Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs.

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.

Long-term follow-up of laryngeal Rhinosporidium seeberi diagnosed by PCR and treated with laser ablation and voriconazole nebulization in a retired thoroughbred polo horse.

A 21-year-old retired polo Argentinian thoroughbred horse from a teaching herd was presented for a routine bronchoalveolar lavage demonstration, during which an incidental finding of a granulomatous mass on the dorsal aspect of the epiglottis was made. Rhinosporidium seeberi was suspected from a histological section obtained from an initial biopsy, and the mass was removed via laser surgery for cytology and PCR. Sequencing of the PCR amplicons confirmed the diagnosis of R. seeberi. A treatment protocol of nebulized voriconazole for 10 d postoperatively was used. Long-term follow-up required 2 more laser surgeries plus oral fluconazole to resolve the remaining fungal spores. However, 2.5 y later, there was no evidence of remaining fungal spores. Key clinical message: Horses from endemic regions can potentially be exposed to R. seeberi. Based on its travel history, this horse may have contracted the infection in South America, California, or Alberta. Treatments administered, including diode laser resection, voriconazole antifungal nebulization, and oral fluconazole administration, were successful but required repeated interventions.

Suivi à long terme du Rhinosporidium seeberi laryngé diagnostiqué par PCR et traité par ablation au laser et nébulisation au voriconazole chez un cheval de polo thoroughbred pur-sang à la retraiteUn cheval thoroughbred argentin de polo retraité de 21 ans, issu d'un troupeau d'enseignement, a été présenté pour une démonstration de lavage broncho-alvéolaire de routine, au cours de laquelle une découverte fortuite d'une masse granulomateuse sur la face dorsale de l'épiglotte a été faite. Rhinosporidium seeberi a été suspecté à partir d'une coupe histologique obtenue à partir d'une biopsie initiale, et la masse a été retirée par chirurgie au laser pour cytologie et PCR. Le séquençage des amplicons PCR a confirmé le diagnostic de R. seeberi. Un protocole de traitement au voriconazole nébulisé pendant 10 jours après l'opération a été utilisé. Le suivi à long terme a nécessité 2 autres interventions chirurgicales au laser et du fluconazole oral pour éliminer les spores fongiques restantes. Cependant, 2,5 ans plus tard, il n'y avait aucune trace de spores fongiques restantes.Message clinique clé:Les chevaux des régions endémiques peuvent potentiellement être exposés à R. seeberi. D'après ses antécédents de voyage, ce cheval pourrait avoir contracté l'infection en Amérique du Sud, en Californie ou en Alberta. Les traitements administrés, notamment la résection au laser à diode, la nébulisation antifongique au voriconazole et l'administration orale de fluconazole, ont été efficaces mais ont nécessité des interventions répétées.(Traduit par Dr Serge Messier).

Clinico-histopathological and molecular detection of small ruminants' papillomaviruses in Iran.

BACKGROUND: Papilloma DNA viruses are one of the viruses that cause skin lesions in ruminants. OBJECTIVES: The clinical, histopathological and molecular characteristics of cutaneous papilloma in ruminants in Iran are to be investigated in this study. METHODS: Samples were collected from 19 small ruminants (5 sheep and 14 goats) with various papillomatosis lesions. The samples taken were studied with histopathological and molecular techniques. RESULTS: In clinical terms, the lesions appeared in different sizes, ranging from 0.5 to 11 cm, and the cauliflower exophytic masses appeared in other parts of the animal's body. In the limbs, most papilloma lesions have been seen (42.1%). In histopathological examination, perinuclear vacuolation epidermal granule layer with various degrees of hypergranulosis, hyperkeratosis, acanthosis, orthokeratosis and parakeratosis were seen. Moreover, all the suspected samples were positive for papillomavirus using the polymerase chain reaction technique. CONCLUSIONS: Although the prevalence of papillomaviruses in Iranian sheep and goats is low, it seems necessary to distinguish them from other viral skin diseases, such as cutaneous contagious ecthyma, using molecular techniques and histopathology.

