Microbiologic evaluation of gallbladder bile of healthy dogs and dogs with iatrogenic hypercortisolism: a pilot study.

BACKGROUND: In people, hypercortisolism (HC) has been associated with acalculous cholecystitis and biliary dyskinesia, which may potentiate ascending biliary infections. In dogs, an association between HC and gallbladder disease recently has been documented, although the role of bacteria remains controversial. Furthermore, there is no information on the gallbladder bile microbial flora in healthy dogs. OBJECTIVES: To investigate the microbial flora in gallbladder bile in healthy dogs, the relationship between iatrogenic hyperadrenocorticism and bactibilia and possible changes in biliary microbial flora after cortisol withdrawal in dogs. ANIMALS: Six control dogs and 6 dogs treated with hydrocortisone. METHODS: Gallbladder bile obtained by percutaneous ultrasound-guided cholecystocentesis was cultured aerobically and anaerobically and examined cytologically before (d0), during (d28, d56, d84), and after (d28p, d56p, d84p) administration of hydrocortisone (8 mg/kg PO q12h). RESULTS: In the control group, 2/42 bile cultures yielded bacterial growth (Enterococcus sp.; Escherichia coli on d0) and 1/42 bile smears had cytological evidence of bacteria (d28). In the HC group, 2/42 bile cultures yielded bacterial growth (Enterococcus sp. on d28; Bacillus sp. on d28p) and 3/42 bile smears had cytological evidence of bacteria (d84, d84, d28p). All dogs remained healthy throughout the study period (168d). CONCLUSIONS AND CLINICAL IMPORTANCE: Based on the results of conventional bacterial culture techniques, gallbladder bile of healthy dogs periodically may harbor bacteria, which do not appear to be clinically relevant. A 3-month period of iatrogenic HC was not associated with bactibilia. A higher prevalence of bactibilia may be detected with micromolecular techniques.

Aetiology of bovine abortion in Switzerland from 1986 to 1995--a retrospective study with emphasis on detection of Neospora caninum and Toxoplasma gondii by PCR.

In a retrospective study, covering the period from 1986 to 1995, tissues of aborted fetuses were re-examined. A total of 347 cases were tested immunohistochemically, among them samples of 223 brains were examined for Neospora caninum, Toxoplasma gondii and Bovine Virus Diarrhoea Virus (BVDV), and 249 placentae for Chlamydiaceae. Two real-time PCR assays, one for N. caninum, and one for T. gondii, were developed. These potential abortion-inducing agents were detected - and confirmed by PCR, except for BVDV - in 16.1% (N. caninum), 0% (T. gondii), 9.9% (BVDV) and 0.8% (Chlamydiales) of the cases examined. Immunohistochemistry proved to be inadequate for the detection of the protozoal epitopes, whereas it was confirmed as a very useful tool for the detection of BVDV. In abortion material, PCR is considered to be more suitable for the detection of protozoa and Chlamydophila abortus, an adequate sampling presupposed.

[Field study of the prevalence and diagnosis of diarrhea-causing agents in the newborn calf in a Swiss veterinary practice area]. / Feldstudie zu Prävalenz und Diagnostik von Durchfallerregern beim neonaten Kalb im Einzugsgebiet einer schweizerischen Nutztierpraxis.

The prevalence of cryptosporidia, rotavirus, bovine coronavirus and Escherichia coli F5 (K99) in dairy calves with diarrhea and in healthy calves was established in a limited area served by a veterinary practice. Immuno-chromatographic rapid tests (FASTest Strips) were applied in the field and their results were compared to the ones obtained with standard methods (modified Ziehl-Neelsen stain, antigen-ELISA and cultivation). In 78% of the calves with diarrhea (n=46) and in 29% of the healthy calves (n=14), one or two agents were isolated. Of the diseased calves, 43% excreted cryptosporidia and in 46% rotavirus was isolated. Bovine corona virus and Escherichia coli F5 (K99) seemed to be of minor importance in the investigated population. Compared to the modified Ziehl-Neelsen stain or the antigen-ELISA, the FASTest Strips CRYPTO and ROTA were of very high diagnostic specificity of 100% each and their diagnostic sensitivity was 75% and 57%, respectively. Due to the low number of cases, the results of the FASTest Strips BCV and E.coli-K99 could not be interpreted. Although the diagnostic sensitivity of the FASTest Strips CRYPTO and ROTA--evaluated with standard methods--was not very high, their use in calves with acute diarrhea is recommended.

