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Original articles
Оригинальные статьи
Russian Journal of Infection and Immunity = Infektsiya i immunitet Инфекция и иммунитет
2019, vol. 9, no. 5-6, pp. 665-670 2019, Т. 9, № 5-6, с. 665-670
ISOLATION OF MYCOBACTERIUM AVIUM subsp. PARATUBERCULOSIS FROM MOUFLON IN BULGARIA
T. Savovaa, R. Petrovaa, V. Valchevab, M. Bonovskab, H. Najdenskib
a National Diagnostic and Research Veterinary Medical Institute, Sofia, Bulgaria b The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Abstract. Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (John's disease) mainly in large and small domestic and wild ruminants, and suspected causative agent in human Crohn's disease. In Bulgaria, paratuberculosis is still poorly researched in both groups of ruminants. We present results of the first in-depth study of mouflon, grown free in one hunting reserve in the Western region of the country. The aim was to prove the presence of MAP in diagnostic materials from regularly hunted or dead mouflon suspected for paratuberculosis. Small intestine and mesenteric lymph nodes (MLN) from 12 hunted and 4 dead mouflon and 10 faecal samples (Fc) were studied in the period of 2009—2013. Typical for paratuberculosis pathomorphological lesions were observed in four mouflon (of 16 examined). The intestinal wall was thickened, strongly folded and soft, with severe hyperemia. The MLN were enlarged, soft, with marbled appearance. The affected section of the ileum showed hyperplasia of the mucous corion and submucosa with diffuse infiltration of epithelioid cells. Lymphadenopathy with atrophy of T and B lymphocytes areas was observed in the mesenteric lymph nodes. For bacteriological isolation of MAP, the tissue and faecal samples were decontaminated with NALC-NaOH, cultured in Middlebrook 7H9 Broth and on Herrold's medium. The Ziehl—Neelsen stained smears and isolates were examined microscopically for acid-fast bacteria. Presence of MAP was observed in tissue samples of 4 (25%) mouflon and in 2 (20%) faecal samples. The same samples were confirmed by the IS900 PCR for the presence of specific for MAP fragments with a commercial amplification kit. The cases of paratuberculosis found at different times in the free-living mouflon in our study prove that the disease exists in Bulgaria and highlight the need for more serious control of the disease among wild and domestic ruminants.
Key words: mouflon, paratuberculosis, pathomorphology, microbiology, PCR.
ВЫДЕЛЕНИЕ MYCOBACTERIUM AVIUM subsp. PARATUBERCULOSIS ИЗ МУФЛОНА В БОЛГАРИИ
Савова Т.1, Петрова Р.1, Вылчева В.2, Боновска М.2, Найденски Х.2
1 Национальный диагностический и научно-исследовательский ветеринарный медицинский институт, София, Болгария 2Институт микробиологии им. Стефана Ангелова, Болгарская академия наук, София, Болгария
Резюме. Mycobacterium avium subsp. paratuberculosis (MAP) является этиологическим агентом паратубер-кулеза (болезнь Джона), главным образом, у крупных и мелких домашних и диких жвачных животных и предполагаемым возбудителем болезни Крона у человека. В Болгарии паратуберкулез все еще плохо
Адрес для переписки:
Магдалена Боновска
26, ул. Акад. Георги Бончев, София 1113, Болгария, Институт микробиологии им. Стефана Ангелова, Болгарская академия наук. Тел.: +3592 979-31-57. E-mail: m_bonovska@abv.bg Виолета Вылчева
26, ул. Акад. Георги Бончев, София 1113, Болгария, Институт микробиологии им. Стефана Ангелова, Болгарская академия наук. Тел.: +3592 979-31-57. E-mail: violeta_valcheva@mail.bg
Библиографическое описание:
Савова Т., Петрова Р., Вылчева В., Боновска М., Найденски Х. Выделение Mycobacterium avium subsp. paratuberculosis из муфлона в Болгарии // Инфекция и иммунитет. 2019. Т. 9, № 5-6. С. 665-670. doi: 10.15789/2220-7619-2019-5-6-665-670
© Savova T. et al., 2019
Contacts:
Magdalena Bonovska
26, Acad. Georgi Bonchev str., Sofia 1113, Bulgaria, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences. Phone:+3592 979-31-57. E-mail: m_bonovska@abv.bg Violeta Valcheva
26, Acad. Georgi Bonchev str., Sofia 1113, Bulgaria, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences. Phone:+3592 979-31-57. E-mail: violeta_valcheva@mail.bg
Citation:
Savova T., Petrova R., Valcheva V., Bonovska M., Najdenski H. Isolation of Mycobacterium avium subsp. paratuberculosis from mouflon in Bulgaria // Russian Journal of Infection and Immunity = Infektsiya i immunitet, 2019, vol. 9, no. 5-6, pp. 665-670. doi: 10.15789/2220-7619-2019-5-6-665-670
DOI: http://dx.doi.org/10.15789/2220-7619-2019-5-6-665-670
изучен в обеих группах жвачных животных. В данной работе представлены результаты первого углубленного исследования муфлонов, выращенных в охотничьем заповеднике в западном регионе страны. Целью исследования было доказать наличие MAP в диагностическом материале от муфлонов при подозрении на паратуберкулез. В период 2009—2013 гг. были исследованы образцы из тонкого кишечника и мезентериальных лимфатических узлов (МЛУ) 12 убитых во время охоты и 4 павших муфлонов и 10 образцов фекалий. Типичные патоморфологические поражения, характерные для паратуберкуле-за, наблюдались у четырех муфлонов (из 16 обследованных). Стенка кишечника была толстой, сильно скрученной и мягкой, с выраженной гиперемией. МЛУ были увеличеными, мягкими, имели мраморный внешний вид. На пораженном участке подвздошной кишки наблюдались гиперплазия слизистой оболочки и диффузная инфильтрация эпителиоидными клетками подслизистой оболочки. В мезентериальных лимфатических узлах наблюдалась лимфаденопатия с атрофией в области Т- и В-лимфоцитов. Для бактериологического выделения MAP образцы тканей и фекалий дезактивировали NALC-NaOH, культивировали в бульоне Миддлбрук 7H9 и на плотной среде Херролда. Мазки, окрашенные по Цилю— Нильсену, и изоляты, исследовали под микроскопом на наличие кислотоустойчивых бактерий. Присутствие MAP наблюдалось в образцах ткани 4 (25%) муфлона и в 2 (20%) образцах фекалий. Присутствие MAP в тех же самых образцах было подтверждено с помощью ПЦР IS900 с коммерческим набором для амплификации специфического для MAP фрагмента. Случаи паратуберкулеза, обнаруженные в нашем исследовании в разное время у свободно живущих муфлонов, показывают, что MAP циркулирует в Болгарии и подчеркивают необходимость более строгого контроля заболеваемости паратуберкулезом среди диких и домашних жвачных животных.
Ключевые слова: муфлон, паратуберкулез, патоморфология, микробиология, ПЦР.
Introduction
Paratuberculosis is one of the oldest diseases in animals, described in 1829 in England and studied by John and Froitam in 1895. They first detected acid-alcohol-resistant bacillus in the intestine of a cow with chronic diarrhea. Twort and Inggram (1912) obtained a pure bacterial culture and gave a general description of the disease, named "John's disease" [8]. At the suggestion of Thorel, the etiological agent was named Mycobacterium avium sub-sp. paratuberculosis (MAP) [24].
The isolation of MAP from the blood culture of patients, from breast milk and patients with ulcerative colitis gives reason to accept MAP as one of the etiological factors in Crohn's disease [6, 15, 16, 17]. This shows zoonotic nature of MAP, although the question still remains controversial [18, 21].
Etiological agent of the disease, M. paratuber-culosis is small, thin, Gram-positive, acid-resistant rod. The bacteria are immobile, do not form spores and capsules, pink stained by Ziehl—Neelsen. MAP grows on an enriched culture media and is very resistant to drying, low temperatures and disinfectants [22, 24, 26]. MAP is a facultative aerobic bacterium belonging to Mycobacterium avium complex [3, 8, 17, 24]. It is an obligate intracellular pathogen that can not replicate outside of animal and human hosts [2, 16, 18]. Characteristics distinguishing MAP from other Mycobacterium species, include its extremely slow growth, its inability to produce the necessary for its growth mycobactin and pos-
session of 14—18 copies of the IS900 insertion element in the MAP genome [4, 5, 20, 22].
Paratuberculosis (John's disease) affects mainly the young domestic and wild ruminants, including mouflon (Ovis aries musimon; wild sheep), most commonly causing hypertrophic enteritis. Diagnosis of John's disease is a difficult and very long process. In wildlife, paratuberculosis usually occurs subclinically without visible symptoms. The affected animals spread the agent in the environment through faeces and so contaminate soil, pastures and water and may be the source of infection for other animals [2, 10, 11, 18, 20].
