Mycoplasma hafezii sp. nov., isolated from the trachea of a peregrine falcon (Falco peregrinus).

Mycoplasma species are well known pathogens in avian medicine, especially in poultry. However, several Mycoplasma species have been regularly found in the respiratory tract of birds of prey which seem to be commensals in these bird species. In previous studies, an unknown Mycoplasma species which caused false positive results in a Mycoplasma meleagridis-specific PCR, was isolated from a tracheal swab of a clinically healthy, captive, adult peregrine falcon (Falco peregrinus). The isolate appeared in typical fried-egg-shaped colonies on SP4 agar plates and was dependent on sterol for growth. Acid was produced from glucose, but no arginine or urea was hydrolysed. The temperature range for growth was 28-44 °C, with an optimum at 37 °C. Strain M26T was serologically distinct from all species of the genus Mycoplasma with 16S rRNA gene sequence similarity ≥94 %. Biochemical, serological and molecular biological properties demonstrate that this organism represents a novel species of the genus Mycoplasma, for which the name Mycoplasma hafezii sp. nov. is proposed; the type strain is M26T (NCTC 13928, DSM 27652).

Soft plastron, soft carapace with skeletal abnormality in juvenile tortoises. Histopathology and isolation of a novel picornavirus from Testudo graeca and Geochelone elegans.

OBJECTIVE: A disease is described in juvenile tortoises (Testudo graeca and Geochelone elegans) consisting mainly of a soft carapace, soft plastron and deformed skeleton. The aim of this study was to determine histopathological lesions and the biological properties of the isolated viruses. MATERIALS AND METHODS: Clinical signs and gross pathology were determined on diseased and healthy appearing tortoises. Paraffin sections were stained with HE, PAS and Prussian Blue and histologically examined. Terrapene heart (TH-1) cell cultures served for virus isolations from 64 tissues and 104 swabs. One isolate (isolate 1243/37 tongue) was used in neutralization tests on 19 sera. RESULTS: Retarded growth and increasingly soft plastron and carapace were the prominent signs in diseased tortoises. Pathological lesions consisted of dilated urinary sac, enlarged kidneys and livers. Histopathologically, hepatic hemosiderosis, hypoplastic anaemia, congestive glomerulonephrosis and osteodystrophy were seen. A novel vi- rus ("virus X") was isolated from 64 organs and 79 of 104 swabs. The isolated viruses were identified as a novel chelonid picornavirus based on cytopathic effect, resistance to chloroform and stability at low pH. Co-cultivation with 5-iodo-2'-deoxyuridine and actinomycin D did not reduce virus titres. Electron microscopically, round, non-enveloped particles (25-30 nm) were detected. Neutralizing antibodies to the isolate 1243/37tongue were present in 17 of 19 sera from seven species of tortoises. CONCLUSION AND CLINICAL RELEVANCE: Nephropathy, osteodystrophy and virus isolations suggest a viral aetiology. Metabolic bone disease is the major differential diagnosis. Further investigations in vivo are needed to evaluate the likely effects of the picornavirus on tortoises.

[Diagnosis of Lawsonia intracellularis using the polymerase chain reaction (PCR) in pigs with and without diarrhea and other animal species]. / Diagnostik von Lawsonia intracellularis mittels Polymerasekettenreaktion (PCR) bei Schweinen mit und ohne Diarrhoe und anderen Tierarten.

