Diagnóstico de babesia caballi em éguas doadoras e receptoras de embrião pela técnica de reação em cadeia polimerase

A piroplasmose equina é uma doença transmitida por carrapatos infectados pelos protozoários Babesia caballi e Theileria equi, conhecida, respectivamente, como Babesiose ou Theileriose. As manifestações clínicas variam desde febre, anemia, apatia, até quadros agudos que podem resultar em morte ou perdas reprodutivas em éguas. O objetivo deste estudo foi identificar as sequências de DNA de Babesia caballi através da técnica de PCR convencional e diagnosticar a ocorrência de piroplasmose equina. Foram coletados 2 mL de sangue de 50 fêmeas equinas das raças Brasileiro de Hipismo (BH), Sem Raça Definida (SRD) e Quarto de Milha (QM), de idades variadas, pertencentes à Haras localizados no nordeste do Brasil. Os animais foram divididos em 2 grupos, sendo 9 animais do grupo das doadoras e 41 animais no grupo das receptoras, o sangue foi enviado ao laboratório de biologia molecular para ser utilizado para detecção de Babesia spp. Das 50 fêmeas equinas testadas nenhuma foi positiva para o protozoário Babesia spp. Resultado do bom manejo sanitário e controle de ectoparasitas de éguas doadoras e receptoras de embriões.

Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species

We describe the development, optimization, and validation of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of Babesia, Bartonella, and Borrelia spp. DNA from several sample matrices, including clinical blood samples from animals and humans, vectors, in-vitro infected human and animal cell lines, and tissues obtained from animal models (infected with Bartonella and/or B. burgdorferi). The multiplex ddPCR assay was able to detect 31 Bartonella, 13 Borrelia, and 24 Babesia species, including Theileria equi, T. cervi, and Cytauxzoon felis. No amplification of Treponema or Leptospira spp. was observed. Sensitivity of 0.2–5 genome equivalent DNA copies per microliter was achieved for different members of the Bartonella and Borrelia genus, depending on the species or matrix type (water or spiked blood DNA) tested. The ddPCR assay facilitated the simultaneous detection of co-infections with two and three vector-borne pathogens comprising four different genera (Babesia, Bartonella, Borrelia, and Theileria) from clinical and other sample sources.

Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt

Equine Piroplasmosis (EP) is an infectious disease caused by the hemoprotozoan parasites Theileria equi, Babesia caballi, and the recently identified species T. haneyi. Hereby, we used a multiplex PCR (mPCR) targeting the 18S rRNA gene of T. equi and B. caballi for the simultaneous detection of EP in Egyptian equids and examined the presence of T. haneyi infections in Egypt. Blood samples from 155 equids (79 horses and 76 donkeys) collected from different governorates of Egypt were examined by mPCR and PCR targeting T. hayeni. The mPCR method revealed a prevalence of T. equi of 20.3% in horses and of 13.1% in donkeys and a prevalence of B. caballi of 1.2% in horses. B. caballi was not detected in donkeys in the current study. The mPCR method also detected coinfections with both species (2.5% and 1.3% in horses and donkeys, respectively). Additionally, we report the presence of T. haneyi in Egypt for the first time in 53.1% of the horse and 38.1% of the donkey tested samples. Coinfection with T. haneyi and T. equi was found in 13.5% of the samples, while infection with the three EP species was found in 1.9% of the samples.

Evaluation of the Inhibitory Effects of Coumermycin A1 on the Growth of Theileria and Babesia Parasites in vitro and in vivo

Coumermycin A1, a coumarin antibiotic, has anticancer, antibacterial, antiviral, and antimalarial activities. We aimed to evaluate the anti-thielerial and anti-babesial activity of coumermycin A1 in mice in vivo. Coumermycin A1 efficacy was determined by the transcription of DNA gyrase, a type II DNA topoisomerase using reverse transcriptase-polymerase chain reaction (RT-PCR) transcription. Coumermycin A1 significantly inhibited the development of preliminary parasitemia (1%). Theileria equi and the Babesia species B. bigemina, B. bovis, and B. caballi were observed with IC50 values of 80, 70, 57, and 65 nM, respectively. Their development was remarkably inhibited at observed concentrations of 10, 25, 50, and 100µM for the studied organisms T. equi, and the Babesia species B. caballi, B. bovis and B. bigemina, respectively. In the subsequent viability test, parasite re-growth was suppressed at 100µM for B. bigemina and B. bovis and at 50 µM for B. caballi and T. equi. Coumermycin A1 Treatment of B. bovis cultures with Coumermycin A1 completely suppressed the transcription of the DNA gyrase subunits B and A genes. In BALB/c mice, the development of Babesia microti was inhibited by 70.73% using 5 mg/kg of Coumermycin A1.

Effects of Methanolic Extract from Turmeric (Curcuma longa) against the In Vitro Multiplication of Several Babesia Species and Theileria equi

Anti-piroplasm drugs currently on the market have proven toxicity to the host and parasite resistance. Plants are possible sources of novel drugs. Subsequently, a novel strategy should be used to find new anti-piroplasm agents that are both effective and safe. In the present study, we have evaluated the effect of turmeric (Curcuma longa) methanolic extract on the in vitro growth of Babesia (B.) bovis, B. divergens, B. caballi, and Theileria (T.) equi. The in vitro inhibitory effectiveness of turmeric was assessed using a fluorescence test. The enhancement in the in vitro inhibitory efficacy of turmeric when administrated in combination with diminazene aceturate (DA) was investigated using in vitro cultures of different piroplasm parasites. Turmeric reduced the in vitro growth of B. bovis, B. divergens, T. equi, and B. caballi with IC50 values of 0.830 0.078, 0.375 0.055, 1.405 0.575, and 0.720 0.090 mg/mL, respectively. An amount of 1 mg/mL turmeric for B. bovis, 0.5 mg/mL turmeric for B. divergens, 1 mg/mL turmeric for T. equi, and 0.5 mg/mL turmeric for B. caballi exhibited 73.43%, 80.065%, 73.47%, and 47.375% inhibitions in the growth of the parasites, respectively. When turmeric was combined with DA, its in vitro inhibitory impact on bovine Babesia and equine Babesia/Theileria parasites was amplified. These findings show that a methanolic extract of turmeric could be a promising medicinal plant for the treatment of babesiosis, especially when administered in conjunction with DA.

