Noise-induced hearing loss (NIHL) is a major concern in the non-manufacturing industries. This study aimed to investigate the occupational noise exposure and the NIHL among Chinese restaurant workers and entertainment employees working in the service industry in Hong Kong.This cross-sectional survey involved a total of 1,670 participants. Among them, 937 were randomly selected from the workers of Chinese restaurants and 733 were selected from workers in three entertainment sectors: radio and television stations; cultural performance halls or auditoria of the Leisure and Cultural Services Department (LCSD); and karaoke bars. Noise exposure levels were measured in the sampled restaurants and entertainment sectors. Each participant received an audiometric screening test. Those who were found to have abnormalities were required to take another diagnostic test in the health center. The “Klockhoff digit” method was used to classify NIHL in the present study.The main source of noise inside restaurants was the stoves. The mean hearing thresholds showed a typical dip at 3 to 6 KHz and a substantial proportion (23.7%) of the workers fulfilled the criteria for presumptive NIHL. For entertainment sectors, employees in radio and television stations generally had higher exposure levels than those in the halls or auditoria of the LCSD and karaoke bars. The mean hearing thresholds showed a typical dip at 6 KHz and a substantial proportion of the employees fulfilled the criteria for presumptive NIHL . Being male, older, and having longer service and daily alcohol consumption were associated with noise-induced hearing impairment both in restaurant workers and entertainment employees.Excessive noise exposure is common in the Chinese restaurant and entertainment industries and a substantial proportion of restaurant workers and entertainment employees suffer from NIHL. Comprehensive hearing conservation programs should be introduced to the service industry in Hong Kong. Occupational NIHL has been regarded as a global public health problem, and systematic research on occupational NIHL was performed as early as the late 19th century However, few studies have been carried out to ascertain the size of the exposed group and the magnitude of the problem in the service sector. Employees who work in Chinese restaurants in Hong Kong are an obvious target group, because noise-generating pressurized gas stoves are frequently used in Chinese restaurants for Chinese-style cooking. At present, little data on noise in Chinese restaurants are available. A systematic and scientific evaluation of noise levels in Chinese restaurants and the collection of baseline prevalence data on the hearing levels of the workers are essential for developing occupational health control measures.In addition to restaurant workers, many studies have shown that there is a risk of hearing loss among musicians and teenagers who listen to loud music The Hong Kong Standard Industrial Classifications code for Chinese restaurants is 641 Service workers: workers in the service areas, including managers, captains, waiters/waitresses, pantry helpers, dim sum sales staff, barbecue counter staff, fish tank caretakers, dish-up, clerks, and accountantsCooks: including chief cooks, second cooks, dim sum cooks, and barbecue cooksDishwashersKitchen workers: workers inside the kitchens other than cooks.Based on an estimated 30% prevalence of noise-induced hearing impairment among the exposed workers Noise assessment included measuring the noise levels in selected working environments, and measuring the cumulative personal noise exposures for some participants with job titles including cook and dishwasher, which are a typical position having a high risk of noise exposure in Chinese restaurants.Environmental noise levels were measured by using precision sound level meters  in various locations where the workers were working. The measurements were then integrated to give an A-weighted sound pressure level (LAeq) for the time period of the measurements.d values). The computing capability of the dosimeter allowed a complete real-time record of the sound levels which the workers were exposed to during the measurement period to be statistically analyzed. The personal exposure data were then transferred from the dosimeters to a personal notebook computer  and they were then processed by using the software supplied with the dosimeters.Regarding cumulative personal noise exposure, “B & K” noise dosimeters  were used to measure the participants' personal daily noise exposure levels  radio and television stations; (2) cultural performance halls or auditoria of the LCSD; and (3) karaoke bars. One radio and television station, 11 halls or auditoria of the LCSD and two karaoke bars were selected, and their employees were invited to participate in this study. The response rate in each sector varied. For the radio and television stations, a total of 428 employees were invited and 262 of them agreed to participate (response rate: 61.2%); for the 11 halls or auditoria of the LCSD, 613 employees were invited and 438 of them agreed to participate (response rate: 71.5%); for the two karaoke bars, 100 employees were invited and 33 of them agreed to participate (response rate: 33.0%). A total of 733 participants were therefore included in the analysis. The study protocol was reviewed and approved by the Survey and Behavioural Research Ethics Committee of Chinese University of Hong Kong. Signed informed consent was obtained from the participant beforehand.Noise assessment referred to the measurements of noise levels for (1) typical radio program presenters who wore headsets during work, and (2) typical participants who did not wear headsets during work.A special method was used to assess the personal noise exposures for radio program presenters who wore headsets while carrying out their duties. The sound pressure levels were measured by using a precision sound level meter  via an artificial ear (CUOI) with the earphones  plugged into the monitor interphase that was parallel in output and subjected to adjustment with the headset worn by the presenters. In other words, we measured the sound levels in the artificial ear, which had the same levels of sound pressure as in the ears of the person who wore the headset.For participants who did not wear headsets, cumulative personal noise exposures were assessed and methods. The methods and equipments were the same as those used for restaurant cooks and dishwashers, described above.The audiometric tests consisted of a screening test in the field and a diagnostic test in the hearing test center. The screening test was performed for all participants . Those who were found to have abnormalities during the screening test were then requested to attend the diagnostic audiometric test.A standard questionnaire was used for all participants during the audiometric tests to collect information such as personal particulars, current and past occupational noise exposures, noise exposures other than from their occupation and the use of ear protectors. An otoscopic examination of the external ear canals and eardrums was then performed and any abnormalities were noted.Audiometric screening examinations were carried out in the field. For all participants, pure tone air conduction hearing thresholds at different frequencies were measured manually by the abridged ascending method following a standard procedure in accordance with BS 6655: 1986. The hearing thresholds were measured for both ears at the following octave frequencies: 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8,000 Hz. A portable audiometer  with earphones  was used to perform the audiometric screening test. The earphones were equipped with audiocups  to enable testing in quiet rooms at workplaces. The rooms used for the audiometric testing were either VIP rooms or the managers' rooms. The background sound pressure levels were monitored by using a precision sound level meter  with a type NX-05 octave band filter unit. Sound levels (LAeq) in these testing rooms varied considerably, ranging from 46–74 dBA. For low frequencies (∼500 Hz), the noise levels of most locations ranged from 44–75 dBA, while at higher frequencies (∼8 KHz) the levels ranged from 16–63 dBA.Participants who were found to have abnormalities (having a hearing threshold >25 dBA at any frequency in either ear) during the screening test were then requested to attend an audiometric diagnostic test. The diagnostic test included the pure tone air conduction hearing thresholds at the following octave frequencies 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8,000 Hz for both ears. Bone conduction audiometry was performed by using the method described by the British Society of Audiology The audiometric diagnostic test was carried out inside a sound-proof audiometric booth  by using a Madsen audiometer  at the Department of Community and Family Medicine, Lek Yuen Health Centre in Shatin, or at the Hong Kong Federation of Trade Unions Clinic  in Mongkok.Objective calibrations of all audiometers were carried out once every six months to adjust the sound pressure output in the left and right earphones by using an artificial ear. Biological calibration checks of audiometer functions were carried out before the start of the tests on each day of fieldwork.et al.The classification of hearing defects used in this study was suggested by Klockhoff The term ‘Presumptive Noise Induced Hearing Loss’ (P-NIHL) was used when high tone hearing impairment  was detected in any subject in this study. There are three levels of P-NIHL, namely, slight, moderate, and severe high-tone loss. According to the above coding classification, P-NIHL could be made up of combinations with “12”, “21”, “13”, “31”, “14”, “41”, “22”, “23”, “32”, “24”, “42”, “33”, “34”, “43”, and “44” when the differences in the average hearing levels at 3,000, 4,000, and 6,000 Hz between the better and poorer ear did not exceed 30 dBA To indicate the prevalence of moderate and severe high tone loss in our study population, we used the term “High Tone Loss” (HTL) in this report, which combined P-NIHL coding “33”, “34”, “43”, and “44”.The double entry of data into computers was done independently by two research assistants and compared subsequently. Any mismatch was corrected after checking the corresponding questionnaire and the test record. Descriptive statistics were used for calculating the means of the continuous variables and the prevalence of the categorical variables. The logistic model was used for calculating the odds ratios. We considered p values of <0.05 to be statistically significant, and 95% confidence intervals were also presented. All data analyses were performed by using the Statistical Package for the Social Science .The mean age (SD) of the 733 entertainment employees was 38.6 (9.4) years, which was younger than that of the 937 restaurant workers (their mean age was 41.9 (11.7) years). There were more females working in the Chinese restaurants. The mean duration (SD) of working in the entertainment sector was 7.6 (7.1) years, while the