Benign cystic mesothelioma of the appendix presenting in a woman: a case report

Donal B O'connor
David Beddy
Muyiwa A Aremu
Benign cystic mesothelioma of the appendix presenting in a woman: a case report
C A S E R E P O R T Open Access
Introduction: Benign cystic mesothelioma or peritoneal inclusion cysts are rare benign abdominal tumors usually occurring in females of reproductive age. These cysts present as abdominopelvic pain or masses but are often found on imaging or incidentally at surgery. They are commonly associated with pelvic inflammatory disease, endometriosis, or ovarian cysts. We report what is, to the best of our knowledge, the first case of a benign cystic mesothelioma complicating a presentation of acute appendicitis. Case Presentation: A 19-year-old Irish Caucasian woman presented with abdominal pain. Imaging suggested appendicitis with abscess formation. She was treated with antibiotics and scheduled for interval appendicectomy. At laparoscopy, an unusual cystic mass was found arising from the appendix. Histology revealed benign cystic mesothelioma.

Introduction

Benign cystic mesothelioma (BCM) or peritoneal inclusion cysts are rare abdominal tumors usually occurring in women of reproductive age. These cysts present as abdominal or pelvic pain or masses but are often found on imaging or incidentally at surgery. There have been many cases described associated with pelvic inflammatory disease, endometriosis, or ovarian cysts. We describe the first case of a benign cystic mesothelioma arising from the appendix and complicating a presentation of acute appendicitis.

Case Presentation

A 19-year-old Irish Caucasian woman presented to the hospital with a three-day history of abdominal pain and fever. The pain was gradual in onset and associated with nausea and one episode of vomiting. She had no urinary symptoms, and her last menstrual period had finished the previous day. She had no surgical history, and her medical history was significant only for viral meningitis two years previously. She denied any history of sexually transmitted disease or recent urinary tract infection. She was not taking regular medications and had no allergies. On examination, her vital signs were normal except for mild pyrexia of 37.4°C. Examination of the abdomen revealed a tender mass in the right iliac fossa.

Laboratory investigations included a white cell count of 10,500 cells/mm, hemoglobin of 13.3 g/dl, and platelets of 212,000/mm. Urea and electrolytes were within normal ranges. Urine analysis was negative for leucocytes and urinary βHCG was negative.

A computed tomography (CT) scan of the abdomen and pelvis was requested and showed a 10.4 × 4.5 × 3.8 cm loculated cystic mass in the right pelvis that appeared to contain the tip of the appendix (Figure 1). The patient remained febrile. Clinically, we made a working diagnosis of an appendix mass but considered a tubo-ovarian abscess as a differential. The patient was treated with intravenous antibiotics, and radiological drainage of the abscess was arranged. An ultrasoundguided drain was placed in the largest locule via the right iliac fossa. Unusually, 30 ml of serous fluid but no pus was aspirated. The drain was removed after three days with no further output. Drained fluid was sent for culture, and peripheral blood cultures showed no growth after 72 hours of incubation. After five days, intravenous antibiotics the patient was clinically well. She was discharged and readmitted two weeks later for an interval diagnostic laparoscopy, as we were now suspicious of a non-infective pathology based on the drain output. The patient consented to an appendicectomy if no other pathology was found.

At laparoscopy, a multiloculated, thin-walled and translucent cystic mass was seen in the right iliac fossa ( Figure 2). Adherent to the cystic mass was a spherical, smooth-walled cyst in continuity with the tip of the appendix. The rest of the appendix, caecum, and large and small bowel appeared grossly normal. Both ovaries and the uterus were visualized and found to be normal. The diagnosis was not clear at this point, but our differential included a mucinous cystadenoma or adenocarcinoma (pseudomyxoma peritonei). The lesion appeared very friable, and we were concerned we would rupture it and contaminate the pelvis with the cyst fluid. We made a decision to convert to an open procedure using a Lanz incision to safely perform an appendicectomy and remove the cystic mass. The incision incorporated the previous drain site.

Macroscopy showed a 12-cm appendix with an attached 4 × 4 × 3 cm smooth cyst containing clear fluid. Numerous smaller translucent cysts up to 0.7 cm in diameter were loosely attached to and easily separated from the larger cyst ( Figure 3). We concluded that the radiological drain had entered one of these cysts. Histological analysis revealed cysts lined with flattened mesothelial cells, and the walls were composed of loose connective tissue with occasional chronic inflammatory cells (Figure 4). These findings were consistent with a histological diagnosis of a multiloculated benign cystic mesothelioma. The appendix showed resolving appendicitis with perforation at the tip. The patient was discharged well on the second postoperative day and was also well at six-week and three-month follow-up.

Discussion

Benign cystic mesothelioma or peritoneal inclusion cysts are rare but well-described benign tumors of unknown etiology. First described by Plaut in 1928 (1), they are cystic mesothelial proliferations. They are thought to be due to an inflammatory reaction. They usually occur in the peritoneal cavity in the abdomen or pelvis, and the most common predisposing factors in the clinical history are previous surgery, pelvic inflammatory disease, or endometriosis. These conditions are believed to interfere with peritoneal reabsorption. This would tend to support a hypothesis of BCM being reactive and inflammatory rather than neoplastic (2). These conditions tend to occur in women of reproductive age, but cases have been reported in men (3). The most common sites are the serosal surfaces of the ovary and uterus, but cases outside the abdomen have been described, including the pleural cavity (4). Typical microscopic findings are a single layer of flattened mesothelial cells sometimes described as a hobnail configuration. Squamous metaplasia and papillae may also be seen (3).

The clinical presentation is usually abdominal or pelvic pain, a mass found clinically or radiologically, or an incidental surgical finding (5). BCM is considered to be a benign inflammatory process; however, malignant transformation has been reported (6). Diagnostic modalities include ultrasound and CT, but preoperative diagnosis is often not conclusive and there are no protocols for diagnostic imaging. The main differentials are ovarian cysts, ovarian tumors (benign or malignant), or cystic lymphangioma. When presenting acutely with signs of infection as in the case described here, pelvic inflammatory disease complicated by abscess would be the most common differential as associations with appendicitis are very rare.

