extfieldsofstudy: Geography Medicine

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Bovine Babesiosis in Turkey: Impact, Current Gaps, and Opportunities for Intervention

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.

Introduction
An important chapter on animal infectious diseases began in 1888 with the sudden deaths of thousands of cows in Romania [1]. At the time, Victor Babes associated the animal deaths with an intraerythrocytic organism, which he named as "haematococcus." These organisms were later identified as protozoan parasites, and renamed Babesia to honor its discoverer [2]. A few years later, two researchers in the US, Smith and Kilbourne, found out that Babesia parasites were transmitted by Ixodid ticks, demonstrating for the first time transmission of a parasite by an arthropod vector [3]. Follow up studies have characterized several species of Babesia as tick-transmitted apicomplexan protozoan hemoparasites with veterinary and human importance, and great economic impact worldwide [4].
Babesiosis is a disease that affects many vertebrate hosts, from humans to bats, as well as farm animals, such as cattle, horses, and small ruminants, and companion animals [5]. There are more than 100 Babesia species reported so far, with different host specificities [6]. Here, we focus on bovine babesiosis, a disease with a particular large impact on cattle worldwide. In addition to major economic losses derived from death of animals and decreased production of meat and dairy products as a result of bovine babesiosis, there are other important costs associated with tick control, diagnosis, and treatments required to prevent the disease. Despite the importance, there is no reliable specific quantification of the impact of bovine babesiosis at the global scale, but only independent regional assessments performed in individual countries, such as Brazil, Argentina, Australia, among others, and the estimated losses are in the order of hundreds of millions of US dollars per year.
Babesia parasites have a complex lifecycle that includes the development of asexual stages in mammalian hosts and sexual stages inside their definitive tick vectors. Two characteristics that define sensu stricto Babesia parasites are their ability to be transmitted transovarially by tick vectors and exclusively infect red blood cells (RBC) in their vertebrate host. These aspects are particularly important for B. bovis, B. bigemina, and B. divergens, the major causative agents of bovine babesiosis [6,7].
Growth of asexual stages of Babesia parasites inside the vertebrate host RBC causes severe intravascular hemolytic anemia, which is a pathognomonic sign of the acute disease and highly debilitating for the host. Additionally, fever, prostration, abortion, and temporary infertility are also common clinical findings during acute infection. Hemoglobinuria is also usually present at the peak of the hemolytic crisis in B. bigemina or B. divergens infection and in late stages of the disease caused by B. bovis. In addition, residues and toxic metabolites released as a result of the infection and RBC destruction can negatively affect host organ systems [6][7][8]. Moreover, B. bovis has the unique ability to evade the cattle immune system by expressing proteins that facilitate cytoadhesion of infected RBC to capillaries, such as in the brain, causing neurological symptoms and generalized organ failure, a feature that results in increased virulence. Altogether, these pathological mechanisms frequently lead to rapid death of cattle during the acute stage of the disease, especially when affecting adult naïve animals.
Upon infection, the immune system of the host responds differentially, depending on the age of the animals. While young animals, less than seven months old, are frequently able to control severe acute babesiosis and can survive re-exposures to the parasites, older than one-year-old animals often succumb rapidly to infection. Features associated with resistance in young animals include early and strong activation of the innate and adaptive immune effectors. Briefly, the parasite expresses molecules able to bind pathogen associated molecular patterns (PAMPs) receptors expressed on the surface of dendritic cells (DC), macrophages, neutrophils, and monocytes, especially TLR9 [9,10], and an immune response is initiated. Cytokines, such as IL-1β, TNF-α, and IL-12, and nitric oxide (NO) are released from monocytes and neutrophils, and chemokines attract immature DC to the site of infection, especially at the spleen. These stimulate natural killer cells (NKs) that release early IFNγ. The mature DC migrating to the spleen presents Babesia antigens to naive T cells. Spleen macrophages are activated by IFNγ, phagocytize infected RBC, and kill the parasites by releasing reactive nitrogen and oxygen intermediates. In turn, cytokines, such as 1L-1β, IL-12, and TNF-α, released from activated macrophages inhibit the growth of B. bovis. Activated CD4 + T cells and specific B-cell producing antibodies are also important in maintaining immunity and overcoming the infection [11][12][13][14]. Despite mounting protective adaptive immune responses, animals that survive acute babesiosis develop persistent infection, which allows transmission and perpetuation of Babesia parasites in endemic areas. These areas usually have elevated prevalence of bovine babesiosis but low numbers of clinical cases due to the establishment of endemic stability, a condition of herd immunity that develops when more than 75% of the animals have acquired protective immunity by exposure to the parasite before one year of age, when animals are less susceptible to the parasites. A highly unstable state may occur, in contrast, when less than 30% of the herd is naïve for the disease [15][16][17].
