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11 páginas, 11 figuras, 4 tablas.-- El Pdf del artículo es la versión pre-print: arXiv:1101.2522v2.-- MAGIC Collaboration: et al., [Context]: 3C 279, the first quasar discovered to emit VHE γ-rays by the MAGIC telescope in 2006, was reobserved by MAGIC in January 2007 during a major optical flare and from December 2008 to April 2009 following an alert from the Fermi space telescope on an exceptionally high γ-ray state. [Aims]: The January 2007 observations resulted in a detection on January 16 with significance 5.4σ, corresponding to a F (>150 GeV) (3.8 ± 0.8) × 10-11 ph cm-2 s-1 while the overall data sample does not show significant signal. The December 2008–April 2009 observations did not detect the source. We study the multiwavelength behaviour of the source at the epochs of MAGIC observations, collecting quasi-simultaneous data at optical and X-ray frequencies and for 2009 also γ-ray data from Fermi. [Methods]: We study the light curves and spectral energy distribution of the source. The spectral energy distributions of three observing epochs (including the February 2006, which has been previously published) are modelled with one-zone inverse Compton models and the emission on January 16, 2007 also with two zone model and with a lepto-hadronic model. [Results]: We find that the VHE γ-ray emission detected in 2006 and 2007 challenges standard one-zone model, based on relativistic electrons in a jet scattering broad line region photons, while the other studied models fit the observed spectral energy distribution more satisfactorily., The support of the German BMBF and MPG, the Italian INFN, the Swiss National Fund SNF, and the Spanish MICINN is gratefully acknowledged. This work was also supported by the Marie Curie program, by the CPAN CSD2007-00042 and MultiDark CSD2009-00064 projects of the Spanish Consolider-Ingenio 2010 programme, by grant DO02-353 of the Bulgarian NSF, by grant 127740 of the Academy of Finland, by the YIP of the Helmholtz Gemeinschaft, by the DFG Cluster of Excellence “Origin and Structure of the Universe”, and by the Polish MNiSzW Grant N N203 390834.

DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.

Hemagglutinating and acetylesterase functions as well as the 124 kDa glycoprotein were present in the highly cell-culture adapted, avirulent bovine coronavirus strain BCV-L9, in the Norden vaccine strain derived from it, and in 5 wild-type, virulent strains that multiplied in HRT-18 cells but were restricted in several types of cultured bovine cells. The BCV-L9 and the wild-type strain BCV-LY-138 agglutinated chicken and mouse erythrocytes. The acetylesterase facilitated break-down of the BCV-erythrocyte complex with chicken but only to a minimal extent with mouse erythrocytes in the receptor-destroying enzyme test. Purified preparations of the vaccine and the wild-type strains agglutinated chicken erythrocytes at low titers and mouse erythrocytes at 128 to 256 times higher titers whereas receptor destroying enzyme activity was detectable only with chicken erythrocytes. When wild-type strains were propagated in HRT cells at low passage levels, they produced 5 x 10(5) to 4.5 x 10(6) plaque forming units per 50 microliters which agglutinated erythrocytes from mice but not from chickens. Diisopropylfluoro-phosphate moderately increased the hemagglutination titers, but completely inhibited the receptor destroying enzyme of purified virus of all strains. It had virtually no influence on the plaque-forming infectivity of the different BCV strains. The acetylesterase of strain BCV-L9 reacting in the receptor-destroying enzyme test was stable for 3 h at 37 and 42 degrees C. It was inactivated within 30 min at 56 degrees C while the hemagglutinin function of this strain was stable for 3 h at 37, 42, and 56 degrees C, but it was inactivated at 65 degrees C within 1 h.

The entire nucleotide sequences of the hemagglutinin/esterase (HE) genes specified by the highly virulent strain LY138 and the avirulent strain L9 of bovine coronavirus (BCV) were determined. These sequences were compared with recently published sequences of the HE genes of the Quebec and Mebus strains. A large open reading frame of 1272 nt encoding a protein of 424 amino acid residues was predicted. The putative esterase active site was conserved in the virulent and avirulent BCV strains, indicating that this domain is probably not a determinant for BCV virulence. Four amino acid substitutions occurred between the HE proteins of BCV-L9 and BCV-LY138 (leu to Pro at 5, Leu to Val at 103, Ser to Pro at 367, and Thr to Asn at 379). Monoclonal antibodies specific for the HE glycoprotein inhibited the hemagglutination and acetylesterase activities of BCV-L9, but showed no inhibitory effect on the acetylesterase activity of BCV-LY138. These results suggest that at least one epitope is located proximal to one of the three strain-specific amino acids. Four S-specific monoclonal antibodies inhibited hemagglutination but not acetylesterase activity of BCV-L9, implying that the S glycoprotein can promote hemagglutination of chicken erythrocytes in addition to the HE glycoprotein.

