Morphological description and molecular characterization of Contracaecum larvae (Nematoda: Anisakidae) parasitizing market-size hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and red drum (Sciaenops ocellatus) farmed in Israel

Nematodes belonging to the genus Contracaecum (family: Anisakidae) are heteroxenous parasites with a complex life cycle. Contracaecum larvae infecting farmed fish and fishery products are economically important causing market rejection in massive infection and may have zoonotic potential. In Israel, Contracaecum larvae have been described morphologically in several fish species; however, none of these descriptions were supported by molecular tools. In 2019–2020, hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and red drum (Sciaenops ocellatus), farmed in polyculture were found to be heavily infected with nematodes referable to Contracaecum larvae. Prevalence of infection in hybrid tilapia and red drum was 53.8% and 40.9%, respectively. A combined (morphological and molecular) approach revealed that both infected fish species were parasitized by the same species of Contracaecum, although larvae in hybrid tilapia were localized in the pericardial cavity whereas in red drum, they were observed in the abdominal cavity. Genetic analysis of internal transcribed spacer rDNA and cox2 mtDNA showed high similarity to unidentified Contracaecum larvae detected in several fish species in Ethiopia, Egypt and Kenya. In this study, molecular and morphological analyses place the possible new species in the C. multipapillatum complex and was provisionally named C. multipapillatum E. Further analyses combining morphological and molecular approaches are required on adult specimens collected from piscivorous birds living in the same area to support the identification of a potentially new species

Invasive parasites and global change: Evidence for the recent and rapid spillover of a potential pathogen of tilapias with a complex, three-host life cycle

Biological invasions pose a serious threat to local flora and fauna and have negative impacts on ecosystems. Invasive parasites can also cause severe losses in aquaculture. In this article, we provide evidence of the recent spillover of an African parasite with a complex, three-host life cycle that has rapidly and successfully established itself in the Middle East, most likely due to the recent migration of its final hosts (great cormorant) from Africa. This case of parasite introduction into a country with intensive aquaculture is also important from an economic point of view, since large (up to 2 cm long) larvae of this parasite, the cyclophyllidean tapeworm Amirthalingamia macracantha (Cestoda) localised in the liver, can be pathogenic to their fish hosts, including farmed and wild fish, as shown by our histopathological examination of heavily infected fish. Since its first detection in Israel in November 2020, the parasite has spread rapidly and is currently found in both migratory (great cormorant, Phalacrocorax carbo) and non-migratory birds (pygmy cormorant, Microcarbo pygmaeus), as well as in fish intermediate hosts, including farmed tilapia in several farms in Israel and wild cichlids. There are numerous examples of the spillover of introduced parasites, including those that parasitise fish of commercial importance, but have a direct life cycle or use only a single intermediate host. Tilapines are the second most important group of farmed fish in the world after carps and are produced mainly in Southeast Asia, Central and South America. The global spread of great cormorants and the early evidence that pygmy cormorant may also harbour A. macracantha pose the risk of further spread of this invasive parasite to other countries and areas. In addition, global warming and reductions in foraging and resting areas near these waters may allow the parasite to complete its life cycle in new hosts

Resistance of Leishmania infantum to allopurinol is associated with chromosome and gene copy number variations including decrease in the S-adenosylmethionine synthetase (METK) gene copy number

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL), a widespread, life-threatening disease. This parasite is responsible for the majority of human VL cases in Brazil, the Middle East, China, Central Asia and the Mediterranean basin. Its main reservoir are domestic dogs which, similar to human patients, may develop severe visceral disease and die if not treated. The drug allopurinol is used for the long-term maintenance of dogs with canine leishmaniasis. Following our report of allopurinol resistance in treated relapsed dogs, we investigated the mechanisms and markers of resistance to this drug. Whole genome sequencing (WGS) of clinical resistant and susceptible strains, and laboratory induced resistant parasites, was carried out in order to detect genetic changes associated with resistance. Significant gene copy number variation (CNV) was found between resistant and susceptible isolates at several loci, including a locus on chromosome 30 containing the genes LinJ.30.3550 through LinJ.30.3580. A reduction in copy number for LinJ.30.3560, encoding the S-adenosylmethionine synthetase (METK) gene, was found in two resistant clinical isolates and four induced resistant clonal strains. Using quantitative real time PCR, this reduction in METK copy number was also found in three additional resistant clinical isolates. Furthermore, inhibition of S-adenosylmethionine synthetase encoded by the METK gene in allopurinol susceptible strains resulted in increased allopurinol resistance, confirming its role in resistance to allopurinol. In conclusion, this study identified genetic changes associated with L. infantum resistance to allopurinol and the reduction in METK copy number identified may serve as a marker for resistance in dogs, and reduced protein activity correlated with increased allopurinol resistance. Keywords: Leishmania infantum, Dogs, Allopurinol, Drug resistance, S-adenosylmethionine synthetas

