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.

Cryptosporidium parvum gp60 subtypes in diarrheic lambs and goat kids from Israel.

Cryptosporidium parvum is the second-most prevalent Cryptosporidium species that infects humans worldwide. In European countries, it is the most prevalent species in sheep, suggesting that these animals are a source of zoonotic infection. Preweaned lambs and goats are particularly susceptible to infection by the parasite and may suffer from severe diarrhea whilst excreting large quantities of infectious oocysts. Fifty fecal samples from preweaned lambs and goats with diarrhea from 35 farms across Israel, found to be Cryptosporidium-positive by microscopy, were tested by PCR and sequence analyses to determine the infective species and subtypes. Cryptosporidium parvum DNA was detected in most samples from both lambs and goats (46/50). Cryptosporidium xiaoi DNA was detected in three samples from kids, with co-infection detected in a single sample. Eleven different C. parvum subtypes were found, 10 in lambs and 5 in goats. All subtypes were from the IIa and IId subtype families, with subtypes IIdA20G1 and IIaA15G2R1 being the most prevalent and widespread. These subtypes were previously found in calves and humans in Israel and are considered the most prevalent C. parvum subtypes in small ruminants globally. These results underline the zoonotic potential of C. parvum from small ruminants and the high subtype diversity compared to previous reports from other Middle Eastern countries. In addition, this is the first report of C. xiaoi in Israel.

Comparison of multiplex copro PCR with coproscopy followed by PCR on recovered eggs for the detection of Echinococcus granulosus and Taenia spp. infection in dogs.

Echinococcosis and taeniasis are important helminth diseases that carry considerable impact on human and animal health. Domestic dogs and other canids are definitive hosts for several parasites of this group, including Echinococcus granulosus, Taenia multiceps, T. ovis, T. hydatigena and E. multilocularis. Detection of infection in dog populations is imperative for estimating the risk to susceptible humans and animals, and for its mitigation through prevention measures in dogs, other animals and humans. To date, identification of taeniid eggs, antigens or DNA in fecal samples are the most practical diagnostic modalities available for canine definitive hosts. Although widely used for this purpose, there is limited information comparing copro PCR and combined coproscopy-PCR protocols for the detection of taeniids. In the current study, a widely used multiplex PCR was performed on a large number of dog fecal samples using DNA extracted directly from feces. The samples were also tested by fecal flotation and coproscopy, eggs were isolated from microscopically-positive samples and extracted DNA was tested using the same multiplex PCR. The total number of taeniid positive samples detected using both methods was 46/317 (14.5%), including 10/317 (3.2%) E. granulosus positive samples. Both copro PCR and coproscopy have identified an equal number of samples as taeniid positive (n = 32). However, for the purpose of identification to species level, the copro PCR was significantly more sensitive than coproscopy followed by PCR on isolated eggs (sensitivity 0.7 vs. 0.41, p = 0.012), with 32/317 (10.1%) and 19/317 (6%) positive samples identified, respectively. The difference in identification of E. granulosus was highly apparent, as the majority of the E. granulosus positive samples (8/10) were detected by the copro PCR only. Coproscopy and egg PCR have identified 5/317 (1.6%) positive samples not detected by the copro PCR, including only a single sample (0.3%) positive for E. granulosus. Adding these positive samples to those identified by the copro PCR did not significantly improve the overall sensitivity (p = 0.074). Therefore, using both copro PCR and coproscopy in parallel may not be advantageous for taeniid detection and identification, at least until the egg PCR is further optimized and performs better. These results should be weighed against the different advantages that coproscopy based approach may offer.

Spinal cord protothecosis causing paraparesis in a dog.

Protothecosis, an infectious disease caused by the green algae Prototheca zopfii and P. wickerhamii, occurs sporadically in domestic animals and humans. Diagnosis of CNS protothecosis is based on neurologic signs that indicate multifocal nervous system lesions and that follow a period of chronic diarrhea and weight loss, cytologic observation of algae in fecal culture or histopathology, and detection of the agent by PCR assay of infected tissues. Here, we report a case of a paraparetic dog with CNS protothecosis that was diagnosed definitively antemortem using CSF cytology, PCR, and DNA sequencing. A 4-y-old mixed-breed dog developed progressive paraparesis that followed weight loss and diarrhea. CSF analysis revealed marked eosinophilic pleocytosis. Prototheca organisms were detected by microscopic examination of the CSF, and speciated as P. zopfii by CSF PCR and DNA sequencing. Other possible causes of paraparesis were ruled out using computed tomography, serology, and CSF PCR. The dog's condition deteriorated despite treatment, developing forebrain and central vestibular system clinical signs, and it was euthanized at the owner's request. Postmortem examination was declined. Our findings indicate that when CNS protothecosis is suspected, antemortem diagnosis can be made using CSF analysis and a PCR assay.

