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Haemoparasitic infections are frequently observed in dogs from tropical regions, including India. The present investigation combined microscopic blood smear examination and PCR assays to assess the occurrence of canine tick-borne diseases (CTBD) from suspected dogs in and around Hisar, Haryana. Using the Giemsa-stained peripheral thin blood smear examination, 15 (12.5%) of the 120 dogs were infected with CTBD, with 5.8%, 3.3%, 2.5%, and 0.8% dogs testing positive for Hepatozoon canis, Ehrlichia canis, Babesia vogeli, and Babesia gibsoni, respectively. Using the PCR assay, CTBD was found to be 64.16% (77/120) in examined dogs. Of the 77 PCR-positive canines, 56 were infected with a single haemoparasite, while 21 were infected with two or more species. H. canis was the most abundant tick-borne pathogen, representing 35%, followed by E. canis 25.8%, B. vogeli 20%, and B. gibsoni 2.5%. The most common co-infection was with H. canis along with E. canis (7.5%). The PCR assay was proven to be more efficient for detecting haemoparasites in dogs compared to blood smear examinations. The study suggests that canine tick-borne diseases are common in Haryana and recommends using PCR-based molecular tests in addition to conventional microscopic examination to diagnose these infections for effective treatment and management of infected canines., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection., (© Wildlife Disease Association 2024.)

Objective: To provide a video tutorial on how to perform preputial scraping for the testing of Tritrichomonas foetus in bulls., Animals: Postpubertal bulls., Methods: A preputial scraping device is inserted in the prepuce, and back-and-forth scraping movements are made to collect smegma and T foetus organisms. The sample is placed in an appropriate media and prepared for shipment to the diagnostic laboratory., Results: Preputial sampling is an effective method for diagnosing T foetus in infected bulls., Clinical Relevance: Bulls are the primary carriers of T foetus, a protozoan parasite responsible for bovine trichomoniasis. By sampling and testing bulls, veterinarians can identify infected animals and implement control measures to prevent the spread of the disease within herds. Trichomoniasis can lead to significant reproductive problems in cattle, including infertility, embryonic death, and abortions. Sampling bulls allows for the detection of infection, enabling prompt intervention to protect the reproductive health of the entire herd. Trichomoniasis outbreaks can result in substantial economic losses for cattle producers due to reduced conception rates, increased calving intervals, and decreased calf crops. Sampling bulls helps to mitigate these losses by identifying and removing infected animals from the breeding pool, thereby minimizing the spread of the disease and its associated reproductive inefficiencies. In many regions, T foetus testing in bulls is a regulatory requirement for cattle movement and trade. Sampling bulls and obtaining negative test results are often necessary for obtaining health certificates and complying with interstate or international movement regulations.

Tritrichomonas foetus is a flagellated and anaerobic parasite able to infect cattle and felines. Despite its prevalence, there is no effective standardized or legal treatment for T. foetus-infected cattle; the vaccination still has limited success in mitigating infections and reducing abortion risk; and nowadays, the diagnosis of T. foetus presents important limitations in terms of sensitivity and specificity in bovines. Here, we characterize the plasma membrane proteome of T. foetus and identify proteins that are represented in different isolates of this protozoan. Additionally, we performed a bioinformatic analysis that revealed the antigenicity potential of some of those proteins. This analysis is the first study to identify common proteins at the plasma membrane of different T. foetus isolates that could be targets for alternative diagnostic or vaccine techniques in the future., (© 2024. The Author(s).)

Isolation of genomic DNA of blood parasites in birds, reptiles, amphibians, and fishes is a challenging task, given that their red blood cells are nucleated; for that reason, parasite genomic DNA is only a fraction of the total extracted DNA, and it is challenging to obtain concentrated high-quality genetic material. Percoll Density Gradient (PDG) and flow cytometry are tools for separating and analyzing cell populations or even a single cell, and both represent potent approaches for isolating avian haemosporidians parasites. Our experimental design included several steps seeking to concentrate the parasite´s DNA. We used blood samples from a Rock pigeon infected with Haemoproteus columbae. After inducing parasite exflagellation and gametogenesis in vitro, we subjected the samples to a Percoll Density Gradient to separate the parasites from the rest of the blood cells. Following centrifugation, the layer containing extracellular parasites underwent a flow cytometry and cell sorting process, during which we selected two different subpopulations of cells for analysis. Based on qPCR analyses, we demonstrate parasite DNA enrichment in Percoll Density Gradient and flow cytometry samples; simultaneously, these samples showed the lowest concentration of Columba livia DNA. However, the concentration of parasite DNA was higher in the PDG than in the cell sorting sample. This study reports the concentration of the Haemoproteus parasite by flow cytometry without DNA-intercalating dyes, and this methodology can serve as a technique for DNA enrichment of blood parasites infecting nucleated red blood cells to improve techniques that allow obtaining complete genomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Case Series Summary: This case series describes six cases involving seven cats naturally infected with Cytauxzoon felis in Indiana, USA. Medical records were retrospectively reviewed and all available information on signalment, history, clinical and diagnostic findings, treatment, outcome and pathology was reported. Cats infected with C felis were domestic shorthairs, were aged between 2 and 9 years and all but one of the cats were male. The seven infected cats originated from five counties in southwestern Indiana. Six of seven cats were found to have acute cytauxzoonosis based on clinical signs, gross pathologic lesions, observation of C felis in tissues and/or detection of C felis DNA. One cat was identified as a subclinical survivor cat with no known clinical history of cytauxzoonosis., Relevance and Novel Information: The reported cases are the first confirmed reports of acute and chronic cytauxzoonosis in cats from Indiana and document an expansion in the range of C felis . Veterinary practitioners in Indiana should consider infection with C felis as a differential diagnosis for cats that present with fever, inappetence, lethargy, depression, dehydration, dyspnea, hemolytic crisis, anorexia or icterus. Administration of approved acaricides to cats currently offers the best protection and control against C felis infection., Competing Interests: Conflict of interestThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium , Haemoproteus ( Parahaemoproteus ), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Musa, Hemberle, Bensch, Palinauskas, Baltrūnaitė, Woog and Mackenstedt.)

In a 2-yr study on prevalence of Haemosporidia in an avian community in Ithaca, New York, USA, we tested the hypothesis that apparent seasonal variation in prevalence is influenced by the detection protocol. We confirmed a higher detection of Haemosporidia using a molecular diagnosis technique (PCR) than by microscopy; this further increased when the PCR test was triplicated. Microscopic examination and PCR techniques have different specificity and sensitivity and therefore different probabilities of detecting hemoparasites. Birds with chronic infections or sampled during winter often have very low parasitemia, and such infections may be missed by microscopy but detected by PCR. Haemosporidian prevalence was higher during the breeding season than during the nonbreeding season regardless of the method used. Detection of Leucocytozoon spp. infection from blood smears using microscopy was challenging., (© Wildlife Disease Association 2024.)