B. bovis and B. bigemina, which are transmitted by Rhipicephalus ticks, are the most important causative agents of bovine babesiosis in tropical and subtropical regions worldwide. In addition, B. divergens is another important Babesia species that is transmitted by Ixodes ticks and affects cattle in Europe and North Africa. Apart from its impact on bovines, B. divergens is especially important as a zoonotic pathogen implicated in human babesiosis in Europe [6][7][8]18]. Acute bovine babesiosis caused by B. bovis is often severe due to cytoadhesion of infected RBC in the lung, kidney, and brain capillaries, which leads to hypotension, respiratory stress syndrome, neurological symptoms, and death [8,19]. In contrast, B. bigemina induces massive hemolytic anemia without causing the symptoms associated with cytoadhesion [4,6,18]. Also of importance as causative agents of bovine babesiosis are B. ovata, B. major, B. occultans, and additional unclassified Babesia that are characterized by their low pathogenicity in cattle compared to B. bovis, B. bigemina, and B. divergens [8,[20][21][22][23][24].
Numerous cases of bovine babesiosis involving most of these mentioned Babesia species have been reported throughout the years (from 1955 to 2020) in Turkey. Despite the overall economic importance of bovine babesiosis worldwide, only relatively few studies have addressed the impact of the disease on the cattle industry in this country, and updates on this important subject have also been overlooked in recent years. Therefore, the focus of this study is on compiling relevant studies to improve our understanding of the effect of bovine babesiosis on the Turkish cattle industry, with the aim of assessing the current status of the disease and identifying research and intervention gaps that can contribute to improved control of this disease. Here we cover not only the major causative agents B. bovis, B. bigemina, and B. divergens, but also less virulent species recently identified in Turkey, such as B. major and B. occultans, among others. In addition, we seek to stimulate basic and applied research on this field that is currently neglected with the expectation that this will result in substantial benefits on food production, animal health, economic growth, and human well-being in Turkey and surrounding countries.

An Overview of the Cattle Industry in Turkey
Turkey is a transcontinental country located in the Northern hemisphere with a territory spanning the Anatolian peninsula in Western Asia and a small portion on the Balkan Peninsula in Southeastern Europe. Geographic coordinates of the country lie at latitude 39 and longitude 35. It is a peninsula with a strategic position as a land connection between Europe and Asia. Considering its location, the country has been also regarded as a natural bridge for transcontinental transmission of tick species and tick-borne diseases (TBD) [25]. Turkey covers an area of 783,582 km 2 with a population of 82 million people. The country's economy is based on modern industry, tourism, and trade, and is also heavily supported by the agricultural sector. Therefore, the presence of emerging tick populations and TBD can pose a serious risk to the cattle industry that may impact the overall economy of Turkey and neighboring countries [25,26].
