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1.  Transmission of Bovine viral diarrhea virus 1b to susceptible and vaccinated calves by exposure to persistently infected calves 
Abstract
Bovine viral diarrhea virus (BVDV) persistently infected (PI) calves represent significant sources of infection to susceptible cattle. The objectives of this study were to determine if PI calves transmitted infection to vaccinated and unvaccinated calves, to determine if BVDV vaccine strains could be differentiated from the PI field strains by subtyping molecular techniques, and if there were different rates of recovery from peripheral blood leukocytes (PBL) versus serums for acutely infected calves. Calves PI with BVDV1b were placed in pens with nonvaccinated and vaccinated calves for 35 d. Peripheral blood leukocytes, serums, and nasal swabs were collected for viral isolation and serology. In addition, transmission of Bovine herpes virus 1 (BHV-1), Parainfluenza-3 virus (PI-3V), and Bovine respiratory syncytial virus (BRSV) was monitored during the 35 d observation period.
Bovine viral diarrhea virus subtype 1b was transmitted to both vaccinated and nonvaccinated calves, including BVDV1b seronegative and seropositive calves, after exposure to PI calves. There was evidence of transmission by viral isolation from PBL, nasal swabs, or both, and seroconversions to BVDV1b. For the unvaccinated calves, 83.2% seroconverted to BVDV1b. The high level of transmission by PI calves is illustrated by seroconversion rates of nonvaccinated calves in individual pens: 70% to 100% seroconversion to the BVDV1b. Bovine viral diarrhea virus was isolated from 45 out of 202 calves in this study. These included BVDV1b in ranch and order buyer (OB) calves, plus BVDV strains identified as vaccinal strains that were in modified live virus (MLV) vaccines given to half the OB calves 3 d prior to the study. The BVDV1b isolates in exposed calves were detected between collection days 7 and 21 after exposure to PI calves. Bovine viral diarrhea virus was recovered more frequently from PBL than serum in acutely infected calves. Bovine viral diarrhea virus was also isolated from the lungs of 2 of 7 calves that were dying with pulmonary lesions. Two of the calves dying with pneumonic lesions in the study had been BVDV1b viremic prior to death. Bovine viral diarrhea virus 1b was isolated from both calves that received the killed or MLV vaccines. There were cytopathic (CP) strains isolated from MLV vaccinated calves during the same time frame as the BVDV1b isolations. These viruses were typed by polymerase chain reaction (PCR) and genetic sequencing, and most CP were confirmed as vaccinal origin. A BVDV2 NCP strain was found in only 1 OB calf, on multiple collections, and the calf seroconverted to BVDV2. This virus was not identical to the BVDV2 CP 296 vaccine strain. The use of subtyping is required to differentiate vaccinal strains from the field strains. This study detected 2 different vaccine strains, the BVDV1b in PI calves and infected contact calves, and a heterologous BVDV2 subtype brought in as an acutely infected calf. The MLV vaccination, with BVDV1a and BVDV2 components, administered 3 d prior to exposure to PI calves did not protect 100% against BVDV1b viremias or nasal shedding. There were other agents associated with the bovine respiratory disease signs and lesions in this study including Mannheimia haemolytica, Mycoplasma spp., PI-3V, BRSV, and BHV-1.
PMCID: PMC1176294  PMID: 16187545
2.  Epidemiological study of enzootic pneumonia in dairy calves in Saskatchewan. 
A field study involving 325 calves from 17 dairy herds in Saskatchewan was conducted to determine the risk of enzootic pneumonia and to assess its association with a number of factors. Two different case definitions of pneumonia were used in the analyses: the first was based on producers' treatment risk (CASE1) and the second was based on semimonthly clinical examinations of calves by the research veterinarian (CASE2). The risk of pneumonia based on CASE1 was 39% and on CASE2 was 29%. The measure of agreement between CASE1 and CASE2 at the calf level of analysis was poor (kappa = 0.24, SE = 0.02) and at the herd level of analysis was moderate (kappa = 0.40, SE = 0.12). The mortality risk from pneumonia was 1.8% and a variety of infectious organisms were isolated from pneumonic lungs. Twenty-seven percent of the calves had inadequate (total IgG < or = 800 mg/dL) levels of passively acquired antibodies as measured by radial immunodiffusion. The proportion of seropositive titers in calves within the first two weeks of age was 94% to parainfluenza 3 virus (PI3V) and bovine respiratory syncytial virus (BRSV), 73% to Pasteurella haemolytica (Ph), 68% to bovine viral diarrhea virus (BVDV), 67% to infectious bovine rhinotracheitis virus (IBRV), 46% to Mycoplasma dispar (Md), 44% to Haemophilus somnus (Hs), and 21% to Mycoplasma bovis (Mb). At the calf level of analysis and after adjusting for clustering, there was a negative association (p = 0.10) between the diagnosis of pneumonia based on CASE2 and total IgG levels and Ph titers (rPh).(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263636  PMID: 8269363
