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Detection of a novel haemoplasma species in fattening pigs with skin alterations, fever and anaemia
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  1. Julia Stadler1,
  2. Julia Ade2,
  3. Mathias Ritzmann1,
  4. Katharina Hoelzle2 and
  5. Ludwig E Hoelzle2
  1. 1 Clinic for Swine, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University Munich, Oberschleissheim, Bavaria, Germany
  2. 2 Institute of Animal Science, Institute of Environmental and Animal Hygiene and Veterinary Medicine, University of Hohenheim Faculty of Agricultural Sciences, Stuttgart, Baden-Wuerttemberg, Germany
  1. Correspondence to Dr Julia Stadler, Clinic for Swine, Ludwig-Maximilians-Universitat Munchen Tierarztliche Fakultat, Munchen, Bayern, Germany; J.Stadler{at}med.vetmed.uni-muenchen.de

Abstract

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.

  • skin alterations
  • high fever
  • normocyctic
  • normochromic anaemia
  • haemotrophic mycoplasma
  • Candidatus M.haemosuis
  • case report
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Introduction

Haemotrophic mycoplasmas (haemoplasmas) are uncultivable, small epicellular, cell wall-less, tetracycline-sensitive bacteria that attach to the surface of host erythrocytes.1 Haemoplasmas were formerly classified in the two genera Eperythrozoon and Haemobartonella within the family Anaplasmataceae. Phylogenetic analyses of the 16S rDNA and RNase P RNA gene led to a reclassification into the family Mycoplasmataceae.2–6

Haemotrophic mycoplasmas are detected in a large number of vertebrate hosts.7–16 Despite the description of host adaptation and strict host specificity for most haemoplasma species, interspecies transmission and zoonotic potential of haemoplasma infections have to be considered.17–20 Until 2017, two porcine haemoplasma species have been described in pigs, that is, Msuis and M parvum. While M parvum is considered non-pathogenic, M suis is known as the major cause of infectious anaemia in pigs (formerly known as eperythrozoonosis).1 21 22 Depending on the host susceptibility and the virulence of M suis, the disease manifestation in pigs varies from acute life-threatening anaemia with high fever, icterus, cyanosis and necrosis in the ears and massive hypoglycaemia to chronic disease associated with mild anaemia, reduced growth rate, poor reproductive performance and increased susceptibility to secondary infections of the respiratory and enteric system as well as asymptomatic infections.1 22–24 M suis and M parvum are phylogenetically closely related, both clustering in the ‘Haemominutum’ group within the phylogenetic cluster of haemoplasmas.5 This ‘Haemominutum’ group mainly represents the former species Eperythrozoon. Recently, a novel porcine haemoplasma species was described in clinically healthy pigs in China with a prevalence of 26.5% and in Korea with a prevalence of 0.01%.25 Phylogenetic analyses revealed that this potentially novel haemoplasma species is closely related to the feline haemoplasma ‘Ca. M. turicensis’, in the ‘Haemofelis’ group which mainly includes the former members of the species Haemobartonella. 5 25 The novel species was provisionally named ‘Candidatus (Ca.) M. haemosuis’.25 In the present case, skin alterations, fever and anaemia were reported in a fattening farm. ‘Ca. M. haemosuis’ was diagnosed in blood samples of affected pigs and spleen sample of a necropsied pig by PCR and subsequent sequencing of the 16S rDNA amplicons.

Clinical report

Case history and clinical findings

The case described here occurred on a fattening farm with 1200 pigs located in a high pig density area in Southern Germany. The farm is single sourced from a 300-sow self-recruiting piglet producing farm in Southern Germany located at a distance of 50 km from the fattening farm. Every 4 weeks, 360 pigs with an average weight of 30 kg which were vaccinated against Porcine Circovirus Type 2 and Mycoplasma hyopneumoniae in the third week of life are delivered. Upon arrival at the fattening farm, pigs are vaccinated with a PRRSV-1 MLV. The fattening farm consists of four barns each containing six unequally sized pens. On the fattening farm, pigs are housed on fully slatted floor in groups of 15 (smaller pens) and 80 (bigger pens) pigs per pen, respectively. Pigs are fed with liquid feed which consists of commercial feed and fresh water from the public supplier. All-in all-out with cleaning and disinfection between groups is performed. During the fattening period, no antimicrobials are used and deworming is routinely performed on arrival at the fattening farm. The average length of the fattening period is 115 days, with an average mortality rate of 1% and average daily weight gain of 780 g.

