Abstract
Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, causing enteritis and chronic granulomatous lymphadenitis, characterized by malabsorption syndrome, its agent is the Mycobacterium avium subsp. paratuberculosis (MAP). Thus, the objective of this work was to identify and characterize MAP in buffalo herds slaughtered in Baixada Maranhense region. Samples of intestines, mesenteric lymph nodes, and ileocecal valves were collected from 115 buffaloes slaughtered at Baixada Maranhense slaughterhouses to perform the diagnosis by histopathological examination using staining with Hematoxylin and Eosin (H&E) and Ziehl-Neelsen, bacterial isolation, and real-time PCR. In the histopathology by H&E staining, there was evidence suggestive of paratuberculosis in 30% (31/115) of the buffaloes. With Ziehl-Neelsen staining, acid-fast bacilli (AFB) were visualized in 27% (26/115) of the tissue samples analyzed. MAP was isolated in 4.3% (5/115) of the fecal samples subjected to bacterial culture. The samples inoculated in HEYM with mycobactin J produced colonies identified with MAP according to their own morphological characteristics such as round, white, smooth and slightly rough, alcohol-acid staining, and slow growth with 8 weeks of incubation and mycobactin dependence. The agent confirmation was performed in five bacterial isolates (4.3%) and 15 (13%) fragments of jejunum, ileum, and mesenteric lymph node by the IS900 real-time PCR technique. The results of the present study demonstrate the subclinical occurrence of paratuberculosis in flocks of buffalo slaughtered in slaughterhouses of Baixada Maranhense.
Keywords: Paratuberculosis, Buffalo, Mycobacterium avium subsp. paratuberculosis, Diarrhea
Introduction
Paratuberculosis (Johne’s disease in cattle) is an infectious, chronic incurable disease of worldwide distribution that affects mainly domestic ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) responsible for the development of enteritis and granulomatous lymphadenitis in hosts [13].
The disease is characterized by chronic and intermittent diarrhea, reduction of feed conversion, reduced productivity, reduced levels of protein and fat in milk, loss of weight at slaughter, high incidence of mastitis, and reduction of fertility [6, 21–23]. The infection occurs through the ingestion of food contaminated with MAP, usually in the first months of life; however, the clinical signs manifest with greater frequency between 2 and 5 years of age [5].
In Brazil, the disease was first reported in buffaloes by Mota et al. [14], in Pernambuco. Later, other cases were registered by Barbosa et al. [2] and Brito et al. [3] in Maranhão, Dalto et al. [7] in Rio Grande do Sul. The subtype Bison MAP was predominant in samples of Minas Gerais, Sao Paulo, Paraiba, Alagoas, and Maranhão [1].
The early and specific diagnosis of paratuberculosis is still a challenge related, in part, to the low sensitivity of existing laboratory tests [19, 20].
In this region, buffalo milk is intended for cheese production and sold as in natura to the population, which can be considered a risk to human health once infected; cattle can make this population susceptible for developing Crohn’s disease. Considering the lack of information on paratuberculosis in buffaloes in Brazil and in Maranhão, the objective of this study was to report the occurrence of subclinical paratuberculosis in buffaloes slaughtered in Baixada Maranhense through anatomopathological techniques, bacterial culture, and PCR for confirmation of Mycobacterium avium subsp. paratuberculosis in intestinal tissue samples and feces.
Materials and methods
Sampling and collection of tissue fragments
This study was conducted with buffaloes slaughtered at the municipal slaughterhouses of Viana and Ararí, MA, situated in Baixada Maranhense (01° 59″–04° 00″ S; 44° 00′–45° 33″ W). Between the months of September 2014 to March 2015, visits were performed fortnightly to slaughterhouses for fragment’s collection of different portions of the small intestine and large intestine (duodenum, jejunum, ileum, ileocecal valve, cecum, and colon) and mesenteric lymph nodes, totaling 115 animals, in which 66 were females and 49 were males with age ≥ 36 months of breeds Murrah, Mediterranean, and their crossbreeds, without obvious clinical signs of paratuberculosis.
