A global systematic review and meta-analysis on the babesiosis in dogs with special reference to Babesia canis.

BACKGROUND: Canine babesiosis is a clinically significant tick-transmitted disease caused by several species of the intraerythrocytic protozoan parasite Babesia, which result in a wide range of clinical manifestations, from mild, transient infection to serious disease and even death. OBJECTIVES: The current study aimed to estimate the global prevalence and associated risk factors of Babesia in dogs. METHODS: Multiple databases (PubMed, Scopus, ProQuest, Web of Science and Google Scholar) were searched for relevant literature published from January 2000 up to December 2022. The statistical analyses were performed based on the R software (version 3.6) meta-package. RESULTS: Out of 23,864 publications, 229 studies met the inclusion criteria. The pooled prevalence of canine babesiosis was 0.120 (95% CI; 0.097-0.146). The highest pooled prevalence was found in Europe (0.207, 95% CI; 0.097-0.344). Among several species, Babesia canis was the most prevalent parasite (0.216, 95% CI; 0.056-0.441). The highest pooled prevalence of Babesia in dogs was observed in the summer season (0.097, 95% CI; 0.040-0.174). CONCLUSIONS: Regular screening and appropriate control strategies are recommended for the prevention of transmission of tick-borne disease transmission among dogs.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. Methods: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. Results: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. Conclusions: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Dog blood parasite infection in upland and lowland communities of northern Thailand: The role of environment and care of dog owners.

Dogs play an important role as hosts and reservoirs for many zoonotic diseases. Ehrlichiosis, babesiosis and hepatozoonosis are a group of canine vector-borne diseases that can be transmitted via ectoparasites from dog to dog and also from dog to humans. This study focused on three main blood parasites of dog (i.e., Babesia spp., Ehrlichia spp. and Hepatozoon spp.) among two different landscape types of eight villages of Santhong Sub-district, Nan Province, Thailand. In this study, 149 dogs were surveyed and blood samples were collected. Blood parasite infections in dogs were assessed using molecular detection approach. Babesia canis vogeli, Babesia gibsoni, Ehrlichia canis and Hepatozoon canis were detected with prevalence of infection at 10.7%, 8.1%, 3.4% and 0.7%, respectively. In terms of landscape type, prevalence of overall blood parasites, particularly Babesia spp. infections were higher in dogs living in upland forested areas (28.3%) compared to dogs from lowland agricultural areas (12.3%). Data obtained from the questionnaires on perceptions of dog owners showed that dogs raised all the time outside owner's house, and those dogs whose owners have never bathed and cleaned were more likely to be exposed to blood parasites. As infected dogs could play an important role as reservoirs of the blood parasites, attitude of dog owners may affect public health in terms of zoonotic disease transmission. Effective control measures and surveillance program of arthropod vectors and blood parasite infection in dogs still need to be advocated to minimize zoonotic disease transmission.

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.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France.

Ticks are major vectors of various pathogens of health importance, such as bacteria, viruses and parasites. The problems associated with ticks and vector-borne pathogens are increasing in mountain areas, particularly in connection with global climate change. We collected ticks (n = 2,081) from chamois and mouflon in 4 mountainous areas of France. We identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata and Dermacentor marginatus. We observed a strong variation in tick species composition among the study sites, linked in particular to the climate of the sites. We then analysed 791 ticks for DNA of vector-borne pathogens: Babesia/Theileria spp., Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, and Rickettsia of the spotted fever group (SFG). Theileria ovis was detected only in Corsica in Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) and Anaplasma phagocytophilum (3 sites) were detected in I. ricinus. Anaplasma ovis was detected at one site in I. ricinus and Rh. sanguineus s.l. SFG Rickettsia were detected at all the study sites: R. monacensis and R. helvetica in I. ricinus at the 3 sites where this tick is present; R. massiliae in Rh. sanguineus s.l. (1 site); and R. hoogstraalii and Candidatus R. barbariae in Rh. bursa in Corsica. These results show that there is a risk of tick-borne diseases for humans and domestic and wild animals frequenting these mountain areas.