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

A 1-year-old major Mitchell's cockatoo ( Lophochroa leadbeateri ) was presented for evaluation of weakness, diarrhea with undigested seeds in the droppings, and weight loss. Leukocytosis with severe heterophilia, monocytosis, and lymphocytosis was noted on the complete blood count. Altered plasma biochemical parameters included a slight increase in creatine kinase and mild hypoproteinemia. Two blood smears before and after 2 days of treatment revealed mild polychromasia and anisocytosis but no blood parasites. Radiographic and computed tomographic imaging of the cockatoo were helpful in identifying airsacculitis, pneumonia, and gastrointestinal motility disorders. The patient died 5 days after treatment for the presenting clinical problems. On the gross postmortem examination, dark red foci in the ventricular muscle layers and 1-3-mm white foci in the myocardium, opaque air sacs, and dark lungs were identified. Histopathologic examination of submitted tissue samples found severe granulomatous ventriculitis and myocarditis with intralesional Haemoproteus species megalomeronts. Qualitative polymerase chain reaction testing for the cytochrome b (cyt b ) gene performed on pooled heart, liver, kidney, and intestinal tissues identified 99.5% homology to Haemoproteus minutus . This case report demonstrates the expansion of the geographic range of H minutus to France and potentially to Belgium, which may compromise breeding and conservation of Australian parrots living outdoors. Challenging diagnosis, rapid disease progression, and the absence of validated treatment protocols for psittacine patients suggest that the use of preventive measures to reduce the presence of insect vectors such as hippoboscid flies and biting midges ( Culicoides ) should be considered. Haemoproteus minutus should be considered and potentially screened by polymerase chain reaction testing on blood samples, especially in the case of highly susceptible avian species (eg, Australian parrots in Europe) that present with sudden weakness, heterophilic leukocytosis, and monocytosis associated with mild anemia.

Bovine trichomonosis (BT), a disease of the bovine urogenital tract, is caused by the protozoan Tritrichomonas foetus (Tf). Tf causes endometritis, infertility, and premature death of the embryo, which generates considerable economic losses. The proteins released can mediate fundamental interactions between the pathogen and the host, triggering factors associated with the symptomatology, immune evasion and pathogenesis characteristic of the species. However, little is known about the profile of the proteins released by Tf. In order to contribute to their knowledge, we performed an isolation protocol and a proteomic profiling of the supernatant (SN) content of six Tf isolates. A total of 662 proteins present in the SN of Tf were detected, out of which 121 were shared by the six isolates, while the remaining 541 were found in at least one of the isolates studied. The comparative analyses using the databases of Tf strain genome K revealed 32.9% of uncharacterized proteins. The bioinformatic analyses showed that the main molecular functions predicted were binding (47.9%) and catalytic activity (38.2%). Additionally, we performed immunodetection assays to evidence the antigenic potential of SN proteins. Interestingly, we observed great ability to detect SN proteins from all six isolates using serum from immunized mice and infected bulls. A complementary mass spectrometry assay allowed us to determine that the proteins that showed the strongest signal intensity in the immunoassays were Grp78 (A0A1J4IZS3) and Ap65 (A0A1J4JSR1). This work represents the first proteomic characterization of Tf SN proteins and their antigenic potential, which might be interesting for the future design of new diagnosis and treatment methods for BT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus , it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Objective: The geographical distribution of feline cytauxzoonosis is expanding in the US. Clinical signs of feline cytauxzoonosis, including lethargy, anorexia, and icterus, are similar to hepatic lipidosis and cholangiohepatitis. Hematologic and serum biochemical abnormality patterns may assist practitioners in prioritizing feline cytauxzoonosis as a differential diagnosis over hepatic lipidosis and cholangiohepatitis., Sample: Hematology and serum biochemical profiles of cats with naturally acquired feline cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis., Procedures: Retrospective search and analysis of the Kansas State Veterinary Diagnostic Laboratory or Kansas State University Veterinary Health Center records between January 2007 and June 2018 for cats with cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis., Results: Patients with acute feline cytauxzoonosis presented with frequent nonregenerative anemia (20/28 [71%]), leukopenia (23/28 [82%]), thrombocytopenia (23/23 [100%]), hyperbilirubinemia (27/28 [97%]), hypoalbuminemia (26/28 [93%]), reduced (18/28 [64%]) or low normal (10/28 [36%]) serum ALP activity, and hyponatremia (23/28 [82%]). Reduced ALP activity was unique to cats with feline cytauxzoonosis relative to hepatic lipidosis and cholangiohepatitis. No correlation between the severity of anemia and the magnitude of hyperbilirubinemia was identified in feline cytauxzoonosis patients., Clinical Relevance: The combination of nonregenerative anemia, leukopenia, thrombocytopenia, hyperbilirubinemia, and reduced serum ALP activity in icteric cats may increase the clinical suspicion, but is not pathognomonic, for acute feline cytauxzoonosis. Hematologic and serum biochemical abnormalities of naturally acquired feline cytauxzoonosis are like those reported with feline bacterial sepsis. Blood smear evaluation for intraerythrocytic Cytauxzoon felis piroplasms, tissue aspirates for schizont-laden macrophages, and/or molecular testing are required to diagnose feline cytauxzoonosis.

Cytauxzoon felis is a hematoprotozoan parasite with a complex life cycle involving a tick-vector and a mammalian host. The mammalian hosts are all felidae but in the bobcat reservoir host, the parasite typically causes only a brief, self-resolving illness followed by a prolonged subclinical infection. In domestic cats, however, infection often leads to an acute febrile illness characterized by severe morbidity and mortality. Diagnosis is based on microscopic identification of parasites or molecular testing. Treatment for ill cats is expensive, difficult, and often unsuccessful. Prevention is quite possible and depends on avoidance of feeding by vector ticks., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The development of new molecular methods has significantly improved the detection and identification of avian haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) compared to microscopic examination. Very large numbers of previously hidden Haemosporida species of a wide range of avian hosts have thus been discovered in the last two decades. However, test parameters of the various detection methods remain largely unevaluated. In this study, the merits of microscopy, multiplex PCR, and nested PCR were compared to identify the infection status of three Malagasy bird species. A total of 414 blood samples of Hypsipetes madagascariensis, Foudia omissa and F. madagascariensis, as well as 147 blood smears, were examined for haemosporidian infection. Thirty-four lineages of haemosporidian parasites could be identified, of which six have been detected for the first time. Microscopy, multiplex and nested PCR showed differences in detection rate, most likely due to low parasitemia of chronically infected birds. The combination of both PCR methods yielded the best results. In particular, detection of multiple infections could be greatly improved and will enable more precise prevalence estimates of individual haemosporidian species in wild birds in the future., (© 2022. The Author(s).)