service duration for restaurant workers was 10.3 (9.5) years. The general characteristics of the entertainment employees and restaurant workers are presented in The main source of noise inside restaurants was from the stoves. Other sources included dishwashing machines and ventilation systems, such as fume extractors. The measurements of environmental noise in different areas are shown in A total of 22 personal dosimetric measurements were taken from 20 cooks and two dishwashers . Higher A total of 34 personal dosimetric measurements of typical entertainment employees who did not wear headsets were taken from various job groups. Noise levels ranged from 74.3 to 101.9 dBA. Filming with headphone (TV) and Carpenter (TV) had the highest exposure to mean noise levels. The results of dosimetric measurements for the entertainment employees are also presented in The noise levels of the radio program presenters who wore headsets are presented in The mean hearing thresholds by frequency for restaurant workers stratified by gender are shown in Panel A in The prevalence of P-NIHL and HTL, were 23.7% (95% CI: 21.0–26.4) and 11.3% (95% CI: 9.3–13.3) among restaurant workers, respectively. For entertainment employees, the corresponding figures were 38.6% (95% CI: 35.1–42.1%) and 7.0% (95% CI: 5.2–8.8%). Our survey examined noise exposures and hearing impairment in Chinese restaurant workers and entertainment employees in Hong Kong. Our results show that noise exposure is common and excessive in Chinese restaurants and in the entertainment industry in Hong Kong. From our representative sample of Chinese restaurant workers we estimated that about 47% of workers in Chinese restaurants are exposed to noise levels of above 85 dB(A). The mean hearing thresholds show a typical dip at 3 to 6 KHz and a substantial proportion of restaurant workers fulfilled the criteria for P-NIHL and HTL (23.7% suffer from P-NIHK and 11.3% suffer from HTL) Regarding the noise exposures and prevalence of hearing loss in the manufacturing industry in Hong Kong, previous studies have shown that 38% of workers were exposed to noise levels of 90 dB(A) or more, and that 19% suffered from HTL, which was higher than the HTL prevalence (7.0%) in our entertainment employees and the HTL prevalence (11.3%) in Chinese restaurant workers, but there are no data on P-NIHL in this study et al. in San Francisco showed that the noise levels generally ranged from 60–80 dB(A), with the peak at 87 dB(A) There is a little information about NIHL for restaurants in other countries. The study conducted by Lebo et al. reported that environmental noises and headphone styles were associated with increased preferred listening levels among the adults using MP3 players Hitherto, there has been limited information on hearing loss on employees in the entertainment industries worldwide. Some overseas studies for employees working at music venues showed that the prevalence generally ranged from 29–50%. However, the sample sizes of these studies were small with there being 14–82 participants in each study Aging has been well documented as an important risk factor for hearing loss. However, presbycusis usually shows a pattern on an audiogram different from that of NIHL. Generally presbycusis shows the greatest threshold shifts at higher frequencies, leading to a downward sloping curve without a “notch” et al. reported an association between hearing levels and smoking Our results show that being male, and older were significantly associated with an increased risk of hearing impairment in both restaurant workers and entertainment employees. These findings are in conformity with previous studies There are several disadvantages in our study which limited us for thoroughly assessing the relationship of NIHL with noise exposures: (1) due to the shortage of resources, information on many potential risk factors could not be collected, and we explored only the associations between NIHL and some demographic factors as well as smoking and drinking. (2) the cross-sectional nature did not allow us to assess any temporal relationship of NIHL with noise level as well as the potential risk factors. Prospective cohort studies are required to thoroughly assess the relationship between sound exposures and NIHL, as well as the effects of other risk factors/confounders on the relationship; and (3) the current measurements of noise exposure levels might not accurately reflect past exposures. Other limitations in our survey include the low response rate for karaoke bars, the non-representative sampling in the entertainment industry, and small sample size of each sub-sector in the entertainment industry. However, because of the wide scope and various job natures in the entertainment industry, a representative sample is difficult to obtain unless a census can be carried out for the whole entertainment industry.Our results have important public health implications. NIHL is permanent and irreversible, but actually it is completely preventable


Ultrasound signal intensity was significantly different among these 3 stages and was well correlated with Mankin scores (R = −0.80) and FTIR parameters related to collagen and proteoglycan content in superficial zone. In conclusion, ultrasound can detect microscopic cartilage deterioration when such changes do not exist macroscopically, reflecting superficial histological and biochemical changes.Recognizing subtle cartilage changes in the preclinical stage of osteoarthritis (OA) is essential for early diagnosis. To this end, the ability of the ultrasound signal intensity to detect macroscopically undetectable cartilage change was investigated. In this study, cartilage of rabbit OA model and human OA samples was examined by macroscopic evaluation, ultrasound signal intensity, histology with Mankin scores, and Fourier transform infrared imaging (FTIRI) analysis. Rabbit OA was induced by anterior cruciate ligament transection and evaluated at 1, 2, 4 and 12 weeks. Twenty human samples were harvested during total knee arthroplasty from OA patients who had macroscopically normal human cartilage (ICRS grade 0) on the lateral femoral condyle. In the animal study, there was no macroscopic OA change at 2 weeks, but histology detected degenerative changes at this time point. Ultrasound signal intensity also detected degeneration at 2 weeks. In human samples, all samples were obtained from macroscopically intact site, however nearly normal (0 Osteoarthritis (OA) is a slow progressive degenerative joint disease and is a leading cause of impaired mobility in the elderly Quantitative ultrasound is a candidate method for detecting subtle changes in the cartilage Our hypothesis is that the US signal intensity is sufficiently sensitive to detect the microscopic degeneration of articular cartilage. Therefore, we determined whether ultrasound can detect macroscopically undetectable histological changes in OA by using a rabbit OA model. We also used human macroscopically intact cartilage samples (ICRS grade 0) to clarify the ability of ultrasound to differentiate cartilage that has such intact surface.All animal studies were conducted in accordance with principles by Kyoto University Committee of Animal Resources, based on International Guiding Principles for Biomedical Research Involving Animals. All procedures for this study were approved by Kyoto University Committee of Animal Resources (Permit Number: Med Kyo 10184). For all human species, ethical approval for this study was granted by the ethics committee of Kyoto University Graduate School and Faculty of Medicine. Written informed consent was provided and obtained from all study participants.Eighteen skeletally matured female Japanese white rabbits  were used. Two rabbits (4 knees) were allocated to 0-week control, and 4 were randomly allocated into 4 groups that were examined at 1, 2, 4, or 12 weeks after surgery. They were individually housed at 22°C and 50% humidity with a 14/10-h light/dark cycle and had free access to food and water. For operations, intravenous pentobarbital sodium (25 mg/kg) was used to induce and maintain general anesthesia. An intraarticular injection (3 mL) of 1% lidocaine to each knee was used and a medial parapatellar incision was made to expose both knee joints. Bilateral anterior cruciate ligaments were exposed, and left anterior cruciate ligament transection (ACLT) was carefully performed. The joint capsule and skin incision were closed. The rabbits were allowed full weight-bearing postoperatively. The animals were sacrificed by an intravenous injection of pentobarbital sodium (100 mg/kg) and were macroscopically evaluated by two orthopaedic surgeons. In brief, cartilage changes were graded on a scale from 0 to 5  Human articular cartilage samples were obtained from a series of total knee arthroplasties of OA. Patients were diagnosed as OA according to the criteria of the American College of Rheumatology The ultrasonic measurement system with noncontact probe has been described previously and provides a quantitative assessment of tissues properties ≤ Mankin score <2), early OA (2≤ Mankin score <6), moderate OA (6≤ Mankin score <10), and late OA (10≤ Mankin score ≤14) Samples were fixed in 4% paraformaldehyde. Animal and Human samples were decalcified with 0.25 mol/L ethylenediaminetetraacetic acid in phosphate-buffered saline solution (pH 7.4) and Morse’s solution (10% sodium citrate and 22.5% formic acid), respectively. Sagittal sections (6-µm thick) were cut, stained with hematoxylin and eosin (HE) and safranin O/fast green (safranin/O). Histological scoring of the cartilage was performed by two blinded investigators and averaged by using the Mankin scoring system with the following 4 categories: cartilage structure (6 points), cartilage cells (3 points), staining (4 points), and tidemark integrity (2 points); normal and severely degenerated cartilage scoring 0 and 14, respectively −1 wavenumber, and a spectral region of 2000∼670 cm−1 was collected. An integrated absorbance area of the carbohydrate region (1150–950 cm−1) and that under the amide I peak (1710–1595 cm−1) were defined as the proteoglycan and collagen contents, respectively FTIRI was used to determine the spatial distribution of proteoglycan and collagen. The paraffin sections were mounted on metal plates and deparaffinized. A Fourier transform infrared spectrometer  coupled to a microscope  was used for data acquisition. Spatial pixel size was 20×20 µm. Spectral resolution was set to 4 cmt-test was used for comparing sham and ACLT sides in the animal study. One-way ANOVA with a post hoc comparison was used to analyze differences among OA stages in the human study. Pearson’s linear correlation coefficient was used to determine correlations between the ultrasound and FTIRI parameters. Spearman’s correlation coefficient was used to determine correlations between the ultrasound parameter and Mankin scores. Statistical significance was set at P<0.05.All values are means ± standard deviation. Student’s The sham side of the LFC showed little OA-like changes throughout the experimental period. At 1 or 2 