Title: Prévalence d'agents pathogènes vectorisés chez des tiques collectées chez des ongulés sauvages (mouflons, chamois) dans 4 zones montagneuses en France. Abstract: Les tiques sont des vecteurs majeurs de différents agents pathogènes d'importance sanitaire, tels que des bactéries, des virus et des parasites. Les problématiques liées aux tiques et aux pathogènes vectorisés augmentent en zones de montagne, en lien notamment avec le réchauffement climatique. Nous avons collecté des tiques (n = 2 081) sur des chamois et des mouflons dans 4 zones montagneuses en France. Six espèces ont été identifiées : Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata et Dermacentor marginatus. Nous avons observé une forte variation de la composition en espèces de tiques entre les sites d'étude, en lien notamment avec le climat des sites. Nous avons ensuite recherché les ADN d'agents pathogènes vectorisés sur 791 tiques : Babesia/Theileria spp, Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, et de Rickettsia du groupe des fièvres boutonneuses (SFG). Theileria ovis a été détecté uniquement en Corse chez Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) et Anaplasma phagocytophilum (3 sites) ont été détectés chez I. ricinus. Anaplasma ovis a été détecté dans un site chez I. ricinus et Rh. sanguineus s.l.. Les Rickettsia SFG ont été détectées dans tous les sites d'étude : Rickettsia monacensis et R. helvetica chez I. ricinus dans les 3 sites où cette tique est présente; R. massiliae chez Rh. sanguineus s.l. (1 site); et R. hoogstraalii et Candidatus R. barbariae chez Rh. bursa en Corse. Ces résultats montrent un risque de transmission de maladies par les tiques pour les personnes et les animaux domestiques et sauvages fréquentant ces zones de montagne.

The prevalence of selected vector-borne diseases in dromedary camels (Camelus dromedarius) in the United Arab Emirates.

Vector-borne diseases (VBDs) affecting dromedary camels (Camelus dromedarius) have considerable importance in the United Arab Emirates (UAE) because of the consequences associated with production decline and economic losses. Our study aimed to determine the prevalence of selected VBDs in camels in the UAE and identify risk factors. This research is currently affected by the low number of epidemiological molecular surveys addressing this issue. Blood samples were obtained from 425 dromedary camels from different locations across the UAE. Whole genomic DNA was isolated, and PCR screening was done to detect piroplasmids (Babesia/Theileria spp.), Trypanosoma spp., and Anaplasmataceae spp. (Anaplasma, Ehrlichia, Neorickettsia and Wolbachia spp.). Amplicons were sequenced, and phylogenetic trees were constructed. Trypanosoma sequences were identified as T. brucei evansi, whereas Anaplasmataceae sequences were identified as A. platys-like. All camels were negative for Babesia/Theileria spp. (0%); however, 18 camels were positive for T. b. evansi (4%) and 52 were positive for A. platys-like (12%). Mixed infection with T. b. evansi and A. platys-like was found in one camel. Statistical analyses revealed that camels with a brown coat colour were significantly more prone to acquire the A. platys-like strain compared with those having a clearer coat. A similar finding was observed when comparing urban moving camels with desert indoor and urban indoor camels. Continuous disease surveillance is required to ensure and maintain the good health status of the camels in the UAE. Nonetheless, the risk of disease outbreak remains if the misuse of drugs continues.

Epidemiological assessment of Anaplasma marginale, Babesia bigemina, and Babesia bovis infections in Colombian creole cattle breeds: A molecular survey in northeastern Colombia.

Anaplasmosis and babesiosis are globally distributed arthropod-borne diseases known for causing substantial economic losses due to their high morbidity and mortality rates. This study aims to assess the frequency and epidemiological features associated with the infection of Anaplasma marginale, Babesia bigemina, and Babesia bovis in three Creole cattle breeds (Chino Santandereano (Chino), Casanareño (CAS), and Sanmartinero (SM)) in northeastern Colombia. Between June 2019 and March 2020, a total of 252 Creole cattle were sampled, with Chino, CAS, and SM accounting for 42.8%, 29.5%, and 29.5% of the samples, respectively. Blood samples were subjected to molecular analysis to detect the DNA of A. marginale, B. bigemina, and B. bovis, using species-specific primers. Additionally, Packed Cell Volume (PCV), total serum proteins, and body condition were evaluated. Molecular analyses revealed the presence of B. bigemina, A. marginale, and B. bovis in 83.7% (211/252; 95% CI = 79.1%-88.3%), 59.9% (151/252; 95% CI = 53.8%-66.1%), and 40.9% (103/252; 95% CI = 34.7%-46.9%) of the samples, respectively, with 69% (174/252; 95% CI = 57.8%-80.3%) exhibiting coinfections. Notably, in infected animals, no significant alterations in PCV, total serum proteins, or body condition were observed. Multivariate analyses indicated a statistically significant association between the frequency of A. marginale infection and the breed and season, with a higher frequency in SM during the rainy season (P < 0.05). To our knowledge, this is the first molecular survey that evaluates multiple arthropod-borne pathogens in Colombian Creole breeds. The results revel a high frequency of B. bigemina and A. marginale infections, coupled with a notable frequency of coinfections, all without significant alteration in the PCV, total serum proteins and body conditions. Our findings enhance the understanding of the epidemiological aspects of arthropod-borne pathogens in Colombian Creole breed and contribute to the improvement of sanitary programs for these animals.