The parasite T. foetus causes trichomonosis in cattle but is generally asymptomatic in males. Thus, many bulls carrying the disease go unnoticed, making the detection of T. foetus in bulls an important aspect for its control. Due to drawbacks posed by its cultivation, PCR is a preferred option for diagnostic laboratories. Most published PCR protocols target the genomic region compring the 18S, 5.8S, and 28S rRNA genes and internal transcribed spacers 1 and 2 (rRNA-ITS region), homologous to that of other Tritrichomonas species. There is minimal information on alternative genetic targets and no comparative studies have been published. We compared a protocol based on the microsatellite TfRE (called H94) and five protocols based on the rRNA-ITS region (called M06, M15, G02, G05, and N02). We also designed and evaluated a novel PCR-based assay on the EF1-alpha-Tf1 gene (called V21). The analytical sensitivity and specificity assays for the PCR protocols were performed according to the World Organisation for Animal Health (OIE) directives and the comparative study was performed with a widely used PCR (M06) on clinical samples from 466 breeding bulls. V21 showed a high degree of agreement with our reference M06 (kappa = 0.967), as well as M15 (kappa = 0.958), G05 (kappa = 0.948), and H94 (kappa = 0.986). Protocols H94 and V21 appear to be good approaches for confirming clinical cases in preputial bull samples when genomic regions alternative to rRNA-ITS are required. By contrast, N02 gave false negatives and G02 false positives., (© 2022. The Author(s).)

A 2-year-old, 4.2 kg, spayed female, Maine coon cat was referred to the veterinary hospital for evaluation of hyporexia, slow growth, and chronic, intermittent, mucoid, bloody, voluminous, and fetid diarrhea. The diarrhea had been observed since the cat was acquired from a cattery at 4 months of age; with acute worsening in the 5 d before presentation. Abdominal palpation revealed moderate pain. Ultrasonographic examination showed thickening of the jejunal wall and ileal loops, increased echogenicity of the jejunal mucosa, and enlargement of the jejunal and ileocolic lymph nodes. Histopathology of full-thickness intestinal biopsies showed moderate, diffuse, lymphoplasmacytic, erosive enteritis with hemorrhage and edema. Diffuse, lymphoplasmacytic, erosive colitis with mild, interstitial fibrosis and hemorrhage was also noted. The ileocecal lymph node biopsy showed eosinophilic lymphadenitis. Based on the immunohistochemical evaluation of intestinal samples with CD3 and CD79a antibodies, a diagnosis of lymphoma was ruled out. Fecal polymerase chain reaction testing was positive for Tritrichomonas foetus . Based on these results, inflammatory bowel disease and trichomonosis were diagnosed. Treatment for the cat included a hypoallergenic diet and an oral omega-3 fatty acid supplement, in conjunction with prednisolone, to manage the inflammatory bowel disease. Ronidazole was administered to target the Tritrichomonas foetus . The cat was clinically normal during a follow-up examination after 6 months of treatment., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background: Cytauxzoon felis is a life-threatening protozoan disease of cats. Identification of schizont-laden macrophages is a point-of-care diagnostic test for acute cytauxzoonosis., Hypothesis/objectives: The primary objective determined cytologic agreement between sample types to diagnose acute cytauxzoonosis. The secondary objective evaluated novices' ability to identify cytauxzoon organisms in blood films and tissue aspirates., Animals: Thirty-eight cats with suspected acute cytauxzoonosis and 5 controls examined postmortem., Methods: Cases were prospectively submitted and collected. Blood film, lymph node, and splenic aspirates were blindly reviewed for sample quality, presence of schizont-laden macrophages, and agreement between sample types. A subset of cases and controls were evaluated by 12 blinded novice observers to determine sensitivity and specificity for identifying organisms in various sample types., Results: Acute cytauxzoonosis diagnosis was made on at least 1 sample type in 28/38 cats. Schizont-laden macrophages were seen on 33% (10/30) of blood films, 56% (19/34) lymph node aspirates, 77% (26/34) splenic aspirates. Schizont-laden macrophages were more likely seen on splenic than lymph node aspirates (McNemar's, P = .03) or blood film (McNemar's, P = <.001). Novice observers were more likely to agree with experts when identifying schizont-laden macrophages in splenic aspirates (sensitivity = 77.1%, specificity = 94.4%) versus lymph node aspirates (sensitivity = 52.8%, specificity = 96.4%) or blood films (sensitivity = 41.7%, specificity = 96.9%)., Conclusions and Clinical Importance: Schizont-laden macrophages are most frequently identified in spleen, even by novice observers. If the diagnosis of acute cytauxzoonosis cannot be confirmed via blood film, then splenic, followed by peripheral lymph node aspirates can be considered., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Feline cytauxzoonosis is a disease caused by Cytauxzoon felis, a protozoan that infects the red blood cells and macrophages. It is responsible for an acute and often fatal disease in domestic cats. The purpose of this study was to investigate the occurrence of C. felis infections in healthy cats. Piroplasm forms were seen in the erythrocytes of 2 cats, and C. felis DNA was identified by polymerase chain reaction (PCR) in one of them. The results demonstrate that erythrocytic piroplasmids associated with tick-borne parasitic protozoa may be found circulating in the blood of healthy cats in Rio de Janeiro. These can be differentiated from the morphologically similar forms of species such as Babesia by analysis of DNA, thereby demonstrating the potential for further studies of feline populations in Brazil., (© American Society of Parasitologists 2021.)

