STUDIES ON MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS ISOLATED FROM SOME EGYPTIAN DAIRY FARMS

STUDIES ON MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS ISOLATED FROM SOME EGYPTIAN DAIRY FARMS

ABDEL MOGHNEY, A.F.; NASHWA, M. HELMY and ARAFA, A.A.

Animal Health Research Institute, Dokki, Giza, Egypt, 12618.

Email: abdelrasheedghanem@yahoo.com                                                             Assiut University web-site: www.aun.edu.eg

INTRODUCTION

Paratuberculosis or Johne’s disease is a chronic debilitating disease that affects a wide range of animal hosts, both domestic and wild. Paratuberculosis has been recognized as a major problem in many livestock production systems (Lombard , 2011).  It is a common disease in all countries with a significant dairy industry (Barrington et al., 2003). Paratuberculosis herd prevalence guesstimates in Europe, United States and south America appeared to be 50%, 21.6% and 70% respectively (Wells and Wagner 2000, Ferrerira et al., 2001, Alfaro et al., 2006, Nielsen  and Toft 2009. Paratuberculosis is present in many species and  regions in Egypt (Salem et al., 2005, Abdel Mogney 2008, Abdel Moghney et al., 2012 and Amin et al., 2015); While in neighbouring countries, studies indicated that the disease is established in some areas of Sudan  (Ibrahim et al., 2004,  Aradaib et al., 2005), and Libya, (Mustafa and  Mugadmi 1986, Ali  and Abdel Salam 1997),  in Tunisian cattle (Gallo et al., 1989).

The complement fixation test and enzyme-linked immunosorbant assay identify antibodes against mycobacterium avium subspecies paratuberculosis while the microscopic examination of Ziehl-Neelsen stained faecal smears , faecal culture, and polymerase chain reaction (PCR) assays are methods to demonstrate the presence of mycobacterium avium subspecies paratuberculosis (Kalis et al., 2002). PCR and sequencing the amplified product are useful tools to predict the origin of a paratuberculosis infection, (Sohal et al., 2010).

The purpose of this study is to diagnose “MAP” infection within some Egyptian dairy farms using traditional diagnostic methods like serology and isolation and molecular methods like real time PCR as confirmatory test , in addition   sequencing of PCR products targeting fragment of the IS900 gene as advanced techniques for tracing infectionʹs source.

MATERIALS and METHODS

This study was carried out during 2009 – 2012, samples"n=1118" were collected from three dairy herds(268 from Alexandria, 350 from  Ismailia and 500 from Mansoura) previously tested  for detection of anti Mycobacterium paratuberculosis antibodies in bovine serum by ELISA test with a main goal of using confirmed and advanced methods to diagnose and trace MAP within herds.

Blood and fecal samples (23 of each) were colleccted from ELISA positive (for paratuberculosis) animals representing 3 Egyptian governorates(Alexandria n= 15, Ismailia n=4, and Mansoura n=4)

- Serum antibody testing: ELISA Paratuberculosis antibody verification kit "Institute Pourquier" which approved by OIE (2004) was used procedure and results interpretation according to kit instructions.

- Bacterial culture: Fecal samples from seropositive animals for paratuberculosis were cultured onto  (HEYM), supplemented with Mycobactin J for up to 4 months.

- Ziehl-Neelsen stain for isolated bacteria to demonstrate the acid fast bacilli  using carbol fuchsin, hydrochloric alchol and methylene blue. Culture and Ziehl-Neelsen staining were done according to OIE terrestrial manual (2004).

- Molecular diagnosis: 23 fecal samples and 11 of isolated bacteria were tested using real time PCR, while 5 isolates were tested using conventional PCR with gel electrophoresis and sequencing of PCR products of the IS900 gene.

Extraction of DNA from faecal samples was performed according to instructions of QIAamp DNA Mini Kit. Extraction of DNA from mycobacterium paratuberculosis isolates was performed according to Sevilla et al. (2005).

Extracted DNA from fecal samples and bacteria were amplified with ADIAVET® PARATB REALTIME PCR Kit which uses primers and a TaqMan probe labeled with FAM, specific of M. paratuberculosis (IS900) for Real time PCR. Amplification of the IS900-based PCR was conducted in 25 μL and under the following conditions: 1 initial cycle of denaturation and activation at 50 °C for 2 minutes, another cycle of denaturation at 95 °C for 10 minutes and 45 cycles of denaturation at 95°C for 30 sec., annealing, extension and quantification at 60°C for 1 minute on thermocycler apparatus “Stratagene, USA”.