Management currently involves surgical resection, but recurrences are well documented. There are no protocols for surgical management, and the literature is based on case reports and small case series. Laparoscopic resections have been described. While laparoscopy is an elegant tool for investigation of masses or pain in women, we believed open surgery to be safer when a malignant process was suspected owing to the possibility of cyst rupture and seeding. Follow-up after surgical resection includes clinical review and ultrasound or CT, but again there are no guidelines.

BCM involving the appendix is very rare. Only four other cases have been reported. Two were in middleaged women presenting with abdominal pain and suspected appendicitis where cysts were found adjacent to but not involving the appendix (7,8). In a third case, a BCM was found incidentally beside an otherwise unremarkable appendix at laparotomy for sigmoid diverticular disease (9). The other case involved a 28-year-old man who presented with appendicitis in which a 25-cm separate cystic mass was found (10). Our patient's case is unique in that the BCM was in direct continuity with the tip of the appendix and presented with both clinical signs and histological evidence of acute appendicitis.

Conclusions

BCM is a rare benign tumor, but surgeons should include it in the differential when investigating abdominal masses or pain in women of reproductive age. This is the first reported case of a BCM arising from the appendix which complicated a presentation of acute appendicitis. Surgery is the authors' recommended treatment, but patients should be advised of the possibility of recurrence.

Figure 1
1Axial CT image showing a loculated cystic mass in the right pelvis which appears to contain the tip of the appendix.

Figure 2
2Laparoscopy. Operative photograph showing thin-walled cystic mass in the right iliac fossa above the appendix.

Figure 3
3Gross specimen of a 4 × 3 × 3 cm thick-walled cyst seen in continuity with the tip of the appendix. Immediately below the 15-cm ruler in the photograph. Membranes of the remainder of the multiloculated cyst after removal from the appendix are seen lying toward the bottom of the photograph. The cyst had ruptured in transit from the operating table to the specimen photography table in the operating room.
© 2010 O'Connor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ConsentThe authors have written informed consent from the patient for the publication in a medical journal of the manuscript and images. A copy of this consent can be made available to the editorial team.Authors' contributions DB O'C drafted and conceived the manuscript, DB assisted in the drafting and editing of the final manuscript, and MA performed critical revisions of the manuscript. DB O'C and MA performed the operation. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Submit your next manuscript to BioMed
Multiple peritoneal cysts and their histogenesis. A Plaut, Arch Pathol. 5Plaut A: Multiple peritoneal cysts and their histogenesis. Arch Pathol 1928, 5:754-756.

Multilocular peritoneal inclusion cysts (so called cystic mesotheliomas). M J Ross, W R Welch, R E Scully, Cancer. 64Ross MJ, Welch WR, Scully RE: Multilocular peritoneal inclusion cysts (so called cystic mesotheliomas). Cancer 1989, 64:1336-1346.

Multicystic mesothelioma: an analysis of pathologic. findings and biologic behaviour in 37 cases. S W Weiss, F A Tavassoli, Am J Surg Pathol. 12Weiss SW, Tavassoli FA: Multicystic mesothelioma: an analysis of pathologic. findings and biologic behaviour in 37 cases. Am J Surg Pathol 1988, 12:737-746.

Pleural multicystic mesothelial proliferation: the socalled multicystic mesothelioma. N J Ball, S J Urbanski, Am J Surg Pathol. 14Ball NJ, Urbanski SJ: Pleural multicystic mesothelial proliferation: the so- called multicystic mesothelioma. Am J Surg Pathol 1990, 14:375-380.

Peritoneal inclusion cysts with mural proliferation: a clinicopathological analysis of six cases. D E Mcfadden, P B Clement, Am J Surg Pathol. 10McFadden DE, Clement PB: Peritoneal inclusion cysts with mural proliferation: a clinicopathological analysis of six cases. Am J Surg Pathol 1986, 10:844-854.

Malignant transformation of "benign" cystic. mesothelioma of the peritoneum. S Gonzalez-Moreno, H Yan, J Surg Oncol. 79Gonzalez-Moreno S, Yan H: Malignant transformation of "benign" cystic. mesothelioma of the peritoneum. J Surg Oncol 2002, 79:243-251.

Benign cystic mesothelioma of the peritoneum: a case report. Y L Suh, W J Choi, J Korean Med Sci. 4Suh YL, Choi WJ: Benign cystic mesothelioma of the peritoneum: a case report. J Korean Med Sci 1989, 4:111-115.

Benign multicystic mesothelioma of the peritoneum (in Italian). P G Betta, F Robutti, G Spinoglio, G Ital Oncol. 9Betta PG, Robutti F, Spinoglio G: Benign multicystic mesothelioma of the peritoneum (in Italian). G Ital Oncol 1989, 9:39-42.

Benign mesothelioma of the appendix: an incidental finding in a case of sigmoid diverticular disease. A Bansal, H D Zakhour, J Clin Pathol. 59Bansal A, Zakhour HD: Benign mesothelioma of the appendix: an incidental finding in a case of sigmoid diverticular disease. J Clin Pathol 2006, 59:108-110.

Benign multicystic mesothelioma of the peritoneum: a case report. A Cavallaro, M Murazio, P Modugno, A Vona, L Revelli, A E Potenza, R Colli, 10.1186/1752-1947-4-394Chir Ital. 54Cavallaro A, Murazio M, Modugno P, Vona A, Revelli L, Potenza AE, Colli R: Benign multicystic mesothelioma of the peritoneum: a case report. Chir Ital 2002, 54:569-572. doi:10.1186/1752-1947-4-394

Benign cystic mesothelioma of the appendix presenting in a woman: a case report. O'connor, Journal of Medical Case Reports. 4394Cite this article asCite this article as: O'Connor et al.: Benign cystic mesothelioma of the appendix presenting in a woman: a case report. Journal of Medical Case Reports 2010 4:394.


