ABSTRACT: Anamnesis: a six-year-old milking Holstein cow (Bos taurus taurus) was diagnosed with stage IV Johne´s
Disease (JD). Clinical and Laboratory findings: the cow suffered intermittent diarrhea during 6 months
with no response to antibiotic treatment. Consequently, the cow was subjected to euthanasia. Treatment
approach: antemortem milk and peripheral blood samples and postmortem colon, mediastinic, mesenteric
lymph nodes, and spleen samples were processed for macrophages isolation. Total DNA was extracted from
macrophages and used to diagnose IS900 of Mycobacterium avium paratuberculosis (MAP) through real time
PCR. The MAP IS900 segment was successfully amplified from cells of all samples, indicating that these
cells were MAP-infected macrophages. Conclusion: macrophages of cows suffering from JD can be used for
amplification of the MAP IS900 segment.RESUMEN: una vaca Holstein de 6 años de edad (Bos taurus taurus) presentó sintomatología de la fase
IV de la enfermedad de Johne. Hallazgos clínicos y de laboratorio: la vaca presentó diarrea intermitente
durante 6 meses sin respuesta al tratamiento con antibióticos. En consecuencia, la vaca fue sometida a eutanasia. Esquema de tratamiento: muestras de leche y de sangre periférica se tomaron ante-mortem;
muestras de la mucosa del intestino, bazo y linfonodos mediastínico y mesentérico, se tomaron post-mortem,
todas para aislamiento de macrófagos. El ADN total de los macrófagos fue usado para la amplificación del
segmento IS900 de Mycobacterium avium paratuberculosis (MAP) por PCR en tiempo real. Conclusión:
los macrófagos de vacas con la enfermedad de Johne pueden ser usados para la amplificación del segmento
IS900 de MAP