Lawsonia (L.) intracellularis, an obligately intracellular bacterium, causes proliferative enteropathy (PE) in swine and, occasionally, in other animals. To determine the spread of the agent among German pig herds pooled fecal samples of five animals each of clinically normal Hessian pig herds collected between november 1998 and february 1999 as well as feces (n = 1684) from individual animals representing 648 herds, sent to our laboratory by veterinarians from all parts of Germany, were tested for L. intracellularis using the polymerase chain reaction (PCR). In addition, fecal samples from diarrhoic foals (n = 46), dogs (n = 57), cats (n = 50), calves (n = 37), hedge hogs (n = 9), seals (n = 8) and one giraffe were also studied. DNA was extracted from feces using high concentrations of chaotropic salt and diatomaceous earth. For PCR, primers flanking a 279 bp fragment of L. intracellularis DNA were used (JONES, G. F., WARD, G. E., MURTAUGH, M. P., LINN, G. (1993), J. Clin. Microbiol. 31, 2611-2615). Amplificates were separated by agarose gel electrophoresis and visualized under UV-light. L. intracellularis was found in 26 (12.8%) samples from 21 (30.0%) of the Hessian pig herds without symptoms of diarrhoea. In feces of pigs with diarrhoea (n = 1684) the agent was present in 431 (25.6%) samples originating from 224 (34.6%) herds. Of the other animal species studied, L. intracellularis was detected in feces of 4 (7.0%) dogs, 2 (5.4%) calves, 3 (33.3%) hedge hogs and in the sample of the giraffe. The remaining species were all tested negative.

Studies of epidemiology and seroprevalence of bovine noroviruses in Germany.

Jena virus (JV) is a bovine enteric calicivirus that causes diarrhea in calves. The virus is approximately 30 nm in diameter and has a surface morphology similar to the human Norwalk virus. The genome sequence of JV was recently described, and the virus has been assigned to the genus Norovirus of the family CALICIVIRIDAE: In the present study, the JV capsid gene encoded by open reading frame 2 was cloned into the baculovirus transfer vector pFastBac 1, and this was used to transform Escherichia coli to generate a recombinant bacmid. Transfection of insect cells with the recombinant baculovirus DNA resulted in expression of the JV capsid protein. The recombinant JV capsid protein undergoes self-assembly into virus-like particles (VLPs) similar to JV virions in size and appearance. JV VLPs were released into the cell culture supernatant, concentrated, and then purified by CsCl equilibrium gradient centrifugation. Purified JV VLPs were used to hyperimmunize laboratory animals. An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and characterized initially with clinical specimens containing defined human noroviruses and bovine diarrheal samples from calves experimentally infected with JV; the ELISA was specific only for JV. The ELISA was used to screen 381 diarrheal samples collected from dairy herds in Thuringia, Hesse, and Bavaria, Germany, from 1999 to 2002; 34 of these samples (8.9%) were positive for JV infection. The unexpectedly high prevalence of JV was confirmed in a seroepidemiological study using 824 serum or plasma samples screened using an anti-JV ELISA, which showed that 99.1% of cattle from Thuringia have antibodies to JV.

Prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern Germany.

Faecal samples from suckling (n = 205) and weaned piglets (n = 82) with diarrhoea from 24 farms in Southern Germany were examined for shedding of important metazoic parasitic, viral and bacterial pathogens using culture, microscopic and electronmicroscopic methods. Escherichia coli isolates were tested further for the enterotoxin genes est-Ia and elt-I by colony blot hybridization. Isospora suis was diagnosed in 26.9% and Cryptosporidium parvum in 1.4% of the piglets investigated. The proportion of coronavirus-positive animals was 13.4% and 4% were positive for rotavirus. It was found that 17.6% of the animals were infected with enterotoxigenic E. coli (ETEC; 10.1% ETEC-ST-Ia and 8.6% ETEC-LT-I, respectively). The occurrence of the pathogens was significantly associated with the age of the animals examined (P < 0.001). Isospora suis was predominantly isolated from suckling piglets (in the second and third week of life), while in weaned piglets (fourth week of life) rotavirus and ETEC were most prevalent. On 22 of the 24 piglet production farms examined at least one of the investigated pathogens was detected. Coronavirus was diagnosed in 66.7%, I. suis in 62.5%, rotavirus in 20.8% and C. parvum in 8.3% of the farms. These results underline the fact that despite the hygienic, technical and immune preventive efforts during the last years, enteropathogens are still common in German piglet production units.

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus.

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.

Demonstration of group C rotaviruses in fecal samples of diarrheic dogs in Germany.