Conventional PCR was conducted on five bacterial DNA extracts (two from Alexandria, two from Ismaelia and one from Mansoura) using two oligonucleotide primers P90B and P91B (Table 1). These primers constructed to target a 413-bp fragment of the IS900 of MAP (Singh et al., 2010).

Table 1: Oligonucleotide sequences of primers specific for MAP.

Gel electrophoresis and sequencing: PCR products of 5µl were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide with final concentration of 0.5µg/ml at 95 V for 30 min in 1x Tris Borate EDTA (TBE) buffer, against GeneRuler™ 100 bp Plus DNA Ladder (Fermentas). Images of the gels were taken on BioDoc Analyze Digital Systems (Biometra, Germany). Gel containing DNA band of the expected size (413bp) was excised and purified with the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturerʼs instruction.

Five purified PCR products were sequenced directly at Gene analysis unit, NL_QP, Animal Health Research Institute, Egypt using the ABI-PRISM® BigDyeTM Terminators v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the ABI PRISM® 3130 genetic analyzer (Applied Biosystems). The sequences were edited with SeqScape® Software Version 2.5(Applied Biosystems). Bacterial sequences in this study were aligned and compared to international M. avium subspecies sequences in genebank using BLAST software, while, the assembly of the consensus sequences ,alignment trimming and phylogenetic tree were performed with the Lasergene DNASTAR group of programs (DNASTAR Inc., Madison,WI).

The sequences of IS900 gene of the 5 Egyptian isolates were submitted to Genbank under accession numbers: KJ173780 to KJ173784.

RESULTS

Phenotypic characteristics: The grown colonies after 4 months incubation, the inoculated HEYM slants, the isolates revealed acid fast bacilli after staining with Ziehl-Neelsen (Fig 1, 2, 3 and Tables 2, 3). The inoculated slants revealed the isolated strains on “HEYM” with mycobactin J, growth were  observed after 4 weeks as small pin point colonies without pigmentation and at the end of observation period (16 weeks), the colonies kept their appearance with larger size and more in number (Fig.1), in addition  HEYM still green except one sample from mansoura and another sample from Ismaelia, the colonies were creamy, and the media changed to faint green  color (Fig.2). Pin point colonies were grown on HEYM. All isolates revealed acid fast bacilli after staining with Ziehl-Neelsen (Fig.3).

Table 2: Results of ELISA, Fecal culture, and Fecal PCR of 23 cattle from (Alexandria, Ismaelia and Mansoura) herds.

Table 3: Comparison between results of fecal culture and culture PCR.

**CT= Cut threshold (interpretation values: positive → ≤ 40 Negative ›42)

Table 4:  percentage of results  for paratuberculosis diagnostic tests.

p/t = positive/total

Figure (5): Phylogenetic analysis of Mycobacterium. avium subsp. Paratuberculosis of Egyptian strains in comparison to other international strains

Based on these results (Fig.1, 2. 3 and Tables 2, 3),  5 isolates selected were for sequencing of IS900 gene. Three of them shared in their phenotypic characteristics, sample no.4 “Alexandria, which was imported from Germany; sample no.27”Alexandria, from the same farm; sample no.21 “Ismaelia”. The other two samples no.3 ”Ismaelia” and no.8 ”Mansoura “ which had creamy colonies and green yellow colored media, Fig.(2) that supports to be  belonging to mycobacterium paratubeculosis type “S” strain. The selection of the 5 isolates for sequencing aimed to genotyping and strain identification and to predict the origin or the source of infection.

Table 5: Sequences comparison of IS900 gene of Egyptian isolated Mycobacterium avium subsp. Paratuberculosis

PCR results (table 2, 3 and Fig 4) confirmed that extracted DNA from fecal and bacteria were mycobacterium avium subspp. Paratuberculosis.

Table 6: percent identity of IS900 gene of Egyptian isolated Mycobacterium avium subsp. Paratuberculosis with some worldwide strains.

Sequencing data of 413 bp amplified product of IS900 gene:

Samples AHRI-3 (Ismaelia) and AHRI-8 (Mansura) showing 98% similarity resulting from a single nucleotide polymorphism (SNP)  at position 237, with a T instead of an G, in comparison to other sequences from Egypt or worldwide. Samples AHRI-4 (Alexandria, imported from Germany), sample AHRI-21 (Ismaelia) and AHRI-27 (Alexandria) showed 100% identity with each other and high similarity to other strains from Europe (Fig. 5) (strains from UK, Spain, Netherlands, Slovakia, Germany and Italy) without any sequence modifications.

