Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Determination of Optimal Phage Load and Administration Time for Antibacterial Treatment.

Using phages as antibacterials is becoming a customary practice in Western countries. Nonetheless, successful treatments must consider the growth rate of the bacterial host and the degradation of the virions. Therefore, successful treatments require administering the right amount of phage (viral load, Vφ) at the right moment (administration time, Tφ). The present protocols implement a machine learning approach to determine the best combination of Vφ and Tφ to obtain the elimination of the target bacterium from a system. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: One bacterium, one phage Alternate Protocol 1: One bacterium, one phage (wrapping function) Alternate Protocol 2: One bacterium, one phage (wrapping function, alternative growing model) Basic Protocol 2: Two bacteria, one phage Alternate Protocol 3: Two bacteria, one phage (launch from terminal).

Occurrence of Mycoplasma parvum in German Pigs of Different Age Groups Using a Novel Quantitative Real-Time PCR Assay

Mycoplasma (M.) parvum is a hemotrophic bacterium circulating in the blood of pigs but is not considered a primary pathogen. Only a handful of studies dealing with this agent have been published since its first description in 1951, and many issues, including epidemiology and the impact of subclinical infections, are yet to be elucidated. This study aimed to establish a M. parvum specific real-time PCR for its detection and quantification in porcine blood and the application of this assay to obtain insights into the occurrence of M. parvum in German pigs. Furthermore, 16S rDNA amplicons of M. parvum positive blood samples were phylogenetically analyzed using MEGA 11 software. The established qPCR targeting the M. parvum glyceraldehyde-3-phosphate dehydrogenase encoding gene (gap) showed a lower detection limit of 10 gene copies per reaction and no cross-reactivity within the specificity test. A total of 36.0% (n = 72) of the sampled fattening pigs, 25.0% (n = 15) of the sows, and 4.37% (n = 8) of the boars tested M. parvum positive. The dendrogram showed the typical allocation of the M. parvum isolates into the "haemominutum group" subgroup within the hemotrophic Mycoplasma species. Both the novel established qPCR and the obtained epidemiological data can serve as an important basis for future studies dealing with M. parvum.

Reassessment of Historical Clinical Trials Supports the Effectiveness of Phage Therapy.

Phage therapy has become a hot topic in medical research due to the increasing prevalence of antibiotic-resistant bacteria strains. In the treatment of bacterial infections, bacteriophages have several advantages over antibiotics, including strain specificity, lack of serious side effects, and low development costs. However, scientists dismissed the clinical success of early clinical trials in the 1940s, slowing the adoption of this promising antibacterial application in Western countries. The current study used statistical methods commonly used in modern meta-analysis to reevaluate early 20th-century studies and compare them with clinical trials conducted in the last 20 years. Using a random effect model, the development of disease after treatment with or without phages was measured in odds ratios (OR) with 95% confidence intervals (CI). Based on the findings of 17 clinical trials conducted between 1921 and 1940, phage therapy was effective (OR = 0.21, 95% CI = 0.10 to 0.44, P value < 0.0001). The current study includes a topic review on modern clinical trials; four could be analyzed, indicating a noneffective therapy (OR = 2.84, 95% CI = 1.53 to 5.27, P value = 0.0009). The results suggest phage therapy was surprisingly less effective than standard treatments in resolving bacterial infections. However, the results were affected by the small sample set size. This work also contextualizes the development of phage therapy in the early 20th century and highlights the expansion of phage applications in the last few years. In conclusion, the current review shows phage therapy is no longer an underestimated tool in the treatment of bacterial infections.

Vitamin C Deficiency in Blood Samples of COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is the most notable pandemic of the modern era. A relationship between ascorbate (vitamin C) and COVID-19 severity is well known, whereas the role of other vitamins is less understood. The present study compared the blood levels of four vitamins in a cohort of COVID-19 patients with different severities and uninfected individuals. Serum concentrations of ascorbate, calcidiol, retinol, and α-tocopherol were measured in a cohort of 74 COVID-19 patients and 8 uninfected volunteers. The blood levels were statistically compared and additional co-morbidity factors were considered. COVID-19 patients had significantly lower plasma ascorbate levels than the controls (p-value < 0.001), and further stratification revealed that the controls had higher levels than fatal, critical, and severe COVID-19 cases (p-values < 0.001). However, no such trend was observed for calcidiol, retinol, or α-tocopherol (p-value ≥ 0.093). Survival analysis showed that plasma ascorbate below 11.4 µM was associated with a lengthy hospitalization and a high risk of death. The results indicated that COVID-19 cases had depleted blood ascorbate associated with poor medical conditions, confirming the role of this vitamin in the outcome of COVID-19 infection.