Characterization and LC-MS/MS based proteomic analysis of extracellular vesicles separated from blood serum of healthy and dogs naturally infected by Babesia canis. A preliminary study.

Canine babesiosis is a rapidly spreading tick-borne disease in Europe, which entails protozoan parasites invading red blood cells. Small extracellular vesicles (EVs) (< 200 nm) were isolated from the serum of 15 healthy and 15 by Babesia canis naturally infected dogs aimed to distinguish EV characteristics and protein profiles. There were no significant differences (P = 0.05) observed in the mean sizes and concentrations of serum EVs between the healthy and canine babesiosis groups. Despite a higher number of Canis lupus proteins detected in EVs from serum of diseased dogs, there were no statistically significant differences (P < 0.05) in the number of protein IDs between the experimental groups. We successfully identified 211 Canis lupus proteins across both experimental groups, of which 147 Canis lupus proteins were validated as being EV-associated. This data set is accessible via the ProteomeXchange PXD047647. EVs isolated from serum of B. canis infected dogs were Cd9+, Cd63+, Cd81+, and Cd82+. Furthermore, 73 Canis lupus proteins were validated as EV-associated and specific for EVs isolated from serum of B. canis-infected dogs. These were predominantly membrane and cytosolic proteins, and innate and adaptive immune system-related proteins, especially those involved in adhesion and proteoglycan mechanisms like integrins. Enrichment was also observed for proteins involved in vascular and cellular responses, including signalling pathways such as VEGF, VEGFR, and the LKB1 network. When only blood-related sites of EV expression were evaluated, the origins of EV proteins were mostly cells of immune system. These were dendritic cells, neutrophils, B cells, monocytes and platelets. In general, proteins were enriched in pathways that collectively regulate various cellular processes, including immune responses, communication, signal transduction, membrane trafficking, and apoptosis. Serum EVs and their protein cargo may have an important role in both the invasion of B. canis and the host's response to the parasitic infection, nevertheless, additional experimental research is warranted. The overall count of identified EV proteins of parasitic origin, meeting cut off criteria of two peptides and 1 % FDR, was relatively low.

Enhancing parasitic organism detection in microscopy images through deep learning and fine-tuned optimizer.

Parasitic organisms pose a major global health threat, mainly in regions that lack advanced medical facilities. Early and accurate detection of parasitic organisms is vital to saving lives. Deep learning models have uplifted the medical sector by providing promising results in diagnosing, detecting, and classifying diseases. This paper explores the role of deep learning techniques in detecting and classifying various parasitic organisms. The research works on a dataset consisting of 34,298 samples of parasites such as Toxoplasma Gondii, Trypanosome, Plasmodium, Leishmania, Babesia, and Trichomonad along with host cells like red blood cells and white blood cells. These images are initially converted from RGB to grayscale followed by the computation of morphological features such as perimeter, height, area, and width. Later, Otsu thresholding and watershed techniques are applied to differentiate foreground from background and create markers on the images for the identification of regions of interest. Deep transfer learning models such as VGG19, InceptionV3, ResNet50V2, ResNet152V2, EfficientNetB3, EfficientNetB0, MobileNetV2, Xception, DenseNet169, and a hybrid model, InceptionResNetV2, are employed. The parameters of these models are fine-tuned using three optimizers: SGD, RMSprop, and Adam. Experimental results reveal that when RMSprop is applied, VGG19, InceptionV3, and EfficientNetB0 achieve the highest accuracy of 99.1% with a loss of 0.09. Similarly, using the SGD optimizer, InceptionV3 performs exceptionally well, achieving the highest accuracy of 99.91% with a loss of 0.98. Finally, applying the Adam optimizer, InceptionResNetV2 excels, achieving the highest accuracy of 99.96% with a loss of 0.13, outperforming other optimizers. The findings of this research signify that using deep learning models coupled with image processing methods generates a highly accurate and efficient way to detect and classify parasitic organisms.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

Molecular identification, risk factor assessment, and phylogenetic analysis of tick-borne pathogens in symptomatic and asymptomatic cattle from South-Eastern Iran.

