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Identification of Mycobacterium avium subspecies paratuberculosis in sheep farms in Bayannaoer, Inner Mongolia, China (short communication)

Abstract

Background

Paratuberculosis is a widespread chronic infection of Mycobacterium avium subspecies paratuberculosis (MAP) that causes significant economic losses to the sheep industry. The current study investigated this disease, which causes diarrhea in sheep, particularly, in Bayannaoer, Inner Mongolia, China. Diagnosis was based on clinical symptoms, pathological autopsy, histopathological inspection, and serological and molecular methods.

Results

MAP was confirmed using polymerase chain reaction using DNA extracted from tissue and fecal samples. Serum samples from 472 individual sheep were obtained to detect antibodies against MAP using an enzyme-linked immunosorbent assay. MAP antibodies were separately detected in 17.86% (35/196) and 18.48% (51/276) of sheep herds at approximately 6 months and ≥ 1 year of age, respectively. The tissue lesion and pathological section results were consistent with paratuberculosis infection.

Conclusions

To our knowledge, this is the first report of Mycobacterium avium subspecies paratuberculosis seroprevalence in Bayannaoer sheep in Inner Mongolia. Our findings show that MAP is not only prevalent, but also a potential threat to this region. Further investigations, including long-term epidemiological surveillance and isolation are needed for the awareness and effective treatment of paratuberculosis in sheep of Inner Mongolia.

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Background

Johne’s disease (JD) or paratuberculosis (PTB) is a chronic, progressive granulomatous enteritis disease that is caused by the zoonotic bacterium, Mycobacterium avium subspecies paratuberculosis (MAP) [1]. The disease primarily affects ruminants, including cattle, sheep, and goats. Paratuberculosis is a public health threat, and it reduces animal productivity and causes significant economic losses in ruminant industries worldwide [2, 3]. The common clinical manifestations are chronic diarrhea or scours, decreased productivity, weight loss and eventually, death of the animal [4]. Many countries have reported that infection with JD in sheep and goats occurs in a more discrete and insidious manner than in other animals. In Europe, the infection rate in sheep and goat herds is approximately 4% [5, 6]. Furthermore, in some Australian flocks, mortality rate reached 20% per annum [7]. An economic study showed that MAP infection reduced the profit efficiency from 84% to 64% in Italian dairy sheep and goat farms [8]. Other countries and regions have also shown varying degrees of economic losses as a result of PTB [9].

In recent years, MAP infection of ruminants in China has occurred rapidly. Some studies have indicated that MAP has become a common pathogen in dairy farms. Notably, the herd-level prevalence of 57.9% in 19 dairy herds in the Shandong province of China was observed [10]. MAP infection in dairy cattle differs with farming modes at the animal and herd level, and farming density could be an important risk factor associated with the presence of MAP-infected cattle [11]. One study showed that the overall MAP seroprevalence in the tested Tibetan sheep was 11.29% [12]. Additionally, recent studies have shown that PTB is widely prevalent in dairy farms in Tai'an, in the Shandong province of China [13]. Therefore, it is evident that research should focus on the prevalence, prevention, and control of PTB.

Moreover, the mutton sheep industry plays an important role in Bayannaoer, which is in western Inner Mongolia in China. Every year, approximately 10% of sheep in this area have intermittent diarrhea, against which pharmaceutical treatments are ineffective. This results in the eventual death of these sick sheep due to exhaustion, which emphasizes the significant negative effect that PTB has on economic development. As such, we diagnosed several sheep herds with PTB through clinical inspection, molecular biological methods, histopathological examination, and serological detection, to understand the epidemiology of MAP in Bayannaoer.

Results

Clinical symptoms and necropsy findings

The sick sheep showed clinical signs of JD, including severe weight loss and chronic diarrhea (Fig. 1 A).

Fig. 1
figure 1

Symptoms and Necropsy of Mycobacterium avium subsp. paratuberculosis infected Sheep. A Sheep with signs of diarrhea and emaciation. B Thickening and corrugation of the intestinal mucosa. C Enlarged and edematous abomasum. D Mesenteric lymph node edema and liquefaction. E Intestinal mucosa exfoliation. F Mesenteric congestion

Necropsy findings in representative dead sheep showed ileal hyperplasia (Fig. 1 B), enlarged and edematous abomasum (Fig. 1C), in addition to mesenteric lymph node edema and liquefaction (Fig. 1D). Mesenteric congestion and intestinal mucosa exfoliation were also found in intestinal lesions (Fig. 1 E, and F).