Three out of 9 fecal samples from diarrheic dogs, which were positive for rotavirus by electron microscopy, revealed RNA-migration patterns identical to those of porcine group C rotavirus when studied by polyacrylamide gel electrophoresis (PAGE). The results were further confirmed by solid phase immuno electron microscopy (SPIEM). This is the first report of the occurrence of group C rotaviruses in dogs.

Isolation of a cytopathogenic virus from a case of porcine reproductive and respiratory syndrome (PRRS) and its characterization as parainfluenza virus type 2.

From a lung of a fetus of a breeding sow showing PRRS-like symptoms a viral agent could be isolated. It was characterized as an enveloped, hemagglutinating RNA virus. Ultrastructural examination of purified virus revealed paramyxovirus-like pleomorphic virions of approx. 200 nm in diameter. The helical nucleocapsids were about 18 nm in diameter. The virus was found to be antigenically related to simian virus 5 (SV5) a prototype strain of parainfluenza virus type 2, but not to bovine respiratory syncytial virus, parainfluenza virus type 1, parainfluenza virus type 3, and Newcastle disease virus as determined by western blot analysis.

The region between the M and S genes of porcine haemagglutinating encephalomyelitis virus is highly similar to human coronavirus OC43.

The nucleotide sequences of the regions between the membrane and spike protein genes of three strains of porcine haemagglutinating encephalomyelitis virus (HEV) were determined. A total of 739 (HEV strain 67N) and 751 (strains NT9 and VW572) nucleotides were sequenced. Two ORFs, potentially encoding proteins of 12.8 and 9.6 kDa, were identified. Pairwise comparisons with the corresponding ORFs in bovine coronavirus (BCV) and human coronavirus (HCV) OC43 revealed sequence similarities of greater than 88.5 percent at the nucleotide and 85.3 percent at the amino acid level for the 12.8 kDa ORF product. For the 9.6 kDa ORF product similarities were greater than 96.9 percent and 95.2 percent, respectively. An additional 12 nucleotide deletion upstream of the 12.8 kDa ORF start codon was found in HEV 67N compared to NT9 and VW572. These results reveal a genomic organization of HEV in the region analysed that is homologous to HCV OC43 but different from BCV.

[Electron microscopic demonstration of viruses in feces of dogs with diarrhea]. / Elektronenmikroskopischer Virusnachweis in Kotproben durchfallkranker Hunde.

4044 stool samples of dogs with diarrhoea were examined by electron microscopy. The samples were sent for routine diagnostics in the years 1988-1993. Over the examination period virus was detected in 32% of the samples. Parvovirus was diagnosed in 17.2% and coronavirus in 12.4% of the cases. The number of parvovirus-positive samples was lower than in former years, whereas the number of coronavirus-positive samples was higher. Other virus particles (paramyxo-, picorna-, calici- and astrovirus or morphologically similar particles as well as rota- and adenovirus) were altogether detected in 2.5% of the samples. This detection rate corresponded to the results of former years. The majority of parvovirus-positive samples (80.7%) was found in animals aged between six weeks and six months. Of the coronavirus-positive samples 56.5% were detected in dogs older than six months of age and 42.5% in animals between six weeks and six months. In puppies up to six weeks viruses were only detected infrequently (parvovirus 4.7%, coronavirus 0.9%).

Genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus OC43 as studied by the use of bovine coronavirus S gene-specific probes.