weeks, there was no visible change at the LFC in the ACLT side. At 4 weeks, some rabbits exhibited slight LFC fibrillation in ACLT side. At 12 weeks, most rabbits exhibited fibrillation at the LFC of ACLT side. The macroscopic scores of the LFC were significantly higher only at 12 weeks .The US signal intensity of the ACLT side decreased with time. At 2 weeks, the US signal intensity of ACLT (0.74±0.20) was significantly lower than that of sham (1.19±0.29). At 4 and 12 weeks, the US signal intensity of the LFC  decreased further and was significantly lower than sham side .At one week, both the ACLT and sham sides showed reduced safranin/O staining. At 2 weeks, although the safranin/O staining of the sham side looked restored, the surfaces of the ACLT group appeared crackled and the safranin/O staining was not restored, and started to show OA-like changes. At 4 weeks, the safranin/O staining of the sham side was completely restored. The ACLT side exhibited fibrillation of the cartilage surface and chondrocyte cloning. At 12 weeks, the sham side appeared normal, whereas the ACLT side exhibited progression of OA-like changes, including fibrillation increase, chondrocyte cloning, and decrease in safranin/O staining. The Mankin scores  of the ATwo specimens were excluded due to unsuccessful decalcification process of the paraffin sections. Mankin scores ranged widely (0 to 8), although all samples were obtained from ICRS grade 0 sites. The samples were classified into the following stages: nearly normal (3 samples), early OA (12 samples), and moderate OA (3 samples). There were no samples which were classified as late stage.Samples in the nearly normal stage had smooth surface, dense safranin/O staining, and columnar chondrocyte arrangements. FTIRI mapping showed Amide I rich area in superficial zone and Carbohydrate region rich area along with Safranin/O staining. In the early OA stage, samples showed slight fibrillation and reduced safranin/O staining and its amide I and carbohydrate region rich area decreased. In the moderate OA stage, fibrillation became obvious and chondrocyte arrangement became random, safranin/O staining decreased, and the amide I or carbohydrate rich area was not seen.US signal intensity of the nearly normal stage was significantly higher than early or moderate stage, and US signal intensity of early stage was significantly higher than moderate stage.US signal intensity was significantly correlated with Mankin scores and its sub categories, including structure, cells, and safranin/O staining, but not tidemark . There wThe US signal intensity was able to detect macroscopically undetectable OA changes in both animal and human samples. In the animal part, there were no macroscopic changes in the ACLT side at 2 weeks; however, the US signal intensity of the ACLT side was already lower than that of the sham side. The histological Mankin scores also showed degenerative changes in the sham side at 2 weeks. Although all the human samples appeared intact, some samples were histologically degenerated. There was a significant difference among the US signal intensity of nearly normal, early OA, and moderate OA samples.In the animal study, the US signal intensity detected cartilage deterioration when changes could only be identified histologically. OA changes were not macroscopically identified until 4 weeks after ACLT. However, upon histological evaluation, changes in OA, such as tiny crackle on the cartilage or decreased safranin/O staining, were observed at 2 weeks. Thus, ultrasound differentiated changes at this early stage.In the human samples, although all cartilage samples were obtained from macroscopically intact areas (ICRS grade 0), Mankin scores varied widely. Three moderate OA samples out of 20 were actually diagnosed as normal by a trained orthopaedic surgeon. In clinical situations, orthopaedic surgeons mainly diagnose cartilage changes macroscopically. Thus, evaluating articular cartilage solely based on external findings is unreliable and more objective and distinct decision standards are required. We think US signal intensity with noncontact probe is possible candidate for objective evaluation of cartilage quality.In human species, the correlation between US signal intensity and the subcategory of Mankin score showed the strongest correlation with structure which was largely from the superficial layer information but no correlation with tidemark which was from the information of deep region. US signal intensity was also strongly correlated with the amide I value of the superficial zone, but not with the whole zone. In terms of the correlation between US signal intensity and carbohydrate region value, correlation of superficial zone was greater than that of whole zone. The correlation of ultrasound reflection to collagen or proteoglycan content is previously reported There are some disagreement concerning the use of US signal intensity Although the current study demonstrates clinically important findings, it has some limitations. First, the number of animals and human samples is limited. In the human study, only 20 samples were evaluated, which is too small for analyzing subgroups including gender or age. Second, instead of biochemical analysis, FTIRI with univariate-based spectral analysis was used to evaluate cartilage and determine the spatial distribution of materials of interest including collagen and proteoglycan. Amide I and carbohydrate lesion values were used to measure collagen and proteoglycan, respectively, although we did not determine their actual amounts. Nowadays, it is reported that multivalent analysis provides more accurate concentration revealing subtle change In conclusion, we showed the ability of ultrasound to distinguish microscopic OA change in both animal and human cartilage, especially reflecting the information of the degeneration of superficial zone. We believe that the ultrasound is a potential method for diagnosing early subtle changes in OA at the preclinical stage.


Fixed laboratory capacity in Africa may be inadequate; mobile microbiological laboratories may address this issue but their utility has seldom been evaluated.During 2012, the Benin Ministry of Health requested mobile microbiological laboratory (LaboMobil®) support following the failure of polysaccharide meningococcal A+C vaccine to prevent an epidemic in five Northern districts. Within four days, the intervention was initiated. A fixed site in Northern Togo, Pasteur Institutes in Côte d’Ivoire and France, and a research laboratory in Burkina Faso provided additional laboratory support.Local laboratories initially reported most cases to have Gram-positive diplococci suggestive of pneumococcal meningitis. The LaboMobil® evaluated 200 cerebrospinal fluid (CSF) samples and 59 stored isolates collected from 149 individuals. Of the 74 individuals with etiologic confirmation, 60 (81%) had NmW135 and 11 (15%) NmX identified; no pneumococci were identified. Testing in France on 30 NmW135 and 3 NmX confirmed the etiology in all cases. All five districts had crossed the epidemic threshold , all had NmW135 identified and four had NmX identified. NmX were identified as X:ST-181:ccST-181∶5-1∶10-1:F1–31 and NmW135 as W:ST-11: ccST-11∶5∶2:F1-1.In an area with limited local laboratory capacity, a mobile microbiology laboratory intervention occurred in four days through the cooperation of four African and one European country. Results were different from those reported by local laboratories. Despite the introduction of serogroup A meningococcal and 13-valent pneumococcal conjugate vaccines, endemic and epidemic meningitis will continue in the region, emphasizing the usefulness of the LaboMobil® in the short and medium term. Neisseria meningitidis (Nm) and Streptococcus pneumoniae (Sp) The African meningitis belt spans from Senegal in the West to Ethiopia in the East Despite these advances, the meningitis belt is likely to continue to experience endemic and epidemic meningitis. NmX Unfortunately, a substantial portion of the meningitis belt population resides in rural areas with limited laboratory capacity This investigation was a public health response to an infectious disease emergency by the Benin Ministry of Health and organizations enlisted by the Ministry of Health to provide assistance. Generalizable knowledge was neither an a priori or a posteriori goal, other than the functioning of a piece of equipment, namely the LaboMobil®. Under these circumstances of public health non-research The LaboMobil® is a mobile microbiology laboratory first developed in 2003, and operational in Burkina Faso since 2005 and Côte d’Ivoire since 2010. The current third version is a Toyota Hilux WS 726 X-Tra Cabine 100 D40 4X pick-up with a Type II container and a heavy-duty shock-mounted chassis . This veHaemophilus influenzae (Hi) type b containing pentavalent vaccine during June 2005, and 13-valent pneumococcal conjugate vaccine (PCV13) during July 2011. For the latter vaccine, coverage during 2011 was estimated as 36% and during 2012 as 98% . Serogroup A meningococcal conjugate vaccine (MCV-A) was introduced in five meningitis belt departments in mass campaigns among persons age 1–29 years during November 2012, well after the current investigation .Benin introduced into their routine Expanded Program on Immunization At the end of 2011, acute bacterial meningitis cases were reported from Northern Benin, which was followed in February 2012 by a mass immunization campaign using NmA+C polysaccharide vaccine among persons age one year and older. Despite this campaign, several areas continued to experience a large number of cases. Reports from the region indicated that some cases were due to serogroup W135 and during week 11, the Benin Ministry of Health requested an intervention with trivalent Nm A+C+W135 polysaccharide vaccine. Four districts in Atacora and one in Bourgou region passed the epidemic threshold  including Nikki during week 7, Tanguiéta during week 9, Materi during week 12, Cobly and Natitingou during week 13 ; the curDuring week 14, the WHO and the Benin Ministry of Health requested the use of the LaboMobil® based in Burkina Faso to assist with the outbreak investigation . SecurinThe district laboratory of Natitingou received cerebrospinal fluid (CSF) and isolates from the districts of Cobly, Materi, and Tanguiéta, as well as Natitingou itself. The three peripheral laboratories tested CSF with Gram stain and then sent the CSF to Natitingou, where culture was performed and isolates preserved in milk placed in a standard freezer of refrigerator. The Nikki laboratory also performed Gram stain and then transported CSF to the local reference laboratory in Parakou; while the LaboMobil® did not perform an intervention in Parakou, the team made a visit to retrieve specimens for Nikki.Neisseria Meningitidis Antisera package insert website. Available: http://www.bd.com/ds/technicalCenter/inserts/S1190_DifcoNeisseria(0703).pdf last accessed January 8, 2013). Stored isolates that could be revived and isolates from CSF were conserved for transportation in trans-Isolate The