The entire nucleotide sequences of the spike glycoprotein (S) genes of the highly virulent bovine coronavirus (BCV) strain BCV-LY138, the avirulent BCV-L9 and related Norden Vaccine (BCV-Vaccine) strains were determined using the polymerase chain reaction (PCR) to amplify cDNAs obtained by reverse transcription of viral RNA, and to produce single strand cDNAs for DNA sequencing. The S gene sequences of these viral strains were compared with those of recently published strains BCV-Mebus, BCV-Quebec, and BCV-F15. An open reading frame of 4092 nucleotides, encoding a protein of 1363 amino acid residues, was found in all six strains. Frameshifts and insertions or deletions were not observed except for the BCV-Fl 5. The S gene sequences were more than 98% conserved overall inspite of different origins of the six viruses. There were 45 to 56 nt differences between the virulent and avirulent groups while there were 6 to 14 nt differences among four avirulent strains. Comparison of the deduced amino acid sequences indicated that the S proteins had typical properties of membrane glycoproteins. Nineteen Winked glycosylation sites were predicted in five strains, and 18 of them were conserved in the avirulent strain BCV-L9. The sequence KRRSRR at the predicted proteolytic cleavage site was identified in five strains while the sequence KRRSVR was found in BCV-Fl 5. Substitutions of few amino acids in the putative fusogenic domains and two prolines at 507 and 567 in the antigenic domains may cause altered immunogenic and other functional properties of the S proteins specified by the virulent and avirulent BCV strains. Nine amino acid substitutions between the virulent and avirulent groups may correlate with BCV virulence.

Monoclonal antibodies (MAbs) against two major structural proteins of the cell-adapted Mebus strain of bovine coronavirus (BCV-L9) were produced and characterized. Seven MAbs reacted with the peplomeric glycoprotein, gp100/S, while three MAbs reacted with the nucleoprotein p53/N in Western blot analysis of BCV polypeptides. MAbs to gp100/S reacted with discontinuous epitopes of gp100/S in Westerns under mild but not under standard denaturing conditions. In contrast, MAbs to p53/N reacted in both types of Westerns, and those epitopes were thus continuous. MAbs to p53/N failed to neutralize BCV infectivity, while 4 MAbs to gp100/S neutralized BCV effectively. Cross reactivity of MAbs to gp100/S specified by five virulent wild-type strains and two high passage, cell-culture-adapted strains in mildly denaturing Westerns and neutralization assays indicated that two epitopes were conserved in all seven strains, while two epitopes of the avirulent strains were not detected in the wild-type strains. Non-neutralizing MAbs of gp100/S reacted with all seven strains in Westerns with the exception of one MAb that was specific for the highly cell-adapted strain BCV-L9.

Expression of bovine coronavirus (BCV) antigen in the plasmalemma of epithelioid human rectal tumor (HRT-18) and fibroblastic bovine fetal spleen (BFS) cell lines was traced by immunofluorescence and immunoelectron microscopy facilitated by colloidal gold. Cytoplasmic fluorescence was first observed at 12 hr postinfection (h.p.i) in infected HRT-18 cultures. This fluorescence coincided with the appearance of cell surface antigen reacting with colloidal gold-labeled antibodies to BCV antigens. At 24 h.p.i the amount of viral antigens at the surface of HRT-18 had increased, although cytoplasmic fluorescence remained constant. Infected BFS cells but not HRT-18 cells formed polykaryons when incubated in the presence of trypsin. One viral antigen in the plasma membrane of BFS cells was thus identified as the S glycoprotein with a fusion domain. In contrast to HRT-18 cells, the overall amount of BCV antigens at the surface of BFS cells remained constant after the onset of fusion. Analysis of the labeling characteristics established that the goldmarked-sites represented de novo expression of BCV antigen in the plasma membrane of infected cells.

Summary The early events in the infection of human rectal tumor cells by bovine coronavirus were investigated by colloidal gold-mediated immunoelectron microscopy and by analysis of the effect of lysosomotropic weak bases on virus yield. Electron microscopic studies revealed sites of fusion between the virus envelope and the plasmalemma but fusion events along intracellular membranes were not observed despite extensive searches. Virion-antibody-colloidal gold complexes were, in fact, endocytosed by synchronously infected cells. These complexes were apparently non-infectious, and they accumulated in vacuoles that resembled secondary lysosomes. Exposure of cells to ammonium chloride or to methylamine during the first hour of infection had little inhibitory effect on the production of infectious virus. Chloroquine treatments were inhibitory but this effect depended on relatively late events in the infectious process. The chloroquine inhibitory step blocked infection of virus adsorbed to cells that were exposed to buffers in the pH range of 4.4 to 8.4. These findings indicate that BCV penetrates its host cell by direct fusion with the plasmalemma and does not require an acidic intracellular compartment for infectious entry.

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization.

Summary The ecology of cytopathic expression of bovine coronavirus (BCV) in HRT-18 cells was analyzed within virus-induced plaques by scanning electron microscopy. Virus replication was cytocidal for many HRT-18 cells, a function enhanced in the presence of trypsin. A monolayer of cells remained that imparted a characteristic turbidity to the plaque. These structurally normal, lysisresistant cells did not stain with fluorescent antibodies specific for BCV antigens, failed to adsorb virus particles or mouse erythrocytes in contrast to the susceptible cells. The survival of cells in the plaque interior reflects a non-productively infected population with evidence of viral persistence.