A SYSTEMATIC REVIEW AND META-ANALYSIS TO ASSESS THE SIGNIFICANCE OF MICROBIAL INFECTIONS IN ZEBRA

Wild equids can harvest multiple-host infectious agents that are able to affect other wildlife species, but also domestic animals and humans. The contact between wild and domestic equids is constantly increasing due to the depletion of natural areas, climate and land-usage changes, which could result in burdensome epidemics. Nevertheless, currently there is a lack of adequate epidemiological data from zebra. Three electronic databases were searched from 10 to 20 March 2021 for publications reporting bacterial, viral and protozoan infections in zebra. Data for a total of 12 nominal variables were extracted from reviewed papers to undergo a qualitative analysis on microbial infections in zebra. Prevalence-reporting studies were subjected to meta-analysis for estimating the pooled prevalence and seroprevalence of infectious agents in wild zebra populations. We identified 29 pathogen species and the most represented were Equine Herpesvirus 1 and 9, Bacillus anthracis, African Horse Sickness virus and Theileria equi. They were reported from all the three zebra species, both in captivity and wilderness. Pooled seroprevalences were estimated for the equine Orbiviruses AHSV (70%; 95%CI: 35-96%) and EEV (21%; 95%CI: 8-38%) and for the equine α -Herpesviruses EHV-1 (72%; 95%CI: 43-93%), EHV-4 (40%; 95%CI: 0-100%) and EHV-9 (58%; 95%CI: 9-98%), and pooled prevalences for the equine piroplasms T. equi (100%; 95%CI: 94-100%) and B. caballi (8%; 95%CI: 0-28%). Zebra is most probably a reservoir from which AHSV, EHV-1 and T. equi can be transmitted to horse populations, potentially causing disastrous epidemics. Zebra can also harvest zoonotic pathogens like B. anthracis, A. phagocytophylum, CCHFV and T. brucei. Other agents like EHV-9, BPV-1 and BPV-2 have the potential to spread from zebra to other wild endangered animal species. We conclude that zebra is an important host for multiple and dangerous pathogens. Alert and epidemiological research should be increased on infectious agents of zebra.

Molecular and microscopic detection of Babesia caballi and Theileria equi among working horses and donkeys in Cairo and Giza provinces of Egypt

Equine piroplasmosis (EP) is an ixodid tick-borne disease caused by Theileria equi and/or Babesia caballi that can lead to severe health issues and economic losses among equine population. This study aimed to determine the prevalence of T. equi and B. caballi among Egyptian equines based on microscopy and conventional PCR. Also, to determine the effect of season, age, and sex of on their prevalence and determining the difference in sensitivity between microscopy and conventional PCR in the diagnosis of EP. This study was carried out on 432 blood samples randomly collected from 146 horses and 286 donkeys during a period from April 2016 to March 2018. Microscopic examination revealed that among horses, 13 (8.9%) and 4 (2.7%) were infected by T. equi and B. caballi respectively. While among donkeys, 22 (7.7%), 16 (5.6%) respectively. While mixed infections were detected in 4 (1.4%) donkeys. There was a statistically nonsignificant relation between prevalence of infection and season and sex of equines but the highest prevalence was recorded in age group less than 5 years old. By conventional PCR, among 64 horses, 15 (23.4%) and 8 (12.5%) were infected by T. equi and B. caballi, respectively. While among 76 donkeys, 36 (47.4%), 16 (21.1%), and 5 (6.6%) were infected by T. equi, B. caballi, and mixed infection, respectively. Our finding proved the existence of T. equi and B. caballi among equines.

Comparing the efficacy of three diagnostic tools (microscopic examination, conventional PCR, and Real-Time PCR) in detecting Theileria equi infection among Egyptian equines

Equine theileriosis represents one of the main and serious health problems affecting equines industry globally, that is caused by tick-borne protozoan parasite called T. equi. This study aimed to assess the sensitivity of three diagnostic tools named: microscopic examination of a blood smear, conventional PCR, and Real-Time PCR (qPCR) to detect T. equi among equine population (n = 116) raised in Giza Governorate, Egypt. Microscopic examination of Giemsa-stained blood smears revealed the infection of 16.4% (19/116) of examined equines by T. equi while conventional PCR and qPCR revealed that 29.3% (34/116) and 43.1% (50/116) of examined equines were infected with T. equi respectively. Our results demonstrated that the qPCR had the highest sensitivity (100%) followed by conventional PCR (68%) while microscopic examination had the lowest sensitivity (38%). Furthermore, the negative predictive value (NPV) of qPCR was the highest (100%) compared to conventional PCR and microscopical examination (80.49% and 68.04% respectively) which revealed that all negative cases detected by qPCR were certainly correct compared to the other two diagnostic assays. Therefore, it is highly recommended to incorporate PCR diagnostic assays (conventional PCR and qPCR) alongside microscopic examination to evaluate the epidemiological status of equine theileriosis.