Investigation on the status of Johne's disease based on agar gel immunodiffusion, ziehl-neelsen staining and nested PCR approach in two cattle farm

) Investigation on the status of Johne's


Introduction
herd as free of infection [3].Serological assays, including agar gel immunodiffusion (AGID) assay, Johne's disease (JD) or paratuberculosis is chronic, enzyme-linked immunosorbent assay (ELISA) and infectious and granulomatous enteritis of ruminants.It complement fixation test have prescribed to detect is caused by Mycobacterium avium subspecies antibodies against Map in animal sera [5].Depending paratuberculosis.It is considered as a threat to the on the antigens used, these assays can yield falsedairy sector, beef/meat industry and livestock trade [1, positive results and may not consistently detect 2] in many countries, including India [3].In spite of infected animals in the early stages of disease [6].The exhaustive research on diagnosis, control and serological tests currently used in Australia by different prevention, for more than 100 years, it remains as a diagnostic laboratories in goats comprise the AGID or challenge to the veterinary profession.The primary gel test and the absorbed ELISA [7].AGID assay has a source of infection in calves is milk, contaminated with higher specificity (>90%) in animals with clinical feces of diseased animals.Clinical signs of JD are slow signs, but its sensitivity is very low (~30%) in early progressive wasting condition, diarrhea, which is stage of infection [8].intermittent at first, becoming progressively more The application of molecular method, for the severe and consistent [4].
diagnosis of JD, is under constant development and Various serological methods have been developed modification.A number of genes and sequences unique for the detection of antibody produced against it.
to Map have been identified over the years.The Diagnostic tests, measuring antibody response, lack insertion element IS900 has been routinely used to sensitivity, particularly in early phase of infection and detect the presence of Map in clinical samples.their use have been restricted to the diagnostic However, sequences related to IS900 like IS902 (Wood confirmation of suspected clinical cases or certifying a pigeon mycobacterium), IS901 (Maa), IS1626 (Maa and M. intracellulare) were reported [9, 10,11] and hence reduces its specificity.Therefore, a positive IS900 PCR should be confirmed by subsequent nested Investigation on the status of Johne's disease based on agar gel immunodiffusion, ziehl-neelsen staining and nested PCR approach in two cattle farm PCR or by a PCR assay targeting another gene.Another (Protein estimation kit, GeneiTM).Antigen was diluted sequence named, f57 has been identified and don't have with PBS up to protein content to 1mg/ml, and kept atany homology with any known sequences [12,13].It is 20ºC in different aliquots.recommended to use f57 as complementary with IS900 Raising hyperimmune sera: Hyperimmune sera were in nested PCR format to eliminate false-positive cases raised in two healthy adult rabbits (approved by [14]. Institute Animal Ethics Committee, No. F.1-53/2004-Based on above fact, present study was designed th J.D (Res), Dated, 10 Jan. 2012) against Map as per the to screen cattle from two distinct farms with entirely methods described by Castelnuovo et al. [15] with different organizational set-up.AGID, Ziehl-Neelsen suitable modifications.Briefly, antigen consisting of (ZN) staining and nested PCR approach targeting an emulsion of 100 mg of whole cells, 5 mg of IS900 and f57 sequences, were used to screen the sonicated antigen and 2 ml of sonicated sediment was serum and fecal samples.Standard antigens for AGID mixed with an equal amount of Freund's incomplete were prepared from 8 week old growth culture of Map adjuvant (Difco).Each of two rabbits was inoculated strain (ATCC 19698) and standard positive control with 0.5 ml of above emulsion subcutaneously at serum was raised in two rabbits against sonicated weekly intervals for 6 weeks.Antibody titer was antigens of Map.th monitored by AGID assay after 5 injection.Rabbits