The paratuberculosis in wild animals was first described in 1922 by Jarmy in an antelope in a zoo, and in 1949 by Dorofeev and Kalacheva in deer populations [3]. The disease in captive and free-range mouflon and other wildlife is described in many countries in Europe and worldwide. According to OIE (World Organisation for Animal Health) documents over the last decades, 44% of member states have reported the presence of paratuberculosis and in 2004 is categorized in sheet B as a disease with serious economic and health consequences [18].
In Bulgaria paratuberculosis in wild animals has been poorly studied. There are no official data on the prevalence of the disease in both domestic and wild ruminants. Because its rare clinical manifestations, the incidence of subclinical forms of the disease is not investigated or monitored. Only single cases in cattle of private farms were described [12]. In the period 2009—2013 was con-
ducted the first in-depth study of diagnostic materials from the hunted and dead wildlife, grown freely in one hunting area in the northwestern region of the country [22].
The aim of this study is to prove the presence of MAP in diagnostic material from legally hunted or dead mouflon suspected for paratuberculosis, which may be a risk for spreading the disease in the population of other wild and domestic ruminants in contact with infected animals or contaminated environment.
Materials and methods
Collection of samples. Samples from proximal and distal parts of the ileum (IL) and mesenteric lymph nodes (MLN) of 12 regularly hunted and 4 found dead mouflon were studied for visible macroscopic lesions of paratuberculosis. Faecal samples (Fc) from 8 of killed and 2 dead animals were collected and examined too.
Histopathologic examination. The examinated tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin and processed routinely for histopathologic examination [12]. Sections (5 ^m thick) were stained with Hematoxilin-Eosin (H&E).
Bacterial cultures. The isolation of Mycobacte-rium avium subsp. paratuberculosis (MAP) from tissue samples (TS) and faeces (Fc) was carried out with commercial kit DNA-Sorb-B (Sacace Biotechnologies, Italy). The tissue smears stained with commercial Ziehl—Neelsen (ZN) Color Kit (Liofilchem, Italy) were observed microscopically. Briefly, the tissue and faecal samples were homogenised in saline solution and decontaminated with NALC-NaOH (N-acetyl-L-cysteine-sodium hydroxide). The sediment obtained after the sample treatment was resuspended in phosphate buffer. 100 ^l of each sample were cultured in 2 tubes with Herrold's medium with Mycobactin J and pyrovate (Remel-Kansas, USA) and Middlebrook 7H9 Broth with polysorbate 80 (Becton&Dickinson, USA). Bacteriological study was conducted on 16 samples of IL, 10 of MLN and 10 of Fc. The samples were grown at 37°C from 3 to 6 months.
PCR detection assay. For isolation and amplification of MAP's DNA from tissue samples and faeces, both DNA-Sorb-B kit and respectively M. paratuberculosis Vet DNA amplification kit (Sacace Biotechnologies, Italy) were used. To confirm the strains identified bacteriologically as MAP, the IS900 PCR-based technique was carried out. DNA kit's control and DNA from MAP 7072 (clinical isolate from deer) were used as positive controls. The size of the PCR products was determined by a DNA marker of 100 bp.
Results
Typical for paratuberculosis pathomorphologi-cal lesions were observed in 25% of mouflon (4 out of 16 examined). In affected animals, hyperplastic changes were observed in the proximal and distal segments of the ileum. The intestinal wall was thickened, strongly folded and soft, with severe hyperemia (fig. 1, see cover II). Intestinal content was scanty with watery to thick whitish consistency (fig. 2, cover II). The MLN were enlarged, soft, with marbled appearance. Their capsule and cut surface had an intensive yellowish-cream color, without bleeding, abscesses or caseous foci.
Histologically the affected section of the ileum showed hyperplasia of the mucous corion and sub-mucosa with diffuse infiltration of epithelioid cells. In the altered parts of the ileum diffuse noncaseat-ing granulomatous enteritis was observed. The normal structure of the intestinal mucosa was altered. The lamina propria was markedly expanded by lymphocytes and scattered macrophages with foamy cytoplasm and absence of Langhans giant cells Intestinal villi were shortened and thickened. Atrophy of the intestinal crypts was observed due to compression from the mononuclear inflammatory cells. The microscopic examination of lymph nodes from mouflon revealed extracellular deposition of amyloid in the form of an amorphous eosinophilic substance. Its accumulation is high and exerts strong pressure on the lymph tissue, causing severe atrophy of T and B lymphocyte areas. The lymph node cortical and para-cortical zones are mainly affected (fig. 3, cover II).
