Nephroblastoma in a Common Mudpuppy Necturus maculosus simultaneously Present with a Mollicute Bacterium of the Genus Acholeplasma.

In March 2017, a wild-caught female common mudpuppy Necturus maculosus from Iowa, USA, with an enlarged posterior abdomen was submitted for diagnostic assessment. The cause of the abdominal distension was a large fluid-filled abdominal mass, diagnosed as a nephroblastoma. Parasites and numerous bacteria were isolated and identified from the mudpuppy but were determined to be incidental. Samples of the neoplasm inoculated onto an American toad Anaxyrus americanus cell line (BufoTad) yielded cytopathic effect during several passages. However, standard molecular testing of the cell culture supernatant failed to identify any viruses. Next-generation sequencing identified the replicating agent as a bacterium of the genus Acholeplasma. Immunohistochemistry confirmed the presence of Acholeplasma within the nephroblastoma, including within tumor cells. This is the first report of nephroblastoma and the second report of neoplasia in this species. The results also suggest that certain bacteria of the genus Acholeplasma might be oncogenic.

Acholeplasma equirhinis sp. nov. isolated from respiratory tract of horse (Equus caballus) and Mycoplasma procyoni sp. nov. isolated from oral cavity of raccoon (Procyon lotor).

We describe two novel species of Acholeplasma sp. strain N93 and Mycoplasma sp. strain LR5794 which were isolated from the nasopharynx of a horse from the United Kingdom and from the oral cavity of a North American raccoon from Canada, respectively. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma and Acholeplasma species. Both strains are facultative anaerobes, resistant to penicillin, and produce acid from glucose but do not hydrolyze arginine and urea. Both strains grew well in microaerophilic and anaerobic atmospheric conditions at 35-37 °C using PPLO (pleuropneumonia-like organisms) medium. Acholeplasma sp. N93 does not require serum for growth. Colonies of both strains showed a typical fried-egg appearance and transmission electron microscopy of bacterial cells revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of several genetic loci. The genetic analysis indicated that Acholeplasma sp. N93 and Mycoplasma sp. LR5794 were most closely related to A. hippikon and A. equifetale, and M. molare and M. lagogenitalium, respectively. However, both novel strains were genetically unique in comparison to other well-known Mycoplasma and Acholeplasma species. Based on the isolation source history, phenotypic, genotypic, and phylogenetic characteristics of these novel strains, we propose the name Acholeplasma equirhinis sp. nov. for Acholeplasma sp. isolated from the nasopharynx of a horse [the type strain is N93T (= DSM 106692T = ATCC TSD-139T = NCTC 14351T)], and the name Mycoplasma procyoni sp. nov. for the Mycoplasma sp. isolated from the oral cavity of a North American raccoon [the type strain is LR5794T (= DSM 106703T = ATCC TSD-141T = NCTC 14309T)].

Evaluation of effects of Mycoplasma mastitis on milk composition in dairy cattle from South Australia.

BACKGROUND: Mycoplasma mastitis is increasingly posing significant impact on dairy industry. Although the effects of major conventional mastitis pathogens on milk components has been widely addressed in the literature, limited data on the effects of different Mycoplasma and Acholeplasma spp. on milk quality and quantity is available. The aim of this study was to determine the casual relationship of Mycoplasma spp. and A. laidlawii to mastitis and compare them to subclinical mastitis caused by conventional mastitis pathogens from a single dairy herd in South Australia; Mycoplasma spp. and A. laidlawii were detected using PCR applied directly to milk samples. The herd had mastitis problem with high somatic cell count and low response rate to conventional antimicrobial therapy. A total of 288 cow-level milk samples were collected aseptically and used in this study. RESULTS: Conventional culture showed a predominance of coagulase-negative staphylococci, followed by coagulase-positive staphylococci, Streptococcus spp., Enterococcus spp., E. coli, and Klebsiella spp. PCR results showed a high prevalence of mycoplasmas (76.7%), including A. laidlawii (10.8%), M. bovis (6.2%), M. bovirhinis (5.6%), M. arginini (2%), and (52.1%) of cows were co-infected with two or more Mycoplasma and Acholeplasma species. Mycoplasma co-infection significantly increased somatic cell counts (SCC) similar to conventional mastitis pathogens and compared to non-infected cows with 389.3, 550.3 and 67.3 respectively; and decreased the milk yield with 29.0, 29.9 and 34.4 l, respectively. Mycoplasma co-infection caused significant increase in protein percentage, and significant decrease in fat percentage and total milk solids, similar to other conventional mastitis pathogens. In contrast, changes in milk composition and yield caused by various individual Mycoplasma species were non-significant. CONCLUSIONS: Mycoplasma mastitis had on-farm economic consequences similar to common conventional mastitis pathogens. Results of our study indicate that co-infection Mycoplasma mastitis caused similar effect on milk composition to other mastitis pathogens and we hope these findings raise the awareness of the importance of their detection on routine diagnostic panels.