Although the importance of cattle has been different in every society throughout the history of humanity, these animals have always had significant importance in several cultures and religions, and still remain an important economic asset in Turkey and worldwide. There are approximately 66 million farm animals in Turkey, and 27% of which (18 million) are cattle. In Turkey, milk production consisted of 90.5% cattle, 6.6% sheep, and 2.5% goat. As for meat production, 89.5% of the total meat produced in Turkey comes from cattle. According to a report from 2018, approximately 1.5 million live animals are imported to Turkey [27]. Some of these animals come from countries that are endemic for cattle ticks, bovine babesiosis, and other cattle TBD, such as Brazil, from where Turkey obtains 42% of its total imported animals [27,28]. Considering that cattle are a very important source of protein, especially meat and milk production, the cattle industry is a significant sector to secure food supply and sustain the economy in Turkey [27]. Assuring constant supply of cattle milk and meat requires keeping high animal sanitary standards and rational strategies for industry development. In addition, particular attention should be placed in controlling diseases that limit cattle production and may compromise public health. This should be extended to the potential introduction of additional animal health risk factors, such as foreign pathogenic organisms. Taking into consideration the social and economic importance of cattle in Turkey, we argue that the development of a national intensive research program on TBD, specifically in bovine babesiosis, and the implementation of informed animal health policies of disease control based on the state-of-the-art knowledge should be considered issues of crucial importance for the country.

Economic Impact of Bovine Babesiosis on the Cattle Industry in Turkey
It is estimated that more than 500 million cattle are at risk of babesiosis worldwide; therefore, this disease poses a major threat to animal health and human livelihood in areas where Babesia parasites and competent tick vectors are present [4]. As an attempt to contain such threat, a radical tick control campaign was launched in the US at the beginning of the 20th century, lasting 40 years and demanding the use of millions of taxpayer's dollars. With this effective, but costly campaign, bovine babesiosis was eradicated in the US, and consequently approximately $3 billion US dollars annually were saved for the livestock industry [6,29,30]. Unfortunately, the success of this approach was not reproduced in other countries that also attempted similar tick eradication [31], and given a current scenario of increased acaricide resistance in ticks and climate change, among other factors, it is unlikely that this achievement can be duplicated elsewhere [30][31][32].
Annual economic losses due to bovine babesiosis and anaplasmosis in the world range from $16.9 million US dollars in Australia, $21.6 million US dollars in South Africa, and $57.2 million US dollars in China [8]. These losses are not only due to animal mortality, but also abortion, decrease in meat and milk production, and disease control costs (e.g., spraying, vaccination, disease treatments, professional veterinary support, and others). In addition, disease-related deaths are frequently observed in naïve cattle imported to regions with enzootic stability for bovine babesiosis [6,8,33], a factor that causes additional economic losses and complicates the attempts to carry out genetic improvement of herds. In this way, preventing clinical cases of bovine babesiosis by strategies based on maintaining enzootic stability may also interfere with the efforts to improve productivity, such as increased weight and milk production, heavily affecting the meat and dairy industries, respectively, which increases production costs [6,8,17].
Turkey's geographic location and climatic conditions, in addition to the country's animal management systems, encourage the occurrence of ticks and TBD [25,26]. The emergence of tick populations and TBD have increased around the globe in the recent years, including in Turkey [25]. Estimation of the amount of TBD drugs sale per year during the disease seasons indirectly shows the importance of these diseases on animal health and in the economy of Turkey [26]. The economic impact of topical theileriosis caused by Theileria annulata, a tick-borne parasite similar to Babesia sp., was estimated at a total annual loss of approximately 600,000 US dollars in Turkey [34]. However, despite being considered as a costly burden, the actual economic impact of bovine babesiosis on the cattle industry in Turkey remains largely unknown. Therefore, a well-designed national surveillance study to evaluate the real impact of the disease on the cattle industry in the country is urgently needed.