BMC Veterinary Research Equine herpesvirus 2 (EHV-2) infection in thoroughbred horses in Argentina

María I Craig
Instituto de Virología
Centro de Investigación en Ciencias Veterinarias y Agronómicas (CICVyA)
INTA
1712) Castelar25Buenos AiresCCArgentina

María E Barrandeguy maríaebarrandeguyfirstname.lastname@example.com
Instituto de Virología
Centro de Investigación en Ciencias Veterinarias y Agronómicas (CICVyA)
INTA
1712) Castelar25Buenos AiresCCArgentina

Fernando M Fernández fernandomfernándezemail@example.com
Instituto de Virología
Centro de Investigación en Ciencias Veterinarias y Agronómicas (CICVyA)
INTA
1712) Castelar25Buenos AiresCCArgentina

BMC Veterinary Research Equine herpesvirus 2 (EHV-2) infection in thoroughbred horses in Argentina
10.1186/1746-6148-1-9BioMed Central Page 1 of 5 (page number not for citation purposes) Research article * Corresponding author
Background: Equine herpesvirus 2 is a gamma-herpesvirus that infects horses worldwide. Although EHV-2 has been implicated in immunosuppression in foals, upper respiratory tract disease, conjunctivitis, general malaise and poor performance, its precise role as a pathogen remains uncertain. The purpose of the present study was to analyse the incidence of EHV-2 in an Argentinean horse population and correlate it with age and clinical status of the animals.Results:A serological study on 153 thoroughbred racing horses confirmed the presence of EHV-2 in the Argentinean equine population. A virus neutralization test showed a total of 79.7 % animals were sero-positive for EHV-2. An increase in antibodies titre with age as well as infection at earlier ages were observed.EHV-2 was isolated from 2 out of 22 nasal swabs from horses showing respiratory symptoms. The virus grew slowly and showed characteristic cytopathic effect after several blind passages on RK13 cells. The identity of the isolates was confirmed by nested PCR and restriction enzyme assay (REA).

Background

Equine herpesvirus 2 (EHV-2) is a slowly growing, cellassociated gamma-herpesvirus. This virus is widespread throughout the equine population and has been isolated from horses of different countries like United Kingdom [1], Japan [2], Australia [3], New Zealand [4], Switzerland [5], Germany [6,7], United States [8,9], Canada [10], Hungary [11] and more recently, from Poland [12]. Although its role as a pathogen is controversial, some authors have reported its association with upper respiratory tract disease, inappetance, lymphadenopathy, immu-nosuppression, keratoconjunctivitis, general malaise and poor performance [8,[13][14][15]7,16].

Equine Influenza Virus and Equine Herpesvirus 4 (EHV-4) are the most common viral agents related to respiratory disease in Argentina (Dr. Barrandeguy, personal communication). These and other respiratory viruses as Adenovirus, Equine herpesvirus 1 (EHV-1), Arteritis Virus and Rhinovirus, are usually checked in the diagnostic routine of our laboratory but no records about EHV-2 isolation or frequency of sero-positive samples were available.

The purpose of the present study was to analyse the presence of EHV-2 in an Argentinean horse population and to correlate its incidence with age and presence of respiratory symptoms.

Results and discussion

Sero-prevalence of EHV-2 was calculated on one hundred and fifty-three (153) thoroughbred racing horses by a neutralization test (NT). Cross reactivity with EHV-5 was not checked in this study. The percentage of sero-prevalence to EHV-2 was 79.7% (122/153). Sera samples were grouped according to the clinical status into animals with symptoms (fever, cough, nasal discharge) or clinically healthy. Again, each of these groups was divided according to the animal age in older or younger than 1 year old. The arithmetical mean of the antibody titres was calculated for each of these four groups. Mean antibody titres between older and younger than 1 year old animals both, with and without clinical symptoms were statistically compared. Mean values for the older horses (1.28 > 1.02) were significantly (p < 0.05) higher than for the younger ones. These results agree with the observations of other authors [17] about the increase in antibodies titre with age. Within the older than 1-year group, the mean titre in the group with clinical symptoms was higher (1.34>1.22) though not significant. However, mean titre values were significantly (p < 0.05) higher (1.19>0.85) in the group with clinical symptoms within the younger than 1 year group (Table 1). This difference might be related to the early exposure to this agent.

Our results suggest the virus is circulating with a high prevalence on the analysed equine population, in accordance with other sero-prevalence data [17,18], and confirm previous reports [19] about the acquisition of EHV-2 at earlier ages.

Taking into account the relatively high percentage of seroprevalence and the association of EHV-2 with respiratory disease [16], the isolation of this viral agent from horses showing different respiratory symptomathology was carries out. Twenty-two (22) nasal swabs from horses, aged between 6 months and 2 years old, displaying respiratory symptoms were checked for the respiratory viruses commonly analysed in the laboratory routine, and none of them resulted positive for these viruses. Only two (2) nasal swab samples, named E1 and E2, showed CPE after the third blind passage. Some authors reported the presence of vacuoles in RK-13 cells infected with EHV-2 isolates [20] while others described various CPE forms depending on the virus isolate and cell type [21]. In our isolate, CPE was characterized by rounded cells, syncytia and vacuolized cell aggregates with suited partial cell membrane fusion. The late development of CPE referred to a slow growing viral agent.

In order to identify these two isolates, two simple techniques like EM and IIFA were first used. The particle morphology observed by EM showed a size corresponding to a typical herpesvirus (data not shown). The IIFA showed fluorescence signals on cells infected with EHV-2 reference strain LK, E1 and E2. Mock-infected cells were negative (data not shown). As the polyclonal antiserum used for the staining could have cross reaction with EHV-5, another member of the gamma-herpesvirinae subfamily that shows growth characteristics similar to EHV-2 [22], a virus specific nested PCR and a restriction enzyme analysis (REA) were performed.

Nested PCR was carried out on E1 and E2 samples as described in Methods. After the second round, both isolations showed a band of around 600 bp (Figure 1), as the one displayed by the positive control.

EHV-5 identity of the isolations was unequivocally excluded by REA. As shown in Figure 2, the patterns after digestion with Hind III and Eco RI of the isolates and a control EHV-5 were completely different.