Impact of cytotoxic agents or apoptosis stimulants on αklotho in MDCK, NRK-52E and HK2 kidney cells.

αKlotho is a transmembrane protein acting as a co-receptor for FGF23, a bone hormone regulating renal phosphate and vitamin D metabolism. αKlotho expression is controlled by PPARγ. Soluble αklotho (sKL) regulates cellular signaling impacting stress resistance and death. αKlotho deficiency causes early onset of aging-associated diseases while its overexpression markedly increases lifespan. Cellular stress due to cytotoxic therapeutics or apoptosis induction through caspase activation or serum deficiency may result in cell death. Owing to αklotho's role in cellular stress and aging, this study explored the effect of cytotoxic agents or apoptosis stimulants on cellular αklotho expression. Experiments were performed in renal MDCK, NRK-52E and HK-2 cells. Gene expression was determined by qRT-PCR, sKL by ELISA, apoptosis and necrosis by annexin V binding and a fluorescent DNA dye, and cell viability by MTT assay. Cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis induction with caspase 3 activator PAC-1 and serum deprivation induced αklotho and PPARG gene expression while decreasing viability and proliferation and inducing apoptosis of MDCK and NRK-52E cells to a variable extent. PPARγ antagonism attenuated up-regulation of αklotho in MDCK cells. In HK-2 cells, αklotho gene expression and sKL protein were down-regulated by chemotherapeutics. SKL serum levels in patients following chemotherapy were not significantly changed. In summary, potentially fatal stress results in up-regulation of αKlotho gene expression in MDCK and NRK-52E cells and down-regulation in HK-2 cells. These results indicate that different renal cell lines may exhibit completely different regulation of αklotho.

Occurrence of 'Candidatus Mycoplasma haemosuis' in fattening pigs, sows and piglets in Germany using a novel gap-based quantitative real-time PCR assay.

BACKGROUND: The appearance of the novel porcine haemotrophic mycoplasma (HM) species 'Candidatus Mycoplasma haemosuis' was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of 'Ca. M. haemosuis' in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained. RESULTS: Our novel qPCR assay successfully detected 'Ca. M. haemosuis' in all blood samples from the 'Ca. M. haemosuis'-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood. 'Candidatus M. haemosuis' was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for 'Ca. M. haemosuis'. Co-infections with M. suis were evident in all age groups. CONCLUSION: 'Candidatus M. haemosuis' infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for 'Ca. M. haemosuis' described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of 'Ca. M. haemosuis' infections.

Update on shedding and transmission routes of porcine haemotrophic mycoplasmas in naturally and experimentally infected pigs.

Horizontal transmission of Mycoplasma suis via parenteral exposure during standard practices or through bites during fightings have been identified as key epidemiological routes. However, as knowledge gaps on other potential shedding and transmission routes exist, the present study combines both laboratory experiments and field surveys to gain new insights into the epidemiology of porcine haemotrophic mycoplasmas. Splenectomised pigs were orally inoculated with a M. suis field strain and investigated for clinical signs related to infectious anaemia of pigs (IAP) and the presence of M. suis in blood, urine and saliva samples by qPCR. All blood samples were negative for M. suis and animals did not show obvious clinical signs of IAP throughout the entire study period. Additionally, urine, nasal and saliva samples from sows of conventional piglet producing farms and semen samples from a boar stud revealed no detection of M. suis and 'Candidatus Mycoplasma haemosuis' by qPCR. Thus, the results indicate that blood-independent transmission routes might be of minor relevance under field conditions.

Clinical, haematological and pathomorphological findings in Mycoplasma suis infected pigs.