Histopathology and bacterioscopy
After the material fixation, in a solution of formalin 10% buffered with calcium carbonate, the dehydration of fragments in baths of absolute alcohol, diaphanization in xylene followed by Paraffin bath at 60 °C, were performed and finally included in paraffin blocks and cut to 5 μm and stained by hematoxylin and eosin (H&E). The animal tissues that showed granulomatous inflammatory infiltrate were stained by the Ziehl-Neelsen technique for acid-alcohol bacilli resistant (BAAR) research. The staining was performed as described by Luna (1968) using commercial phenicated fuchsin (LABORCLIN®).
Bacterial culture
Approximately 100 g of feces was collected directly from rectal ampoule, packed in identified plastic bags, chilled, and transported to the laboratory of Infectious Diseases of the Veterinary Medicine Course of UEMA. After homogenization, 1.5 to 2 g of feces was diluted in 35 mL of sterile distilled water in a 50-mL Falcon tube type with lid and subjected to agitation and then kept at rest for sedimentation for 30 min at room temperature. Then, 5 mL of supernatant was transferred to new type 50-mL Falcon tube and centrifuged at 2800 rpms for 15 min. After centrifugation, the supernatant was discarded and added to 25 mL of hexadecyl-pyridium chloride monohydrate at 0.9% (Sigma Chemical Co., St Louis, Mo); it remained for 18 h at a room temperature and the tubes were later centrifuged again and the supernatant was discarded. The sediment was resuspended in 1 mL of antimicrobial solution (2000 mg vancomycin, 2000 mg, nalidixic acid, and 3 mg amphotericin B) and kept at 37 °C in an oven for 18 h. A total of 0.1 mL of the mixture was sowed in the medium of specific cultivation of Herrold with egg yolk (HEYM – Herrold’s egg yolk medium) containing mycobactin J (Allied Monitor, Inc., Fayette, MO, USA) [18]. The media were kept in oven at 37 °C for a period of 6 months under biweekly observation.
DNA extraction
After visual examination of the Herrold cultivation media, smear of colonies morphologically compatible with the MAP followed by Ziehl-Neelsen staining was performed. The cultivation samples positive in the staining and tissue fragments (jejunum, ileum, and mesenteric lymph nodes) were subjected to qPCR to detect the region IS900 of the MAP at the Laboratory of Domestic Animals Bacteriose of UFRPE. Then, the DNA extraction was performed using a commercial kit Wizard SV Genomic DNA Purification System (Promega®), following the manufacturer’s instructions.
Polymerase chain reaction in real time
After the DNA extraction, the amplification reactions were performed in a final volume of 25 mL containing 5 μL of genomic DNA; 1 μL of specific primers for S900 at 10 μM (DF: 5′-GACGACTCGACCGCTAATTG-3′ and DR-1: 5′-CCGTAACCGTCATTGTCCAG-3′); 5.5 μL of Milli-Q ultrapure water; and 12.5 μL of QuantiFast SYBR Green PCR Kit (PCR-QIAGEN® mixture) according to the supplier’s protocol. The thermal profile from the reaction’s steps was performed in a thermocycler “Rotor-Gene Q” (QIAGEN) with initial denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 20 s and 60 °C for 30 s. The software “Rotor-Gene Q Software v1.7” was used to accompany the reaction and perform the interpretation of the results of the qPCR. For positive reaction control, the DNA of a MAP strain provided by the National Laboratory Agriculture of Minas Gerais, identified as “Nakajima1991,” was used, whereas for negative control, sterile ultrapure water was used.
Results
Macroscopic findings
The lymphatic vessels from the mesentery and intestinal serosa had vacriceal aspect, with prominent and dilated features, were observed (64/115). The mesenteric lymph nodes were hypertrophied, damp and shiny, and protracted when cut, with the medullary region in general having dark brown color. In 55/115 of the mesenteric lymph nodes, there was the presence of irregular areas with multinodular appearance and whitish nodules with varying diameters in the cortical region with multinodular appearance with varying diameters. Furthermore, it was noted the presence of milky liquid in the medullary sinus in 53/115 and bloody content in 04/115 samples (Fig. 1). Of the 115 samples collected, 66/115 showed thickening and wrinkling of the intestinal wall, assuming a “cerebroid” aspect in various segments of the small intestine. In 46/115 of the animals, areas of intense erythema accompanied by mucous serous content, with a yellowish coloration in their intestinal mucous membranes, were observed.
Fig. 1.