Materials and Methods
were bled one week after the last injection, and serum was stored at -20°C.Animal population and sample collection: Cattle from two distinct farms were selected for present study.
Agar gel immunodiffusion assay: One-percent agarose A. Unorganized cattle farm: locally, it was called gel was prepared in PBS (pH 7.4) containing sodium Gaushala or unproductive cattle rehabilitation center.It azide (0.02%, w/v).Gel was cast into sterile plastic was situated in the village Barsana, Mathura (Uttar petriplates and allowed to solidify at 4°C for 1 h in the Pradesh).In India aged, unproductive, chronically humid chamber.Wells of 3 mm diameter were punched infected animals were not slaughtered but sent to this out in a hexagonal pattern with six peripheral wells for type of farms.These animals were maintained by nonsera and one centre well for the antigen at equi-distance profit, non-government organization until death.A of 5 mm between them.The central well was filled with total of 160 sick cattle were selected.These cattle were sonicated antigen with optimum concentration (1 having history of chronic diarrhea and ill health.Blood mg/ml).The test sera was charged in duplicate in six and fecal samples were collected from those animals.
peripheral wells and incubated at 4°C overnight in the B. Organized cattle farm: this farm was maintained by humid chamber along with positive control.Gels were GB Pant Agriculture University, Pantnagar (Uttarakhand), examined after 24 and 48 h and in suspected cases after for research and educational purposes.Randomly, 140 72 h.White precipitation line between antigen and sera cattle were selected.Sera was separated from wells was taken as positive where as an absence of a individual blood samples and kept in cold chain.
precipitation line was recorded as negative.

Similarly individual fecal sample, collected directly
Processing of fecal samples: Fecal samples were decofrom the rectum, was kept inside a plastic bag and ntaminated to remove contaminant microorganism as stored in cold chain.Details of serum and fecal samples the procedure mentioned OIE terrestrial manual [1].collected in respect of age and sex are mentioned in Hexadecylpyridium chloride (HPC) at final concentable 1, 2 and 3. tration of 0.75% (W/V) was used for decontamination Bacterial strain and preparation of antigens: Standard of fecal samples.Briefly, 1 g feces and 20 ml of sterile culture of Mycobacterium avium subspecies distilled water was vortex in 50 ml tube for half an hour paratuberculosis (ATCC 19698) was procured from to make a homogenous mixture.The larger particles Biological Product Division, Indian Veterinary Research were allowed to settle for 30 min at room temperature.

Institute, India. It was maintained in Lowenstein
The uppermost 5 ml of faces suspension was Jenson (LJ) medium containing mycobactin J.For transferred to a 50 ml tube containing 20 ml of 0.9% large-scale production of antigen; the culture was HPC and tubes were inverted several times to assured grown as a surface pellicle on Watson and Reid uniform distribution and allowed to stand undisturbed synthetic broth containing mycobactin J and incubated for 18 h at room temperature.Undisturbed sediments for eight weeks at 37°C.Bacterial growth was killed by were used for ZN staining.heating at 72°C for 2 h and separated by filtration.The cells were washed thrice with PBS and centrifuged to Ziehl-Neelsen staining: Smears were prepared from get cell mass.It was re-suspended in PBS containing 100 µl sediment of decontaminated fecal samples and 0.2mM phenyl methyl sulfonyl fluoride and sonicated air dried.It was heat fixed by passing slide 4-5 times at 16 µ amplitude for 45 min with intermittent breaks.
over flame and stained with strong solution of Carbol Sonicated preparation was centrifuged at 12000x g for fuchsin with the help of heat for five minutes without 1 h at 4°C.The supernatant was filtered through 0.22 boiling.Thereafter, smear was decolorized with 3% µm membrane filter.The protein content of the solution acid alcohol and subsequently counter stained with 5% was estimated by the bicinchoninic acid method malachite green.Slides were screened in an oil immersion field of a light microscope.Results were denaturation (94°C) for 4 min; followed by 40 cycles interpreted as acid-fast positive if minimum four field (25 cycles for 2nd run) of denaturation (94°C), in oil immersion had at least five bacilli per field, as annealing (68°C) and extension (72°C), each for 45 morphology seen in positive slide prepared in parallel sec; and at last, final extension (72°C) for 10 min was with samples with Map strain (ATCC 19698), given.otherwise it was considered as acid-fast negative.
PCR products were analyzed by electrophoresis in 1X Tris-acetate EDTA (TAE) for 2 h at 50 V.PCR

Extraction of genomic DNA from fecal samples:
products along with DNA marker were loaded in 2% Approximately, 200 mg fecal samples was used to (w/v) agarose gel, made in 1X TAE containing 0.5 isolate genomic DNA by using QIAamp Stool DNA kit µg/ml (w/v) ethidium bromide.Separation of DNA (Qiagen®) according to manufacturer's instruction.
was visualized by ultraviolet at 260 nm.Extracted DNA were stored at -20°C for further use.