3.  The incidence of Mycoplasma dispar, Ureaplasma and conventional Mycoplasma in the pneumonic calf lung. 
This report describes the incidence of Mycoplasma dispar, ureaplasma and conventional (large colony) mycoplasma isolated from the pneumonic lungs of groups of young calves and the identification to species level of mycoplasmas in mixed populations with the aid of the indirect fluorescent antibody test. Pneumonic lung tissue yielded one or more mycoplasma species from 88% of the 153 calves cultured. The mycoplasmas identified and percent of the calves with lungs positive for each species were: M. dispar (56%), ureaplasma (44%), Mycoplasma bovis (37%), Mycoplasma arginini (33%) and Mycoplasma bovirhinis (23%). Conventional mycoplasmas isolated from two calves (1%) could not be identified using the antisera available.
PMCID: PMC1320011  PMID: 398234
4.  The virulence of T-mycoplasmas, isolated from various animal species, assayed by intramammary inoculation in cattle 
The Journal of Hygiene  1973;71(1):163-170.
The virulence of T-mycoplasmas for cattle was tested by examining their ability to produce mastitis in cows. It was found that both virulent and avirulent strains of T-mycoplasmas can be isolated from cattle. All of four strains from pneumonic calf lungs and a strain from a case of bovine kerato-conjunctivitis caused mastitis but only two of four strains isolated from the urogenital tract of cows were virulent. None of the human, simian or canine T-mycoplasmas examined were able to cause mastitis in cattle. However, a bovine strain was found to be capable of causing mastitis in goats. Virulent and avirulent strains from the same and different species contain common antigens detected by the metabolic inhibition test. Pathogenicity could not be shown to be characteristic of any particular serotype. The possibility is raised of some species barrier being responsible for the inability of non-bovine strains to infect cattle.
PMCID: PMC2130429  PMID: 4632825
5.  Association of Mycoplasma ovipneumoniae Infection with Population-Limiting Respiratory Disease in Free-Ranging Rocky Mountain Bighorn Sheep (Ovis canadensis canadensis)▿  
Journal of Clinical Microbiology  2007;46(2):423-430.
Bronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniae was detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniae in the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniae was isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniae PCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P < 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniae antibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P < 0.001]). M. ovipneumoniae is strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.
doi:10.1128/JCM.01931-07
PMCID: PMC2238132  PMID: 18057131
6.  Respiratory disease in a colony of rats 
The Journal of Hygiene  1972;70(3):387-407.
Mycoplasma pulmonis was isolated from the pneumonic lung of a rat. Two groups of mycoplasma-free rats were inoculated, one with a culture of the M. pulmonis strain which had been cloned four times (group A) and the other with a lung homogenate of the rat from which the strain had been isolated (group B). A third group (C) consisted of uninoculated control animals. Each group was kept in strict isolation and allowed to breed so that the progeny was naturally exposed to any pathogens present in the inoculated animals. After different periods of exposure, rats were autopsied, respiratory tracts and inner ears were cultured for mycoplasmas and bacteria, and sera were tested for complement-fixing antibodies to murine mycoplasmas.
In group-A rats, M. pulmonis was consistently isolated from the inner ears or lungs from 50 to 715 days after exposure. Complement-fixing antibody to M. pulmonis was detected 20 days after inoculation, but in the naturally exposed progeny antibody took longer than 50 days to develop. Antibodies to the other known mycoplasmas of murine origin, M. arthritidis and M. neurolyticum, were never found. Purulent otitis interna was consistently found from day 55 onwards, while lung lesions were first observed at 85 days and persisted to 715 days. Pulmonary lesions developed more slowly in inoculated parents than in exposed progeny. Similar results were found in group-B rats, which were examined up to 441 days after inoculation. Uninoculated group-C rats were examined up to 768 days of age, but M. pulmonis was not recovered; of the 54 animals whose serum was tested all were negative to the three species of mycoplasmas, except one which had a titre of 16 with M. pulmonis. Pneumonia, bronchiectasis or lymphoreticular hyperplasia were not seen in any of these control rats. Bacterial respiratory pathogens were not isolated from rats in any of the groups, nor was antibody to Sendai virus detected.
The results suggest that M. pulmonis alone can cause pneumonia and bronchiectasis in rats since mechanical carry-over of another pathogen with the initial cloned inoculum is very unlikely and there was no evidence for the participation of any other rat pathogen. The respiratory disease induced by the cloned culture was comparable with that induced by the lung homogenate, and with the well-known syndrome of chronic respiratory disease and bronchiectasis in the rat.
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PMCID: PMC2130201  PMID: 4506989
7.  Mycoplasma ovipneumoniae - A Primary Cause of Severe Pneumonia Epizootics in the Norwegian Muskox (Ovibos moschatus) Population 
PLoS ONE  2014;9(9):e106116.
