Trop Anim Health Prod DOI 10.1007/s11250-014-0639-9
SHORT COMMUNICATIONS
Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius) Lidia E. Mederos-Iriarte & José B. Poveda & Carlos G. Poveda & Orestes M. Vega-Orellana & Carlos Gutiérrez & Juan A. Corbera & Ana S. Ramírez
Accepted: 6 July 2014 # Springer Science+Business Media Dordrecht 2014
Abstract In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders. Keywords Mollicutes . Mycoplasma . Camel . Camelus dromedarius . Dromedary L. E. Mederos-Iriarte : J. B. Poveda : C. G. Poveda : O. M. Vega-Orellana : A. S. Ramírez (*) Unidad de Epidemiología y Medicina Preventiva, Universidad de Las Palmas de Gran Canaria, Trasmontaña s/n, Arucas, 35416 Las Palmas, Spain e-mail:
[email protected] C. Gutiérrez : J. A. Corbera Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Trasmontaña s/n, Arucas, 35416 Las Palmas, Spain
Introduction The Canarian Archipelago (Spain) has approximately 2,000 camels (Camelus dromedarius), which are used as a tourist attraction. This is the largest camel population in Europe (Gutiérrez et al. 2005). Although many advances have been reported in camel medicine during the last two decades, a small amount of information is found in scientific literature concerning mycoplasma (Cl/Mollicutes) and mycoplasmosis in camels. Mollicutes comprise a group of wall-less prokaryotes and are among the smallest self-replicating organisms (Brown et al. 2010). To the authors’ knowledge, there are only two scientific papers related to the isolation of mycoplasmas in camels. Elfaki et al. (2002) isolated Mycoplasma arginini from pneumonic lesions, while Refai (mentioned in Wernery and Kaaden (1995)) recovered Acholeplasma (A.) laidlawii, Acholeplasma oculi, and M. arginini all from healthy animals. Another study unsuccessfully tried to implicate the presence of mycoplasmas with arthritic joints in camels (Bani Ismail et al. 2007). Serology could detect previous exposure to mycoplasmas. Although not confirmed, serological testing involves the following mycoplasma diseases which may be present in camels: contagious bovine pleuropneumonia (Egwu and Aliyu 1997), contagious caprine pleuropneumonia (mentioned in Wernery and Kaaden (1995)), and respiratory diseases caused by Mycoplasma mycoides subsp. capri (Ur-Rahman et al. 2006). This study was undertaken in order to detect, isolate, and identify mollicutes from camels.
Material and methods Sample Samples were taken in two stages. Initially, eight apparently healthy camels (C. dromedarius) were sampled from a farm
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(150 camels) located on the Canary Islands (Spain), five females and three males. Sterile swabs were used to take saliva and ear smears and conjunctival and vaginal secretions from female animals, whereas only conjunctival secretions were taken from male animals. In the second stage, 22 vaginal samples from different healthy female camels were taken from the same farm. In addition, one sample was taken from another herd (60 animals). All of them were taken in duplicate. Information about animal age and reproductive stage was registered when possible.
Culture conditions In the first phase, swabs were inoculated directly into two different broth culture media; PH medium (Kirchhoff and Rosengarten 1984) and PH medium supplemented with 10 % urea. Samples were incubated at 37ºC as soon as possible (within 3 h after collection). After 24 h, samples were filtered through 0.45-μm pore size sterile membranes and subcultured into the same medium. As soon as swirls of growth appeared, the cultures were inoculated onto PH agar medium (Kirchhoff and Rosengarten 1984) or carried out 2 weeks later in the absence of growth. Plates were observed with a microscope until growth. Negative growth was reported when no colonies were seen for 2 weeks. Subcultures were done in SP4-II medium (Ramírez et al. 1997). Single colonies were selected for cloning and subcultured three times. In the second phase of sampling, the culture medium used for transporting the samples was PH medium supplemented with 10 % urea. The cultures were filtered 24 h later through 0.45-μm pore size sterile membranes and subcultured into SP4-II medium.
Biochemical tests A growth inhibition test using digitonin was performed. Mycoplasmas were examined for their ability to ferment glucose and mannose, to hydrolyse arginine and urea, and for the reduction of tetrazolium chloride as well as film and spot production on SP4-II medium (Poveda, 1998).
DNA preparation and PCR DNA was extracted from cloned and uncloned broth cultures using silica as described elsewhere (Tola et al. 1997). Generic Mycoplasma PCR was done as described by Van Kuppeveld et al. (1992). The intergenic spacer region (ISR) between 16S and 23S ribosomal DNA (rDNA) of the samples was amplified using a published protocol (Ramírez et al. 2008). The virtually complete 16S rRNA gene sequence was determined as previously described by Yavari (2010).