Prevention of antibiotic-associated metabolic syndrome in mice by intestinal alkaline phosphatase.

AIMS: To examine whether co-administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice. METHODS: A total of 50 mice were allocated to four treatment groups after weaning. Mice were treated with azithromycin (AZT) ± IAP, or with no AZT ± IAP, for three intermittent 7-day cycles. After the last treatment course, the mice were administered a regular chow diet for 5 weeks and subsequently a high-fat diet for 5 weeks. Body weight, food intake, water intake, serum lipids, glucose levels and liver lipids were compared. 16S rRNA gene pyrosequencing was used to determine the differences in microbiome composition. RESULTS: Exposure to AZT early in life rendered mice susceptible to MetS in adulthood. Co-administration of IAP with AZT completely prevented this susceptibility by decreasing total body weight, serum lipids, glucose levels and liver lipids to the levels of control mice. These effects of IAP probably occur as a result of changes in the composition of specific bacterial taxa at the genus and species levels (e.g. members of Anaeroplasma and Parabacteroides). CONCLUSIONS: Co-administration of IAP with AZT early in life prevents mice from susceptibility to the later development of MetS. This effect is associated with alterations in the composition of the gut microbiota. IAP may represent a novel treatment against MetS in humans.

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms.

Mycoplasma mastitis is a contagious and costly disease of dairy cattle that significantly affects animal health and milk productivity. Mycoplasma bovis is the most prevalent and invasive agent of mycoplasma mastitis in dairy cattle, and early detection is critical. Other mycoplasma have been isolated from milk; however, the role and prevalence of these species as mastitis pathogens are poorly understood. Routine screening of milk for mycoplasma by bacteriological culture is an important component of a farm control strategy to minimize a herd mycoplasma outbreak, but phenotypic methods have limited ability to speciate mycoplasma, affecting how farms and practitioners can understand the role and effect of species other than M. bovis in herd health. Fastidious mycoplasma culture can be lengthy and inconclusive, resulting in delayed or false negative reports. We developed and validated a multitarget PCR assay that can in the same day confirm or reject a presumptive positive mycoplasma culture found upon bacteriological testing of clinical specimens, further discriminate between Acholeplasma and Mycoplasma, and identify M. bovis. Coupled with sequence analysis isolates can be further identified as bovine mycoplasma Mycoplasma arginini, Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovirhinis, Mycoplasma bovigenitalium, Mycoplasma californicum, Acholeplasma laidlawii, and Acholeplasma oculi. Assay validation included analysis of 845 mycoplasma representing these species and 30 additional bacterial species obtained from routine milk submissions to the Quality Milk Production Services from New York State farms and veterinary clinics between January 2012 and December 2015. Among 95 herds, we found 8 different Mycoplasma species and 3 different Acholeplasma species, with an overall prevalence of M. bovirhinis of 1%, A. oculi of 2%, M. arginini of 2%, M. californicum of 3%, M. canadense of 10%, M. bovigenitalium of 10%, A. laidlawii of 11%, M. alkalescens of 17%, and M. bovis of 78%. More than one mycoplasma was found in 14% of the herds tested, and both M. bovis and Acholeplasma were found in 6% of the farms. Incorporation of the validated molecular diagnostic assay into routine bacteriological screening as a supportive confirmation and identification tool will lead to an improved assessment of Mycoplasma and Acholeplasma prevalence data, which will facilitate increased knowledge about the role of these mycoplasma in mastitis.