BACKGROUND: Mycoplasma suis (M. suis) belongs to the group of haemotrophic mycoplasmas and is known as the causative agent of infectious anaemia in pigs. In the last few years valuable insights into the mechanism of adhesion and invasion, shedding patterns and cell tropism of M. suis were gained by the use of new molecular techniques. However, details on M. suis induced lesions as well as the distribution of M. suis in different organs are still lacking. Therefore, seven splenectomised pigs were experimentally infected and clinical and laboratory investigations as well as a detailed histopathological examination were performed. Detection and quantification of M. suis DNA in blood and various tissue samples was done using a quantitative real-time PCR. RESULTS: During the course of experimental infection, periodically occurring signs of infectious anaemia of pigs including severe icteroanaemia, fever, apathy and anorexia were observed. In addition, dermatological manifestations such as haemorrhagic diathesis presenting as petechiae occurred. The most important haematological alterations were normochromic, normocytic anaemia, hypoglycaemia as well as increased bilirubin and urea concentrations. Necropsy revealed predominant evidence of haemolysis with consecutive anaemia, as well as disseminated intravascular coagulation. M. suis was found in all investigated tissues with the highest copy numbers found in the kidneys. In Giemsa stained sections M. suis was only detected red blood cell (RBC)-associated. CONCLUSION: In the present study, no RBC independent sequestration of M. suis was detected in organs of experimentally infected pigs. Pathological findings are most likely resulting from haemolysis, consecutive anaemia as well as from disseminated intravascular coagulation and subsequent organ impairments.

Distribution of the Warmblood Fragile Foal Syndrome Type 1 Mutation (PLOD1 c.2032G>A) in Different Horse Breeds from Europe and the United States.

Warmblood fragile foal syndrome (WFFS) is an autosomal recessive disorder caused by a single nucleotide variant in the procollagen-lysine-2-oxoglutarate-5-dioxygenase 1 gene (PLOD1:c.2032G>A, p.Gly678Arg). Homozygosity for the PLOD1 variant causes an Ehler-Danlos-like syndrome, which has to date only been reported in warmblood breeds but the WFFS allele has been also detected in the Thoroughbred. To investigate the breed distribution of the WFFS allele, 4081 horses belonging to 38 different breeds were screened. In total, 4.9% of the horses representing 21 breeds carried the WFFS allele. The affected breeds were mainly warmbloods, with carrier frequency as high as 17% in the Hanoverian and Danish Warmblood. The WFFS allele was not detected in most non-warmblood breeds. Exceptions include WFFS carriers in the Thoroughbred (17/716), Haflinger (2/48), American Sport Pony (1/12), and Knabstrupper (3/46). The origin of the WFFS allele remains unknown. The Arabian breed and specifically the stallion Bairactar Or. Ar. (1813), whose offspring were reported to have a similar phenotype in the 19th century, were hypothesized as the origin. DNA from a museum sample of Bairactar Or. Ar. showed that he did not carry the mutated allele. This result, together with the genotypes of 302 Arabians, all homozygous for the reference allele, does not support an Arabian origin of the WFFS allele. Our extensive survey shows the WFFS allele to be of moderate frequency and concern in warmbloods and also in breeds where it may not be expected.

Study of the antibiotic residues in poultry meat in some of the EU countries and selection of the best compositions of lactic acid bacteria and essential oils against Salmonella enterica.

In this study, the presence of antibiotics (ANB) residues was evaluated in poultry meat purchased from German and Lithuanian markets. In addition, the antimicrobial activity of 13 lactic acid bacteria (LAB) strains, 2 essential oils (EO) (Thymus vulgaris and Origanum vulgare L.), and their compositions were tested for the purpose of inhibiting antibiotic-resistant Salmonella spp. ANB residues were found in 3 out of the 20 analyzed poultry meat samples: sample no. 8 contained enrofloxacin (0.46 µg/kg), sample no. 14 contained both enrofloxacin and doxycycline (0.05 and 16.8 µg/kg, respectively), and sample no. 18 contained enrofloxacin (2.06 µg/kg). The maximum residue limits (MRLs) for the sum of enrofloxacin and ciprofloxacin and for doxycycline in the poultry muscle are 100 µg/kg. Finally, none of the tested poultry meat samples exceeded the suggested MRLs; however, the issue of ANB residues still requires monitoring of the poultry industry in Germany, Poland, and Lithuania, despite the currently established low ANB concentrations. These findings can be explained by the increased use of alternatives to ANB in the poultry industry. Our results showed that an effective alternative to ANB, which can help to reduce the occurrence of antibiotic-resistant salmonella, is a composition containing 1.0% of thyme EO and the following LAB strains: Lactobacillus plantrum LUHS122, Enteroccocus pseudoavium LUHS242, Lactobacillus casei LUHS210, Lactobacillus paracasei LUHS244, Lactobacillus plantarum LUHS135, Lactobacillus coryniformins LUHS71, and Lactobacillus uvarum LUHS245, which can be recommended for poultry industry as components of feed or for the treatment of surfaces, to control the contamination with Salmonella strains. However, it should be mentioned that most of the tested LAB strains were inhibited by thyme EO at the concentrations of 0.5 and 1.0%, except for LUHS122, LUHS210, and LUHS245. Finally, it can be noted that the agents responsible for the inhibitory effect on Salmonella are not the viable LAB strains but rather their metabolites, and further studies are needed to identify which metabolites are the most important.