[Incidence of zoonoses in petting zoos and evaluation of hygiene measures to prevent the transmission to humans]. / Vorkommen von Zoonosen in Streichelzoos und Beurteilung der Hygienemassnahmen für die Ubertragung auf den Menschen.

In summer 2003, a study was performed in thirty Swiss petting zoos with the objective to determine the prevalence of zoonotic agents, and to describe hygiene measures implemented to reduce the risk of human infection. Fecal samples from different animal species were collected from the floor of pens to determine the prevalence of Salmonella spp., Campylobacter spp., verocytotoxin producing E. coli/ VTEC and Francisella tularensis. A questionnaire on hygiene measures, number of animals per species, housing system, care procedures and feeding was administered to every petting zoo to estimate exposure of visitors to zoonotic microorganisms. In total, 423 fecal samples were examined. Of these samples, 41 were positive for Campylobacter spp., which were mainly isolates from pigs and poultry (35% positive samples from each species). In pigs, 50% of the positive samples (6 samples) were typed as C. jejuni. The others were typed as C. coli (3) and C lan' (3), respectively. Five poultry isolates were typed as C. jejuni, and two as C. coli. Two samples were positive for Salmonella spp. Salmonella typhimurium was isolated from a goat, the other isolate could not be identified by serotyping. Neither Francisella tularensis nor verocytotoxin producing E. coli/ VTEC were found. The low prevalence of zoonotic microorganisms in Swiss petting zoos could be attributed to the cleanness of enclosures and animals, low stocking rates and good animal care. However, there is room for improvement concerning visitors' information on hygiene and hand washing. Furthermore, a strict separation between picnic - areas and animals should be enforced.

Diagnostic investigation into the role of Chlamydiae in cases of increased rates of return to oestrus in pigs.

Cervical swabs and serum samples were taken from Swiss herds of sows with high rates of irregular return to oestrus (group A) and from control herds without reproductive problems (group B. The genital tracts of 21 slaughtered sows of group A were also examined. The swabs and genital tracts were screened for Chlamydiae by a new 16S rRNA PCR and the sera by an ELISA for Chlamydiaceae lipopolysaccharide. Chlamydophila (Cp) abortus was isolated from seven of the 65 swabs taken from group A but from none of the 128 swabs taken from group B. Chlamydia suis was present in swabs from both groups A (1.5 per cent) and B (2.3 per cent). In addition, Cp abortus was detected in 33.3 per cent of the genital tracts. Of the 193 sera tested, 61.7 per cent were positive, with no significant difference between group A (52.3 per cent) and group B (66.4 per cent). Chlamydia-like organisms were detected in 28.2 per cent of the swabs from group A and in 22 per cent of those from group B.

Diagnosis of porcine proliferative enteropathy: detection of Lawsonia intracellularis by pathological examinations, polymerase chain reaction and cell culture inoculation.

A total of 21 pigs aged 7-17 weeks with clinical symptoms suggestive for Porcine Proliferative Enteropathy were examined for Lawsonia intracellularis by analysing the following parameters: (i) intestinal gross and histological lesions, (ii) presence of comma-shaped bacteria in enterocytes by Warthin-Starry and a modified Ziehl-Neelsen stain, (iii) PCR amplification of L. intracellularis DNA from intestinal mucosa by using two oligonucleotide primer pairs targeting a 255-bp DNA fragment of the 16S rDNA-gene and a 319-bp DNA fragment of the L. intracellularis chromosome. Specificity of PCR reactions was confirmed by using DNA extracted from the L. intracellularis reference strain N343 (ATCC 55672) as well as by DNA sequence comparisons of PCR amplification products with data bank entries. Intestinal gross lesion indicative for PPE were observed in 20 pigs (95.2%). For all 21 pigs, the L. intracellularis aetiology was confirmed by histological as well as bacterioscopical examinations. Specific PCR amplification products were obtained from 20 pigs (95.2%). Taking PCR positivity as the definite criterion, L. intracellularis was diagnosed in 20 pigs from 11 herds in seven Swiss cantons (Argovia, Berne, Fribourg, Grisons, Lucerne, Schwyz, Thurgovia). To grow L. intracellularisin vitro, the cell culture method of Lawson et al. (J. Clin. Microbiol. 1993: 31, 1136-1142) was adopted. Inocula prepared from heavily infected fresh and frozen ileal mucosa of 15 pigs were cultured in rat enterocytic IEC-18 cells (ATCC CRL 1589). Six cell culture passages of 10 days each were completed. The reference strain N343 was examined for cultivability, accordingly. Except for occasional specific PCR amplifications from cell cultures up to the second passage, any indications for growth of L. intracellularis in IEC-18 cells were not found.

