Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States.

We report 2 autochthonous feline thelaziosis cases caused by the eyeworm Thelazia callipaeda and discuss the spread among dogs in the northeastern United States. Phylogenetic analysis suggests the parasite was introduced from Europe. Adopting a One Health approach is needed to limit further spread of T. callipaeda eyeworms in North America.

Q3 lab-on-chip real-time PCR for the diagnosis of Leishmania infantum infection in dogs.

Leishmaniasis is a vector-borne disease caused by many Leishmania spp. which infect humans and other mammalian hosts. Leishmania infantum is the main agent of canine leishmaniasis (CanL) whose diagnosis is usually confirmed by serological and molecular tests. This study aimed to evaluate the clinical and analytical sensitivities of a lab-on-chip (LOC) real-time PCR applied on the portable Q3-Plus V2 platform (Q3 qPCR) in the detection of L. infantum. The Q3 qPCR performance was assessed by comparing the quantification cycle (Cq) values with those obtained using the qPCR run on a CFX96 Real-Time System (CFX96 qPCR). A total of 173 DNA samples (extracted from bone marrow, lymph node, blood, buffy coat, conjunctival swab, and skin) as well as 93 non-extracted samples (NES) (bone marrow, lymph node, blood, and buffy coat) collected from dogs were tested with both systems. Serial dilutions of each representative DNA and NES sample were used to assess the analytical sensitivity of the Q3 qPCR system. Overlapping Cq values were obtained with the Q3 qPCR and CFX96 qPCR, both using DNA extracted from L. infantum promastigotes (limit of detection, <1 promastigote per milliliter) and from biological samples as well as with NES. However, the Q3 qPCR system showed a higher sensitivity in detecting L. infantum in NES as compared with the CFX96 qPCR. Our data indicate that the Q3 qPCR system could be a reliable tool for detecting L. infantum DNA in biological samples, bypassing the DNA extraction step, which represents an advance in the point-of-care diagnostic of CanL.

Candida auris from the Egyptian cobra: Role of snakes as potential reservoirs.

Candida auris represents one of the most urgent threats to public health, although its ecology remains largely unknown. Because amphibians and reptiles may present favorable conditions for C. auris colonization, cloacal and blood samples (n = 68), from several snake species, were cultured and molecularly screened for C. auris using molecular amplification of glycosylphosphatidylinositol protein-encoding genes and ribosomal internal transcribed spacer sequencing. Candida auris was isolated from the cloacal swab of one Egyptian cobra (Naja haje legionis) and molecularly identified in its cloaca and blood. The isolation of C. auris from wild animals is herein reported for the first time, thus suggesting the role that these animals could play as reservoirs of this emerging pathogen. The occurrence of C. auris in blood requires further investigation, although the presence of cationic antimicrobial peptides in the plasma of reptiles could play a role in reducing the vitality of the fungus.

Candida auris represents one of the most urgent threats to public health. In this study, we reported for the first time the isolation of C. auris from snake thus suggesting the role of these animals as reservoirs of this emerging pathogen.

Microfilaruria of Dirofilaria immitis in a dog from Italy.

Dirofilaria immitis is a mosquito-borne nematode-causing canine heartworm disease, with adult worms localized in the pulmonary arteries and right heart. In rare cases, ectopic migration might occur, and adults and blood circulating microfilariae can be found in unusual organs or fluids (e.g., eyes, abdominal cavity, bone marrow, and urine). A 17-year-old mixed-breed female dog was presented in a private veterinary clinic in Italy for hematuria and dysuria. Physical examination showed cardiac mitral murmur with marked respiratory distress and cyanotic mucous membranes after handling. Abdominal ultrasounds revealed a non-specific chronic cystopathy, while the echocardiography showed enlargement of the right heart associated with tricuspid insufficiency and mitral regurgitation, with the presence of an adult filariae in the right ventricular chamber. Circulating microfilariae were observed in the blood smear and molecularly identified as D. immitis. Unusual microfilaruria was detected in the urine sediment. Data presented raise awareness about the occurrence of microfilariae in unusual locations, such as the bladder, suggesting the need of a thorough clinical and laboratory assessment where D. immitis is endemic.

Prevention of heartworm infection in dogs using a combination of moxidectin, imidacloprid and praziquantel: evidence from a randomized clinical trial.