Maldonado Estrada, Juan Guillermo

Ramírez García, René

Revista Colombiana de Ciencias Pecuarias

English

Biblioteca Digital del Sistema de Bibliotecas de la Universidad de Antioquia

219Ramírez-García R et al. Macrophages infected with MAP in a cow with johne´s diseaseRev Colomb Cienc Pecu 2013; 26:219-225Detection of macrophages infected with Mycobacterium aviumsubspecies paratuberculosis in a cow with clinical stage IV of Johne's disease. A case report¤Detección de macrófagos infectados con Mycobacterium avium subspecies paratuberculosis en una vaca en fase clínica IV de la enfermedad de Johne. Reporte de un casoDetecção de macrófagos infectados com Mycobacterium avium paratuberculosis subespécie em uma vaca com uma fase clínica IV da doença de Johne. Um relato de casoRené Ramírez-García, MV, MSc; Juan G Maldonado-Estrada*, MVZ, MS, PhD.Grupo de investigación CENTAURO, Escuela de Medicina Veterinaria, Facultad de Ciencias Agrarias Universidad de Antioquia, Medellín, Colombia(Received: October 22, 2012; accepted: January, 29, 2013)SummaryAnamnesis: a six-year-old milking Holstein cow (Bos taurus taurus) was diagnosed with stage IV Johne´s Disease (JD). Clinical and Laboratory findings: the cow suffered intermittent diarrhea during 6 months with no response to antibiotic treatment. Consequently, the cow was subjected to euthanasia. Treatment approach: antemortem milk and peripheral blood samples and postmortem colon, mediastinic, mesentericlymph nodes, and spleen samples were processed for macrophages isolation. Total DNA was extracted from macrophages and used to diagnose IS900 of Mycobacterium avium paratuberculosis (MAP) through real time PCR. The MAP IS900 segment was successfully amplified from cells of all samples, indicating that these cells were MAP-infected macrophages. Conclusion: macrophages of cows suffering from JD can be used foramplification of the MAP IS900 segment.Key words: intracellular bacteria, real time PCR, resident macrophages, susceptible hostResumenAnamnesis: una vaca Holstein de 6 años de edad (Bos taurus taurus) presentó sintomatología de la fase IV de la enfermedad de Johne. Hallazgos clínicos y de laboratorio: la vaca presentó diarrea intermitentedurante 6 meses sin respuesta al tratamiento con antibióticos. En consecuencia, la vaca fue sometida a ¤	 To cite this article: Ramírez-García R, Maldonado-Estrada JG. Detection of macrophages infected with Mycobacterium avium subspecies paratuberculosis in acow with clinical stage IV of the disease. A case report. Rev Colomb Cienc Pecu 2013; 26:219-225.*	 Corresponding author: Juan G Maldonado-Estrada. Escuela de Medicina Veterinaria, and Sede de Investigación Universitaria (SIU) Calle 62 # 52-59, Lab-233. Universidad de Antioquia. 050011 Medellín, Colombia. E-mail: juanguimal@gmail.comRevista Colombiana de Ciencias PecuariasClinical CaseRamírez-García R et al. Macrophages infected with MAP in a cow with johne´s disease220Rev Colomb Cienc Pecu 2013; 26:219-225eutanasia. Esquema de tratamiento: muestras de leche y de sangre periférica se tomaron ante-mortem;muestras de la mucosa del intestino, bazo y linfonodos mediastínico y mesentérico, se tomaron post-mortem, todas para aislamiento de macrófagos. El ADN total de los macrófagos fue usado para la amplificación del segmento IS900 de Mycobacterium avium paratuberculosis (MAP) por PCR en tiempo real. Conclusión:los macrófagos de vacas con la enfermedad de Johne pueden ser usados para la amplificación del segmento IS900 de MAP.Palabras clave: bacteria intracelular, hospedero susceptible, macrófagos residentes, PCR en tiempo realResumoAnamnese: uma vaca Holstein com 6 anos de idade (Bos taurus taurus) apresentou os sintomas da fase IV da doença de Johne. Achados clínicos e laboratoriais: a vaca teve diarreia intermitente por seis mesessem resposta ao tratamento com antibióticos. Por conseguinte, a vaca foi submetida à eutanásia. Abordagem de tratamento: amostras de leite e de sangue periférico foram retiradas ante-mortem, enquanto as amostrasda mucosa intestinal, baço e linfonodos mesentéricos e mediastinais foram tomadas todas post-mortem para o isolamento de macrófagos. O ADN total de macrófagos foi utilizado para amplificação do segmento IS900 do Mycobacterium avium paratuberculosis (MAP) por PCR em tempo real. Conclusão: macrófagos isoladosa partir de vacas com doença de Johne podem ser utilizados para a amplificação da segmento IS900 do MAP.Palavras chave: bactérias intracelulares, hospedeiro suscetível, macrófagos residentes, PCR em tempo realIntroductionParatuberculosis or Johne’s disease (JD) is causedby the intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP). MAP causes a devastating chronic disease in the