Results
Nested polymerase chain reaction: Oligonucleotides used were taken from published literature [14].
Agar gel immunodiffusion assay: Samples from two Amplification of both sequences was based on the cattle farms were screened for JD using cytoplasmic nested PCR approach; PCR product of first run was antigens, obtained after sonication of standard Map nd strain (ATCC 19698) along with sera raised in rabbit used as the template for the 2 amplification.Briefly, against sonicated antigens of Map as positive control.for 1st run; primers IS900 F (5'-GGGTTGATC TGG Sera samples of cattle, from organized farm, did not ACAATGACGGTTA-3') and IS900 R (5'-AGC GCG show any visible precipitating band in AGID assay.GCACGGCTCTTGTT-3') were used for amplification However, cattle from unorganized cattle farm (38.75%) of IS900, whereas, primers, F57 F (5'-CCTGTC TAA were markedly infected with this disease.Particularly, TTCGATCACGGACTAGA-3') and F57 R (5'-TCA in female animals, 40.00% of the total screened GCTATTGGTGTACCGAATGT-3') for amplification nd samples found to be infected with this disease (Table-1, of f57.In 2 amplification, IS900 FN (5'-GGAG GT Figure -1).Most of the positive cases in female animals GGTTGTGGCACAACCTGT-3') and IS900 RN (5'were from the age group > 6 yrs (48 out of 113), CGATCAGCCACCAGATCGGAA-3') were used to followed by 3 to < 6 yrs (10 out of 22) and < 3 yrs (4 out anneal the amplified product of IS900 from first run. of 20), respectively.Infection level in male animals Similarly, F57 F and F57 RN (5'-TGGTGT ACC from organized farm were unable to trace because, GAATGTTGTTGTCAC-3') were used to anneal the number of sera samples included in the study was too amplified product of f57 amplification of first run.
st nd less to detect even a single case of JD.PCR reaction mixtures for 1 and 2 amplification of IS900 and f57 were made in total volume of 25 µl.
Ziehl-Neelsen staining: Similar to AGID assay, feces Final concentrations of different constituents in from male animals from organized and unorganized reaction mixture were made respectively as, tris-Cl (pH cattle farms were failed to demonstrate, even a single 9); 10 mM, KCl; 50 mM, MgCl2; 1.6 mM, dNTP mix; animal shedding acid fast bacilli (AFB).As mentioned 800 µM total; primers; 0.8 µM each; Red Taq in previous section, lesser numbers of sera samples polymerase; 0.5 U, triton-X100; 0.01% and DNA included in the present study reduced the chances of templates; 10-20 ng.For positive control, DNA detection of animals shedding AFB.However, in isolated from standard Map strain (ATCC 19698) were female animals, 26.45% and 3.57% fecal samples used whereas, nuclease-free water in negative control.
respectively, from unorganized cattle farm and Amplification of both sequences were performed in organized dairy farm, were shedding AFB (  3a), 452bp (2 run, IS900; Figure 3b), 432bp (1 nd studies were not statistically viable.Standard statistical run, f57; Figure 3c) and 424bp (2 run, f57; Figure 3d) procedure was not followed for sampling and herd taken as positive for Map, provided that, no amplicons selection.Even though, samples collected from the in negative controls.Overall, 14.37% and 0.71% animals which had clinical sign suggesting JD. animals respectively, in unorganized cattle farm and Therefore, in terms of proportion, diagnosed cases organized dairy farm were diagnosed JD positive secured higher prevalence than the endemic level.(Table-3).Although, 14.14% and 0.71% females, Till today, countrywide survey of Map infection respectively in unorganized cattle farm and organized has not been undertaken so far, but investigations by dairy farm were harbored Map bacilli.In unorganized different workers [18,19,20] indicated, it is endemic in cattle farm, maximum number of female animals in the domestic livestock.In the present study, 38.75% age group > 6 yrs (15 out of 113) were positive, animals in unorganized cattle farm were diagnosed JD followed by 3 to < 6 yrs (4 out of 22) and < 3 yrs (3 out positive by AGID.In same groups, Nested PCR of 20) respectively.Whereas, in case of organized dairy confirmed 14.37% positive animals, whereas 25.62% farm, only one female of age group of > 6 yrs was animals shed AFB.Kaur et al. [21] identified even positive for JD.Similarly, only one male animal of less higher number of animals positive for JD in similar than 3 yrs was diagnosed as JD positive from unorganized kind of investigation.She detected 71% animals cattle farm.Complex organic matters, toxic components like by using indigenous ELISA, diagnosed 25.8% animals phenol in feces make DNA isolation difficult.Some of infected.In domestic animals, different workers have them have been identified as polymerase inhibitors estimated 20-30% animals infected with JD [22, 23, 24, [38]  but AGID also detected clinical or advance stage of complex interaction of bacilli with immune system disease.Noticeable humoral responses are generated in delay the shedding of bacilli in feces [28].Therefore, animal at later stage of infection [31].So it may be diagnostic used to detect bacilli in feces and humoral predicted that the level of infection both in organized responses in blood gives more positive results in older and unorganized cattle farm may higher as diagnosed animals or in clinical stage of disease.Cell-mediate by PCR or AGID.immune response predominate [29, 30,31] in the initial Conclusions stage of infection.If animal fails to resist the multiplication of bacilli, it switches its immune It was evidenced that an organized farm, with response toward humoral [32], which are detectable by better hygiene and management practices, showed all commonly implied serological tests.Vazquez et al.
lesser occurrence of JD in cattle in comparison to [33] make out an observation; serologically positive unorganized farm.In unorganized farm, a large number case, infected early in life will produce a noticeable of cattle were sero-positive with AGID assay.humoral response in around 3-4 yrs of age.All these However, all the sero-positive cattle did not shad AFB accounts for high level of infection diagnosed in older when examined by ZN stain.Again, the cattle smears animals (In present case, it was > 6 yrs age).
which were positive for AFB did not always contain Among the tests used, AGID performed well in DNA of Map organism.This indicated that, all serounorganized cattle farm.However, in organized dairy positive cattle might not be the efficient shedder of farm, same test didn't recognize even a single positive Map and mare detection of AFB in fecal smears did not animal.Although, 3.57% animals shed AFB and 0.71% always indicate the presence of Map organism.Cattle are Map positive (nested PCR) in organized dairy farm.
infected with JD were mostly in the age group of six Mere detection of AFB in feces will not warrant Map; it years and above.may be other common inhabitants of intestine.

