Pulmonary Mycoplasmosis in an Axis Deer (Axis Axis) From a Wildlife Reserve in Argentina

doi:10.21203/rs.3.rs-4863846/v1

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Case Report

Pulmonary Mycoplasmosis in an Axis Deer (Axis Axis) From a Wildlife Reserve in Argentina

https://doi.org/10.21203/rs.3.rs-4863846/v1

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A 4-year-old male axis deer (Axis axis) died after several weeks of progressive weight loss and severe respiratory distress. A field necropsy was performed, and lung tissue was submitted for histopathology. Microscopically, there was severe, subacute to chronic, diffuse, suppurative bronchopneumonia with multiple caseonecrotic foci and bronchiectasis. Immunohistochemical (IHC) analysis using polyclonal antibodies for Mycoplasma bovis revealed positive immunoreactivity. The histopathological, and IHC findings observed in this deer are remarkably similar to pulmonary mycoplasmosis of cattle. These findings represent the first report of M. bovis-associated pneumonia in the axis deer and suggest that deer develop pulmonary disease and lung lesions like mycoplasmosis in domestic cattle.

caseonecrotic bronchopneumonia

cervid

diagnostic immunohistochemistry

Mycoplasma spp.

respiratory disease

Mycoplasma species have been associated with economically important diseases affecting ruminants worldwide, especially causing respiratory diseases in cattle and small ruminants (Caswell & Archambault, 2007). Mycoplasma bovis (M. bovis) is most commonly recognized as a cause of pneumonia, arthritis, and mastitis in cattle (Ayling et al., 2004, Caswell et al., 2010), However, there are few documented cases of mycoplasmosis by M. bovis or isolation/molecular identification of this bacterium in other ruminants like sheep, goats, and deer (Egwu et al., 2012; Register et al., 2019; Deeney et al., 2021). This study describes a case of pulmonary mycoplasmosis associated with M. bovis in an axis deer (Axis axis) in South America.

In July 2023, tissue samples from a 4-year-old, male axis deer (Axis axis) from a private wildlife reserve located in Tucuman province, Argentina (26°15'03.6" S; 65°31'08.3" W) were analyzed in the Specialized Veterinary Diagnostic Service (SVDS) of the National Institute of Agriculture Technology (INTA) in Salta, Argentina. Deer at this wildlife reserve are maintained for recreation and educational activities. During the last few months, the reserve had experienced deer mortality due to respiratory disease and weight loss at a rate of 1 or 2 deer per month. Eight adult deer were held within a collective barn (Fig. 1-A) and fed alfalfa hay, cracked corn, and a vitamin/mineral mix. They were dewormed twice a year using oral albendazole and injectable ivermectin. For two weeks, one of these deer exhibited hyperthermia (40°C), reduced feed consumption, decreased social interaction, and respiratory distress characterized by rales, distress, cough, and rapid shallow breathing and progressive weight loss. The deer was treated with one dose of injectable oxytetracycline (Terramicina L.A., Zoetis) and one dose of injectable dexamethasone, but showed no improvement of the clinical signs and died shortly after.

After natural death, field necropsy of the diseased deer was performed. Lung sections were fixed in 10% neutral buffered formalin solution and submitted to the SVDS for diagnosis. These sections were then embedded in paraffin wax at the SVDS, sectioned at 3 µm, and stained with hematoxylin and eosin (H&E) following standard staining procedures for histopathological examination.

Selected formalin fixed paraffin embedded (FFPE) tissue sections were used in an immunohistochemical (IHC) assay designed to detect intralesional tissue antigens of M. bovis as described (Adegboye et al., 1995), using the rabbit anti-M. bovis polyclonal antibody applied for 12h at a dilution of 1:5000. Epitope retrieval was done by autoclaving in citrate buffer. The IHC assay was done with the commercial CytosScanTM HRP detection kit (Cell Marque) detection system following the manufacturer's recommendations. Negative controls were prepared by replacing the primary antibody with non-immune rabbit serum.

The molecular detection of Mycoplasma spp. was done using selected FFPE tissue sections to amplify the 16S-23S rRNA intergenic spacer region in a nested PCR assay as described (Tang et al., 2000), with the primers designed by Nakagawa et al. (1992) and Harasawa et al. (1993). Total DNA extraction was conducted using the Puri-Prep S commercial kit (Inbio Highway, Argentina) following the manufacturer's guidelines. To ascertain the species of Mycoplasma, the PCR products obtained were purified using the Puriprep-GP Kit from Inbio Highway, quantification and sequencing using the ABI 3130xl platform from Applied Biosystems, employing the inner primers detailed by Harasawa et al. (1993).