Occurrence and genotyping of Enterocytozoon bieneusi in flying squirrels (Trogopterus xanthipes) from China.

Enterocytozoon bieneusi is an obligate intracellular microsporidian parasite with a worldwide distribution. As a zoonotic pathogen, E. bieneusi can infect a wide range of wildlife hosts through the fecal-oral route. Although the feces of flying squirrels (Trogopterus xanthipes) are considered a traditional Chinese medicine (as "faeces trogopterori"), no literature is available on E. bieneusi infection in flying squirrels to date. In this study, a total of 340 fresh flying squirrel fecal specimens from two captive populations were collected in Pingdingshan city, China, to detect the prevalence of E. bieneusi and assess their zoonotic potential. By nested PCR amplification of the ITS gene, six specimens tested positive, with positive samples from each farm, with an overall low infection rate of 1.8%. The ITS sequences revealed three genotypes, including known genotype D and two novel genotypes, HNFS01 and HNFS02. Genotype HNFS01 was the most prevalent (4/6, 66.7%). Phylogenetic analysis showed that all genotypes clustered into zoonotic Group 1, with the novel genotypes clustering into different subgroups. To our knowledge, this is the first report of E. bieneusi infection in flying squirrels, suggesting that flying squirrels could act as a potential reservoir and zoonotic threat for E. bieneusi transmission to humans in China.

Title: Occurrence et génotypage d'Enterocytozoon bieneusi chez les écureuils volants (Trogopterus xanthipes) de Chine. Abstract: Enterocytozoon bieneusi est un parasite microsporidien intracellulaire obligatoire présent dans le monde entier. En tant qu'agent pathogène zoonotique, E. bieneusi peut infecter un large éventail d'hôtes sauvages par la voie fécale-orale. Bien que les excréments d'écureuils volants (Trogopterus xanthipes) soient considérés comme un ingrédient de médecine traditionnelle chinoise (comme « faeces trogopterori ¼), aucune littérature n'est disponible à ce jour sur l'infection par E. bieneusi chez les écureuils volants. Dans cette étude, un total de 340 spécimens fécaux frais d'écureuils volants provenant de deux populations captives ont été collectés dans la ville de Pingdingshan, en Chine, pour détecter la prévalence d'E. bieneusi et évaluer leur potentiel zoonotique. Par amplification PCR nichée du gène ITS, six échantillons se sont révélés positifs, avec des échantillons positifs dans chaque ferme, et un taux d'infection global faible, à 1,8 %. Les séquences ITS ont révélé trois génotypes, dont le génotype D connu et deux nouveaux génotypes, HNFS01 et HNFS02. Le génotype HNFS01 était le plus répandu (4/6, 66,7 %). L'analyse phylogénétique a montré que tous les génotypes se regroupaient dans le groupe zoonotique 1, les nouveaux génotypes se regroupant en différents sous-groupes. À notre connaissance, il s'agit du premier rapport d'infection par E. bieneusi chez des écureuils volants, ce qui suggère que les écureuils volants pourraient agir comme un réservoir potentiel et une menace zoonotique pour la transmission d'E. bieneusi aux humains en Chine.

Prevalence of Dichelobacter nodosus in western Austrian sheep flocks: Comparison of bacterial cultures, clinical foot rot and lameness with PCR and analysis of sample pooling for PCR diagnosis.