affected animal. The prevalence and incidence of JD ranges from 5% to 100% (McNab et al, 1991). Our research group documented the presence of JD in the northern dairy region of Antioquia province (Colombia) based on clinical, pathological (Ramirez et al, 2011), and molecular results (Zapata-Restrepo et al, 2010; Fernandez-Silva et al, 2011b). Macrophages infected with MAP can circulate and re-circulate from Peyer’s patches of infected intestinal mucosato other tissues via blood and lymph compartments (Mutharia et al, 2010; Bimczok and Rothkötter, 2006). Consequently, we wondered if it would be possible to take advantage of this recirculation process for early detection and diagnosis in cows. The main objective of this work was to evaluate the usefulness of circulating and resident macrophages for ampli cation of the IS900 sequence of MAP.Patient examinationAnamnesisA six-year-old lactating Holstein cow (Bos taurus taurus) from the University of Antioquia’sdairy herd was diagnosed with stage IV JD. The farm is located at 2200 m above sea level in a high-humidity region at San Pedro de los Milagros (Antioquia, Colombia). The farm is enzootic for JD as reported by Ramírez et al (2011). The cow suffered from recurrent episodes of profuse diarrhea with progressive weight loss for 6 months. Clinical ndingsThe cow’s body condition score (BCS) was1.5 (scale 1 to 5) and showed severe depression. Cardiac frequency was 80 beats/min. The animal was tachypneic and evidenced diarrhea. It had not eaten for several days (Figure 1). Rectaltemperature was 39.5 °C. Mucosae were pale and dry. Lack of intestinal motility was also evidenced at auscultation of abdominal quadrants. The last 221Ramírez-García R et al. Macrophages infected with MAP in a cow with johne´s diseaseRev Colomb Cienc Pecu 2013; 26:219-225milking record of the cow—taken 1 month before the clinical exam—indicated a yield of 20 kg milk per day. Based on the enzootic status of the farm, anamnesis, and clinical exam, the cow was diagnosed as clinically compatible with stage IV JD. In addition, the cow had tested positive when evaluated for antibodies against MAP by ELISA test. This test was performed as reported by Fernández-Silva et al (2011a) and the speci cresult of this cow was taken from the of cial recordreported to the staff of the farm. The farm and veterinarian staff decided to euthanize the cow. Blood samples were taken ante-mortem in sterile vacutainer tubes containing citrate as anticoagulant. A milk sample was obtained by manual milking in sterile 50 ml Coning centrifuge tubes (Corning, NY,USA). Samples were transported under refrigerationto the laboratory and processed to isolate mononuclear cells as previously reported (Zapata-Restrepo et al, 2010). Figure 1. Ante mortem aspect of a cow affected by Johne’s Disease at clinical stage IV. A, B: Lateral and caudal views from where the clinical stage can be observed. The cow was then euthanized by intrathecal injection of 25 ml lydocaine through the foramen magnum. Signi cant carcass alterations were notobserved at the macroscopic examination during necropsy but a generalized pale condition suggested anemia. Abundant brown fat was observed around the pericardium near the insertion of the aorta adventitia. Adherences were present at the left costal side of the pericardium. Lung atelectasis was present. The heart was cardiomegalic and marked atrophy of the spleen was observed. The liver borders were rounded, bile ducts were thick, and several adult forms of Fasciola hepaticawere observed in the bile ducts. Mediastinal and mesenteric lymph nodes were hypertrophic (Figure2A). Jejunum and ileum were normal (Figure 2B),but the terminal ileum and the colon were thickened and the macroscopic aspect of intestinal mucosa was typical of JD (Figures 2C and 2D).Several mediastinic and mesenteric lymph nodes, a spleen sample (5 x 5 cm), and transversal colon segments were taken during post-mortem examination. Samples were imbibed in Phosphate Buffer Saline (PBS, 1mM, pH = 7.2; Sigma-Aldrich-Aldrich, St. Louis, MO, USA) containing1% penicillin/streptomycin and amphotericin B. Samples were kept on ice and transported to the laboratory in 75 cm2 Falcon culture asks (Falcon,San Jose, CA, USA). After necropsy the carcass wasdeposited into a septic fossa according to the farm’ssanitary protocol, which includes deep underground disposition (1.5 m) followed by abundant lime covering and complete carcass burial. Tissue samples and secondary lymphoid tissues were cut and processed by mincing with dissecting scissors sterilized in PBS with added antibiotics as previously indicated. Samples were processed for macrophage isolation according to the protocol by