As it was specified by others authors [23,24], EHV-2 was described as a virus with a high genomic variation. In our experiments, the restriction patterns of the isolates were different to those of T400 and LK4. Moreover, E1 and E2 resulted identical to each other and very similar to strain H40. Both, E1 and E2 belonged to the same stud farm and were probably epidemiologically related.

Conclusion

Although EHV-2 has been reported in equine populations worldwide, no reports about its prevalence in Argentina were available. In this study, serological data and virus isolation provided a clear evidence for the presence of this agent on the studied horse population. In horses younger than 1 year old but not in the older ones, serological data correlated with the presence of clinical symptoms. Though viral agents associated with respiratory disease were discarded, no bacterial analysis was done in the studied samples. Further studies are necessary to correlate EHV-2 with the respiratory disease. The EHV-2 identity of the isolated viruses was finally confirmed by the use of a nested PCR and a restriction enzyme assay. This is the first report of EHV-2 isolation in Argentina and adds new data to the EHV-2 distribution map.

Methods

Cells

RK-13 (ATTCC CCL 37) cell monolayers were grown with Eagle 's-minimum essential medium (MEM-E) supplemented with 100 UI/ml penicillin, 60 µg/ml streptomycin, 50 µg/ml gentamycin and 10% FCS. Subsequently, the cells were maintained with MEM-E supplemented with same concentrations of antibiotics and 2% FCS.

Virus

Samples

Sera Serum samples were obtained from one hundred and fifty three (153) thoroughbred racing horses, aged between 6 months and 2 years old.

Nasal swabs

Nasal swabs from twenty-two horses showing respiratory symptoms like nasal discharge, cough and fever, were used in our research. Samples were collected with a cotton swab and transferred to a tube containing 3 ml of E-MEM supplemented with 2% FCS and antibiotics. Clarification was performed by centrifugation at 3000 rpm (Hermle Z 513 K) for 15 min. Each sample was aliquoted and stored at -70°C until use.

Nested PCR on nasal swab samples from foals with respira-tory symptoms Figure 1 Nested PCR on nasal swab samples from foals with respiratory symptoms. Nested PCR amplification products from two equines (E1 and E2) showing respiratory symptoms were analysed by gel electrophoresis. DNA was extracted from original nasal swabs; DNA from the suspension was used as template in the first round. Genomic DNA from EHV-2 strain LK was used as a PCR positive control (PCR+) and H 2 0 as negative control (PCR -). DNA marker φX-174 Hae and λ-RF/Hind III is seen in lane M.

Restriction enzyme assay profile of EHV-2 isolates and refer-ence strains Figure 2 Restriction enzyme assay profile of EHV-2 isolates and reference strains. EHV-2 isolates (E1 and E2), EHV-2 strains H40, T400, and LK4, and EHV-5 strain P48, were amplified in RK-13 cell monolayer. Viral DNA was purified by Genomic-tip 20/G column, digested with Hind III and Eco RI and analysed by gel electrophoresis. DNA marker φX-174 Hae III and λ-RF/Hind III is seen in lane M.

Neutralization test

This assay was performed on 96 wells RK-13 cell monolayer, where serial four-fold dilutions of complement-inactivated serum were incubated with 100 ID 50 of LK strain for 4 hs at 37°C as described elsewhere [25]. After 7 incubation days, antibody titres were calculated by Reed and Muench formula [26]. Sera that protected 100% of the cell monolayer at the lower dilution assayed were considered positive. An EHV-2 positive horse reference serum used as positive control, was kindly supplied by Dr. Borchers.

Indirect immunofluorescence assay

Trypsinised virus infected cells were seeded on IFA micro slides and fixed with ice-cold acetone for 30 min. A polyclonal EHV-2 specific rabbit serum (341 EDV 8301, NVSL Ames Iowa) was added and incubated in a moist chamber al 37°C for 1 h. Fluorescein conjugated rabbit antiserum (KPL Kikergaard and Perry laboratories) was used to reveal the reaction. EHV-2 LK strain infected RK-13 and non-infected cells were used as positive and negative assay controls, respectively.

Electron microscopy

Cell cultures showing 70% CPE were trypsinised and sonicated for 30 seconds in a Sonicator XL (Hert System) and spinned at 3000 × g during 10 minutes to eliminate cell debris. The supernatant was centrifuged at 14000 × g for 1 h 30 minutes and the resulting pellet was resuspended with a saline buffer solution and stained with 2% phosphotungstate acid.

Virus isolation

All nasal swab samples were previously checked for the presence of equine viruses associated with respiratory disease. Equine adenovirus, rhinovirus, arteritis virus, equine herpesvirus 1 and 4, were checked by isolation in cell tissue culture while equine influenza virus was assayed using embryonated eggs. Once it was confirmed that all the samples were negative for the mentioned viruses, the presence of EHV-2 was analysed. Briefly, 0.5 ml of each sample was inoculated onto confluent RK-13 monolayers in 25 cm 2 flask (NUNC™, Denmark). After an adsorption period at 37°C for 1 h, the inoculum was removed and cells were maintained at 37°C at an atmosphere of 5% CO 2 for 7 days with periodic microscopical examination. After this time period, a blind passage was made if CPE was absent. Cell monolayers were thawed and freezed three times and an aliquot of the suspension was passed on to fresh cell culture. The samples were considered negative after the forth-blind passage.

Nested PCR

The nested PCR used in this study amplifies a 620 bp sequence located upstream of the ORF coding for vIL-10 [27].

Original nasal swab samples were centrifuged for 10 min at 15,000 × g as described elsewhere [27]. Briefly, resulting pellets were diluted in 50 µl digestion buffer (50 mM Tris pH 8.5; 1 mM EDTA; 0.5% Tween 20) plus 1.5 µl Proteinase K (10 mg/ml, Sigma) and incubated at 56°C for 3 h, boiled for 10 min at 95°C to inactivate the proteinase K and the supernatant used for nPCR. PCR conditions were optimised using purified viral DNA from the EHV-2 LK strain obtained as described below. The PCR mix (50 µl) contained 0.2 mM dNTPs, 0.4 µM of each primer [27], 1.5 U Taq polymerase (QIAGEN), 1 × reaction buffer (QIA-GEN) and 10 µl of DNA suspension obtained from the original nasal swab sample. The optimal annealing temperature for each of the two primer pairs was 60°C. Cycling was carried out using a Biometra trio-thermblock (Biometra ® ) with 35 amplification cycles consisting of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. For the second round of this nested PCR, 3 µl from the first PCR reaction were amplified with the inner pair of primers using the same cycle as before. Purified viral DNA from the EHV-2 LK strain was used as a PCR positive control.

