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Abstract Background Stephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans) intermediate host. Despite being relatively common, little attention has been given to a thorough description of S. stilesi lesions and the potential integration of pathological and molecular diagnostic findings to confirm infection. Methods To characterize the cutaneous lesions caused by S. stilesi in cattle (Bos taurus taurus and Bos taurus indicus), skin of the ventral abdominal midline was collected from 22 animals during postmortem examination. Skin samples were processed for histology, transmission electron microscopy (TEM), DNA extraction, PCR, and Sanger sequencing targeting molecular markers cytochrome oxidase c subunit 1 (cox1), 12S, 18S rDNA, and 28S rDNA. Results Macroscopically, lesions ranged from 5 × 4 cm to 36 × 10 cm, consisting of one large single lesion, or two to four ovoid areas at the ventral abdominal midline, surrounding the umbilicus. Each lesion presented as ulcerative dermatitis with dry, serocellular crusts, or alopecic and lichenified areas. Histologically, eosinophilic, neutrophilic, and ulcerative dermatitis with furunculosis, folliculitis, and epidermal hyperplasia was observed. Cross sections of adult nematodes were identified in ~ 60% of the cases (n = 13) within intact follicles, sebaceous ducts, crusts, and areas of furunculosis. Stephanofilaria first-stage larvae (L1) were observed in five cases within “vitelline membranes” in the superficial dermis and crusts. Ultrastructurally, the L1 cross sections were compounded of smooth multilayered cuticle and somatic cells. The “vitelline membrane” is a tri-layered membrane where L1 are suspended in a matrix. Stephanofilaria stilesi DNA was found in 5 out of the 13 cases in which adults or L1 were histologically observed (38%) and in 1 out of the 9 cases without adults or L1 present (11%). Phylogenetic analyses suggest a closer relationship of the genus Stephanofilaria with Thelazioidea, instead of the family Filariidae (Filarioidea), in which it has been historically allocated. Conclusions Our study improved the characterization of lesions and described ultrastructural findings of S. stilesi and highlights that molecular tools should be utilized in combination with histology for improved diagnostic resolution. Graphical Abstract

Background: Stephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans) intermediate host. Despite being relatively common, little attention has been given to a thorough description of S. stilesi lesions and the potential integration of pathological and molecular diagnostic findings to confirm infection. Methods: To characterize the cutaneous lesions caused by S. stilesi in cattle (Bos taurus taurus and Bos taurus indicus), skin of the ventral abdominal midline was collected from 22 animals during postmortem examination. Skin samples were processed for histology, transmission electron microscopy (TEM), DNA extraction, PCR, and Sanger sequencing targeting molecular markers cytochrome oxidase c subunit 1 (cox1), 12S, 18S rDNA, and 28S rDNA. Results: Macroscopically, lesions ranged from 5 × 4 cm to 36 × 10 cm, consisting of one large single lesion, or two to four ovoid areas at the ventral abdominal midline, surrounding the umbilicus. Each lesion presented as ulcerative dermatitis with dry, serocellular crusts, or alopecic and lichenified areas. Histologically, eosinophilic, neutrophilic, and ulcerative dermatitis with furunculosis, folliculitis, and epidermal hyperplasia was observed. Cross sections of adult nematodes were identified in ~ 60% of the cases (n = 13) within intact follicles, sebaceous ducts, crusts, and areas of furunculosis. Stephanofilaria first-stage larvae (L1) were observed in five cases within "vitelline membranes" in the superficial dermis and crusts. Ultrastructurally, the L1 cross sections were compounded of smooth multilayered cuticle and somatic cells. The "vitelline membrane" is a tri-layered membrane where L1 are suspended in a matrix. Stephanofilaria stilesi DNA was found in 5 out of the 13 cases in which adults or L1 were histologically observed (38%) and in 1 out of the 9 cases without adults or L1 present (11%). Phylogenetic analyses suggest a closer relationship of the genus Stephanofilaria with Thelazioidea, instead of the family Filariidae (Filarioidea), in which it has been historically allocated. Conclusions: Our study improved the characterization of lesions and described ultrastructural findings of S. stilesi and highlights that molecular tools should be utilized in combination with histology for improved diagnostic resolution. [ABSTRACT FROM AUTHOR]

Over the last century, only two cases of ocular oxyspirurosis were recorded in primates, both in zoos, and two species were described: in Berlin, Germany, Oxyspirura (O.) conjunctivalis from the lemurid Microcebus murinus, later also found in the lorisid Loris gracilis; in Jacksonville, Florida, O. (O.) youngi from the cercopithecid monkey Erythrocebus patas. In the present case from the Moscow zoo, oxyspirurosis was recorded in several species of Old World lemuriforms and lorisiforms, and some South American monkeys. i) The intermediate host was discovered to be a cockroach, as for O. (O.) mansoni, a parasite of poultry. The complete sequence identity between ITS-1 rDNA from adult nematodes of the primate and that of the larval worms from the vector, Nauphoete cinerea, confirmed their conspecificity. ii) Parasites from Moscow zoo recovered from Nycticebus c. coucang were compared morphologically to those from other zoos. The length and shape of the gubernaculum, used previously as a distinct character, were found to be variable. However, the vulvar bosses arrangement, the distal extremity of left spicule and the position of papillae of the first postcloacal pair showed that the worms in the different samples were not exactly identical and that each set seemed characteristic of a particular zoo. iii) The presence of longitudinal cuticular crests in the infective stage as well as in adult worms was recorded. Together with several other morphological and biological characters (long tail and oesophagus, cockroach vector), this confirmed that Oxyspirura is not closely related to Thelazia, another ocular parasite genus. iv) The disease in the Moscow zoo is thought to have started with Nycticebus pygmaeus imported fromVietnam, thus the suggestion was that Asiatic lorisids were at the origin of the Moscow set of cases. The natural host(s) for the Berlin and Jacksonville cases remain unknown but they are unlikely to be the species found infected in zoos. Consequently the notion of type hosts is artificial and the three agents of oxyspirurosis are provisionally placed in the taxon O. (O.) conjunctivalis.

Abstract: This study investigated genetic variability within the ‘eyeworm’Thelazia callipaeda (Nematoda: Thelazioidea) from Europe and Asia by polymerase chain reaction (PCR)-coupled sequencing and mutation scanning of the mitochondrial cytochrome c oxidase subunit 1 gene (cox 1). Eight different sequence variants of cox1 (haplotypes) were determined for the 50 individual adult specimens of T. callipaeda (from dogs, foxes or cats from Italy, Germany and the Netherlands and from dogs from China and Korea). Nucleotide variation (0.3–2%) was detected at 23 of 649 positions in the cox1. Six of these positions were invariable among all 37 individuals from Europe and among the 13 individuals from Asia (irrespective of host origin) but differed (five G↔A and one C↔T changes) between Europe and Asia. PCR-based single-strand conformation polymorphism (SSCP) analysis of the most variable portion (v-cox1) of the cox1 was validated (for a subset of samples) as a tool to rapidly screen for genetic (haplotypic) variability. The results for the SSCP analysis and sequencing were concordant, indicating that the mutation scanning approach provides a useful tool for investigating the population genetics and molecular ecology of T. callipaeda. [Copyright &y& Elsevier]