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Copyright of Revista Biológico Agropecuaria Tuxpan is the property of Revista Biologico Agropecuaria Tuxpan and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. Methods Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. Results Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. Conclusions Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances. Graphical Abstract

Background: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. Methods: Nilgai were needle inoculated with ≈108B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. Results: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. Conclusions: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances. [ABSTRACT FROM AUTHOR]

Abstract Bovine babesiosis, caused by different Babesia spp. such as B. bovis, B. bigemina, B. divergens, and B. major, is a global disease that poses a serious threat to livestock production. Babesia bovis infections are associated with severe disease and increased mortality in adult cattle, making it the most virulent agent of bovine babesiosis. Babesia bovis parasites undergo asexual reproduction within bovine red blood cells, followed by sexual reproduction within their tick vectors, which transmit the parasite transovarially. Current control methods, including therapeutic drugs (i.e., imidocarb) have been found to lead to drug resistance. Moreover, changing environmental factors add complexity to efficient parasite control. Understanding the fundamental biology, host immune responses, and host–parasite interactions of Babesia parasites is critical for developing next-generation vaccines to control acute disease and parasite transmission. This systematic review analyzed available research papers on vaccine development and the associated immune responses to B. bovis. We compiled and consolidated the reported vaccine strategies, considering the study design and rationale of each study, to provide a systematic review of knowledge and insights for further research. Thirteen studies published since 2014 (inclusive) represented various vaccine strategies developed against B. bovis such as subunit, live attenuated, and viral vector vaccines. Such strategies incorporated B. bovis proteins or whole live parasites with the latter providing the most effective prophylaxis against bovine babesiosis. Incorporating novel research approaches, such as "omics" will enhance our understanding of parasite vulnerabilities. Graphical Abstract

The hard tick Ixodes ricinus transmits a variety of zoonotic pathogens, including Babesia divergens, the most common cause of bovine babesiosis in northern Europe. In endemic areas, cattle are rarely clinically affected, as animals up to the age of nine months are resistant against relevant clinical disease and develop protective premunity. However, outbreaks in immunologically naïve herds may lead to considerable losses. Such an outbreak with a high mortality rate occurred in 2018 on a northern German beef production farm, as previously reported. The present study provides an update on the epidemiological situation and management strategy of the farm. In spring 2022, blood samples were taken from 46 animals for PCR and serological testing before pasture turnout. Although no clinical cases had been noticed since 2019, B. divergens DNA was detected by quantitative real-time PCR (qPCR), followed by amplification and sequencing of the 18S rRNA gene, in 6.5% (3/46) of cattle blood samples. Presence of anti-B. divergens antibodies was confirmed in 26.1% (12/46) of animals, while further 10.9% (5/46) had a borderline antibody titre. The antibody status of 23 of these animals had already been determined in 2018 and/or 2020, revealing fluctuating titre patterns indicative of repeated pathogen exposure. Moreover, 457 questing I. ricinus specimens collected on the farm’s pastures and 83 I. ricinus specimens detached from cattle were screened for Babesia spp. DNA by qPCR, followed by 18S rDNA amplification and sequencing. Endemisation of B. divergens was confirmed by 0.9% (4/457) positive questing I. ricinus, while the ticks detached from cattle were Babesia-negative. The farm’s management strategy includes annual metaphylactic treatment with imidocarb dipropionate during the main tick exposure period in spring. However, the antibody titre fluctuations and the persistent infections at the end of the housing period indicate that the absence of clinical disease is primarily due to a rising level of premunity. Metaphylactic treatment with imidocarb seems to be a suitable management option to protect newly acquired immunologically naïve animals. The endemisation of B. divergens is also of public health significance, as the pastures are located close to a tourist destination in a popular hiking area.

Bovine babesiosis, caused by different Babesia spp. such as B. bovis, B. bigemina, B. divergens, and B. major, is a global disease that poses a serious threat to livestock production. Babesia bovis infections are associated with severe disease and increased mortality in adult cattle, making it the most virulent agent of bovine babesiosis. Babesia bovis parasites undergo asexual reproduction within bovine red blood cells, followed by sexual reproduction within their tick vectors, which transmit the parasite transovarially. Current control methods, including therapeutic drugs (i.e., imidocarb) have been found to lead to drug resistance. Moreover, changing environmental factors add complexity to efficient parasite control. Understanding the fundamental biology, host immune responses, and host–parasite interactions of Babesia parasites is critical for developing next-generation vaccines to control acute disease and parasite transmission. This systematic review analyzed available research papers on vaccine development and the associated immune responses to B. bovis. We compiled and consolidated the reported vaccine strategies, considering the study design and rationale of each study, to provide a systematic review of knowledge and insights for further research. Thirteen studies published since 2014 (inclusive) represented various vaccine strategies developed against B. bovis such as subunit, live attenuated, and viral vector vaccines. Such strategies incorporated B. bovis proteins or whole live parasites with the latter providing the most effective prophylaxis against bovine babesiosis. Incorporating novel research approaches, such as "omics" will enhance our understanding of parasite vulnerabilities. [ABSTRACT FROM AUTHOR]

