Copromicroscopic and molecular assays for the detection of cancer-causing parasitic nematode Spirocerca lupi

Spirocerca lupi (Nematoda, Spirurida) is a life-threatening parasitic nematode of dogs that is presently emerging in several countries. Nonetheless, canine spirocercosis is neglected and underestimated, mainly due to diagnostic limitations inherent to clinico-pathologic, diagnostic imaging and laboratory methodologies. Given the significant benefit of improved diagnosis, the present work evaluated the reliability of a recently described copromicroscopic approach, the FLOTAC technique, as well as a PCR-based assay with that of traditional coproscopic techniques to diagnose S. lupi infection. Ninety-four faecal field samples were collected from two endemic areas (i.e. 29 and 65 from Kenya and Israel, respectively) and processed using different coproscopic examination techniques. In particular, set I (Kenyan samples) comprised the modified flotation with Sheather's sugar solution and merthiolate-iodine-formalin technique, while set II (Israeli samples) comprised a flotation technique with zinc sulphate solution, a modified sugar flotation procedure and the FLOTAC method. All samples were also subjected to a semi-nested PCR protocol specific for a region internal to the mitochondrial cytochrome c oxidase subunit 1 gene of S. lupi. The coproscopic examinations showed low sensitivity and high variability, demonstrating the unreliability of the conventional methods for detecting S. lupi eggs. Nonetheless, the FLOTAC technique scored the highest number of positives and significantly higher number of S. lupi eggs per microscopic field compared to the other coproscopic methods. Additionally, of the coproscopically negative samples, 9 (45%) Kenyan and 21 (38.2%) Israeli samples scored molecularly positive using the PCR-based approach. The potential implications and perspectives for canine spirocercosis of these coproscopic and molecular diagnostic methodologies evaluated herein are discussed.     

Haplotypic variability within the mitochondrial gene encoding for the cytochrome c oxidase 1 (cox1) of Cylicocyclus nassatus (Nematoda, Strongylida): evidence for an affiliation between parasitic populations and domestic and wild equid hosts

This study investigated the genetic variability within Cylicocyclus nassatus (Nematoda, Strongylida, Cyathostominae) collected from different domestic and wild hosts (i.e. horse, donkey, Przewalskii horse, tarpan and Turkmen kulan) and localities in Europe and/or USA. The ribosomal Internal Transcribed Spacer 2 (ITS2) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were PCR-amplified and sequences characterized from seventy individual parasitic specimens. While ITS2 displayed 0-0.6% variation rate among all individual adult specimens of C. nassatus examined, 22 different sequence variants (haplotypes) of cox1 were detected. Nucleotide variation was detected at 75 of the total 689 positions (overall 10.8% rate of intraspecific nucletidic difference) in the cox1, with the absence of invariable positions among specimens collected from each equid species or country. Conversely, two haplotypes were detected in horses from USA and in donkeys of Italy and Ukraine, respectively. The absence of haplotypes shared by the equid species suggests an affiliation of C. nassatus populations to their specific host. The results of the present study demonstrated that the characterization of mitochondrial regions may have important implications for studying the genetic structure and biology of equine cyathostomes, and to exploit taxonomic issues and practical implications related to the spread of anthelmintic resistance.     

Magnetic resonance imaging findings of spinal intramedullary spirocercosis

Spirocerca lupi is a nematode infecting dogs in tropical and subtropical areas. Aberrant S. lupi migration to different body organs, including the spinal cord, has been documented. To date, the diagnosis of aberrant spinal cord migration was made at post-mortem examination or as an incidental finding, during spinal surgery. We describe two dogs with acute asymmetric paraparesis that were subsequently diagnosed with spinal cord spirocercosis. In magnetic resonance (MR) images of the spine, T2 hyperintense lesions were seen in the spinal cord of both dogs. The lesions appeared isointense on T1-weighted images and focal enhancement was detected after gadolinium administration. The MR imaging findings were compatible with focal inflammation, presumably along the parasite migration tract. Gross and microscopic pathologic findings confirmed the diagnosis of aberrant spinal intramedullary migration of S. lupi in one dog, and in the other dog, the clinical and imaging findings were supportive of this diagnosis.     