Taguchi method was used to optimize loop mediated isothermal amplification tests aimed to amplify segments of the elongation factor 1a1 (tf-ef1a1), the 5,8 ribosomal gene (tf-5,8 r) or the beta tubulin 2 (tf-btub2) from the protozoan parasite Tritrichomonas foetus. L9 orthogonal array and quadratic loss functions that penalize deviations from prediction values revealed the effect of amplification reaction components. Analysis of variance (ANOVA) decomposed the contribution of individual factors to a small Ct. Confirmation experiments established that optimum conditions were predictable, verifiable and reproducible. Primers concentration conditioned the non-specific amplification of tf-ef1a1 while betaine and magnesium concentration contributed to accelerate the time to reach a positive threshold in tf-ef1a1, tf-5,8 r and tf-btub2. The general strategy of simple and robust experimental design holds potential as a general optimization protocol for LAMP tests in every diagnostic laboratory., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Noting lipidomic changes following the parasitism of migrating birds, the metabolic needs of which are primarily fueled by lipids, can deepen our understanding of host-parasite interactions. We identified lipids of migrating Northern saw-whet owls (Aegolius acadicus) using collision-induced dissociation mass spectrometry, compared the lipidomic signatures of hemoparasite-infected and noninfected individuals, and performed cross-validation analyses to reveal associations between parasite infection and lipid levels. We found significantly lower levels of lipid classes phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) in infected Northern saw-whet owls than in the noninfected individuals. Conversely, we found higher levels for certain lysoPS and lysoPE species, and variable lipid level changes for free fatty acid (FFA) species. Reporting lipidomic changes observed between hemosporidian-infected and noninfected Northern saw-whet owls can strengthen our understanding of the mechanisms governing parasite proliferation in this species. Furthermore, our analysis indicated that lipidomic signatures are better predictors of parasite infection than the log-adjusted mass/wing chord body index, a metric commonly used to assess the influence of hemosporidia infection on the health of birds. Establishing a lipidomic profile for Northern saw-whet owls that provides baseline lipid levels during fall migration may assist future studies assessing causes of reductions in breeding brought about from subtle differences in behaviors such as delayed migration., (© American Society of Parasitologists 2021.)

An 8-year-old, spayed female Rottweiler dog, under immunosuppressant treatment for protein-losing enteropathy (PLE) and intestinal lymphangiectasia, was presented for anorexia, poor general conditions and episodes of diarrhea. A subcutaneous mass between the caudal abdominal mammary glands was found. A fine-needle aspiration cytology was performed and revealed the presence of inflammatory cells mixed with pear- or round-shaped microorganisms with cytomorphological features of flagellated protozoan trophozoites, belonging to Trichomonadida order. The final diagnosis of Tritrichomonas foetus infection was achieved with molecular analysis (Real-Time PCR) and sequencing. T. foetus has often been reported as a pathogenic agent in cattle and cats, and rarely reported in dogs, where its pathogenetic role is poorly understood. To the author's knowledge, this is the first case of T. foetus infection described in a location different from the gastrointestinal tract in a dog., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vector-borne pathogens are responsible for serious emerging diseases and Rangelia vitalii, the etiologic agent of canine rangeliosis, is one of the most pathogenic tick-borne pathogens for dogs in South America. This protozoan is transmitted by the Amblyomma aureolatum tick bite and the clinical features associated to the disease are fever, hemolytic anemia, jaundice, hepatosplenomegaly and bleeding from natural orifices, mainly from the ear egde. The reports of canine rangeliosis in Argentina are scarce. In the present study we report the detection of Rangelia vitalii in a naturally infected dog from Gualeguay, Entre Ríos, Argentina with history of tick infestation and clinical signs compatible with rangeliosis. An initial blood sample was positive to piroplasmids by blood smear examination and the molecular amplification of a fragment of the 18SrRNA gene. Sequencing of the fragment confirmed the pathogen identity. After treatment with imidocarb dipropionate, the clinical signs remitted and the blood smear tested negative., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The use of a sensitive and accurate parasite detection methodology is crucial in studies exploring prevalence of parasites in host populations or communities, and uncertainty in identifying parasite genera and/or lineages may limit the understanding of host-parasite interactions. Here, we used a multistate occupancy approach that accounts for imperfect detection to assess whether sex and breeding season influenced the prevalence of a specific Haemoproteus lineage (TARUF02) in a white-lined tanager population. Likewise, we explored whether the probability of detecting the target parasite in an infected bird using PCR and sequencing analyses may be influenced by season and host sex. We found little evidence that sex influenced the probability of an individual host being infected by a haemosporidian parasite. Conversely, we found that the probability of infection by Haemoproteus TARUF02 was ~30% higher during the breeding season, reflecting a higher prevalence of this parasite in this season. The probability that PCR detects DNA of haemosporidian parasite was higher for female birds, suggesting that they are more prone to be parasitized with parasitemia levels that are more successfully detected by molecular analysis. Sequencing successfully determined the Haemoproteus TARUF02 lineage in 60% of samples collected during the breeding season and 84% of samples collected during the non-breeding season. Understanding the ecology of hosts and aspects of their physiology that may influence the parasite infection is essential to better understanding of hemoparasite infections and how parasites influence their native hosts, through decreasing reproductive success, lifespan, and/or survival.

Endoparasitic infections are associated with morbidity in cats. This study aimed to investigate the occurrence of endoparasites among cats of different life stages in the city of Rio de Janeiro, Brazil. The samples were analyzed individually by macroscopic exploration for proglottids and centrifugal-flotation. Stool samples were obtained from household cats (n = 57) and shelter cats (n = 336). Endoparasites were detected in 50.64% of the samples. Among household and shelter cats, 21.05% and 55.66% were infected with endoparasites, respectively. In household cats, the most prevalent endoparasites were Ancylostoma spp. (in 25.0%) and Strongyloides spp. (in 25.0%), followed by Toxocara spp. (in 16.67%), Dipylidium caninum (in 16.67%), Cystoisospora spp. (in 8.33%), and Uncinaria spp. + Ancylostoma spp. (in 8.33%). In shelter cats, the most prevalent endoparasite was Ancylostoma spp. (in 29.41%), followed by Cystoisospora spp. (in 26.20%) and Toxocara spp. (in 16.58%), as well as Cystoisospora spp. + Toxocara spp. (in 8.02%); Ancylostoma spp. + Toxocara spp. (in 11.76%); Cystoisospora spp. + Ancylostoma spp. (in 3.74%); Cystoisospora spp. + Toxocara spp. + Ancylostoma spp. (in 3.21%); and Dipylidium caninum + Ancylostoma spp. (in 0.53%). Endoparasitic infections in cats underscore the need for preventive veterinary care and routine coproparasitologic tests.