Clinical problems in the herd started beginning of May 2017, when approximately 30% of the pigs showed reduced feed intake and high fever (>40°C) 2 weeks after arrival. The herd veterinarian initiated treatment of affected pigs with cefquinomsulfat (2 mg/kg body weight/q 24 hours) via intramuscular injection for three consecutive days and dexamethasone (0.05 mg/kg body weight/q 24 hours) for two consecutive days. However, treatment did not result in improvement of clinical signs. The Clinic for Swine was involved into the clinical problem of the farm by the herd attending veterinarian end of May 2017. Clinical signs observed during the herd visit were characterised by apathy, fever and skin alterations. Clinical examination of the skin revealed pallor, icterus, petechia, ecchymosis and cyanosis of ears, ventral abdomen and legs. In several pigs, generalised skin reactions like small round pink to purple raised wheals in common with urticaria and haemorrhagic diathesis were observed (figure 1). Clinical signs also included necrosis of the edge and tip of the ears. Further anamnesis revealed no changes of housing, water supply or the feeding procedure. During the herd visit, an elevated temperature of 26.0°C–27.0°C was evident in all fattening barns. Subsequently, in the affected age group the mortality increased to 4.8% and a prolonged fattening period (132 fattening days) and decreased average daily weight gain of 680 g were recorded.

Figure 1

Fifteen-week-old pig (ID 82/17) with cutaneous haemorrhages on the ears (A) and on the entire body (B), housed at the University Clinic for swine for further investigations.

Diagnostic methods and laboratory findings

For diagnostic work-up, blood samples (EDTA-anti-coagulated blood and serum) from 20 pigs with skin alterations and/or fever were collected during the farm visit by the University Clinic for Swine for haematological, PCR and serological investigations. Blood samples were investigated for Classical and African swine fever (PCR and ELISA), PCV-2 (PCR26), PCV-3 (PCR27), M suis (PCR28), PRRSV (PCR29) and Sarcoptes scabiei var. suis (ELISA). All samples were negative for Classical and African Swine Fever, PCV-2 and PCV-3, M suis, PRRSV and Sarcoptes scabiei var. suis. Haematological parameters, that is, red blood cell count, haemoglobin, packed cell volume (PCV), mean corpuscular volume, mean corpuscular haemoglobin (MCH) and MCH concentration were determined using the Scil Vet ABC tool (Scil Animal Care Company GmbH). Analysis of haematological parameters revealed normocytic, normochromic anaemia in 11 out of 20 blood samples (table 1). A haemoplasma-specific 16S rDNA PCR30 was performed at the Institute of Animal Science, University of Hohenheim, Germany. Seven out of 21 blood samples were 16S rDNA PCR positive. Sequencing of the 16S rDNA amplicons revealed a 99% identity to the 16S rDNA of a novel porcine haemoplasma species (accession no. JX489601) which has so far been found in China25 and Korea.31 In addition, all blood samples were further investigated using a novel quantitative PCR (qPCR) specific for the novel porcine haemoplasma species (‘Candidatus (Ca.) M. haemosuis’; paper in preparation). Briefly, we used primers targeting the ‘Ca. M. haemosuis’ gap gene (CMhsuisF 5′-TGCTTTGGCTCCTGTGGTTA-3′ and CMhsuisR 5′- GCAGCAGCACCTGTAGAAGTA-3′; accession no. MN896988). ‘Ca. M. haemosuis’ DNA was detected with the StepOne System (Applied Biosystems) using Fast SYBR Green Master Mix and a melting curve analysis was performed after each PCR run. The lower limit of detection was 0.45 fg of the plasmid DNA per reaction (CT <35 cycles) corresponding to 10 genome equivalents per reaction and 2.5×103 bacteria per millilitre of blood. The PCR was considered highly specific as no cross-amplification was detected using DNA-derived several haemotrophic and non-haemotrophic Mycoplasma species (including M suis, M parvum and M hyopneumoniae) as well as other porcine bacterial pathogens. We further included plasmid DNA standard dilutions containing ‘Ca. M. haemosuis’ gap amplicons in each run to quantify ‘Ca. M. haemosuis’ DNA. Table 1 includes the qPCR results. All 16S rDNA PCR-positive samples also reacted qPCR positive.