[Abortion in small ruminants in Switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions]. / Aborte beim kleinen Wiederkäuer in der Schweiz: Untersuchungen während zwei Ablammperioden (1996-1998) unter besonderer Beachtung des Chlamydienabortes.

Abortion cases of 144 goats und 86 sheep were investigated etiologically during 2 lambing seasons (1996/1997, 1997/1998). Macroscopic inspection of fetus and placenta was completed by histopathology and bacteriological isolation of agents. In addition, immunohistologically the following antigens were labeled in formalin-fixed and paraffin-embedded tissue sections: Toxoplasma gondii, Neospora caninum, Chlamydophila abortus (formerly Chlamydia psittaci serovar 1) and Border Disease Virus. From farms with abortions caused by Chlamydophila abortus specific data were recorded. In 75% of abortion cases in sheep and in 59% of cases in goats an etiologic diagnosis could be substantiated. Chlamydophila abortus is the most commonly involved agent in the etiology of caprine and ovine abortion (sheep 39%, goats 23%), followed by Toxoplasma gondii (sheep 19%, goats 15%) and Coxiella burnetti (sheep 1%, goats 10%). All other agents are of minor importance. An infectious cause of abortion based on histopathologic findings without isolation of agents was observed in sheep (10%) and goats (21%). Malformation occurred in sheep (2%) and goats (3%) and lesions suggestive for Vitamin E/Selenium deficiency were seen in goats only (2%).

Demonstration of heterogeneous genotypes of Taylorella equigenitalis isolated from horses in six European countries by pulsed-field gel electrophoresis.

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.

[Purulent osteomyelitis in fattening pigs]. / Eitrige Osteomyelitis beim Mastschwein.

Purulent osteomyelitis caused by Arcanobacterium pyogenes was diagnosed in three pigs aged between 3 and 4 months by radiological and pathological findings. Osteomyelitis was localized in metaphysis and/or epiphysis of limb bones. The prevalence of osteomyelitis in swine seems to be underestimated because inspection of limb bones is not a routine procedure either at slaughter or at necropsy. Osteomyelitis may also have consequences for meat cutting. Osteomyelitis can be controlled by prophylactic procedures.

Experimental enteric infection of gnotobiotic piglets with a Chlamydia psittaci strain of avian origin.

The pathogenicity of a Chlamydia psittaci isolate of pigeon origin was assessed using a litter of gnotobiotic piglets. At 3 days of age, six piglets were inoculated intragastrically with egg-grown chlamydiae, the remaining six pigs were sham-inoculated. The animals were observed for clinical signs, and they were killed and necropsied sequentially between 4 and 15 days of age. Clinical manifestations consisted of slight softening of the faeces between 6 and 10 days post-inoculation (DPI). Immunohistochemistry revealed chlamydial replication predominantly in the small intestine, initially within villous enterocytes, after 4 DPI mostly in the lamina propria. Histopathology showed villous atrophy and increased numbers of inflammatory cells in the gut up to 6 DPI. Chlamydial stages of normal morphology were identified within enterocytes using transmission electron microscopy. An enzyme-linked immunosorbent assay (ELISA) run on faecal samples revealed shedding of chlamydial antigen from 3 until 11 DPI. Systemic dissemination of Chlamydia occurred to a limited extent according to polymerase chain reaction and immunohistochemistry results of several extraintestinal organs. Corresponding histopathological changes were minimal. Sera of all pigs were negative for anti-chlamydial antibodies using a complement fixation test. In conclusion, inoculation of this isolate in gnotobiotic piglets resulted in a productive enteric infection with mild lesions, weak systemic dissemination, and faecal shedding, indicating the pig as a potential host for avian chlamydiae.

Occurrence of chlamydiae in the genital tracts of sows at slaughter and their possible significance for reproductive failure.

