus, Enterobacter and Staphylococcus species. Macdonald and others (2003) and Foster and others (2004a, b) described similar findings. All the bacterial species isolated in the present study except Bordetella bronchiseptica, which is considered a primary pathogen, have Veterinary Record (2006) 158, 269-270

Feline urinary tract pathogens: prevalence of bacterial species and antimicrobial resistance over a 10-year period

The purpose of this retrospective study was to identify bacterial species in cats with bacterial urinary tract infections (UTIs) and to investigate their antimicrobial susceptibilities over a 10-year period. Three hundred and thirty cultures from 280 cats were included in the study. The mean age of affected cats was 9.9 years; female cats with bacterial UTIs were significantly older than male cats with UTIs. The most common pathogen identified was Escherichia coli (42.3 per cent), followed by Streptococcus species (19.3 per cent), Staphylococcus species (15.6 per cent), Enterococcus species (6.6 per cent) and Micrococcaceae (5.8 per cent). Forty specimens (12.1 per cent) yielded growth of more than one isolate. Streptococcus and Enterococcus isolates were resistant to a significantly higher number of antimicrobial agents than E coli and Staphylococcus species isolates. Applying the formula to select rational antimicrobial therapy, bacterial isolates were most likely to be susceptible to nitrofurantoin, amoxicillin clavulanic acid, enrofloxacin and gentamicin. The antimicrobial impact factor for nitrofurantoin increased significantly over the 10-year period, whereas there was no significant change in antimicrobial impact factors for doxycycline, trimethoprim-sulfamethoxazole, gentamicin, enrofloxacin, cephalothin and amoxicillin clavulanic acid. The detected changes in in vitro antimicrobial efficacy could help to develop hospital-specific guidelines for antimicrobial use to prevent the further development of resistance in feline uropathogens.

Prospective study of bacteraemia in acute haemorrhagic diarrhoea syndrome in dogs

In dogs with idiopathic acute haemorrhagic diarrhoea syndrome (AHDS), a serious loss of intestinal mucosal barrier integrity occurs. However, the incidence of bacterial translocation in dogs with idiopathic AHDS is not known. Thus, the objectives of this prospective study were to identify the incidence of bacteraemia, to evaluate the frequency of septic events and the influence of bacteraemia on various clinical and laboratory parameters, duration of hospitalisation and survival of dogs with idiopathic AHDS. The study included 87 dogs with idiopathic AHDS. Twenty-one healthy dogs served as control group. To evaluate clinical significance of bacterial translocation, blood culture results were compared between patients and controls. Clinical and laboratory parameters were compared between patients with positive and negative blood cultures. There was no significant difference in either incidence of bacteraemia between patients with idiopathic AHDS (11 per cent) and controls (14 per cent) or in severity of clinical signs, laboratory parameters, duration of hospitalisation or mortality between blood culture-positive and culture-negative dogs with idiopathic AHDS. The results of this study suggest that the incidence of bacteraemia in dogs with idiopathic AHDS is low and not different from that of healthy control dogs. Bacteraemia does not influence the clinical course or survival and thus antibiotic treatment is not indicated to prevent sepsis.