Restriction enzyme assay

Viral DNA was obtained from RK-13 cells infected with EHV-2 isolates and also with EHV-2 reference strains H40, LK4, T400 and EHV-5 (P48). Viruses were pelleted from cell culture supernatant by centrifugation through a 30% sucrose cushion at 24,000 × g for 1 h 30 min. Pellets were resuspended with DNA buffer (10 mM MgCl2, 2 mM CaCl2, 200 mM Tris pH 7.4) and treated with RNAse (10 mg/ml) and DNAse I (2 mg/360 µl) at 37°C for 1 h. Subsequently, the pellets were treated with EDTA (0.2 M), 1% sodium dodecyl sulphate and proteinase K (10 mg/ml) (Boehringer, Manheim). Finally, DNA was purified by Genomic-tip 20/G column (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Aliquots of 200 ng of the obtained purified viral DNA were digested overnight at 37°C with Eco RI and Hind III. The fragments were separated by electrophoresis through 1% agarose gel and stained with ethidium bromide.

Statistical analysis

Two (2) ways ANOVA [28] followed by post hoc comparisons (Tukey HSD for unequal N, Spiotvoll/Stoline test) [29] were used.

List of abbreviations

Table 1 :
1Distribution of serum samples according to age and clinical statusWith symptoms
Antibodies
Clinically healthy
Antibodies

Animals
Sero-negative
Sero-positive
(Mean titre) a
Sero-negative
Sero-positive
(Mean titre) a

Older than 1 year old
5
3 2
1 . 3 4
3
4 0
1 . 2 2
Younger than 1 year old
3
26
1.19
18
26
0.85*
Total (n)
66
87

n: number of samples
a: arithmetical media of Ab titre (Reed and Muench)
*: Significantly different (p < 0.05)

EHV-2, equine herpesvirus 2; EHV-5, equine herpesvirus 5; E-MEM, Eagle's minimum escential medium; EM, electron microscopy; IIFA indirect immunofluorescence assay; Ab, antibody; PCR, polymerase chain reaction; REA, restriction enzyme assay; NT, neutralization test; CPE, cytopathic effect; vIL-10, viral interleukin 10; ORF, open reading frame.
(page number not for citation purposes)
Publish with Bio Med Central and every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours -you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentral
AcknowledgementsThis work was supported by the Technology Link Agreement INTA-Haras and a CONICET fellowship.The authors thank Dr. Kerstin Borchers for useful comments and critical review of this manuscript. Technical assistance of O. Zabal, D. Compaired and T. Leiskau is gratefully acknowledged. We thank Dr. A. Bolondi for his assistance in electron microscopy.Authors' contributionsM.I. Craig carried out the experimental work and drafting of the manuscript. M.E. Barrandeguy carried out the data collection procedure and conceived the study. F.M. Fernández coordinated the project. All authors read and approved the final manuscript.
Equine herpesvirus. G Plummer, A P Waterson, 19Plummer G, Waterson AP: Equine herpesvirus. Virol 1963, 19:412-416.

Cytopathogenic equine orphan (CEO) virus in horse kidney cell culture I. Isolation and properties. Y Kono, K Kobayashi, Natl Inst An Health Q Japan. 4Kono Y, Kobayashi K: Cytopathogenic equine orphan (CEO) virus in horse kidney cell culture I. Isolation and properties. Natl Inst An Health Q Japan 1964, 4:10-20.

Equine herpesvirus 1. Isolation and characterization of equine rhinopneumonitis virus and other equine herpesvirus from horses. M J Studdert, A J Turner, J E Peterson, Aust Vet J. 46Studdert MJ, Turner AJ, Peterson JE: Equine herpesvirus 1. Isola- tion and characterization of equine rhinopneumonitis virus and other equine herpesvirus from horses. Aust Vet J 1970, 46:83-89.

Isolation of equine herpesvirus from horses with respiratory disease. G W Horner, R Hunter, J D O'flaherty, L G Dickinson, N Z Vet J. 24Horner GW, Hunter R, O'Flaherty JD, Dickinson LG: Isolation of equine herpesvirus from horses with respiratory disease. N Z Vet J 1976, 24:171-176.

Characterization of a new herpes-like virus isolated from foal kidney. A Karpas, Ann Inst Pasteur. 110Karpas A: Characterization of a new herpes-like virus isolated from foal kidney. Ann Inst Pasteur, Paris 1966, 110:688-696.

K Petzoldt, Die Virusausscheidg beim Stutenabort (Rhinopneumonitis). Arch Exp Vetarmed. 21Petzoldt K: Die Virusausscheidg beim Stutenabort (Rhinop- neumonitis). Arch Exp Vetarmed 1967, 21:115-119.

The association of EHV-2 infection with keratitis and research on the occurrence of equine coital exanthema (EHV-3) of horses in Germany. P Thein, Equine infectious disease IV Edited by: Bryans JT. Gerber H. Princeton New JerseyVeterinary PublicationsThein P: The association of EHV-2 infection with keratitis and research on the occurrence of equine coital exanthema (EHV-3) of horses in Germany. In Equine infectious disease IV Edited by: Bryans JT, Gerber H. Princeton New Jersey: Veterinary Publications; 1978:33-41.

Isolation of herpesvirus from equine leucocytes: comparison with equine rhinopneumonitis virus. L J Kemeny, J E Pearson, Can J Comp Med. 34Kemeny LJ, Pearson JE: Isolation of herpesvirus from equine leu- cocytes: comparison with equine rhinopneumonitis virus. Can J Comp Med 1970, 34:59-65.