Tick-borne pathogens (TBPs) represent a substantial threat to cattle globally, exerting adverse impacts on production, health, and economic viability. This study delves into the prevalence and implications of TTBPs in cattle sourced from resource-limited smallholder livestock farms situated in southeastern Iran, proximate to Afghanistan and Pakistan. Blood and tick specimens were systematically collected from a cohort of 230 cattle, comprising 150 asymptomatic and 80 symptomatic individuals. Genomic DNA isolated from blood samples underwent rigorous examination for the presence of key TBPs, including Anaplasma marginale, A. phagocytophilum, A. bovis, A. centrale, Babesia bigemina, and Theileria annulata, utilizing multiple genetic markers. Nucleotide sequence analysis facilitated the reconstruction of phylogenetic relationships. The study also evaluated various potential risk factors, such as clinical status, gender, age, breed, tick infestation, and management practices, to elucidate their associations with TTBPs. Among the cattle cohort, a staggering 87.8% (202/230) tested positive for at least one pathogen. Prevalence statistics encompassed A. marginale (72.2%), T. annulata (68.3%), A. phagocytophilum/A. platys-like complex (66.1%), A. centrale (16.7%), B. bigemina (10.0%), and A. bovis (6.1%). Remarkably, mixed infections involving two, three, and four pathogens were detected in 23%, 52.1%, and 2.2% of animals, respectively. Notably, all asymptomatic cattle were positive for at least one TBP. Tick infestation was observed in 62.2% (143/230) of cattle, predominantly caused by Hyalomma anatolicum (82.5%), Rhipicephalus (Boophilus) annulatus (13.1%), and R. sanguineus sensu lato (4.4%). Risk factors linked to TBPs encompassed tick infestation, older age, and crossbred animals. Clinical presentations among symptomatic cattle encompassed fever, anemia, weight loss, anorexia, jaundice, and enlarged superficial lymph nodes. This study underscores the pivotal role of asymptomatic carriers in the propagation of TTBPs within endemic regions. Furthermore, it emphasizes the potential for the implementation of molecular diagnostics to unmask subclinical infections, thereby affording the opportunity for targeted interventions aimed at ameliorating the burden of TTBPs in resource-constrained smallholder dairy farms.

Challenges of animals shelters in caring for dogs infected with Leishmania and other pathogens.

The incidence of human Visceral Leishmaniasis (VL) has decreased in Brazil; however, the number of areas reporting human and canine cases has increased, with Leishmania infantum usually preceding human infection. This study aimed to analyze the profile of infectious diseases that are endemic for both human and canine VL, in dogs housed in a shelter located in the state of Rio Grande do Norte, Northeast Brazil. Data was obtained between November/2021 to April/2022. All dogs residing at the shelter (98 dogs) were examined and blood was collected for testing for L. infantum, Ehrlichia canis, and Babesia sp. Statistical analyses considered the clinical and laboratory findings. Of the 98 animals, approximately 43% were positive for L. infantum antibodies, 19% were positive for L. infantum kDNA, and 18% were L. infantum positive by culture. Greater levels of anti-leishmania antibodies were observed in dogs with symptoms suggestive of VL. The dogs tested positive for E. canis (19/98) and B. canis (18/98). Lutzomyia longipalpis was captured inside the shelter, representing 74.25% (n = 225) of whole sandflies in the dog shelter. Concomitant infection by L. infantum and E. canis increased the odds of death. Treatment of VL included the use of allopurinol (n = 48) and miltefosine (n = 8). Treated animals showed more signs of Leishmania infection. Tickborn parasites and Leishmania were prevalent in sheltered dogs in a VL-endemic area, which increases the odds of death and poses an additional challenge for caring for abandoned dogs and at the same time setting protocols to manage reservoirs of L. infantum.

A small proportion of Zebu genetic background in crossbred calves may not be enough to improve resistance against natural bovine Babesia spp. infections.