Gross evaluation revealed severe, bilateral, anteroventral consolidation and discoloration with numerous small, tan, caseous nodules randomly distributed throughout the affected area that involved approximately 50% of the total pulmonary mass (Fig. 1-B). No other gross lesions were observed in any of the major organ systems.

Histopathological evaluation of the lung samples revealed severe bronchopneumonia characterized by numerous, multifocal to coalescing, areas of caseous necrosis surrounded by neutrophils, macrophages and fewer lymphocytes and plasma cells (Fig. 1-C). The pulmonary parenchyma between the areas of caseous necrosis had edema, intraalveolar macrophages and fewer neutrophils, plasma cells and lymphocytes (Fig. 1-D). There was moderate hyperplasia of the bronchiolar-associated lymphoid tissue (BALT).

The IHC assay revealed strongly positive immunoreactivity at the center and the periphery of the necrotic lesions (Fig. 1-E; Fig. 1-F).

Mycoplasma spp. PCR was negative from FFPE tissue blocks.

In this case, the diagnosis of pulmonary mycoplasmosis was established based on the characteristic gross and histopathological lesions observed in the lungs and the detection of bacterial antigen within the lesions by IHC. The pattern of pulmonary disease observed in this case and a previous report (Dyer et al., 2004) show that deer can develop the same type of M. bovis-related lesions as described in cattle (Margineda et al., 2017; Oliveira et al., 2019). These findings suggest that M. bovis is a natural disease of the axis deer and that it should be considered as a differential diagnosis of pulmonary diseases of deer.

The non-amplification of Mycoplasma spp. by molecular testing (PCR) can be related to the technical difficulties associated with the adequate extraction of total high quality viable DNA from FFPE.

Although respiratory illnesses are markedly prevalent among deer populations, only a limited number of studies have extensively explored the causes and their possible interactions. Like young cattle reared in production settings, farm-raised fawns are particularly vulnerable to bacterial and viral pneumonia triggered by environmental stressors, often resulting in substantial mortality rates (Margineda et al., 2017; Brooks, 2010).

One mortality study in 160 farm-raised white-tailed deer (Odocoileus virginianus) noted bronchopneumonia was the most common cause of death (39/160; 24,4%). Affected deer ranged in age from 10 days to 8.2 years. Pneumonic lungs from 36 of the 39 were processed for aerobic, anaerobic, and mycoplasma bacterial culture. Mycoplasma spp. was isolated from the lungs of 3 deer (3/36; 8,33%) (Hattel et al., 2004). Viral pneumonias caused by parainfluenza (Dastjerdi, et al., 2022), coronavirus (Dastjerdi, et al., 2022), astrovirus (Wang et al., 2020), and adenoviruses have also been described (Hoskins et al., 2023) in deer.

M. bovis is widely recognized as a significant contributor to pneumonia of cattle worldwide. In natural cases, the characteristic pulmonary pattern is exudative bronchopneumonia with caseous, and coagulative necrosis surrounded by a mixture of inflammatory cells (Margineda et al., 2017; Cantón et al., 2022). Calves experimentally infected show predominantly purulent bronchitis with lymphocytic peribronquial accumulations (Rodríguez et al., 1996; Caswell & Archambault, 2007). In natural clinical cases in cattle, intralesional tissue antigens of M. bovis were observed at the margins of areas with coagulation necrosis, within the necrotic exudate, engulfed by phagocytic cells (Margineda et al., 2017; Oliveira et al., 2019), and within bronchial epithelial cells (Oliveira et al., 2019). In the experimentally induced disease in cattle, antigenic material was detected in airway epithelial cells, inflammatory cells, and the walls of the alveoli (Rodríguez et al., 1996).

There are documented cases of M. bovis pneumonia in captive white-tailed Deer (Dyer et al., 2004) and free-ranging mule deer (Odocoileus hemionus) (Register et al., 2019). Recently, Malmberg et al. (2020) reported a high-mortality outbreak of respiratory diseases by M. bovis in free-ranging Pronghorn (Antilocapra Americana). To the best of our knowledge, this study represents the first documentation of M. bovis pneumonia in an axis deer from the Southern Hemisphere. The presence of M. bovis in deer is significant as it shows evidence of mycoplasma circulation between various animal species.