Pictures of small intestine of buffalo, where subserous prominent, dilated, tortuous of variceal aspect lymphatic vessels (a, arrows), mesenteric lymph node chain with increase in size and volume (b, arrows), a prolonged mesenteric lymph node protracted when cut with dark and cortical medullary region with clear area with accumulation of milky liquid (c, arrows)
Small intestine
In the abdominal cavity, the intestinal loops were thickened, with girdled, and/or cerebroid aspect handles of the intestine are observed, to the opening of the duodenum mucosa with irregular and thickened, creased, or wrinkled surfaces. In certain animals, it was noticed hyperemic areas followed by hemorrhagic petechiae; there was a brown colored pasty exudates and clear intestinal content. The jejunum also showed a thickened, wrinkled mucosa, with similar appearance to cerebral gyri, with diffuse irregular areas; in general, the lesions are restricted to the final third of the jejunum extending to the final portion of the ileum. Accumulation of viscous secretion of light-brown color was noted. In the ileum, a thickened wall was evidenced, with girdled and or cerebroid aspect; proliferative aspect was also visible in the mucosa, which gave an aspect often wrinkled or pleated, frequently diffuse reddish areas and/or multiple hemorrhagic petechiae (Fig. 2).
Fig. 2.
Pictures of buffalo’s small intestine, showing dilated and thickened wall with cerebroid aspect (a, arrows), thickened duodenal mucosa with wrinkled appearance (b, arrow), presence of a brownish liquid content (c, arrow), ileus with thickened and wrinkled, dark brown mucosa (d, arrows)
Large intestine
The cecum was distended, with thickened mucosa (63/115), in 49/115 showed reddish regions with multiple hemorrhagic petechiae of granular characteristic, which gives reticulated aspect. The ileocecal valve (24/115) was edematous, with thickened mucosa and transverse folds that had microgranular appearance on the surface. In 20/115, the colon exhibited, sharp, thickening, and evident pleating. It was noted in certain cases (50/115) reddish areas and granular aspect of the mucosa (Fig. 3).
Fig. 3.
Pictures of buffalo’s large intestine, denoting the ileocecal valve increased in volume and edematous (a, arrow), cecum with dilated and wrinkled mucosa with irregular and reddish areas (b, arrow), colon with pleating and reddish areas in the mucosa (c, arrow)
Bacterioscopy and bacterial culture
Bacterial colonies were observed morphologically similar to MAP (white, round, smooth and slightly rough, alcohol-acid stain resistant, and slow growth with 8 weeks of incubation), in 4.3% (5/115) of fecal samples. Acid-alcohol bacilli resistant (BAARs) were detected in feces samples, mesenteric lymph nodes, and mucosa of the ileum in 22% (26/115) (Fig. 4). In the other inoculated samples, there was no bacterial growth after 24 weeks of cultivation and/or contamination made in the observation of typical colonies of MAP inexplicit.
Fig. 4.
Pictures of bacterial colonies typical of Mycobacterium avium subsp. paratuberculosis, of buffaloes’ fecal samples (a, arrows), acid-alcohol bacillus resistant of buffaloes’ fecal samples (b, arrow), in Herrold with egg yolk (HEYM – Herrold’s egg yolk medium) containing mycobactin J. Mucosa of the ileum and mesenteric lymph node with numerous alcohol-resistant acid bacillus (c, arrows), Ziehl-Neelsen staining, obj. 400×
Histopathology
The histological lesions restricted to the regions of the small intestine were characterized by inflammatory infiltrate granulomatosis in which there was the presence of Langhans giant cells and mononuclear infiltrate on the submucosa (Fig. 5). Atrophy and fusion of villi in the small intestine sharply and multifocally were also observed. The lymphoid follicles in Peyer’s patches, ileocecal valve, and large intestine showed inflammatory infiltration of macrophages and giant cells. In the mesenteric lymph nodes, epithelioid cells and giant cells were observed in the cortical region and spinal cord.
Fig. 5.
Pictures of intestinal mucosa (jejunum) granulomatous lesion with intense infiltrate of epithelioid macrophages cells and giant cells in the buffalo’s mesenteric lymph node mucosa (a, arrows); diffuse infiltration of epithelioid macrophages and Langhans-type giant cells (b arrows). Subclinical paratuberculosis, Baixada Maranhense, MA, 2015. Hematoxylin and eosin. Obj 200×
Polymerase chain reaction in real time
In qPCR, the genetic sequence IS900 specific of MAP in 4.3% (5/115) samples of bacterial isolation and in 13% (15/115) of the fragments of jejunum, ileum, and the tested mesenteric lymph nodes were amplified. Table 1
Table 1.