The sensitivity of direct faecal examination, direct faecal flotation, modified centrifugal faecal flotation and centrifugal sedimentation/flotation in the diagnosis of canine spirocercosis

Several faecal examination techniques have shown variable sensitivity in demonstrating Spirocerca lupi (S. lupi) eggs. The objective of this study was to determine which faecal examination technique, including a novel modified centrifugal flotation technique, was most sensitive to diagnose spirocercosis. Ten coproscopic examinations were performed on faeces collected from 33 dogs confirmed endoscopically to have spirocercosis. The tests included a direct faecal examination, a faecal sedimentation/flotation test, 4 direct faecal flotations and 4 modified faecal centrifugal flotations. These latter 2 flotation tests utilised 4 different faecal flotation solutions: NaNO3 (SG 1.22), MgSO4 (SG 1.29), ZnSO4 (SG 1.30) and sugar (SG 1.27). The sensitivity of the tests ranged between 42% and 67%, with the NaNO3 solution showing the highest sensitivity in both the direct and modified-centrifugal flotations. The modified NaNO3 centrifugal method ranked 1st with the highest mean egg count (45.24 +/- 83), and was superior (i.e. higher egg count) and significantly different (P < 0.05) compared with the routine saturated sugar, ZnSO4 and MgSO4 flotation methods. The routine NaNO3 flotation method was also superior and significantly different (P < 0.05) compared with the routine ZnSO4 and MgSO4 flotation methods. Fifteen per cent (n = 5) of dogs had neoplastic oesophageal nodules and a further 18% (n = 6) had both neoplastic and non-neoplastic nodules. S. lupi eggs were demonstrated in 40% of dogs with neoplastic nodules only and 72.9% of the dogs with non-neoplastic nodules. The mean egg count in the non-neoplastic group (61) was statistically greater (P = 0.02) than that of the neoplastic group (1). The results show that faecal examination using a NaNO3 solution is the most sensitive in the diagnosis of spirocercosis. The modified centrifugal flotation faecal method using this solution has the highest egg count. The study also found that dogs with neoplastic nodules shed significantly fewer eggs than dogs with non-neoplastic nodules.     

Evaluation of doramectin for the treatment of experimental canine spirocercosis

The nematode Spirocerca lupi is primarily a parasite of dogs, which causes typical lesions of esophageal nodular granulomas, aortic aneurysms and spondylitis. In order to evaluate the therapeutic effect of doramectin on experimental canine spirocercosis, seven beagle dogs experimentally infected with 40 infectious S. lupi larvae (L(3)) were treated with doramectin. Treatment was commenced following endoscopic visualization of esophageal granulomas, and typical S. lupi eggs were detected in the feces. The treatment protocol included six treatments of doramectin (400 microg/kg subcutaneously) at 2 weeks intervals, followed by monthly injections until the disappearance of the esophageal granulomas or the end of the study (768 days post-inoculation).Eggs could not be found on fecal examinations 3-10 days after the first or second doramectin treatment. In addition, a gradual decrease in size of granulomas was noticed in all seven dogs during the course of the study. Esophageal granulomas had completely resolved in six of the seven dogs between day 35 and day 544 post-initial doramectin treatment, by day 35 in one dog (after three treatments), by day 43 in two dogs (after four treatments), by day 98 in one dog (after seven treatments), by day 460 in one dog (after 18 treatments) and by day 544 in another dog (after 21 treatments). In one dog, remnants of S. lupi granulomas could still be seen 544 days post-initiation of treatment with doramectin. Multiple subcutaneous injections of doramectin (400 microg/kg) were shown to be effective and safe in the treatment of canine spirocercosis.     