Introduction: Live in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia-susceptible adult cattle. Thus, the use of vaccines based on welldefined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain. Methods and results: Herein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P<0.05) monocytosis, with concomitant neutropenia and CD4+ leukopenia, in peripheral blood early after vaccination. Also, vaccinated animals developed a specific signature of pro- and anti-inflammatory cytokine expression in peripheral blood and significant levels of IgM, total IgG, IgG1, and IgG2 against the B. bovis immunodominant antigen RAP-1 CT. Strikingly, none of the vaccinated animals showed any signs of acute babesiosis after challenge with Vir-S74-T3Bo. In contrast, control adult cattle (n=5) showed pathognomonic symptoms of acute babesiosis, and significant decrease (P<0.05) in lymphocytes, monocytes, and neutrophils, starting on day 7 post-challenge. All control animals developed severe acute disease and were euthanized on days 10 through 12 days post-challenge. Discussion and conclusion: Evidence from this study indicates that Att-S74-T3Bo safely protects highly susceptible adult cattle against challenge with a homologous virulent strain of B. bovis. In conclusion, Att-S74-T3Bo may be considered as a potential efficient and sustainable attenuated candidate vaccine strain to control acute bovine babesiosis in highly susceptible adult cattle. Future studies should focus on increasing the number of animals vaccinated, duration of immunity, and efficacy of this attenuated strain against heterologous virulent parasite strains. [ABSTRACT FROM AUTHOR]

IntroductionLive in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia-susceptible adult cattle. Thus, the use of vaccines based on well-defined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain.Methods and resultsHerein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P

In this paper, the model for bovine babesiosis epidemic transmission is analyzed using a fractional operator with a Mittag-Leffler kernel. The existence and uniqueness of the solution of the considered model is studied using real analysis. The Hyers–Ulam (HU) stability is investigated with the help of nonlinear functional analysis. The numerical results of the proposed model are deduced through the Adams–Bashforth technique, which is based on the two-step Lagrangian interpolation method. All results are simulated for a few fractional orders to observe the dynamics of the proposed model. [ABSTRACT FROM AUTHOR]

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies. [ABSTRACT FROM AUTHOR]

Wildlife plays an important role in the emergence of livestock diseases, and their movements can complicate disease management efforts. One of the most significant vector‐borne diseases of livestock worldwide is bovine babesiosis, spread by cattle fever ticks (CFTs; Rhipicephalus [=Boophilus] microplus and Rhipicephalus [=Boophilus] annulatus). Although CFTs were eradicated from the United States by 1943, bovine babesiosis and CFTs are prevalent in México. Recently, management of CFTs in the Texas–México region has been complicated by the presence of free‐ranging, exotic nilgai antelope (Boselaphus tragocamelus). Nilgai are abundant in this region and are competent hosts for CFTs. The goal of this study was to better understand nilgai movements and space use to inform CFT treatment strategies. We analyzed hourly locations from 40 GPS‐collared nilgai in Cameron County, TX, USA, from April 2019 to September 2020. We assigned each nilgai a movement behavior using the net squared displacement metric. We estimated nilgai home range sizes at different temporal scales (monthly, seasonally, and overall) using Brownian bridge movement models. We observed movement patterns consistent with residency (52.5%), seasonal movers (17.5%), dispersal (5%), and unclassified (25%). Resident nilgai had an average maximum axial distance of 7.8 km, while two young females made separate dispersal movements of about 40 km within a year. Overall, nilgai had large and highly variable home ranges: annual median home range estimate for females was 563 ha (range = 105–1545 ha), and for males, it was 937 ha (range = 221–1602 ha). Peak nilgai movements occurred during crepuscular hours, and median hourly movement for females was 57 m/h, and for males, it was 66 m/h. Nilgai home ranges and long‐distance movements have the potential to overlap multiple ranches, as the typical ranch size in South Texas ranges from 250 to 6000 ha. Resident nilgai were more likely to maintain local infestations of CFT. Dispersal events took place during peak tick season, demonstrating the potential for nilgai to aid in the spread of CFT. Understanding these behaviors will help the CFT Eradication Program develop more efficacious treatment strategies to treat infestations in nilgai. [ABSTRACT FROM AUTHOR]

Copyright of Revista Cientifica de la Facultade de Veterinaria is the property of Universidad del Zulia, Facultad de Ciencias Veterinarias and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The aim of this study was to identify the presence of tick-borne pathogens as the probable cause of reproductive problems in cows in a commercial dual-purpose cow operation living in tropical conditions in Northern Veracruz, Mexico. As part of the reproductive control of the herd, ultrasonography was performed periodically, and the presence of three open (non-pregnant) cows with historical infertility problems was detected. The ovaries from the two open cows and the mummified foetus of a third one were removed, and two sets of tissue samples were collected. Histopathology analysis revealed the presence of follicular abnormalities. The DNA tests revealed the presence of Anaplasma marginale, Babesia bigemina and/or Babesia bovis. In conclusion, the present work represents the first report of tick-borne pathogens in ovarian tissue samples and a mummified foetus of infertile cows in tropical conditions in Northern Veracruz, Mexico, highlighting the possibility that many reproductive problems in tropical conditions could be caused by tick-borne pathogens.