Competent Tick Vectors for Babesia Parasites Identified in Turkey
Tick vectors are essential components for the completion of the lifecycle of Babesia parasites. Thus, competent ticks must provide the environment required for sexual reproduction, which occurs in their midgut, and for invasion of tick eggs by the kinete stage of parasites that circulates in the tick hemolymph, an event that ultimately guarantees transovarial transmission of Babesia. A large number of Ixodid tick species are listed as competent Babesia vectors in the literature [5]. Of these, 22 were confirmed vectors for 18 different Babesia species that infect livestock, companion animals, and humans [5]. Identification of pathogen DNA in adult ticks cannot be accepted alone as evidence of vector competence, and more detailed studies on tick-Babesia interactions are needed to establish the tick competence. Additionally, the presence of Babesia DNA in the salivary glands, eggs, and unfed larvae, though more convincing, also requires confirmation as a measure of tick competence [5]. B. bovis and B. bigemina are transmitted by R. annulatus, R. microplus, and R. geigyi ticks found in tropical and temperate regions of the world. B. bigemina can also be transmitted by R. decoloratus and R. evertsi, making it the most common Babesia species infecting cattle in Africa [8,35]. B. divergens are transmitted mainly by I. ricinus, which develops only in moisture-saturated microhabitats [19]. B. occultans, B. major, B. orientalis, and B. ovata are transmitted by Hyalomma rufipes, Haemaphysalis punctata, R. haemaphysaloides, and Hae. longicornis, respectively [5]. A summary of known competent tick vectors implicated in bovine babesiosis is shown in Table 1, where we highlight the species present in Turkey. To date, R. annulatus, R. bursa, R. turanicus, R. sanguineus, Hy. anatolicum, Hy. dromedari, Hy. detritum, Hy. excavatum, Hy. marginatum, Hy. rufipes, Hy. aegyptium, Dermacentor marginatus, D. niveus, I. ricinus, and Hae. parva ticks have been reported infesting cattle in Turkey [25,46], and some of them were associated with transmission of cattle Babesia parasites (Table 1). In a study using ticks collected from cattle in the Black Sea region, Babesia parasites were reported in Hy. marginatum, Hy. Excavatum, and R. turanicus at the rates of 3.5%, 2.3%, and 6.6%, respectively [47]. Babesia sp. Kayseri 1, a novel parasite isolate, was identified in Hy. marginatum feeding on cattle in the Kayseri province located in Central Anatolia [48]. B. bigemina was also reported in unfed larvae from R. annulatus in this same province [48]. In another study in the same region, B. bigemina was found in tick populations of R. annulatus, R. turanicus, Hy. marginatum, and Hy. Anatolicum, whereas B. bovis positive samples were detected in Hy. marginatum ticks [49]. B. occultans was reported in Hy. marginatum and R. turanicus, as well as in their eggs, and thus, these findings suggest that this later tick can also be a competent vector for B. occultans [50]. In another study, B. occultans was identified in questing Hy. marginatum [51]; however, despite the findings, effective transmission of Babesia by these ticks remains to be demonstrated in Turkey.
Collectively, currently available data indicate the presence and expansion of tick populations in Turkey. In addition, most of these tick species have been shown to be competent in transmitting Babesia parasites implicated in bovine babesiosis. Considering the current environmental changes and the importance of the cattle industry in Turkey, epidemiological and entomological studies focused on ticks associated with Babesia transmission are urgently needed in the country. Given the absolute dependence of ticks for parasite survival, identifying all competent vectors for Babesia species circulating in the country and a more complete understanding of the dynamics of the Babesia-tick interactions will be essential to achieve improved control of bovine babesiosis in Turkey.

Current Control Methods
Live, attenuated, blood based Babesia vaccines, combined with babesicidal drugs and strategies to control tick vectors, are commonly used to prevent and treat bovine babesiosis worldwide, especially in endemic areas. Here we present a brief description of these methods and their applications and importance for the control of bovine babesiosis in Turkey.

Anti-Babesia Vaccines
Live vaccines based on attenuated parasites are widely used to control bovine babesiosis in many countries, such as Australia, Israel, and Argentina [8,10,17]. B. bovis attenuated vaccine strains are produced by quick serial passages of infected blood in splenectomized calves (ranging from 23-30 animals) [52]. In contrast, B. bigemina vaccine strains are produced by slow serial passages (3 to 16-week intervals) in intact spleen calves [53]. Attempts were also made to produce a live vaccine for B. divergens in splenectomized calves or intact gerbils. However, treatment with babesicidal drugs was needed to prevent clinical manifestations after vaccination [18], and due to its poor efficacy, this anti-B. divergens live vaccine is no longer in use [18]. The mechanisms involved in the process of attenuation of Babesia parasites during passages in calves still remain unknown, but recent genomic and transcriptomic comparative analyses among parental virulent and their derived attenuated vaccine strains have shed some light on the presence of differences in the patterns of expression of a limited number of genes [54]. For instance, expression of the spherical body protein 2t11 (SBP211t) was found upregulated in attenuated vaccine strains of B. bovis [55], suggesting that comparing the levels of expression of this gene among strains could be correlated with virulence. Hence, it would be of relevance to compare the pattern of expression of SBP2t11 among local parasite strains isolated in Turkey in order to select parasites that could be candidates for the development of live vaccines.
