Mycoplasma DNA Research Articles

Detection of Mycoplasma DNA Using Conventional Polymerase Chain Reaction in Canine Abortion, Stillbirth, and Neonatal Mortality Cases in Central Italy

Mycoplasma spp. has been involved in canine infertility, but research on this topic is limited and shows conflicting results, as it has also been isolated from vaginal swabs of healthy dogs. This study aimed to retrospectively research Mycoplasma DNA by a conventional dual Polymerase Chain Reaction from 114 cases of canine abortion, stillbirth, and neonatal mortality that occurred in Central Italy. In addition, four fetal membranes from dystocic dams were analyzed. In total, 7 out of 114 cases (6.14%) and one of the fetal membranes tested positive for Mycoplasma DNA. From five of them (62.5%), other microorganisms were identified, particularly Canid herpesvirus-1 (2/8, 25%) and Escherichia coli, Staphylococcus aureus, and/or Staphylococcus pseudintermedius (3/8, 37.5%), notoriously responsible for infertility in bitches or neonatal mortality. In two different litters, only one puppy of each one tested positive for Mycoplasma DNA. Additionally, Mycoplasma DNA was detected in fetal membranes collected during a cesarean section of a bitch whelping Mycoplasma-negative puppies, supporting that Mycoplasma spp. is part of the normal genital microflora of the female. The detection of Mycoplasma DNA in association with other major pathogens and its detection in the female genital tract without transmission to puppies support the hypothesis that Mycoplasma belongs to the autochthonous genital microflora or, at most, may play a secondary role in canine abortion and neonatal mortality.

Detection, genetic diversity, and factors associated with infection by hemotropic mycoplasmas in rodent and marsupial populations in regions of the states of Rio de Janeiro and Parana, Brazil

Hemotropic mycoplasmas, also known as hemoplasmas, are found in several animal species around the world. New species and genotypes of mycoplasmas have been described recently but much more remains to be discovered, especially in wildlife. The objective of this study was to investigate the presence of hemotropic mycoplasmas and diversity of the 16S rDNA sequence fragment in populations of small nonflying mammals in regions with different phytophysiognomies in the states of Rio de Janeiro and Parana, Brazil, as well as to evaluate environmental and animal factors influencing infection rates. Of the 257 animals sampled, 33.8% (n=87) were considered positive by conventional PCR. Of the 230 samples from wild rodents (Cricetidae), 36.5% (n=84) amplified hemotropic mycoplasma DNA, followed by synanthropic rodents (Muridae) (12.5%, n= 2/16), and marsupials (Didelphidae) (9.0%, n= 1/11). The regions with the highest frequency of positivity were Ponta Grossa (46.5%, n=27/58) and Cruz Machado (46.1%, n= 24/52), followed by Nova Friburgo (30.8%, n= 33/107), and Lidianopolis (12.5%, n=5/40). Oligoryzomys had the highest rate of infection (78.5%), statistically different from Oxymycterus spp. (40.0%), Akodon spp. (27.0%) and Sooretamys (8.3%). Males had a higher prevalence of Mycoplasma spp., being 3.97 times more likely to be positive for hemoplasma than females. Phylogenetic analysis grouped the sequences from this study into two clades, A and E, together with other hemoplasma sequences previously detected in small wild and synanthropic rodents from Brazil, Hungary, and Japan. Haplotype network reveals exclusive haplotypes for wild and synanthropic rodents. Our study reveals considerable genetic diversity suggesting the circulation of at least two species among sequences from this study and highlights the importance of considering environmental and biological factors in the epidemiology of these infections in wild mammals.

Molecular Survey and Genetic Characteristics of Vector-Borne Pathogens in Domestic Dogs from Four Regions of China.