Abstract Wildlife plays an important role in the emergence of livestock diseases, and their movements can complicate disease management efforts. One of the most significant vector‐borne diseases of livestock worldwide is bovine babesiosis, spread by cattle fever ticks (CFTs; Rhipicephalus [=Boophilus] microplus and Rhipicephalus [=Boophilus] annulatus). Although CFTs were eradicated from the United States by 1943, bovine babesiosis and CFTs are prevalent in México. Recently, management of CFTs in the Texas–México region has been complicated by the presence of free‐ranging, exotic nilgai antelope (Boselaphus tragocamelus). Nilgai are abundant in this region and are competent hosts for CFTs. The goal of this study was to better understand nilgai movements and space use to inform CFT treatment strategies. We analyzed hourly locations from 40 GPS‐collared nilgai in Cameron County, TX, USA, from April 2019 to September 2020. We assigned each nilgai a movement behavior using the net squared displacement metric. We estimated nilgai home range sizes at different temporal scales (monthly, seasonally, and overall) using Brownian bridge movement models. We observed movement patterns consistent with residency (52.5%), seasonal movers (17.5%), dispersal (5%), and unclassified (25%). Resident nilgai had an average maximum axial distance of 7.8 km, while two young females made separate dispersal movements of about 40 km within a year. Overall, nilgai had large and highly variable home ranges: annual median home range estimate for females was 563 ha (range = 105–1545 ha), and for males, it was 937 ha (range = 221–1602 ha). Peak nilgai movements occurred during crepuscular hours, and median hourly movement for females was 57 m/h, and for males, it was 66 m/h. Nilgai home ranges and long‐distance movements have the potential to overlap multiple ranches, as the typical ranch size in South Texas ranges from 250 to 6000 ha. Resident nilgai were more likely to maintain local infestations of CFT. Dispersal events took place during peak tick season, demonstrating the potential for nilgai to aid in the spread of CFT. Understanding these behaviors will help the CFT Eradication Program develop more efficacious treatment strategies to treat infestations in nilgai.

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle. [ABSTRACT FROM AUTHOR]

The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (106 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (106 RBC/calf). All Att-S74-T3Boinfected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4+ lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFa, CXCL10, IFNg, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 postinoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (107 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P<0.05) parasite load in peripheral blood compared to animals previously exposed to Att-S74-T3Bo. No significant alterations in the profile of leukocytes and cytokines were observed in Att-S74-T3Bo-inoculated after Vir-S74-T3Bo infection. In conclusion, data demonstrate novel changes in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle. [ABSTRACT FROM AUTHOR]

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle.

The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (106 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (106 RBC/calf). All Att-S74-T3Bo-infected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4+ lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (107 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P

Abstract Babesiosis is a tick-borne disease with global impact caused by parasites of the phylum Apicomplexa, genus Babesia. Typically, acute bovine babesiosis (BB) is characterized by fever, anemia, hemoglobinuria, and high mortality. Surviving animals remain persistently infected and become reservoirs for parasite transmission. Bovids in China can be infected by one or more Babesia species endemic to the country, including B. bovis, B. bigemina, B. orientalis, B. ovata, B. major, B. motasi, B. U sp. Kashi and B. venatorum. The latter may pose a zoonotic risk. Occurrence of this wide diversity of Babesia species in China may be due to a combination of favorable ecological factors, such as the presence of multiple tick vectors, including Rhipicephalus and Hyalomma, the coexistence of susceptible bovid species, such as domestic cattle, yaks, and water buffalo, and the lack of efficient measures of tick control. BB is currently widespread in several regions of the country and a limiting factor for cattle production. While some areas appear to have enzootic stability, others have considerable cattle mortality. Research is needed to devise solutions to the challenges posed by uncontrolled BB. Critical research gaps include risk assessment for cattle residing in endemic areas, understanding factors involved in endemic stability, evaluation of parasite diversity and pathogenicity of regional Babesia species, and estimation of whether and how BB should be controlled in China. Research should allow the design of comprehensive interventions to improve cattle production, diminish the risk of human infections, and increase the availability of affordable animal protein for human consumption in China and worldwide. In this review, we describe the current state of BB with reference to the diversity of hosts, vectors, and parasite species in China. We also discuss the unique risks and knowledge gaps that should be taken into consideration for future Babesia research and control strategies.