LaboMobil® team received fresh CSF from local clinicians and retrieved and tested stored CSF (if visibly cloudy) and stored isolates that had been collected previously. CSF and isolates were analyzed by classical microbiological methods for Gram stain and culture. CSF also underwent antigen detection via latex agglutination  which detected Sp, Hib, and Nm A, C, and W135/Y. Isolates underwent meningococcal serogrouping using monovalent Difco™ Nm antiserum tests for serogroups A, C, W135, and X  analysis crgA for Nm, bexA for Hi, lytA for Sp, siaD for Nm genogroups B, C, Y, X, and W135, and mynB for NmA. The Pasteur Institute, Paris, France performed molecular analysis according to European Meningococcal Disease Society (EMGM) recommendations The Pasteur Institute in Côte d’Ivoire used a multiplex PCR technique which involved gene amplication of The Benin Ministry of Health collected information on reported acute bacterial meningitis for the intervention districts from week 1 through week 26 of 2012. The peak weekly and cumulative attack rates per 100,000 population were, respectively, for Cobly 15.5 (week 14) and 125; for Materi 17.3 (week 12) and 112; for Natitingou 10.5 (week 13) and 46.8; for Nikki 11.7 (week 8) and 94.7; and for Tanguiéta 23.8 (week 14) and 211 .The LaboMobil® evaluated 185 CSF samples and 59 isolates that had been collected and stored in Natitingou district laboratory before arrival as well as 15 CSF samples that were collected during the period the LaboMobil® was on site. Of the total 200 CSF specimens, the LaboMobil® staff performed latex agglutination on 40 and culture on 60 while the Pasteur Institute in Côte d’Ivoire performed PCR on 171 CSF specimens. Of the 59 stored isolates, LaboMobil® staff attempted to perform culture on all 59 but only 40 (68%) were viable. Of 11 NmX identified at the Natitingou Laboratory, LaboMobil® staff confirmed all 11 using monovalent antiserum; four of these 11 had associated CSF available with one having NmX confirmed by PCR. Of 21 NmW135 isolates stored at the Natitingou Laboratory that had associated CSF available, PCR testing confirmed 15 (71%) as NmW135. Nine stored CSF (three associated with NmX and six associated with NmW135) were negative by PCR.The 200 CSF specimens and 59 stored isolates came from 149 individual patients, as some patients had up to four CSF samples collected. Among these 149 individual patients, an etiological agent was confirmed for 74 (50%) . Of the Four of the five districts  were confirmed to be experiencing an epidemic during weeks 9 to 17, 2012 . In all Of the 48 total isolates (37 NmW135 and 11 NmX), 33 (30 NmW135 and 3 NmX) were tested at the Pasteur Institute, Paris, France and serogroups were confirmed for all 33. Genotyping documented that the three NmX were X :ST-181 :ccST-181∶5-1∶10-1 :F1-31; of 15 genotyped NmW135, all were W: ST-11: cc ST-11∶5∶2 :F1-1.Of the 48 isolates, antibiotic sensitivity testing was performed in Dapaong, Togo for 19 NmW135 and 4 NmX; all were sensitive to ceftriaxone, chloramphenicol, and rifampicin. The Pasteur, Institute, Paris, France confirmed this sensitivity pattern for all 23 isolates.One of the local district laboratories limited testing to Gram stain on CSF and results were compared to results from the LaboMobil® intervention using latex agglutination, culture, or PCR. This district laboratory reported 46 Gram-positive cocci via CSF microscopy of which LaboMobil® intervention identified 19 as NmW135, 3 as NmX, and 24 as negative. The same laboratory reported seven Gram-negative diplococci of which the LaboMobil® intervention identified six as NmW135 and one as negative. For four CSF samples, the laboratory identified no organisms while the LaboMobil® intervention identified two NmW135 and two Hi.The LaboMobil® results confirmed the appropriateness of the earlier decision by the Benin Ministry of Health to implement Nm A+C+W135 polysaccharide vaccine mass campaigns, which was done during week 18. By the time vaccine arrived, all five evaluated districts had a weekly attack rate below 10 per 100,000 persons and thus were no longer considered to be experiencing an epidemic .http://www.wahooas.org/?lang=en, last accessed January 8, 2013).The current study involved an outbreak in Benin, the use of a mobile laboratory unit assigned to the Burkina Faso Ministry of Health, African reference laboratories in Togo and Côte d’Ivoire, and international support from AMP and the Pasteur Institute, Paris, France. Remarkably, the intervention was organized within four days of funding commitment. This level of regional collaboration would be impressive anywhere in the world. It was made possible by a common interest in identifying and limiting meningitis outbreaks as well as earlier efforts at cross-border cooperation by the World Health Organization  as well as the West African Health Organization  would be difficult to conduct in any scenario limited to reinforcing fixed rural African laboratories, and thus can be considered an added value of the mobile feature of the LaboMobil®.While the LaboMobil® intervention occurred quickly and results were used rapidly to support the Ministry of Health’s decision on vaccine use, the vaccine intervention occurred late in the epidemic and thus its public health usefulness likely was limited, as suggested by the epidemic curve in the Figure. The usefulness of mobile laboratory interventions could be increased if they were fully integrated into Ministry of Health Infectious Disease Surveillance and Response systems, and thus available for deployment to rural areas at the start of an epidemic. However, even under these circumstances, difficulties remain. Outbreaks often are of short duration, particularly at the village or district level, and may be shorter for serogroup W135 than serogroup A. Additionally, insufficient W135 containing vaccine stocks are available, and none are routinely stored in affected countries.The development of conjugate vaccines against meningococcal serogroup A and up to 13 pneumococcal serotypes likely will prove of great benefit to the inhabitants of the meningitis belt. However, this is not the end of the needed work. We and others have reported previously the existence of serogroup W135 We support a long-term goal of adequate fixed laboratory and epidemiological capacity in all districts, which clearly offers the best chance of identifying and responding quickly to outbreaks. Unfortunately, many laboratories and surveillance systems in the meningitis belt suffer from insufficient funding, human resources, equipment, supplies, and training. For example, despite use of culture in Natitingou district laboratory, this laboratory did not have capacity to evaluate antibiotic susceptibility or have a system in place to ship biological samples to a reference laboratory. Moreover, in one of the largest district hospitals, care providers based case management on Gram stain results, which often were not correct.Over the short- and medium-term, mobile laboratory units can play an important role by responding quickly to outbreaks. The delay of 15 days from request to first CSF analysis could be reduced substantially by local availability of a mobile laboratory and support staff. Mobile laboratories also can support studies by allowing the inclusion of rural populations, train local laboratory technicians, and support the rapid transport of specimens to local and international reference laboratories. These roles will be particularly important for surveillance that occurs in the context of new vaccine introductions, given the questions that remain about duration of protection, serogroup/type replacement, disease caused by etiologies not included in vaccines, geographic inequities in vaccine distribution, and other issues.


Chronic exposure to high level of inorganic arsenic in drinking water has been associated with Type 2 Diabetes (T2D). Most research has been ecological in nature and has focused on high levels of arsenic exposure with few studies directly measuring arsenic levels in drinking water as an index of arsenic exposure. The effect of low to moderate levels of arsenic exposure on diabetes risk is largely unknown thus our study is adding further knowledge over previous works.>30 years and were living in Bangladesh and had continuously consumed arsenic-contaminated drinking water for at least 6 months. T2D cases were diagnosed using glucometer following the new diagnostic criteria (Fasting Blood Glucose > 126 mg/dl) from the WHO guideline (WHO 2006), or a self-reported physician diagnosis of type 2 diabetes. Association between T2D and chronic arsenic exposure was estimated by multiple logistic regression with adjustment for age, sex, education, Body Mass Index (BMI) and family history of T2D.This cross sectional study was conducted in 1004 consenting women and men from 1682 eligible participants yielding a participation rate of 60%. These participants are aged A total of 1004 individuals participated in the study. The prevalence of T2D was 9% (95% CI 7-11%). After adjustment for diabetes risk factors, an increased risk of type 2 diabetes was observed for arsenic exposure over 50 μg/L with those in the highest category having almost double the risk of type 2 diabetes . For most levels of arsenic exposure, the risk estimates are higher with longer exposure; a dose–response pattern was also observed.These findings suggest an association between chronic arsenic exposure through drinking water and T2D. Risks are generally higher with longer duration of arsenic exposure. The risk of T2D is highest among those who were exposed to the highest concentration of arsenic for more than 10 years. Arsenic is a recognised carcinogen and toxicant ,2. It haPrevious studies reported a possible association between chronic ingestion of arsenic in drinking water and T2D ,6,12, alAt least, 20% of the rural population of Bangladesh has been exposed to naturally occurring arsenic through drinking water . From thBangladesh is divided into 64 districts, with each district divided into sub-districts named upazilas, and each upazila further divided into a number of unions. Each union consists of a number of villages, each with a number of tube wells from which the inhabitants draw their water for daily usage including drinking and cooking.The study was conducted from January to July 2009. Ethics approval was obtained from the Bangladesh Medical Research Council (BMRC) and the Human Research Ethics Committee (HREC), The University of Newcastle, Australia.