Canine vector-borne diseases are widely distributed around the world. They are transmitted by arthropods, and many seriously threaten the health of animals and humans. In China, our knowledge of Ehrlichia, Hepatozoon, and Mycoplasma species circulating in dogs is still poorly understood. Therefore, the aim of this study was to understand the prevalence and genetic characteristics of canine Ehrlichia spp., Hepatozoon spp., and Mycoplasma spp. in Chongqing (southwest), Fujian (southeast), Shandong (southeast), and Hubei (central) Provinces of China. Blood samples from healthy pet dogs were processed to detect Ehrlichia, Hepatozoon, and Mycoplasma DNA with PCR. Haplotype and phylogenetic analyses were performed on 18S rRNA sequences. Among 306 dogs, no Ehrlichia spp. or Mycoplasma spp. were detected, whereas one Hepatozoon sp. was detected in 10 (3.27%) of the animals. Only Hepatozoon canis was identified and was endemic to Chongqing (2.46%) and Hubei (8.77%). A haplotype analysis identified eight haplotypes among the H. canis isolates. A phylogenetic analysis showed that the H. canis isolates in this study clustered into four clades, together with isolates from different countries and hosts, forming a large group that was clearly separate from other Hepatozoon species. These findings provided new information on the epidemiological characteristics of canine vector-borne diseases in China and will be helpful in the development of efficient measures to safeguard the health and well-being of companion animals and their owners.

The association of host and vector characteristics with Ctenocephalides felis pathogen and endosymbiont infection.

Surveillance of the fleas and flea-borne pathogens infecting cats is important for both human and animal health. Multiple zoonotic Bartonella and Rickettsia species are known to infect the most common flea infesting cats and dogs worldwide: Ctenocephalides felis, the cat flea. The ability of other flea species to transmit pathogens is relatively unexplored. We aimed to determine cat host and flea factors independently associated with flea Bartonella and Rickettsia infection. We also assessed flea and cat infection by flea-host pair and location. To accomplish these aims, we performed qPCR for the detection of Bartonella, hemotropic Mycoplasma, Rickettsia, and Wolbachia DNA using paired cat and flea samples obtained from free-roaming cats presenting for spay or neuter across four locations in the United States. A logistic regression model was employed to identify the effect of cat (sex, body weight, geographic location, and Bartonella, hemotropic Mycoplasma, and Rickettsia spp., infection) and flea (clade and Rickettsia and Wolbachia infection) factors on C. felis Bartonella clarridgeiae infection. From 189 free roaming cats, we collected 84 fleas: Ctenocephalides felis (78/84), Cediopsylla simplex (4/84), Orchopeas howardi (1/84), and Nosopsyllus fasciatus (1/84). Ctenocephalides felis were phylogenetically assigned to Clades 1, 4, and 6 by cox1 gene amplification. Rickettsia asembonensis (52/84) and B. clarridgeiae (16/84) were the most common pathogenic bacteria detected in fleas. Our model identified host cat sex and weight as independently associated with B. clarridgeiae infection in fleas. Rickettsia asembonensis (52/84), Rickettsia felis (7/84) and Bartonella henselae (7/84) were detected in specific clades: R. felis was detected only in Clades 1 and 6 while B. henselae and R. asembonensis were detected only in Clade 4. Wolbachia spp., also displayed clade specificity with strains other than Wolbachia wCfeT only infecting fleas from Clade 6. There was poor flea and host agreement for Bartonella spp., infection; however, there was agreement in the Bartonella species detected in cats and fleas by geographic location. These findings reinforce the importance of considering reservoir host attributes and vector phylogenetic diversity in epidemiological studies of flea-borne pathogens. Widespread sampling is necessary to identify the factors driving flea-borne pathogen presence and transmission.

Identification of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma arginini by culture, PCR, and histopathology in pneumonic lungs of slaughtered goats in Mashhad, Iran.