Abstract Background The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. Methods The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. Results We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Bovine babesiosis, caused by Babesia bovis, is an economically significant tick-borne disease that imposes restrictions to livestock production worldwide. Current methods to control bovine babesiosis have severe limitations and novel approaches, including transmission-blocking vaccines, are needed. Members of the widely conserved CCp family are multidomain adhesion proteins containing LCCL motifs, which are differentially expressed on gametocytes of apicomplexans, including Babesia spp. and Plasmodium spp. While Plasmodium parasites contain 6 distinct CCp genes, only three members (CCp 1-3) were previously identified in B. bovis. In this study, we describe the identification and characterization of two novel non-canonical members of the CCp gene family in B. bovis, named CCp5 and FNPA. The genes were identified in silico by TBLASTN using P. falciparum CCp family domains as queries. Unlike CCp1-3, the B. bovis CCp5 and FNPA proteins lack the LCCL canonical domain but contain other typical multidomain adhesion motifs which are present in classical CCp proteins. In addition, the B. bovis CCp5 and FNPA are in synteny with known CCp genes in related apicomplexans. Sequence analysis of these two proteins demonstrated high sequence conservation among B. bovis different isolates. Transcription, immunoblot, and immunofluorescence analyses demonstrated expression of CCp5 and FNPA in blood and in vitro induced sexual stages of B. bovis. The FNPA, in contrast to CCp5, has a predicted transmembrane domain, suggesting that it might be expressed in the surface of sexual stage parasites. Altogether, finding of this study support FNPA as a possible target of a transmission-blocking vaccine against B. bovis.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen. [ABSTRACT FROM AUTHOR]

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.

Abstract Background Bovine babesiosis is caused by protozoan parasites of the genus Babesia and presents a wide spectrum of clinical manifestations. Disease severity depends on the type of Babesia species infection. Generally, B. bovis and B. bigemina are considered as the causative agents of bovine babesiosis; in addition, Babesia ovata and B. major are a group of benign bovine piroplasms. Therefore, species identification is important for diagnosis, epidemiological investigations and follow-up management. Methods Real-time PCR combined with high resolution melting (RT-PCR-HRM) analysis was used to detect and discriminate four Babesia species infective to cattle, including Babesia bovis, B. bigemina, B. major and B. ovata. The melting profiles and melting temperatures (Tm) of the amplicon targeting 18S rRNA revealed differences that can discriminate the four Babesia spp. Sensitivity and specificity of the analytical method were evaluated using 50 blood samples collected from experimentally infected cattle and 240 blood samples from areas where bovine babesiosis is an issue. Results RT-PCR-HRM analysis allowed to detect and discriminate four Babesia spp. (B. bovis, B. bigemina, B. major and B. ovata), which were responsible for bovine babesiosis in China. The protocol was validated with DNA samples from experimentally infected cattle and field infection in cattle. Conclusions Our results indicate that RT-PCR-HRM is a fast and robust tool for the simultaneous detection and discrimination of four Babesia species that are responsible for bovine babesiosis in China. This approach is applicable for both field and experimental samples, thus it could be useful in epidemiological investigations and diagnoses of bovine babesiosis.

Babesiosis is a tick-borne disease with global impact caused by parasites of the phylum Apicomplexa, genus Babesia. Typically, acute bovine babesiosis (BB) is characterized by fever, anemia, hemoglobinuria, and high mortality. Surviving animals remain persistently infected and become reservoirs for parasite transmission. Bovids in China can be infected by one or more Babesia species endemic to the country, including B. bovis, B. bigemina, B. orientalis, B. ovata, B. major, B. motasi, B. U sp. Kashi and B. venatorum. The latter may pose a zoonotic risk. Occurrence of this wide diversity of Babesia species in China may be due to a combination of favorable ecological factors, such as the presence of multiple tick vectors, including Rhipicephalus and Hyalomma, the coexistence of susceptible bovid species, such as domestic cattle, yaks, and water buffalo, and the lack of efficient measures of tick control. BB is currently widespread in several regions of the country and a limiting factor for cattle production. While some areas appear to have enzootic stability, others have considerable cattle mortality. Research is needed to devise solutions to the challenges posed by uncontrolled BB. Critical research gaps include risk assessment for cattle residing in endemic areas, understanding factors involved in endemic stability, evaluation of parasite diversity and pathogenicity of regional Babesia species, and estimation of whether and how BB should be controlled in China. Research should allow the design of comprehensive interventions to improve cattle production, diminish the risk of human infections, and increase the availability of affordable animal protein for human consumption in China and worldwide. In this review, we describe the current state of BB with reference to the diversity of hosts, vectors, and parasite species in China. We also discuss the unique risks and knowledge gaps that should be taken into consideration for future Babesia research and control strategies. [ABSTRACT FROM AUTHOR]