We report control of magnetic anisotropy in epitaxial $(\mathrm{Ga},\mathrm{Mn})\mathrm{As}$ by anisotropic strain relaxation in patterned structures. The strain in the structures is characterized using reciprocal space mapping by x-ray techniques. The magnetic anisotropy before patterning of the layer, which shows biaxial easy axes along [100] and [010], is replaced by a hard axis in the direction of large elastic strain relaxation and a uniaxial easy axis in the direction where pseudomorphic conditions are retained.

The complete genome sequences are reported here of two field isolates of bovine coronavirus (BCoV), which were isolated from respiratory and intestinal samples of the same animal experiencing fatal pneumonia during a bovine shipping fever epizootic. Both genomes contained 31028 nucleotides and included 13 open reading frames (ORFs) flanked by 5′- and 3′-untranslated regions (UTRs). ORF1a and ORF1b encode replicative polyproteins pp1a and pp1ab, respectively, that contain all of the putative functional domains documented previously for the closest relative, mouse hepatitis virus. The genomes of the BCoV isolates differed in 107 positions, scattered throughout the genome except the 5′-UTR. Differences in 25 positions were non-synonymous and were located in all proteins except pp1b. Six replicase mutations were identified within or immediately downstream of the predicted largest pp1a-derived protein, p195/p210. Single amino acid changes within p195/p210 as well as within the S glycoprotein might contribute to the different phenotypes of the BCoV isolates.

Over the last few years numerous respiratory bovine coronaviruses (RBCV) were isolated from respiratory infections of cattle including two shipping fever epizootics (Storz et al., 1996; Storz et al., 2000). The RBCV strain 98TXSF-110-LU (LU) and the enteric strain 98TXSF-110-ENT (ENT) were isolated from lung and intestinal samples, respectively, from an animal experiencing fatal respiratory disease during a 1998 shipping fever epizootic. We obtained and compared the entire genomic sequences of the LU and ENT isolates to delineate nucleotide (nt) and amino acid (aa) differences between them. In addition, we analyzed the leader-mRNA junction sequences of the S and 12.7 kDa mRNAs specified by different BCV strains.

Numerous respiratory bovine coronaviruses (RBCV) were isolated recently from nasal swab samples and lung tissues of feedlot cattle with acute respiratory tract disease. These newly emerging RBCV isolates exhibited distinct phenotypic features that differentiated them from enteropathogenic bovine coronaviruses (EBCV). The RBCV strains had a receptor-destroying enzyme function mediated by acetylesterase (AE) activity of the haemagglutinin-esterase (HE) glycoprotein. The HE genes of wild-type EBCV strain LY138 and RBCV strains OK-0514 (OK) and LSU-94LSS-051 (LSU) were cloned, sequenced and transiently expressed in COS-7 cells. The enzymic properties of HE proteins in COS-7 cellular extracts and in purified virus preparations were assayed at room temperature, 37 degrees C and 39 degrees C by two different assays. One assay used p-nitrophenyl acetate (PNPA) as substrate and detected serine-esterase activity; the second assay monitored AE function with bovine submaxillary mucin (BSM) as substrate. The PNPA tests confirmed that HE proteins of EBCV and RBCV were functionally expressed in transfected COS-7 cells. Time-dependent determination of the AE activity of purified RBCV OK and LSU particles showed lower AE activity at 39 degrees C than at 37 degrees C, whereas the purified EBCV LY particles retained full AE activity at both 37 degrees C and 39 degrees C. Transiently expressed RBCV HE exhibited a marked reduction of AE activity after 40 min of assay time at 37 degrees C. In contrast, the AE activity of the transiently expressed EBCV HE remained stable beyond 40 min. The deduced amino-acid sequences of the HE proteins specified by the RBCV strains OK and LSU contained specific amino-acid changes in comparison with the EBCV LY strain, which may be responsible for the observed enzymic differences. These results are consistent with the hypothesis that RBCV strains have evolved to selectivelyreplicate in respiratory tissues and that HE may play a role in this tissue tropism.

A monoclonal antibody-based capture enzyme-linked immunosorbent assay (ELISA) was developed to detect respiratory bovine coronavirus (RBCV) antigens in nasal swabs collected from cattle showing signs of respiratory tract disease following shipping. These samples had been previously tested for RBCV by inoculation of G clone cultures of human rectal tumor cells (HRT-18G) and for bovine herpes virus 1, parainfluenza virus 3, bovine adenovirus, bovine respiratory syncytial virus, and bovine viral diarrhea virus on other specifically permissive cell cultures. RBCV has not previously been recognized as an important etiological factor in the bovine respiratory disease complex of feedlot cattle. Thirty of 100 samples tested positive for RBCV antigen by capture ELISA in contrast to 38 of 100 samples that yielded RBCV isolates in G clone cells. Samples yielding other bovine respiratory viruses in the absence of RBCV were negative in the capture ELISA, which was based on the use of a single monoclonal antibody that recognizes one RBCV epitope on the S glycoprotein with the broadest reactivity with different strains of RBCV tested. Some RBCV strains may not be detected by this ELISA, which may account for the higher percentage of RBCV-infected cattle detected by RBCV isolation. However, the ELISA was simple to perform, sensitive, and specific and was more rapid than virus isolation. This assay will be useful for processing large numbers of field samples in future epidemiologic and diagnostic studies of RBCV infections of cattle.