The efficacy of milbemycin oxime against pre-adult Spirocerca lupi in experimentally infected dogs

The aim of this investigation was to determine the efficacy of milbemycin oxime in preventing the oesophageal encapsulation of Spirocerca lupi, following the experimental infection of dogs. Two studies were conducted which involved a total of 21 purpose-bred Beagles. Each dog was infected with approximately 40, third stage infective S. lupi larvae. The larvae were dissected from scarabaeid beetles that had been collected from areas endemic for spirocercosis. In the first study, milbemycin oxime (minimum dose 0.5mg/kg body weight) was administered to seven dogs on day 30 post-infection. Seven other dogs served as untreated controls. In the second study, milbemycin oxime (also at a minimum dose of 0.5mg/kg body weight) was administered to four of seven infected dogs on day 28 post-infection. Treatment was repeated at 14- or 28-day intervals. All of the dogs, from both studies, were euthanized 168 or 169 days after infection. All S. lupi were recovered, and lesions in the thoracic aorta and oesophagus were described and quantified. A single treatment with milbemycin oxime was 79.8% effective in preventing the establishment of S. lupi in the oesophagus. This treatment significantly (p<0.05) reduced both the number of S. lupi within the oesophagus and the size of the oesophageal nodules. The efficacy of anthelmintic treatment was increased to 100% when repeat doses of milbemycin oxime were administered at 14- or 28-day intervals. These repeat treatments completely prevented the establishment of S. lupi within the oesophagus and thereby averted the development of oesophageal nodules. As expected, none of the treatment protocols reduced S. lupi related damage within the aorta because the administration of milbemycin oxime only began after the larvae had completed their first stage of migration.     

First reports of autochthonous eyeworm infection by Thelazia callipaeda (Spirurida, Thelaziidae) in dogs and cat from France

Thelazia callipaeda (Spirurida, Thelaziidae) is a small nematode living in the conjunctival sac of domestic and wild carnivores, rabbits and humans causing lacrimation, epiphora, conjunctivitis, keratitis and even corneal ulcers. The first autochthonous cases of thelaziosis affecting four dogs and one cat living in South Western France (Dordogne area) are reported and described. Nematodes recovered from the animals were morphologically identified as T. callipaeda and a partial region of the cytochrome oxidase c subunit 1 gene (cox1) was amplified by PCR from nematode specimens (from two dogs and the cat). In each case, this was shown to have an identical sequence to the haplotype 1 (h1) of T. callipaeda. So far, the arthropod acting as intermediate host of T. callipaeda eyeworms has not been identified in France although it might be Phortica variegata (Steganinae, Drosophilidae) as recently described in Italy.     

Canine spirocercosis: clinical,diagnostic, pathologic,and epidemiologic characteristics

The nematode Spirocerca lupi is a parasite of dogs with beetles of several species serving as intermediate hosts. The medical records of 50 dogs diagnosed with spirocercosis at the Hebrew University Veterinary Teaching Hospital (HUVTH) in Israel during 1991-1999 were retrospectively reviewed and compared to a control group (n=100). There was a seven-fold increase in the annual number of dogs diagnosed with spirocercosis during these years while the hospital caseload increased by 80%, indicating an emerging outbreak of this infection. Dogs from the greater Tel Aviv area were at the highest risk of being diagnosed with spirocercosis with 74% of the cases originating from this region compared to only 17% of the controls. The disease appeared to have a primarily urban pattern of distribution with a significantly higher percentage (P=0.025) of dogs from cities versus rural areas, as compared to the control group. Sixty-two percent of the cases were diagnosed during the colder months of December through April. The median age of infected dogs was 5 years, with dogs 1 year old or younger at the lowest risk of being diagnosed with spirocercosis. Large breeds were at a higher risk of infection in comparison to small breeds and the Labrador Retriever was significantly over represented (P=0.027) in the study group compared to the control population. The most common signs were vomiting or regurgitation (60%), pyrexia (24%), weakness (22%), respiratory abnormalities (20%), anorexia (18%), melena (18%) and paraparesis (14%). A caudal esophageal mass was identified by radiography in 53% of the dogs and spondylitis of the thoracic vertebrae in 33%. Fecal flotation was positive for S. lupi eggs in 80% of the dogs, and endoscopy was found to be the most sensitive diagnostic procedure and allowed diagnosis in 100% of the examined dogs. Fifty-three percent of the dogs were anemic and creatine kinase (CK) activities were elevated in 54%. Necropsy of 14 dogs revealed esophageal or gastric granulomas in 13 dogs, and an esophageal osteosarcoma in a single animal. Aortic aneurysms were found in six (43%) dogs. Out of 24, 15 dogs (63%) for which follow-up information was available died or were euthanized within 1 month of admission. The case-fatality rate decreased toward the end of the study period when improved therapy with avermectins became available.     