In this paper, analysis and modeling of bovine babesiosis disease are designed with fractional calculus. The solution for a bovine babesiosis disease and tick populations fractional order system is determined using the Caputo and Atangana–Baleanu–Caputo (ABC) fractional derivatives. Applying the homotopy analysis method and the Laplace transform with polynomial homotopy, the analytical solution of the bovine babesiosis disease has obtained. Furthermore, using an iterative scheme by the Laplace transform, and the Atangana–Baleanu fractional integral, special solutions of the model are obtained. Uniqueness and existence of the solutions by the fixed‐point theorem and Picard–Lindel of approach are studied. Numerical simulation has been established for both Caputo and ABC fractional derivative of the proposed system is carried out. The numeric replications for diverse consequences are carried out, and data attained are in good agreement with theoretical outcomes, displaying a vital perception about the use of the set of fractional coupled differential equations to model babesiosis disease and tick populations. [ABSTRACT FROM AUTHOR]

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as “housekeeping” genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.

Abstract Serum and DNA samples from 15 naturally infected calves in Seropédica, Brazil, were obtained quarterly from birth to 12 months of age, in order to longitudinally evaluate their humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis. Anti-B. bovis IgG antibodies were detected by an indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Using DNA amplification, sequencing and phylogenetic analysis, the genetic diversity of B. bovis was assessed based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 0, 3 and 5 sequences of the msa-1, msa-2b and msa-2c genes were obtained, respectively. The present study demonstrated that the msa-2b and msa-2c gene sequences amplified from blood DNA of B. bovis-positive calves were genetically diversified. These data emphasize the importance of conducting deeper studies on the genetic diversity of B. bovis in Brazil, in order to design diagnostic antigens and vaccines in the future.

Abstract Background Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis. Results The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas. Conclusions The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

• Babesia bovis has a complex life cycle that includes development within a mammalian host and tick vector. • Differentially expressed B. bovis genes were found in mammalian blood and tick stages. • The genome assembly of B. bovis was improved by closing a substantial gap of 139 kbp on chromosome 1. • The RNA-Seq datasets derived from mammalian blood and tick stages greatly improved the annotation of the B. bovis genome. Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis , a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis. [ABSTRACT FROM AUTHOR]

Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other. [ABSTRACT FROM AUTHOR]

Bovine babesiosis is a tick‐transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick‐hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species‐specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C‐terminal region of rhoptry‐associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa‐stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis. [ABSTRACT FROM AUTHOR]

The apical membrane antigen 1 (AMA‐1) is a protein of the micronemes that is present in all organisms of the phylum Apicomplexa; it has been shown that AMA‐1 plays an essential role for parasite invasion to target cells. It has been reported that AMA‐1 is conserved among different isolates of Babesia; however, it is unknown whether the protein contains conserved B‐cell epitopes and whether these epitopes are recognized by antibodies from cattle in endemic areas. In this research, using an in silico analysis, four peptides were designed containing exposed and conserved linear B‐cell epitopes from the extracellular region of Babesia bovis AMA‐1. The selected peptides were chemically synthesized, and then each peptide was emulsified and used to immunize two bovines per peptide. The antibodies produced against these peptides were able to recognize intra‐erythrocytic parasites in an IFAT, except peptide 4, which was insoluble. The synthetic peptides were covalently fixed to the wells of an ELISA plate and incubated with sera from B. bovis naturally infected cattle. Peptides P2AMA and P3AMA were recognized by the sera of naturally infected cattle from different regions of Mexico. Statistical analysis showed that the ELISA test for peptides P2AMA and P3AMA had a concordance of 91.2% and 61.1% compared to the IFAT, a sensitivity of 94.56% and 71.74%, and a specificity of 76.19% and 14.2%, respectively. The presence of antibodies in bovine sera from endemic areas that bind to the identified peptides indicates that AMA‐1 from B. bovis has conserved B‐cell epitopes involved in the immune response under natural conditions. However, to propose their use as vaccine or diagnostics candidates, a further characterization of the humoral immune response elicited in cattle by these peptides is needed. [ABSTRACT FROM AUTHOR]

A stochastic model for Bovine Babesiosis (BB) including ticks, and both juvenile and adult cattle is developed. This model is formulated by a system of continuous-time Markov chains (CTMCs) that is derived based on an extension of the deterministic ordinary differential equation model developed by Saad-Roy et al. (Bull Math Biol 77:514–547, 2015). The nonlinear CTMC model is approximated by a multitype branching process, giving a theoretical estimate of the probability of an outbreak of BB. Unlike the deterministic dynamics where the basic reproduction number is a sharp threshold parameter, the stochastic model indicates that there is always a positive probability of disease extinction within the cattle population. For parameter values from Colombia data, conditional probability distributions are numerically obtained for the time to disease extinction or outbreak, and are found to depend on the host type at the initiation of infection. The models with and without the inclusion of juvenile cattle are compared, and our result highlights that neglecting juvenile bovine in the models may lead to faulty predictions of critical disease statistics: particularly, it may underestimate the risk of infection. Endemic disease prevalence in adult cattle is examined for certain parameter values in the corresponding deterministic model. Notably, with long-lasting immunity, increased tick to juvenile infectivity decreases the proportion of infectious adults. [ABSTRACT FROM AUTHOR]