The main objective of cattle breeders in tropical and subtropical regions is to acquire animals with taurine-productive traits adapted to the broad weather range of these regions. However, one of the main challenges on using taurine genetics in these areas is the high susceptibility of these animals to tick-borne diseases. Consequently, the present study evaluated from 10 November 2021-19 April 2022, the over 13 assessments, the Babesia bovis and Babesia bigemina DNA loads and the IgG anti-B. bovis and anti-B. bigemina levels in Angus (n = 17, 100% Taurine) and Ultrablack (n = 14, ∼82% taurine and 18% Zebu) calves. Data were analyzed using a multivariate mixed model with repeated measures of the same animal including the fixed effects of evaluation, genetic group, sex, Babesia spp., and their interactions. The repeatability values were estimated from the (co)variances matrix and expressed for each species. The correlations between the DNA loads (CNlog) and IgG titers (S/P) values for the two species were also estimated using the same model. Regarding the specific IgG antibody titers for both Babesia spp., no significant differences were observed between the two genetic groups. However, for B. bovis and B. bigemina DNA loads, Ultrablack calves presented significantly higher values than Angus calves. Under the conditions evaluated in this study, our findings suggest that the low percentage of Zebu genetic in the Ultrablack breed was insufficient to improve resistance against babesiosis. Further studies must demonstrate if the low percentages of Zebu genetics in Taurine breeds can modify the susceptibility to babesiosis infections.

Seasonal dynamics and genetic characterization of bovine arthropod-borne parasites in Nan Province, Thailand with molecular identification of Anaplasma platys and Trypanosoma theileri.

Virulent species or strains of hematophagous borne pathogens such as Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp., are lethal to susceptible animals or reduce their productivity on a global scale. Nonetheless, efforts to diagnose the causative agents and assess the genotypic profiles as well as quantify the parasite burden of aforementioned parasites across seasons remain limited. Therefore, the present investigation sought to elucidate the genotypic composition of Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp. The findings revealed heightened infection rates during the summer, manifesting a correlation between Trypanosoma spp. infection and seasonal fluctuations. Among the identified pathogens, Anaplasma marginale emerged as the most dominant species, while the occurrence of Anaplasma platys in Thai cattle was confirmed via the sequencing of the groEL gene. Moreover, the study successfully identified two lineages of Trypanosoma theileri. The findings of this investigation offer valuable insights that can inform the development of preventive strategies for vector-borne diseases, such as considering the appropriate use of insect repellent, mosquito or insect nets, or eliminating breeding places for insects in each season.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

Assessment of Babesia spp. prevalence in various domestic animals across Southern Punjab, Pakistan.

Parasitic diseases, notably babesiosis, exert a substantial impact on the global cattle industry, posing challenges to commerce, economies, and human health. This study, conducted in Southern Punjab, Pakistan, aimed to assess the prevalence of Babesia spp. across various livestock species using microscopic and PCR methods. A total of 180 blood samples (60 from each district) were systematically collected from apparently healthy animals, with 36 samples obtained from each domestic animal species, including camel, cattle, buffalo, goat, and sheep, noting that 12 samples were collected from each district for each animal species. Overall prevalence was determined to be 32.8% (59/180), with varying rates among species: 25.0% in cattle, 41.66% in buffalo, 30.55% in goats, 33.3% in sheep, and 33.3% in camels. Microscopic examination revealed slightly varied infection rates among large and small domestic animals (22.2%), while PCR results indicated a 32.8% overall infection rate in both large and small domestic animals, with no statistical significance. District-wise analysis showed regional variations, with Muzaffargarh recording a prevalence rate of 23.33% through microscopic examination, while Lodhran and Bahawalpur recorded 21.67%. PCR results revealed higher rates (38.33%, 26.67%, and 33.33%, respectively), underlining the importance of employing PCR for accurate detection. Examining ruminant types, large ruminants exhibited a 32.4% infection rate, while small domestic animals showed 33.3%, with no significant difference (p=0.897). District-wise prevalence showcased significant variation, with Muzaffargarh demonstrating a 25% prevalence, Lodhran 22%, and Bahawalpur 22%, through microscopic examination. PCR results displayed 38.33%, 27%, and 33.3%, respectively, with no statistical significance. Detailed analysis of individual districts highlighted variations in infection rates among camels, cattle, buffalo, goats, and sheep. The binomial test indicated significant differences through microscopic analysis (P=0.011) but non-significant variations through PCR (P=0.065), emphasizing the precision of PCR. Regional variations in prevalence, notably with Punjab exhibiting the highest frequency (33.87%) and KPK the lowest (13.24%), suggest potential influences from varying veterinary practices and environmental factors. This study underscores the pivotal role of PCR alongside microscopy for accurate babesiosis diagnosis. These findings contribute to the broader understanding of babesiosis prevalence, emphasizing the necessity of advanced molecular techniques for informed control measures.

Transmission risk evaluation of transfusion blood containing low-density Babesia microti.

Background: Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness. Purpose: This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti. Methods: Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA. Results: The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/µL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti. Conclusion: Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

Expression of sex-specific molecular markers by Babesia bovis gametes.

BACKGROUND: Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. METHODS AND RESULTS: We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. CONCLUSIONS: Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.