Detection of a novel haemoplasma species in fattening pigs with skin alterations, fever and anaemia.

BACKGROUND: In a fattening farm in Southern Germany, skin alterations (urticaria, haemorrhagic diathesis) and high fever were observed in 30% of the pigs 2 weeks after arrival. Feed intake was severely compromised in affected pigs. METHODS: After detailed clinical observation, blood samples from affected pigs were collected for haematological, PCR and serological investigations. In addition, pathological investigations were performed on one pig. RESULTS AND CONCLUSION: Analysis of blood parameters revealed a normocytic, normochromic anaemia. A novel porcine haemoplasma species was detected in blood samples of affected pigs and spleen sample of the necropsied pig by PCR. Phylogenetic analyses based on the 16S rDNA showed 99% identity to a novel porcine haemoplasma ('Candidatus (Ca.) M. haemosuis') species which has recently been described in China. Interestingly, this is the first report of 'Ca. M. haemosuis' in pigs with clinical signs resembling those of Mycoplasma (M) suis and the first description of this novel haemoplasma species outside Asia. On-farm affected pigs were treated with oxytetracycline and non-steroidal anti-inflammatory drugs. Clinical signs improved after implementation of treatment and optimisation of management procedures. This case might indicate that other porcine haemoplasma species than M suis can induce fever and skin alterations and may have an economic impact on affected farms.

Symposium report: emerging threats for human health - impact of socioeconomic and climate change on zooanthroponosis in the Republic of Sakha (Yakutia), Russia.

Population growth, socio-cultural and economic changes as well as technological progress have an immediate impact on the environment and human health in particular. Our steadily rising needs of resources increase the pressure on the environment and narrow down untainted habitats for plants and wild animals. Balance and resilience of ecosystems are further threatened by climate change, as temperature and seasonal shifts increase the pressure for all species to find successful survival strategies. Arctic and subarctic regions are especially vulnerable to climate change, as thawing of permafrost significantly transforms soil structures, vegetation and habitats. With rising temperature, the risk of zoonotic diseases in the Republic of Sakha (Yakutia) has also increased. As vegetation periods prolong and habitats broaden, zoonotic pathogens and their vectors find more favourable living conditions. Moreover, permafrost degradation may expose historic burial grounds and allow for reviving the vectors of deadly infections from the past. To assess the current state of knowledge and emerging risks in the light of the "One Health" concept, a German-Russian Symposium took place on 13 August 2018 in Yakutsk, Russian Federation. This symposium report presents the main findings generated from presentations and discussions.

Recommended rejection of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov. [Gupta, Sawnani, Adeolu, Alnajar and Oren 2018] and all proposed species comb. nov. placed therein.

The consensus of the members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes is that recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales, specifically involving introduction of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceaefam. nov., Mycoplasmoidaceaefam. nov., Mycoplasmoidalesord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov., and all proposed species or subspecies comb. nov. placed therein, should be rejected because they violate one or more essential points of the International Code of Nomenclature of Prokaryotes.

Detection of Mycoplasma suis in pre-suckling piglets indicates a vertical transmission.

BACKGROUND: Transmission of Mycoplasma (M.) suis mainly occurs via iatrogenic or zootechnical manipulations or due to ranking fights. Other transmission routes including ingestion of secretes/excretes; blood-sucking arthropods and intra-uterine transmission have thought to play an epidemiological role without being experimentally proven. To investigate a vertical transmission of M. suis under field conditions blood samples from pre-suckling piglets and their corresponding dam were examined for M. suis by quantitative polymerase chain reaction (qPCR) in 21 farms in Southern Germany. RESULTS: A total of 14.35% of the 474 blood samples from pre-suckling piglets reacted qPCR positive. Additionally, M. suis was detected in 65 (31.25%) of the 208 sows at farrowing. On farm level, 16 (76.2%) of the 21 farms had at least one M. suis positive animal. M. suis positive farms had an average of 0.41 more stillborn piglets per litter than M. suis negative farms (p = 0.007). CONCLUSION: The present study provides further insights into M. suis infection dynamics as it is the first detection of M. suis in piglets immediately after birth prior to colostrum intake and the first large scale investigation of M. suis in sows at farrowing.

Species-Specific Conservation of Linear Antigenic Sites on Vaccinia Virus A27 Protein Homologs of Orthopoxviruses.

