A novel bacterial symbiont in the nematode Spirocerca lupi
© Gottlieb et al.; licensee BioMed Central Ltd. 2012
Received: 17 January 2012
Accepted: 25 June 2012
Published: 5 July 2012
The parasitic nematode Spirocerca lupi (Spirurida: Thelaziidae), the canine esophageal worm, is the causative agent of spirocercosis, a disease causing morbidity and mortality in dogs. Spirocerca lupi has a complex life cycle, involving an obligatory coleopteran intermediate host (vector), an optional paratenic host, and a definitive canid host. The diagnosis of spirocercosis is challenging, especially in the early disease stages, when adult worms and clinical signs are absent. Thus, alternative approaches are needed to promote early diagnosis. The interaction between nematodes and their bacterial symbionts has recently become a focus of novel treatment regimens for other helminthic diseases.
Using 16S rDNA-based molecular methods, here we found a novel bacterial symbiont in S. lupi that is closely related to Comamonas species (Brukholderiales: Comamonadaceae) of the beta-proteobacteria. Its DNA was detected in eggs, larvae and adult stages of S. lupi. Using fluorescent in situ hybridization technique, we localized Comamonas sp. to the gut epithelial cells of the nematode larvae. Specific PCR enabled the detection of this symbiont's DNA in blood obtained from dogs diagnosed with spirocercosis.
The discovery of a new Comamonas sp. in S. lupi increase the complexity of the interactions among the organisms involved in this system, and may open innovative approaches for diagnosis and control of spirocercosis in dogs.
Vector-borne helminthic diseases, such as onchocerciasis and lymphatic filariasis, are major human diseases in endemic areas. Novel treatment approaches have been recently focusing on the interaction between the causative helminth agent and its bacterial symbiont. Consequently, antibiotics, such as doxycycline, are used instead of, or with, anti-helminthic drugs for treatment [1, 2]. However, because of difficulties in application, various bacterial targets are constantly studied . This approach has also been adopted in veterinary helminthic diseases, such as bovine onchocerciasis and canine heartworm disease [4–6].
Spirocercosis is a vector-borne helminthic disease, mostly affecting carnivores, especially canids [7, 8]. It is caused by the esophageal nematode Spirocerca lupi (Spirurida: Thelaziidae) that has a wide distribution, but is mostly prevalent in warm, humid areas. The exact annual number of dogs affected annually worldwide has never been assessed. However, the disease has a wide distribution in the Mediterranean basin, Africa, Central and South America .
Diagnosis of spirocercosis is always challenging, because the clinical signs are variable and occur in advanced disease stages. Most animals are thus diagnosed only in the advanced stage of the disease, once nodules containing adult to egg shedding worms, are present in the esophagus . The diagnosis of the disease in its early stages, prior to formation of esophageal nodules and egg shedding, is currently difficult and is almost impossible.
Recent studies have shown a relationship between bacterial symbionts of the genus Wolbachia and filarial pathogenic nematodes . Wolbachia which is estimated to infect 66% of arthropods and nematodes  can manipulate various aspects of its arthropod hosts’ biology . Wolbachia was found to be an obligatory symbiont of certain filarial nematodes, with a possible role in the pathogenesis and immune response to filarial infection in the mammalian host [4, 5, 15, 16].
In the current study, we tested for the presence of Wolbachia species and other specific symbionts in the nematode S. lupi, and detected a novel and stable infection in the worm. Our findings are expected to promote further understanding of the interactions among various organisms in complex systems such as spirocercosis, and may have clinical implications, because this stable bacterial infection can potentially be used for novel simple diagnostic methods of this disease and aid in its prevention and treatment.
Results and discussion
Identification of novel bacterial symbiont in S. lupi from the Thelazioidea super family
Phylogenetic analysis of the S. lupi symbiont
At present, the role that the identified Comamonas sp. plays in the biology of the nematode remains unknown, and so is its potential role in canine spirocercosis. A recent study, however, showed that benign infection with S. lupi induces an immune response that is atypical to chronic helminthic infection, but rather suggests a bacterial infection .
Localization of Comamonas sp. within S. lupi
Detection of S. lupi-derived Comamonas sp. in blood samples of infected dogs
In the present study, we detected an additional organism, a bacterial symbiont of the genus Comamonas, within the causative agent of spirocercosis, the nematode S. lupi. Recently, microbial symbiosis has been repetitively shown to be a driving force in the biology and evolution of many organisms. The present study adds yet additional evidence of this trend, in a highly complex system. Resolution of the complex interactions among the different organisms involved in the spirocercosis system may lead to novel, applicable methods for the early diagnosis, prevention and treatment of canine spirocercosis, in a similar manner as has been applied when the interaction between Wolbachia spp. symbionts with their filarial nematode hosts has been elucidated [3, http://a-wol.com].
Adult S. lupi worms were obtained from esophageal nodules of dogs diagnosed with spirocercosis at the Hebrew University Veterinary Teaching Hospital, at necropsy, and stored in −20°C pending analysis. Larvae (L2 and L3) were dissected under a stereoscope from O. sellatus beetles, isolated in the laboratory from dog fecal dungs, collected in a public park located in a S. lupi-endemic area in Central Israel . These were either stored in absolute ethanol at −20°C, or freshly used. S. lupi eggs were concentrated through floatation , and stored as described above.