Detection and differentiation of avian mycoplasmas by surface-enhanced Raman spectroscopy based on a silver nanorod array.

Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array-surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.

Rapid and sensitive detection of Mollicutes in cell culture by polymerase chain reaction.

Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.

Isolation of acholeplasmas from horse feces.

Acholeplasmas were detected in five of 96 feces samples from clinically normal horses. Three of the five strains isolated were identified as A. equifetale, one as A. hippikon, and one was serologically identical with the Acholeplasma strain 881.

Examination of uterine biopsies from infertile cows for the presence of viruses and mycoplasmas.

Uterine biopsies from 45 infertile cows were examined for the presence of mycoplasmas, bovine viral diarhoea--mucosal disease virus, bovine syncytial virus and other viruses. No viruses were detected. Ureaplasmas were isolated from four cows, two clinically normal and two with nodular vulvovaginitis.

Pathogenicity of some Mycoplasma and Acholeplasma species in the lungs of gnotobiotic calves.

Cloned cultures of 16 strains, representing nine different species of Mycoplasma and Acholeplasma, were inoculated intratracheally into gnotobiotic calves. Strains of M bovirhinis, M canadense, M verecundum, A axanthum and A modicum did not produce visible pneumonic lesions and were not reisolated from the lungs. Strains of M alkalescens and M arginini colonised the lower respiratory tract but failed to produce visible pneumonia. M bovigenitalium (strain M991/70) and M dispar (strain Gri226) both colonised the respiratory tract and induced pneumonic lesions estimated to involve up to 8 per cent (M bovigenitalium) and 17 per cent (M dispar) of the lung. Histologically M bovigenitalium produced a cuffing pneumonia and M dispar produced a interstitial alveolitis.

A survey of Mycoplasma infections in domestic poultry.

A survey of mycoplasma infections of chickens, turkeys and ducks was made on tissues from a variety of sources and birds of various ages, and from pipped and dead-in-shell turkey embryos. The tissues examined consisted mainly of respiratory tissue and the cloaca and contents and also a small number of joint lesions and other tissues. From chickens, mycoplasmas were isolated from a total of 138 tissues with Mycoplasma gallisepticum in preponderance. This was followed by M gallinarum, untyped organisms, M synoviae and a number of other mycoplasmas and Acholeplasma laidlawii. From turkeys, poults and embryos, mycoplasmas were recovered from 164 tissues with M meleagridis in preponderance. This was followed by M gallisepticum, serovar I, M synoviae and a number of other species including untyped isolates. From ducks, M gallisepticum and M anatis were recovered in equal numbers. Mycoplasma infections with more than one species occurred in the same tissue in all species of stock but especially in turkeys.

Isolation of Acholeplasmatales from rabbit faeces.

Acholeplasma laidlawii was isolated from the faeces of 23.5% and 24% of groups of 51 conventional and 45 specified-pathogen-free (SPF) rabbits respectively. Isolation of the organism from individual animals could often be repeated, suggesting that infection was not merely transient. Two further acholeplasmas were isolated from two SPF rabbits. One was serologically related to Acholeplasma modicum. The other could not be identified and may be a new species.

Genital mycoplasma infections.

Since 1937, 13 Mycoplasma species, two Acholeplasma species, and one Ureaplasma species have been isolated from humans. Six of these have the urogenital tract as the primary site of colonisation but others, which have the oropharynx and respiratory tract as the primary site, are found occasionally in the urogenital tract because of orogenital contact. Mycoplasma hominis was the first to be isolated and is most strongly associated with bacterial vaginosis (BV), together with a variety of other bacteria. Its involvement in pelvic inflammatory disease (PID) and other conditions may be as part of BV, although when isolated in pure culture from the blood of women who have postpartum or postabortal fever there is no reason to suspect its aetiological role. There is evidence for an aetiological role for Ureaplasma urealyticum organisms (ureaplasmas) in acute non-gonococcal urethritis (NGU) and particularly chronic NGU in men, but they rank third to Chlamydia trachomatis and M. genitalium. Whether the association of ureaplasmas with miscarriage and preterm labour is in the context of BV is not clear. Of no doubt, however, is the ability of ureaplasmas to cause septic arthritis in hypogammaglobulinaemic patients and there is evidence that they may cause some cases of sexually acquired reactive arthritis. The advent of polymerase chain reaction technology has seen an advance in the understanding of the role of M. genitalium; there is strong evidence that it is one of the causes of both acute and chronic NGU independent of C. trachomatis. There is some support for the role of M. genitalium in PID, but this needs to be substantiated. Other mycoplasmas, for example M. fermentans, M. pivum, M. primatum, M. penetrans, M. spermatophilum and even M. pneumoniae have the capacity to cause urogenital tract disease but there is no evidence to indicate that they do so.