The vaccinia virus (VACV) A27 protein and its homologs, which are found in a large number of members of the genus Orthopoxvirus (OPXV), are targets of viral neutralization by host antibodies. We have mapped six binding sites (epitopes #1A: aa 32-39, #1B: aa 28-33, #1C: aa 26-31, #1D: 28-34, #4: aa 9-14, and #5: aa 68-71) of A27 specific monoclonal antibodies (mAbs) using peptide arrays. MAbs recognizing epitopes #1A-D and #4 neutralized VACV Elstree in a complement dependent way (50% plaque-reduction: 12.5-200 µg/mL). Fusion of VACV at low pH was blocked through inhibition of epitope #1A. To determine the sequence variability of the six antigenic sites, 391 sequences of A27 protein homologs available were compared. Epitopes #4 and #5 were conserved among most of the OPXVs, while the sequential epitope complex #1A-D was more variable and, therefore, responsible for species-specific epitope characteristics. The accurate and reliable mapping of defined epitopes on immuno-protective proteins such as the A27 of VACV enables phylogenetic studies and insights into OPXV evolution as well as to pave the way to the development of safer vaccines and chemical or biological antivirals.

Phytate degradation, intestinal microbiota, microbial metabolites and immune values are changed in growing pigs fed diets with varying calcium-phosphorus concentration and fermentable substrates.

The present study assessed effects of diets containing varying calcium-phosphorus (CaP) concentration and fermentable substrates on digestibility of diets, intestinal microbiota and immune system using 32 crossbred pigs (initial BW 54.7 kg). In a 2 × 2 factorial arrangement, pigs were fed either a corn-soybean meal (CSB) or corn-field pea (CFP) diet with either low [-] (4.4 g Ca/kg; 4.2 g total P/kg) or high [+] (8.3 g Ca/kg; 7.5 g total P/kg; supplemented with monocalcium phosphate) CaP content for a period of 9 weeks. In week 8, blood samples were taken, and at the end of the trial, all pigs were euthanized to collect digesta and mesenteric lymphoid tissue. Apparent total tract digestibility (ATTD) of P was greater (p < 0.05) for pigs fed the CaP+ and CFP diets than CaP- and CSB diets. The myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6 ) concentration in jejunal digesta was higher (p < 0.05) for CaP+ than in CaP- fed pigs. In addition, caecal and faecal InsP5 isomer concentration were greater (p < 0.05) for CSB than CFP diets. In the caecum, gene copy numbers of saccharolytic bacteria, such as Eubacterium rectale and Roseburia spp., as well as SCFA concentration were higher (p < 0.05) for CaP+ than CaP- diets. In particular, innate immune cell numbers, such as natural killer cells, dendritic cells, monocytes and neutrophils, were greater (p < 0.05) for CaP+ than CaP- fed pigs. Diets high in CaP resulted in higher abundance of potential beneficial bacteria and might promote the first line of defence enhancing the activation of the cellular adaptive immune response, thereby possibly decreasing the risk for intestinal disturbances. These results strongly suggest that both, CaP supply and dietary ingredients differing in fermentability, may beneficially affect gut health through increase in SCFA-producing bacteria and/or bacteria with anti-inflammatory properties.

Hemotrophic mycoplasma in Simmental cattle in Bavaria: prevalence, blood parameters, and transplacental transmission of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii.

BACKGROUND: The significance of hemotrophic mycoplasma in cattle remains unclear. Especially in Europe, their epidemiological parameters as well as pathophysiological influence on cows are lacking. The objectives of this study were: (1) to describe the prevalence of 'Candidatus Mycoplasma haemobos' ('C. M. haemobos') and Mycoplasma wenyonii (M. wenyonii) in Bavaria, Germany; (2) to evaluate their association with several blood parameters; (3) to explore the potential of vertical transmission in Simmental cattle; and (4) to evaluate the accuracy of acridine-orange-stained blood smears compared to real-time polymerase chain reaction (PCR) results to detect hemotrophic mycoplasma. A total of 410 ethylenediaminetetraacetic acid-blood samples from cows from 41 herds were evaluated by hematology, acridine-orange-stained blood smears, and real-time PCR. Additionally, blood samples were taken from dry cows of six dairy farms with positive test results for hemotrophic mycoplasma to investigate vertical transmission of infection. RESULTS: The period prevalence of both species was 60.24% (247/410), C. M. haemobos 56.59% (232/410), M. wenyonii 8.54% (35/410) and for coinfection 4.88% (20/410). Of the relevant blood parameters, only mean cell volume (MCV), mean cell hemoglobin (MCH), and white blood cell count (WBC) showed differences between the groups of infected and non-infected individuals. There were lower values of MCV (P < 0.01) and MCH (P < 0.01) and higher values of WBC (P < 0.05) in 'C. M. haemobos'-infected cows. In contrast, co-infected individuals had only higher WBC (P < 0.05). In M. wenyonii-positive blood samples, MCH was significantly lower (P < 0.05). Vertical transmission of 'C. M. haemobos' was confirmed in two calves. The acridine-orange-method had a low sensitivity (37.39%), specificity (65.97%), positive predictive value (63.70%) and negative predictive value (39.75%) compared to PCR. CONCLUSIONS: 'Candidatus Mycoplasma haemobos' was more prevalent than M. wenyonii in Bavarian Simmental cattle, but infection had little impact on evaluated blood parameters. Vertical transmission of the infection was rare. Real-time PCR is the preferred diagnostic method compared to the acridine-orange-method.