Recovery of adenoviruses and slow herpesviruses from horses having respiratory tract infection. A W Roberts, D L Whitenack, G R Carter, Am J Ve t Res. 35Roberts AW, Whitenack DL, Carter GR: Recovery of adenovi- ruses and slow herpesviruses from horses having respiratory tract infection. Am J Ve t Res 1974, 35:1169-1172.

Infectious causes of equine respiratory disease on Ontario Standardbred racetracks. J Sherman, J Thorsen, J A Barnum, W R Mitchell, D G Ingram, J Clin Microbiol. 5Sherman J, Thorsen J, Barnum JA, Mitchell WR, Ingram DG: Infec- tious causes of equine respiratory disease on Ontario Stand- ardbred racetracks. J Clin Microbiol 1977, 5:285-289.

Isolation of equine herpesvirus type 2 from foals showing respiratory symptoms. V Pàlfi, S Belák, T Molnár, Zentralbl Veterinarmed B. 25Pàlfi V, Belák S, Molnár T: Isolation of equine herpesvirus type 2 from foals showing respiratory symptoms. Zentralbl Veteri- narmed B 1978, 25:165-167.

Equine herpes virus 2 infection in horse populations in Poland. A Rusczcyk, A Cywinska, M W Banbura, Acta Virol. 483Rusczcyk A, Cywinska A, Banbura MW: Equine herpes virus 2 infection in horse populations in Poland. Acta Virol 2004, 48(3):189-192.

Equine herpesviruses: antigenic relationships and deoxyribonucleic acid densities. G Plummer, C R Goodheart, M J Studdert, 8Infect ImmuPlummer G, Goodheart CR, Studdert MJ: Equine herpesviruses: antigenic relationships and deoxyribonucleic acid densities. Infect Immu 1973, 8:621-627.

Comparative aspects of equine herpesviruses. M J Studdert, Cornell Vet. 64Studdert MJ: Comparative aspects of equine herpesviruses. Cornell Vet 1974, 64:94-122.

Evidence of respiratory tract infection induced by equine herpesvirus type 2 in the horse. J R Blakeslee, R G Olsen, Mc Allister, E S Fassbender, J Dennis, R , Can J Microbiol. 21Blakeslee JR, Olsen RG, Mc Allister ES, Fassbender J, Dennis R: Evi- dence of respiratory tract infection induced by equine her- pesvirus type 2 in the horse. Can J Microbiol 1975, 21:1940-1946.

Passive immunization of foals to prevent respiratory disease caused by equine herpesvirus type 2. S Belák, V Pàlfi, S Tuboly, L Bartha, Zentralbl Veterinarmed B. 27Belák S, Pàlfi V, Tuboly S, Bartha L: Passive immunization of foals to prevent respiratory disease caused by equine herpesvirus type 2. Zentralbl Veterinarmed B 1980, 27:826-830.

Studies on equine herpesviruses 3. The incidence in Queensland of three different equine herpesvirus infections. T J Bagust, R R Pascoe, T J Harden, Aust Vet J. 48Bagust TJ, Pascoe RR, Harden TJ: Studies on equine herpesvi- ruses 3. The incidence in Queensland of three different equine herpesvirus infections. Aust Vet J 1972, 48:47-53.

Prevalence of antibodies to herpesvirus types 1 and 2, arteritis and infectious anemia viral antigens in equine serum. T C Mc Guire, T B Crawford, J B Henson, Am J Vet Res. 35Mc Guire TC, Crawford TB, Henson JB: Prevalence of antibodies to herpesvirus types 1 and 2, arteritis and infectious anemia viral antigens in equine serum. Am J Vet Res 1974, 35:181-185.

Studies on equine herpesvirus 5. Isolation and characterization of slowly cytopathic equine herpesviruses in Queensland. T J Harden, T J Bagust, R R Pascoe, P B Spradbow, Austr Vet J. 50Harden TJ, Bagust TJ, Pascoe RR, Spradbow PB: Studies on equine herpesvirus 5. Isolation and characterization of slowly cyto- pathic equine herpesviruses in Queensland. Austr Vet J 1974, 50:483-488.

The prevalence of equid herpes virus 2 infections. The Vet Rec. P L Roeder, G R Scott, 96Roeder PL, Scott GR: The prevalence of equid herpes virus 2 infections. The Vet Rec 1975, 96:404-405.

Equine herpesvirus 2 (Equine Cytomegalovirus). G F Browning, M J Studdert, Vet Bull. 58Browning GF, Studdert MJ: Equine herpesvirus 2 (Equine Cytomegalovirus). Vet Bull 1998, 58:775-790.

Equine herpesviruses 2 and 5: comparisons with other members of subfamily gamma-herpesvirinae. C T Agius, M J Studdert, Adv Virus Res. 44Agius CT, Studdert MJ: Equine herpesviruses 2 and 5: compari- sons with other members of subfamily gamma-herpesviri- nae. Adv Virus Res 1994, 44:357-379.

Physical mapping of a genome of equine herpesvirus type 2 (equine cytomegalovirus). G F Browning, M J Studdert, Arch Virol. 104Browning GF, Studdert MJ: Physical mapping of a genome of equine herpesvirus type 2 (equine cytomegalovirus). Arch Virol 1989, 104:77-86.

Isolations of equine herpesvirus type 2 (equine gammaherpesvirus 2) from foals with keratoconjunctivitis. P N Collinson, J L O'reilly, N Ficorilli, M J Studdert, J Am Vet Med Assoc. 205Collinson PN, O'Reilly JL, Ficorilli N, Studdert MJ: Isolations of equine herpesvirus type 2 (equine gammaherpesvirus 2) from foals with keratoconjunctivitis. J Am Vet Med Assoc 1994, 205:329-331.

The use of neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2). A Nordenghran, B Kingeborn, A Lindholm, M Merza, J Vet Diagn Invest. 13Nordenghran A, Kingeborn B, Lindholm A, Merza M: The use of neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2). J Vet Diagn Invest 2001, 13:389-393.