To investigate the susceptibility of polarized epithelioid human rectal tumor (HRT-18G) cells to bovine coronaviruses (BCV) isolated from enteric (EBCV) and respiratory (RBCV) tract infections.Cells of the G clone of HRT-18 were grown to confluent monolayers on permeable supports, and were directionally infected at the apical and basolateral domains with 3 wild-type BCV strains, RBCV-LSU-94LSS-051-2, RBCV-OK-0514-3, and EBCV-LY138-2, and 1 cell culture-adapted strain, EBCV-L9-80. Sequential cytopathic changes were microscopically monitored. Medium samples for titration of hemagglutinins and viral infectivity were collected directionally from both domains of the infected cell cultures at various intervals.Polarized epithelioid HRT-18G cells from apical domains had maximal susceptibility to infection with the EBCV and RBCV strains, and those from basolateral surfaces had minimal susceptibility. Titers of hemagglutinins and infective progeny BCV reached 1,280 hemagglutinin units and 4.2 x 10(8) plaque-forming units/ml for apical samples, but were minimal for basolateral samples. Asymmetric virus release occurred through the apical surfaces of the HRT-18G cells by 12 hours after infection when cell fusion as a sign of cytopathic changes began. When cells were infected basolaterally, progeny virions released from apical surfaces reinfected the target cells from the apical domains and induced cytopathic changes were delayed about 12 hours, compared with changes detectable in apically exposed cultures.EBCV and RBCV, isolated from cattle, had marked tropism for polarized epithelioid HRT-18G cells. Entry of BCV into the polarized HRT-18G cells was effected maximally through the apical domains and minimally through the basolateral domains. Release of progeny BCV occurred preferentially from the apical domains.

To analyze the antigenic cross reactivity of various proteins of strains of Chlamydia pecorum, C psitaci, and C trachomatis. SAMPLES AND PROCEDURES: Strains FC-Stra and LW-613 of C pecorum, strains B577, Fitz-9, and 68C of C psitaci, and strain LGV-2 of C trachomatis were studied. Strains of C pecorum were propagated in Georgia bovine kidney cells, and other chlamydial strains were propagated in L cells or Georgia bovine kidney cells. Partially purified chlamydial elementary bodies propagated in RAG cells, a BALB/c cell line cloned from a renal adenocarcinoma of BALB/c mice, were used to immunize BALB/c mice for production of monoclonal antibodies. Rabbits were inoculated with yolk sack-propagated, purified elementary bodies to produced polyclonal antisera. The reaction of monoclonal antibodies and polyclonal antisera with chlamydial proteins was analyzed by use of immunoblot techniques.Two monoclonal antibodies reacted with a 90-kd protein of C psittaci and a 94-kd protein of C pecorum strains. One monoclonal antibody reacted strongly with a 67-kd protein of C pecorum and strain B577 of C psittaci, but weakly with proteins of strains 6BC and LGV-2. Another monoclonal antibody reacted with a 46-kd protein of 2 C pecorum strains and of strain B577 of psittaci, but not with those of strains 6BC and LGV-2. Two monoclonal antibodies reacted with a 20-kd protein of C pecorum and a 22-kd protein of C psittaci and LGV-2 strains. Polyclonal antisera reacted similarly with the proteins identified by monoclonal antibodies in the various chlamydial strains.Reactions of several monoclonal antibodies with chlamydial antigens indicated that 67- and 46-kd proteins contain genus- and species-specific epitopes, respectively; a 94-kd protein of C pecorum is homologous to a 90-kd protein of C psittaci and C trachomatis strains; and a 20-kd protein of C pecorum corresponds to a 22-kd protein of C psittaci and C trachomatis.

To monitor the prevailing viral respiratory tract infections in cattle after transportation to feedlots.100 cattle with signs of respiratory tract disease on arrival at 2 feedlots.Nasal swab samples were obtained from each animal and were used for inoculation of defined cell culture systems that detected bovine viruses known to cause respiratory tract infections, as well as viruses previously not recognized as respiratory pathogens for cattle.Bovine respiratory coronaviruses were isolated from 38 of the 100 cattle, including 6 of 50 cattle from California, 22 of 31 cattle from Oklahoma, 6 of 11 cattle from Texas, and 4 of 8 cattle of unknown origin. Parainfluenza 3 viruses also were isolated from 5 California cattle, but other bovine viruses were not detected.The high rate of coronavirus isolations from feedlot cattle with signs of respiratory tract disease implied wide distribution and high susceptibility among cattle to this infection, which had not been detected by use of viral isolation systems in previous etiologic evaluations of feedlot cattle affected with bovine respiratory disease complex.