INTRODUCTION: Ovine foot rot is a highly contagious and multifactorial claw disease, caused by Dichelobacter nodosus (D. nodosus) and is the main cause of lameness in sheep. The aim of this cross-sectional study was to determine the prevalence of D. nodosus in western Austria both at animal and farm levels. Real-time PCR was evaluated in comparison with clinical and bacteriological investigations from interdigital foot swabs to detect D. nodosus-infected animals. In addition, the use of pooled four-foot swabs to detect foot rot was determined. In course of the study a total of 3156 sheep from 124 farms were examined for lameness and clinical signs of foot rot. The found flock prevalence of D. nodosus was 30,65 % with bacterial culture showing a sensitivity of 75,0 % and a specificity of 100,0 % (p < 0,001) respectively, compared with PCR. Furthermore, clinical foot rot scores (Ckorr = 0,87; p < 0,001) and lameness scores (Ckorr = 0,71; p < 0,001) highly correlated with the detection of D. nodosus by PCR. The result showed that the clinical examination can be used to identify animals infected with D. nodosus in flocks, but PCR must be used to confirm the diagnosis. D. nodosus could be detected equally well with risk-based pools-of-five samples as with undiluted samples (p < 0,001), suggesting that a pool-of-five samples might be a suitable and cost-effective method for detecting D. nodosus in sheep flocks. This study provides an overview of foot rot in Tyrolean sheep flocks and outlines the possibilities and limitations of the various diagnostic tools for D. nodosus. Further studies to investigate possible influencing factors, including alpine pasturing, management factors and biosecurity predisposing to foot rot are necessary for the design of effective future control programs in alpine regions.

INTRODUCTION: Le piétin ovin est une maladie des onglons hautement contagieuse et multifactorielle, causée par Dichelobacter nodosus (D. nodosus) qui constitue la principale cause de boiterie chez les ovins. L'objectif de cette étude transversale était de déterminer la prévalence de D. nodosus dans l'ouest de l'Autriche, tant au niveau de l'animal que de l'exploitation. La PCR en temps réel a été évaluée en comparaison avec les examens cliniques et bactériologiques effectués à partir d'écouvillons des espaces interdigités pour détecter les animaux infectés par D. nodosus. En outre, l'utilisation d'un pool d'écouvillons des quatre membres pour détecter le piétin a été déterminée. Au cours de l'étude, un total de 3156 moutons provenant de 124 fermes ont été examinés pour détecter des boiteries et des signes cliniques de piétin. La prévalence de D. nodosus dans les troupeaux était de 30,65 %, la culture bactérienne montrant une sensibilité de 75 % et une spécificité de 100 % (p < 0,001), respectivement, par rapport à la PCR. En outre, les scores cliniques de piétin (Ckorr = 0,87; p < 0,001) et les scores de boiterie (Ckorr = 0,71; p < 0,001) étaient fortement corrélés avec la détection de D. nodosus par PCR. Les résultats montrent que l'examen clinique peut être utilisé pour identifier les animaux infectés par D. nodosus dans les troupeaux mais que la PCR doit être utilisée pour confirmer le diagnostic. D. nodosus a pu être détecté aussi bien avec des pools de cinq échantillons basés sur le risque qu'avec des échantillons non dilués (p < 0,001), ce qui suggère qu'un pool de cinq échantillons pourrait être une méthode appropriée et rentable pour détecter D. nodosus dans les troupeaux de moutons. Cette étude donne un aperçu du piétin dans les troupeaux de moutons tyroliens et souligne les possibilités et les limites des différents outils de diagnostic pour D. nodosus. D'autres études visant à examiner les facteurs d'influence possibles, y compris les pâturages alpins, les facteurs de gestion et la biosécurité prédisposant au piétin, sont nécessaires pour la conception de futurs programmes de contrôle efficaces dans les régions alpines.

First report of occurrence of Babesia gibsoni in captive Indian wolves.

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves.

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation.

BACKGROUND: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis. METHODS: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis. RESULTS: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection. CONCLUSIONS: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

Molecular detection of Toxoplasma gondii in domestic and wild guinea pigs (Cavia spp.) from the Marangani district in Cuzco, Peru.