A number of Mycoplasma spp., often referred to as the Mycoplasma mycoides (Mm) cluster can produce respiratory tract infections in goats; however, only Mycoplasma capricolum subspecies capripneumoniae (Mccp) is considered to causecontagious caprine pleuropneumonia. Isolation and identification of M. capricoluum subspecies capripneumoniae and M. arginini from the pneumonic lungs of slaughtered goats and their association with pathological changes. Lungs of 2000 goats slaughtered at an industrial abattoir in Mashhad, Iran, were examined for the presence of gross pneumonic lesions. Fifty affected lungs were selected for pathology, culture, and molecular (PCR) studies for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and replicated using genus and species specific primers for Mycoplasma. Grossly, consolidation and dark red to grey discoloration in the cranioventral to caudal lobes in fibrinopurulent bronchopneumonia and rubbery texture associated with rib impressions on the costal surfaces of the diaphragmatic lobes in interstitial pneumonia were observed. Histopathologically, bronchointerstitial pneumonia in 40 (80%), and fibrinopurulent bronchopneumonia in 10 (20%) of affected goats were diagnosed. The evidence of Mycoplasma growth such as turbidity and Mycoplasma colonies on the Mycoplasma agar plates was observed in 2 (4%) of samples. Genus-specific Mycoplasma DNA was identified in 11 (22%) of samples. Of them, 3 (6%) and 3 (6%) of tissue lung samples were positive for M. capricolum subspecies capripneumoniae and M. arginini, respectively, by PCR. Our results showed that M. capricolum subspecies capripneumoniae and M. arginini were the two agents that can involve lung consolidation and pneumonia in goats.

The prevalence and species of Mycoplasma identified from dairy goats in Taiwan

Except for Mycoplasma capricolum subsp. capripneumoniae, which only causes pneumonia, Mycoplasma species can cause otitis, arthritis, septicemia, mastitis, pneumonia and encephalitis in goats. After a mycoplasma outbreak was recorded in Taiwan in 2016, an increasing number of mycoplasma infection cases have been observed. It is important to understand how many Mycoplasma species are currently prevalent in Taiwan. In this study, 57–61 bulk milk samples were collected from dairy goats in Taiwan every year from 2017 to 2020 to identify the presence of mycoplasma DNA fragments by polymerase chain reaction (PCR). The mycoplasma species were identified based on colony characteristics, PCR, and DNA sequences. In total, 39 out of 236 samples (16.52%) tested mycoplasma positive. The annual mycoplasma prevalence rates from 2017 to 2020 were 24.59% (15/61), 8.20% (5/61), 17.54% (10/57) and 15.79% (9/57), respectively. Mycoplasma mycoides subsp. capri (n = 19), Mycoplasma putrefaciens (n = 10), Mycoplasma ovipneumoniae (n = 5) and Mycoplasma bovis (n = 5) were present in Taiwan. No M. capricolum subsp. capripneumoniae was detected. Except for the large drop in 2018, the prevalence of mycoplasma infection declined to a consistent level for the final two years of the study. However, the seasonal pattern is still worth investigating. This is the first survey of mycoplasma infection in goats, and we believe that a larger survey is still needed to understand the pathogenicity and diversity of these mycoplasma isolates.

Occurrence of urogenital mycoplasmas in men with the common genitourinary diseases

Many serious and fatal infections with urogenital mycoplasmas in immunocompromised patients have been reported. M. genitalium is recognized as a cause of male urethritis and other common genitourinary diseases. The aim of the study was to estimate prevalence of urogenital mycoplasmas which can cause complications in men with common genitourinary diseases. Study included 85 men with genitourinary tract carcinoma (n = 35), urolithiasis (n = 36), and BPH (benign prostatic hyperplasia) (n = 14). The control group consisted of 50 healthy men. FVU (first void urine) samples were examined by PCR for the presence of urogenital mycoplasmas DNA. Occurrence of urogenital mycoplasmas was significantly more common in study group compared with control 24/85 (28.2%) and 7/50 (14%), respectively (p = 0.05). In men with urolithiasis, positive results for mycoplasmas DNA were significantly more frequent than in control: 33.3% vs. 14% (p < 0.05). In patients with urolithiasis DNA of U. urealyticum was most often found, while in the genitourinary carcinoma and BPH groups, U. parvum was more frequent. Incidence of M. fermentans was also significantly higher in the urolithiasis group vs. control (p = 0.03). A higher percentage of positive results for urogenital mycoplasma DNA in study group has been found. Further studies are required to confirm the role of urogenital mycoplasmas in the development of infectious complications among patients with urolithiasis, genitourinary carcinoma, and BPH.