A simple method of estimating fifty per cent end points. L Reed, H Muench, Am J Hyg. 27493Reed L, Muench H: A simple method of estimating fifty per cent end points. Am J Hyg 1938, 27:493.

Distribution and relevance of equine herpesvirus type 2 (EHV-2) infections. K Borchers, U Wolfinger, M Goltz, H Broll, H Ludwig, Arch Virol. 142Borchers K, Wolfinger U, Goltz M, Broll H, Ludwig H: Distribution and relevance of equine herpesvirus type 2 (EHV-2) infec- tions. Arch Virol 1997, 142:917-928.

. R R Sokal, Fj: Rohlf, Biometry, 1981Freeman, New York2nd editionSokal RR, Rohlf FJ: Biometry. 2nd edition. Freeman, New York; 1981:1-859.

B J Winer, Statistical Principles in Experimental Design. New YorkMac Graw Hill9072nd editionWiner BJ: Statistical Principles in Experimental Design. 2nd edition. Mac Graw Hill, New York; 1971:907.


Zn(II) AND Cu(II) UPTAKE FROM ACID SOLUTIONS WITH MICROCAPSULES CONTAINING A NON-SPECIFIC ALKYL PHOSPHONIC EXTRACTANT

Fernando Valenzuela
Facultad de Ciencias Químicas y Farmacéuticas
Laboratorio de Operaciones Unitarias
Universidad de Chile
Vicuña Mackenna 20P.O.Box 233SantiagoChile

Mehrdad Yazdani-Pedram
Facultad de Ciencias Químicas y Farmacéuticas
Laboratorio de Química de Polímeros
Universidad de Chile
Olivos 1007P.O.Box 233SantiagoChile

Claudio Araneda
Facultad de Ciencias Químicas y Farmacéuticas
Laboratorio de Operaciones Unitarias
Universidad de Chile
Vicuña Mackenna 20P.O.Box 233SantiagoChile

Carlos Basualto
Facultad de Ciencias Químicas y Farmacéuticas
Laboratorio de Operaciones Unitarias
Universidad de Chile
Vicuña Mackenna 20P.O.Box 233SantiagoChile

Eiji Kamio
Department of Chemical Engineering and Material Science
Doshisha University
610-0321Kyotonabe, KyotoJapan

Kazuo Kondo
Department of Chemical Engineering and Material Science
Doshisha University
610-0321Kyotonabe, KyotoJapan

Zn(II) AND Cu(II) UPTAKE FROM ACID SOLUTIONS WITH MICROCAPSULES CONTAINING A NON-SPECIFIC ALKYL PHOSPHONIC EXTRACTANT
CopperZincExtractantHydrometallurgyMicrocapsules
It is studied the uptake of Zn(II) and Cu(II) from acid aqueous solutions using microcapsules containing 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester, a nonspecific extractant, as an alternative method for metals separation in place of current techniques as ion-exchange solid resins or solvent extraction. The microcapsules employed in this study were prepared by an in situ radical polimerization method. The amount of extractant held within each microcapsule was determined by potentiometric titration. Many experimental tests were carried out to measure the hydrometallurgical capacity of the microcapsules to remove these metallic ions from the aqueous solutions.

INTRODUCTION

Among the most serious difficulties of mining and metallurgy activities, it is possible to indicate a) the generation in its processes of a great variety of aqueous waste solutions and b) the natural formation of acid mine drainages due to geochemical oxidation and microbial causes [1].These resulting solutions are usually high in acidity and dissolved metals, becoming an environmental problem that is very difficult to clean up. However, these solutions can also constitute a potential source of many valuable and scarce metals. The technical challenge is how to remove them from this type of solutions by means of simple and economical manner. In this respect, we have come studying during many years different alternatives for metal recovery and uptake from dilute mine waters, including current solvent extraction in mixer-settler reactors [2], the use of both supported liquid membranes [3][4][5] and surfactant liquid membranes [6,7]. This short communication reports in advance on the uptake of zinc and copper ions from acid solutions using a novel method based on the use of microcapsules containing non-specific commercial extractant. This technique would present advantages in many cases with respect to other conventional methods [8,9].

Experimental procedure and results

As extractant was employed the commercial compound PC-88A (2ethylhexylphosphonic acid mono-2-ethylhexyl ester) supplied by Daihachi Chemical Industry Co. which was used without further purification. Styrene and divinylbenzene were used as monomer to prepare the microcapsules. Grade chemical, toluene and benzoyl peroxide were utilized as a diluent and a polymerization initiator, respectively. All other chemicals, including sodium silicate and minerals acids were of reagent grade and used as received. The metals-containing aqueous solutions were prepared by dissolving appropriate amount of each metal-sulfate salts in distilled water whose pH was adjusted with sulfuric acid and measured in a pH meter (Corning 320).

The synthesis of microcapsules were carried out in a batch-type reactor having a 500 cm 3 capacity. A mixture of distilled water and sodium silicate was added as the continuous phase being stirred by magnetics bars driven by synchronous motor. When the temperature of this aqueous solution reached a value of 70ºC, it was added the organic solution composed of the monomers, the polymerization initiator and the organophosphorus acid extractant, mixture that behaves as the dispersed phase. Both phases (continuous and dispersed) were intensely agitated for almost 3 h in an inert nitrogen atmosphere, enough to attain the experimental conditions for the polymerization reaction. Once obtained, the polymerized microcapsules were repeatedly washed with distilled water and then dried at room temperature. Moreover, the following potentiometric titration method was accomplished to measure the amount of the organophosphorous extractant retained within each microcapsule: 1.0 g of the microcapsules was mixed with 50 cm 3 of methanol during a week, being after separated from the organic liquid by filtration. The alcoholic solution was divided into portions 10 cm 3 each, which were titrated with a 0.052 mol/L NaOH solution. From a hydrometallurgical point of view, it is necessary to test the capacity of microcapsules to remove metallic ions from acid aqueous solutions. Then, the next step was to carry out some metal uptake equilibrium experiments. They were made in a batchwise by contacting in a Polyscience orbital-shaker apparatus 0.2 g of microcapsules containing the extractant and 20 cm 3 of the aqueous solution containing zinc and copper ions. The sorption runs were carried out during 24 h enough to allow equilibrium to be reached. Once filtrated the metal concentration in the resulting aqueous solution was measured by atomic absorption spectrophotometry in a Perkin Elmer 3110 apparatus.