Polymerase chain reaction (PCR) MAP detection

The deoxyribonucleic acids (DNA) from fecal and ileum samples of sheep with diarrhea were used as templates. The results showed that the fragment size amplified by the nested PCR (L/AV) targeting IS900 was 298 bp, consistent with the expected size (Fig. 2).

Fig. 2
figure 2

PCR products from the IS900[L/AV] nested PCR. M: DNA marker 2000 bp. Lane 1-Lane3: Positive sample of feces; Lane 4-Lane3: Positive sample of mesenteric lymph node samples; Lane 7: negative sample

Incidence of MAP in sheep

A total of 472 serum samples collected from different sheep farms were tested using enzyme-linked immunosorbent assay (ELISA). The sheep were either approximately 6 months or ≥ 1 year old, and the detection rates of sheep in these two stages were similar. A total of 17.86% (35/196) and 18.48% (51/276) were separately tested positive for MAP in the two age groups (Table 1).

Table 1 The total number (n) of animals at two different age groups (6 months and ≥ 1 year old) with the number of positive MAP samples, and the positive MAP detection rate (%) sampled from each herd in Bayannaoer, Inner Mongolia, China

Histopathological assessment

The heart, spleen, lung, kidney, intestinal, and mesenteric lymph nodes of the sheep were stained with hematoxylin and eosin (H & E) to detect MAP infection. The myocardial fibers were swollen, thickened, and partially broken, and the transverse striations were not obvious. Moreover, there were scattered calcifications of different sizes (Fig. 3A). The endothelial cells of glomerular capillaries were partially necrotic, with red-stained silk reticular fibrin and a small amount of red blood cells distributed between them; a small amount of red blood cells was also seen in the renal capsule. In addition, the epithelial cells of the renal tubules were swollen and partially necrotic, and protein tubules appeared in most of the lumens (Fig. 3B). Many spleen lymphocytes were necrotic and significantly bleeding, especially around the splenic nodules, forming a red halo (Fig. 3C). The intestinal mucosa was thickened; many inflammatory cells were distributed in the lamina propria (Fig. 3D), and some lymphocytes in the mesenteric lymph nodes were necrotic and scattered in granulomas of different sizes. Moreover, epithelioid cells and lymphocytes were observed in these granulomas (Fig. 3E). The pulmonary interstitium widened, and many inflammatory cells were distributed. Most of the alveoli were dilated, and the alveolar wall ruptured (Fig. 3F).

Fig. 3
figure 3

Pathological changes of the diseased sheep. A Myocardial fiber swelling, thickening and calcification. B Renal tubulointerstitial injuries . C Massive necrosis of spleen lymphocytes. D Thickening of the intestinal mucosa. E Necrosis of mesenteric lymph nodes. F Pathological changes of lung tissues

Discussion

Johne’s disease is one of the most economically important diseases affecting ruminants worldwide, and it is a growing concern in the sheep and goat industries [14]. The disease is caused by the zoonotic pathogen MAP, that poses a challenge to public health [3]. The sheep farming and industrial wool spinning industries are particularly developed in Bayannaoer. Therefore, MAP could act as a threat to workers in these industries and to public health security. In 2011, the flocks in this region suffered from intermittent diarrhea and eventually died of exhaustion. Most of them were adult sheep, and the main symptoms were similar to those reported in previous studies—elevated body temperature, dark green and thick feces [15, 16]. The course of the disease was 10–30 days, over which the sick sheep gradually lost weight and their eyelids became white. This brought huge economic losses to local herdsmen; therefore, it is essential to control the spread of this disease. However, clinical signs in sheep or goats are not a reliable indicator of the presence or absence of MAP infection [17]. Weight loss is the predominant clinical sign in infected sheep and goats. In sheep, the period of weight loss differs from one animal to another. Softening of the faeces or diarrhoea occurs only in 20% of the cases at the end stages of the disease [18]. Detailed epidemiological investigations are important to understand the regional prevalence and potential threats of MAP. MAP can also induce bacteremia, which was unevenly distributed in the filtering organs of the sheep [19]. Attention should be paid to MAP infection in young sheep. Two clinical syndromes have been described in red deer: sporadic disease with low morbidity and high mortality in adult populations, and severe outbreaks in young deer (8–15 months old) resulting in both high morbidity and high mortality [20]. Young animals, especially those under 6 months, have a higher risk of infecting MAP, but the risk decreases thereafter [21]. Research also shows that calves inoculated with MAP at an earlier age had more severe tissue lesions [22].