High within-host genetic variation of the nematode Spirocerca lupi in a high-density urban dog population

The nematode worm Spirocerca lupi has a cosmopolitan distribution and can cause the death of its final canid host, typically dogs. While its life cycle, which involves a coprophagous beetle intermediate host, a number of non-obligatory vertebrate paratenic hosts and a canid final host, is well understood, surprisingly little is known about its transmission dynamics and population genetic structure. Here we sequenced cox1 to quantify genetic variation and the factors that limit gene flow in a 300 km(2) area in South Africa. Three quarters of the genetic variation, was explained by differences between worms from the same host, whereas a quarter of the variation was explained by differences between worms from different hosts. With the help of a newly derived model we conclude that while the offspring from different infrapopulations mixes fairly frequently in new hosts, the level of admixture is not enough to homogenize the parasite populations among dogs. Small infrapopulation sizes along with clumped transmission may also result in members of infrapopulations being closely related.     

Global situation of foot-and-mouth disease (FMD)--a short review

This article reviews the actual world FMD situation. In 2000, fifty nine countries officially reported outbreaks of FMD. The disease occurred in Europe (Greece), Asia (Russia, Mongolia, Bangladesh, Cambodia, China, Japan, Laos, Nepal, Pakistan, Philippines, Republic of Korea, Taiwan, Thailand, Vietnam, Iran, Iraq, Turkey, in Caucasian region--Georgia, Azerbaijan and Armenia as well as in Kazakhstan, Kyrgyzstan, Turkmenistan and Tajikistan), Africa (Egypt, Kenya, Mauritania, South Africa, Tanzania, Uganda, Malawi, Namibia, Zambia and Zimbabwe) and in South America (Brazil, Colombia, Uruguay, Bolivia, Peru, Ecuador and Venezuela). In 2001, FMD was still spreading throughout the endemic regions and appeared in some of the west European countries--Great Britain, The Netherlands, France and Ireland. In South America, FMD occurred in Argentina, Uruguay, Brazil and Colombia. In Asia the FMD spread in Turkey, Iran, Afghanistan, Georgia, Azerbaijan, Mongolia, Kuwait, Bahrain, Yemen, Qatar, United Arab Emirates, Oman, Iran, Bhutan, Nepal, Malaysia, Philippines, Thailand and Taiwan. The FMD situation in Africa was unclear, but probably most countries in West, East and South Africa were affected. The most recent data of the OIE from May 2002 confirmed FMD outbreaks in population of pigs in Republic of Korea.     

猬迭宫绦虫线粒体cox3基因的克隆及序列分析

本研究旨在阐明猬迭宫绦虫湖南分离株线粒体细胞色素c氧化酶第Ⅲ亚基（cox3）基因部分序列（pcox3）的遗传变异情况。利用聚合酶链反应（PCR）扩增猬迭宫绦虫的pcox3,应用ClustalX 1.81程序对序列进行比对,同时利用DNAStar 5.0中的Megalign程序进行同源性分析,再用Phylip 3.67程序MP法绘制系统发育树,并与GenBank中已知猬迭宫绦虫相应基因序列进行比对分析。结果显示,所获得的pcox3序列长度一致,均为264 bp,湖南分离株与已知猬迭宫绦虫位于同一分支,来自湖南省的猬迭宫绦虫pcox3序列有微小差异。本研究结果为猬迭宫绦虫进一步的分类、鉴定和遗传变异研究奠定了基础     

Maternal origins,population structure and demographic history of ten Chinese indigenous goat breeds from Yunnan