> 40% of the tube wells contaminated with arsenic >50 μg/L, of which 25% had arsenic more than 250 μg/L were considered as a high arsenic contaminated area for this study. A union with no tube wells containing arsenic at more than 50 μg/L was considered as a low arsenic contaminated area. These areas were chosen purposively to ensure the study population’s exposure to a wide range of arsenic that enabled us to study the effect of various levels of arsenic on T2D. Comilla and Jhenidah are 100 km south-east and 180 km south-west from Dhaka city, the capital of Bangladesh, respectively. One hundred households were randomly selected from each of the seven unions. Interviewers visited all selected households in each area. All individuals present during the household visit who fulfilled eligibility criteria were recruited in the study after their informed verbal consent was given. Eligibility criteria were as follows: aged >30 years, history of drinking water from any tube well for a least 6 months continuously, and resident in the study area for at least the preceding 6 months. Those aged less than 30 years or those with a chronic medical condition except T2D were deemed ineligible.After careful review of the Bangladesh Arsenic Mitigation Water Supply Project’s (BAMWSP) national survey report, arsenic contaminated areas were selected from the Kandirpar, Gobindogonj, Uttarda and Modaffargonj union of the Laksam upazila, and the Jolmuttar union of the Monohargonj upazila of Comilla; the Sundorpur and Durgapur unions of the Kaliganj upazila of Jhenidah were chosen as low arsenic contaminated areas. A union having Six trained field workers conducted a door to door survey to identify potential participants from the randomly selected households. Since the literacy rate is low in the selected sampling areas in Bangladesh, the study information sheet was read directly in front of potential participants, and the objectives and expectations of the study were comprehensively explained. Trained field workers then obtained informed verbal consent from the participants.Interviewers collected information on socio-demographics, and drinking water use history by face to face interview using a structured questionnaire. The questionnaire was developed by the researchers and pretested in a neighbouring village similar to the study area. All participants underwent a full body examination by a well-trained physician under sunlight to investigate the presence of arsenical skin lesions i.e., melanosis, leucomelanosis and keratosis. Standing height and weight of each participant were measured with the subjects wearing light clothes and not wearing shoes. Weight was measured in kilograms using bathroom scales (Precision 500gm) that were calibrated daily and zeroed before each measurement. Height was measured in centimetres using a locally made wooden height board with a metal measurement tape attached. All measurements were made according to standard WHO procedures . FastingA single well water measurement was used to characterize chronic arsenic exposure for each participant. We measured the arsenic content of each tubewell water. In case of participants who changed their arsenic contaminated tubewell following BAMWSP survey, we collected water samples from the previous tubewell as a measure for arsenic exposure. This however, only affected 20 participants. We also collected self-reported information on duration of drinking water from these selected tube wells. Cumulative arsenic  exposure was calculated by multiplying the arsenic content of the tube well with duration of use of the well. Drinking water samples were collected from the selected tube wells, transferred and analysed following standard procedure [2) [Information on socio-demographic characteristics such as age, sex, income, education and occupation, marital status, drinking water history, smoking history, height, weight, arsenical skin lesions, family history of T2D and hypertension were collected. BMI was also calculated using the standard method (weight in kg / height in meter2) .> 126 mg/dl or a self-reported physician diagnosis of type 2 diabetes [Participants were classified as having T2D according to the WHO standard diagnostic criteria: Fasting Blood Glucose diabetes . FastingData were analysed using Stata statistical analysis software, version 10 . Frequency tables and summary statistics were obtained to check missing data, out of range values and to assess distributions of continuous variables, and logic checks were undertaken.<50 μg/L and >50 μg/L, as 50μg/L is the maximum permissible limit of arsenic in drinking water for Bangladesh [<50 μg/L and >50 μg/L, and >50 μg/L arsenic concentration was sub categorized as 51–250 μg/L and >250 μg/L [>18.5 as normal), and presence of arsenical skin lesions were included in the initial model. Only those variables found to be associated at P<0.25 level of significance in the initial model were included in the final multiple regression model. Additionally, we included BMI in the final regression model although it was not statistically significant at P <0.25 level of significance in the initial model as BMI is a known confounder for T2D. To estimate the likelihood ratio for model comparison adjusting for the clustering effect of tube well (household using the same well), we fitted a random effect logistic regression model using tube well id as the random effect variable. We used the random effect models to estimate likelihood ratio to compare between nested models while adjusting for clusters. After we came up with the final set of relevant covariates we refit a multiple logistic regression model where confidence intervals were estimated using the Huber/White robust ‘sandwich’ estimator of variance, which is based on the within cluster correlation observed in the data. Cuzick's nonparametric test for trend was used to test for trend across ordered groups.Multiple logistic regressions were done to determine the association between chronic arsenic exposure and T2D, adjusting for potential confounders. For these regression analyses, exposure was initially categorised as ngladesh ; seconda250 μg/L . Initial2) of 20.3 (±4.5). Overall, 14.2% of the participants were current smokers, and mean duration of smoking was 22.6 years (±12.9). A total of 1004 drinking water samples were collected and analysed. All tubewell water contained arsenic above the minimum detection limit (3 microgram). An average of 5.5 (±2.2) persons was using each drinking water sample, with a range of 1 to 18 persons. Mean arsenic concentration of the tubewells were 159 μg/L (±198.5) ranging from 10 to 1401 μg/L. The overall prevalence of T2D among the study participants was 9% (95% CI 7%-11%), while 4.7% of the participants reported a family history of type 2 diabetes. Only 38% of the participants with T2D were already diagnosed, with the remaining 62% being diagnosed on blood tests during the study. Diabetic individuals were older and more likely to be current or ex-smokers  for more than 10 years.More females than males participated in this study. This is because data was collected during the morning when many of the males were out of the home for their daily work. The overall prevalence of T2D is higher in this study than that reported previously in rural Bangladesh . The preStudies in Taiwan, Bangladesh, and Mexico have all shown an association between high levels of inorganic arsenic in drinking water and increased risk of T2D ,6,26-28.<35 μg/g creatinine after adjustment for potential confounders [2) in 55% and exposure to <300μg/L of arsenic in 77% of the participants. The low prevalence of diabetes among arsenic exposed people in the Chen Y et al study might be due to the use of glycated hemoglobin (HbA1C) and glucosuria to identify diabetic individuals, rather than fasting blood glucose and 2-hour blood glucose that were used to identify diabetics in another study [Only a limited number of studies have examined the association of low or moderate exposure to inorganic arsenic with diabetes risk ,30. An efounders . Howeverfounders . On the er study . InaccurThe mechanism by which arsenic causes T2D is still largely undefined.Animal model experimentation suggests that arsenic impairs pancreatic β cell function, particularly affecting insulin synthesis and secretion ,33. The Important strengths of this study include the use of a large population sample, the direct measurement of arsenic in the subjects’ drinking water, and the adjustment for relevant diabetes risk factors such as age, sex, education, body mass index (BMI) and family history of diabetes. While the public health and research implications of this study are important, some caveats must also be considered. One limitation of this study is that biomarkers of arsenic exposure were not measured. Also, we excluded people with chronic medical conditions which may be related to arsenic exposure, although this would tend to bias towards the null and therefore indicates robustness of our results.Other limitations include the use of a glucometer to measure fasting glucose as this may give inaccurate results. Validity of this glucometer has not been tested earlier. Therefore, there may be misclassification of T2D. Use of field screening tests to diagnose T2D in relation to explore association with chronic arsenic exposure has been a concern. A few previous studies reported an association between T2D and chronic arsenic exposure using field screening tests such as glucometer for blood, and glucometric strips for glucosuria ,37. LaboAlso many other factors need to be considered in any population studies as diabetes risk increases with age, obesity and individual’s physical activity . AnotherOur result adds to the growing body of work indicating an association between chronic arsenic exposure and T2D and provides a graded, dose–response relationship. Such an effect could yield insights into the pathophysiology of diabetes and indicate potential future preventive and therapeutic targets in diabetic patients and warrants future larger prospective studies.CCEB, Centre for Clinical Epidemiology and Biostatistics; CHAD, Centre for Health and Development; ICMH, Institute of Child and Mother Health; T2D, Type 2 Diabetes; WHO, World Health Organization; BMI, Body Mass Index; CI, Confidence Interval; OR, Odds Ratio; μg/L, Microgram per liter; BMRC, Bangladesh Medical Research Council; BAMWSP, Bangladesh Arsenic Mitigation Water Supply Project; FIHG-AAS, Flow injection-hydride generation atomic absorption spectrometry; ml, Millilitre; HCl, Hydrochloric Acid; KI, Potassium Iodide; FBG, Fasting blood glucose; TX, Texas; USA, United States of America; Kg/m2, Kilogram per square meter; HbA1C, Glycosylated haemoglobin A1C or a type of haemoglobin that increases in diabetes; HREC, Human Research Ethics Committee.We declare that we have no conflict of interests.MRI contributed to the conception of the project, proposal writing, data analysis and manuscript writing. IK contributed to conception of the project, proposal writing, data collection and review of the manuscript. SMNH contributed to project supervision and data collection. MM contributed to data analysis and manuscript writing. CD contributed to data analysis and manuscript writing. JA contributed to data analysis and manuscript writing. RP contributed to conception of the project and manuscript writing. MS contributed to project supervision, data collection and manuscript writing. SA contributed to project supervision and manuscript writing. AHM contributed to the conception of the project, proposal writing, data analysis and manuscript writing. All authors read and approved the final manuscript.