Infertility in dairy cows – Possible bacterial and viral causes

In this research uterine swab and biopsy samples were collected from 40 infertile dairy cows kept at five dairy cattle farms in Hungary. Samples were tested for bacteria including Coxiella burnetii chlamydiae, Mycoplasma and Ureaplasma, and for the viruses Bovine herpesvirus 1 (BoHV-1) and Bovine viral diarrhoea virus (BVDV). Chlamydiaceae DNA was detected by real-time PCR in 22/40 (55%) samples. Coxiella burnetii DNA was detected in 3/40 (7.5%) cases by real-time PCR. Mycoplasma and Ureaplasma DNA was found in 2/40 (5%) and 4/40 (10%) cows, respectively. BVD and BoHV-1 DNA was not detected in any samples. Escherichia coli as a recognised uterine pathogen was found in two cases. The following potential uterine pathogens were found: Bacillus licheniformis (one case), non- haemolytic streptococci (five cases), Histophilus somni (two cases) and Candida krusei (two cases). Blood samples were collected at same time as swab samples from all 40 cows, and their examination for C. burnetii antibodies by ELISA revealed seropositivity in 26/40 cows (65%). Histological examination of the uterine biopsy samples showed the presence of mild lympho-histiocytic infiltration in the mucosain 22 cases (59%). Moderatelympho-histiocytic infiltration of the endometrium was evident in 13 cases (35%), while in two cases (6%) severe inflammatory cell infiltration of the endometrium with lympho-histiocytes and neutrophil granulocytes was found. Although no statistical correlation could be demonstrated between the severity of histological lesions of the endometrium and the uterine pathogenicity of the bacteria (P = 0.8555), endometritis of a certain severity grade and/or a recognised or potential uterine pathogen were found in all samples. The latter may play a role in the development of infertility either collectively or independently.

Testing Hybridoma Cells for Mycoplasma Contamination.

Because mycoplasmas are a diverse group of organisms and are difficult to culture, several different strategies for detecting mycoplasma contamination have been developed. To date, no one test is suitable for detecting all of the possible mycoplasmas that may contaminate hybridoma or myeloma cultures. Therefore, it is sensible to consider using several methods. The most commonly used techniques are described here. Mycoplasma screening by growth on microbial medium can be performed on agar plates or in broth culture. Cultures are grown under both aerobic and anaerobic conditions because some common strains of Mycoplasma prefer the lack of oxygen (Mycoplasma buccale, Mycoplasma faucium, M. orale, and M. salivarium). Mycoplasma colonies form a characteristic "fried egg" appearance on agar plates, and this is the diagnostic feature used to confirm mycoplasma contamination. The colonies are small and are most easily seen with an inverted microscope. A quicker method for testing for mycoplasma takes advantage of the DNA-intercalating dye Hoechst 33258. Fixed cells are stained with the dye, and contaminated cultures are detected by the bright, punctate cytoplasmic staining of the Mycoplasma DNA. Finally, commercial kits for the detection of mycoplasmas using colorimetric assays or reporter cells are also described.

Control tactics and evaluation of clinical efficacy of therapy in children with bronchial asthma associated with mycoplasma infection