Despite their relative efficacy, current live vaccines have several limitations, and their use for the control of bovine babesiosis is somehow considered a double-edged sword. On one side, live vaccines remain the most effective way of controlling acute bovine babesiosis, especially considering that there are no safer alternative subunit vaccines available against the disease. Additionally, a study performed in Argentina suggested that the systematic application of live Babesia vaccines in calves might render a benefit-cost ratio between 4.6 and 9.0 [56]. Ojeda et al. 2010 [57] also reported high levels of protection conferred by the vaccine (93%) and demonstrated a ratio of ill/healthy (vaccinated:non-vaccinated) animals of 1:6.5. On the other side of the sword, production of live vaccines is expensive and laborious, cold chain is needed, and contamination with infectious agents and reversion to virulence of vaccine strains remain a possibility. In addition, live vaccines may also lead to transmission of the vaccine strains by ticks, which can contribute to the generation of antigenic diversity among field strains and affect endemic stability. To prevent these problems, live vaccines lacking genes required for sexual reproduction of the parasite using transfection or gene editing technologies can be alternatively developed [58]. Other options available to overcome the various disadvantages of live vaccines are to develop formulations containing killed parasites, soluble parasite antigens (SPAs), and recombinant protective parasite antigens [59]. SPAs are released into peripheral blood during Babesia infection, and promising results in B. canis and B. rodhaini studies suggest that these antigens are potential candidates for developing similar vaccines against other Babesia species [60]. Thus, SPAs prepared from supernatants of in vitro cultures have shown promising results against B. bigemina [61], B. canis [62], B. divergens [63], and B. orientalis [64]. However, on the downside, it was found that vaccines based on B. bovis SPAs were more effective in protecting against challenge with homologous than heterologous strains, raising concerns about their use as universal vaccines.
Although many studies have been conducted to develop subunit vaccines against babesiosis based on recombinant proteins, a commercial-grade product has still not been obtained so far. The use of eukaryotic instead of prokaryotic expression systems mixed with new-generation adjuvants has been recommended in order to increase vaccine efficacy [59]. Recombinant proteins can also be used in the development of transmission-blocking vaccines (TBV), based on parasite sexual stage proteins that are usually exclusively expressed in ticks [58,65,66]. Testing of candidate antigens for TBV against Plasmodium [67] have suggested the notion that such vaccines may also be efficient against Babesia [59]. Several previously characterized B. bovis sexual and tick-stage antigens, such as HAP2 [66], CCp1-3 [65], and 6 Cys [58,68], are also being currently considered as candidates for TBV. In addition, recently characterized B. bigemina sexual-stage antigens are also considered as possible candidates for the development of TBV [69,70]. A possible future effective vaccination strategy would be a combination of a TBV, which can block the development of sexual stage parasites in ticks to prevent parasite transmission in endemic regions, with a blood-stage live subunit vaccine to prevent acute babesiosis. Hence, this combination of blood and tick-stage antigens can provide effective protection while, at the same time, diminishing the load of Babesia circulating in tick vectors in endemic areas. Yet, these vaccines will still require the pending definition of protective and well-conserved blood and tick stage antigens. An important body of work has been also developed in the search of blood stage vaccine candidate antigens, using several approaches. Thus, the B. bovis RAP-1 [71], RRA [4], MSA-2 [72], TRAPs [73], and AMA-1 [74,75] proteins, among others, are currently being investigated as a component of subunit vaccines. It would be important to determine the degree of conservation of such candidate antigens in B. bovis strains isolated in Turkey as well as the presence of equivalent homologous proteins expressed by other cattle Babesia parasites common in this country.