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.

Cryptosporidium parvum subtypes from diarrheic dairy calves in Israel.

Cryptosporidium are protozoan parasites with worldwide distribution, infecting a wide range of terrestrial and aquatic animals, as well as humans. Cryptosporidium parvum is the most important zoonotic species and is the primary cause of cryptosporidiosis in preweaned calves, a highly prevalent, economically important disease. Extensive subtyping of C. parvum from infected humans and animals has expanded current understanding of the parasites' epidemiology. Israel has a highly developed dairy sector with intensive, zero-grazing operations. While C. parvum has been found in dairy calves throughout the country, and subtype data from human patients have also been published, subtype data from animals, and in particular preweaned ruminants, are lacking. We carried out an initial study of Cryptosporidium species and subtypes from preweaned diarrheic calves. Cryptosporidium species were determined in 71 fecal samples from 43 different dairy farms using 18S rRNA PCR, and subtyping of C. parvum based on the 60-kDa glycoprotein (gp60) sequences was done on one sample per farm. C. parvum was the only species found, with eight different subtypes belonging to the zoonotic IIa and IId families. Subtype IIaA15G2R1 was the most prevalent and widespread, found in 50% of the farms over a wide geographical distribution. Our results confirm the presence of subtypes IIaA15G2R1 and IIdA20G1, which were previously found in human patients in Israel, also in Israeli calves. In addition, subtype IIaA12G1R1 is reported here for the first time in an animal. These findings demonstrate the value of monitoring C. parvum subtypes in animal samples, and suggest that the role of calves as well as other young ruminants in the transmission of zoonotic cryptosporidiosis in Israel should be further studied.

Invasive Amirthalingamia macracantha (Cestoda: Cyclophyllidea) larvae infecting tilapia hybrids in Israel: a potential threat for aquaculture.

Larvae (metacestodes) of gryporhynchid tapeworms (Cestoda: Cyclophyllidea) are reported for the first time from the liver of tilapia hybrids (Oreochromis aureus × O. niloticus) reared in earth ponds in northeastern Israel (along the Jordan River). This is the first record of Amirthalingamia macracantha (Joyeux & Baer, 1935), a parasite of cormorants (Phalacrocoracidae), outside Africa and outside the tropics. Larvae found in the liver of tilapias (Cichlidae) were identified to species level because they possessed 20 massive rostellar hooks of 3 types, with the 4 largest hooks measuring almost 500 µm. Molecular data confirmed species identification. The possible route of introduction to Israel of this African parasite, which is large (length up to 2 cm) and potentially pathogenic for cultivated tilapias, is discussed.

Establishing Babesia bovis-Free Tick Colony Following Treatment of the Host with Diminazene Aceturate (Berenil).

Babesia bovis is a widely-spread tick-borne hemoparasite of cattle with major economic and animal welfare consequences. Rhipicephalus (Boophilus) annulatus is a one-host tick which transmits bovine babesiosis in the Middle East and Africa. Laboratory rearing of ixodid ticks is essential for the investigation on ticks or tick-borne diseases. Establishing a tick colony in the laboratory usually originates from ticks harvested in the field, which may be naturally infected with various pathogens. This especially applies to carriage of B. bovis as it is highly prevalent in endemic areas and is transmitted transovarially in ticks. Here, we describe the use of diminazene aceturate (Berenil) in order to establish laboratory colonies of Babesia-free R. annulatus, from ticks collected in the field. Ticks collected in the field were kept until oviposition and hatched larvae were introduced to naïve calves, which led to infection of the calves with B. bovis. Calves were then treated with diminazene aceturate several times until the engorged ticks dropped. The eggs and larvae collected from these ticks were parasite-free, as demonstrated both by infection of splenectomized calves and by PCR. This suggested protocol is a useful tool to create parasite-free tick colony and may, theoretically, also be beneficial to reduce parasite circulation in the field, although not recommended, as resistance to diamenizene aceturate might develop.

The Effect of Vaccination with Neospora caninum Live-Frozen Tachyzoites on Abortion Rates of Naturally Infected Pregnant Cows.