The uncontrolled course of bronchial asthma (BA) in children and insufficient efficacy of standard therapy regimens may be due to underestimated infectious factors. The objective: to study specific parameters of the course and treatment of mycoplasma infection, improve monitoring over BA therapy in children of the tender and preschool age. Subjects and methods. 320 children with BA in the age from 1 to 7 years old were followed up. In this work, Mycoplasma pneumoniae (FH), Mycoplasma hominis (H-34), Ureaplasma urealyticum (serotype 8), Mycoplasma fermentans (PG18) and Mycoplasma arthritidis (PG6) were used, they were cultured on a liquid medium for cultivation of mycoplasmas and ureaplasmas. To isolate CIC from blood serum samples, we used the method of precipitation with 3.5% polyethylene glycol (PEG, 6000 Da), hemagglutination assays and IFA were used to identify mycoplasma antigens, mycoplasma DNA was detected by PCR with InterLabService diagnostic kits. The data of 47 patients with prolonged mycoplasma antigenemia were assessed at the baseline and in 1.5-3 months after the treatment course of azithromycin.Results. 320 blood serum samples from children with BA were tested, and the detection rate by hemagglutination assays of M. pneumoniae antigens was 60.9%, M. hominis – 43.4%, U. urealyticum – 44.8%, M. arthritidis – 29.7%, M. fermentrans – 45.3%. The assessment of relationship between of M. pneumoniae, M. hominis and asthma exacerbation showed that antigens of M. pneumoniae and M. hominis were found in 216 children (single or associated). After treatment with azithromycin, the frequency of BA exacerbations within 3 months decreased by 2.4 times, as well as there was a reduction in the number of samples positive for antigens and DNA of mycoplasma in a free state and within CIC. The persistence of antigens, DNA of M. pneumoniae and M. hominis before treatment of 47 children was 80.9 and 66.0% of cases, after treatment with azithromycin – 31.9 and 25.5% of cases, respectively (p &lt; 0.001). Within CIC isolated from the blood serum of patients, antigens to M. pneumoniae and M. hominis before treatment were detected by IFA in 63.8 and 70.2% of children, after treatment – in 31.9 and 23.4%, respectively. p &lt; 0.001. In blood samples, DNA of M. pneumoniae and M. hominis was detected by PCR before treatment in 8.5 and 34.0%; after treatment in 6.4% (p = 0.318) and 19.1% of cases, respectively (p = 0.009), and within CIC isolated from blood serum, in 27.7 and 48.9% of cases before treatment and 8.5 and 34.0% after it, respectively (p = 0.009).

Detection of Infectious Agents in Samples from Reptiles Presented at Veterinary Clinics in Poland

This study screened reptiles presented to specialized veterinary practices in Poland for DNA and RNA of six pathogens and evaluated connections between infections and animal husbandry. Four clinics with veterinarians specializing in exotic animal medicine collected swabs from the soft palate, cloaca, and any lesions from reptiles during routine visits. All samples were screened for the presence of herpesvirus, adenovirus, ranavirus, and mycoplasma DNA. Snake samples were also screened for reptarenavirus and nidovirus RNA. A short questionnaire was filled out by the owner(s) that included questions about the animal and its husbandry. Samples were collected from 254 animals; 107 (42.1%) tested positive for at least one pathogen. The most common agents detected were mycoplasmas (29.1% positive) and adenoviruses (10.6% positive); no rana- or reptarenaviruses were detected. Herpes and nidoviruses were each found in a few cases (2 and 13, respectively). Mycoplasmas were most often detected in Russian tortoises (Testudo horsfieldii; 88.9% positive) and green tree pythons (Morelia viridis; 76.9% positive). Adenoviruses were detected primarily in bearded dragons (Pogona vitticeps; 35.2% positive). Nidoviruses were found only in pythons in the genera Python and Morelia. Herpesviruses were detected in green iguanas (Iguana iguana). There was no correlation between husbandry and mycoplasma or virus detection. Although the detection of many of these pathogens corresponds to findings in other countries, several (high rates of mycoplasma-positive snakes and herpesvirus-positive iguanas) were surprising and may reflect specific disease dynamics within Poland.

Widespread Infection with Hemotropic Mycoplasmas in Free-Ranging Dogs and Wild Foxes Across Six Bioclimatic Regions of Chile.