The aim of this study was to evaluate the efficacy of a topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% for the prevention of Dirofilaria immitis (Leidy, 1856) infection in dogs. For this purpose, a randomized and controlled clinical trial was conducted between August 2021 and October 2022, in the municipality of Goiana, state of Pernambuco, north-eastern Brazil, where heartworm is highly prevalent. Of the 213 dogs initially sampled (baseline), 68 (31.9%) were positive for adult antigens (SNAP 4Dx Plus, Idexx) and/or microfilariae (modified Knott's test). On day 0, 140 negative dogs were randomly included in the treatment and control groups, 70 animals each. During the study, 60 dogs (34 treated and 26 untreated) were removed for different reasons. At the end of the study (day 360 ± 2), 36 treated and 44 untreated were sampled and included in the efficacy calculation. The efficacy against the development of adults and microfilariae was 84.7%, with only one treated dog being positive for adult antigens but negative for microfilariae. On the other hand, eight untreated dogs were positive for adult antigens and/or microfilariae, resulting in a significant difference in the number of positives between groups (Chi-square test = 4.706, df = 1, P = 0.0301). Remarkably, the efficacy against the appearance of D. immitis microfilariae was 100% (i.e., all treated dogs negative) and three untreated dogs were positive for microfilariae. The topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% significantly reduced the risk of D. immitis infection in treated dogs as compared with untreated dogs, in a highly endemic area in north-eastern Brazil.

Detection of Trypanosoma evansi in jaguars (Panthera onca): insights from the Brazilian Pantanal wetland.

Trypanosoma evansi is a widespread and neglected zoonotic parasite that affects domestic and wild animals, causing a disease commonly known as "surra." The Brazilian Pantanal wetland is recognized as an enzootic area for this protozoan, yet recognizing the importance of reservoir hosts also in order to prevent zoonotic outbreaks. This study aimed to assess the occurrence of T. evansi in jaguars (Panthera onca) from the Brazilian Pantanal wetland and explore associated clinical and hematological manifestations. A total of 42 animals were screened by PCR and sequenced for species identification when positive. Trypanosoma evansi was detected in six free-ranging jaguars (six positive animals of 42 captures and 16 recaptures), representing the first molecular evidence of such infection in this animal species. Our findings suggest that jaguars may act as reservoir hosts of T. evansi in the Brazilian Pantanal wetland. The better understanding of the role of wildlife in the epidemiology of T. evansi is also of importance to future reintroduction and translocation programs toward wildlife conservation efforts.

Dirofilaria immitis and Onchocercidae spp. in wild felids from Brazil.

Among the species described within the Onchocercidae family, Dirofilaria immitis is regarded as the most common worldwide, causing severe and often fatal conditions in dogs, cats, and occasionally humans. Dirofilaria spp. are vectored by mosquitoes, simulids, and culicoids, with their epidemiology dependent on the geographical distribution of competent vectors. Eight species of Dirofilaria have been reported so far in Brazil, of which six parasitize non-human primates, deer, procyonids, and marsupials. Here, we investigated the occurrence of Onchocercidae in wild felids (i.e., Panthera onca, Puma concolor, Herpailurus yagouaroundi, Leopardus geoffroyi, Leopardus guttulus, Leopardus pardalis, Leopardus wiedii, Leopardus munoai) from different locations in Brazil. Overall, 82 samples (n = 63 blood; n = 19 tissues) were molecularly screened for cytochrome c oxidase subunit-1 (cox1) gene. Four (i.e., 4.8%) wild felid samples were positive, and at BLAST analysis, the obtained sequences showed varying percentage of nucleotide identity with the genera Brugia (i.e., 87-88%), Setaria (i.e., 89%), and D. immitis (i.e., 94.4%). Phylogenetic analyses clustered sequences obtained into three distinct clades, one with D. immitis and the remaining two with other Onchocercidae spp. Data herein obtained highlight the need for a more comprehensive understanding of the diversity and biology of Onchocercidae in South America in order to assess the potential impact that these species may have for domestic and wild animals, as well as humans.

Rickettsia conorii Subspecies israelensis in Captive Baboons.

Hamadryas baboons (Papio hamadryas) may transmit zoonotic vector-borne pathogens to visitors and workers frequenting zoological parks. We molecularly screened 33 baboons for vector-borne pathogens. Three (9.1%) of 33 animals tested positive for Rickettsia conorii subspecies israelensis. Clinicians should be aware of potential health risks from spatial overlapping between baboons and humans.