To investigate the antigenic diversity of lipo-oligosaccharides of Haemophilus parasuis.Immunoblot assays were done with monoclonal and polyclonal antibodies on whole-cell lysates. Individual colonies of H parasuis strains H 54, H 53, and H 128 were tested for reactivity with lipo-oligosaccharide-specific monoclonal antibodies after a single passage on chocolate agar, and colonies of strain H 54 were analyzed after 10 passages. Colony blot tests were used to screen H parasuis strains for spontaneously occurring antigenic variation in their lipo-oligosaccharides.Eight H parasuis strains were separated into 4 lipo-oligosaccharide serovars on the basis of immunoblot reactions with 3 polyclonal rabbit antisera. Nine monoclonal antibodies against lipo-oligosaccharides of a lipo-oligosaccharide-serovar I strain reacted with all tested serovar I strains but failed to react with other H parasuis strains.Variations in the antigenic reactivity after 1 or 10 passages on chocolate agar were not observed. The serovar I lipo-oligosaccharide strains included virulent as well as avirulent H parasuis strains, indicating that these epitopes do not correlate directly with virulence properties of H parasuis.

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 nonstructural 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 BCVLY138. 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 BCVLY138 than to HCV‐OC43. © 1994 Wiley‐Liss, Inc.

Design-basis transuranic (TRU) waste to be emplaced in the Waste Isolation Pilot Plant (WIPP) in southeastern New Mexico may generate significant quantities of gas, which may affect the performance of the WIPP with respect to regulations for radioactive and/or chemically hazardous waste constituents. We are developing a model to predict gas generation in WIPP disposal rooms during and after filling and sealing. Currently, the model includes: (1) oxic and anoxic corrosion of steels and other Fe-base alloys, including passivation and depassivation; (2) microbial degradation of cellulosics with O2, NO3-, FeO(OH) , SO42-, or CO2as the electron acceptor; (3) α radiolysis of brine; (4) consumption of CO2and, perhaps, H2S by Ca(OH)2(in cementitious materials) and CaO (a potential backfill additive). The code simulates these processes and interactions among them by converting reactants (steels, cellulosics, etc.) to gases and other products at experimentally observed or estimated rates and plotting temporal reaction paths in three-dimensional phase diagrams for solids in the Fe-H2O-CO2-H2-H2S system.

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and by the morphologic features and ultrastructure of intracellular inclusions. Amplified chlamydial ompA DNA fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (RB) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet RB were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 RB, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the RB as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.

A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.

The complete nucleotide sequences of the hemagglutinin/esterase (HE) genes of human coronavirus (HCV) strain OC43 and bovine respiratory coronavirus (BRCV) strain G95 were determined from single-stranded cDNA fragments generated by reverse transcription of virus-specific mRNAs and amplified by polymerase chain reaction. An open reading frame of 1272 nucleotides was identified as the putative HE gene by homology to the bovine coronavirus HE gene. This open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kDa. Ten potential N-linked glycosylation sites were predicted in the HE protein of HCV-OC43 while nine of them were present in BRCV-G95. Fourteen cysteine residues were conserved in the HE proteins of both viruses. Two hydrophobic sequences at the N-terminus and the C-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. The predicted HE protein of HCV-OC43 was 95% identical to the HEs of BRCV-G95 and other bovine coronaviruses, and 60% identical to the HEs of mouse hepatitis viruses. Phylogenetic analysis suggests that the HE genes of coronaviruses and influenza C virus have a common ancestral origin, and that bovine coronaviruses and HCV-OC43 are closely related.

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 degrees C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.

The primary disease mechanism in infections of calves with enteropathogenic bovine coronaviruses (BCV) involves cytocidal interaction with differentiated enterocytes. Cytopathic expression of BCV infection of cultured cells depends on the viral strain and the cell type. BCV-induced cell fusion occurred under neutral or alkaline but not acidic conditions. Lysosomotropic bases did not significantly reduce virus yield, suggesting that productive BCV infection was not mediated by endocytosis but rather by direct fusion of the viral envelope with the plasma membrane. Trypsin cleavage of the 185 kDa S glycoprotein into 100 and 110 kDa subunits was required for cell fusion and productive infection of cultured BFS cells with the cell-adapted, avirulent strain BCV-L9. This strain has a wide host cell range in vitro while several wild-type, virulent strains were restricted to HRT-18 cells. Antigenic and genomic comparisons of the avirulent and virulent strains revealed corresponding differences. Some monoclonal antibodies against S reacted with both virulent and avirulent strains, while others reacted only with the avirulent strain BCV-L9. Nucleotide sequences of the S genes indicated that amino acid substitutions in cleavage sites, antigenic regions, and putative fusion domains occurred and differentiated BCV strains.

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.

Chlamydial isolates of bovine origin were serotyped by a plaque reduction method. Of the two major serotypes observed, type 1 included isolates from bovine abortion and enteric infections, whereas type 2 isolates were associated with polyarthritis or encephalomyelitis. These two serotypes were identical to those with a similar disease distribution previously observed in isolates of ovine origin. The two groups did not cross-react and they were serologically unrelated to chlamydiae of avian origin. Thus, it appears that many chlamydial isolates causing intestinal infections or abortion in sheep or cattle are closely related antigenically, as are those producing polyarthritis, encephalomyelitis, and conjunctivitis, and that the two groups are distinct.