Sequencing and sequence analysis Sequencing was conducted by “Servicio de Genética y Diagnóstico Molecular” Universidad de Las Palmas de Gran Canaria (Spain), using BigDye version 3.1 chemistry (Applied Biosystems) on an ABI PRISM 3100 sequencer (Applied Biosystems). Both strands of DNA for all PCR amplicons were sequenced at least twice. The resulting chromatograms were examined in Finch TV (version 1.3.1 Geospiza Inc.). The forward- and reverse-complemented sequences were compared to produce a consensus sequence using MEGA (version 5). The sequences obtained were compared to the sequences held at GenBank, using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih. gov/Blast.cgi).
Results The study was conducted in two stages. Initially, a total of 23 samples were taken. From them, mycoplasma isolation was only achieved from one of the vaginal secretion samples in a tube with PH medium supplemented with urea. Subcultures were only possible to maintain the culture in SP4-II medium (Ramírez et al. 1997). The isolate was cloned and named as Mycoplasma (M.) sp. Jamara-1. In the second stage, 23 vaginal samples were tested, obtaining PCR-positive results from ten of them, including the sample from the second farm. Mycoplasma isolation was successful in nine out of ten samples. The 16S–23S DNA intergenic spacer region PCR amplicon from the ten positive cloned samples was obtained, showing that they had the same length as Mycoplasma sp. Jamara-1 (data not shown), a single 500-bp ISR PCR amplicon band. All of the biochemical tests gave the same profile: sterols were required for growing; arginine was hydrolyzed, but not the urea. Glucose and mannose were not used, tetrazolium was not reduced, and film and spots production were not present. Mycoplasma DNA was successfully extracted from cells in the liquid medium. The 16S rDNA PCR products were obtained from Mycoplasma sp. Jamara-1 isolate and sent for sequencing. After the BLAST search, the highest sequence similarity found was 97 % for the Mycoplasma sp. Mirounga ES2805-ORL and 96 % with Mycoplasma californicum and Mycoplasma bovigenitalium, among others, while the ISR sequence showed a similarity of 78 % for Mycoplasma lipofaciens and 77 % for Mycoplasma fermentans. Five randomly selected samples were sent for sequencing their ISR, resulting in a similarity with Jamara-1 strain of 99–100 %. When the Mycoplasma sp. Jamara-1 DNA was extracted before cloning, the ISR PCR produced two bands (400– 500 bp) (data not shown). The shorter sequence was dominant
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being able to be analyzed (Mycoplasma sp. Jamara-2). A similarity of 90–91 % was seen with Mycoplasma indiense and Mycoplasma orale. Age information was obtained from 17 out of the 27 female camels, including ten positive animals. The ages ranged from 6 to 20 years, being the mean age in positive animals 15.2 years old and 9 in negative animals. Most of the sampled animals were pregnant. Mycoplasma sequences determined here were assigned in GenBank with accession numbers HF548852 for the Jamara-1 16S rRNA gene sequence and HF548850 (Jamara-1) and HF548851 (Jamara-2) for the ISR sequences.
Discussion In total, mycoplasmas were detected in 11 samples by molecular biology comprising two possible new Mycoplasma spp. One of them (strain Jamara-1) could be cultured in ten of the 11 positive samples and a second one (strain Jamara-2) detected only in the same sample, as Jamara-1 apparently is an uncultivable mycoplasma. Failure in maintaining the culture could mean that the lost mycoplasma was a highly fastidious mycoplasma type. Most of the mycoplasmas have intra-species variability in the ISR that goes from 0 to 6 % (Ramírez et al. 2008); in our study, the variability was as small as 0–1 %. This information suggests that these two mycoplasmas probably are novel species. Eight of the positive animals were 12 years or older, while only two of the younger nine adults were positive. Although this age gap is not statistically significant, it seems that the detection of these Mycoplasma species is more probable in older animals. Several Mollicutes species can produce infertility in other mammals, but also, they have been detected in asymptomatic animals (De la Fe et al. 2009). Reproductive efficiency of camels is considered low and it is known that infectious organisms are related to it, being uterine infections the most common cause of reproductive failure in Camelidae (Tibary et al. 2006). Nevertheless, mycoplasma isolations could not be associated with reproductive disorders, any disease, or clinical sign. Nowadays, all assessed females are pregnant, and so far, no reproductive problems have been detected. Currently, the pregnant females are being monitored to evaluate the course of their pregnancy. In conclusion, these mycoplasmas which have been isolated from apparently healthy camels could be new species. Further molecular and serological studies would be necessary to describe Mycoplasma sp. Jamara-1 as a new species. The identification of new mycoplasmas gives significance to our
results and provides information to the poorly researched bacterial ecosystem of camels. Acknowledgments This work was supported by a project from Gobierno de Canarias (P2007/046), Spain. Conflict of interest The authors declare that they have no competing interests.
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