Isolation and identification of mycoplasmas from pig lungs in Singapore.

A total of 656 lung specimens comprising 196 grossly pneumonic lungs from clinically diseased pigs and 230 grossly pneumonic and normal lungs from abattoirs were cultured for mycoplasmas. Mycoplasmas or acholeplasmas were recovered from 102 lung specimens, of which 28 were serologically identified (by disc growth inhibition and agar gel diffusion tests) as Mycoplasma suipneumonia, 14 as M hyorhinis, 35 as Acholeplasma granularum and 4 as A laidlawii. In addition, 15 isolates were unclassified and six were not typed. Mycoplasmas isolated from the suspect enzootic pneumonic lungs: slaughterhouse pneumonic lungs: normal lungs were in the ratio of 3:2:1. M suipneumonia, A granularum and A laidlawii were isolated from both normal and pneumonic lungs of pigs while M hyorhinis was only isolated from pneumonic ones. A close antigenic relationship between M suipneumonia and A granularum was demonstrated.

Ovine infectious keratoconjunctivitis: a microbiological study of clinically unaffected and affected sheep's eyes with special reference to Mycoplasma conjunctivae.

In a field survey of ovine infectious keratoconjunctivitis, the microbiological flora of 240 clinically unaffected eyes from sheep in 10 flocks was compared with the flora of an equivalent number of clinically affected eyes from 12 natural outbreaks of the disease. Totals of 16 and 17 genera of bacteria were recovered from unaffected and affected eyes, respectively. Staphylococcus, bacillus and branhamella were isolated significantly more often than the other genera of bacteria, in both the unaffected and affected eyes (P less than 0.05). Branhamella ovis and Escherichia coli occurred more frequently in affected eyes, and Staphylococcus aureus occurred more frequently in severely than mildly affected eyes. The genera Mycoplasma and Acholeplasma were isolated from both groups, and Mycoplasma conjunctivae occurred in 92 affected eyes (38.3 per cent), and 27 unaffected eyes (11.3 per cent).

Mycoplasmas and acholeplasmas isolated from ducks and their possible association with pasteurellas.

Two hundred and sixty-three cases of clinically diseased ducks of all ages were examined for the presence of mycoplasmas. Mycoplasmas and acholeplasmas belonging to more than eight serogroups were cultured from 68 of them, and comprised 12 M anatis, one M columbinasale, two M gallinaceum, two M gallinarum, nine M synoviae, three unidentified Mycoplasma species, 37 Acholeplasma laidlawii and one unclassified acholeplasma belonging to each of serogroups 7 and 8. They were identified by biochemical characterisation, disc growth inhibition and agar gel diffusion tests. Fifty-three (78 per cent) of the isolates occurred with species of Pasteurella: 33.8 per cent with Pasteurella anatipestifer, 32.4 per cent with P multocida and 11.8 per cent with both P anatipestifer and P multocida. Nine of the isolates (13.2 per cent) were in pure culture and six (8.8 per cent) with other agents. Of the ducks negative for mycoplasmas 33.3 per cent were infected with P anatipestifer, 25.1 per cent with P multocida and 14.4 per cent with both P anatipestifer and P multocida. There was no correlation between the infections with mycoplasmas and P anatipestifer but there was a weak association between the infections with mycoplasmas, especially M anatis and P multocida.

Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000.