Bovine abortion causes considerable economic losses to the livestock industry worldwide and is of concern for public health and food safety, given that many abortigenic infectious agents of cattle are zoonotic. Despite its importance, the etiological diagnosis of abortion in cattle is challenging both for veterinary practitioners and laboratory technicians, partly due to the difficulty in recovering aborted fetuses under extensive field conditions for pathological and microbiological diagnostic investigation, and in the early identification of aborted dams. Neospora caninum is a cosmopolitan protozoon identified as one of the main abortigenic agents in cattle worldwide. In this study we propose a comparative seroepidemiological approach for the diagnosis of abortion by N. caninum in dairy cattle. Samples from 12 to 93 cows/heifers with and without recent history of abortion (cases and controls) in four commercial dairy farms were tested. The ratio of controls to cases tested varied from 1:1 to 4.6:1. All samples (n=230) were analyzed by three commercial ELISA kits for the detection of anti-N. caninum antibodies. In all four dairy farms, the proportion of seropositive cows and/or heifers per kit was significantly higher in the cases than in the controls (Odds Ratios=5.13 to 36, p=0.0002 to 0.0485). The agreement among the three kits varied from weak to strong (Coheńs kappa coefficients=0.58 to 0.83). We conclude that, despite the imperfect agreement between these kits, all of them allowed to arrive at similar conclusions regarding the statistical association between N. caninum seropositivity and abortion, thus representing a useful tool for the diagnostic approach at the population level under field conditions., (Copyright © 2019 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background: Fecal polymerase chain reaction (PCR) testing for Tritrichomonas foetus is considered the most sensitive means for diagnosis of infection but results could be influenced by fecal collection technique and prior use of antimicrobial drugs., Objectives: To establish any association between fecal collection technique or treatment history and results of fecal PCR testing for T. foetus., Animals: Fecal samples from 1717 cats submitted by veterinarians between January 2012 and December 2017., Methods: This study used a retrospective analysis. T. foetus PCR test results from 1808 fecal samples submitted for diagnostic testing were examined for their association with method of fecal collection and prior antimicrobial treatments. Data were collected from sample submission form., Results: Positive T. foetus PCR test results were obtained for 274 (16%) cats. Fecal samples collected via fecal loop had increased probability of positive PCR test results (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.31-3.17, P = .002) compared to samples collected by colonic flush. There was no association between PCR test results and treatment history, treatment type, or prior treatment with ronidazole. After an initial positive PCR test, 4/19 (21%; 95% CI 2.7%-39.4%) cats treated with ronidazole had a second positive test result., Conclusions and Clinical Importance: Results of this study support that fecal samples collected by loop might be better for PCR diagnosis of T. foetus infection. Lack of association of ronidazole with PCR test results and a 21% all-potential-causes failure rate of ronidazole in cats with preconfirmed infection are important limitations to use of this drug., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Internal parasites are a major concern in livestock production because they can impact the health and well-being of animals clinically and subclinically, and ultimately cause significant production loss. Among these internal parasites are nematodes, tapeworms, flukes, and coccidian protozoans. This review focuses on the diagnostic tests that are routinely performed by veterinarians and diagnostic laboratories, but also highlights recently developed tools that may improve diagnostic capabilities, including molecular and immunodiagnostic tests. Overall, diagnostic tests for parasites of livestock are an integral part of health management practices, and for assessing individual animal and herd health., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Abortion is a major source of economic losses in cattle breeding. Abortion occurs due to a wide range of causes, but infections are the most frequently diagnosed. However, establishing an aetiological diagnosis remains challenging due to the large variety of bacteria, protozoa, viruses, and fungi that have been associated with abortion in cattle. Economic restraints limit the range of diagnostic methods available for routine diagnostics, and decomposition of the conceptus or lack of proper fetal and/or maternal samples further restrict the diagnostic success. In this study, we report recent diagnostic findings from bovine abortions in Denmark, a country that has a large dairy sector and is free from most infectious agents causing epizootic abortion in cattle. The aims of the study were: (i) to identify infectious causes of bovine abortion in Denmark, (ii) to categorise the diagnostic findings based on the level of diagnostic certainty, and (iii) to assess the diagnostic rate. Due to economic restraints, only a limited panel of routine diagnostic methods were available. Placentas and/or fetuses from mid- to late-term abortions and stillbirths (n = 162) were submitted to the Danish National Veterinary Institute between January 2015 and June 2017. The aborted materials were examined macroscopically, histologically, and by bacterial culture. Maternal blood samples were tested for bovine viral diarrhoea virus (BVDV) antibodies., Results: The likely aetiology of the abortion was diagnosed in 52 cases, resulting in a diagnostic rate of 33%. The most common cause was protozoal infection (19%) followed by infection with Trueperella pyogenes (3%), Staphylococcus aureus (2%), and non-haemolytic Escherichia coli (2%). Lesions in fetuses with a protozoal infection were consistent with neosporosis. In many cases (38%), inflammatory changes were found in the placenta and/or fetal organs but no specific aetiology was identified. Neither infection with Brucella spp. nor maternal BVDV antibodies were detected. The majority of submitting herds (92%) were each represented by fewer than three abortion cases over the study period., Conclusions: Protozoal infection, most likely neosporosis, was the most commonly diagnosed cause of abortion and the only one associated with potential epizootic abortion events. Despite using a reduced number of diagnostic methods in comparison to other abortion studies, the diagnostic rate of this study was within the range reported in an earlier Danish study, as well as in recent international studies. The low number of submitted cases per herd and the sparse anamnestic information provided at submission hampered conclusions on the potential epizootic character of the abortion events in question.

The aim of this study was to verify the presence and identify the species of haemosporidian parasites in eared doves (Zenaida auriculata) in Brazil. Two hundred and eleven male and female eared doves were trap-captured in four different regions of Londrina city, in southern Brazil. Whole blood was collected in EDTA tubes through heart puncture after euthanasia in a CO2 chamber. A nested PCR targeting the mitochondrial cytochrome b gene (cyt b) of Haemoproteus spp./Plasmodium spp. was performed, followed by an enzymatic digestion to identify the genus. Phylogenetic trees were constructed to determine the closely related species. Out of 211 eared doves, 209 (99.05%) were positive for Haemoproteus spp. and/or Plasmodium spp. RFLP analysis showed that 72.72% (152/209) of eared doves were positive only for Haemoproteus spp., 6.22% (13/209) were positive only for Plasmodium spp., and 21.05% (44/209) of eared doves had mixed infections. Genetic analysis found four samples that were homologous with Haemoproteus multipigmentatus and one that was homologous with Plasmodium sp. This is the first molecular study of hemoparasites from eared doves in Brazil, and it is also the first description of H. multipigmentatus and Plasmodium spp. infection in eared doves in Brazil.

One captive musophagid bird at a zoological garden in Japan showed clinical symptoms and was found to be infected with avian haemosporidia. We subsequently collected blood from all musophagid birds kept in the garden and examined for avian haemosporidia using both microscopic and molecular examination. Only Haemoproteus gametocytes were observed in the blood of two Guinea turaco (Tauraco persa). Three genetic lineages of Haemoproteus were identified from three Guinea turacos and one genetic lineage of Leucocytozoon was identified from a grey plantain-eater (Crinifer piscator). Detected Haemoproteus lineages were all identical and completely different from those previously reported in Japan, suggesting that these birds were infected in their original habitat. This is the first record of Haemoproteus infection in Guinea turacos.