In this study, we provided a detailed description of the clinical inspection and pathological changes, as well as molecular biology techniques and ELISA to diagnose naturally occurring PTB cases. We found that approximately 10% of sheep were infected in February and March; notably, most infections occurred in August and fewer infections were observed in winter. This cycle is almost the same every year. The main symptoms were emaciation and general weakness with intermittent diarrhea in infected sheep, which is consistent with the infection of paratuberculosis [18].

Paratuberculosis was confirmed by PCR using DNA extracted from mesenteric lymph node tissues and feces. At the histopathological level, the tissues and organs of the sheep, in this study, showed different degrees of lesions at the late stage of the disease. Serum samples of different sheep flocks were subjected to a MAP ELISA, and the seroprevalence was found to be 17.86% (35/196) and 18.48% (51/276), respectively, for the two different age groups. This prevalence was lower than the 19.5% seroprevalence in sheep in the Eastern Province of Saudi Arabia [23]; however, it was higher than the 11.9% seroprevalence in the Tibetan sheep in China [12], and 3.25% in female sheep in Tunisia [24]. These data show that PTB may be a risk factor in Inner Mongolia; however, detailed statistical analysis is needed.

MAP causes chronic diarrheic intestinal infections, which are difficult to treat, in domestic animals, including ruminants [25]. Several aminoglycosides are active against several species of mycobacteria, including MAP [26]. In this study, gentamicin was used in combination with lincomycin to treat diseases associated with MAP. We found that the symptoms in sheep were relieved after treatment for the first time; however, diarrhea occurred approximately 30 days after the first treatment. At this time, the use of therapeutic drugs was ineffective, and the sick sheep continued to suffer from diarrhea and subsequently died of exhaustion. The results demonstrated that some products effectively inhibited bacterial growth, and since these findings are applicable to the veterinary field, these products may become available for veterinary use [27, 28]. Further research may substantiate the efficacy of pharmaceuticals for the treatment and control of PTB.

Conclusion

In conclusion, this study is the first to estimate the epidemiology of PTB in sheep in Bayannaoer, Inner Mongolia, China. Our results showed that MAP was prevalent in this region. This information will provide a comprehensive view for the prevention and control of MAP infection in Inner Mongolia sheep.

Methods

Sampled animals and Sample collection

This study was carried out during October 2020, a total number of 472 sheep from 13 small to middle-sized sheep farms were sampled. The sampling proportion of the sheep was not less than 50% for the 6-month old sheep, and the proportion of sheep over 1 year old was not more than 5%. In addition, 196 sheep at approximately 6 months old and 276 sheep at ≥ 1 year old were selected. Sheep owners were interviewed about all treatments, therapeutic agents, and incidences of MAP in their sheep flocks. In this study, aminoglycosides were used to treat the disease, such as intramuscular injection of gentamicin (4 mg/kg) combined with oral electrolytic multidimensional preparation and lincomycin (500 mg oral), twice a day for 3 consecutive days. Three sheep that were extremely weak and were suspected of having JD were euthanized. The study was in compliance with ARRIVE guidelines. Serum and tissues samples, including ileum and mesenteric lymph node samples, were collected. Three milliliters of blood were collected from the jugular vein of each animal using a vacutainer. Sera were collected in Eppendorf tubes and stored at –20 °C until use [24]. Necropsy findings of the carcasses showed emaciated intestinal mucosa, enlarged lymph nodes, and occasional serous fat atrophy. The samples were separated into two parts, immediately placed in sterile plastic bags, placed in cooling boxes, and transported to the microbiology laboratory. Samples of the carcasses were subjected to tissue staining and DNA extraction.

PCR detection of MAP

DNA was extracted from the feces and mesenteric lymph node samples of sick sheep and subjected to PCR for MAP detection. DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) and from tissue using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China). The primers targeting IS900 were designed as follows: L1 (5’-CTTTCTTGAAGGGTGTTCGG-3’) and L2 (5’ -ACGTGACCTCGCCTCCAT-3’), AV1(5’-ATGTGGTTGCTGTGTTGGATGG-3’) and AV2 (5’-CCGCCGCAATCAACTCCAG-3’) and were used for the first round of PCR and nested PCR, respectively, as previously described [29]. For the first round of PCR, conditions were as follows: 94°C for 2 min, followed by 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with a final elongation step at 72°C for 3 min. The second PCR round used a 10× dilution of the products of the first round as templates, and the reaction parameters were carried out according to the primary reaction.

ELISA assays

Serum samples were collected from sheep farms in Bayannaoer, with herd sizes ranging from 300 to 500. The samples were analyzed using a commercial indirect ELISA kit. The tests were performed according to the manufacturer’s instructions (ID-VET, Montpellier, France). Sera with sample to positive (S/P) ratios ≤ 75% were scored as MAP-negative, while those with ratios ≥ 85% were considered MAP-positive. Moreover, 75%< S/P% <85% were scored as “suspect” and treated as negative for data analysis.