Table 1

Results of haematological examination and qPCR investigation

In addition, one pig (ID 82/17) with severe skin alterations and fever was selected and forwarded to the University Clinic for Swine for further diagnostic work-up. Pathological investigations were performed at the Institute of Veterinary Pathology, Ludwig-Maximilians-University Munich, Germany. Postmortem examination revealed multifocal generalised cutaneous haemorrhages and pallor of mucus membranes. Gross lesions were further characterised by icterus of the liver and vascular endothelium as well as ascites. Microscopic examination of the liver revealed a splenic sinus hyperplasia and a generalised follicular hyperplasia. In addition, multifocal lymphoplasmacytic dermatitis was microscopically found. An aerobic and anaerobic bacteriological examination from various organs (brain, spleen, liver, kidney, heart, lung, lymph nodes and intestines) was performed (Institute of Infectious Diseases and Zoonosis, Ludwig-Maximilians-University Munich, Germany) and revealed growth of commensal pathogens (lungs: Rothia nasimurium, Aerococcus viridans; intestines: Bacillus subtilis, Enterococcus faecalis). In addition, organ samples (tonsils, lungs, lymph nodes, spleen and kidneys) were investigated at the Institute of Infectious Diseases and Zoonosis, Ludwig-Maximilians-University Munich, Germany for the presence of PRRSV by PCR and PCV-2, PCV-3 by PCR and in situ hybridisation for PCV-2 and at the Institute of Animal Science, University of Hohenheim, Germany for porcine haemoplasma by PCR as previously described. All samples were negative for PRRSV, PCV-2 and PCV-3. Spleen samples of the necropsied pig were negative for M suis but positive for the novel porcine haemoplasma species.

Further steps and outcome of the case

Pigs with skin alterations and fever were each treated twice with intramuscular injection of long-acting oxytetracycline (20 mg/kg body weight, q 48 hours) and metamizol (20 mg/kg body weight q 24 hours). In addition, to reduce predisposing factors the farmer was advised to decrease the barn temperature. The clinical signs improved after implementing treatment with oxytetracycline and adaptation of barn temperature. Moreover, blood samples (n=11) were collected from sows at the piglet producing farm and investigated for M suis and ‘Ca. M. haemosuis’ by qPCR. Despite qPCR-negative results for both M suis and ‘Ca. M. haemosuis’, the farmer of the fattening farm decided to change the piglet supplier. No new cases of skin alterations and fever occurred after the fattening farm purchased piglets from a new supplier located in Northern Germany.

Discussion

The present report describes a case of skin alterations and fever in a fattening farm in Southern Germany caused by the novel porcine haemoplasma species (‘Candidatus (Ca.) M. haemosuis’). Haemotrophic mycoplasma can induce haemolytic anaemia in a wide range of mammalian species. Clinical manifestations can range from life-threatening haemolytic anaemia to mild chronic anaemia, ill thrift, infertility and immune suppression as well as clinically inapparent infections.32–34 Until 2017, only two species have been described in pigs: M parvum and M suis. Research has mainly focused on the latter, as M parvum is regarded non-pathogenic.21 35 In 2017, a novel haemoplasma species was identified in apparently clinically healthy pigs in China25 and recently in Korea in one pig without further information on the clinical manifestation.31 This novel haemoplasma species was most closely related to a feline haemoplasma species, ‘Ca. M. turicensis’. To the best of the author’s knowledge, this is the first report on the occurrence of ‘Ca. M. haemosuis’ in clinically affected pigs. In the present case report, clinical signs, haematological alterations and lesions resembling an acute M suis infection were found. However, M suis could not be confirmed by PCR investigation.

Cutaneous manifestations in terms of urticaria and haemorrhagic diathesis as observed in the present case have also been described after experimental infection with M suis.32 36 Interestingly, dermatological disorders including erythematous maculopapular and vesicular rashes are frequently found in human medicine as complication of M pneumoniae infections.37 During the last decades, intravasal coagulation and subsequent consumption coagulopathy was regarded as the primary cause of haemorrhagic diathesis during acute M suis infection.24 However, more recent research indicates that interaction between M suis and endothelial cells resulting in endothelial damage, adhesion to the endothelium and vascular occlusion might be responsible for the development of haemorrhagic diathesis.38 Further research is certainly needed to elucidate the exact pathogenesis responsible for cutaneous manifestation.