The protozoan parasite Tritrichomonas foetus may cause severe diarrhea in cats all over the world. In order to evaluate the methodology in coprological molecular diagnosis of feline tritrichomonosis, we compared previously published ("old") and newly developed ("novel") loop-mediated isothermal amplification (LAMP) (targeted to the T. foetus β-tubulin and the elf1α 1 gene, respectively) as well as an old conventional and an old and novel real-time PCR (all targeted to overlapping regions of T. foetus rDNA) assays regarding their diagnostic sensitivities and specificities. Here, the novel real-time PCR yielded the best methodical performance in that a sensitivity with a detection limit of <0.1 trophozoites (corresponding to ca.<0.13 trophozoites per mg feces) and a maximal specificity for diagnosis of Tritrichomonas spp. was achieved. The other test systems exhibited either an approximately 10-times lower sensitivity (<1 trophozoite corresponding to ca.<1.3 trophozoites per mg feces) (conventional PCR and both LAMP assays) or a lower specificity (old real-time PCR). Conversely, the diagnostic performance assessed with clinical fecal samples from cats demonstrated identical sensitivities (8 of 20 samples tested were positive) for the novel PCR and both LAMP assays. Diagnostic sensitivities were significantly higher than those found for the old real-time (5 positive samples) and conventional PCR (6 positive samples), respectively. Accordingly, our data suggested the novel PCR and both LAMP assays to be well suited molecular tools for direct (i.e. without including an in vitro cultivation step) coprological diagnosis of tritrichomonosis in cats. Interestingly, relative high (novel LAMP, 7 positive samples) to at least moderate (old LAMP, 6 positive samples and 1 sample with equivocal score) diagnostic sensitivities were also achieved by testing clinical samples upon simple visual inspection of colorimetric changes during the LAMP amplification reactions. Accordingly, both LAMP assays may serve as practical molecular tools to perform epidemiological studies on feline (and bovine as well as porcine) tritrichomonosis under simple laboratory conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine trichomoniasis is a notifiable, reproductive disease of cattle caused by the parasite Tritrichomonas foetus. Culturing with modified Diamond's medium (MDM) is required to increase the low number of organisms received from a preputial sample, but is limited in application to remote areas as it requires continuous cold chain storage. This study utilized lyophilization to sustain the viability of MDM during transport in lieu of a continuous cold chain. All lyophilized MDM was able to sustain T. foetus after storage for 42 days at 24 °C, and the results demonstrated that lyophilized MDM was equally as viable as refrigerated liquid MDM. Storage of lyophilized MDM at room temperature for 1 and 7 days did not impact T. foetus yield, both with and without exposure to light. A limitation of the lyophilized MDM was demonstrated with a significant decrease in T. foetus yield when the media was stored at 37 and 58 °C. The lyophilization of MDM provides a robust method of transporting and storing medium prior to reconstitution and inoculation, for use in T. foetus diagnosis and surveillance in remote areas.

Background: Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37-42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1-10 target DNA copies in less than 20 minutes., Methods: We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye., Results: The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20-25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field., Conclusions: Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

Background: Avian haemosporidian parasites can cause severe disease in their hosts due to excessive exo-erythrocytic merogony and anaemia caused by blood stages. Notably, the development of megalomeronts by species of Haemoproteus and Leucocytozoon has been associated with mortalities in birds. Diagnosis of lethal infections is currently accomplished by the detection of parasites' tissue stages in histological sections combined with PCR and sequencing. However, sequences frequently are not reliably obtained and the generic discrimination of exo-erythrocytic tissue stages based on morphological characters is challenging. Therefore, the present study aimed at developing specific molecular probes for the identification of Haemoproteus spp. and Leucocytozoon spp. in histological sections using chromogenic in situ hybridization., Methods: Parasite subgenus-specific oligonucleotide probes were designed to target the 18S ribosomal RNA of Haemoproteus species (subgenus Parahaemoproteus) and Leucocytozoon spp. (subgenus Leucocytozoon) and were in situ hybridized to sections from formalin-fixed, paraffin-embedded tissue samples determined positive for these parasites by PCR and histopathology. To confirm the presence of parasites at sites of probe hybridization, consecutive sections were stained with haematoxylin-eosin and examined., Results: Parahaemoproteus- and Leucocytozoon-specific probes labelled erythrocytic and exo-erythrocytic stages of Haemoproteus spp. and Leucocytozoon spp., respectively. Binding of probes to parasites was consistent with detection of the same exo-erythrocytic meronts in consecutive haematoxylin-eosin-stained sections. Cross-reactivity of the probes was ruled out by negative chromogenic in situ hybridization when applied to samples positive for a parasite of a genus different from the probes' target., Conclusions: Chromogenic in situ hybridization using 18S ribosomal RNA-specific oligonucleotide probes reliably identifies and discriminates Haemoproteus and Leucocytozoon parasites in tissue sections and enables unequivocal diagnosis of haemosporidioses.