Exposure to estrogen-mimicking chemicals during critical periods of development, such as infancy, may have adverse effects. However, these effects can be difficult to characterize in most epidemiologic studies. For example, growth of reproductive organs may be susceptible to estrogenic chemicals, but measuring it requires skilled ultrasound examination; timing of pubertal onset may be altered, but observing it requires long-term follow up. To address the need for a simple marker of response to estrogenic exposures in infants, we propose a novel application of a classic marker of estrogen response in adult women: cytological evaluation of urogenital epithelial cells. In this cross-sectional study of 34 female and 41 male infants, we demonstrate that epithelial cells can be obtained from swabs of the vaginal introitus  and urethral meatus , as well as from spun urine, and that these cells respond to differential estrogenic conditions, as indicated by the relative abundance of the superficial epithelial cell type. To model varying estrogen exposure, we sampled from infants who were either newborn , or 12 weeks old (12W) (negligibly exposed to estrogen). Newborns had a higher percentage of superficial cells (%S), as compared to 12W (mean ± standard error: 8.3 ± 1.8 vs. 0.9 ± 0.2) (p < 0.01), consistent with an estrogen response. This difference in %S from newborn to 12W was observed similarly for swab (-7.6 ± 1.7) and urine (-7.3 ± 2.6) specimens and for males (-9.6 ± 2.9) and females (-5.2 ± 2.1). Examination of urogenital epithelial cells can successfully demonstrate estrogen response in both sexes, using cell specimens collected from either swab or urine sampling. In future studies, this simple, non-invasive method may be applied to assess whether estrogen-mimicking chemicals produce an estrogenic response in infants.  Exposure to these chemicals during critical periods of development, such as infancy, is of particular concern. However, while it is possible to measure an infant or child’s response .  For exresponse . AccordiThe epithelial cells of the vagina and urethra are additional markers of estrogen response that are simple to collect and analyze. Papanicolaou  first obUrogenital epithelial cells in infants should exhibit responsiveness that is similar to that seen in adults. Superficial cells, for example, should be found in higher proportions in the neonatal period, due to high levels of exposure to maternal estrogen at term, than they are later in infancy, after the infant is no longer exposed to maternal estrogen, and endogenous estrogen production is low. Urethral epithelial cells collected from urine specimens ,13 and vThe National Institute of Environmental Health Sciences (NIEHS) Epidemiology Branch maintains a protocol for the collection and evaluation of specimens from healthy children. Studies on this protocol are usually small, involve minimal data collection, and produce study records that cannot be linked to the child once the study is complete. This overall protocol was cleared by the Institutional Review Board (IRB) at NIEHS, and specific uses of it are reported yearly. Subject recruitment for this study was carried out by the Division of Gastroenterology, Hepatology and Nutrition at the Children’s Hospital of Philadelphia (CHOP), and the specific study protocol was cleared by the CHOP IRB. A parent provided informed, written consent for the infant’s participation.  Infants born between 37 and 41 weeks gestation, with a birth weight between 5 and 10 pounds, no known acute or chronic health condition, and no reported use of soy-based infant formula were eligible. All subjects were aged either ≤7 days (“newborn”) or 12 weeks (± 3 days) to represent high estrogen exposure and low estrogen exposure, respectively. This design is intended to correspond to the natural model of high maternal estrogen exposure near term birth, and low endogenous estrogen exposure later in infancy ,17. ThirTM  Preservative and stored at room temperature until processing. Epithelial cells were collected from the vaginal introitus in female subjects, or the meatus of the urethra in male subjects using a saline-moistened Polyester –tipped swab. The swab was rubbed on the vaginal introitus or urethral meatus for 5 seconds, and was then placed in a centrifuge tube with 10 ml of SurePathTM Preservative equal to the volume of the specimen was added to each cup. Specimens were thoroughly mixed and stored under refrigeration until processing. Urethral cells were collected from bagged urine samples obtained from both female and male infants . The urine bag was firmly adhered to clean perineal skin and remained on the infant for up to 1 hour, or until a 50 ml specimen was obtained. Once collected, specimens were drained from the bag into clean, sterile urine cups and placed on ice. For specimen volumes > 45 ml, the volume was split between two cups. A volume of SurePathTM preservative by vortexing. The resuspended specimens were then processed by the standard GYN SurePathTM processing procedure, and the slides stained with Sure Path PrepStainTM . Specimen processing was completed at TriCore Reference Laboratories .Tubes containing the vaginal or urethral swabs were agitated to ensure transfer of the collected specimen from the swab to the liquid. Both swab and urine specimens were then poured into centrifuge tubes and centrifuged for 10 minutes at 2000 rpm. The supernatants were decanted, and the remaining cell buttons mixed with SurePathOne pathologist  evaluated all specimens with an Olympus BX51 light microscope, using the 40x objective to accurately assess the nuclear chromatin pattern. When possible, he counted 300 cells; if a specimen was hypocellular, he counted 100, or all the cells if a specimen contained only 50 - 99. Specimens with fewer than 50 cells or those that had inflammatory cells, bacterial or yeast overgrowth, or cytolysis were excluded. We also excluded 3 urine specimens with abundant small basal cells of probable urothelial origin. These cells, which come from the bladder, are not estrogen responsive. A large number of such cells would distort counts and possibly obscure an estrogenic effect. The NIEHS Pathologist was unaware of the age and sex of the child from whom the specimens were obtained and could not link a swab specimen to its paired urine pellet specimen; he was aware of the study design and its hypotheses. Swab and urine specimens were examined in separate batches, with all swab specimens evaluated first, and then all urine specimens. Each squamous cell in vaginal and urethral swab specimens was categorized into the following standard cytological types ,18,19: • Basal/parabasal cells: relatively small cells with scant to moderate amounts of cytoplasm and large, round, centrally located nuclei containing vesicular chromatin.• Intermediate cells: ranging in size from slightly larger than basal/parabasal cells to the size of superficial cells, with abundant, thin, transparent cytoplasm and nuclei containing fine vesicular chromatin. • Superficial cells: similar to intermediate cells with abundant cytoplasm, but with small, pyknotic nuclei and no discernible chromatin pattern. Cells in urine specimens, alternatively, were categorized only as “superficial” or “other,” since it was difficult to distinguish parabasal and intermediate epithelial cells of distal urethral origin from those of less responsive regions of the urogenital tract. A subset of specimens was also scored by an independent pathologist in the processing laboratory . The processing laboratory was not blinded to the pairing of the swab and urine specimens and implemented less stringent inclusion criteria than the NIEHS pathologist. One hundred cells from each slide were counted and evaluated as described by Naib , using c Both swab and urine specimens were evaluated on the basis of the percent of superficial cells (%S), defined as the number of superficial cells divided by the total number of cells counted, similar to the “pyknotic index” used in previous studies . For theOf 141 processed specimens representing 75 infants, 94 specimens, representing 58 infants were successfully scored by the NIEHS Pathologist  . Most ofSuperficial and intermediate cells, indicated by large size and abundant cytoplasm, predominate in both swab and urine newborn samples , with suFor newborn swab and urine specimens , the perOur data analysis did not support any two- or three-way interactions between age, collection method, and sex . For example, the decrease in %S from newborn to 12 weeks was not statistically different for swab (-7.6 ± 1.7) and urine (-7.3 ± 2.6) specimens (p = 0.91) as well as for males (-9.6 ± 2.9) and females (-5.2 ± 2.1) (p = 0.22).  Consequently, the main effects alone summarize the important features of the data . The meaUsing specimen evaluations from the processing laboratory gave results that were broadly similar to those based on the NIEHS Pathologist’s readings. Among the 131 specimens evaluated, representing 70 subjects, 119 (91%) were scored; eight of the 12 excluded specimens (67%) were from newborn males. Like the NIEHS Pathologist’s findings, these laboratory evaluations showed a significant difference in the percent of superficial cells between newborn and 12-week specimens . Notably, these mean percentages tended to be higher than those generated by the NIEHS Pathologist, particularly for the 12-week specimens. Expectedly, results from the processing lab indicated no difference between sexes  or between swab and urine collection methods . However, uniquely among newborn males , the mean percent of superficial cells was 3.6% greater (p < 0.01) for swab specimens than for urines.  In this study, we used the framework of the natural hormonal decline in early infancy  to demon While novel to the study of environmental endocrine disruption, estrogen-responsive epithelial cells have been well understood for decades and have been proposed as clinically informative biological endpoints in children. A predominance of superficial cells in the neonatal vaginal mucosa was noted as early as 1938 ; and, inOur study replicates the basic findings of several of these early studies and provides many practical considerations for study design, field application and sample analysis. In this respect, we observed that sample quality varied by sex, age at sampling, and collection method. Newborn males were prone to low cell counts and inflammation; urine specimens in this subpopulation were particularly problematic. These difficulties with newborn male sampling and specimen interpretation were possibly related to circumcision, but our data did not include circumcised state. In addition, newborn male specimen quality may have been compromised due to repeat sampling of the urinary meatus epithelial tissue: for example, if a swab was collected prior to a urine sample, the post-swab urethral tissue may have been depleted of informative cells at the time of urination. In a broader research context, however, it is important to note that newborns were sampled in this exercise only for the purpose of capturing a known period of high estrogen exposure. For future studies interested in elucidating responsiveness to exogenous estrogen exposures, this method would most suitably be applied to older infants, with low natural estrogen exposures. We also observed that two different scoring approaches yielded similar results. The NIEHS Pathologist tended to count more cells per specimen than the processing laboratory and applied more stringent inclusion criteria. As a result, the NIEHS Pathologist provided high quality readings on a limited number of specimens. Conversely, by using less stringent criteria, the processing laboratory’s results better represented the complete study sample. However, the laboratory’s results may have been more prone to cell-type misclassification, including false identification of cells as superficial due to ”pseudomaturation” (pyknotic nuclei), an effect of inflammation ,11. SuchIn this study, we have illustrated the potential for urogenital epithelial cells to be informative markers of estrogen response in healthy, term, normal birth weight infants. Additional study is needed to determine how epithelial cell characteristics might depend on gestational age, birth weight, and method of delivery, as well as demographic characteristics such as race. Ultimately, though, our findings support the extension of established cytological methods as a simple, inexpensive and minimally invasive way to detect a young child’s estrogenic response. We are currently conducting a study of infants fed soy formula who have relatively high exposure to the plant estrogens genistein and daidzein. We are using the method described here, as well as organ volume and concentrations of serum sex hormones, to establish the utility of different measures of estrogen response. As a result of this work, we hope that future studies will have methods, usable in infants, which credibly demonstrate biologically meaningful exposure to estrogens, including environmental endocrine disruptors.