The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004–2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection.
doi:10.1371/journal.pone.0106116
PMCID: PMC4157772  PMID: 25198695
8.  Enzootic Pneumonia of Pigs: Identification of a Causative Mycoplasma in Infected Pigs and in Cultures by Immunofluorescent Staining 
Immunofluorescent staining has been used to identify Mycoplasma hyopneumoniae in smears of broth cultures, in infected pig testicle cell cultures, and in frozen cut sections of pneumonic lungs from field and experimentally produced cases of enzootic pneumonia. In the pneumonic pig lung, fluorescent staining was limited to the surface of the bronchial and bronchiolar epithelium and to the contained exudate. In a series of trials using experimentally infected pigs fluorescence was not detected until 25 days post-infection and was regularly seen in pigs killed thereafter. Porcine immune globulin precipitated from the serum of experimentally infected pigs and conjugated with fluorescein isothiocyanate was reactive and specific for the detection of M. hyopneumoniae. Immune globulin conjugates prepared from the serum of hyperimmunized rabbits were reactive but in some cases produced a faint non-specific staining of frozen tissue sections. No such non-specific reactions were noted on stained culture smears or cell cultures.
Fluorescence was not seen in known positive preparations stained with non-immune pig globulin conjugates or in preparations from uninoculated cell cultures or pigs, stained with non-immune or immune globulin conjugates.
Mycoplasma hyorhinis was detected by immunofluorescent staining with homologous conjugates, in smears of broth cultures and in tissue sections from pigs with polyserositis.
Immunofluorescent staining was found to be species specific and useful for the early species identification of mycoplasma isolated from pigs.
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PMCID: PMC1319418  PMID: 4246002
9.  Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease 
The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in 2 groups of stocker calves with acute respiratory disease. Both studies used calves assembled after purchase from auction markets by an order buyer and transported to feedyards, where they were held for approximately 30 d. In 1 study, the calves were mixed with fresh ranch calves from a single ranch. During the studies, at day 0 and at weekly intervals, blood was collected for viral antibody testing and virus isolation from peripheral blood leukocytes (PBLs), and nasal swabs were taken for virus isolation. Samples from sick calves were also collected. Serum was tested for antibodies to bovine herpesvirus-1 (BHV-1), BVDV1a, 1b, and 2, parainfluenza 3 virus (PI3V), and bovine respiratory syncytial virus (BRSV). The lungs from the calves that died during the studies were examined histopathologically, and viral and bacterial isolation was performed on lung homogenates. BVDV was isolated from calves in both studies; the predominant biotype was noncytopathic (NCP). Differential polymerase chain reaction (PCR) and nucleic acid sequencing showed the predominant subtype to be BVDV1b in both studies. In 1999, NCP BVDV1b was detected in numerous samples over time from 1 persistently infected calf; the calf did not seroconvert to BVDV1a or BVDV2. In both studies, BVDV was isolated from the serum, PBLs, and nasal swabs of the calves, and in the 1999 study, it was isolated from lung tissue at necropsy. BVDV was demonstrated serologically and by virus isolation to be a contributing factor in respiratory disease. It was isolated more frequently from sick calves than healthy calves, by both pen and total number of calves. BVDV1a and BVDV2 seroconversions were related to sickness in selected pens and total number of calves. In the 1999 study, BVDV-infected calves were treated longer than noninfected calves (5.643 vs 4.639 d; P = 0.0902). There was a limited number of BVDV1a isolates and, with BVDV1b used in the virus neutralization test for antibodies in seroconverting calves' serum, BVDV1b titers were higher than BVDV1a titers. This study indicates that BVDV1 strains are involved in acute respiratory disease of calves with pneumonic Mannheimia haemolytica and Pasteurella multocida disease. The BVDV2 antibodies may be due to cross-reactions, as typing of the BVDV strains revealed BVDV1b or 1a but not BVDV2. The BVDV1b subtype has considerable implications, as, with 1 exception, all vaccines licensed in the United States contain BVDV1a, a strain with different antigenic properties. BVDV1b potentially could infect BVDV1a-vaccinated calves.
PMCID: PMC227002  PMID: 12146890
10.  Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review. 
Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.
PMCID: PMC2590553  PMID: 6382831
11.  Epizootic Pneumonia of Bighorn Sheep following Experimental Exposure to Mycoplasma ovipneumoniae 
PLoS ONE  2014;9(10):e110039.
Background
Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia.
Methodology/Principal Findings
In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy.