Results of diagnostic tests performed on MAP naturally infected buffaloes, in slaughterhouses, Baixada Maranhense, Brazil
| Animals | Ziehl-Neelsen | Bacterial culture | Real-time (qPCR) bacterial culture | Real-time (qPCR) fabrics |
|---|---|---|---|---|
| 1 | + | − | − | + |
| 2 | + | − | − | + |
| 3 | + | + | + | + |
| 4 | + | − | − | − |
| 5 | + | − | − | − |
| 6 | − | − | − | − |
| 7 | + | − | − | − |
| 8 | − | − | − | − |
| 9 | + | − | − | + |
| 10 | + | − | − | + |
| 11 | + | − | − | + |
| 12 | + | − | − | − |
| 13 | + | + | + | + |
| 14 | + | − | − | + |
| 15 | + | − | − | + |
| 16 | + | − | − | + |
| 17 | + | + | + | + |
| 18 | + | − | − | + |
| 19 | + | + | + | + |
| 20 | + | + | + | + |
| 21 | + | − | − | + |
| 22 | + | − | − | − |
| 23 | − | − | − | − |
| 24 | + | − | − | − |
| 25 | − | − | − | − |
| 26 | + | − | − | − |
| 27 | + | − | − | − |
| 28 | − | − | − | − |
| 29 | + | − | − | − |
| 30 | + | − | − | − |
| 31 | + | − | − | − |
| Total | 26/115 (22%) | 05/115 (4.3%) | 05/115 (4.3%) | 15/115 (13%) |
Discussion
In the case of paratuberculosis in buffaloes, the records include Italy [11] and India [17]. In Brazil, having several reports of the disease affecting the cattle through the country, clinical cases of paratuberculosis in buffalo species have been reported in some regions of the country; being the first case described in the Northeast region, the State of Pernambuco by Mota et al. [14] and afterwards by Dalto et al. [7] who reported a flock of buffaloes infected by MAP in the South region, in the State of Rio Grande do Sul, subsequently by Barbosa et al. [2] in the state of Maranhão.
In the present study, the natural occurrence of 13% (n = 15/115) of the disease in buffaloes surpassed results of Sivakumar et al. [18] in India and Barbosa et al. [2] in the State of Maranhão; this high rate is associated with the extensive buffaloes rearing in Baixada Maranhense, i.e., there is no sanitary and zootechnical control and at least segregation of animals by age, favoring transmission of MAP for young animals, since the infection occurs in the first days of life [19]. There was, probably, no control over the calves’ milk supply.
Another key factor in the spread of paratuberculosis in flock studied was associated with the great agglomeration of these animals in the extensive shallow lakes, which are formed in the period of drought in Baixada Maranhense. These “natural pools” remain almost the whole day, feeding and pouring their excreta in these locations, facilitating the transmission of (MAP) via orofecal, especially of females for young animals at the time of breastfeeding. This peculiarity inherent in Baixada Maranhense favors the maintenance and dissemination of the agent in the studied buffalo herds.
A striking feature in this study was the presence of focal granulomas in the mesenteric lymph nodes of adult buffaloes, being this finding reported only in cattle calves in the early stages of the disease [4, 10]. This denotes that the paratuberculosis behaves in a peculiar way in these animals and that the infection was active. Another important alteration, already reported in cattle and buffaloes by several authors [7, 16], was the granulomatous mesenteric lymphadenitis rich in epithelioid macrophages and giant cells, with the presence of BAAR.
In the intestine in general, the cerebroid and girdled appearance in the mucosa, resulting from the accentuated thickening of the intestinal wall, were attributed to severe transmural inflammatory infiltration, mainly in the mucosa and submucosa of the jejunum and ileum. Macroscopic lesions in the small intestine, which correspond histologically to enteritis, are usually described in paratuberculosis [2]. These lesions were observed in buffaloes, in varying degrees of intensity, from mild to severe. It is worth mentioning that they are not always present and that the variation in the intensity of these lesions may be related to the stage of infection, the immune status of the animal, the individual susceptibility, and the MAP strain [7].