Yunnan as a frontier zone that connects China with South and Southeast Asia, has 11 well‐recognized goat breeds. However, the knowledge about maternal origins, population structure and demographic history of Chinese indigenous goats from Yunnan is limited. In this study, we analysed a 481‐bp fragment of first hypervariable segment (HVSI) of the mitochondrial DNA (mtDNA) control region sequences of 749 individuals from 10 Yunnan indigenous goat breeds, of which 556 sequences were newly determined. There were 110 polymorphic sites that defined 158 haplotypes among all sequences. The haplotype and nucleotide diversity of these breeds ranged from 0.782 ± 0.079 to 0.982 ± 0.015 and from 0.028 ± 0.003 to 0.043 ± 0.005, respectively. Phylogenetic analysis identified two lineages A and B, of which the lineage A had higher frequency (68.1%) and distributed in all Yunnan breeds. We combined previously reported sequences with our sequences belonging to the lineage B and detected two subclades B1 and B2, in which the B1 subclade shared individuals from Eastern Asia, Southeast Asia and Southern Asia. Given higher level of diversity and more unique haplotypes, the B2 subclade probably originated from Southwestern China. The haplotype network, analysis of molecular variance (AMOVA) and a Mantel test revealed no significant phylogeographic structuring among Yunnan goat breeds. This can be explained by high gene flow and genetic admixture among these breeds from different geographic regions in Yunnan. Additionally, both the lineages A and B reflected different demographic histories. This study will provide a scientific basis for the conservation and utilization of Yunnan indigenous goats.     

Spirocercosis-associated esophageal sarcomas in dogs. A retrospective study of 17 cases (1997-2003)

Seventeen client-owned dogs diagnosed with spirocercosis-associated esophageal sarcomas were retrospectively reviewed. The most common clinical signs noticed were vomiting and/or regurgitation (94%), lethargy and depression (59%), pyrexia and anorexia (41% each). Leukocytosis (82%) and microcytic hypochromic anemia (30%) were the most common hematological abnormalities. Caudal thoracic masses were demonstrated on survey radiographs of 13/15 of the dogs and thoracic spondylitis was detected in 12/15 dogs. Spirocerca lupi eggs were detected in 2/8 patients and worms were demonstrated on 1/11 at necropsy. Ten cases underwent surgical attempt to remove the tumors. In six of them partial esophagectomy (PE) was performed and all of them survived the immediate postoperative hospitalization. Five of the cases that underwent PE also received chemotherapy after surgery (doxorubicin (Adriamycin, Upjohn)) with an average survival time of 267 days. The histopathological results of the esophageal tumors were osteosarcoma (9), fibrosarcoma (5) and undifferentiated sarcoma (1). In areas endemic to spirocercosis, regurgitation or vomiting in dogs and microcytic hypochromic anemia and neutrophilia warrant ruling out esophageal sarcomas. Proper surgical treatment could prolong the dogs' lifespan for months, and improve their quality of life.     

湖南省猬迭宫绦虫的线粒体cox1和nad1基因的序列测定及种系发育分析

本研究旨在阐明猬迭宫绦虫湖南分离株线粒体细胞色素c氧化酶第I亚基(cox1)基因部分序列(pcox1)和烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位l基因(nad1)部分序列(pnad1)的遗传变异情况,并用pcox1和pnad1序列重构猬迭宫绦虫与其它绦虫的种群遗传关系。利用聚合酶链反应(PCR)扩增猬迭宫绦虫的pcox1和pnad1,应用ClustalX1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树,同时利用DNAStar5.0中的Megalign程序进行同源性分析。结果显示所获得各样品株的pcox1和pnad1序列长度一致,分别为396和566bp,湖南分离株与已知猬迭宫绦虫位于同一分枝。由于猬迭宫绦虫pcox1和pnad1序列种内相对保守,种间差异较大,故均可作为种间遗传变异研究的标记,从而为猬迭宫绦虫的种群体遗传学研究和其相关疾病的诊断奠定基础。     

Treatment of esophageal spirocercosis with milbemycin oxime

Six medium sized mixed breed dogs treated with milbemycin oxime (11.5mg on days 0, 7 and 28 and then monthly) stopped shedding Spirocerca lupi ova after 3-44 days. There was no evidence of subsequent shedding in repeated tests (about 5/dog) carried out from 31 to 133 days after initiation of treatment. Esophageal nodules disappeared in all dogs within 95-186 days and radiographic signs resolved within 85-127 days in the five dogs that were studied. This preliminary data shows milbemycin oxime deserves further evaluation for the treatment of spirocercosis.     