Histopathological examination

Tissue samples from the heart, kidney, spleen, lungs, ileum and mesenteric lymph nodes were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5-μm thickness and stained with H & E.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Abbreviations

DNA:

Deoxyribonucleic acid

ELISA:

Enzyme linked immunosorbent assay

JD:

Johne’s disease

MAP:

Mycobacterium avium subspecies paratuberculosis

PCR:

Polymerase Chain Reaction

PTB:

Paratuberculosis

References

  1. Stevenson K. Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review. Vet Res. 2015;46(1):64.

    Article  Google Scholar 

  2. Barkema HW, Orsel K, Nielsen SS, Koets AP, Rutten V, Bannantine JP, et al. Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection. Transbound Emerg Dis. 2018;65(Suppl 1):125–48.

    Article  Google Scholar 

  3. Kuenstner L, Kuenstner JT. Mycobacterium avium ssp. paratuberculosis in the Food Supply: A Public Health Issue. Front Public Health. 2021;9:647448.

    Google Scholar 

  4. Johansen MD, de Silva K, Plain KM, Whittington RJ, Purdie AC. Mycobacterium avium subspecies paratuberculosis is able to manipulate host lipid metabolism and accumulate cholesterol within macrophages. Microb Pathog. 2019;130:44–53.

    CAS  Article  Google Scholar 

  5. Nielsen SS, Toft N. A review of prevalences of paratuberculosis in farmed animals in Europe. Prev Vet Med. 2009;88(1):1–14.

    Article  Google Scholar 

  6. Whittington R, Donat K, Weber MF, Kelton D, Nielsen SS, Eisenberg S, et al. Control of paratuberculosis: who, why and how. A review of 48 countries. BMC Vet Res. 2019;15(1):198.

  7. Windsor P. Managing control programs for ovine caseous lymphadenitis and paratuberculosis in Australia, and the need for persistent vaccination. Vet Med. 2014;5:11–22.

    Google Scholar 

  8. Sardaro R, Pieragostini E, Rubino G, Petazzi F. Impact of Mycobacterium avium subspecies paratuberculosis on profit efficiency in semi-extensive dairy sheep and goat farms of Apulia, southern Italy. Prev Vet Med. 2017;136:56–64.

    Article  Google Scholar 

  9. Fiorentina P, Martino C, Mancini Y, De Iorio MG, Williams JL, Minozzi G. Using Omics Approaches in the Discovery of Biomarkers for Early Diagnosis of Johne’s Disease in Sheep and Goats. Animals. 2021;11(7):1912.

    Article  Google Scholar 

  10. Yue R, Liu C, Barrow P, Liu F, Cui Y, Yang L, et al. The isolation and molecular characterization of Mycobacterium avium subsp. paratuberculosis in Shandong province, China. Gut Pathog. 2016;8(1):9.

  11. Liu X, Li J, Yang X, Wang D, Wang J, Wu J. The seroprevalence of Mycobacterium avium subspecies paratuberculosis in dairy cattle in Xinjiang, Northwest China. Ir Vet J. 2017;70(1).

  12. Ma J, Tian A, Zheng W, Zou Y, Zhang Y, Yang Z. First report of bovine viral diarrhea virus and Mycobacterium avium subspecies paratuberculosis infection in Tibetan sheep (Ovis aries) in Tibetan Plateau, China. Trop Anim Health Pro. 2019;51(3):719–22.

  13. Cheng Z, Liu M, Wang P, Liu P, Chen M, Zhang J, et al. Characteristics and Epidemiological Investigation of Paratuberculosis in Dairy Cattle in Tai’an, China. BioMed Res Int. 2020;2020(11):1–7.

  14. Windsor PA. Paratuberculosis in sheep and goats. Vet Microbiol. 2015;181(1-2):161–9.

    CAS  Article  Google Scholar 

  15. Eslami M, Shafiei M, Ghasemian A, Valizadeh S, Al Marzoqi AH, Shokouhi Mostafavi SK, et al. Mycobacterium avium paratuberculosis and Mycobacterium avium complex and related subspecies as causative agents of zoonotic and occupational diseases. J Cell Physiol. 2019;234(8):12415–21.

    CAS  Article  Google Scholar 

  16. Aitken JM, Phan K, Bodman SE, Sharma S, Watt A, George PM, et al. A Mycobacterium species for Crohn's disease? Pathology. 2021;53:818–23.