Three isolates, Isamelia (one sample) and Alexandria (two samples) showed 100% identity. While the results indicated that isolates of Ismaelia herd were unidentical (samples AHRI-3, AHRI-21).

DISCUSSION

In the present study, clinical stage of paratuberculosis was the predominant sign, ELISA results showed high titre (SP%= 142.9-295) of immunoglobuline G1 antibodies for MAP (table 2), which coincide with (Stabel 2000, Coussens 2001). The late stage of subclinical phase of mycobacterium avium subspecies paratuberculosis was characterized by production of immunoglobulin G1 antibodies, predominates, this shift in the predominant immune response is often associated with progression to clinical disease. The fecal culture of animals with highest ELISA S/P showed more colonies per culture tube which indicate that cows are heavy fecal shedders with high risk factors on environment and susceptible cows (Fecteau and  Whitlock 2010). While the negative cultured samples 11/23 (Table 3) may be due to that shedding of microorganisms was below the limit of detection at the time of sampling . These results support the need for caution when interpreting negative results from a single fecal culture (Barrington et al., 2003).  

Direct amplification of  fecal MAP DNA by real time PCR (table 2), revealed 91.30% agreement (table 4) with ELISA where there were two samples negative which may be attributed to presence of inhibitory substances in fecal matter or due to intermittently shedding of MAP in feces with differing concentrations (Coussens, 2001 and  David and Göran 2006).

Although, animals of Ismaelia & Mansoura farms (table 2) were clinically infected, fecal samples of 2 cases showed negative results by culture method on HEYM with mycobactin J while by real time PCR showed positive results. So, additional of other  culture media like BACTEC may be useful in future further study. The results agree with Pierre et al. (2010) who mentioned that high-throughput PCR tests have the potential to provide rapid (less than 2 days) detection of M. avium subsp. Paratuberculosis at a cost comparable to or less than that of conventional culture (16-20 weeks).

Sixty percent of Alexandria fecal culture (9/15) were negative. Poor sensitivity may be due to small numbers of  bacilli in subclinical stage of disease or the treatment of samples with   hexadocylepyridenium chloride (HPC) for decontamination before culture. MAP, though culture is a sensitive because live undamaged bacilli are necessary for culture but only qualitatively preserved DNA are required for PCR (Altwegg 1995). Moreover the difference between positive fecal culture (n=12) with acid fast bacilli on staining with ziehl-neelsen (Table 3) and culture PCR (n=10) was unclear since the two cases showed weak positive with Fecal PCR . Overall fecal samples for PCR revealed 91.3% (21/23) positive, otherwise 2 negative samples were in complete agreement with results of culture results.

In the present study the isolated strains were phenotypically (growth on HEYM and mycobactin dependent) and genotypically (by the presence of multiple copies of an insertion element IS900) classified as mycobacterium avium subspecies paratuberculosis (Collins and de Lisle 1986, Thorel   et al., 1990).

The five selected MAP isolates from 3 separate geographical locations which confirmed by amplification of 413 bp of IS900 gene specific for bacteria using conventional PCR. Three isolates, Isamelia (one sample) and Alexandria (two samples) shows 100%  identity  (table 5) which reflects the same origin of animals in the farms from those governorates. However Ismaelia farm has 2 unidentical isolates (samples 3, 21) that reflect importation of animals from different sources or introducing new animals from other farms. These results indicate presence of 2 different strains from Mycobacterium avium subsp. paratuberculosis circulating in those three herds according to the current study (Fig.4 ). Unique (SNPs) were recently reviewed and DNA sequencing for the detection of polymorphism has been considered to be promising for the differentiation of MAP strains (Castellanos    et al., 2009). Source tracking is one of the most common applications for molecular epidemiological methods (Zadoks and Schukken 2006). Strain typing can elucidate from where the infectious agent originated and may help to understand ways and risk factors for transmission, therefore sample AHRI-4 “Alexandria” has a special attention as it was imported from Germany and transferred to the farm after quarantine period. DNA sequencing of Sample AHRI-4 showed  100% identity with European strains (fig.4), which was in complete agreement with Fernandez-Silva et al. (2012). Mycobacterium avium subspecies paratuberculosis isolated from symptomatic dairy cattle in Germany is similar to those previously determined in other parts of Europe. Based on these data, the main possibility for disease occurrence is the transmission of new infected animals either by importation or purchasing from infected source otherwise the mechanical transmission.

RECOMMENDATION

Establishment of strict control measures for mycobacterium avium subspecies paratuberculosis infections by the Egyptian authorities is very important.

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