Authors' contributions
Standard managemental husbandry practices followed in organized dairy farm limit JD to undetectable level.
AM and PD planned and designed the study.AM, NK It has been pointed out by different workers, important and AKN collected the samples from farms.AM and management practices influence the JD occurrence in a PD maintained and culture the Map as surface pellicle 14.Vansnick, E., de Rijk, P., Vercammen, F., Geysen, D., on broth, AM and KK produced the sonicated antigens
used in later steps.Shedding of Map bacilli in 25].In contrast, some authors detected comparatively feces are erroneous except, the clinical and advance lower prevalence in different animal farms [26, 27].stage of disease [39].Therefore, PCR amplification of Maximum numbers of animals in the age group > 6 yrs shed AFB, Map and produced detectable humoral DNA isolated has given lower efficacy.Not only PCR immune response.Long incubation period and
Number of JD positive case from unorganized cattle farm and organized dairy farm with respect to age and sex.AGID assay was used to screen sera samples from both farms.Figure in parentheses indicate percentage, P = No. of positive samples, T = Samples tested.AGID assay, a portion of petri plate showing white precipitation line between cytoplasmic antigens of Map and antibodies from cattle sera.

Table - 3
. Number of JD positive case from unorganized cattle farm and organized dairy farm with respect to age and sex.Nested PCR approach, targeted to both IS900 and f57 sequences, was used to screen the fecal samples.Figure in parentheses indicate percentage, P = No. of positive samples, T = Samples tested.