In the duodenum, polypoid structures on the surface of the thickened mucosa were observed; in histological cuts of these areas, the findings corresponded to the proliferation of duodenal and intestinal glands, who gave “arboriform” aspect to the mucosal surface. These areas had inflammatory infiltration similar to other sections of the intestine, but at a lower intensity, and no structure was verified that could suggest a parasitosis or another pathological process.
The Ziehl-Neelsen staining proved to be satisfactory in the identification of BAAR in tissues of duodenum, jejunum, ileum, and mesenteric lymph nodes in animals with the absence of clinical signs, being that, in this study, all the slaughtered buffaloes were from properties without confirmed cases of paratuberculosis. However, five animals with macroscopic lesions, characteristics of paratuberculosis, were negative in ZN. According to Mota et al. [14] and Dalto et al. [7], this can be justified by the segment analyzed that in this region, mild injuries can be visualized and that generally the bacterial load is lower, due to possible changes in the cell wall during the tissues preparation and by the processing. Due to this fact, many organisms cannot stain with fuchsin making it difficult the bacterium visualization [6]. When the bacterial load is low, the bacilli may not be detected on microscopic examination, which could explain the negative results to the Ziehl-Neelsen staining and positive in other techniques.
The results obtained in this study demonstrated that fecal cultivation has a good specificity when used as a diagnostic method for the identification of animals with no clinical signs of paratuberculosis. This test favors the control of new outbreaks and dissemination of infection in the flock [15], since the diagnosis in the subclinical stage is difficult, due to the tests low sensitivity developed until now. In highly infected flocks, some adult animals can ingest large quantities of mycobacteria along with food and/or water, eliminate them in feces, and these are detected in the cultivation [24].The time of the colonies growth that are compatible with MAP in feces samples varies between 8 and 12 weeks which was similar to that reported by Sivakumar et al. [18] in India, who detected six buffaloes positive in the bacteriological cultivation, after 12 weeks of incubation.
The “real-time” PCR proved to be an important tool in the identification of genetic elements, in addition to considerably increase the tests’ sensitivity for the detection of animals with subclinical infection, and studies conducted by Logar et al. [12] and Timms et al. [21].
In the present study, 100% of the isolates of feces was positive in “real-time” PCR and reported by Yadav et al. [9] and Sivakumar et al. [18] in fecal culture of buffaloes in northern India; however, due to the techniques of protocol adopted, there may have been interference in the amplification and identification of genetic material derived from tissues subjected for analysis in this study. According to Whittington et al. [25], some tissue samples from intestines and mesenteric lymph nodes may be negative when analyzed in a first moment, however, when examined after treatments with different substances, which could minimize the inhibitory effects for the amplification of the DNA chain, resulted positive. In our study, the tissues were not subjected to a second PCR analysis, and perhaps for this reason, the percentage of positive samples in this technique has been relatively low.
The use of molecular tools, bacteriological cultivation associated with macroscopic, and microscopic findings has been effective on confirmatory diagnosis of MAP in buffaloes, according to Espeschit et al. [8], when reporting in his review article about Paratuberculosis in Latin America, several works with this methodology. However, there is an advantage as the association of those tests makes the costly cost of diagnosis to the animal owner.
The subclinical paratuberculosis presents a high prevalence among the buffaloes flocks slaughtered in slaughterhouses in Baixada Maranhense. The association of diagnostic techniques allows greater accuracy in the confirmation of this disease and increases the sensitivity and specificity in the diagnosis of subclinical disease in buffaloes. It was possible to observe lesions in the small intestine, large intestine, and mesenteric lymph nodes, the jejunum, ileum, and ileocecal valve, the most affected regions in buffaloes with absence of clinical signs of paratuberculosis. According to the results of this study, it is evident that paratuberculosis is a disease that causes serious economic losses to the buffaloes flock of Maranhão. Therefore, there is need for deployment of a national control program since there are no concrete actions from official defense service for paratuberculosis, even though it is considered an immediate notification illness from any confirmed case.
Acknowledgments
The authors would like to thank the Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) by encouraging aimed at the IECT Project - 008/17.
Funding information
The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa e ao Desenvolvimento Tecnológico do Maranhão (FAPEMA) for funding this research.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethics approval
This study was approved by the Committee on Ethics and Animal Experimentation (CEEA/CVMP/UEMA), according to process no. 17/2014, approved on 24/11/2015.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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