Toxoplasmosis is a foodborne disease caused by the protozoan Toxoplasma gondii, and transmitted to humans by eating raw or undercooked meat, mainly. Poultry, beef, and pork are the main meats consumed in Peru; despite this, guinea pig meat is also widely consumed. For this reason, the objective of this study was to molecularly detect T. gondii in domestic and wild guinea pigs from the Marangani district in Cuzco, Peru, and identify some risk factors associated with this pathogen. DNA was extracted from the brain tissue samples of guinea pigs (30 domestic and 30 wild), and PCR protocols were used to amplify the internal transcribed spacer (ITS-1) region and a 529 bp fragment from the T. gondii genome. T. gondii DNA was detected in 14 (23.3%) guinea pigs. T. gondii frequency was 33.3% in domestic guinea pigs and 13.3% in wild guinea pigs. Our results demonstrated that guinea pigs represent an important source for T. gondii infection in human populations in this locality.

Prevalence and molecular characterization of Giardia duodenalis in companion dogs, domestic livestock and wildlife in the Jordan Basin, Israel.

Giardiasis is a small intestinal disease caused by the zoonotic parasite, Giardia duodenalis. This study presents the molecular findings of G. duodenalis infection in companion dogs, domestic livestock and wildlife in the Northern Jordan Basin, Israel. Identification of G. duodenalis was accomplished by nested PCR (nPCR) targeting the 18S rRNA gene. Samples were collected from water (five samples from four sources of which one was recycled water), as well as feces from wolves (Canis lupus) (n = 34), jackals (Canis aureus) (n = 24), wild boars (Sus scrofa) (n = 40), cattle (Bos taurus) (n = 40), dogs (Canis lupus familiaris) (n = 37) and nutria (Mayocastor coypus) (n = 100). All positive samples were sequenced and a phylogenetic tree was drawn using the Bayesian Inference (BI) algorithm. Differences in G. duodenalis prevalence between the different hosts were analyzed by Pearson's chi-square (p < 0.05). Of the total 275 fecal samples, 36 were positive for G. duodenalis (13%). Frequency rates among different animal species was highest in wolves (32.3%), whilst rates in wild boars (22.5%), dogs (16.2%), cattle (12.5%) and jackals (4.2%), were observed to be significantly lower (p < 0.001). Three out of 5 recycled water (RW) samples were G. duodenalis positive. Three clusters with high posterior probabilities (PP) were found in the BI: Cluster 1: samples from wolves, wild boars, water and cattle together with database sequences of assemblages A, B and F, Cluster 2: samples from dogs, nutria and a jackal with sequences from assemblage D and Cluster 3: samples from cattle, wild boars, wolves and dogs with sequences from assemblage C and D. We suggest that wolves serve as reservoirs of G. duodenalis in this region. The finding of Giardia in RW suggests that this vehicle may further contaminate crops intended for human consumption as this water source is used for agricultural irrigation.

Investigating the infection dynamics and molecular detection of Cryptosporidium in Buffaloes in Sylhet, Bangladesh.

Cryptosporidiosis, a zoonotic infection impacting both livestock and humans, is inadequately understood in terms of its prevalence and transmission dynamics involving buffaloes in Bangladesh. This research, conducted in the Sylhet division, aimed to explore the prevalence and potential risk factors influencing Cryptosporidium spp. in the faecal samples of 392 buffaloes. Detection of the parasite utilized modified Ziehl-Neelsen staining, with molecular identification achieved through nested PCR (nPCR). The comprehensive analysis revealed 9.18% (36/392) prevalence at the individual animal level and 40.48% (17/42) at the herd level. Age-based analysis revealed fluctuating infection rates of Cryptosporidium spp. in buffaloes across distinct age brackets, with rates of 22.61% in those aged 0-6 months, 5.00% in those aged 6-12 months, and 1.03% in those aged 12-18 months. Diarrheic buffaloes showed a significantly (p < 0.001) higher infection rate (26.67%; 28/105) compared to non-diarrheic buffaloes (2.79%; 8/287). In risk factor analysis, binary logistic regression revealed that buffaloes aged 0-6 months were experiencing a likelihood that is 14.84 times higher to be affected by Cryptosporidium in contrast to their older counterparts (OR = 14.85; p = 0.02). Additionally, diarrhoeic buffaloes were found to be more susceptible to Cryptosporidium compared to healthy buffaloes (OR = 17.50; p < 0.001). A higher stocking density was associated with an increased likelihood of infection in buffaloes (OR = 11.20; p = 0.01). The results of this study emphasize the necessity for targeted interventions, considering factors like diarrheic condition and stocking density, to effectively manage and control cryptosporidiosis in Bangladesh.

Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt.

BACKGROUND: Tick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene. RESULTS: We collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries. CONCLUSION: This study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A.

BACKGROUND: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. METHODS: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. RESULTS: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. CONCLUSIONS: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Identification and characterization of a novel circovirus in Iberian lynx in Spain.

Circoviruses cause severe disease in pigs and birds. Canine circovirus has thus far only been associated with respiratory and gastrointestinal disorders and systemic disease in dogs. The Iberian lynx (Lynx pardinus) is one of the most endangered carnivores in Europe and the most endangered felid worldwide. Exploring the virome of these animals may be important in terms of virus discovery and assessing the interspecies-circulation of viruses from related carnivores. In this study, 162 spleen samples from Iberian lynx were screened for CRESS DNA viruses. Overall, 11 (6.8%) of 162 samples tested positive using a consensus PCR. Partial rep sequences were tightly related to each other (96.6-100%). Specific molecular protocols were designed on the partial rep sequences of the novel virus, Iberian lynx-associated circovirus-1 (ILCV-1). By screening a subset of 45 spleen samples, the infection rate of ILCV-1 in Iberian lynxes was 57.8% (26/45). ILCV-1 strains formed a separate cluster intermingled with bat, rodent, mongoose, and felid circoviruses. The genome of the novel virus displayed the highest nucleotide identity (64.3-65.3%) to mongoose circoviruses, thus representing a novel candidate circovirus species. The detection of these viruses in the spleen tissues could suggest systemic infection in the animal host. Overall, these findings suggest that this novel circovirus is common in the Iberian lynx. Further studies are warranted to assess the possible health implications of ILCV-1 in this endangered species.

First report of Leishmania tropica in domestic and wild animal hosts in hyperendemic areas of human cutaneous leishmaniasis in western Yemen: a neglected tropical disease needing One Health approach.

Cutaneous leishmaniasis (CL), a neglected tropical disease, is a major public health concern in Yemen, with Leishmania tropica identified as the main causative agent. This study aims to investigate the occurrence and distribution of Leishmania parasites in domestic and wild animals in CL endemic areas in the western highlands of Yemen. A cross-sectional study was conducted in the Utmah District of western Yemen. Blood and skin scraping specimens were collected from 122 domestic and wild animals and tested for the Leishmania DNA using internal transcribed spacer 1 (ITS1) nested polymerase chain reaction. Phylogenetic analyses were performed on 20 L. tropica sequences obtained from animals in this study and 34 sequences from human isolates (collected concurrently from the same study area) retrieved from the GenBank. Overall, L. tropica was detected in 16.4% (20/122) of the examined animals, including 11 goats, two dogs, two bulls, one cow, one donkey, one rabbit, one rat and one bat. None of the examined cats and sheep was positive. The animal sequences were segregated into four different L. tropica haplotypes, with the majority of the animal (15/20) and human (32/34) sequences composed of one dominant haplotype/genotype. These findings represent the first confirmed evidence of natural L. tropica infections in different kinds of domestic and wild animals in western Yemen, suggesting these animals potentially have a role in the transmission of CL in Yemen. Therefore, a One Health approach is required for the effective prevention and control of this devastating disease among endemic populations.

Detection of Toxoplasma gondii infection in buffaloes (Bubalus bubalis) and cattle (Bos taurus) at the Tabriz abattoir, Iran.