In the present case, all ‘Ca. M. haemosuis’-positive pigs showed a normocytic, normochromic anaemia, usually observed during natural and experimental M suis infection.32 39 40 Interestingly, in cats severe anaemia was only detected after co-infection with M haemofelis and ‘Ca. M. haemominutum’ and not after infection with ‘Ca. M. turicensis’ or ‘Ca. M. haemominutum’ alone.41 Macroscopic examination of one pig in the present case revealed similar results as observed after infection with M suis (icterus, ascites, liver alterations, pale mucus membranes).42 However, pathognomonic findings during necropsy were neither detected by Dent et al 42 nor in our investigations. The field case illustrates that this novel porcine haemoplasma species might pose a pathogenic potential for pigs. However, considering the high prevalence reported by Fu et al 25 in apparently asymptomatic carrier pigs, it might be speculated that the clinical presentation described here was exacerbated by predisposing factors as elevated barn temperature observed during the farm visit. It is well known that non-infectious and infectious co-factors support the clinical manifestation of M suis infections.1 The potential of this novel haemoplasma species to induce haemolytic anaemia, corresponding clinical signs and pathological lesions needs to be further investigated in experimental studies.

Several transmission routes for M suis have been described in literature including blood-dependent and blood-independent horizontal transmission routes.1 43 A recent publication also indicates the possibility of vertical transmission under field conditions.32 However, despite extensive research on transmission routes of M suis within the last decades, the exact mechanism of inter-transmission and intra-transmission routes needs to be further evaluated. To elucidate the potential source of introduction of this novel haemoplasma species in the present case, blood samples from 11 sows were tested for M suis and ‘Ca. M. haemosuis’ by qPCR at the piglet producing farm. The sample size was based on previous investigations determining the intra-herd prevalence of M suis in a German pig population.40 However, as data on the intra-herd prevalence of ‘Ca. M. haemosuis’ are lacking so far, and clinical signs disappeared after the change of the piglet producing farm, the sample size might have been insufficient to detect ‘Ca. M. haemosuis’.

The diagnosis of haemoplasma infections is impaired by the lack of an in vitro cultivation system. However, different techniques are applied for the diagnosis of M suis.28 39 44–46 The formerly used microscopic detection of the agent in chemically stained blood smears has been mainly replaced by more specific, sensitive, reproducible and reliable modern molecular techniques. Next to the establishment of different PCR assays, serological diagnostic methods based on whole-cell ELISA or recombinant ELISAs have been widely used for prevalence studies in recent years. For the differentiation of porcine haemoplasma species, phylogenetic analyses based on 16S rDNA sequences or species-specific PCRs are needed. As both methods are not always available for routine diagnostic, it is supposed that infections with this novel haemoplasma species are underdiagnosed. In this report, the novel haemoplasma species was identified by a haemoplasma-specific 16S rDNA PCR30 and subsequent sequencing of the 16S rDNA amplicons. Additional diagnostic investigations using a newly established qPCR (paper in preparation) confirmed the results of the conventional PCR and sequencing. Bacterial loads in blood ranged from 3.08×102 to 3.96×107Ca. M. haemosuis’ cells/ml blood and were thus within the range found for M suis in feeder pigs in Germany (range 1.2×102 to 1.1×1010 M suis cells/ml blood).40 Due to the lack of vaccination against haemoplasma, treatment with oxytetracycline combined with a reduction of infectious and non-infectious predisposing factors are currently the method of choice.

Conclusion

In conclusion, the present case report resembles the first description of ‘Ca. M. haemosuis’ in pigs outside Asia. The occurrence of skin alterations, fever and anaemia in ‘Ca. M. haemosuis’-positive pigs indicates that this novel haemoplasma species might induce clinical signs comparable with M suis. However, further studies are certainly needed to assess the prevalence of this novel porcine haemoplasma and gain more information on transmission routes as well as its impact on the European swine industry.

Acknowledgments

The author would like to thank the colleagues from the Clinic for Swine at the Centre for Clinical Veterinary Medicine, Ludwig‐Maximilians-University, Germany for their support. The collaboration of the farmer is also highly appreciated.

References

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Footnotes

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as online supplementary information.

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