  17. Whittington RJ, Sergeant E. Progress towards understanding the spread, detection and control of Mycobacterium avium subsp paratuberculosis in animal populations. Aust Vet J. 2001;79(4):267–78.

    CAS  Article  Google Scholar 

  18. Carrigan MJ, Seaman JT. The pathology of Johne's disease in sheep. Aust Vet J. 2010;67(2):47–50.

    Article  Google Scholar 

  19. Bower KL, Begg DJ, Whittington RJ. Tissue localisation of Mycobacterium avium subspecies paratuberculosis following artificially induced intracellular and naked bacteraemia. Vet Microbiol. 2013;162(1):112–8.

    Article  Google Scholar 

  20. Mackintosh CG, de Lisle GW, Collins DM, Griffin J. Mycobacterial diseases of deer. New Zeal Vet J. 2004;52(4):163–74.

    CAS  Article  Google Scholar 

  21. Karuppusamy S, Kirby GM, Mutharia L, Tripathi BN. An update on Mycobacterium avium subspecies paratuberculosis antigens and their role in the diagnosis of Johne’s disease. World J Microbiol Biotechnol. 2019;35(8):120.

    Article  Google Scholar 

  22. Mortier RAR, Barkema HW, Bystrom JM, Illanes O, Orsel K, Wolf R, et al. Evaluation of age-dependent susceptibility in calves infected with two doses of Mycobacterium avium subspecies paratuberculosis using pathology and tissue culture. Vet Res. 2013;44(1):94.

  23. Elsohaby I, Fayez M, Alkafafy M, Refaat M, Al-Marri T, Alaql FA, et al. Serological and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis (MAP) from Sheep, Goats, Cattle and Camels in the Eastern Province, Saudi Arabia. Animals. 2021;11(2):323.

    Article  Google Scholar 

  24. Khamassi KM, Romdhane R, Sassi L, Amami A, Rekik M, Benzarti MH. Seroprevalence of anti-Mycobacterium avium subsp. paratuberculosis antibodies in female sheep in Tunisia. Vet Med Sci. 2020;6(3):393–8.

    Google Scholar 

  25. Cirone KM, Lahiri P, Holani R, Tan YL, Arrazuria R, De Buck J, et al. Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages. Cell Tissue Res. 2020;379(1):207–17.

    CAS  Article  Google Scholar 

  26. Fecteau M, Whitlock RH. Treatment and Chemoprophylaxis for Paratuberculosis. Vet Clin N Am-food A. 2011;27(3):547–57.

    Article  Google Scholar 

  27. Wong SYY, Grant IR, Friedman M, Elliott CT, Situ C. Antibacterial Activities of Naturally Occurring Compounds against Mycobacterium avium subsp. paratuberculosis. Appl Environ Microbiol. 2008;74(19):5986–90.

    CAS  Article  Google Scholar 

  28. Ali ZI, Saudi AM, Albrecht R, Talaat AM. The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall. J Dairy Sci. 2019;102(6):4935–44.

    CAS  Article  Google Scholar 

  29. Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, et al. Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease. J Clin Microbiol. 2003;41(7):2915–23.

    CAS  Article  Google Scholar 

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Acknowledgments

We would like to thank Yatu Ji and Xin Li for their support during sample collection. The authors are also grateful to the farmers that participated in the survey.

Funding

The study was supported by grants from the performance incentive and guidance program of the Chongqing scientific research institutions (cstc2019jxjl80016).

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Authors and Affiliations

Authors

Contributions

Yu Y D was primarily responsible for the writing of this manuscript, and participated in detection and statistical analyses. Shen K F and Fu L Z were responsible for study design. Shen K F and Zheng H carried out ELISA. Zhang S H, Xu G Y, Xu D F, and Li B performed sample collection. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Kefei Shen or Lizhi Fu.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Committee on Animal Ethics of Chongqing Academy of Animal Sciences (permission number Cqaa2019004) and carried out in accordance with the approved guidelines. The animals sampled in this study are owned by private sheep farmers. The sheep owners consented to the use of their animals in the study under supervision of qualified veterinarians. Informed consent - Owners gave informed consent for their animals’ inclusion in the study. The study was in compliance with ARRIVE guidelines.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Yu, Y., Zhang, S., Xu, G. et al. Identification of Mycobacterium avium subspecies paratuberculosis in sheep farms in Bayannaoer, Inner Mongolia, China (short communication). BMC Vet Res 18, 281 (2022). https://doi.org/10.1186/s12917-022-03293-6

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Keywords

  • Sheep
  • Paratuberculosis
  • ELISA
  • PCR
  • Histopathology