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Simple Summary: We developed a new test to detect Babesia aktasi, a parasite that infects goats, using a method called semi-nested PCR. This method focuses on a specific part of the parasite's DNA to ensure accuracy. We checked the test against several other similar parasites to make sure it only detected B. aktasi, which it did successfully. To see how sensitive our test is, we used blood samples with known amounts of the parasite and found that our test could detect even very low levels of infection. Our results show that this new test is both highly accurate and sensitive, making it a valuable tool for identifying B. aktasi infections in goats. This new PCR method provides a reliable tool for detecting B. aktasi in goats, which is crucial for managing and preventing the spread of this infection, ultimately protecting goat health and improving agricultural productivity. We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10−8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, an experimental investigation was conducted to assess the pathogenicity of Babesia aktasi in immune-suppressed sheep. For this purpose, five immune-suppressed lambs under one year of age were infected by intravenous injection of fresh blood containing approximately 9.2% and 12% parasitemia of B. aktasi. Following parasite injection, the lambs were monitored daily for clinical and microscopic findings of babesiosis for 30 days. Throughout this period, no clinical and microscopic signs of babesiosis were observed in the lambs. Of the five recipient lambs, two tested negative for B. aktasi in nested PCR up to 30 days post-infection. Among the remaining three lambs, two were PCR positive on the first day, and the other one remained positive until the fourth day post-infection. DNA sequencing confirmed that the PCR positivity in the recipient lambs originated from the inoculum. These findings indicate that immune-suppressed lambs do not appear to be susceptible to infection with B. aktasi, which is highly pathogenic to immune-suppressed indigenous goats. Our survey in the Mediterranean region of Türkiye revealed high prevalence of Babesia aktasi in goats, while no molecular evidence of the parasite was found in sheep grazing in the same pasture. We hypothesized that the parasite may not be infectious to sheep. To test this hypothesis, the present study was designed to evaluate the susceptibility of Akkaraman sheep breed to B. aktasi infection. Fifteen mL of fresh blood infected with B. aktasi was injected into immune-suppressed lambs (n = 5). The recipient lambs were monitored daily for clinical signs of babesiosis over 30 days, and blood was collected for microscopic and molecular diagnostic evaluation. The lambs did not display clinical and parasitological signs of babesiosis. Two out of five recipient lambs were nested PCR-negative for B. aktasi over 30 days post infection. Out of the remaining three lambs, two were PCR positive on the first day, and one recipient was positive until the fourth day post infection. DNA sequencing confirmed that the PCR positivity in the recipient lambs originated from the inoculum. These findings revealed that immune-suppressed sheep do not appear to be susceptible to infection with B. aktasi that is lethal to immune-suppressed indigenous goats. [ABSTRACT FROM AUTHOR]

Simple Summary: The tick species involved in the transmission of Babesia aktasi, which is widespread in the Mediterranean region, is unknown. However, studies have shown that Rhipicephalus bursa is the most common tick species in the regions where the prevalence of B. aktasi is widespread. This finding increases the possibility that R. bursa may serve as a vector for the transmission of B. aktasi and encourages the evaluation of the vector competence of R. bursa. For this purpose, clinical babesiosis was induced in an immune-suppressed indigenous donor goat. Babesia spp.-free R. bursa larvae (n = 2000) and adults (n = 25) obtained from laboratory colonies fed on the donor goat. Following oviposition, PCR analysis of engorged female carcasses and engorged nymphs revealed the presence of B. aktasi, whereas no positivity was found in unfed larvae and adult ticks. The subsequent developmental stages of these ticks were used to infest three additional immune-suppressed goats. No clinical signs of babesiosis were observed in the infested goats. Moreover, molecular analysis did not detect DNA in the goats. These results demonstrated that R. bursa does not transmit B. aktasi, neither transovarially nor transstadially. A recent molecular survey revealed a high prevalence of Babesia aktasi in indigenous goats from the Mediterranean region of Türkiye, coinciding with heavy Rhipicephalus bursa infestations. This geographical overlap has raised the possibility that R. bursa may serve as a vector for the parasite. To evaluate the potential of R. bursa to serve as a vector for the parasite, an experimental study was conducted in indigenous goats. An immune-suppressed donor goat was intravenously injected with 15 mL of the cryopreserved B. aktasi stabilate, resulting in severe clinical babesiosis and parasitemia. Subsequently, R. bursa larvae and adults derived from Babesia-free laboratory colonies were allowed to feed on the infected donor goat. After oviposition, engorged female carcasses, representative engorged nymphs, unfed larvae, and adult pools were used for DNA extraction and PCR analysis. No PCR positivity was detected in any of the DNA samples, except for those with engorged female carcasses and nymphs. Three immune-suppressed recipient goats were infested with the unfed immature and mature ticks consuming the blood of a donor infected with B. aktasi. No clinical or parasitological findings were encountered in the recipient for 40 days post-infestation. These findings indicated that R. bursa was not a competent vector for B. aktasi. [ABSTRACT FROM AUTHOR]

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10−8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

Our survey in the Mediterranean region of Türkiye revealed high prevalence of Babesia aktasi in goats, while no molecular evidence of the parasite was found in sheep grazing in the same pasture. We hypothesized that the parasite may not be infectious to sheep. To test this hypothesis, the present study was designed to evaluate the susceptibility of Akkaraman sheep breed to B. aktasi infection. Fifteen mL of fresh blood infected with B. aktasi was injected into immune-suppressed lambs (n = 5). The recipient lambs were monitored daily for clinical signs of babesiosis over 30 days, and blood was collected for microscopic and molecular diagnostic evaluation. The lambs did not display clinical and parasitological signs of babesiosis. Two out of five recipient lambs were nested PCR-negative for B. aktasi over 30 days post infection. Out of the remaining three lambs, two were PCR positive on the first day, and one recipient was positive until the fourth day post infection. DNA sequencing confirmed that the PCR positivity in the recipient lambs originated from the inoculum. These findings revealed that immune-suppressed sheep do not appear to be susceptible to infection with B. aktasi that is lethal to immune-suppressed indigenous goats.

A recent molecular survey revealed a high prevalence of Babesia aktasi in indigenous goats from the Mediterranean region of Türkiye, coinciding with heavy Rhipicephalus bursa infestations. This geographical overlap has raised the possibility that R. bursa may serve as a vector for the parasite. To evaluate the potential of R. bursa to serve as a vector for the parasite, an experimental study was conducted in indigenous goats. An immune-suppressed donor goat was intravenously injected with 15 mL of the cryopreserved B. aktasi stabilate, resulting in severe clinical babesiosis and parasitemia. Subsequently, R. bursa larvae and adults derived from Babesia-free laboratory colonies were allowed to feed on the infected donor goat. After oviposition, engorged female carcasses, representative engorged nymphs, unfed larvae, and adult pools were used for DNA extraction and PCR analysis. No PCR positivity was detected in any of the DNA samples, except for those with engorged female carcasses and nymphs. Three immune-suppressed recipient goats were infested with the unfed immature and mature ticks consuming the blood of a donor infected with B. aktasi. No clinical or parasitological findings were encountered in the recipient for 40 days post-infestation. These findings indicated that R. bursa was not a competent vector for B. aktasi.