Blood samples of 626 rural dogs, 140 Andean foxes (Lycalopex culpaeus), and 83 South American grey foxes (L. griseus) from six bioregions of Chile spanning 3000 km were screened for Mycoplasma DNA by conventional PCR and sequencing. Risk factors of infection were inferred using Generalized Linear Mixed Models and genetic structure by network analyses. Overall, Mycoplasma haemocanis/Mycoplasma haemofelis (Mhc/Mhf) and Candidatus Mycoplasma haematoparvum (CMhp) observed prevalence was 23.8% and 12.8% in dogs, 20.1% and 7.2% in Andean foxes, and 26.5% and 8.4% in grey foxes, respectively. Both hemoplasmas were confirmed in all the bioregions, with higher prevalence in those where ticks from the Rhipicephalus sanguineus species group were absent. Candidatus M. haematominutum and a Mycoplasma sp. previously found in South American carnivores were detected in one fox each. Although the most prevalent Mhc/Mhf and CMhp sequence types were shared between dogs and foxes, network analysis revealed genetic structure of Mhc/Mhf between hosts in some regions. Male sex was associated with a higher risk of Mhc/Mhf and CMhp infection in dogs, and adult age with CMhp infection, suggesting that direct transmission is relevant. No risk factor was identified in foxes. Our study provides novel information about canine hemoplasmas with relevance in distribution, transmission routes, and cross-species transmission.

Mycoplasma and Ureaplasma Molecular Testing Does Not Correlate with Irritative or Painful Lower Urinary Tract Symptoms.

For patients with persistent irritative lower urinary tract symptoms, such as dysuria and urinary frequency, evaluation for the atypical organisms Ureaplasma and Mycoplasma has been a common part of care. However, these species are genitourinary colonizers and have not been established as causative pathogens in chronic lower urinary tract symptoms. We therefore sought to evaluate diagnostic testing patterns for Ureaplasma and Mycoplasma and characterize the associations of these bacteria with irritative lower urinary tract symptoms using molecular detection techniques. Ureaplasma/Mycoplasma testing patterns for 2019 were assessed using an anonymized data repository. Clean catch urine specimens (179) were collected prospectively from female and male patients with and without irritative lower urinary tract symptoms. Quantitative polymerase chain reaction evaluated urinary Ureaplasma and Mycoplasma DNA concentrations, while next-generation sequencing assessed the relative abundance of Ureaplasma and Mycoplasma within the urinary bacterial population. Ureaplasma/Mycoplasma testing was common, with 575 tests performed in 2019 in our community hospital system. In our cohort, Ureaplasma and Mycoplasma were identified in similar proportions in symptomatic and asymptomatic subjects: 25% of female controls and 27% of females with lower urinary tract symptoms and 9.5% of asymptomatic males and 3.3% of men with symptoms (p=0.87 and p=0.91 for females and males, respectively). Regression analysis revealed that both abundance and concentrations of Mycoplasmataceae correlated negatively with a range of irritative lower urinary tract symptoms, including dysuria and urethral pain. A statistically significant negative correlation of Ureaplasma/Mycoplasma levels with a variety of lower urinary tract symptoms suggests that polymerase chain reaction-based Mycoplasmataceae detection has little diagnostic benefit in assessment of chronic irritative urinary symptoms.

Flea-borne pathogens in the cat flea Ctenocephalides felis and their association with mtDNA diversity of the flea host

Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected populations.

Molecular identification of vector-borne organisms in Ehrlichia seropositive Nicaraguan horses and first report of Rickettsia felis infection in the horse

Certain vector-borne organisms serve as etiological agents of equine disease. After previously identifying a new Ehrlichia species in horses from Mérida, we aimed to determine the infection frequency and screen for a wide range of vector-borne organisms from 93 tick-exposed, Ehrlichia seropositive horses in this region. PCR assays were performed to identify infection by organisms within the following genera: Anaplasma, Babesia, Bartonella, Ehrlichia, Leishmania, Mycoplasma, Neorickettsia, Rickettsia and Theileria. Overall, 90/93 horses (96.8%) were infected with one or more vector-borne organisms. Ninety (96.8%) horses were infected with Theileria equi and 21 (26.8%) with Babesia caballi. Nine (9.7%) horses were infected with the novel Ehrlichia species previously designated H7, reported in horses from Nicaragua and Brazil. Two horses (2.2%) were infected with Rickettsia felis. Anaplasma, Bartonella, Leishmania, Mycoplasma, or Neorickettsia species DNA was not amplified from any horse. Ticks collected from horses infected with vector-borne organisms were identified as Amblyomma cajennense sensu lato and Dermacentor nitens. Horses in Mérida are infected by a range of vector-borne organisms, including B. caballi, T. equi, Ehrlichia species H7, and R. felis. To the authors’ knowledge, this constitutes the first report of molecular detection of R. felis in horses.