Prevention of Thelazia callipaeda Reinfection among Humans.

Thelazia callipaeda is a zoonotic vector-borne nematode that infects and causes eye disease among a wide range of domestic and wild mammals, including humans. We describe an unusual case of reinfection by this nematode in Serbia and call for a focus on preventive measures in endemic areas.

Elimination of Dirofilaria immitis Infection in Dogs, Linosa Island, Italy, 2020-2022.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease-free area.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

In vitro maintenance of the endosymbiont Wolbachia of Dirofilaria immitis.

Wolbachia has an obligatory mutualistic relationship with many onchocercid nematodes of the subfamilies Dirofilariinae and Onchocercinae. Till date, no attempts have been made for the in vitro cultivation of this intracellular bacterium from the filarioid host. Hence, the current study attempted cell co-culture method using embryonic Drosophila S2 and the LD cell lines to cultivate Wolbachia from Dirofilaria immitis microfilariae (mfs) harvested from infected dogs. Microfilariae (mfs = 1500) were inoculated in shell vials supplemented with Schneider medium using both cell lines. The establishment and multiplication of the bacterium were observed during the initial inoculation, at day 0 and before every medium change (from days 14 to 115). An aliquot (50 µl) from each time point was tested by quantitative real-time PCR (qPCR). Comparing the average of Ct values, obtained by the tested parameters (i.e., LD/S2 cell lines and mfs with/without treatment), the S2 cell line without mechanical disruption of mfs provided the highest Wolbachia cell count by qPCR. Despite the maintenance of Wolbachia within both S2 and LD-based cell co-culture models for up to 115 days, a definitive conclusion is still far. Further trials using fluorescent microscopy and viable staining will help to demonstrate the cell line infection and viability of Wolbachia. Use of considerable amount of untreated mfs to inoculate the Drosophilia S2 cell lines, as well as the supplementation of the culture media with growth stimulants or pre-treated cells to increase their susceptibility for the infection and development of a filarioid-based cell line system are recommended for the future trials.

Tracking the existence of Dioctophyma renale in Parthian Empire of Iran (247 BC-224 AD).

Paleoparasitological investigation in the archeological site of the Caspian Sea littoral revealed the presence of human Dioctophyma renale infection, a ubiquitous kidney-residing helminth of mammals, during the Parthian Empire of Iran (247 BC-224 AD). This study reports the oldest human infection with giant kidney worm in the Middle East, mainly in an area where the infection is reported nowadays. The paucity of records throughout the literature suggests that the same species of parasite occurred with low frequency in the past, as well. The cemetery in the archeological site of Kiasar represented a unique opportunity of studying human and animal parasites of the Parthian Empire in Iran.

Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile.

The genus Spirocerca includes nematodes that parasitize the stomach and the oesophagus of carnivores, chiefly canids. Herein, we provide new data about the morphological, histopathological, and molecular characterization of Spirocerca sp. in Andean foxes (Lycalopex culpaeus) in Chile. Intact immature worms, identified as Spirocerca sp., were recovered in the lumen of the stomach from two foxes. Histologically, worms morphologically consistent with spirurid nematodes were present within the wall of the stomach and surrounded by nodular areas of inflammation with central necrotic debris. Molecular analysis of the cox1 gene yielded 19 sequences and 5 nucleotide sequence types with 99.95 to 99.98% similarity, being shared between both foxes. Nucleotide similarity ranged from 93.1 (with genotype 2 of S. lupi and S. vulpis) to 95.8% (with genotype 1 of S. lupi), a higher similarity than noted from sequences of S. lupi from an Andean fox from Peru (91.0 to 93.3%). However, the Poisson Tree Processes for species delineation did not support the existence of a new species Spirocerca. Phylogenetic and nucleotide analyses suggest that these specimens belong to a new variant or genotype of S. lupi or to a cryptic species. Whether the presence of the worms in the stomach has to do with genotypic differences in parasites or host or some combination is uncertain. Spirocerca lupi has never been found in Chilean dogs and must be investigated.

The jaguar (Panthera onca) as a potential new host of Dracunculus sp.