BACKGROUND: Toxoplasma gondii is a widely prevalent zoonotic protozoan parasite in humans and warm-blooded animals worldwide. Infection of humans by this parasite can result in severe clinical symptoms, particularly in individuals with congenital toxoplasmosis or immunocompromised patients. Contamination mainly occurs through foodborne routes, especially the consumption of raw or undercooked meat from animals. OBJECTIVES: The aim of this study was to use PCR to detect T. gondii in tissues and organs of buffaloes and cattle slaughtered at Tabriz slaughterhouse, in Iran. METHODS: Fifty grams of heart, thigh, diaphragm and tongue from 50 buffaloes and 100 cattle slaughtered at the Tabriz industrial slaughterhouse were selected for sampling using a combination of convenience sampling. The samples were tested using a previously published PCR method. RESULTS: Out of the 150 animal samples, T. gondii was detected in 10 (6.7%, 95%CI: 3.2-11.9), including one buffalo (2%, 95%CI: 0.1-10.6) and nine cattle (9%, 95%CI: 4.2-16.4). There was no statistically significant difference in the rate of T. gondii infection among cattle based on age and sex (p > 0.05). CONCLUSIONS: The results indicated a potential risk of T. gondii transmission to humans through the consumption of infected meat. Therefore, appropriate and effective preventive measures should be taken to limit the transmission of this parasite to humans, and the consumption of raw and undercooked meat should be discouraged.

Molecular survey of Piroplasmida, Hepatozoon spp. and Anaplasmataceae in anemic and thrombocytopenic dogs from Uruguay.

Canine tick-borne diseases, such as babesiosis, rangeliosis, hepatozoonosis, anaplasmosis and ehrlichiosis, are of veterinarian relevance, causing mild or severe clinical cases that can lead to the death of the dog. The aim of this study was detecting tick-borne protozoan and rickettsial infections in dogs with anemia and/or thrombocytopenia in Uruguay. A total of 803 domestic dogs were evaluated, and 10% were found positive (detected by PCR) at least for one hemoparasite. Sequence analysis confirmed the presence of four hemoprotozoan species: Rangelia vitalii, Babesia vogeli, Hepatozoon canis and Hepatozoon americanum, and the rickettsial Anaplasma platys. The most detected hemoparasite was R. vitalii, followed by H. canis and A. platys. This is the first report of B. vogeli in Uruguay and the second report of H. americanum in dogs from South America. The results highlight the importance for veterinarians to include hemoparasitic diseases in their differential diagnosis of agents causing anemia and thrombocytopenia.

Development of PCR-based genetic test for detection of TTF1 mutation causing abortion in Holstein Friesian cattle.

A stop-gain mutation (rs715966442; BTA11: 1,02,463,944 nucleotide position) in transcription termination factor, RNA polymerase I (TTF1) gene causes abortion in Holstein Friesian (HF) cattle. A PCR-restriction fragment length polymorphism (PCR-RFLP)-based genetic test has been developed and validated to screen the TTF1 mutation locus in HF cattle. The mutation locus was screened in 80 HF and HF crossbreds using the protocol, which revealed two animals as carriers of the mutant TTF1 allele. The test employed is cost-effective, rapid and precise and can be utilized as an effective tool for the screening of TTF1 mutation carriers in HF cattle population.

Occurrence of gastrointestinal nematodes in lambs in Norway, as assessed by copromicroscopy and droplet digital polymerase chain reaction.