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

The indirect fluorescent antibody test (IFAT) is the most frequently used test to conduct seroepidemiological studies so far, and it is regarded as the "gold standard" test for the serological diagnosis of bovine babesiosis. The aim of the present study was to compare the enzyme-linked immunosorbent assay (ELISA) and the rapid immunochromatography test (ICT) for use in the serological diagnosis of cattle exposed to B. bovis in Mexico. The evaluation of test performance was carried out with 30 positive and 30 negative reference sera. A total of 72 bovine sera samples collected from cattle in a region with endemic bovine babesiosis were analyzed by ELISA and ICT, and the results were compared with those of IFAT. Kappa value (k) was also calculated to determine the agreement between tests. The sensitivity and specificity of ELISA for detecting antibodies against B. bovis were 87% (26/30) and 80% (24/30), respectively. The sensitivity and specificity of ICT for detecting antibodies against B. bovis were 90% (27/30) and 83.3% (25/30), respectively. The overall concordance determined for ELISA and ICT was 94.4% (68/72) and 98.6% (71/72), respectively, when the results were compared with those of IFAT. ICT was more sensitive and specific in this comparative study, showing good strength of agreement (k = 0.79) with respect to IFAT. ICT combines a strip-based assay system that is fast, practical, and sensitive for detection of antibodies to B. bovis, which suggests that it could be applied in the field without requiring any laboratory equipment for its use and interpretation of test results.

Abstract Background Bovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future. Results To isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro. Conclusion Babesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.

Abstract Background Bovine babesiosis, mainly caused by Babesia bovis and B. bigemina, is a huge threat to the livestock industry. In Indonesia, the current distribution of the disease is unknown due to a lack of scientific study. Methods In the present study, 487 blood samples were collected from cattle with different breeding and age groups in a broad geographical area across the archipelago. The presence of antibodies and current infections of B. bovis and B. bigemina were determined using enzyme-linked immunosorbent assay (ELISA), immunochromatographic test (ICT), and nested PCR (nPCR) targeting B. bovis SBP-4 and B. bigemina RAP-1a genes. Sequence analysis was performed to the amplicon of B. bovis SBP-4, B. bigemina RAP-1a, and internal transcribed spacer (ITS) region of ribosomal RNA of both Babesia species. Results In total, B. bovis positives were detected by ELISA, single-ICT, dual-ICT and nPCR in 340 (69.8%), 317 (65.1%), 307 (63.0%) and 247 (50.7%) samples, respectively. For B. bigemina, the positive samples were detected in 134 (27.5%), 130 (26.7%), 127 (26.1%) and 93 (19.1%), respectively. Furthermore, mixed infections were found in 125 (25.7%), 113 (23.2%), 109 (22.4%) and 52 (10.7%) samples, respectively, which occurred only by chance and were not influenced by additional factors. The obtained nucleotide sequences of B. bovis SBP-4 and B. bigemina RAP-1a genes showed a high homology with other isolates from different countries. Further nucleotide sequence analysis using ITS region showed a great genetic diversity of B. bovis isolates among sampling locations; a lower diversity was found in B. bigemina ITS isolates. Conclusions These data revealed the current distribution of B. bovis and B. bigemina infection in cattle in Indonesia. The rate of infection varied among sampling locations, cattle breeds and age groups. Furthermore, B. bovis ITS isolates from Indonesia were found to be more genetically diverse than B. bigemina ITS isolates. The data presented in this study are necessary to develop an effective strategy for controlling the disease in the country.

Cattle fever ticks (CFT), vectors of bovine babesiosis and anaplasmosis, were eradicated from the United States by 1943, but are frequently reintroduced from neighboring border states of Mexico via stray cattle and wildlife hosts including white-tailed deer (Odocoileus virginianus) (WTD) and nilgai antelope (Boselaphus tragocamelus). Nilgai antelope are exotic bovids from India that are hosts of CFT, have large home ranges as compared to WTD, thus have the potential to spread CFT through the landscape. Currently, there are no methods to control CFT on nilgai. Odor lures were evaluated to determine if nilgai could be attracted to a central point for development of control methods. Four treatments, nilgai offal a natural odor lure was used as the positive control; and compared to three artificial odors; screw worm lure, volatile fatty acids, citronella oil. Studies were conducted on a free-ranging population of nilgai at the East Foundation's Santa Rosa Ranch (Kenedy Co., near Riviera, Texas, USA). Game cameras were used to document visitation to the lures. In the ten randomly placed transects, 110 nilgai and 104 WTD were photographed. Offal had significantly more visits by nilgai (71% of total visits) than screwworm (15%), VFA (11%), and citronella (4%). For WTD, there was no significant difference in visitation at the lure treatments.