Summary Bovine Herpesvirus Type 4: Studies on the Biology and Spread in Cattle Herds and in Insemination Bulls A serological survey with approx. 3,000 bovine sera revealed that 18.4% of the animals tested from West Germany and 8.7% from the USA harboured antibodies against bovine herpesvirus-4. A large number of BHV-4 reactors was found among healthy bulls in different German bull studs. In one stud with almost 70% seropositive animals an incidence rate of ca. 5% was observed. Virus could not be isolated from the semen of some of these animals. No correlation between the number of BHV-4 positive animals and respiratory or genital distress, Infectious Bovine Rhinotracheitis or Malignant Catarrhal Fever was detectable. The virus has a comparatively long replication cycle in tissue culture and a wide host cell range. Eight isolates were indistinguishable by serological means and no cross-reactions with BHV-1, —2, —3 and —6 were detectable, when tested by indirect fluorescence assays. Using this test, virus specific antigens could be stained in the nucleus of infected cells from 10 hours p. i. onwards and four hours later also in the cytoplasm. Zusammenfassung Eine serologische Untersuchung mit etwa 3000 Rinderseren zeigte, das in der Bundesrepublik 18,4%, in den USA 8,7% aller getesteten Tiere Antikorper gegen das bovine Herpesvirus-4 besasen. Auffallig viele Reagenten befanden sich dabei unter den gesunden Bullen verschiedener deutscher Besamungsstationen. In einer Station mit fast 70% Reagenten traten in einem Jahr knapp 5% Neuinfektionen auf. Versuche, aus dem Sperma dieser Tiere Virus zu isolieren, blieben erfolglos. Zusammenhange zwischen der Anzahl BHV-4-positiver Tiere und dem Auftreten von Erkrankungen des Respirations- oder Genitaltraktes, infektioser boviner Rhinotracheitis und bosartigem Katarrhalfieber liesen sich nicht ermitteln. In vitro zeichnet sich dieses Virus durch eine relativ langsame Replikation und ein breites Wirtszellspektrum aus. Wahrend 8 untersuchte Isolate serologisch einheitlich reagierten, liesen sich Kreuzreaktionen mit BHV-1, —2, —3 und —6 im indirekten Fluoreszenzantikorpertest nicht feststellen. Mit Hilfe dieses Testes waren virusspezifische Antigene in infizierten Zellen ab 10 Stunden p.i. im Kern, 4 Stunden spater auch im Cytoplasma nachweisbar.

Summary Polykaryon formation in bovine fetal spleen (BFS) cells infected with bovine coronavirus L9 occurred only in media supplemented with trypsin. A single 1 to 2 h trypsin treatment 10 h and later after infection induced formation of polykaryons. Trypsin treatment at pH 7.5 and 8.0 induced polykaryons while treatments at lower or higher pH levels did not. Cell fusion activity was partially suppressed by the presence of antibody.

The growth characteristics of Chlamydia psittaci serotypes 1 and 2 (ovine and bovine origin) were studied in mouse L cells. Formation of inclusions and yield of infectious progeny for serotype 1 were maximal when host cells were treated with cycloheximide and the pH in the cell culture medium was 7.2-7.4. The number of cells that contained inclusions and the infectivity yield for serotype 2 were maximal when the pH was 6.6-7.0. Treatments with diethylaminoethyl dextran and cycloheximide increased inclusion formation in serotype 2 but decreased the yield per infected cell. For both serotypes, centrifugation of the chlamydiae onto monolayers infected 50-1,200 times the number of cells infected with use of stationary adsorption. An enumeration method for L-cell infectious units was developed that gave higher titers than the chicken embryo 50% lethal dose after yolk sac inoculation.

Indirect enzyme-linked immunosorbent assays were applied to differentiate Chlamydia (C.) psittaci -specific IgG1 and IgG2 levels in 143 individual serum samples from cattle with naturally occurring chlamydial infections and in 190 sequential serum samples from 26 experimentally infected pregnant cows, calves, and a bull. The mean IgG1:IgG2 ratio of naturally infected cattle was 1:4 indicating a significant (p C. psittaci and Coxiella (C.) burnetii simultaneously. The predominance of C. psittaci -specific IgG2, in contrast to the predominance of C. burnetii -specific IgG1 detected in these same individual serum samples under identical conditions, indicates that the ability to preferentially produce either IgG1 or IgG2 was not limited in these individual cattle. A transient yet significant IgG1 response was also developed in cows following chlamydia-induced abortions (immunotype 1) or in cattle infected with the polyarthritis-serositis-encephalomyelitis agents (immunotype 2). IgG1 levels decreased faster than IgG2 levels. These findings have diagnostic implications and identify the need for determining the immunoglobulin classes and subclasses of the humoral immune responses of animals and man to chlamydial infections.

Summary Biological properties and restriction enzyme patterns of the slowly replicating herpesviruses isolated from cattle affected with different diseases in North America and Europe were analysed. These virus isolates induced identical plaques that developed within 7 to 9 days in bovine foetal spleen cells and within 5 days in actively growing Georgia bovine kidney cells. These virus isolates were found to be antigenically related when tested in the indirect immunofluorescence test, and antigenic relationships with bovine herpesvirus 1 (BHV-1), BHV-2, BHV-3 or BHV-6 were not detected. The genomes of these strains were shown to have virtually identical cleavage sites when treated with restriction enzymes EcoRI, BamHI, SstII, SphI and HindIII. The resulting restriction enzyme patterns differed strikingly from those of BHV-1, BHV-2, BHV-3 and BHV-6. Because the herpesviruses tested become enveloped on the nuclear as well as on endoplasmic membranes, a process through which they induce cytoplasmic vesicles filled with enveloped viral particles, and because of the unique cytoplasmic inclusions that are induced, we classify them tentatively as bovine cytomegaloviruses.