BACKGROUND: Gastrointestinal nematodes (GINs) have a major impact on sheep production, health, and welfare worldwide. Norway is no exception, but there are only a few studies on the prevalence of GINs in Norwegian sheep. The aim of this study was to investigate the current occurrence of the most important nematodes in sheep flocks in Norway. Faecal samples were collected from flocks in 2021/2022, mainly from three geographical regions in Norway, i.e., northern, eastern, and western. In each of 134 flocks included, individual samples from 10 lambs (autumn) were pooled. Third stage larvae (L3) were cultivated and harvested (Baermann method) from the pooled samples. The DNA was then extracted and further analysed using droplet digital PCR (ddPCR). This enables assessment of the proportions of the three most important nematode species/genera, i.e., H. contortus, T. circumcincta, and Trichostrongylus. The fractional abundance/relative proportion of each species/genus was assessed by performing duplex assays with universal strongyle and species/genus-specific primers and probe sets. In addition, the occurrence of Nematodirus eggs was assessed by standard faecal egg counts (i.e., McMaster method). RESULTS: Of the 134 flocks sampled, 24 were from the northern region, 31 from eastern, and 71 from western Norway. In addition, some flocks from central (n = 7), and southern (n = 1) Norway were included. Among the sampled flocks, T. circumcincta occurred most commonly (94%), followed by H. contortus (60%) and Trichostrongylus (55%), and Nematodirus (51%). In general, mixed infections were observed, with 38% and 18% of flocks infected with three or all four genera, respectively. CONCLUSIONS: The results of this study indicate that GINs are widespread in Norway. Teladorsagia circumcincta seems to be present in most flocks based on this screening. Moreover, the results show that Nematodirus spp. infect lambs throughout the country, predominantly N. battus, and indicate that this nematode has become more abundant, which could lead to an increase in nematodirosis.

Dermatophytosis in cats: A comprehensive study on diagnostic methods and their accuracy.

Background: Dermatophytosis is a contagious fungal infection that affects mainly cats. It poses significant challenges in veterinary medicine due to its zoonotic potential and impact on animal and public health. Rapid and reliable diagnosis is crucial for preventing the spread of the disease, guiding treatment decisions, and monitoring disease control efforts. Although there are several studies on diagnostic methods in feline dermatophytosis, the comparison between them from the same sample lacks data. The absence of a universally accepted gold standard diagnostic method highlights the need for a multifaceted approach to diagnosing feline dermatophytosis. Aim: This study aims to assess the accuracy and efficacy of different diagnostic techniques comprehensively. Methods: For this, 48 samples of cats were analyzed by dermoscopy, direct hair examination, fungal culture using various media (Mycosel, Sabouraud, and Dermatophyte Test Medium), and polymerase chain reaction (PCR). Results: Direct examination and dermoscopy yielded unsatisfactory results. Mycosel and Sabouraud were suboptimal. DTM demonstrated superior selectivity, making it the most reliable among traditional methods. PCR was the top performer, exhibiting singular sensitivity, specificity, and accuracy. Conclusion: The study suggests that PCR may be the preferred choice for diagnosing feline dermatophytosis in clinical practice, especially when rapid and accurate results are essential.

Molecular detection of Histoplasma capsulatum in organ samples from bats randomly captured in urban areas of Araraquara, São Paulo state, Brazil.

The mycosis histoplasmosis is also considered a zoonosis that affects humans and other mammalian species worldwide. Among the wild mammals predisposed to be infected with the etiologic agent of histoplasmosis, bats are relevant because they are reservoir of Histoplasma species, and they play a fundamental role in maintaining and spreading fungal propagules in the environments since the infective mycelial phase of Histoplasma grows in their accumulated guano. In this study, we detected the fungal presence in organ samples of bats randomly captured in urban areas of Araraquara City, São Paulo, Brazil. Fungal detection was performed using a nested polymerase chain reaction to amplify a molecular marker (Hcp100) unique to H. capsulatum, which revealed the pathogen presence in organ samples from 15 out of 37 captured bats, indicating 40.5% of infection. Out of 22 Hcp100-amplicons generated, 41% corresponded to lung and trachea samples and 59% to spleen, liver, and kidney samples. Data from these last three organs suggest that bats develop disseminated infections. Considering that infected bats create environments with a high risk of infection, it is important to register the percentage of infected bats living in urban areas to avoid risks of infection to humans, domestic animals, and wildlife.