As laws change around the United States, wildlife that were once kept as companion animals are now often confiscated by local authorities. They are then euthanized unless a home is found for them at a sanctuary. Wolf sanctuaries are, therefore, becoming increasingly important for their conservation and management. However, little data is available on best practices for the health management of captive wolves, including data on parasitic diseases. Our objective was to assess the prevalence of parasites of captive wolves combining classical coprological techniques and immunoassays based on the detection of coproantigen of selected canid parasites. Fecal samples of 39 animals were collected upon observation of individual animals defecating. All samples were processed using the Fecal Dx® tests, a suite of coproantigen ELISAs for detection of ascarid, hookworm, whipworm, and Giardia (IDEXX Laboratories Inc.). Out of the 39 samples, 38 were processed using the double-centrifugation sugar flotation (DCSF) and 34 using a modification of the Baermann technique. Twenty-eight samples (71.8%) were positive for hookworm, and none positive for the other parasites tested using coproantigen ELISA. Ancylostoma sp. (26, 68.4%), Eucoleus boehmi (13, 34.2%), and Trichuris sp. (2; 5.3%), and Sarcocystis sp. (13, 34.2%) were detected using DCSF. No metastrongyloid lungworm larvae were found. The Cohen's kappa index (0.97) showed excellent agreement between the hookworm coproantigen ELISA and the DCSF using feces preserved in ethanol for a short period of time. This study provides a baseline on the parasites of captive wolves, and shows that recent innovative diagnostics in veterinary parasitology, developed and optimized for dogs, may be used for assessing the health of wolves., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rangeliosis, caused by protozoan Rangelia vitalii, is transmitted by the tick Amblyomma aureolatum. The disease is characterized by hemolytic and hemorrhagic disorder and has been described in dogs and other wild canids. The aim of this study was to compare clinicopathological findings and laboratory results of a Rangelia infection in a crab-eating fox (Cerdocyon thous) with those of canine rangeliosis. The zoo of Universidade de Caxias do Sul, received a crab-eating fox with marked jaundice in mucous membranes, dark-colored stools and neurological signs. The animal underwent an ear tip smear examination and blood collection for complete blood counts, serum biochemistry and PCR. Free-living and intraerythrocytic pyriform structures consistent with R. vitalii were found in the blood smear of the ear tip. The erythrogram revealed normocytic normochromic anemia, moderate macrocytosis, polychromasia and metarubricytosis. The leukogram revealed leukocytosis with neutrophilia and monocytosis, as well as severe thrombocytopenia. Serum biochemistry showed hypoproteinemia, hypoalbuminemia and elevated levels of urea and creatinine. The treatment was performed with imidocarb hydrochloride and dexamethasone, however 24 h after initiation of treatment the animal died. Macroscopic examination revealed jaundice, subcutaneous edema, enlarged superficial lymph nodes, splenomegaly, and hemorrhage of internal organs. Histological sections of the cerebellum, lung, pancreas, intestine and heart were consistent with R. vitalii infection of the vascular endothelium. Pathological and hematological findings were similar to those found in infected dogs, with clinical presentation characterized by hemolytic anemia and hemorrhage. The description of this case showed that C. thous does not only serve as reservoir of R. vitalii but may also develop disease., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Disease is an increasing threat for marine bivalves worldwide. Recently, a mass mortality event (MME) impacting the bivalve Pinna nobilis was detected across a wide geographical area of the Spanish Mediterranean Sea and linked to a haplosporidian parasite. In 2017-2018, mass mortality events affecting the pen shell Pinna nobilis were recorded in two different regions of Italy, Campania and Sicily, in the Tyrrhenian Sea (Mediterranean Sea). Histopathological and molecular examinations of specimens showed the presence of Haplosporidium sp. in only one specimen in one area. Conversely, in all of the surveyed moribund animals, strong inflammatory lesions at the level of connective tissue surrounding the digestive system and gonads and linked to the presence of intracellular Zhiel-Neelsen-positive bacteria were observed. Molecular analysis of all of the diseased specimens (13) confirmed the presence of a Mycobacterium. Blast analysis of the sequences from all of the areas revealed that they were grouped together with the human mycobacterium M. sherrisii close to the group including M. shigaense, M. lentiflavum and M. simiae. Based on pathological and molecular findings, it is proposed that a mycobacterial disease is associated with the mortality episodes of Pinna nobilis, indicating that, at this time, Haplosporidium sp. is not responsible for these events in Campanian and Sicilian waters.

Noble pen shell or fan mussel, Pinna nobilis Linnaeus (1758), protected since 1992, was incorporated into the Spanish Catalogue of Threatened Species (Category: Vulnerable, Royal Decree 139/2011). The status is presently in the process of being catalogued as critically endangered, pending approval by Spanish Government (https://www.mapama.gob.es/es/biodiversidad/participacion-publica/Borrador&#95;OM&#95;situacion&#95;critica.aspx). The International Union for the Conservation of Nature (IUCN) alerted the countries of the Mediterranean basin to the "emergent situation" due to serious mortality events suffered by the fan mussel, putting it in serious risk of extinction. Thus, emergency actions have been implemented by Spanish authorities in which several research institutes from all over the country are involved. The parasite, Haplosporidium pinnae, was recently characterized by histology, TEM, SEM and molecular biology techniques and it was considered responsible for the mass mortality of P. nobilis in the Mediterranean Sea. In this context, the aim of this study has been to develop species-specific quantitative PCR (qPCR) protocol carrying out a fast, specific and effective molecular diagnose of H. pinnae. In this sense, the detection limit for qPCR was equal to 30 copies of SSU rDNA / ng of DNA using plasmid alone and when 100ng DNA of non-infected oyster were added. The qPCR assay revealed that 94% of the 32 analysed mantle tissues of fan mussel were infected by H. pinnae, showing a high sensitivity and specificity for its detection (100% if we don't consider negative and too much degraded samples). This technique will allow us to make quicker follow-ups of the disease, allowing us to get a better understanding of its evolution in order to help in the rescue of P. nobilis populations., Competing Interests: The authors declare that no competing interests exist.

Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of C t -values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype 'feline'. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bonamia spp. parasites threaten flat oyster (Ostrea spp.) farming worldwide. Understanding test performance is important for designing surveillance and interpreting diagnostic results. Following a pilot survey which found low Bonamia sp. intensity in farmed Ostrea angasi, we tested further oysters (n = 100-150) from each of three farms for Bonamia sp. using heart smear, histology and qPCR. We used a Bayesian Latent Class Model to assess diagnostic sensitivity (DSe) and specificity (DSp) of these tests individually or in combination, and to assess prevalence. Histology was the best individual test (DSe 0.76, DSp 0.93) compared to quantitative polymerase chain reaction (qPCR) (DSe 0.69, DSp 0.93) and heart smear (DSe 0.61, DSp 0.60). Histology combined with qPCR and defining a positive from either test as an infected case maximized test performance (DSe 0.91, DSp 0.88). Prevalence was higher at two farms in a high-density oyster growing region than at a farm cultivating oysters at lower density. Parasite intensities were lower than in New Zealand and European studies, and this is probably contributed to differences in the performance of test when compared to other studies. Understanding diagnostic test performance in different populations can support the development of improved Bonamia surveillance programs., (© 2018 John Wiley & Sons Ltd.)

Tritrichomonas foetus is a parasite that has been definitively identified as an agent of trichomonosis, a disease characterized by chronic diarrhea. T. foetus colonizes portions of the feline large intestine, and manifests as chronic and recurrent diarrhea with mucus and fresh blood, which is often unresponsive to common drugs. Diagnosis of a trichomonad infection is made by either the demonstration of the trophozoite on a direct fecal smear, fecal culture and subsequent microscopic examination of the parasite, or extraction of DNA in feces and amplification by the use of molecular tools. T. foetus is commonly misidentified as other flagellate protozoa such as Giardia duodenalis and Pentatrichomonas hominis. Without proper treatment, the diarrhea may resolve spontaneously in months to years, but cats can remain carriers of the parasite. This paper intends to serve as a source of information for investigators and veterinarians, reviewing the most important aspects of feline trichomonosis, such as trichomonad history, biology, clinical manifestations, pathogenesis, world distribution, risk factors, diagnosis, and treatment.