Reported fatal cases of bovine babesiosis (syn.: piroplasmosis, red water fever) in cattle were analyzed to identify spatial and temporal clusters of their incidence in the Austrian province of Styria. Data were collected within a governmental babesiosis compensation program. Diagnosis was performed using a standardized necropsy protocol. Between 1998 and 2016, a total of 1257 cases of fatal babesiosis were registered and compensated. Within the study interval, annual numbers of fatal babesiosis differed significantly among municipalities. Spatiotemporal analysis covering the entire study period revealed one high-risk cluster in the western and central northern region of Styria and a low-risk cluster in the southeastern part of Styria. Annual temporal analysis demonstrated that cases accumulated in June. Annual spatial analysis revealed consistently that cases mainly occurred in the western and central northern regions, whereas they occurred rarely in the southeastern regions. These results should increase awareness and facilitate protective actions against ticks during certain time periods and geographic areas. [ABSTRACT FROM AUTHOR]

Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum , another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina , the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina -infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion. [ABSTRACT FROM AUTHOR]

Babesia bovis is the etiological agent of bovine babesiosis, a disease transmitted by Rhipicephalus microplus, which affects cattle herds in tropical and subtropical regions of the world, causing significant economic losses due to decreasing meat and milk yield. This study used molecular techniques to determine the occurrence and genetic diversity of B. bovis, based on the genes encoding the spherical body protein (sbp-2) and the merozoite surface antigens (MSAs) genes, in a herd of 400 Nellore (Bos indicus) sampled from beef cattle farms in the Pantanal region, state of Mato Grosso do Sul, Midwestern Brazil. The results of the nested PCR assays based on the sbp-2 gene indicated that 18 (4.5%) calves were positive for B. bovis; out of them, while 77.7% (14/18) were positive for the B. bovis msa-2b fragment, 66.6% (12/18) were positive for the msa-2c fragment. The phylogenetic analysis based on the maximum likelihood method using 14 sequences from msa-2b clones and 13 sequences from msa-2c clones indicated that the sequences detected in this study are clearly distributed in different cladograms. These findings corroborated the diversity analysis of the same sequences, which revealed the presence of 14 and 11 haplotypes of the msa-2b and msa-2c genes, respectively. Furthermore, the entropy analyses of amino acid sequences revealed 78 and 44 high entropy peaks with values ranging from 0.25 to 1.53 and from 0.27 to 1.09 for MSA-2B and MSA-2C, respectively. Therefore, the results indicate a low molecular occurrence of B. bovis in beef cattle sampled in the Brazilian Pantanal. Despite this, a high degree of genetic diversity was found in the analyzed B. bovis population, with possibly different haplotypes coexisting in the same animal and/or in the same studied herd. [ABSTRACT FROM AUTHOR]

Bovine babesiosis is a global tick-borne disease that causes important cattle losses and has potential zoonotic implications. The impact of bovine babesiosis in Turkey remains poorly characterized, but several Babesia spp., including B. bovis, B. bigemina, and B. divergens, among others and competent tick vectors, except Rhipicephalus microplus, have been recently identified in the country. Bovine babesiosis has been reported in all provinces but is more prevalent in central and highly humid areas in low and medium altitude regions of the country housing approximately 70% of the cattle population. Current control measures include acaricides and babesicidal drugs, but not live vaccines. Despite the perceived relevant impact of bovine babesiosis in Turkey, basic research programs focused on developing in vitro cultures of parasites, point-of-care diagnostic methods, vaccine development, “omics” analysis, and gene manipulation techniques of local Babesia strains are scarce. Additionally, no effective and coordinated control efforts managed by a central animal health authority have been established to date. Development of state-of-the-art research programs in bovine babesiosis to address current gaps in knowledge and implementation of long-term plans to control the disease will surely result in important economic, nutritional, and public health benefits for the country and the region.

In Uganda, bovine babesiosis continues to cause losses to the livestock industry because of shortages of cheap, quick, and reliable diagnostic tools to guide prescription measures. In this study, the presence of antibodies to Babesia bigemina and Babesia bovis in 401 bovine blood samples obtained from eastern and central areas of Uganda were detected using enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic test strips (ICTs). The ELISA and ICT test used targeted the B. bigemina C-terminal rhoptry-associated protein (RAP-1/CT17) and B. bovis spherical body protein-4 (SPB-4). Using ELISA, single-ICT and dual-ICT, positive samples for B. bovis were detected in 25 (6.2%), 17 (4.3%), and 14 (3.7%) samples respectively, and positive samples for B. bigemina were detected in 34 (8.4%), 27 (6.7%), and 25 (6.2%), respectively. Additionally, a total of 13 animals (3.2%) had a mixed infection. The correlation between ELISA and single-ICT strips results revealed slight agreement with kappa values ranging from 0.088 to 0.191 between both methods, while the comparison between dual-ICT and single-ICT results showed very good agreement with kappa values >0.80. This study documented the seroprevalence of bovine babesiosis in central and eastern Uganda, and showed that ICT could, after further optimization, be a useful rapid diagnostic test for the diagnosis of bovine babesiosis in field settings.