Sane and sound: a serologic and molecular survey for selected infectious agents in neozootic Egyptian geese (Alopochen aegyptiacus) in Germany

Aquatic birds can act as vectors and reservoir hosts for pathogens relevant for wild birds and poultry as well as human health. In this study, we address the questions (1) if the Egyptian goose (Alopochen aegyptiacus), as one of the most successful neozootic bird species in Europe, carry infectious agents that are relevant for poultry and wild birds and (2) if seasonal prevalences of these infectious agents differ from those of native geese species. In 2015 and 2016, up to 190 Egyptian geese from Western Germany were investigated serologically for antibodies (Ab) against influenza A viruses (IAV), Avian avulavirus 1 (AAvV-1), aviadenoviruses, Duck atadenovirus A (syn.: egg drop syndrome 1976 virus) (EDSV), and West Nile virus (WNV). Ab were detected against IAV in 6.1% (10/164), against AAvV-1 in 2.4% (4/165), against EDSV in 15.2% (16/105), and against aviadenoviruses in 0.86% (1/116) of the geese blood samples, respectively. None of the birds had Ab against WNV (0/84). PCR-based techniques (cloacal and/or pharyngeal swabs) were applied for the presence of IAV, AAvV-1, Mycoplasma spp., and Riemerella anatipestifer. Riemerella DNA was detected in the pharyngeal swabs with an overall prevalence of 70.3% (104/148). Neither Mycoplasma DNA nor IAV or AAvV-1 RNA could be detected in the pharynx or cloaca of the examined birds. Our study shows that Egyptian geese are frequent carriers of Riemerella anatipestifer and furthermore provides serological evidence of exposure to IAV, AAvV-1, and EDSV. It is one of very few studies on infectious agents of neozootic bird species. Comparing our results from a neozootic non-migratory goose species with published results from native migratory geese species (bean goose (Anser fabalis) and white-fronted goose (A. albifrons)) and another neozootic non-migratory goose species (Canada goose (Branta canadensis)), we found differences in the seroprevalence of viral pathogens. Native goose species show higher seroprevalences of IAV and AAvV-1, whereas neozootic non-migratory geese reveal higher seroprevalences for EDSV. The findings are discussed in the frame of seasonal variations in selected infectious agents, differences in sampling periods, and contrasting movement ecology of the different geese species.

Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine

Mycoplasmas are potential contaminants that introduce undesirable changes in mammalian cell cultures. They frequently contaminate cell substrates and other starting materials used for manufacturing cell-derived biologics, such as vaccines and pharmaceutical products. Mycoplasma purity testing of live vaccines, active ingredients, raw material, and seed lots is required during vaccine production. Previously, testing using a time-consuming, costly 28-day culture assay, which lacks sensitivity for species that do not grow in culture, was required in the European Pharmacopoeia (Ph. Eur). But now nucleic acid amplification techniques (NATs) can be used. NATs provide rapid results and are sensitive. We evaluated the sensitivity and specificity of a commercially-available NAT to detect individual mycoplasma DNA in a veterinary modified live vaccine using five reference strains recommended by the Ph. Eur. Our results showed that this NAT-based method can be used to detect mycoplasma in spiked live vaccine, without interference from the vaccine components, with a limit of detection of 10 CFU/mL, as required by the Ph. Eur. Its specificity was demonstrated since no mycoplasmas were detected in non-spiked vaccine. This method is undergoing validation as a replacement for the conventional culture method in the production of veterinary live vaccines.