Conclusions/Significance
Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.
doi:10.1371/journal.pone.0110039
PMCID: PMC4193846  PMID: 25302992
12.  Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran 
A total of 30 Pasteurella multocida strains isolated from 333 pneumonic and apparently health slaughter cattle were examined for capsule biosynthesis genes and 23 virulence-associated genes by polymerase chain reaction (PCR). The disc diffusion technique was used to determine antimicrobial resistance profiles among the isolates. Of the isolates, 23 belonged to capsular type A, 5 to capsular type D and two isolates were untypeable. The distribution of the capsular types in pneumonic lungs and in apparently health lungs was statistically similar. All virulence genes tested were detected among the isolates derived from pneumonic lungs; whereas isolates derived from apparently health lungs carried 16 of the 23 genes. The frequently detected genes among isolates from pneumonic lungs were exbD, hgbA, hgbB, ompA, ompH, oma87, and sodC; whereas tadD, toxA, and pmHAS genes occurred less frequently. Most of the adhesins and superoxide dismutases; and all of the iron acquisition and protectin proteins occurred at significantly (p ≤ 0.05) higher frequencies in isolates from pneumonic lungs. Isolates from apparently healthy lungs didn't carry the following genes; hsf-1, hsf-2, tadD, toxA, nanB, nanH, and pmHAS. One adhesion (hsf-1) and two iron acquisition (exbD and tonB) genes occurred at significantly (p ≤ 0.05) higher frequencies among capA isolates. All the P. multocida isolates were susceptible to ciprofloxacin, co-trimoxazole, doxycycline, enrofloxacin, nitrofurantoin, and tetracyclines. Different proportions of the isolates were however resistant to ampicillin, amoxicillin, erythromycin, lincomycin, penicillin, rifampin, streptomycin, and florfenicol. Our results reveal presence of virulence factors (VFs) in P. multocida strains isolated from symptomatic and asymptomatic bovids. A higher frequency of the factors among isolates from symptomatic study animals may suggest their role in pathogenesis of P. multocida-associated bovine respiratory disease (BRD). The results further reveal occurrence of antimicrobial resistance among some isolates. Control strategies for this pathogen, which could include development of an effective vaccine, are warranted so as to mitigate the social and economic consequences attributable to natural infections with this bacterium.
doi:10.3389/fmicb.2014.00536
PMCID: PMC4196553  PMID: 25352839
Pasteurella multocida; virulence factors; antimicrobial resistance; cattle; Iran
13.  Enzootic Pneumonia in Pigs: Propagation of a Causative Mycoplasma in Cell Cultures and in Artificial Medium 
Three strains of a new species of mycoplasma were recovered from pneumonic pig lungs, known free of Mycoplasma hyorhinis, by prolonged incubation in pig testicle cell cultures. The three strains produced a characteristic cytopathic effect in the cell cultures. A highly enriched meat-infusion-broth medium was evolved and permitted regular propagation of these organisms. Pneumonia could consistently be produced by intratracheal inoculation of pigs with the mycoplasma propagated in the enriched broth medium or in cell cultures. The mycoplasma were recovered from the lungs of experimentally infected pigs by inoculation into the broth medium. Comparative studies of the pneumonia producing mycoplasma and of M. hyorhinis were carried out in cell cultures, broth media, and in pigs infected experimentally by different routes. The morphological characteristics of the mycoplasma, grown in the different media, are described and illustrated.
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PMCID: PMC1319314  PMID: 4237289
14.  Further observations on the problem of isolating Mycoplasma suipneumoniae from field cases of enzootic pneumonia in pigs 
The Journal of Hygiene  1970;68(2):313-325.
In previous work in this laboratory, Mycoplasma suipneumoniae was recovered in liquid medium from 13% of individual cases and 18% of outbreaks of enzootic pneumonia in pigs. In the work now described, however, these recovery rates, when judged by the same criteria, were 45 and 75%, respectively. As there was evidence to suggest that this second series of pneumonic cases was less suitable for cultural examination than the first series, some of the other factors that might have improved the recovery rate were investigated.
Some improvement was probably achieved by inoculating the liquid medium with three or four different dilutions of pneumonic tissue, each dilution always being in duplicate, and by incubating the inoculated tubes for over 3 weeks before discarding them.
A second advantage could have derived from the fact that all batches of liquid medium were tested for their ability to support the growth of M. suipneumoniae before being used to culture field material.
The effect of varying one constituent at a time was observed in controlled experiments: different batches of pig sera had a marked, variable effect on the growth of both M. suipneumoniae and Mycoplasma hyorhinis; medium made with purchased Hartley's broth was found to be superior to medium incorporating broth made in this laboratory, more so for the growth of M. suipneumoniae than M. hyorhinis; the incorporation of yeast extract made in this laboratory gave a marginal advantage for the growth of M. hyorhinis; and both mycoplasmas grew equally well in medium containing or lacking thallium acetate.