The bacteriological study of MLN and IL tissue samples and faeces showed primary growth of single colonies at 5—6 weeks of the culturing on the selective Herold's medium. In the presence of specific growth we observed clearly separated, very small, convex, soft, colorless, translucent and humid colonies. In abundant growth colonies remained small, round, smooth and glossy (fig. 4, cover II). On the third month of culturing on the solid medium the colonies became opaque, gray-whitish, rough and dry. On the Middlebrook 7H9 Broth bacteria have formed a thick, whitish veil. MAP was isolated from tissue samples of the ileum and MLN of 3 hunted and 1 dead mouflon with pathomorphologi-cal lesions and from 2 faecal samples. Other tissue samples and faeces, showed no growth on culture media and after 6 months cultivation (tabl.). The microscopic observation of Zieh—Neelsen (ZN) colored smears found immobile, short pink-red colored rods, clustered on heaps or located severally (fig. 5, cover II). The presence of MAP in the same samples was confirmed by IS900 PCR kit. PCR analysis showed a presence of specific 209-bp DNA fragment (fig. 6). The other samples were PCR negative (tabl.).
1 2 3 4 5 6 К M 7 8 9 10 11 12 13 14
Figure 6. 209 bp-PCR products from bacterial isolates and tissue samples from the mesenteric lymph nodes (MLN) and small intestines (SI) of mouflons
1-3 — bacterial isolates from MLN; 4-6 — bacterial isolates from the small intestine; K — positiv control MAP 7072 (clinical isolate from deer); M — molecular weight marker; 7-8 — fecal samples; 9-11 — tissue specimens ofMLN; 12, 14 — tissue samples of the intestine; 13 — DNA kit's control
Discussion
In wildlife disease usually remains hidden and animals are only carriers and emitters of mycobacteria, which hinders the diagnosis of paratubercu-losis. In the examined samples pathoanatomical changes were observed in the small intestine and mesenteric lymph nodes. Proliferation of intestinal mucosa varied significantly from less pronounced to clearly visible changes. Similar lesions in domestic and wild ruminants with paratuberculosis were described by many authors [3, 7, 8, 9, 12, 14, 20, 26].
The histological examination is the first opportunity for detection of subclinical cases of the disease and in wild animals it is very important. In the altered parts of the intestinal mucosa we observed infiltration of a variable numbers of diffusely located macrophages, lymphocytes and plasma cells significantly expanding lamina propria, thickened
intestinal villi and necrotic debris in intestinal crypts. In the MLNs we observed lymphadenopathy. Many authors reported similar histological findings in sheep, goats, cattle and wild animals suffering from paratuberculosis [2, 7, 12, 14, 26]. In the affected tissues, some of authors observed also the presence of a single or large amount of multinucleated Langhans giant cells. We have not found giant cells, but only diffuse deposition of an amorphous, eosin-ophilic extracellular substance and atrophy of the T and B lymphocyte areas. Such results are not unusual and depend on the species and age of the animals, the development stage and intensity of the infection as well as the prevalence of MAP in the wildlife.
After microscopic examination of tissue and faecal smears we observed pink-red rods located singly or clustered on heaps, resembling morphology many other mycobacteria. Therefore, the finds can only direct us to the disease. The bacteriological method, although time consuming, still remains a "gold standard" for detection of MAP because it does not give false-positive results [11, 18, 19]. Unlike other mycobacteria, MAP is best developed for the nutrient medium containing mycobactin J, a growth factor that bacteria can't produce on their own [1, 3, 18, 26]. Such is the Herald's medium, which we have also successfully used. The infectious agent was isolated from the altered tissues of the IL and MLN of 4 (25%) mouflons. The isolation of MAP from faecal samples is also an important moment in the diagnosis of the disease. Although the method is considered to be highly specific and sensitive test to detect infected animals in herds [11], it has limited sensitivity for subclinically infected and free-living wild animals. In our study MAP was isolated from only two faecal samples (20%). Established bacteriological findings corresponded to those described by other
Table. Overview of data obtained from mouflons samples and types of analyses
Animal species Tested animals MAP positive animals (No.) Type of sample Analyzed samples Type of analysis
Autopsy findings (+) Histology findings (+) Microscopy (+) Culture (+) PCR (IS900) (+)
Mouflons hunted 12 З SI 12 З З З З З
MLN 10 З З З З З
Fc 8 2 2 2
dead 4 1 SI 4 1 1 1 1 1
MLN 4 1 1 1 1 1
Fc 2 0 0 0
Total No. 16 4 Ts/Fc 30/ 10 8 8 10 10 10
SI — small intestine; MLN — mesenteric lymph nodes; Fc —faeces; Ts —tissue samples (SI + MLN).
authors [2, 20, 23]. The MAP isolation from tissues and faeces of mouflon was confirmed by the PCR which is recommended by the World Organisation for Animal Health [18]. Based on genomic analysis Englund et al. [5] reveal that this element is also present in other mycobacterial species. Harris and Barletta [8], Vansnick et al. [25] noted that most of the used IS900 PCR primers did not differentiate MAP from other members of the M. avium complex because they give identical DNA amplicons. More recent research by other authors describe different variants for detection of paratuberculosis bacteria and most of them are also based on the IS900 insertion element, which is accepted as a standard molecular marker for MAP [5, 20, 23, 26].