Ramirez-Garcia et al (personal communication, 2012). Tissues were minced with surgical scissors in petri dishes with added PBS and RPMI-1640 complete medium. The supernatant was recovered and centrifuged in a 1.077 density Ficoll gradient(Sigma, St. Louis, MO, USA) at 800 x g/40 min.Blood and milk samples were processed directly for isolation of mononuclear cells by centrifugation in 1.077 Ficoll gradient at 800 x g/40 min. After gradient Ramírez-García R et al. Macrophages infected with MAP in a cow with johne´s disease222Rev Colomb Cienc Pecu 2013; 26:219-225centrifugation, the buffy-coats were recovered with a sterile plastic Pasteur pipette and washed three times in PBS at 400 x g/10 min. Pellets were recovered from mesenteric lymph nodes, spleen, mediastinal lymph nodes, blood, and milk. Cell pellets were re-suspended in RPMI-1640 complete media (Mediatech Inc., Herndon, VA, USA) and cultured at 37 °C/2hours in sterile polypropylene petri dishes. The non-adherent mononuclear cells were removed after this incubation period while the adherent cells were washed twice with RPMI-1640 complete media and cultured under standard conditions at 37 °C/5% CO2in a humidi ed atmosphere.The cells obtained by this protocol were bovine macrophages according to CD14 expression (PE-conjugated mouse anti-human CD14 moAb [clone61D3]) (Biosurce, San Diego, CA, USA) andphagocytosis of polypropylene spheres coupled to Phycoerythrin (CaliBRITE PE-beads; Becton-Dickinson, CA, USA). Plastic-adherent cells werecultured for at least 48 hours, after which time cells showed the characteristic pattern of macrophages. After 48 hours of macrophages culture cells were processed for total DNA isolation using the DNA Qiagen-mini kit (Qiagen, TX, USA) according tothe manufacturer´s instructions. The DNA purity and concentration was evaluated in a Nanodrop® ND1000 spectrophotometer (Thermo FisherScienti c, Wilmington, DE, USA); samples werestored at -80 °C until processing. Figure 2. Post mortem gross macroscopic findings of the gut in a cow affected by Johne’s Disease at clinical stage IV. (A) Mesenteric lymph node draining in the duodenum; (B) Aspect of mucosa in a segment of the jejunum; (C) and (D) View of mucosa in colon segments exhibiting the characteristic brain-like aspect of JD. 223Ramírez-García R et al. Macrophages infected with MAP in a cow with johne´s diseaseRev Colomb Cienc Pecu 2013; 26:219-225Ampli cation of IS900 MAP fragment by real time PCR was performed using DNA samples obtained from all sources of macrophages as previously indicated, according to the protocol by O’Mahony and Hill (2002), with modi cations in thethermal pro le as follows: hot start at 15 min/95°C(instead of 10 min); 40 cycles (instead of 50) of ampli cation phase at 30 sec/95°C (instead of 15sec), 10 sec/55°C (instead of 30 sec/60°C) and 30 sec/72° (instead of 12 sec). Primers used for IS900ampli cation were: (FW) 5´CGA CGT GTC CTTACACAG C 3´; and reverse (RW) 5G´GTATG GTTTCATGT GGT T 3´. Real time PCR were processedin a Rotor-GeneTM 6000 real time rotary analyzer(Corbett Research, Australia). The PCR curves and orescence normalization were established by theThreshold value (Ct). LinREGPCRTM software wasused for the ef ciency calculation. All samples wereprocessed in triplicate and data were analyzed by the –(ΔΔct) method (Livak and Schmittgen, 2001). TheGAPDH gene was used as control for calculationof ΔCt. The results were analyzed using the Rotor-Gene 6000 series 1.7 software (Corbett Research,Sydney, Australia). The IS900 segment of MAP was ampli ed from all samples of macrophages evaluated(Figure 3).The strongest real time-PCR signal of IS900MAP fragment was observed in macrophages isolated from mesenteric lymph nodes (Figure 3B)and colon mucosa (Figure 3D), whereas the lowestsignal was obtained from mediastinal lymph nodes (Figure 3A), milk (Figure 3C) or peripheral bloodmacrophages (data not shown). The Ct value for the IS900 sequence was 0.003 whereas it was 0.001 for the GAPDH gene. Ct was 9 and 16 for 1/10and 1/100 positive control samples, respectively, whereas Ct of samples from macrophages ranged between 16 and 24.Figure 3. Real time PCR results for amplification of the IS900 segment of MAP. Melting data is shown for DNA of macrophages isolated from mediastinal lymph node (A), mesenteric lymph node (B), milk (C), and colon (D). Note the high melting curve for colon samples. C+, positive control (ATCC Mycobacterium avium paratuberculosis reference Strain # 19698) diluted 1/10 or 1/100. C-, negative control.Ramírez-García R et al. Macrophages infected with MAP in a cow with johne´s disease224Rev Colomb Cienc Pecu 2013; 26:219-225Treatment approachThe cow was subjected to euthanasia