Neosporosis is a major cause of abortions in cattle worldwide. Recently a live attenuated vaccine showing promising results in preventing abortions, when administered at mid-pregnancy to seropositive cows, was developed. In this study, vaccination with 2 × 108 live frozen N. caninum tachyzoites (NcIs491) was used to immunize naturally infected seropositive pregnant dairy dams. The study was performed under field conditions in four herds, and a follow-up of three subsequent pregnancies was analyzed. A total of 1136 cows were serologically examined. Total seroprevalence was 41.4%, with 25.1% of the cows having titers of 1:800 or higher. Abortion rates were significantly higher in cows with high antibody titers (≥1:800) for two consecutive pregnancies. Vaccination was administered to 114 out of 285 cows with antibody titers higher than 1:800. Immunization resulted in lower abortion rates at three of the farms. Vaccine efficacy ranged from -19.8% to 75% at different farms, with overall efficacy of 28.4% in all four farms and overall efficacy of 58.2% in the three farms with positive results. Our results showed different vaccine efficacy in studied farms, suggesting that frozen live vaccination may generally be an effective method to control neosporosis in cattle.

Experimental Infection of Calves with Transfected Attenuated Babesia bovis Expressing the Rhipicephalus microplus Bm86 Antigen and eGFP Marker: Preliminary Studies towards a Dual Anti-Tick/Babesia Vaccine.

Bovine babesiosis, caused by Babesia bovis and B. bigemina, is a major tick-borne disease of cattle with global economic impact. The disease can be prevented using integrated control measures including attenuated Babesia vaccines, babesicidal drugs, and tick control approaches. Vaccination of cattle with the Rhipicephalus microplus Bm86-based recombinant vaccine reduces the fitness of R. microplus and R. annulatus, but several booster inoculations are required to maintain protection. Herein, we generated a stable transfected strain of B. bovis expressing an enhanced GFP (eGFP) and a chimeric version of Bm86 (B. bovis/Bm86/eGFP). The eGFP was expressed in the parasite cytoplasm, whereas Bm86 was displayed on the surface of merozoites. Three splenectomized calves experimentally infected with B. bovis/Bm86/eGFP showed mild signs of acute disease and developed long-lasting antibody responses to B. bovis and native Bm86. No evidence of sequestration of parasites in the cerebral capillaries was found upon postmortem analysis, confirming attenuation of the strain. This is the first report of transfected B. bovis expressing the tick antigen Bm86 on the merozoite surface that elicits an antibody response to native Bm86. These results represent a proof of concept for a novel live, attenuated, tagged dual-vaccine approach to attempt simultaneous control of babesiosis and tick infestation.

Leishmania infection in cats and dogs housed together in an animal shelter reveals a higher parasite load in infected dogs despite a greater seroprevalence among cats.

BACKGROUND: An outbreak of leishmaniosis was studied in cats and dogs housed together with no separation in an animal shelter in Israel. METHODS: The study included recording of clinical signs, serology for Leishmania infection by ELISA, PCR of blood for Leishmania DNA by ITS1 HRM and kDNA PCR, parasite quantification, and trapping of sand flies around the shelter. RESULTS: Thirty-seven % (22/60) of the dogs and 75% (50/67) of the cats were seropositive to L. infantum with a significantly higher seropositivity rate in the cat population (χ2 = 42.160, P < 0.0001). Twenty-five percent (15/60) of the dogs were positive for Leishmania by blood PCR, 12% by the Leishmania ITS1 HRM PCR and 22% by kDNA PCR. Of the cats, 16% (11/67) were positive by kDNA PCR and none by ITS1 HRM PCR. All the PCR-positive animals were infected by L. infantum verified by DNA sequencing and there was no significant difference between the PCR-positivity in the dog and cat populations. Altogether, 43% (26/60) of the dogs and 79% (53/67) of the cats were positive by serology or PCR for L. infantum. The average Leishmania parasite load in the blood of PCR-positive dogs (42,967 parasites/ml) was significantly higher than in PCR-positive cats (1259 parasites/ml) (t(12) = 2.33, P = 0.037). Dogs that were positive by the Leishmania ITS1 HRM PCR and kDNA PCR had significantly higher parasite loads than dogs positive only by the kDNA PCR (t(11) = - 3.186580, P < 0.009). No significant effect was found for FIV seropositivity on Leishmania infection in the cats (χ2 = 0.506, P = 0.777). A higher percentage of Leishmania-positive dogs showed clinical signs compatible with leishmaniosis compared to Leishmania-positive cats (100 vs 52.8%, χ2 =15.242, P < 0.0001). Phlebotomus perfiliewi, a proven vector of L. infantum, comprised 92% of trapped sand flies. CONCLUSIONS: Comparisons of populations of cats and dogs exposed to sand flies and L. infantum under the same conditions indicated that although a high rate of exposure was detected in cats as manifested by a significantly greater degree of seropositivity, dogs had significantly higher blood parasite loads, and were likely to be more infectious to sand flies than cats.