Blood samples were obtained from dogs diagnosed with spirocercosis through esophageal endoscopy and presence of eggs in the feces, and from puppies aged 2 to 4 months, housed in a breeding farm. Puppies were chosen as negative control because they were housed in a restricted kennel, and were thus unexposed to feces of other dogs.
DNA extraction, PCR, clone library and sequencing
DNA of adult S. lupi worms was extracted using hexadecyltrimethyl ammonium-bromide (CTAB) buffer , and were used in PCR with the 16S rDNA (rrs) gene primer set, targeting most known eubacteria (27F-1494R; ), under the following reaction conditions: 3 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 55°C, 1.5 min at 72°C; and 5 min at 72°C. The PCR products were run on 1% agarose gel, and were later extracted and cloned into pGEM-T easy vector (Promega, Madison, WI, USA), and transformed into competent Escherichia coli. Plasmids from 10 inserted clones were extracted from the gel and sequenced (HyLabs, Rehovot, Israel). As a control for DNA quality, PCR analysis was performed using primers for the S. lupi-specific cytochrome oxidase subunit 1 gene (cox1) as previously described .
Direct probing of known invertebrate symbiont
DNA of S. lupi was used in PCR with specific primers and conditions to identify Wolbachia, Cardinium and Rickettsia spp., as previously described . DNA extracted from Bemisia tabaci, harboring Wolbachia and Rickettsia spp., and from Plagiumerus diaspidis containing Cardinium sp. were used as positive controls.
Denaturating gradient gel electrophoresis (DGGE)
PCR was performed on adult S. lupi DNA using primers GC-clamp 341 F-907R, targeting the bacterial rrs gene, with PCR conditions permitting its amplification from most known bacteria . DGGE was performed using a 40% to 60% urea/formamide gradient for standard reactions. After the electrophoresis, gel was incubated in ethidium-bromide solution (250 ng/ml) for 10 min, rinsed in distilled water, and photographed under UV illumination. Bands were extracted, and sent for direct sequencing (HyLab, Rehovot, Israel).
Nine nearly-full length sequences of the rrs gene of Comamonas sp. were obtained from five adult S. lupi worms. All clones were sequenced from both directions. Sequences were edited using DNAMAN software (Lynnon Corporation, Canada) and a consensus sequence was determined. The Comamonas sp. rrs sequence was aligned, using MUSCLE 3.7, with other published Comamonas spp. sequences, selected based on BLAST results, and based on their invertebrate host origin. The rrs gene sequence of Verminephrobacter eiseniae was used as an out group. A maximum-likelihood tree was constructed using PhyML 3.0 software. Bootstrap analyses with 1000 re-samplings were performed to test branching robustness. The tree was illustrated using TreeDyn 198.3. All software packages are available at http://www.phylogeny.fr/.
Direct probing of Comamonas sp.
To confirm the presence of Comamonas sp. in the various S. lupi developmental stages (eggs, larvae and adults), and in blood samples obtained from S. lupi- infected dogs, a diagnostic PCR was planned. Based on the rrs sequence established, specific primers were designed; /ComF323/ 5’-CCTCGGGTTGTAAACTGCTT-3’ and /ComR1393/ 5’-TCTCTTTCGAGCACGAATCC-3’. The primers were used in a standard PCR, under the following conditions: 3 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 58°C, 1 min at 72°; and a final 5 min at 72°C. The PCR product size was expected to be ca. 1000 bp. Positive and negative PCR products were retested using semi-nested PCR, with the forward primer /ComNest F/ 5’- ACTGCCATTGTGACTGCAAG-3’ and the ComR1393 reverse primer, with PCR conditions as described above, resulting in ca. 600 bp product. Three PCR products from each sample category were directly sequenced in order to confirm the Comamonas specific sequence.
Fluorescent in-situ hybridization (FISH)
FISH was performed as previously described . Briefly, larvae were fixed in Carnoy’s fixative (6:3:1 parts of chloroform: ethanol: acetic acid), and later hybridized with the rrs-based designed probe: Com-probe /Cy3/ 5’- TGTGCTACTAGAGCGGCTGA-3’, in hybridization buffer. Since intact larvae could not uptake the probe, larvae were first ruptured using sterile insect pins, and their content was removed from the cuticle. Specimens were viewed under an IX81Olympus FluoView500 confocal microscope. Signal specificity was confirmed based on sequence comparison in the ‘Probe Match’ function in the Ribosomal Database Project website (http://rdp.cme.msu.edu/), and using a no-probe control, and hybridization to a non-target nematode, Trichinella spiralis.
Samples (nematodes and blood) were obtained from S. lupi-infected dogs presented to the Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem with their owners' consent, during diagnosis, treatment and necropsy. Samples obtained from control dogs were obtained with their owner's consent. This study was approved by the Institutional Committee of Animal Handling and Experimentation.
We would like to Dr. Shachar Naor and Dr. Zippi Prize for their technical assistance in the laboratory work.
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