Some batches of medium were, by chance, markedly selective for the growth of M. suipneumoniae compared with M. hyorhinis. As the full reasons for this were not known, attempts were made to develop selective media in a more direct way. One such medium contained 5% pig serum and 15% horse serum, and a second was of similar composition, except that the pig serum used inhibited preferentially the growth of M. hyorhinis compared with M. suipneumoniae. Both media markedly favoured the growth of M. suipneumoniae when tested separately with cultures of M. suipneumoniae and M. hyorhinis. The second medium yielded M. suipneumoniae when inoculated with a 10-1 dilution of a culture of M. suipneumoniae and a 10-2 dilution of a culture of M. hyorhinis, whereas a standard batch of liquid medium, similarly inoculated with M. suipneumoniae did not yield this mycoplasma until the M. hyorhinis culture included in the inoculum was diluted to 10-6.
Both selective media, when tested on a small number of field cases, gave improved isolations of M. suipneumoniae compared with the routine batches of liquid medium used initially.
Considerable difficulty was experienced in producing a sufficiently high level of antibodies to M. hyorhinis in pig sera and to M. suipneumoniae in rabbit sera. This exacerbated the problem of isolating and identifying M. suipneumoniae from field cases of enzootic pneumonia by this cultural method.
PMCID: PMC2130801  PMID: 5270206
15.  The passive haemagglutination test for the detection of Mycoplasma suipneumoniae and the possible diagnosis of enzootic pneumonia of pigs 
The Journal of Hygiene  1970;68(2):327-336.
Fourteen cases of enzootic pneumonia, nearly all of which had presented diagnostic difficulties using the metabolic-inhibition test, were re-examined using specific pig antisera in the passive haemagglutination test (PHA). All proved positive for Mycoplasma suipneumoniae, indicating that the test, used in this manner, might be particularly valuable for routine diagnosis.
The PHA test was also used to demonstrate antibody to M. suipneumoniae in pneumonic tissue and the associated bronchial lymph nodes.
To allay our concern that cross-reactions might interfere with this and other serological tests—the complement-fixation test (CF) and precipitation in agar-gel—the specificity of our reagents and the antigenic relationships of Mycoplasma hyorhinis, Mycoplasma granularum, mycoplasma B3, Mycoplasma hyopneumoniae and three strains of M. suipneumoniae (including cloned and uncloned isolates of the J strain) were studied in various ways. Antibodies to medium constituents occurred in rabbit antiserum but did not present a problem with pig antisera. These antibodies were successfully absorbed from the rabbit antisera but it was not possible to remove medium constituents from the antigens used to produce antisera in rabbits by repeated washing.
By all these tests, the main species of mycoplasmas studied seemed to be antigenically distinct. No major antigenic differences between the three strains of M. suipneumoniae were revealed by the PHA test and the CF test; a slight difference in the precipitation lines of one of these strains (MG) in agar-gel might have indicated an antigenic variation or been a measure of some other factor.
PMCID: PMC2130806  PMID: 4988048
16.  THE INTERACTION IN VITRO OF MYCOPLASMA PULMONIS WITH MOUSE PERITONEAL MACROPHAGES AND L-CELLS 
Methods have been devised for establishing infection in vitro of mouse macrophages and fibroblasts with Mycoplasma pulmonis. The mycoplasmas attached to the cells and under appropriate cultural conditions grew into a lawn of microorganisms covering most of the cell surface. The mycoplasmas grew abundantly on fibroblasts cultured in minimal essential medium containing 20% fetal calf serum; supplementation of this medium with heart infusion broth was necessary to obtain similar growth on macrophages. The infection of these cells appeared to be essentially an extracellular process; only rarely were partially degraded mycoplasmas seen with phagocytic vacuoles. The addition to heavily infected macrophage cultures of low concentrations of anti-mycoplasma antibody stimulated rapid, massive phagocytosis of the surface microorganisms. In sharp contrast, the same antiserum had no discernable effect on the mycoplasma-fibroblast relationship. The antibody effect in the macrophage system was apparently a direct opsonic one rather than an indirect result of microbial killing, since the mycoplasmas in macrophage or fibroblast cultures incorporated labelled thymidine into DNA after the addition of antiserum to the medium. The phagocytic event and the subsequent fate of the mycoplasmas were studied in detail after the addition of antibody to the macrophage cultures. Phase-contrast cinemicrophotography revealed membrane ruffles surrounding the surface mycoplasmas and disappearance from view of the organisms; 10–30 min later translucent grapelike clusters were seen in large phagocytic vacuoles. On electronmicroscopic study the surface mycoplasmas were surrounded by pincers-like projections of the macrophage. Numerous mycoplasmas were seen in phagocytic vacuoles; in the early minutes after the addition of antibody the intracellular mycoplasmas appeared normal, but within 2 hr they appeared partially degraded with a central electron-lucent area and electron-opaque deposits at the microbial cell margin. 24 hr after the addition of antiserum, digestion of the mycoplasmas was nearly complete; the cells appeared normal except for large residual bodies composed of amorphous moderately dense material and increased lipid deposits. Degradation of mycoplasmas within macrophages was also studied using infected cultures in which the mycoplasmas, but not the macrophages, had incorporated tritiated thymidine into DNA. The appearance of large amounts of acid-soluble radiolabel after phagocytosis stimulated by antibody confirmed the degradation of the intracellular mycoplasmas.