due to the advanced clinical stage of the disease. For thisreason the treatment schedule is not reported here.DiscussionTypical ndings of stage IV JD were observedat the clinical exam and gross ndings at necropsy,con rmed by ampli cation of the IS900 fragment in DNA samples isolated of macrophages from all tissues, peripheral blood, and milk. The cow was bred in a dairy farm that was enzootic for JD as demonstrated by clinical, histopathological (Ramirez et al, 2011), serological (Fernandezet al, 2011b), and molecular (Zapata et al, 2010; Fernandez et al, 2011a) evidence. During 6 months the cow had presented intermittent episodes of stage III and IV JD that were treated according to standard procedures with apparent transient recovery for several weeks, followed by episodes of diarrhea. The cow had also a Fasciola hepatica infection that could be related to an immunosuppressive condition (considering its chronicity, evidenced by the gross ndings in thebiliary ducts and liver). The IS900 MAP fragment was successfully ampli ed in DNA obtained from circulatingmacrophages (peripheral blood and milk) and resident macrophages (lymph nodes, colon, and spleen), suggesting that MAP uses the key processes in the physiology of leukocyte traf cking (Bimczokand Rothkötter, 2006) for its dissemination through all tissues in the affected animal (Cheville et al,2001). Colon mucosa and mesenteric lymph nodes displayed the strongest signal of MAP genome ampli cation (Figure 3). This nding probablyrepresents the normal consequence of a high ampli cation rate of MAP in macrophages presentin the lamina propria of the bowel and suggests that circulation and recirculation of macrophages is a mechanism by which MAP disseminates throughout the organism.ConclusionsBovine macrophages isolated from lymphoid tissues, peripheral blood, and milk samples obtained from cows suffering stage IV JD can be used for the ampli cation of the MAP IS900 segment.AcknowledgementsThe authors wish to thank Dr. Carlos Muskus (PECET Group, University of Antioquia), TheColombian Institute for Tropical Medicine (CES University), Giovanny Torres, and Ronald Pelaez(Medellín, Colombia). Gratitude is also extended tothe University of Antioquia (CODI, ConvocatoriaMediana Cuantía 2007) for the nancial support ofthis work.ReferencesBimczok D, Rothkötter HJ. Lymphocyte migration studies. Vet Res 2006; 37:325-338.Cheville NF, Hostetter J, Thomsen BV, Simutis F, VanloubbeeckY, Steadham E. Intracellular traf cking of Mycobacterium avium ss. paratuberculosis in macrophages. Dtsch Tierarztl Wochenschr 2001; 108:236-243.Fernández-Silva JA, Abdulmawjood A, Akineden O, Bülte M.Serological and molecular detection of Mycobacterium aviumsubsp. paratuberculosis in cattle of dairy herds in Colombia. Trop Anim Health Prod 2011a; 43:1501-1507.Fernández-Silva JA, Abdulmawjood A, Bülte M. Diagnosisand molecular characterization of Mycobacterium avium subsp. paratuberculosis from dairy cows in Colombia. Vet Med Int 2011b; 2011:352-561.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402-408.McNab WB, Meek AH, Martin SW, Duncan JR. Associations between dairy production indices and lipoarabinomannan enzyme-immunoassay results for paratuberculosis. Can J Vet Res 1991; 55:356-361.Mutharia LM, Klassen MD, Fairles J, Barbut S, Gill CO.Mycobacterium avium subsp. paratuberculosis in muscle, lymphatic and organ tissues from cows with advanced Johne’sdisease. Int J Food Microbiol 2010; 136:340-344.O’Mahony J, Hill C. A real time PCR assay for thedetection and quantitation of Mycobacterium avium subsp. 225Ramírez-García R et al. Macrophages infected with MAP in a cow with johne´s diseaseRev Colomb Cienc Pecu 2013; 26:219-225paratuberculosis using SYBR Green and the Light Cycler. JMicrobiol Methods 2002; 51:283-293.Ramírez NF, Rodríguez B, Fernandez JA. Diagnóstico clínicoe histopatológico de paratuberculosis bovina en un hato lechero en Colombia. Rev MVZ Córdoba 2011; 16:2742-2753.Zapata-Restrepo M, Arroyave-Henao O, Ramírez-GarcíaR, Piedrahita-Ochoa C, Rodas-González JD, Maldonado-Estrada JG. Identi cation of Mycobacterium avium subspecies paratuberculosis by PCR techniques and establishment of control programs for bovine paratuberculosis in a dairy herds. Rev Colomb Cienc Pecu 2010; 23:17-27.

Detección de macrófagos infectados con Mycobacterium avium subspecies paratuberculosis en una vaca en fase clínica IV de la enfermedad de Johne. Reporte de un caso

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Detección de macrófagos infectados con Mycobacterium avium subspecies paratuberculosis en una vaca en fase clínica IV de la enfermedad de Johne. Reporte de un caso

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