Live vaccines are not currently under production, and therefore, not being utilized in Turkey to prevent bovine babesiosis, and acaricides aimed at controlling ticks is the only method for the disease prevention in use in the country, as described in detail below. However, it needs to be considered that a Babesia-control strategy based solely on targeting vector control appears unrealistic due to multiple factors that include the constant emergence of acaricide resistant tick strains, the multiplicity of competent vectors for these parasites, global climate change that favors the geographic expansion of ticks, and the negative impact that acaricides may have on the environment, among others.

Anti-Tick Control Strategies
Acaricides have been applied for centuries to reduce the harmful effects of ticks and TBD on their vertebrate hosts. Although they have shown efficacy in controlling ticks, acaricides have numerous disadvantages, such as contamination of animal products and the environment. As a control strategy against babesiosis, acaricides are used to decrease or prevent tick infestation of susceptible hosts. However, with the gradual global emergence of acaricide-resistant ticks, the need for the development of new effective drugs and anti-tick vaccines are needed worldwide [17,76]. Yet, despite the implications derived from the emergence of acaricide resistant tick populations, such reports remain unavailable in Turkey. Currently, formamidines, synthetic pyrethroids, phenyl pirazoles, and macrocyclic lactones are used for tick control in the country [25]. The target site for organophosphates and carbamates are acetylcholinesterases that break down the neurotransmitter acetylcholine, while the targets for pyrethroids are voltage-gated Na + channel regulatory proteins of the nerve membrane [77]. Alternatively, the use of anti-tick vaccines surge as an economical and environmentally sustainable approach. Two commercial anti-tick vaccines, TickGARD and Gavac, also known as Bovimune Ixovac in Mexico (http://www.lapisa.com), against R. microplus have been used around the world [78]. While apparently Gavac continues to be commercialized in Cuba (Heber Biotec S.A., Havana, Cuba), and more recently Bovimune Ixovac in Mexico (Lapisa, S.A. de C.V., La Piedad, Mexico), TickGARD is no longer commercially available [79]. These subunit anti-tick vaccines are based on the concealed tick midgut glycoprotein antigen Bm86, which has shown efficacy in reducing the number of engorged female ticks, their weight and fertility capacity, and also in decreasing larval infestation in subsequent tick generations. Cost-effectiveness analysis showed a 60% reduction in the number of acaricide treatments, together with the control of tick infestations and transmission of babesiosis, which resulted in savings of US $23.4 animal/year [80]. A comprehensive vaccination program in Venezuela reported that the Gavac™ vaccine reduced the use of chemical acaricides by 83.7%. The vaccine program was also reported to reduce 81.5% of the estimated cost of conventional chemical tick control procedures [81].
Despite their initially demonstrated efficacy, Bm86 vaccines did not show consistent protection when applied in diverse geographic locations around the world [82]. This lack of effectiveness may be in part due to antigenic polymorphisms, patterns of expression in the ticks, or intrinsic characteristics of the Bm86 antigen, such as conformation and post-translational modifications. It is important to highlight that none of these Bm86-based anti-tick vaccines are used or commercialized in Turkey. Additional tick antigens, such as Bm91 and Bm95 (alternative polymorphic versions of Bm86 isolated from an Argentinian R. microplus strain resistant to vaccination with Bm86), among others, have been characterized and also considered for vaccines [76,83]. Whether Bm86 vaccines or other vaccine based on Bm86 alternative antigens may contribute to tick control in Turkey remains unknown since none of them has been tested in a field trial and are unavailable for use in the country. Tick genome analysis, together with the integration of transcriptomics, proteomics, and metabolomics datasets will surely facilitate the identification of effective protective antigens. Recently, "omics" technologies have shown to be effective approaches for the characterization of tick-host-pathogen molecular interactions, and the potential for vaccine development has emerged. These vaccines should be designed considering tick vector species and susceptible hosts [84]. Studies have shown that the "vaccinomics" approach to select protective antigens (Silk and Subolesin) is valid, and it has been shown that vaccines based on tick proteins involved in vector-pathogen interactions can be used for controlling tick infestation and pathogen infection [85,86].