The aim of this study was to investigate further the role of chlamydiae as pathogens in the genital tracts of sows at slaughter. Genital tracts of 101 randomly selected sows were collected and specimens of genital tract localizations were systematically examined for chlamydiae using immunohistochemistry and PCR. In the genital tracts of 10 sows, Chlamydia psittaci DNA was detected by PCR, and was further typed as 'serotype 1' in nine cases and as avian strain 6 BC in one animal. However, all specimens examined by immunohistochemistry were negative for chlamydiae. Pooled samples of scalding tank water were additionally investigated for 95 animals. Of these samples, 63.2% contained chlamydial DNA, mostly C. trachomatis, and in one sample C. psittaci 'serotype 1'. Although in most cases contamination through influx of faecally contaminated scalding water is a possible reason for the positive PCR results in the genital tract, latent infection cannot be excluded. In conclusion, the results obtained suggest that chlamydiae are of no or only minor importance in the examined group of Swiss breeding sows. Nevertheless, the role and significance of chlamydiae as pathogens in porcine reproductive disorders remain unresolved and require further investigation.

Experimental Chlamydia psittaci serotype 1 enteric infection in gnotobiotic piglets: histopathological, immunohistochemical and microbiological findings.

The enteric pathogenicity of the ovine C. psittaci serotype 1 isolate S26/3 was assessed using a litter of gnotobiotic piglets. In one group, eight piglets were inoculated at 3 days of age; at 10 days, two of these were re-inoculated. In a second group, six animals were mock-inoculated at 3 days of age as negative controls; subsequently, at 10 days, three of these piglets were inoculated with C. psittaci. The animals were observed for clinical signs, killed and necropsied sequentially between 4 and 17 days of age. At necropsy, specimens were collected for histopathology, immunohistochemistry and serology. Clinical manifestations consisted of sporadic slight softening of faeces observed between 8 and 12 days post inoculation (d.p.i.) in pigs inoculated at 3 days of age and between 4 and 6 d.p.i. in those inoculated at day 10. Histopathological changes were minimal and inconsistent and occurred almost exclusively in the small intestine in pigs of 15 days of age and older; they consisted of a slight shortening of villi, of a small number of tongue-shaped villi and of villous fusions. Immunohistochemistry revealed small numbers of chlamydial inclusions in the small intestinal enterocytes of only five pigs, all killed within 5 d.p.i. An ELISA run on faecal samples collected daily after inoculation from six of the pigs showed that chlamydial antigen was excreted in the faeces. In pigs inoculated at 3 days, chlamydial antigen was detected inconsistently before, and consistently after 9 d.p.i. Pigs inoculated at 10 days excreted antigen consistently after inoculation until the end of their observation period (8 d.p.i.). Infective chlamydiae were detected from the faeces of inoculated piglets using Vero cell cultures. Sera of all pigs were negative for anti-chlamydial antibodies using a complement fixation test. In conclusion, enteric pathogenicity of C. psittaci serotype 1 in a litter of gnotobiotic piglets proved minimal. The results, therefore, indicate that serotype 1 C. psittaci is not likely to cause enteric disease in conventionally reared pigs. Nevertheless, a potential role of swine in the epidemiology of this agent should be considered with regard to spread of Chlamydia to other species.

[Pestivirus as causative agent of abortion and perinatal mortality in cattle and sheep in Switzerland]. / Ursächliche Beteiligung von Pestiviren an Aborten und perinatalen Todesfällen bei Rindern und Schafen in der Schweiz.

The causal involvement of bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) infection in bovine and ovine abortion and perinatal mortality remain unclear. From 1992 until 1994, 213 bovine and 31 ovine foetuses as well as 36 calves and 25 lambs which had died perinatally were investigated. Tissue samples were tested for the presence of pestiviruses and serum or fluid from the body cavities were analysed for the presence of pestivirus antibodies. Detection of pestiviruses was performed by (i) cell culture isolation, (ii) antigen ELISA and (iii) immunohistochemical staining for viral antigen. For antibody-testing an indirect ELISA was used. In nine bovine foetuses and in two calves BVDV was isolated. Pestiviruses, most likely BDV were detected in one ovine foetus and three lambs. In 6% of the bovine and 11% of the ovine foetuses anti-pestivirus antibodies were detected. However, clinical features and history of bovine cases did not show a correlation with the diagnostic results, In contrast, the presence of central nervous system signs in neonatal lambs and the detection of BDV was correlated.

Chlamydiae in porcine abortion.