Quantitative analysis of Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos" infections in cattle using novel gapN-based realtime PCR assays.

Hemotrophic mycoplasmas (HMs) are associated with anemia and other disease complexes in a wide range of livestock and wild animals. Two bovine HM species have been identified to date, i.e. Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos'. The study aim was to develop quantitative real-time PCR assays (qPCRs) to detect and quantify M. wenyonii and 'C. M. haemobos' and to apply these assays to DNA samples extracted from bovine blood collected in Germany (n = 220) from 22 herds. The qPCR assays specific for M. wenyonii and 'C. M. haemobos' were designed using the gapN of the respective hemoplasma species as gene target which encodes the NADP-dependent glyceraldehyde 3-phosphate dehydrogenases (GAPN). The sensitivity of both assays was 10 genome equivalents per reaction, corresponding to 2500 genome equivalents per ml blood. No cross-reactivity with non-target bovine HMs. and other bovine pathogens was observed. Bovine HM DNA was detected in 137 samples (62.27%) with 118 samples (53.64%) being positive for 'C.M. haemobos' and 19 samples (8.64%) being positive for M. wenyonii. Thereof, 11 animals (5.00%) were co-infected with both bovine HM species. The found herd prevalence for `C. M. haemobos` was 100.00%, and for M. wenyonii 36.36% with mean bacterial loads of 3.7 × 107 `C. M. haemobos`/mL blood and of 4.29 × 105M. wenyonii/mL blood respectively. Clinical and economic relevance of bovine HM species should be goal of future studies for which the novel gapN qPCR assays can serve as a valuable diagnostic tool.

Dietary changes in nutritional studies shape the structural and functional composition of the pigs' fecal microbiome-from days to weeks.

BACKGROUND: The possible impact of changes in diet composition on the intestinal microbiome is mostly studied after some days of adaptation to the diet of interest. The question arises if a few days are enough to reflect the microbial response to the diet by changing the community composition and function. The present study investigated the fecal microbiome of pigs during a time span of 4 weeks after a dietary change to obtain insights regarding the time required for adaptation. Four different diets were used differing in either protein source (field peas meal vs. soybean meal) or the concentration of calcium and phosphorus (CaP). RESULTS: Twelve pigs were sampled at seven time points within 4 weeks after the dietary change. Fecal samples were used to sequence the 16S rRNA gene amplicons to analyse microbial proteins via LC-MS/MS and to determine the SCFA production. The analysis of OTU abundances and quantification values of proteins showed a significant separation of three periods of time (p = 0.001). Samples from the first day are used to define the 'zero period'; samples of weeks 1 and 2 are combined as 'metabolic period' and an 'equilibrium period was defined based on samples from weeks 3 and 4. Only in this last period, a separation according to the supplementation of CaP was significantly detectable (p = 0.001). No changes were found based on the corn-soybean meal or corn-field peas administration. The analysis of possible factors causing this significant separation showed only an overall change of bacterial members and functional properties. The metaproteomic approach yielded a total of about 9700 proteins, which were used to deduce possible metabolic functions of the bacterial community. CONCLUSIONS: A gradual taxonomic and functional rearrangement of the bacterial community has been depicted after a change of diet composition. The adaptation lasts several weeks despite the usually assumed time span of several days. The obtained knowledge is of a great importance for the design of future nutritional studies. Moreover, considering the high similarities between the porcine and human gastrointestinal tract anatomy and physiology, the findings of the current study might imply in the design of human-related nutritional studies.