Nematode species of the genus Dracunculus (Spirurida: Dracunculoidea) infect tissues and body cavities of reptiles, domestic and wild carnivores, and humans. The definitive hosts acquire the infection by ingesting intermediate (i.e., cyclopoid copepod) or paratenic (i.e., amphibians and fishes) hosts. Here we report the jaguar (Panthera onca) as a potential new host for Dracunculus sp. The nematode was collected from an ulcerated cutaneous nodule on the left anterior limb of a female jaguar in the municipality of Miranda, Mato Grosso do Sul state, Brazil. Based on the morphology of first stage larvae collected from a small fragment of the uterus of the adult nematode, the species was identified as Dracunculus sp. Reichard, 1759. Additionally, the morphological identification was molecularly confirmed by sequencing the cox1 gene. This report advocates for further investigations into the transmission cycle of this parasite in the Brazilian Pantanal wetland, considering the role of wildlife hosts and the zoonotic potential of Dracunculus species in that area.

Pathological findings associated with Dipetalonema spp. (Spirurida, Onchocercidae) infection in two species of Neotropical monkeys from Brazil.

Among vector-borne helminths, filarioids of the genus Dipetalonema (Spirurida: Onchocercidae) localize in several tissues and body cavities of several animal species, causing mild to moderate lesions. The pathological findings associated with Dipetalonema spp. infection in Neotropical monkeys from southern Brazil are herein described, along with a fatal case due to filarial polyserositis and entrapment of an intestinal segment. At necropsy, nematodes were observed in abdominal and thoracic cavities, or in the pericardium of 37 (31.3%) out of the 118 individuals examined (i.e., 35 Alouatta guariba clamitans and two Sapajus nigritus). In addition, at histology, 27.0% of positive animals presented microfilarie (inside blood vessels of lung, spleen, liver, and brain) and 8.1% presented adult nematodes in the heart, lung, and liver. In two cases, cross-sections of filarioids were associated with areas of epicardial thickening with intense fibrosis and pyogranulomatous inflammation in the brain, heart, liver, lungs, or spleen. The DNA fragment was amplify using the cox1 gene, sequenced and analyzed to identify the nematode species collected; presence of Wolbachia was assessed in the filarioids using the 16S rRNA gene. At BLAST analysis of the cox1 gene, 10 sequences showed 91.7% nucleotide identity with Dipetalonema gracile, and two with D. gracile (98.5%) and Dipetalonema graciliformis (98.3%). Phylogenetic analyses clustered sequences of the cox1 obtained in this study in two clades corresponding with the host species. Wolbachia sp. endosymbiont was detected in four samples. Data herein reported provide a description of pathological lesions associated with the infection by Dipetalonema spp., suggesting that they may cause disease in Neotropical monkeys. In addition, a better understanding of diversity and biology of Dipetalonema spp. in South America is needed to assess the impact they may cause in native non-human primates from Brazil.

Detection of Endosymbiont Candidatus Midichloria mitochondrii and Tickborne Pathogens in Humans Exposed to Tick Bites, Italy.

During 2021, we collected blood and serum samples from 135 persons exposed to tick bites in southern Italy. We serologically and molecularly screened for zoonotic tickborne pathogens and only molecularly screened for Candidatus Midichloria mitochondrii. Overall, 62 (45.9%) persons tested positive for tickborne pathogens. Coxiella burnetii was detected most frequently (27.4%), along with Rickettsia spp. (21.5%) and Borrelia spp. (10.4%). We detected Candidatus M. mitochondrii DNA in 46 (34.1%) participants who had statistically significant associations to tickborne pathogens (p<0.0001). Phylogenetic analysis of Candidatus M. mitochondrii sequences revealed 5 clades and 8 human sequence types that correlated with vertebrates, Ixodes spp. ticks, and countries in Europe. These data demonstrated a high circulation of tickborne pathogens and Candidatus M. mitochondrii DNA in persons participating in outdoor activities in southern Italy. Our study shows how coordinated surveillance among patients, clinicians, and veterinarians could inform a One Health approach for monitoring and controlling the circulation of tickborne pathogens.

Comparison of virulence factors and susceptibility profiles of Malassezia furfur from pityriasis versicolor patients and bloodstream infections of preterm infants.