Formalin-fixed, paraffin-embedded fetal livers and lungs from 139 cases of swine abortion were investigated retrospectively for chlamydiae by means of immunohistochemistry. Using a genus-specific antibody, chlamydial antigen was found in eight livers obtained from five (3.6%) abortion cases from different herds. All lung sections were negative. Chlamydiae were also labeled in five of the eight positive livers using a monoclonal antibody against immunotype 1 of Chlamydia psittaci; the remaining three livers were negative. No reactivity was seen using an antibody specific for C. trachomatis. Chlamydiae should be considered a cause of abortion in sows in Switzerland. Porcine abortigenic strains identified in this study differed immunologically from intestinal strains (known to be mainly C. trachomatis) but shared similarities with abortigenic chlamydiae of ruminants.

[The occurrence of spiral-shaped bacteria in the abomasum of cattle]. / Untersuchungen über das Vorkommen von spiralförmigen Bakterien im Labmagen des Rindes.

The goal of this study was to determine whether Helicobacter pylori or similar bacteria are present in the abomasum of cows. The abomasa of 112 clinically healthy cows were examined at slaughter. Prior to macroscopic examination, samples for bacteriological and histological examination were obtained from the fundus and from the pylorus. Bacteriological examination of the abomasal mucosa included the urease test, the microscopic examination of a Gram's stained smear, and culture on various solid media. Samples from the pylorus (63) were more often positive in the urease test than those from the fundus (35). Examination of Gram's stained smears revealed two groups of suspicious microorganisms; spiral-shaped and rod-shaped bacteria, whereby the latter could not be differentiated morphologically from Helicobacter pylori. Spiral-shaped bacteria were more often isolated from the pylorus (101 samples) than from the fundus (30 samples). The bacteria that resembled Helicobacter pylori were found in seven samples from the pylorus and in seven samples from the fundus. Helicobacter pylori was not cultured in any of the abomasal samples. Tissue samples from the fundus and pylorus were stained with hemalum and eosin and with silver according to Warthin-Starry. All but one abomasum had diffuse infiltration of lymphocytes and plasma cells. Lymphocytic follicles were observed in 109 abomasa. Neutrophils were seen in four abomasa, eosinophils in 37 and parasitic lesions in 20. As in the Gram's stained smears, spiral-shaped and rod-shaped bacteria were seen in silver-stained smears. Spiral-shaped bacteria were found in the pylorus of 96 abomasa and in the fundus and pylorus of one abomasum. The rod-shaped bacteria could not be differentiated from Helicobacter pylori by light microscopy. They were seen in glandular lumina of the superficial region of the mucosa in 97 abomasa. They were limited to the pylorus and fundus in 16 and 59 cases, respectively, and occurred in both these areas in 23 cases. The results of this study indicate that spiral-shaped bacteria may be found frequently in the bovine abomasum. Further investigations are required to determine whether these bacteria are associated with the inflammatory lesions that were observed and whether they play a role in the pathogenesis of abomasal ulcers.

Intestinal Chlamydia in finishing pigs.

Gut and blood samples from 119 finishing pigs derived from 11 farms were collected during routine slaughter at an abattoir. Sections of formalin-fixed, paraffin-embedded tissues were labeled immunohistochemically using genus-specific, mouse monoclonal antibody against chlamydial lipopolysaccharide; goat polyclonal antiserum against the major outer membrane protein of Chlamydia trachomatis; and mouse monoclonal antibody against the ovine abortion subtype of C. psittaci. Gut samples from 33 of 111 (29.7%) individual pigs stained positive with the genus-specific monoclonal antibody, and of these 30 of 32 (93.7%) also reacted with the C. trachomatis-specific antiserum. Labeled inclusions were restricted to mature enterocytes of the large intestine in 33 of 111 cases. Infection of small intestinal enterocytes was noted in only one of 82 ileal samples. The blood samples were tested for antichlamydial antibodies by enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT). With ELISA, 95 of the 115 sera tested (82.6%) yielded positive antichlamydial reactions. With CFT, 34 of the 119 sera tested (28.6%) were unequivocally positive (> or = 1:10, 100% binding), and 10 (7.6%) yielded doubtful positive reactions (1:10, 50-75% binding). Positive ELISA and CFT titers showed poor agreement (kappa = 0.112), whereas the agreement between positive findings by immunohistochemical labeling and CFT was fair (kappa = 0.205).