We used an IS900amplification kit, recommended as a specific for M. avium complex, respectively for MAP [22]. The resulting PCR products coincided with the established specific patomorphologi-cal changes and the isolated bacterial culture from the affected tissues of the four mouflons and the two faecal samples, indicating that the amplicons were MAP-specific (tabl.).
The paratuberculosis refers to an "open infection" with large excretion of causative agent in the environment. Contamination of soils, plants, pastures, water and air after excretion of MAP with
References
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The paratuberculosis cases detected over different periods in free-living mouflon in our study shown that the disease exists in Bulgaria. The results indicate the need for further research on hunting reserves in the country and their neighboring cattle farms. This would clarify the prevalence of the disease among wildlife and the potential risk for the domestic ruminants.
Acknowledgements
The authors thank the Veterinary Research Institute Brno, Czech Republic for providing MAP isolate 7072 from deer. We are grateful to Dr. Aneta Trifonova and Dr. Ivan Todev from the National Research Station of Game Management, Biology and Pathology, Sofia, Bulgaria for providing the diagnostic matherials from mouflons.
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Авторы:
Савова Т., доцент, к.в.м.н., зав. Национальной референс-лабораторией по туберкулезу животных в Национальном диагностическом научно-исследовательском ветеринарном медицинском институте «Проф. Д-р Г. Павлов», София, Болгария;
Петрова Р., главный ассистент, к.в.м.н., зав. Национальной референс-лабораторией по сибирской язве и бешенству у животных, кафедра патоморфологии Национального диагностического научно-исследовательского ветеринарного медицинского института «Проф. Д-р Г. Павлов», София, Болгария;
Вылчева В., главный ассистент, к.б.н., Институт микробиологии им. Стефана Ангелова, отдел инфекционной микробиологии, лаборатория зоонозов и бактериальной вирулентности, Болгарская академия наук, София, Болгария; Боновска М., профессор, к.в.м.н., Институт микробиологии им. Стефана Ангеловаа, отдел инфекционной микробиологии и лабораторией зоонозов и бактериальной вирулентности, Болгарская академия наук, София, Болгария; Найденски Х., член-корреспондент, профессор, д.в.м.н., директор Института микробиологии им. Стефана Ангелова, зав. отделом инфекционной микробиологии и лабораторией зоонозов и бактериальной вирулентности, Болгарская академия наук, София, Болгария.
Поступила в редакцию 11.02.2019 Отправлена на доработку 05.03.2019 Принята к печати 26.03.2019
Authors:
Savova T., Associate Professor, PhD (Veterinary Medicine),
Head of National Reference Laboratory for Animal Tuberculosis
at the National Diagnostic Research Veterinary Medical Institute
"Prof. Dr. G. Pavlov", Sofia, Bulgaria;
Petrova R., Assistant Professor, PhD (Veterinary Medicine),
Head of the National Reference Laboratory for Anthrax
and Rabies in Animals, Department of Pathomorphology
at the National Diagnostic Research Veterinary Medical Institute
"Prof. Dr. G. Pavlov", Sofia, Bulgaria;
Valcheva V., Assistant Professor, PhD (Biology), The Stephan
Angeloff Institute of Microbiology at the Bulgarian Academy
of Sciences, Department of Infectious Microbiology, Laboratory
of Zoonosis and Bacterial Virulence, Sofia, Bulgaria;
Bonovska M., Professor, PhD (Veterinary Medicine), The Stephan
Angeloff Institute of Microbiology at the Bulgarian Academy
of Sciences, Department of Infectious Microbiology, Laboratory
of Zoonosis and Bacterial Virulence, Sofia, Bulgaria;
Najdenski H., Corresponding Member, Professor, DS (Veterinary
Medicine), Director of the Stephan Angeloff Institute of Microbiology
at the Bulgarian Academy of Sciences, Head of Department
of Infectious Microbiology and Laboratory of Zoonosis and Bacterial
Virulence, Sofia, Bulgaria.
Received 11.02.2019 Revision received 05.03.2019 Accepted 26.03.2019