The genomic relationship of porcine hemagglutinating encephalomyelitis virus (HEV) to bovine coronavirus (BCV) and human coronavirus (HCV) strain OC43 was examined by dot blot hybridization assays. Two BCV S gene-specific probes were generated by polymerase chain reaction from the avirulent L9-strain of BCV. Probes were located in the S1 and the S2 region of the peplomeric (S) glycoprotein gene. The S1 probe (726 bp) hybridized with BCV and HCV-OC43, but not with HEV under moderate stringency hybridization conditions (50 degrees C). Only slight signals were present with mouse hepatitis virus (MHV) and no signals were observed with feline infectious peritonitis virus (FIPV) or canine coronavirus (CCV). At high stringency conditions (60 degrees C) the S1 probe hybridized with BCV only. Using the S2 probe (680 bp) under moderate stringency conditions, hybridization signals were obtained with BCV, HCV-OC43 and HEV (strains 67N, NT9, VW572). The signals obtained by the three HEV strains were altogether weaker than with BCV and HCV-OC43. The S2 probe did not react with MHV, FIPV and CCV. At high stringency the S2-specific probe hybridized with BCV and HCV-OC43 but did not hybridize with HEV. Nucleotide sequence analysis of the region covering the S2 probe in HEV revealed 92.6% nucleotide sequence homology to BCV and 91.9% to HCV-OC43. In contrast, the region covering the S1 probe in HEV could not be amplified using the BCV S1-specific primers. The hybridization and sequencing results thus indicate a closer genomic relationship between BCV and HCV-OC43 than there is between HEV and BCV or HCV-OC43 respectively.

Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child.

The coronavirus strain HECV-4408 was isolated from diarrhea fluid of a 6-year-old child with acute diarrhea and propagated in human rectal tumor (HRT-18) cells. Electron microscopy revealed coronavirus particles in the diarrhea fluid sample and the infected HRT-18 cell cultures. This virus possessed hemagglutinating and acetylesterase activities and caused cytopathic effects in HRT-18 cells but not in MDBK, GBK and FE cells. One of four S-specific monoclonal antibodies reacted in Western blots with HECV-4408, BCV-L9 and BCV-LY138 but not with HCV-OC43, and two reacted with BCV-L9 but not with HECV-4408, BCV-LY138 and HCV-OC43. One S-specific and two N-specific monoclonal antibodies reacted with all of these strains. cDNA encompassing the 3' 8.5 kb of the viral RNA genome was isolated by reverse transcription followed by polymerase chain reaction amplification had size and restriction endonuclease patterns similar to those of BCV-L9 and BCV-LY138. In contrast, the M gene of HCV-OC43 differed in restriction patterns from HECV-4408 and BCV. A genomic deletion located between the S and M within the non-structural genes of HCV-OC43 was not detected in HECV-4408. DNA sequence analyses of the S and HE genes revealed more than 99% nucleotide and deduced amino acid homologies between HECV-4408 and the virulent wild-type BCV. Forty-nine nucleotide and 22 amino acid differences were found between the HE genes of HECV-4408 and HCV-OC43, while only 16 nucleotide and 3 amino acid differences occurred between the HE genes of HECV-4408 and BCV-LY138. We thus conclude that the strain HECV-4408 is a hemagglutinating enteric coronavirus that is biologically, antigenically and genomically more closely related to the virulent BCV-LY138 than to HCV-OC43.

Comparison of the S genes and the biological properties of respiratory and enteropathogenic bovine coronaviruses.

The nucleotide sequence of the S gene of the bovine respiratory coronavirus (BRCV) strain G95, which was isolated from nasal swabs of a calf suffering from respiratory disorders, was determined and compared with the S gene of the enteropathogenic bovine coronavirus (BECV) strain LY138. Sequence analysis revealed 98.7% nucleotide and 98.3% deduced amino acid identities between the S genes of BRCV-G95 and BECV-LY138 without any deletions or insertions. Nucleotide substitutions were distributed randomly throughout the gene. Five monoclonal antibodies specific for the S protein distinguished BRCV-G95 from BECV-L9, but failed to differentiate it from BECV-LY138 in Western blots under denatured and native conditions. BRCV-G95 induced cytopathic changes in cell cultures that were similar to BECV-LY138 but different from BECV-L9. These results suggest that strain BRCV-G95 is more closely related to the virulent strain BECV-LY138 than to the avirulent, cell culture-adapted strain BECV-L9.

Characterization of a reovirus isolate from a rattle snake, Crotalus viridis, with neurological dysfunction.