PMCID: PMC2138893  PMID: 4943930
17.  Cytomegalovirus Pneumonitis Is a Risk for Bronchiolitis Obliterans Syndrome in Lung Transplantation 
Rationale: Cytomegalovirus pneumonitis is one of the most prevalent opportunistic infections after lung transplantation. Early studies reported that cytomegalovirus pneumonitis was a risk factor for chronic allograft dysfunction. More recently, in the era of routine prophylaxis and ganciclovir treatment, the adverse impact of treated cytomegalovirus pneumonitis on bronchiolitis obliterans syndrome has been challenged.
Objectives: We hypothesized that cytomegalovirus pneumonitis contributes to adverse outcomes in the current antiviral era. We sought to define the impact of treated cytomegalovirus pneumonitis on bronchiolitis obliterans syndrome and survival in a large single-center cohort (n = 231) of consecutive patients undergoing lung transplantation from 2000 to 2004, all receiving short-course ganciclovir prophylaxis.
Methods: Transbronchial biopsies were performed at defined intervals with prospective cytomegalovirus immunostaining on every biopsy (n = 1,887). Cox proportional hazards models were used to assess the relationship between treated cytomegalovirus pneumonitis and clinical outcomes.
Measurements and Main Results: Forty-nine (21%) recipients developed cytomegalovirus pneumonitis a median of 106 days after transplantation. Treated cytomegalovirus pneumonitis within the first 6 months after transplantation significantly increased the risk for bronchiolitis obliterans syndrome (P = 0.001; hazard ratio, 2.19; 95% confidence interval, 1.36–3.51) and post-transplantation death (P = 0.02; hazard ratio, 1.89; 95% confidence interval, 1.11–3.23). This risk persisted when cytomegalovirus pneumonitis was considered as a time-dependent predictor as well as in multivariable models controlling for other risk factors.
Conclusions: Cytomegalovirus pneumonitis affects more than 20% of lung transplant recipients. Despite treatment, it increases the risk for bronchiolitis obliterans syndrome and death. More effective preventive strategies for cytomegalovirus pneumonitis are needed to improve long-term outcomes after lung transplantation.
doi:10.1164/rccm.200911-1786OC
PMCID: PMC2894412  PMID: 20167845
lung transplantation; cytomegalovirus; bronchiolitis obliterans
18.  The microbial flora of the respiratory tract in feedlot calves: associations between nasopharyngeal and bronchoalveolar lavage cultures. 
The upper and lower respiratory tracts of 59 feedlot calves with clinical signs of naturally occurring respiratory disease (cases) and 60 comparison (control) animals were cultured before treatment, using nasopharyngeal swabs (NPS) and bronchoalveolar lavage (BAL). The most prevalent organisms were Pasteurella multocida and Mycoplasma bovis. Isolations of P. multocida from NPS and BAL fluid were found to be significantly associated with morbidity (p less than or equal to 0.05), but the frequency with which other organisms were isolated from the nasopharynx and lungs was similar in cases and controls. There was evidence of moderate agreement between NPS and BAL isolates at the individual calf level using the kappa statistic, (range of kappa values = 0.47-0.61) but the variability of the kappa statistics was large. Therefore, in an individual calf NPS cultures did not accurately predict BAL cultures. The NPS and BAL culture results were quite similar at the group level, however.
PMCID: PMC1263480  PMID: 1790489
19.  Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey 
This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.
doi:10.1007/s11250-013-0394-3
PMCID: PMC3776281  PMID: 23494576
M. ovipneumoniae; PCR; Culture; Pneumonia; Sheep; Lamb
20.  Previously Uncharacterized Mycoplasma Isolates from an Investigation of Canine Pneumonia 
Infection and Immunity  1970;1(1):1-7.
Mycoplasmas recovered from the respiratory tract and genitourinary system of dogs, with and without respiratory infection, have been characterized by biological and immunological methods. Some of the isolates were indentified as being similar to the three species of canine mycoplasmas described earlier under the designation Mycoplasma spumans, M. canis, and M. maculosum. Other mycoplasmas placed in three groups (A, C, and D) were found to be clearly distinct from the three classified species. Group A strains fermented glucose but not mannose and were serologically distinct from other canine mycoplasmas recovered in this study. These strains were subsequently found to be biologically and serologically related to a previously reported, but unclassified, canine mycoplasma. Group D strains differed in some biological properties but were serologically related. These were found to be nonfermenting mycoplasmas representing isolations from the throat and bladder of dogs. They were serologically distinct from other canine mycoplasmas and were apparently unrelated to other known mycoplasma serotypes. Group C mycoplasmas were recovered only from the lungs of dogs. Within the group, they differ in some immunological properties but appear to be serologically distinct from other canine strains. They can also be separated from other dog strains in their ability to ferment glucose and mannose. Group B strains were found to have biological properties similar to M. canis strains but seemed to be only partially related to this serotype when examined in several serological techniques. It is suggested that these strains might represent antigenic variants of M. canis.