Between 1990 and 2000, more than 1600 mycoplasmas and the related acholeplasmas were identified from ruminant animals by the Mycoplasma Group at the Veterinary Laboratories Agency--Weybridge. Mycoplasma bovis was the most commonly identified pathogen, mostly from pneumonic calves but occasionally from cattle with mastitis and arthritis. Mycoplasma canis was first isolated in Britain in 1995 from pneumonic calves and the number of isolates increased to 18 per cent of the total mycoplasmas isolated from cattle in 1999. The ELISA for antibodies to M. bovis detected 1971 positive samples (22 per cent) among 8959 serum samples, mainly from pneumonic cattle. Other mycoplasmas identified included Mycoplasma dispar from the lungs of cattle with respiratory disease, and Mycoplasma bovigenitalium from the reproductive tract of cows with vulvovaginitis and infertility. Mycoplasma bovirhinis and Acholeplasma species were found commonly but are thought to be more opportunistic than pathogenic. In sheep and goats, the majority of Mycoplasma species isolated were identified as Mycoplasma ovipneumoniae from pneumonic sheep, Mycoplasma conjunctivae from sheep with keratoconjunctivitis, and the ubiquitous Mycoplasma arginini.

[Prevalence of Mycoplasma orale as a contaminant of cell cultures in Argentina]. / Prevalencia de Mycoplasma orale como contaminante de los cultivos celulares en la Argentina.

Over a period of 4 years 200 cell cultures were analysed for the presence of mycoplasma. Cultures were established cell lines from different origins, namely monkey, mouse and human, hybrid cell cultures and primary cultures. The cultures belonged to various research and industrial laboratories located in different areas of the country. Seventy per cent of investigated cultures were found to be contaminated with mycoplasma using a DNA fluorescent stain. Fifty cultures, selected at random out of the contaminated cultures, were further investigated to identify the prevalent serotype. For that purpose immunofluorescent reactions were performed using immune sera raised against several mycoplasma strains routinely found among contaminated cultures. Forty one cultures were contaminated with a single type of mycoplasma, whereas in the remaining nine, two or even three serotypes were detected. Mycoplasma orale II contaminated 40% of single infected cultures, followed by M. hyorhinis and A. laidlawii-A (12% each), M. arginini (5%), M. orale III (8%), A. laidlawii-B (2%). We were unable to serotype the remaining positive cultures, because of the lack of a full battery of immune sera against all known serotypes. The prevalence of M. orale in mycoplasma contaminated cultures thus far tested, indicates that human handling would be the main source of infection. This situation could be modified by avoiding mouth pipetting and adopting good microbiological techniques.

[Lung tissue alterations following experimental infection of mice with acholeplasmae isolated from simians with hematosarcoma]. / Izmeneniia legochnoi tkani pri éksperimental'nom zarazhenii myshei akholeplazmami, vydelennymi ot obez'ian s gematosarkomoi.

This paper is devoted to the study of pathogenic acholeplasmae potencies. Two strains of acholeplasmae isolated from the blood and organs of Papio hamadryas suffering from hemoblastosis and a standard Ach. laidlawii A. strain were subjected to the comparative study with the use of a "pulmonary model" in experiments on mice. Morphological investigations showed affections of the pulmonary tissue characterized by stage-by-stage inflammatory-destructive changes analogous to the lesions found in "mycoplasmosis of the lungs". Prolonged inflammatory changes in the pulmonary tissue were maintained by a long-term persistence of acholeplasmae in it. A conclusion was drawn that acholeplasmae isolated from monkeys suffering from hematosarcoma had marked pathogenic action on the pulmonary tissue of mice. Strain differences in the pathogenic activity of the strains under study were revealed.

[Biochemical and serologic study of Acholeplasmae isolated from monkeys]. / Biokhimicheskoe i serologicheskoe izuchenie akholeplazm, vydelennykh ot obez'ian.

Biochemical and serological properties of mycoplasmas isolated from the blood, feces and parenchymatous organs of monkeys have been studied to determine their species. It was established that the isolated strains belong to the family Acholeplasmatoceae. The study of their biochemical properties in different tests has revealed the presence of 5 biochemically heterogeneous groups. Their serological properties suggest that 13 out of 45 strains are identical to the reference strain of A. laidlawii A, and all other strains have been classified as new Acholeplasma species which have never been isolated from monkeys before.