A virus isolate from the brain of a rattle snake with central nervous system (CNS) symptoms was identified as a reovirus. The isolate did not agglutinate pig erythrocytes and was not neutralized by antisera against avian reovirus S1133 and mammalian reovirus type 3. The cytopathic effect in Vero cells was characterized by formation of syncytial giant cells. Electrophoretic analysis of the genome revealed 10 segments of dsRNA. The isolate displayed characteristics distinct from avian and mammalian reoviruses.

Localization of vasoactive intestinal polypeptide-receptor-immunoreactivity in human salivary glands.

A monoclonal VIP-receptor antibody derived from a human adenocarcinoma cell line (HT 29) was used in combination with immunogold silver staining for the immunohistochemical demonstration of VIP-receptor (rec) immunoreactivity (IR) in paraffin-embedded human salivary glands (parotid, palatal, labial glands). VIP-rec-IR was localized to mucous endpieces of labial and--to a lesser extent--palatal glands, intercalated ducts of the parotid gland, and excretory ducts of all glands investigated. The findings correlate well with known effects of VIP on mucous release and electrolyte transport in salivary glands. Lack of VIP-rec-IR at serious acini may point to immunologically different receptor subtypes in these glands.

[Rabbit hemorrhagic disease (RHD)--comparative diagnostic studies using the hemagglutination test and electron microscopy]. / Rabbit Haemorrhagic Disease (RHD)--Vergleichende diagnostische Untersuchungen mittels Hämagglutinationstest und Elektronenmikroskopie.

A total of 56 liver specimens from rabbits with symptoms of rabbit haemorrhagic disease were tested for virus by electron microscopy (EM) and haemagglutination (HA). Both methods simultaneously gave positive or negative results in 28 or 22 cases, respectively. Divergent results were obtained in only 6 samples. Five of them were positive by EM but negative by HA and in one specimen with a HA-titer of 1:32 virus could not be detected.

[Virologico-serologic studies in horses with respiratory tract diseases]. / Virologisch-serologische Untersuchungen bei Pferden mit Atemwegserkrankungen.

Of 1081 acute and chronically respiratory diseased as well as clinically normal horses 824 sera and 257 paired serum samples collected 1986 and 1987 were tested for antibodies against several different respiratory viruses such as influenza virus A/equi 1 and 2 (Influenza 1 a. 2), equine herpesvirus type 1/4 (EHV 1/4), mammalian reovirus type 1-3 (Reovirus 1-3), equine rhinovirus type 1 (ERV 1), equine adenovirus type 1 (EAdV 1), and equine arteritis virus (EAV). The investigations resulted in an antibody prevalence of 57.2% (Influenza 1), 59.5% (Influenza 2), 81.5% (EHV 1/4), 50.3% (Reovirus 1), 43.0% (Reovirus 2), 75.9% (Reovirus 3), 97.6% (EAdV 1), 82.5% (ERV 1) and 8.7% (EAV). With exception of EAV and EAdV 1 the ratios usually were higher in diseased animals than in clinically normal horses. Antibodies to EAV and EAdV 1 were present in all groups to almost the same amount. Of 257 horses with acute respiratory illness 3 showed a significant rise of the antibody titer against Influenza 1, 30 against Influenza 2, 54 against EHV 1/4, 1 against Reovirus 1 and 3, respectively, 11 against EAdV 1 and 26 against ERV 1.

The innervation of the great saphenous vein: an immunohistochemical study with special regard to regulatory peptides.

The human great saphenous vein is often used in by-pass surgery. In spite of its importance little is known about the neuronal regulation and innervation of this vessel. On the other hand, therapeutic phlebectomy of the saphenous vein provides a good basis for investigations on the nervous supply of human veins. In former studies the human great saphenous vein has been proved to be a richly innervated portion of the low pressure vascular system. We studied the innervation of the proximal femoral saphenous vein by immunohistochemical methods and with special regard to regulatory neuropeptides. A peptidergic innervation mainly localized along the vasa vasorum and associated with immunoreactivity of substance P and CGRP was found which yields evidence for a mechanosensory innervation of this vascular segment.