Babesia Drug-Control Strategies
Imidocarb dipropionate and diminazen acetate are the most common drugs used in the treatment of babesiosis in cattle and other animals. Despite their efficacy in controlling acute disease in some cases, these drugs are expensive and leave residual metabolites in milk and meat. Continuous or inappropriate use of these drugs can also lead to parasite resistance [87]. As a result of all these adversities, research efforts have been focused on developing new alternative, effective, and affordable drugs with low toxicity for the control of bovine babesiosis. It has been reported that strategies based on the combination of chemotherapeutics are significantly more effective at eliminating parasites compared to single-drug treatment, which also has the potential to induce greater parasite drug resistance [88]. In addition, combined chemotherapy decreases toxic side effects by reducing the dosages of individual medications (not reported for imidocarb and diminazen) [89]. In recent years, many drugs, such as nimbolide, gedunin, enoxacin, luteolin, pyronaridine tetraphosphate, nitidine chloride, camptothecin, tulathromycin, trifluralin, 17-DMAG, thymoquinone, clofazimine, carfilzomib, and doxorubicin hydrochloride, have been investigated in vitro against Babesia parasites [90][91][92][93][94][95][96][97][98][99]. Despite promising results, none of these drugs are used in the field as commercially available treatments for bovine babesiosis.
Another important consideration that limits the use of chemotherapeutics, besides their elevated cost and the possible development of drug resistance, is their potential to interfere with the development of herd anti-Babesia immunity in endemic areas. Thus, even when this approach may be effective in the treatment of acute bovine babesiosis, the use of chemotherapeutics is not considered an ideal control method. Noteworthy, the only chemotherapeutic drug licensed for use in Turkey to treat bovine babesiosis is imidocarb dipropionate [100] complemented at times with supportive treatments, such as iron preparations, dextrose, B vitamins, and fluid replacement, which are especially important in acute severe cases.
Considering the current progresses and drawbacks associated with the available anti-Babesia vaccines, babesicidal drugs, and tick control measures, we suggest the design and implementation of pro-active national and international collaborative programs to assess the current situation, identify current gaps, and design improved control strategies. As an example, identification, characterization and integration of local strain of Babesia parasites that cause bovine babesiosis in Turkey in the development of novel control measures for the disease can greatly increment the preparedness of the country to adequately manage this important animal health concern.

Diagnosis of Bovine Babesiosis
Although clinical symptoms, season, and tick infestation may be suggestive of the occurrence of bovine babesiosis, confirmatory diagnosis using different techniques needs also to be applied. In addition, the use of diagnostic tests has an important value for epidemiological studies and to evaluate the need for intervention measures based on the status of the herds, especially to determine whether there is a situation of enzootic stability or instability. Direct identification of Babesia infected-RBC using microscopy has been used traditionally to diagnose bovine babesiosis and is still used today as a practical and inexpensive tool. In the acute period of the disease, the number of parasites in RBC usually increases to a level where they can be readily detected microscopically. This is the case especially for Babesia parasites that typically cause high parasitemia, such as B. bigemina. Yet, this is not usually the case of acute B. bovis infection where infected RBC are hard to find in stained blood films, since they can sequester in large numbers in blood capillaries, in an apparent effort to avoid passing through lymphoid organs, such as the spleen. Additionally, analysis of stained blood films in sub-clinically infected large herds, especially in epidemiological studies, is cumbersome, poorly sensitive, and of limited value. Therefore, more sensitive methods, such as serological assays and molecular tests, should be used. While serological assays are designed to detect immune responses (antibodies) produced by exposed hosts, molecular tests, such as standard PCR, nested-PCR, real-time quantitative PCR, reverse line blot (RLB), and loop-mediated isothermal amplification (LAMP), are aimed at revealing the presence of parasite DNA in the vertebrate host [18,87]. Thus, there is a short window of time at the onset of acute infection (approximately 12-15 days post-infection) when antibodies may not be detected by current serological methods. In turn, this is the stage marked by rapid and unchecked expansion of the parasite in the host, and then, the infection can be promptly detected by molecular methods. However, one possible drawback of PCR methods is the occurrence of false positives due to the persistence of DNA for a short time after effective treatment [101]. Conversely, persistently infected animals may have non-detectable levels of parasite DNA, but detectable levels of circulating anti-Babesia antibodies. Therefore, a serological test must be designed based on species-specific antigens that are able to elicit long-term responses and high antibody titers. Yet, serological tests may often have some disadvantages, such as the possible occurrence of poor immune responses in target animals resulting in undetectable levels of antibodies, limited sensitivity of the tests, and cross reactions between parasite species that may turn the tests non-specific. Molecular methods to directly detect the parasite in the field may overcome some of these downsides. RLB has been a preferred method in recent years for the detection of TBD, such as theileriosis, babesiosis, and anaplasmosis, due to its high sensitivity and ability to detect multiple species simultaneously [87]. The use of this technique has allowed the identification of new species and genotypes of Babesia parasites worldwide, including Turkey [102,103].