First molecular identification of Aelurostrongylus abstrusus in a cat presenting severe respiratory disease from Israel.

Feline lung worm infection is increasingly reported in recent years, and recognized as a cause for respiratory disease in cats. Aelurostrongylus abstrusus is regarded as the most prevalent cause of such cases. Infective L3 larvae carried in gastropods and paratenic hosts infect felines, developing to adult worms that reside in the lungs' parenchyma and may cause verminous pneumonia. The L1 larvae hatch from eggs deposited in the lung, and are released to the environment by either feces or sputum. While the majority of epidemiological information regarding A. abstrusus originates in European countries, recent studies have shown that it is also found around the Mediterranean basin, as far east as Turkey and Cyprus. A local domestic cat from Israel showing signs of respiratory illness was diagnosed with aelurostrongylosis, confirmed by both morphological and molecular tools. Presence in Israel of this nematode was previously reported in 1949, with no further mentions since. ITS-2 sequence of the isolated larvae was highly similar to that of A. abstrusus from domestic cats from Italy. These findings show that distribution of A. abstrusus stretch to the eastern shores of the Mediterranean, and that this nematode should be considered as a cause for respiratory disease in cats in Israel and the surrounding countries.

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.

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 < 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 < 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.

Induction of allopurinol resistance in Leishmania infantum isolated from dogs.

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 in-vitro induction of allopurinol resistance in Leishmania infantum 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 in-vitro resistance to allopurinol, which is likely to occur in natural infection.

Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology.

BACKGROUND: Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in dogs. This study describes dogs infected with these Leishmania spp. prevalent in the Middle East and North Africa, and compares the serological response of dogs infected with Leishmania infantum, L. major or L. tropica to whole promastigote antigen enzyme-linked immunosorbent assay (ELISA) of each species and to rK39 dipstick. RESULTS: Leishmania major infection in a 5-month-old male dog was associated with alopecic and ulcerative periocular and limb skin lesions which responded to allopurinol treatment. Infection was detected by skin and blood polymerase chain reaction (PCR) and confirmed by DNA sequencing but the dog was seronegative. Leishmania tropica infection was detected in a 3-month-old female dog co-infected with Babesia vogeli and Anaplasma platys and with no skin lesions. PCR and DNA sequencing of the blood and parasite culture were positive for L. tropica. Sera from 11 dogs infected with L. infantum, L. major or L. tropica were reactive with all three Leishmania spp. antigens except for sera from a dog with L. major infection. No significant differences were found between reactivity of dog sera to the antigen of the infecting species, or to the other Leishmania spp. antigens. Sera from dogs infected with L. infantum and L. tropica were positive with the rK39 antigen kit, while dogs with L. major infection were seronegative. CONCLUSIONS: Skin lesions in L. major infected dogs from this study and previous reports (n = 2) were ulcerative and located on the muzzle, feet and foot pads and not associated with generalized lymphadenomegaly and splenomegaly. In previous L. tropica infections, skin lesions were proliferative mucocutaneous in young dogs (n = 2), or associated with widespread dermatitis, lymphadenomegaly and splenomegaly in older dogs with similarity to L. infantum infection (n = 2). This study suggests that ELISA serology with whole promastigote antigen is not distinctive between L. infantum, L. major and L. tropica canine infections and that some L. major infections are not seropositive. PCR with DNA sequencing should be used to discriminate between canine infections with these three species.

Leishmania major infection in a dog with cutaneous manifestations.