There is continued reliance on conventional veterinary drugs including anthelmintics, to some of which resistance has developed. Loss of indigenous technical knowledge (ITK) from societies affects the opportunities for utilization of ethnopharmacological practices unless properly documented. This study was conducted to identify common traditional practices using medicinal plants against helminthosis and other livestock diseases in Mpigi and Gulu districts of Uganda.Seven focus group discussions with ten farmers per group plus 18 key informant interviews were held in each district from August to November 2011. Ranking was used to quantify disease burdens and to identify priority livestock and breeds. Samples of each plant were submitted to Makerere University herbarium for identification and documentation. The local name, relative availability and International Union for Conservation of Nature (IUCN) status were recorded.2 = 24; p < 0.001). The scientific, English, Luganda and Acholi names of all plants and their availability within the communities are documented herein.Seventy six farmers in Mpigi and 74 in Gulu were interviewed. Theileriosis and helminthosis were the most common disease conditions in cattle and goats, respectively. Forty plant species within 34 genera from 22 botanical families were identified, with 20 of these used against helminthosis. Other plants treated wounds and ecto-parasites, theileriosis, retained placenta and bovine ephemeral fever. Non-plant practices (7) and plants cited were used in combination depending on availability. Males older than 40 years had most ethnopharmacological knowledge. Most plants  were common, but 10 were rare. IUCN status was not evaluated for 95% of these plants. Conventional and traditional drug use in Gulu and Mpigi districts was different (χThis is the first detailed livestock-related ethnopharmacological study in Gulu district. Farmers in Uganda are still using a variety of practices to treat livestock ailments. Scientific validation and evaluation of conservation status are urgently needed to ensure future availability and knowledge about these plant resources. In Ugan forward ,17, freq forward , to whic forward -20.The World Health Organization estimates that about 80% of the population in developing countries depends on traditional medicine for their primary health care needs . Most ofThere are a number of studies in Uganda which have documented livestock species-specific ethnopharmacological practices in different parts of the country ,27-33. HA cross-sectional survey was conducted from August to November 2011 in two districts: Mpigi and Gulu. Mpigi district is located in the central region between 00° 13′ 48″ North and 32° 19′ 48″ East coordinates. The headquarters are located 37 km west of the capital Kampala, and it is part of the intensive livestock/cattle farming corridor in Uganda  techniques . These iIn Mpigi, seven FGDs were conducted in Kammengo, Bwanya, Mpigi Town Council, Bulamu, Konkoma and Jeza villages. In Gulu, seven FGDs were held in Bobi, Unyama, Awach, Purong, Ombachi and Custom Central villages. These villages were selected because they represented the peri-urban and rural agricultural production systems. Seventy six and 74 farmers were interviewed in Mpigi and Gulu, respectively, with an average of 10 people  per group. Participants were livestock farmers owning at least five ruminants and with more than five years of livestock farming experience. A standard interview guide was used to ensure uniformity of the data collected during the FGDs, after the participants had signed a consent form. The discussions were taped and kept in the local languages (Luganda and Acholi) but later translated for the researchers.In each district, nine KIIs were held, four with veterinary extension workers and five with community ITK persons, using a semi-structured interview guide. Both in the KIIs and FGDs information was gathered on priority livestock and prevailing diseases including helminthosis. Perceived causes, disease diagnosis, treatment methods, plant sources, herbal preparations and dosages were documented. The conservation efforts and challenges encountered while using herbal medicine were also documented.To understand how diseases were diagnosed and treated, participants were involved in practical scoring and ranking of diseases and their treatment. The participants were provided with 100 bean seeds each, which they used to indicate differences in availability of the plant species or varieties mentioned, and changes in burden of disease with livestock species and general priorities. This helped to quantify some items in the checklists and achieve agreement on a given variable within the group.http://www.iucnredlist.org) and Convention on International Trade in Endangered Species (CITES) classification (http://www.cites.org).A local name was attached to each plant, and information on its relative availability was recorded . ReferenThe data were transcribed and entered into Microsoft Excel 3.0. During analysis, data were summarized into major themes by content analysis . DescripA total of 150 participants were interviewed in seven FGDs conducted in each district. Of these, 87 (58%) were female and 63 (42%) male. On average, the farmers interviewed were 36 ± 9.8 years old (mean ± standard deviation). Whereas 15 (10%) participants had no formal education, 83 (55%) had primary and 62 (35%) secondary education level. The average number of goats was 8.6 ± 3.9 with a minimum of 2 and maximum of 56 goats. The average cattle number was 3.2 ± 2.9 with a maximum of 49. Twenty percent (n = 150) of the farmers had only goats. The average household land size was 2.5 ± 2.9 acres (range 1–30) in Mpigi and 6.5 ± 3.5 acres (range 3–50) in Gulu district.FGDs in Mpigi revealed that the priority livestock was local breeds of cattle followed by chicken Table . Most >.Theileriosis or East coast fever (ECF), known as “Amakebe” in Luganda and “Oding ding” in Acholi, was ranked by the farmers as the most common cattle disease in both districts Table . HelmintTreatment depended on availability of funds to procure the conventional drugs (49%), availability of veterinary services 22%), knowledge of alternatives such as ITK (15%), cost attached to the animal (10%) and seriousness of the condition (6%). Owing to low availability of service providers, 55% of the farmers treated their own animals. The majority (60%) of the farmers used the conventional drugs to treat their animals especially cattle, pigs and chicken . Farmers in Mpigi mentioned six plants (no Acholi names specified in Table Hyparrhenia rufa (“Ajuu”) not used in Mpigi. Different plant names in English, Luganda and Acholi were given , scarcity of the plant resources 30%) and lower demand from other farmers (10%). There was a significant difference between conventional and traditional drug use in Gulu and Mpigi districts (χ0% and loEuphorbiaceae (15%) followed by Solanaceae (13%) were the commonest plant families. Twenty plants were used to treat helminthosis, wounds and ecto-parasites (8), theileriosis (6), retained placenta (5), bovine ephemeral fever (4), and one each for Newcastle disease, uterine prolapse, constipation and retained placenta. Some plants were indicated for more than one disease/condition. The resource persons (90%) acquired the ITK through inter- and intra-generation oral tradition within the family. Most plants were used in fresh form. A few rare plants (10%), tree leaves and roots were preserved in dried powder or whole form and constituted when needed. The oral route (80%) for systemic conditions and topical application (15%) for ecto-parasite control were the most common modes of administration  of farmers with knowledge of ITK said and believed the practices and plants were efficacious and safe, showing minimal side effects. Doses therefore were not standardized but indicated roughly. For example, a Euphorbia tirucalli L. which was categorised as least concern (LC) and Carica papaya L. as vulnerable (VU). Categorisation by CITES indicated family Euphorbiacaea and Aloe vera as plants traded with caution  of shrubs and forest trees were obtained within 2 – 6 km. The access distances were shorter in Mpigi district and the longest distance was 5 km from designated forests. Herbs followed by shrubs were the most common plant types Figure . A graphon Table .Several challenges in using traditional knowledge and herbal medicine in treatment of livestock diseases were mentioned. The top three ranked were:1. Most of the plants were used in combination, and limited access to some rare ones prohibited the full scale use of such ITK.2. Most treatments were not openly shared, with few people having the knowledge.3. The doses were not well standardized, with wide variations in treatment duration and amounts to administer.This is the first detailed survey of livestock ethnopharmacological practices from Gulu district. Evidently, there was lower utilization of ethnopharmacological practices in Gulu district compared to Mpigi. In Gulu district, younger farmers (<40 years) portrayed less knowledge, possibly as a result of the breakdown in the ‘traditional social safety nets’ breakdown as an outcome of war or insurgencies in the district . The rolThe current study established 40 plants from 34 genera and 22 families, and 7 non-plant practices, used to treat livestock diseases. A previous global study documented that more than 119 conventional drugs prescribed were derived from plants. The bioactivity of 74% of 119 plant-derived drugs was discovered during validation studies of documented traditional uses of the respective plants ,25.More females than males participated, and a majority of the respondents at least had primary level education. Women have better health seeking behaviour than men, and herbal medicine is practiced in a similar pattern ,27. AccoThe numbers of plants and preparation methods described in this study were restricted to the prioritised diseases/conditions for the animal species identified Table . In addiThe most common mode of preparation was water extraction (75%), where the plant parts were crushed and mixed with water before drenching. This is similar to previous findings  that shoDifferent studies have documented usage of some of these plant species in different parts of Uganda and Kenya ,32,41,42Knowledge levels decreased by age, and the people younger than 40 years were less knowledgeable, which is similar to findings in Kenya . Also siIn Mpigi and Gulu districts, farmers used a variety of 40 plants and seven non-plant practices to treat livestock and poultry diseases. More than half of these plants were readily available in their environment while 25% were rare, and thus their use is compromised. The ethnoveterinary knowledge level was low among people below 40 years, especially in Gulu which may be attributed to the negative impact of prolonged war on the traditional social safety nets which are crucial in imparting ITK to the young folks. This was exhibited by fewer farmers practicing ITK in Gulu District. This calls for heightened sensitization about use and conservation of these plant resources in this district. Validation of efficacy of these ethnopharmacological products is also imperative to enhance full-scale use of these products in livestock production.ECF: East coast fever; FGDs: Focus group discussions; ITK: Indigenous technical knowledge; KIIs: Key informant interviews.The authors declare that they have no conflict of interest.All authors contributed equally to this work. IN and KI took the lead in design and field implementation of the study, whereas JH, RA, and DO supervised the work and supported the writing process. JH, RA and IN contributed to data analysis and presentation. RA, KI and JH supported the editing process. All authors read and approved the final manuscript.


Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one , and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six  belonging to subclass Apostomatida, the other clustered with peritrich ciliate  Vampyrophrya, Gymnodinioides and HyalophysaVampyrophrya has been widely discovered to live as symbionts on copepod hosts around the world. For example, V. pelagica has been reported on Acartia tonsa, A. longiremis, Centropages hamatus, C. typicus, Corycaeus sp., Eucalanus sp., Eurytemora sp., Labidocerca aestiva, Oncaea minuta and Paracalanus sp. in North Carolina V. pelagica on several harpacticoid copepods from a sandy, intertidal beach in the river Ythan estuary, Aberdeenshire Copepods are the most numerous metazoans distributed globally in the aquatic ecosystem Gymnodinioides and Hyalophysa, is exuviotrophic , the other group, represented by Vampyrophrya, is histophagous, feeding on the tissues of hosts by entering the body through the wound created by their predators Epistylis daphniae shortened the survival time of copepods in a food-deficient environment As copepods are abundant and widely distributed, the symbiotic ciliates on copepods have the potential to play an important role in affecting the ecological efficiency and carbon flux in the global ocean. The symbiotic apostomes mainly contain two groups. The first group, represented by Given their involvement in the process of material transfer in grazing and detritus food chains and potential threat to the well-being of the food web So far, most of the research on marine copepod symbionts is limited to morphological observations of their presence using Chatton-Lwoff silver-impregnation technique or by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) Molecular technique can be a powerful tool in addressing the genetic diversity of ciliates due to its sensitivity and specificity Copepod samples were collected using a plankton net  by gently towing ∼1–2 m below the surface for about 2 minutes. The contents of the net were transferred to a 10-L insulated container with natural seawater from the sampling location before being transferred to the lab for processing. Copepods were sampled from a wide range of geographic locations, including temperate regions  and subtropical/tropical regions  . No specThalassiosira weissflogii (at 104 cells mL−1) as food for 3 days. The diatom was cultured in f/2 medium  at 25°C on a 12∶12 h light: dark cycle at ∼100 µmol photons m–2s–1. During the feeding period, every 6 hours the copepods were transferred to a piece of 10-µm mesh and rinsed 3–5 times with fresh FSW to clear off any attached detritus, and then transferred to fresh seawater enriched with T. weissflogii again to purge out any residual natural food from the gut. After this procedure was repeated for about 10 times, the copepods were transferred into FSW and kept for two more days to empty the gut content. The copepods were then preserved in 2% Utermöhl’s solution, sorted, and identified to species, and stored at 4°C before DNA extraction as mentioned above.Live copepod individuals were brought directly to the laboratory and transferred into a 500-mL beaker containing 200-mL autoclaved 0.45µm-filtered seawater (FSW) supplied with diatom −1 proteinase K) at 55°C for 48 hours. DNA was extracted and purified essentially as described previously The sorted copepods were serially rinsed five times with FSW followed by one final wash in autoclaved ultrapure water to remove salts, and then examined under a compound microscope to check if there were any visible organisms/debris attached on the surface. Five copepod individuals of the same species were combined and homogenized thoroughly in microfuge tubes using disposable micropestles and incubated with 0.5 ml DNA buffer , with a corresponding PCR amplicon of ∼0.8 kb in length, was chosen in this study. Copepod DNA samples from starved as well as in situ fixed copepods were used as the templates for PCR using the following touch-down program: an initial denaturing step at 94°C for 1 min, 5 cycles of denaturing at 95°C for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 40 s, followed by 30 cycles of denaturing at 95°C for 10 s, annealing at 62°C for 30 s, and extension at 72°C for 40 s, and an final elongation step of 7 min at 72°C. PCR products were purified and cloned; more than 50 clones for in situ sample were randomly picked and sequenced as reported We searched GenBank database for copepod 18S rDNA sequences and aligned them using CLUSTAL W (1.8) Sequences obtained were searched against GenBank database using the Basic Local Alignment Search Tool (BLAST). The best hits were aligned with the sequences obtained in this study using CLUSTAL W (1.8) Temora turbinata, Acartia erythraea and A. pacifica, named as starved copepods) survived the treatment, which were subjected to DNA extraction, PCR and cloning. For T. turbinata, 28 resultant clones were sequenced and 27 of them shared the highest nucleotide identity (96–99%) to apostome ciliate Vampyrophrya pelagica 18S rDNA (EU503539). We also sequenced a few clones for A. erythraea and A. pacifica, and V. pelagica-like sequences were also detected  were identified based on morphological characteristics . No obvidetected .Subeucalanus subcrassus) samples. Several ciliate sequences obtained from Acartia tonsa in Long Island Sound and Maine were of planktonic ciliate species (S. Lin and H. Zhang unpubl.), but all the rest were closest (88–99%) to the V. pelagica-like sequences found from the starved copepods described above or other known symbiotic ciliates, but distant (>20% difference) from other organisms and were hence considered as symbiotic ciliate sequences here. Totally, 362 symbiotic ciliate sequences were obtained from all the in situ fixed copepod samples, 357 of which were close to those of apostome ciliates Vampyrophrya/Gymnodinioides/Hyalophysa; the remaining five were close to that of the peritrich ciliate Vorticella gracilis (GQ872429) (for details see next section). Among the 10 species of copepod hosts fixed in situ, A. erythraea, A. pacifica, Centropages dorsispinatus, and T. turbinata appeared to harbor higher abundances of symbiotic ciliates, because the 18S rDNA sequences of symbiotic ciliates accounted for over 50% of the total non-copepod eukaryotic 18S rDNA sequences obtained, while the relative abundance of symbiotic ciliate 18S rDNA in the other 6 copepod species ranged from 7–43% (DNA samples isolated from the 11 in situ copepod samples (i.e. containing food in the gut) of those10 species  were alsom 7–43% .Groups I-VI). Group I (27 sequences) was basal to the other apostome sequences with strong support and relatively longer branches in the phylogenetic trees, and was closest to the amphipod-hosted ciliate Gymnodinioides pitelkae, suggesting that this group may be a member of another order in subclass Apostomatida. Group II consisted of only one sequence from C. sinicus close to G. pitelkae with strong NJ bootstrap support. Group III sequences were clustered with V. pelagica (>99% identity) with moderate bootstrap in both the NJ and ML trees. Group IV mainly consisted of sequences retrieved from copepods C. tenuiremis and Paracalanus parvus from Daya Bay, China with moderate to strong NJ and ML supports; these sequences shared 92–95% identity with Vampyrophrya/Gymnodinioides/Hyalophysa, indicating this may be a new group close to Foettingeriidae. Group V sequences were closest to V. pelagica (90–97% nucleotide identity). This group could be further divided into three subgroups: sequences from T. turbinata (Va), from C. tenuiremis (Vb), and from both C. tenuiremis and P. parvus (Vc), in which Vb and Vc contained more diverse sequences with longer branches in the trees. Group VI contained almost identical ciliate sequences from 8 copepod species collected from temperate regions of North Atlantic Ocean, and temperate to tropical regions of the western Pacific Ocean; all these sequences shared highest identity (97%) to V. pelagica. The five sequences from C. tenuiremis and P. parvus collected from Daya Bay, China, formed a separate group (Group VII) within subclass Peritrichia, and shared 95–96% identity to Vorticella gracilis.The 391 ciliate 18S rDNA sequences obtained were clustered to collapse redundancy, yielding 43 unique sequences. These sequences were aligned with those of the representatives from the major orders (or subclasses) of class Oligohymenophorea that were significant hits in the BLAST analysis of our obtained sequences. Two dinoflagellate 18S rDNA sequences were included as the outgroup. Both NJ and ML trees gave similar tree topologies . Most ofGroupVI sequences were most abundant among all symbiotic ciliate sequences obtained. They were detected in 8 different copepod species and occupied >50% of the total ciliate sequences obtained from the copepod in most of the cases  were infested with only one group of ciliates, mainly from GroupVI  each harbored 4–5 different groups of ciliates. Therefore, some copepod hosts were infested with different genotypes of ciliates while other hosts were infested with fewer types.Of the ten copepods species fixed on-site, three , the temperate region in the Southern Yellow Sea (North Pacific), while Groups IV and V only in DYB and SYB, the tropical/subtropic regions in the South China Sea (North Pacific). On the contrary, Group VI ciliates were found in all sampling sites ranging from temperate to tropical waters and from both the Atlantic and the Pacific. Groups I and III also occurred in both temperate and tropical oceans, but only in the Pacific.Our data also showed some geographic pattern of the ciliate groups. Vampyrophrya) are associated with copepods around the world V. pelagica, demonstrating that molecular technique is powerful in addressing the symbiotic ciliate genetic diversity due to its sensitivity and specificity and will be useful for more systematically investigating host and geographic specificity of these ciliates.Most of the previous studies on marine copepod symbionts were carried out using morphological observations by silver-impregnation technique or SEM and TEM, which are time-consuming and require specific equipment, and were limited to the equipped laboratories Vampyrophrya/Gymnodinioides/Hyalophysa, apostome ciliates known to be crustacean symbionts; the other 5 sequences showed the highest identity to the peritrich ciliate Vorticella gracilis, a member of the Vorticella genus known to live on copepods This study is the first attempt to systematically investigate occurrence of ciliate symbionts on a variety of copepods collected from different geographic locations using molecular method. Symbiotic ciliates were detected successfully on all but one copepod samples. Geographically, these copepod samples covered from tropical to temperate in the Pacific Ocean and two distinct temperate marine environments in the Atlantic Ocean. Of the 391ciliate sequences obtained, 386 were close to T. weissflogii, a diatom suitable as diet for copepods, to purge the gut contents T. turbinata samples, 27 apostome ciliate sequences were clustered into four groups, indicating this species harbored a high diversity of ciliates. We also examined the in situ fixed copepod samples including this species. In addition to the highly diverse 18S rDNA sequences from ingested food (S. Lin and H. Zhang unpubl.), sequences of apostome ciliates were also detected from the in situ fixed copepods , planktonic ciliates known to be food of copepods were also detected, but in much lower abundances (S. Lin and H. Zhang unpubl.). Real-time quantitative PCR should be conducted in future studies to quantify the abundance of these ciliates. No ciliate sequences were obtained from S. subcrassus DNA, in contrast to the result by Ohtsukaet al. (2004), which showed presence of ciliate symbionts on this copepod. This may be due to the real absence of ciliate symbionts on S. subcrassus samples in this study, or inadequate sequencing depth.The apostome and peritrich ciliates have been reported to be symbionts of a wide variety of copepods all over the world e.g., , and moscopepods . These tV. pelagica, which would probably only be grouped to few morphotypes microscopically due to the complex life cycles of apostome ciliates and the difficulties in morphological observation. However, the molecular analysis revealed that these apostome ciliate assemblages were diverse. These sequences could be divided into six distinct groups. Except Group III that appeared to be V. pelagica, other five groups represented distinct genotypes (or even multiple species) of previously undocumented symbiotic ciliates, as there is no current "cutoff" that can be used to interpret morphological differences (and species criteria) with sequence differences. The diversity of ciliate symbionts differed among copepod host species. A. erythraea, C. tenuiremis, P. parvus and T. turbinata were each infested with different (4–5) groups of ciliates, while other hosts  were only infested with a particular group of ciliates (Group IV)  . We alsoSuctorian ciliates of class Phyllopharyngea have been reported to live as the epibionts on crustaceans especially pelagic copepods Group VI ciliates were the most abundant and ubiquitous, having been found in all but one species (C. sinicus) of copepods collected from all the different locations in this study  that are cosmopolitan in association with copepods.Among the ciliate sequences obtained, is study , 4. ThisWhile planktonic ciliates are generally important in consuming phytoplankton and recycling the nutrients in aquatic environment, symbiotic ciliates heavily living on the abundant copepods can also be important players in the food chain The ciliate 18S rDNA sequence data have been deposited at GenBank under accession numbers JX417888-JX417930.