PMCID: PMC415846  PMID: 16557682
21.  Can lung function measurements be used to predict which patients will be at risk of developing interstitial pneumonitis after bone marrow transplantation? 
Thorax  1992;47(6):421-425.
BACKGROUND: Lung function often deteriorates after bone marrow transplantation for haematological malignancies. Whether pulmonary function measurements are useful for monitoring patients' progress after transplantation and for alerting clinicians to the development of pneumonitis is uncertain. METHODS: Serial pulmonary function measurements were made in 39 patients with a haematological malignancy, and the values from 18 recipients of T cell depleted allogeneic (n = 17) or autologous (n = 1) bone marrow transplants who developed interstitial pneumonitis were compared retrospectively with values from 21 recipients of allogeneic (n = 17) or autologous (n = 4) transplants who did not develop pneumonitis. Lung function was measured at the onset of a further 18 episodes of pneumonitis. RESULTS: Measurements made before transplantation showed no difference in forced expiratory volume in one second (FEV1), transfer factor for carbon monoxide (TLCO), or total lung capacity between the two groups, but the forced vital capacity (FVC) was slightly higher in those who developed pneumonitis (mean (SD)% predicted 104 (12)) than in those who did not (93 (17%)). Six weeks and three months after transplantation all pulmonary function measurements had fallen slightly in both groups but TLCO had fallen considerably more in those who later developed pneumonitis, being 71% (SD 11%) and 77% (7%) of pretransplant values in patients who later developed pneumonitis compared with 109% (38%) and 96% (26%) in those who did not. All lung function measurements were significantly lower at the onset of pneumonitis than three months after transplantation, even in patients with no abnormal signs and a normal chest radiograph. CONCLUSIONS: Serial measurements of gas transfer before and after bone marrow transplantation may be useful for predicting which patients will be at risk of developing pneumonitis and may help to diagnose pneumonitis in breathless patients with no abnormal signs.
PMCID: PMC463805  PMID: 1496500
22.  Absence of Inflammation and Pneumonia during Infection with Nonpigmented Yersinia pestis Reveals a New Role for the pgm Locus in Pathogenesis▿  
Infection and Immunity  2009;78(1):220-230.
Yersinia pestis causes primary pneumonic plague in many mammalian species, including humans, mice, and rats. Virulent Y. pestis strains undergo frequent spontaneous deletion of a 102-kb chromosomal DNA fragment, known as the pigmentation (pgm) locus, when grown in laboratory media, yet this locus is present in every virulent isolate. The pgm locus encodes, within a high-pathogenicity island, siderophore biosynthesis genes that are required for growth in the mammalian host when inoculated by peripheral routes. Recently, higher challenge doses of nonpigmented Y. pestis were reported to cause fatal pneumonic plague in mice, suggesting a useful model for studies of virulence and immunity. In this work, we show that intranasal infection of BALB/c mice with nonpigmented Yersinia pestis does not result in pneumonic plague. Despite persistent bacterial colonization of the lungs and the eventual death of infected mice, pulmonary inflammation was generally absent, and there was no disease pathology characteristic of pneumonic plague. Iron given to mice at the time of challenge, previously shown to enhance the virulence of pgm-deficient strains, resulted in an accelerated disease course, with less time to bacteremia and lethality, but lung inflammation and pneumonia were still absent. We examined other rodent models and found differences in lung inflammatory responses, some of which led to clearance and survival even when high challenge doses were used. Together, the results suggest that the Y. pestis pgm locus encodes previously unappreciated virulence factors required for the induction of pneumonic plague that are independent of iron scavenging from the mammalian host.
doi:10.1128/IAI.00559-09
PMCID: PMC2798233  PMID: 19841077
23.  Enzootic Pneumonia of Pigs: Complement-Fixation Tests for the Detection of Mycoplasma Antibodies in the Serum of Immunized Rabbits and Infected Swine 
The direct, the modified direct and the indirect complement-fixation tests were investigated as methods for the detection of antibodies for the enzootic pneumonia mycoplasma and for Mycoplasma hyorhinis in the serum of infected pigs and of immunized rabbits.
