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.

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.

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.

Antimicrobial Susceptibility from a One Health Perspective Regarding Porcine Escherichia coli from Bavaria, Germany.

Antimicrobial resistance is one of the most crucial One Health topics worldwide. Consequently, various national and international surveillance programs collect data and report trends regularly. Ceftiofur, colistin and enrofloxacin belong to the most important and critical class of anti-infective medications in both human and veterinary medicine. In the present study, antimicrobial resistance was analyzed using the epidemiological cut-off (ECOFF) value on 6569 Escherichia coli isolated from pigs in Bavaria, Germany, during five years, from 2016 to 2020. The statistically relevant results regarding antimicrobial resistance revealed a decrease for colistin, an increase for enrofloxacin, and a constant level for ceftiofur. In Germany, the usage of all three antimicrobial substances in livestock has fallen by 43.6% for polypeptides, 59.0% for fluoroquinolones and 57.8% for the 3rd + 4th generation cephalosporines during this time. Despite the decline in antimicrobial usage, a reduction regarding antimicrobial resistance was solely observed for colistin. This finding illustrates that in addition to the restriction of pharmaceutical consumption, further measures should be considered. Improved biosecurity concepts, a reduction in crowding, and controlled animal movements on farms may play a key role in finally containing the resistance mechanisms of bacteria in farm animals.

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.

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.

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.

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.