Acknowledgements

The authors would like to thank the staff of the animal health group of the private wildlife reserve “Dr. Carlos Pellegrini” (National Wildlife Authorization N°4023 and Provincial Wildlife authorization N°17-18-001) and Aldana Emeli Nieva Rojas for assistance with histopathological techniques.

Funding

This study was supported by grants of Consejo de Investigación de la Universidad Católica de Salta (Proy- Res 698/2020) and INTA (Instituto Nacional de Tecnología Agropecuaria).

CRediT authorship contribution statement

A. Avellaneda-Cáceres: Methodology, Investigation, Writing original draft, review & editing, Visualization, Conceptualization, Formal analysis. C. A. Margineda: Methodology, Investigation, Writing review & editing, Validation. F. Fernández: Writing review & editing, Visualization. D. M. Medina: Methodology. S. Diab: Methodology, Investigation, Writing review & editing, Supervision, Formal analysis. F. H. P. Silva: Writing review & editing. G. V. Sandoval: Methodology. S. A. Headley: Writing review & editing. J. F. Micheloud: Methodology, Investigation, Writing original draft, review & editing, Supervision, Formal analysis.

Ethics declarations

Ethics approval

No approval from an Animal Ethics Committee was required for this study since it is based on the investigation of a dead Axis deer from the private wildlife reserve “Dr. Carlos Pellegrini”. This reserve is authorized to perform investigations (National Wildlife Authorization N°4023 and Provincial Wildlife authorization N°17-18-001)

Institutional review board statement

Not applicable.

Informed consent statement

Not applicable.

Consent to participate

Not applicable.

Consent to publish

Private wildlife reserve “Dr. Carlos Pellegrini” (National Wildlife Authorization N°4023 and Provincial Wildlife authorization N°17-18-001) consented to the publication of this case.

Competing interests

The authors declared no potential conflicts of interest for this article's research, authorship, and publication.

Data availability

No datasets were generated or analysed during the current study.