In spite of the increasing medical interest in Malassezia yeasts, the virulence factors of Malassezia furfur causing bloodstream infections (BSI) were never investigated. Therefore, phospholipase (Pz), lipase (Lz), hemolysin (Hz), biofilm production, and in vitro antifungal susceptibility profiles were evaluated in M. furfur strains, isolated from both pityriasis versicolor (PV) patients (n = 18; Group 1) or from preterm infants BSI (n = 21; Group 2). All the test stains exhibited Pz activity, whereas 92.3% and 97.4% of strains exhibited Lz and Hz activities, respectively. Pz, Lz, and Hz activities were higher (i.e., lower values) within Group 1 strains (i.e., 0.48, 0.40, and 0.77) than those within Group 2 (i.e., 0.54, 0.54, and 0.81). The biofilm production was higher within Malassezia isolates from Group 2 (0.95 ± 0.3) than from Group 1 (0.72 ± 0.4). Itraconazole and posaconazole were the most active drugs against M. furfur, followed by amphotericin B and fluconazole. The minimum inhibitory concentrations (MIC) values varied according to the origin of M. furfur strains being statistically lower in M. furfur from Group 1 than from Group 2. This study suggests that M. furfur strains produce hydrolytic enzymes and biofilm when causing PV and BSI. Data show that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur BSI, whereas lipase and hemolytic activities might display a synergic role in skin infection.

There is no information on the virulence factors of M. furfur involved in invasive infections. Our data suggest that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur blood-stream infections.

Description of Joyeuxiella pasqualei (Cestoda: Dipylidiidae) from an Italian domestic dog, with a call for further research on its first intermediate host.

Although Joyeuxiella pasqualei is frequently detected in cats from Mediterranean Europe, information on its biology is still scarce. This cestode is relatively less frequently reported in dogs, possibly because it is often misdiagnosed with the better-known Dipylidium caninum. The occurrence of J. pasqualei proglottids in a dog living in a closed environment triggered us to delve into the biology of this cestode by collecting biological samples from lizards and a road-killed cat. Two reptile species, Podarcis siculus (Lacertidae), and Tarentola mauritanica (Geckonidae) were also collected in the garden and its surroundings. In addition, experimental infections with eggs obtained from gravid proglottids were performed in laboratory mice, and Tenebrio molitor (Coleoptera: Tenebrionidae) beetles. Proglottids from the dog's feces and adult cestodes detected at necroscopy of a cat were morphologically identified as J. pasqualei. Two out of 13 T. mauritanica collected in the garden had natural infections of J. pasqualei cysts in the liver and attached to the intestine. All P. siculus lizards and experimentally infected rodents and beetles were negative. DNA sequences obtained from J. pasqualei showed the highest nucleotide similarities with Versteria sp., Echinococcus sp., Raillietina sonini, Taenia polyacantha and D. caninum. Data herein provided show the inability of rodents to become infected by direct ingestion of gravid proglottids, suggesting a need for an invertebrate first intermediate host in the life cycle. Thus, more research study is advocated to better understand the biology of J. pasqualei such as its first intermediate host and its mechanism of transmission in reptiles and rodents.

Onchocerca lupi in imported dogs in the UK: implications for animal and public health.

BACKGROUND: Onchocerca lupi is a filarial nematode affecting dogs, and occasionally cats and humans, in continental Europe, North Africa, the Middle East, and the USA. Adult worms are usually found in periocular nodules and enucleation is sometimes required if the infection fails to respond to other treatment options. CASE PRESENTATION: Here, we report the presence of O. lupi in the UK for the first time. Of two dogs re-homed from continental Europe, one developed an ocular nodule seven years after arrival from Portugal. The conjunctival perilimbal mass in its left eye was surgically removed but despite anthelminthic treatment, a further nodule developed in the same eye six months later. In the second case - a dog imported from Romania 12 months earlier - a perilimbal mass was excised from the left eye and prior anthelminthic treatment was supplemented with oral prednisolone and doxycycline. However, nodules recurred, and the left globe was subsequently enucleated. Conjunctival hyperaemia then appeared in the right eye and neither additional anthelminthic treatment nor removal of worm masses failed to prevent the further development of lesions. Excised adult worms were identified in both cases as O. lupi based on morphological characteristics, as well as PCR and sequencing of cytochrome c oxidase subunit I and 12S rRNA gene fragments. CONCLUSION: O. lupi parasitosis can apparently remain cryptic in dogs for several years before any clinical signs manifest. Moreover, the progression of infection can be highly aggressive and recalcitrant to both surgical intervention and anthelminthic treatment. Increasingly, former stray dogs of unknown infection status are entering the UK, raising both veterinary and public health concerns.