Bovine babesiosis caused by protozoan parasites Babesia bovis and B. bigemina is one of the most important causes of losses for the livestock industry in tropical and subtropical regions of the world. Therefore, highly sensitive and specific tools for these hemoparasites detection and monitoring are required, especially in carrier animals, in which low parasite levels were usually present. In this context, qPCR assays have been successfully and fairly used in last years. Aiming to improve the performance of Babesia levels monitoring by qPCR, some of main aspects of this methodology that may influence results were tested: DNA extraction kits, whole blood EDTA pre-treatment, blood source (tip of tail or jugular vein), erythrocytes isolation, FTA card interference and qPCR system of detection. Under our experimental conditions, both EDTA pre-treatment and FTA card application have no influence on the sensitivity of detection, and two DNA extraction kits provided higher sensitivity compared to others. As expected, blood samples collected from the tip of tail vessels presented higher levels of B. bovis DNA compared to those obtained from the jugular vein, and erythrocytes processed isolated has also improved the sensitivity compared to whole blood. Moreover, both qPCR assays here developed using hydrolysis probes for B. bovis and B. bigemina detection, presented enhanced reproducibility compared to qPCR assays using intercalating dye system. Even, qPCR for B. bigemina using hydrolysis probe here developed presented higher sensitivity compared to intercalating dye system. This study has contributed to the improvement of molecular diagnosis of bovine babesiosis, which may improve epidemiological studies related to these pathogens. [ABSTRACT FROM AUTHOR]

Water buffaloes ( Bubalus bubalis ) are raised in tropical and subtropical regions of the world, and act as hosts of Babesia bovis parasites and the tick vector Rhipicephalus microplus . As no clinical cases of B. bovis- infection have been reported, we hypothesized that, unlike bovines, water buffaloes respond asymptomatically to an acute infection. To test this hypothesis, we inoculated two groups of 24-month-old Mediterranean breed water buffaloes with 10 8 erythrocytes infected with two Argentine B. bovis isolates: BboM2P (n = 5) or BboS2P (n = 5). These strains displayed mild (BboM2P) or high (BboS2P) pathogenicity in Bos taurus calves of the same age (n = 5 and n = 1, respectively), when tested in parallel. In water buffaloes, no changes in body temperature were observed with both strains, and no hematocrit changes were detected in BboM2P-inoculated animals. In contrast, in the BboS2P-inoculated water buffalo group significant but relatively minor reductions in haematocrit values were noted compared to the infected bovine. The parasitemia attained in water buffaloes was considerably lower than in bovines and could only be detected by nested PCR, or indirectly via serology, whereas in most bovines, it could also be detected in Giemsa-stained smears under the light microscope. Our results show that water buffaloes present no or significantly mitigated clinical symptoms to B. bovis infections and suggest that they are able to substantially reduce and/or eliminate B. bovis parasites from circulation by an efficient innate immune mechanism. [ABSTRACT FROM AUTHOR]

Babesia bigemina is one of the most prevalent species causing bovine babesiosis around the world. Antigens involved in host cell invasion are vaccine targets for this disease but are largely unknown in this species. The invasion process of Babesia spp. into erythrocytes involves membrane proteins from the apical complex. A protein stored in the micronemes, called Micronemal Protein 1 (MIC-1), contains a sialic acid binding domain that participates in the invasion process of host cells and is a vaccine candidate in other apicomplexan parasites. It is not known if there is a homologous gene for mic-1 in B. bigemina. Therefore, the aim of this study was to characterize the mic-1 gene homologue in Babesia bigemina. A gene was found with a microneme adhesive repeat (MAR) domain in the predicted amino acid sequence. Transcription was determined by reverse transcription polymerase chain reaction (RT-PCR). Subsequently, antibodies against peptides containing conserved B-cell epitopes were used to confirm the expression of MIC-1 in intraerythrocytic merozoites. The presence of anti MIC-1 antibodies in cattle naturally infected with B. bigemina was determined and up to 97.4% of the cattle sera (113 out of 116) identified MIC-1 using enzyme-linked immunosorbent assay (ELISA) methods. Finally, antibodies against MIC-1 were able to block 70% merozoite invasion in-vitro. [ABSTRACT FROM AUTHOR]

Abstract Background Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S. Methods We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency. Results We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635). Conclusions These findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.