Antigens of the immunotype 1 strain B-577 of Chlamydia psittaci, which were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes were used to probe sequential serum samples of cattle with experimentally induced or naturally occurring chlamydial infections. Applying IgGl- and IgG2-specific markers in an enzyme immunoassay procedure, a predominance of IgG2 reactions with different proteins was determined. The interaction of IgGl with antigens such as the genus-specific lipopolysaccharide and the major outer membrane protein was usually limited to periods immediately following overt clinical disease. Some other antigens like the 60,000 and 62,000 D proteins, for example, were recognized by both subclasses over the entire period of investigation. This indicates that it may be possible to determine the phase of infection through analysis of the IgGl and IgG2 responses with the Western blot technique. The different IgGl and IgG2 responses of cattle infected with different strains of Chlamydia psittaci as well as the diverse reactions of cattle from different herds with naturally occurring chlamydial infections further indicate that it may be feasible to distinquish the strains causing these chlamydial infections using different antigens in the Western blot technique. The results obtained by this method may have implications for the production of a subunit vaccine as well as for serodiagnostic purposes.

"There is a consensus that jejunoileal bypass is no longer an acceptable operation for the treatment of morbid obesity because of well-documented serious complications." Over the past 3 years, experience with the gastric stapling procedure has shown it to be a safe and effective operation for the surgical treatment of morbid obesity. The purpose of the operation is to establish a very small proximal gastric reservoir with a tiny outlet for passage of digested food into the distal stomach. By permitting a comfortable intake of only 2 ounces at a time, the procedure affords an average weight loss of 8 pounds per month. At the Naval Regional Medical Center, Oakland, California (NRMC-O), a new approach to gastric stapling using a transthoracic, transdiaphragmatic technique has been initiated. The transthoracic approach offers the following advantages over the transabdominal approach: (1) better and easier exposure, (2) less chance of splenic injury, (3) a thinner layer of subcutaneous fat through which to dissect, (4) decreased problem with postoperative ileus, and (5) decreased incidence of wound infection and dehiscence. Twenty patients have undergone this procedure at NRMC-O over the past 16 months with no significant morbidity and no deaths. Th average operative time is 2 hours and the average hospital stay in 8 days. Beneficial metabolic and endocrine effects of weight loss have been recorded through our computerized data bank. The data confirm that gastric stapling is a safe and effective procedure and that the transthoracic approach offers ease of exposure and minimizes postoperative complications.

Initiation of autonomous parvovirus replication depends on the S phase of host cells actively traversing the cell cycle. The parvoviruses Lu III and H-1 inhibit synchronized cells from entering mitosis, implying that parvoviruses rapidly shut down cell cycle traverse during G2 phase. Bovine parvovirus (BPV) did not inhibit the entry into mitosis of hydroxyurea synchronized bovine fetal spleen cells. Mitotic indexes of infected cultures were as much as 60-fold higher than those of mock-infected controls. Mitosis in control and infected cultures peaked at 10 hr after infection corresponding to the end of the BPV eclipse period. Cytopathic changes, including morphological alteration of mitotic chromosomes, were detected in mitotic cells from infected cultures by light and electron microscopy. Arrest of BPV-infected cells in mitosis may explain these results. Not all infected cells were killed in mitosis, since some developed intranuclear inclusions and became pycnotic as nucleated, interphase cells. Inclusion formation was coincident with viral morphogenesis in interphase nuclei at 16 to 18 hr postinfection, late in the viral replication cycle. The cell cycle stage at which parvovirus-infected cells are arrested and cytopathic events ensue may be determined by the cellular progression rate from S phase through G2 and M.

One-step growth curves of a bovine enterovirus and the virus of bovine diarrhea (BVD) in singly and doubly infected bovine embryonic kidney (BEK) and bovine embryonic spleen (BES) cells were determined. In BEK cells, bovine enterovirus infectivity increased exponentially in both tissue culture fluid and cells 4 to 10 hours after inoculation and then levelled off. In BES cells, the exponential increase lasted 14 to 17 hours. In the growth curve of the BVD virus in BEK cells, the extracellular and cell-associated viruses increased exponentially until hour 12, increasing gradually thereafter. In BES cells, BVD virus infectivity increased exponentially 24 hours after infection and then levelled off. The peak infectivity in BES cells was reached 24 hours before that in BEK cells. Growth of bovine enterovirus was studied in cells of BEK and BES monolayers preinfected with the BVD virus for 20 hours. The infectivity titer in preinfected BEK cells was approximately 2 logs less than in preinfected BES cells. A decreased yield in infectivity was observed compared to the bovine enterovirus one-step growth curve.