The etiological structure of diseases in frequently ill children depending on age

The urgency of the problem of frequently ill children is associated with a significant number of patients who can be assigned to this category. Objective: to present the nosological and etiological structure of diseases in frequently ill children depending on age. Subjects and methods. 243 children aged 1 to 17 years with recurrent respiratory infections were followed up. The children were examined using routine clinical, laboratory, and instrumental examinations. The etiological diagnosisincluded bacteriological examination of the upperrespiratory tract microflora; determination of IgM and IgG antibodies against Chlamydophila pneumoniae, Mycoplasma pneumoniae, Epstein–Barr virus(EBV), and cytomegalovirus(CMV) in blood by enzyme immunoassay and that of Chlamydia and Mycoplasma DNA in pharyngealswabs and that of CMV, EBV and human herpesvirustype 6 DNA in blood by polymerase chain reaction. Results. Recurrentrespiratory diseases were found to be associated with herpesvirusinfection in 75% of the children aged 1 to 6 years, with Streptococcus, Mycoplasma, and Chlamydia infections in 16, 10, and 4% of those aged 3 to 6 years, respectively, and to be accompanied by abnormal lymphoid tissue proliferation in 84% of the children aged 3 to 6 years, with the abundant growth of bacterial opportunistic pathogensin one half of the patients. Despite the continuing importance of infectious agents(herpesviruses and streptococci in 29 and 20%, respectively), the chronically ill school-aged children displayed a high incidence of somatic diseases: chronic tonsillitis (43%) and chronic sinusitis (14%) in children aged older than 12 years and allergic rhinitis (23%) in those aged 7 to 17 years. The symptoms of autonomic vascular dystonia were observed in 21% of the patients; cardiac arrhythmias were diagnosed in 14%.

Mixed infections and inflammatory ophthalmic diseases: clinical and laboratory observations

Over the period 2013-2016, 34 patients (14 men and 20 women) with different inflammatory processes in the eye who appeared DNA-positive for mycoplasmas (Mycoplasma hominis, Ureaplasma urealyticum) and/or chlamydiae (Chlamydia trachomatis) (PCR testing of tear fluid and/or urine) were followed up. All patients were examined for intensive production of herpesvirus, adenovirus, and enterovirus DNA in biological fluids. After being consulted by related specialists, all the patients started local and systemic (antibacterial and antiviral) therapy. In the end of the latter, laboratory tests were repeated. Among all the clinical forms, anterior segment inflammation (i.e. of conjunctiva, cornea, and the anterior vascular tract) prevailed - 76%. In most patients, mycoplasmas and/or chlamydiae formed associations with herpesviruses (n=19; 56%). Bacterial DNA alone (mycoplasma and/or chlamydia) was detected in 12 cases (35%). In 4 cases, mycoplasma and/or chlamydia DNA was detected in tear fluid, in 19 patients - in urine, and in 10 patients - in both secreta. Local and systemic causal treatment enabled resolution of the complaints and symptoms and yielded negative results of follow-up laboratory tests. More than a half of the patients demonstrated concomitant viral-bacterial infection (22 cases). The presence of bacterial/viral DNA in biological secreta, as revealed by PCR, reflects the systemic nature of the infection process and, thus, necessitates engagement of related specialists (dermatologists, urologists, gynecologists).

Mycoplasma Diversity in Arctic Permafrost

Viable bacterial cells and its genetic material can be stably maintained in Arctic permafrost for a long geological time. Because of the seasonal melting of permafrost strata, it cannot be excluded an access to the surface of ancient highly invasive species with increased pathogenicity. Mycoplasmas are very successful pathogens in humans, mammals, birds, insects, and plants, with high genome plasticity and ability to avoid immune response of host organism. The metagenomic approach allowed us to predict mycoplasma diversity in the Arctic permafrost. The number of mycoplasma DNA fragments in soil deposits of comparable age (∼30,000 years) and origin (the late Pleistocene Ice Complexes) is not so abundant compared with other microorganisms, but it is enough for a chance in the presence of living mycoplasmal cells in permafrost. DNA fragments of human, animal, insect, and plant pathogens were identified. The “ubiquitous” mycoplasma Acholeplasma laidlawii is the undisputed leader in the number of identified sequences in all three metagenomes. It may indicate a higher adaptive capacity and more powerful metabolic potential of A. laidlawii among Mollicutes.