Allopurinol Resistance in <i>Leishmania infantum</i> from Dogs with Disease Relapse

<div><p>Background</p><p>Visceral leishmaniasis caused by the protozoan <i>Leishmania infantum</i> is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of <i>Leishmania</i> strains from dogs to allopurinol has not been described before in clinical studies.</p><p>Methodology/Principal Findings</p><p>Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of <i>L</i>. <i>infantum</i> isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group) showed an average half maximal inhibitory concentration (IC50) of 996 μg/mL. A significantly lower IC50 (<i>P</i> = 0.01) was found for isolates from ten dogs before treatment (NT group, 200 μg/mL), as well as for five isolates obtained from treated dogs in remission (TA group, 268 μg/mL). Axenic amastigotes produced from isolates of the TR group also showed significantly higher (<i>P</i> = 0.002) IC50 compared to the NT group (1678 and 671 μg/mL, respectively). The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (<i>P</i> = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol).</p><p>Conclusions</p><p>This is the first study to demonstrate allopurinol resistance in <i>L</i>. <i>infantum</i> and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant <i>L</i>. <i>infantum</i> strains may enhance uncontrolled transmission to humans and to other dogs.</p></div

Promastigote growth curves of strains from the non-treated (NT) and treated relapsed (TR) groups.

<p>No significant differences were found between promastigote counts for any of the isolates on respective days (Repeated measures ANOVA with Greenhouse-Geisser tests, <i>P =</i> 0.784).</p

Vector-borne pathogens in dogs from Costa Rica: first molecular description of Babesia vogeli and Hepatozoon canis infections with a high prevalence of monocytic ehrlichiosis and the manifestations of co-infection

Infection with canine vector-borne pathogens was evaluated in dogs from four different regions of Costa Rica by PCR. Demographic data, clinical signs, packed cell volume values, and the presence of tick infestation were recorded for each dog. Forty seven percent (69/146) of the dogs were infected with at least one pathogen and 12% were co-infected with two pathogens. Ehrlichia canis was detected in 34%, Anaplasma platys in 10%, Babesia vogeli in 8%, and Hepatozoon canis in 7.5% of the blood samples. No infection was detected with Leishmania spp. in blood, skin scrapings or conjunctival swabs. Thirty percent of the dogs presented at least one clinical sign compatible with vector-borne disease, and of those, 66% were infected with a pathogen. Subclinical infections were determined in 58% of the infected dogs including 82% (9/11), 58% (29/50), 42% (5/12) and 36% (5/14) of the dogs with H. canis, E. canis, B. vogeli and A. platys infections, respectively. A distinct relationship was found between infection and anemia. The mean PCV values were 34.4% in dogs with no infection, 31.5% in those who had a single infection and 23% in those with co-infection. Co-infected dogs had significantly lower PCV values compared to non-infected and single-infected dogs (p<. 0.0001). Thirty five percent (51/146) of the dogs were infested with ticks, 82% of them were infested with Rhipicephalus sanguineus sensu lato and 18% with Amblyomma ovale. Dogs infected with A. platys, B. vogeli, or E. canis were significantly associated with R. sanguineus s.l. infestation (p<. 0.029).This is the first description of infections with B. vogeli and H. canis in Costa Rica as well as in Central America. The results of this study indicate that multiple vector-borne pathogens responsible for severe diseases infect dogs in Costa Rica and therefore, increased owner and veterinarian awareness are needed. Moreover, prevention of tick infestation is recommended to decrease the threat of these diseases to the canine population.Se evaluó la infección con patógenos caninos transmitidos por vectores en perros de cuatro regiones diferentes de Costa Rica mediante la PCR. Para cada perro se registraron datos demográficos, signos clínicos, valores de volumen de células empaquetadas y la presencia de infestación de garrapatas. El cuarenta y siete por ciento (69/146) de los perros estaban infectados con al menos un patógeno y el 12% estaban coinfectados con dos patógenos. Se detectó Ehrlichia canis en el 34%, Anaplasma platys en el 10%, Babesia vogeli en el 8% y Hepatozoon canis en el 7,5% de las muestras de sangre. No se detectó ninguna infección con Leishmania spp. en la sangre, los raspados de piel o los hisopos conjuntivos. El 30% de los perros presentaron al menos un signo clínico compatible con la enfermedad transmitida por vectores, y de ellos, el 66% estaban infectados con un patógeno. Se determinaron infecciones subclínicas en el 58% de los perros infectados, incluyendo el 82% (9/11), el 58% (29/50), el 42% (5/12) y el 36% (5/14) de los perros con infecciones de H. canis, E. canis, B. vogeli y A. platys, respectivamente. Se encontró una relación distinta entre la infección y la anemia. Los valores medios de PCV fueron del 34,4% en los perros sin infección, del 31,5% en los que tenían una sola infección y del 23% en los que tenían una coinfección. Los perros coinfectados tuvieron valores de PCV significativamente más bajos comparados con los perros no infectados y con los infectados solos (p<. 0,0001). El treinta y cinco por ciento (51/146) de los perros estaban infestados de garrapatas, el 82% de ellos estaban infestados con Rhipicephalus sanguineus sensu lato y el 18% con Amblyomma ovale. Los perros infectados con A. platys, B. vogeli o E. canis estaban significativamente asociados con la infestación de R. sanguineus s.l. (p<. 0,029). Esta es la primera descripción de las infecciones con B. vogeli y H. canis en Costa Rica así como en América Central. Los resultados de este estudio indican que múltiples patógenos transmitidos por vectores, responsables de enfermedades graves, infectan a los perros en Costa Rica y, por lo tanto, es necesario aumentar la conciencia de los propietarios y los veterinarios. Además, se recomienda la prevención de la infestación por garrapatas para disminuir la amenaza de estas enfermedades para la población canina.Universidad Nacional, Costa RicaEscuela de Medicina Veterinari