Accurate detection and identification are essential components for epidemiological, ecological, and evolutionary surveys of avian haemosporidian parasites. Microscopy has been used for more than 100 years to detect and identify these parasites; however, this technique requires considerable training and high-level expertise. Several PCR methods with highly sensitive and specific detection capabilities have now been developed in addition to microscopic examination. However, recent studies have shown that these molecular protocols are insufficient at detecting mixed infections of different haemosporidian parasite species and genetic lineages. In this study, we developed a simple, sensitive, and specific multiplex PCR assay for simultaneous detection and discrimination of parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon in single and mixed infections. Relative quantification of parasite DNA using qPCR showed that the multiplex PCR can amplify parasite DNA ranging in concentration over several orders of magnitude. The detection specificity and sensitivity of this new multiplex PCR assay were also tested in two different laboratories using previously screened natural single and mixed infections. These findings show that the multiplex PCR designed here is highly effective at identifying both single and mixed infections from all three genera of avian haemosporidian parasites. We predict that this one-step multiplex PCR assay, being convenient and inexpensive, will become a widely used method for molecular screening of avian haemosporidian parasites.

Hepatozoon is a genus of protozoa belonging to the phylum Apicomplexa. Ticks are the vectors for the members of this genus. The protozoans infect a wide variety of mammals, birds, reptiles, and amphibians. The domestic and wild felids are also susceptible to Hepatozoon infection. A five-year-old female cat was presented to the Adnan Menderes University, Faculty of Veterinary Medicine Animal Hospital, with a 1-month history of inappetence and weight loss. In a physical examination, weakness, depression, anorexia, lymphadenopathy, icteric mucous membranes, abdominal distension, and fever were detected. Laboratory analysis revealed anemia, neutrophilic leukocytosis, and increased serum total bilirubin and indirect bilirubin concentrations. Hepatozoonosis was diagnosed by the observation of Hepatozoon spp. gamonts within neutrophils in Giemsa-stained blood smears and confirmed by polymerase chain reaction (PCR). This is the first report about the molecular identification of hepatozoonosis in a cat from Turkey.

Arthropod-borne pathogens are medically important because of their ability to cause diseases in their hosts. The purpose of this study was to detect the occurrence of Ehrlichia spp., piroplasmids and Hepatozoon spp. in dogs with anemia and thrombocytopenia in southern Brazil. EDTA-whole blood was collected from 75 domestic dogs presenting anemia or/and thrombocytopenia from Guarapuava, state of Paraná, Brazil. DNA samples were subjected to conventional PCR assays for Ehrlichia spp. (dsb), piroplasmids (18S rRNA) and Hepatozoon spp. (18S rRNA), followed by sequencing and phylogenetic analyses. Among the 75 dogs, one (1.33%) was positive for Hepatozoon sp. and six (8%) were positive for piroplasmids in 18S rRNA cPCR assays. None of the dogs showed positive results in Ehrlichia spp.-cPCR targeting dsb gene. The phylogenetic analyses revealed that three piroplasm sequences were clustered with Rangellia vitalii, while one sequence was grouped with B. vogeli. The only sequence obtained from Hepatozoon spp.-PCR protocol was pooled with H. canis. Therefore, there is urgent need for differential molecular diagnosis of the two piroplasm species cited as etiological agents in clinical cases of canine hemoparasitic diseases, given the higher pathogenic potential of R. vitalii than of B. vogeli.

Genomic sequencing of avian haemosporidian parasites (Haemosporida) has been challenging due to excessive contamination from host DNA. In this study, we developed a cost-effective protocol to obtain parasite sequences from naturally infected birds, based on targeted sequence capture and next generation sequencing. With the genomic data of Haemoproteus tartakovskyi as a reference, we successfully sequenced up to 1000 genes from each of the 15 selected samples belonging to nine different cytochrome b lineages, eight of which belong to Haemoproteus and one to Plasmodium. The targeted sequences were enriched to ∼10 4 -fold, and mixed infections were identified as well as the proportions of each mixed lineage. We found that the total number of reads and the proportions of exons sequenced decreased when the parasite lineage became more divergent from the reference genome. For each of the samples, the recovery of sequences from different exons varied with the function and GC content of the exon. From the obtained sequences, we detected within-lineage variation in both mitochondrial and nuclear genes, which may be a result of local adaptation to different host species and environmental conditions. This targeted sequence capture protocol can be applied to a broader range of species and will open a new door for further studies on disease diagnostics and comparative analysis of haemosporidians evolution., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Testing for Tritrichomonas foetus and exclusion of infected animals is an effective way of improving the reproductive efficiency in a herd. Conventional PCR is inherently more specific than the culture method and quantitative PCR can significantly increase the detection limit. Loop Mediated Isothermal DNA Amplification (LAMP) is gaining interest because the method does not require expensive equipment, specificity and sensitivity can be as high as quantitative PCR. The object of this study was to develop a sensitive and friendly test for point-of-care detection of T. foetus. The LAMP test that targeted T. foetus elongation factor 1 alpha 1 sequences showed high specificity. Sensitivity was 100-1000 times higher than that reached through culture, polymerase chain reaction or with a previously developed LAMP for 5.8 ribosomal sequences. Moreover, T. foetus detection could be performed without DNA purification from infected cervical vaginal mucus (CVM) or smegma samples. The tf-ef1a1 LAMP method was tested for field detection with paper strips soaked in CVM from infected cows and the results were observed 90 min later. Direct detection of T. foetus in CVM with the tf-ef1a1 LAMP showed high sensitivity and specificity, and an overall diagnostic odds ratio of 56 (CI: 13.3-235.0). The tf-ef1a1 LAMP showed great potential for diagnosis and control of T. foetus in resource-challenged regions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study reports two clinical cases of avian haemosporidian infection caused by a Haemoproteus sp., involving a snowy owl (Bubo scandiacus) and a goshawk (Accipiter gentilis), at a zoo. The snowy owl died after presenting with anorexia, depression and lethargy. A blood smear with Wright's staining confirmed Haemoproteus infection. Necropsy of the snowy owl revealed hypertrophy of the internal organs, including the liver, gallbladder, kidney and adrenal glands. The goshawk showed anorexia, depression and a lowered head position, and was diagnosed with a Haemoproteus infection based on a blood smear. The goshawk was completely cured by treatment with a combination of atovaquone and proguanil hydrochloride. Both cases showed decreased erythrocytes, hemoglobin and hematocrit values on complete blood count.