SummaryPsittacosis - lymphogranulomatrachoma agents were isolated from conjunctival scrapings from 9 of 17 sheep representing 3 different flocks afflicted to 90% with follicular conjunctivitis. The PLT agent isolated from follicular conjuctivitis was specifically related to those causing polyarthritis. Ten to 25% of the sheep with eye lesions had polyarthritic signs and the PLT agents were isolated from the blood of 6 of the 17 sheep tested. Parenteral inoculation resulted in follicular conjunctivitis and polyarthritis.

In assessing the host cell range of bovine parvoviruses, these viruses were found to replicate optimally in actively dividing bovine fetal lung and spleen cells. Other primary bovine fetal cells supported growth to a lesser extent, but bovine line cells and line cells of other animal species tested did not. Minimal infectivity remained after passage of bovine parvovirus in cells from chicken embryos and guinea pig fetuses. During bovine parvovirus replication in bovine fetal lung and spleen cells, production kinetics of infectious virus and hemagglutinins were determined. An eclipse period of 16 h occurred, and viral release from cells was not detected until 30 h after inoculation of bovine fetal lung cells and 36 h after inoculation of bovine fetal spleen cells. Cell-associated virus titers were always higher than extracellular virus titers. Hemagglutinins were detected in parallel to infectious virus.

Tritiated uridine was selectively incorporated into the cytoplasm but not the nucleus of BEK cells infected with bovine enterovirus. This was accompanied by complete shutdown of cellular RNA synthesis. Bovine embryonic spleen cells infected with the BVD virus incorporated3H-uridine into the nucleus and cytoplasm, while uninfected cells showed only nuclear and nucleolar incorporation. Bovine embryonic spleen cells that were preinfected with BVD virus for 20 hours and then superinfected with bovine enterovirus showed complete absence of nuclear and nucleolar labelling in the early stages of superinfection with the bovine enterovirus, but nuclear labelling returned in reduced amounts 8 to 10 hours after superinfection and persisted throughout the remainder of the double infection ending in cell death.

Abstmct. Ultrastructural changes induced by chlamydia1 infection of niucosal cells of the ileum of ncwborn calves after oral inoculation were investigated. Depending on the stage of chlamydia1 development, the organelles of all infected cells became damaged. The damage was degenerative and included vesiculation of microvilli and swelling of the terminal web of absorptive epithelial cells. The mitochondria were swollen and had fragmented cristae. The endoplasmic reticulum was dilated and vesiculated, and infected cells gradually lost their ribosomes. The lateral junctional complex between infected cells became occasionally dislocated and fragmented. The basal lamina was thrown into folds, became discontinuous and separated from the basal border of the epithelial cells. The nuclei of infected cells were affected last, lost their chromatin pattern, and ultimately became pyknotic. Enteric disease represents a serious problem for the newborn of many different animal species and man. The newborn encounters numerous infectious agents during the postnatal period when the intestinal microbial flora is being established. Intestinal infections with viruses and other obligate intracellular parasites such as chlamydiae are also known to occur during this period. Chlamydia1 agents have been isolated from feces of clinically normal young calves [ 151 and from specimens of newborn calves with diarrhea and polyarthritis [ 121. After oral inoculation with specific chlamydia1 strains, young calves developed diarrhea and died rapidly because of water and electrolyte losses, but only minor gross and histopathologic lesions were detected in the intestine [5]. Since chlamydiae replicate by going through a welldefined developmental cycle, infected cells can be identified ultrastructurally. The purpose of this investigation was to analyze ultrastructural changes induced by chlamydiae in the ileal mucosa of calves that developed diarrhea after oral inoculation.

The uptake and release of chlamydiae in infections of highly specialized intestinal, absorptive epithelial cells of the ileum of newborn calves were studied. Infection occurred only through the brush border of the enterocytes. The chlamydial elementary bodies were adsorbed onto the microvilli, a process that induced pinocytosis of the elementary bodies and formation of endosomes containing one dense-centered chlamydial form. The endosomes moved towards the supranuclear region of the enterocytes, and the cell membrane entrapping the chlamydial agent gradually dissolved so that inclusions with reticulate and dense-centered forms appeared to be free in the cytoplasm. After replication, chlamydiae were shed into the intestinal lumen in three different ways. Some infected enterocytes ruptured and liberated their contents into the lumen. In other instances, entire infected cells were extruded into the lumen, where their membranes degenerated to release chlamydial forms. In the third mode of release, chlamydiae were liberated in cytoplasmic fragments surrounded by cell membranes.

After oral inoculation of calves with chlamydial agents, infection of cells in the abomasum and the lower portions of the gastrointestinal tract occurred. The range of different cells infected in the ileum was analysed by electron microscopic examination. In decreasing order of frequency, the following types of cells were found to contain chlamydiae at different stages of the developmental cycle: absorptive epithelial cells at the tip of the villi, absorptive epithelial cells of the intervillous zone, macrophages of the intestinal lamina propria, endothelial cells of the central lacteals, enterochromaffin cells and goblet cells of the villi, fibroblasts of the lamina propria, rarely undifferentiated cells of the crypts of Lieberkuhn. Chlamydial forms were seldom seen in neutrophilic leukocytes which were increased in number in the neigh bourhood of chlamydia-infected cells. Plasma-cells were numerous in the intestinal lamina propria. These cells often contained degenerating chlamydial forms in cytoplasmic phagosegrosomes.