BACKGROUND: Leishmania major is a main cause of cutaneous leishmaniasis in humans in an area that stretches from India through Central Asia, the Middle East, to North and West Africa. In Israel, it is a common infection of humans with rodents as the reservoir hosts and Phlebotomus papatasi as its sand fly vector. FINDINGS: A 6 months old spayed female mixed breed dog was referred to the Hebrew University Veterinary Teaching Hospital with a large ulcerative dermal lesion on the muzzle, and lesions in the foot pads and left hind leg. Histopathology of a skin biopsy found chronic lymphohistiocytic dermatitis with the presence of Leishmania spp. amastigotes in the muzzle. Physical examination indicated that the dog was overall in a good clinical condition and the main findings were the skin lesions and enlarged prescapular lymph nodes. Complete blood count and serum biochemistry profile were within reference ranges. Serology by ELISA was positive for Leishmania spp. and PCR of the prescapular lymph node was positive by an ITS1 region PCR-high resolution melt analysis. However, the melt curve and subsequent DNA sequencing indicated that infection was caused by L. major and not L. infantum, which is the main causative agent of canine leishmaniosis in the Mediterranean region. DNA was extracted from the paraffin embedded muzzle biopsy and PCR with sequencing also indicated L. major. The dog's young age and the absence of hyperglobulinemia and anemia were not typical of L. infantum infection. The dog was treated with allopurinol and the skin lesions improved and later disappeared when the dog was re-evaluated. CONCLUSIONS: This is the first molecularly-confirmed case of L. major infection in a dog. Two previous reports of L. major in dogs originated from Saudi-Arabia and Egypt in 1985 and 1987 were confirmed by enzymatic biochemical techniques. Serology for L. infantum was positive probably due to the well documented serological cross-reactivity between Leishmania spp. Although dogs and wild carnivores are not considered main reservoirs for L. major, the possibility of clinical canine disease and their potential as secondary hosts should be investigated in areas endemic for human L. major infection.

Molecular characterization of Theileria orientalis from cattle in Ethiopia.

This study reports the first molecular characterization of Theileria orientalis in local breeds of cattle in Ethiopia. A conventional PCR utilizing major piroplasm surface protein (MPSP) gene and an established multiplexed tandem PCR (MT-PCR) were used to characterize T. orientalis and to assess the infection intensity, respectively. Of 232 blood samples tested, T. orientalis DNA was detected in only 2.2% of samples using conventional PCR; two genotypes buffeli (1.3%; 3/232) and type 5 (0.9%; 2/232) of T. orientalis were detected. Phylogenetic analysis revealed that the buffeli MPSP sequences from Ethiopia were closely related to those reported from Kenya, Sri Lanka and Myanmar, and type 5 sequences from Ethiopia grouped with those from Korea, Japan, Vietnam and Thailand. A higher number of samples (3.9%; 9/232) were test-positive by MT-PCR and four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected. The average intensity of infections with genotypes buffeli (DNA copy numbers 11,056) and type 5 (7508) were significantly higher (P<0.0001) than the pathogenic genotype ikeda (61 DNA copies). This first insight into T. orientalis from cattle in Ethiopia using MPSP gene provides a basis for future studies of T. orientalis in various agroclimatic zones and of the impact of oriental theilerosis on cattle in this and other countries of Africa.

Allopurinol Resistance in Leishmania infantum from Dogs with Disease Relapse.

BACKGROUND: Visceral leishmaniasis caused by the protozoan Leishmania infantum 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 Leishmania strains from dogs to allopurinol has not been described before in clinical studies. METHODOLOGY/PRINCIPAL FINDINGS: Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of L. infantum 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 (P = 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 (P = 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 (P = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 µg/mL allopurinol). CONCLUSIONS: This is the first study to demonstrate allopurinol resistance in L. infantum 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 L. infantum strains may enhance uncontrolled transmission to humans and to other dogs.

Exposure to Leishmania spp. and sand flies in domestic animals in northwestern Ethiopia.

BACKGROUND: Human visceral leishmaniasis caused by Leishmania donovani is considered an anthroponosis; however, Leishmania-infected animals have been increasingly reported in L. donovani foci, and the role of these animals as reservoirs for human L. donovani infection remains unclear. METHODS: We conducted a study of domestic animals (goats, sheep, cows, dogs, and donkeys) in three L. donovani foci in northwestern Ethiopia. Domestic animals were screened for Leishmania DNA and for anti-L. donovani IgG. Serum anti-sand fly saliva antibodies were used as a marker of exposure to the vector sand fly, Phlebotomus orientalis. RESULTS: Of 546 animals tested, 32 (5.9%) were positive for Leishmania DNA, with positive animals identified among all species studied. Sequencing indicated that the animals were infected with parasites of the L. donovani complex but could not distinguish between L. infantum and L. donovani. A total of 18.9% of the animals were seropositive for anti-L. donovani IgG, and 23.1% of the animals were seropositive for anti-P. orientalis saliva IgG, with the highest seroprevalence observed in dogs and sheep. A positive correlation was found between anti-P. orientalis saliva and anti-L. donovani IgGs in cows, goats, and sheep. CONCLUSIONS: The detection of L. donovani complex DNA in the blood of domestic animals, the reported seroprevalence to the L. donovani antigen, and the widespread exposure to sand fly saliva among domestic animals indicate that they are frequently exposed to Leishmania infection and are likely to participate in the epidemiology of Leishmania infection, either as potential blood sources for sand flies or possibly as parasite hosts.