In Table 1 are listed the different experimental conditions employed to prepare the microcapsules, the mass of microcapsule obtained and the results of the metallurgical tests for zinc and copper removal from an acid aqueous solution. In Table 1 St, DVB and MC denote styrene, divinylbenzene and microcapsule respectively. It is observed that the production of microcapsules was enhanced as the proportion of divinylbenzene monomer was increased. However, as the amount of extractant used in the synthesis was the same in all cases, the content of the phosphonic compound held in the microcapsules resulted to be lower as the microcapsules yield increased since the weak acid-type extractant was dispersed in a higher mass of capsules. Furthermore, it was observed a quite good uptake of Zn(II) and a regular extraction of Cu(II), what can be explained considering that the zinc extraction runs were carried out at a pH value for feed solution where this extractant present a maximum activity [7]. However, the copper uptake runs were accomplished using an initial pH for feed solution around 3.8 looking for improve this metal extraction with PC-88A under a acidity condition where is not efficiently extracted [2].

In Figure 1 are shown the potentiometric titration curves for many microcapsules synthesized varying the proportion between both monomers, styrene and divinylbenzene. It can be appreciated that the higher the proportion of divinylbenzene employed to prepare the microcapsules the lower the acid extractant held in them, confirming the dispersion effect of the extractant when a bigger amount of product was obtained. Therefore, in order to determine the actual capacity of microcapsules to extract metals, it is necessary to determine the dependence between the amount of metal adsorbed in each gram of microcapsule and the content of extractant held in them. This can be analyzed by observing the both most right-handed columns in Table  1 which show the results observed for Zn(II) and Cu(II) ions sorptions expressed in that manner. It is appreciated that the zinc uptake from the acid solution was very high no matter the content of extractant in the microcapsules, meanwhile the copper sorption was increased as the content of the phosphonic compound in the capsule was increased. It is clear that the extractant maintains its high metal-loading capacity in the microcapsules in an improved operation system with respect to the conventional liquidliquid-extraction method today widely industrially used.

It was not measured any extractant leak from microcapsules under the pH condition employed to run the experiments, probably due to the hydrophobicity of the phosphonic extractant what would disable its loss from the microcapsules. This point is being studied in detail in these days for a broad acidity range. The scope of this study also considers to measure the maximum metal adsorption capacities in the microcapsules using a Langmuir or Freundlich-type isotherm model and relate these results with the characteristics of the surface and porosity of microcapsules. However, the most important point is to study the analysis of the possible metal sorption mechanism in the microcapsule based on a mass transfer with chemical reactionprocess, what it will be reported shortly in a full communication.

Until this point of the study we can indicate that the extractant microcapsulization procedure is easy in a high efficiency process carried out using a simple reactor. From the viewpoint of an industrial application of this technique to remove metallic ions, the target must be pointed out to the uptake of heavy metal from mine and industrial waste dilute solutions. Probably by using a reactor based on a continuous column packed with microcapsules containing the appropriate selective or collective extractant.

Fig 1 .-
1Microcapsules preparation: potentiometric titration curves
ACKNOWLEDGMENTSThe authors express their thanks to Daihachi Chem. Co. for providing PC-88A extractant. The authors also thank the Japan-Chile Visit Program (JSPS-CONICYT Nº RC 20437001) by the financial assistance.
. P A Weber, W A Stewart, W M Skinner, C G Weisener, J E Thomas, R S Smart, Applied Geochemistry. 191953P.A. Weber, W.A. Stewart, W.M. Skinner, C.G. Weisener, J.E. Thomas, R.S. Smart, Applied Geochemistry 19, 1953 (2004).

. F Valenzuela, J Andrade, J Sapag, C Tapia, C Basualto, Minerals Engineering. 8893F. Valenzuela, J. Andrade, J. Sapag, C. Tapia, C. Basualto, Minerals Engineering, 8, 893 (1995).

. J Marchese, F Valenzuela, C Basualto, A Acosta, Hydrometallurgy. 72309J. Marchese, F. Valenzuela, C. Basualto, A. Acosta, Hydrometallurgy, 72, 309 (2004).

. F Valenzuela, M A Vega, M F Yañez, C Basualto, J Membrane Sci. 204385F. Valenzuela, M.A. Vega, M.F. Yañez, C. Basualto, J Membrane Sci. 204, 385 (2002).

. F Valenzuela, C Salinas, C Basualto, J Sapag-Hagar, C Tapia, J , Chilean Chemical Society4879F. Valenzuela, C. Salinas, C. Basualto, J. Sapag-Hagar, C. Tapia, J.Chilean Chemical Society, 48, 79 (2003).

. F Valenzuela, C Fonseca, C Basualto, O Correa, C Tapia, J Sapag, Minerals Engineering. 1833F.Valenzuela, C. Fonseca, C. Basualto, O. Correa, C. Tapia, J. Sapag, Minerals Engineering 18, 33 (2005).

. F Valenzuela, J Auspont, C Basualto, C Tapia, J Sapag, Chem. Eng. Research and Design. 83247F. Valenzuela, J. Auspont, C. Basualto, C. Tapia, J. Sapag, Chem. Eng. Research and Design, 83, 247 (2005).

. E Kamio, M Matsumoto, K Kondo, J. Chem. Eng. Japan. 35178E. Kamio, M. Matsumoto, K. Kondo, J. Chem. Eng. Japan, 35, 178 (2002).

. E Kamio, M Matsumoto, F Valenzuela, K Kondo, Ind. Eng. Chem. Res. 442266E. Kamio, M. Matsumoto, F. Valenzuela, K. Kondo, Ind. Eng. Chem. Res., 44, 2266 (2005).

* ) To whom correspondence should be addressed E-mail: fvalenzu@uchile. cl* ) To whom correspondence should be addressed E-mail: firstname.lastname@example.com