Induction of allopurinol resistance in <i>Leishmania infantum</i> isolated from dogs

<div><p>Resistance to allopurinol in zoonotic canine leishmaniasis has been recently shown to be associated with disease relapse in naturally-infected dogs. However, information regarding the formation of resistance and its dynamics is lacking. This study describes the successful <i>in-vitro</i> induction of allopurinol resistance in <i>Leishmania infantum</i> cultured under increasing drug pressure. Allopurinol susceptibility and growth rate of induced parasites were monitored over 23 weeks and parasite clones were tested at selected time points and compared to their parental lines, both as promastigotes and as amastigotes. Allopurinol resistance was formed in strains from two parasite stocks producing a 20-fold rise in IC₅₀ along three distinct growth phases. In addition, characteristic differential clustering of single nucleotide polymorphisms (SNP) was found in drug sensitive and resistant parasite clones. Results confirm that genetic polymorphism, as well as clonal heterogeneity, contribute to <i>in-vitro</i> resistance to allopurinol, which is likely to occur in natural infection.</p></div

Spirocerca vulpis sp. nov. (Spiruridae: Spirocercidae): description of a new nematode species of the red fox, Vulpes vulpes (Carnivora: Canidae)

Previous studies have reported nematodes of the Spirocercidae family in the stomach nodules of red foxes (Vulpes vulpes) described as Spirocerca sp. or Spirocerca lupi (Rudolphi, 1819). We characterized spirurid worms collected from red foxes and compared them to S. lupi from domestic dogs by morphometric and phylogenetic analyses. Nematodes from red foxes differed from S. lupi by the presence of six triangular teeth-like buccal capsule structures, which are absent in the latter. Additionally, in female worms from red foxes, the distance of the vulva opening to the anterior end and the ratio of the glandular-to-muscular oesophagus lengths were larger than those of S. lupi (P &lt; 0.006). In males, the lengths of the whole oesophagus and glandular part, the ratio of the glandular-to-muscular oesophagus and the comparison of the oesophagus to the total body length were smaller in S. lupi (all P &lt; 0.044). Phylogenetic analyses revealed that S. lupi and the red foxes spirurid represent monophyletic sister groups with pairwise nucleotide distances of 9.2 and 0.2% in the cytochrome oxidase 1 and 18S genes, respectively. Based on these comparisons, the nematodes from red foxes were considered to belong to a separate species, for which the name Spirocerca vulpis sp. nov. is proposed

Allopurinol susceptibility of <i>L</i>. <i>infantum</i> parental strains and clones at different time points during induction of resistance.

<p>(A, B) Results for promastigotes, numbers indicate IC₅₀ values of the post-thaw parent cultures at the respective time points. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005910#pntd.0005910.s001" target="_blank">S1 Table</a>. (C) Allopurinol percent inhibition results for intracellular amastigotes. An induced resistant culture, 5 of its respective clones and its drug-free control culture were tested for each parental line (NT4.L and NT5.L) at one time point (day 104 and 86, respectively). Values indicated by distinct letters differ significantly (Tukey HSD test, p<0.05).</p