Only the modified direct complement-fixation test in which the guinea-pig complement is supplemented with fresh, normal unheated calf serum was suitable for the detection of mycoplasma antibodies in sera of infected swine. Based on the close correlation between the production of typical lung lesions in experimentally infected pigs and the appearance of significant serum antibody titres, the modified direct complement-fixation test provides for the first time a sensitive, specific in vitro method for the detection of enzootic pneumonia in the live pig. This test also permitted the in vitro differentiation of the mycoplasma causing enzootic pneumonia from M. hyorhinis which causes polyserositis.
Antibodies in the sera of rabbits were demonstrable by the ordinary direct complement-fixation test. However, in contast to the observation made with swine sera, only a slight quantitative antigenic difference between the enzootic pneumonia mycoplasma and M. hyorhinis was seen when the tests were performed with rabbit serum antibodiies.
PMCID: PMC1319297  PMID: 4234787
24.  Mycoplasma agalactiae subsp. bovis in pneumonia and arthritis of the bovine. 
The pneumonic lungs of 42 cattle from 26 feedlots were examined for the presence of mycoplasma, pathogenic bacteria and viruses. Four animals representative of two lots failed to yield mycoplasma. One of these yielded the virus of infectious bovine rhinotracheitis and Pasteurella hemolytica, the other yielded only P. P. multocida. Nine animals in eight lots yielded Mycoplasma sp.: five of these were M. bovirhinis, two were M. arginini and two were untypable. All of these animals yielded one or more of P. hemolytica, P. multiocida, infectious bovine rhinotracheitis virus or bovine virus diarrhea virus. Twenty-five of 29 animals in 16 lots yieled M. agalactiae subsp. bovis from lung tissues. The same organism was recovered from the arthritic joints of 12 of these animals. Eight of the 25 animals yielded no other pathogen and all of these had not received any treatment. Nine of the 25 M. agalactiae subsp. bovis positive animals also yielded one or more of P. hemolytica, P. multocida, Corynebacterium pyogenes or infectious bovine rhinotracheitis virus. Bacteriological and virological studies were not completed for the remaining eight of the 25 positive animals. In five lots of cattle which had not received medication for pneumonia and for arthritis only M. agalactiae subsp. bovis was recovered. Twenty-five grossly normal lungs obtained from normal cattle at the time of slaughter were cultured and all were negative. The possible role of M. agalactiae subsp. bovis in pneumonia and arthritis was discussed.
PMCID: PMC1277697  PMID: 832194
25.  Factors predicting radiation pneumonitis in locally advanced non-small cell lung cancer 
Radiation Oncology Journal  2011;29(3):181-190.
Purpose
Thoracic radiotherapy is a major treatment modality of stage III non-small cell lung cancer. The normal lung tissue is sensitive to radiation and radiation pneumonitis is the most important dose-limiting complication of thoracic radiation therapy. This study was performed to identify the clinical and dosimetric parameters related to the risk of radiation pneumonitis after definitive radiotherapy in stage III non-small cell cancer patients.
Materials and Methods
The medical records were reviewed for 49 patients who completed definitive radiation therapy for locally advanced non-small cell lung cancer from August 2000 to February 2010. Radiation therapy was delivered with the daily dose of 1.8 Gy to 2.0 Gy and the total radiation dose ranged from 50.0 Gy to 70.2 Gy (median, 61.2 Gy). Elective nodal irradiation was delivered at a dose of 45.0 Gy to 50.0 Gy. Seven patients (14.3%) were treated with radiation therapy alone and forty two patients (85.7%) were treated with chemotherapy either sequentially or concurrently.
Results
Twenty-five cases (51.0%) out of 49 cases experienced radiation pneumonitis. According to the radiation pneumonitis grade, 10 (20.4%) were grade 1, 9 (18.4%) were grade 2, 4 (8.2%) were grade 3, and 2 (4.1%) were grade 4. In the univariate analyses, no clinical factors including age, sex, performance status, smoking history, underlying lung disease, tumor location, total radiation dose and chemotherapy were associated with grade ≥2 radiation pneumonitis. In the subgroup analysis of the chemotherapy group, concurrent rather than sequential chemotherapy was significantly related to grade ≥2 radiation pneumonitis comparing sequential chemotherapy. In the univariate analysis with dosimetric factors, mean lung dose (MLD), V20, V30, V40, MLDipsi, V20ipsi, V30ipsi, and V40ipsi were associated with grade ≥2 radiation pneumonitis. In addition, multivariate analysis showed that MLD and V30 were independent predicting factors for grade ≥2 radiation pneumonitis.
Conclusion
Concurrent chemotherapy, MLD and V30 were statistically significant predictors of grade ≥2 radiation pneumonitis in patients with stage III non-small cell lung cancer undergoing definitive radiotherapy. The cutoff values for MLD and V30 were 16 Gy and 18%, respectively.
doi:10.3857/roj.2011.29.3.181
PMCID: PMC3429901  PMID: 22984669
Non-small cell lung cancer; Radiation therapy; Radiation pneumonitis; Dosimetric factor

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