Adegboye, D. S., Hallbur, P. G., Cavanaugh, D. L., Werdin, R. E., Chase, C. C., Miskimins, D. W., & Rosenbusch, R. F. (1995). Immunohistochemical and pathological study of Mycoplasma bovis-associated lung abscesses in calves. Journal of Veterinary Diagnostic Investigation, 7(3), 333–337. https://doi.org/10.1177/104063879500700306
Ayling, R.D., Bashiruddin, S.E., Nicholas, R.A.J. (2004). Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000. Vet. Rec. 155, 413–416. https://doi.org/10.1136/vr.155.14.413
Brooks, J. W. (2010). A comprehensive study of the health of farm-raised white-tailed deer (Odocoileus virginianus) with emphasis on respiratory tract infection by Fusobacterium spp. PhD dissertation. Pennsylvania State University. https://etda.libraries.psu.edu/catalog/10972
Cantón, G., Llada, I., Margineda, C., Urtizbiría, F., Fanti, S., Scioli, V., ... & Tamiozzo, P. (2022). Mycoplasma bovis-pneumonia and polyarthritis in feedlot calves in Argentina: First local isolation. Revista Argentina de Microbiología, 54(4), 299-304. https://dx.doi.org/10.1016/j.ram.2022.02.005
Caswell JL, Bateman KG, Cai HY, Castillo-Alcala F. (2010). Mycoplasma bovis in respiratory disease of feedlot cattle. Vet Clin North Am Food Anim Pract., 26, 365-379. http://dx.doi.org/10.1016/j.cvfa.2010.03.003
Caswell, J. L., & Archambault, M. (2007). Mycoplasma bovis pneumonia in cattle. Animal health research reviews, 8(2), 161–186. https://doi.org/10.1017/S1466252307001351
Dastjerdi, A., Floyd, T., Swinson, V., Davies, H., Barber, A., & Wight, A. (2022). Parainfluenza and corona viruses in a fallow deer (Dama dama) with fatal respiratory disease. Frontiers in Veterinary Science, 9, 1059681. https://doi.org/10.3389/fvets.2022.1059681
Deeney, A.S., Collins, R. & Ridley, A.M. (2021). Identification of Mycoplasma species and related organisms from ruminants in England and Wales during 2005–2019. BMC Vet Res 17, 325. https://doi.org/10.1186/s12917-021-03037-y
Dyer, N.W., Krogh, D.F., & Schaan, L.P. (2004). Pulmonary mycoplasmosis in Farmed White-tailed Deer (Odocoileus virginianus). Journal of Wildlife Diseases, 40 (2), 366–370. https://doi.org/10.7589/0090-3558-40.2.366
Egwu, G.O., Adamu, M., Mshelia, G.D. and Bukar-Kolo, Y.M. (2012). Isolates of Mycoplasma mycoides subspecies mycoides (SC) in small ruminants in Sahel zone of Nigeria and its implications on disease control. African Journal of Biotechnology, 11, 6396–6401.
Harasawa, R., Mizusawa, H., Nozawa, K., Nakagawa, T., Asada, K., & Kato, I. (1993). Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions. Research in Microbiology, 144(6), 489-493. https://doi.org/10.1016/0923-2508(93)90057-9
Hattel, A. L., Shaw, D. P., Love, B. C., Wagner, D. C., Drake, T. R., & Brooks, J. W. (2004). A retrospective study of mortality in Pennsylvania captive white-tailed deer (Odocoileus virginianus): 2000-2003. Journal of Veterinary Diagnostic Investigation, 16(6), 515–521. https://doi.org/10.1177/104063870401600605
Hoskins, E., Hoffman, J., Ferro, P. J., Diaz-Delgado, J., Porter, B. F., Gomez, G., & Cliften, P. (2023). Deer mastadenovirus B pneumonia in a white-tailed deer fawn. Journal of Veterinary Diagnostic Investigation, 35(5), 543-546. https://doi.org/10.1177/10406387231179140
Malmberg, J. L., O'Toole, D., Creekmore, T., Peckham, E., Killion, H., Vance, M., Ashley, R., Johnson, M., Anderson, C., Vasquez, M., Sandidge, D., Mildenberger, J., Hull, N., Bradway, D., Cornish, T., Register, K. B., & Sondgeroth, K. S. (2020). Mycoplasma bovis Infections in Free-Ranging Pronghorn, Wyoming, USA. Emerging infectious diseases, 26(12), 2807–2814. https://doi.org/10.3201/eid2612.191375
Margineda, C.A., Zielinski, G.O.; Jurado, S.B., Ferrella, A., Mozgovoj, M.V., & López, A. (2017). Mycoplasma bovis pneumonia in feedlot cattle and dairy calves in Argentina. Brazilian Journal of Veterinary Pathology, 10 (2), 79-86. http://doi.org/10.24070/bjvp.1983-0246.v10i2p79-86
Nakagawa, T., Uemori, T., Asada, K., Kato, I., & Harasawa, R. (1992). Acholeplasma laidlawii has tRNA genes in the 16S-23S spacer of the rRNA operon. Journal of Bacteriology, 174(24), 8163-8165. https://doi.org/10.1128/jb.174.24.8163-8165.1992
Oliveira, T. E. S., Pelaquim, I. F., Flores, E. F., Massi, R. P., Valdiviezo, M. J. J., Pretto-Giordano, L. G., Alfieri, A. A., Saut, J. P. E., & Headley, S. A. (2019). Mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows. Transboundary and emerging diseases, 67(2), 82-93. https://doi.org/10.1111/tbed.13223
Register, K. B., Jelinski, M. D., Waldner, M., Boatwright, W. D., Anderson, T. K., Hunter, D. L., Hamilton, R. G., Burrage, P., Shury, T., Bildfell, R., Wolff, P. L., Miskimins, D., Derscheid, R. J., & Woodbury, M. R. (2019). Comparison of multilocus sequence types found among North American isolates of Mycoplasma bovis from cattle, bison, and deer, 2007-2017. Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 31(6), 899–904. https://doi.org/10.1177/1040638719874848
Rodríguez, F., Bryson, D. G., Ball, H. J., & Forster, F. (1996). Pathological and immunohistochemical studies of natural and experimental Mycoplasma bovis pneumonia in calves. Journal of Comparative Pathology, 115(2), 151-62. https://doi.org/10.1016/S0021-9975(96)80037-5
Tang, J., Hu, M., Lee, S., & Roblin, R. (2000). A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture. Journal of Microbiological Methods, 39(2), 121–126. https://doi.org/10.1016/s0167-7012(99)00107-4
Wang, L., Shen, H., Zheng, Y., Schumacher, L., & Li, G. (2020). Astrovirus in White-Tailed Deer, United States, 2018. Emerging Infectious Diseases, 26(2), 374–376. https://doi.org/10.3201/eid2602.190878

No competing interests reported.

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Pulmonary Mycoplasmosis in an Axis Deer (Axis Axis) From a Wildlife Reserve in Argentina

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Abstract

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1. Introduction

2. Case report

3. Materials and methods

4. Results

5. Discussion and conclusion

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