Spirocerca lupi isolated from gastric lesions in foxes (Vulpes vulpes) in Sicily (Italy)

Spirocerca lupi (Rudolphi 1809) is a cosmopolitan nematode of dogs and wild carnivores. In the past it has been reported in Italy, mainly in southern regions and in Sicily, where the parasite was observed in foxes in 2005. The parasite typically produces nodular masses in the oesophagus and thoracic aorta. During the 2003-2004 hunting season, the authors investigated a total of 55 foxes (Vulpes vulpes) hunted or killed by car accidents in the provinces of Palermo and Agrigento. All the foxes were subjected to necropsy and 6 (9.16%) had S. lupi nodules located exclusively in the gastric wall. The nature of the nodules was determined by opening them and detecting the nematodes inside, which were identified as S. lupi. Some of the nodules were characterized anatomopathologically and histopathologically. The formation of the parasitic nodule in the stomach only suggests a deviation from the route commonly followed by the nematode to reach the oesophagus, the elective anatomical site for completion of its lifecycle. This survey gives a contribution to the epidemiology of this parasite which is severely outdated in Italy and highlights some distinctive features of the life cycle and parasite migration.     

Cytochrome oxidase c subunit 1 polymorphisms show significant differences in distribution between a laboratory maintained population and a field isolate of Cooperia oncophora

A 474 bp fragment of the mitochondrial cytochrome oxidase c subunit 1 (cox1) of Cooperia oncophora was cloned and sequenced. The overall nucleotide diversity of the cox1 fragment varied from 0.5 to 2.0% between individuals. Two nucleotide substitutions were found within two RsaI endonuclease restriction sites and were used in a PCR-based restriction fragment length polymorphism (PCR-RFLP) assay to asses the intra-population variation of C. oncophora. Testing 816 individuals revealed the existence of three different haplotypes, having either both (type I) or only one (types II and III) RsaI site. Laboratory maintained individuals obtained at different time points after infection showed no significant difference in the distribution of the three haplotypes. Neither was there a difference in the distribution between male and female worms, confirming that the mitochondrial genome of C. oncophora is also maternally inherited. Nevertheless, there was a significant difference in the prevalence of the RsaI point mutation in the cox1 gene between the laboratory maintained population of C. oncophora and a Dutch field isolate, indicating that these RFLPs can be used to study genetic variation within or among C. oncophora populations.     

New insights into the morphology, molecular characterization and identification of Baylisascaris transfuga (Ascaridida, Ascarididae)

Species ranked within the genus Baylisascaris (Ascaridida, Ascarididae) have been implicated in clinical and subclinical intestinal diseases in their natural hosts (e.g., raccoons and bears) as well as in life-threatening larva migrans syndromes in a number of incidental hosts, including humans. Following the diagnosis of Baylisascaris transfuga infestation in two captive polar bears, living in the zoo park of Pistoia (Tuscany, Italy), nematodes (n=300; both sexes) have been characterized by morphological and molecular methods by sequencing and analysing ribosomal (large ribosomal DNA (28S) and internal transcribed spacer region 1 and 2 (ITSs)) and mitochondrial (cytochrome c oxidase subunit 1 (cox1) and cytochrome c oxidase subunit 2 (cox2)) target regions. In addition, seven faecal samples were collected from the animal enclosure and submitted to copromicroscopic and molecular examination. All nematodes were morphologically identified as B. transfuga and their main distinctive features are here presented. No variation in size and nucleotide polymorphisms was detected within each target sequence among all samples analysed. These data contribute to facilitate an accurate diagnosis of this little known nematode infestation in order to apply appropriate anthelmintic strategies.     

Highly pathogenic H5N1 avian influenza virus: Cause of the next pandemic?

Since 1997, when human infections with a highly pathogenic (HP) avian influenza A virus (AIV) subtype H5N1 – previously infecting only birds – were identified in a Hong Kong outbreak, global attention has focused on the potential for this virus to cause the next pandemic. From December 2003, an unprecedented H5N1 epizootic in poultry and migrating wild birds has spread across Asia and into Europe, the Middle East, and Africa. Humans in close contact with sick poultry and on rare occasion with other infected humans, have become infected. As of early March 2007, 12 countries have reported 167 deaths among 277 laboratory-confirmed human infections to WHO. WHO has declared the world to be in Phase 3 of a Pandemic Alert Period. This paper reviews the evolution of HP AIV H5N1, molecular changes that enable AIVs to infect and replicate in human cells and spread efficiently from person-to-person, and strategies to prevent the emergence of a pandemic virus.