Classic microscopic, serological, and molecular methods have been used for the diagnosis of bovine babesiosis in Turkey [25,26]. Although a set of reliable Babesia-diagnostic tests is available nowadays, most of these techniques have disadvantages, such as requiring training and expertise, which makes them difficult to use in field conditions [87]. Alternative rapid and simple point of care strip tests could be developed for this purpose, which requires no previous training for their use, and utilized for the diagnosis of babesiosis, as previously described [104]. Although many studies on strip methods based on immunochromatography or lateral flow test have been done recently [105,106], there is no commercial product available to date worldwide and in Turkey as well, and developing such tests in the future may become important to aid at campaigns designed for the control of bovine babesiosis in this country.

Current Status of Bovine Babesiosis in Turkey
Bovine babesiosis has been currently reported in all geographic regions of Turkey. Detection of parasites in the country first began with microscopic description of B. bigemina in 1890 [107]. After that, serological studies based on Indirect Fluorescent Antibody Technique (IFAT) revealed exposure to B. bovis, B. bigemina, and B. divergens parasites in different regions of Turkey [108][109][110].
Molecular techniques, such as PCR and RLB, have been recently used to investigate the presence of bovine babesiosis in the country [100,111,112]. Combined serology and molecular results have shown that bovine babesiosis caused by B. bovis, B. bigemina, B. divergens, B. major, and B. occultans is widespread in several areas of Turkey (Table 2 and Figure 1). Microscopic, serological and molecular methods have shown a wide range prevalence of Babesia in cattle in Turkey. The wide range of variation in prevalence described in Table 2 may be due to the use of distinct survey methods with different sensitivities and specificities and/or to real differences among the sets analyzed. These observations underscore the fact that no standardized and systematic survey on bovine babesiosis was so far performed in the country. As a result, the prevalence of bovine babesiosis in Turkey remains unknown. B. bovis was first reported by Mimioglu et al. 1969 [113] in cattle in the Black Sea region. Subsequent studies showed that the incidence of B. bovis by microscopic analysis ranged between 0.2-9% in the Central Anatolian Region, 3.7-29.5% in the Black Sea Region, 1.5% in the South East Anatolia Region and 34.8% in the Marmara Region. Serological studies focused on B. bovis have demonstrated evidence for parasite exposure in herds in all Turkish regions with incidence ranging from 0.6 to 59%. A serological study conducted in 3,773 cattle from all provinces revealed a B. bovis prevalence of 34% [110]. In addition, direct assays for B. bovis have revealed that the prevalence for B. bovis ranged between 0.4-12.7% in Turkey (Table 2 and Figure 1).

Current Gaps on Bovine Babesiosis Research in Turkey and Suggestions for Intervention
Causative agents of transboundary animal diseases, such as bovine babesiosis, can be easily transported among countries with the increase in intercontinental animal trade [147][148][149]. The number of animals imported to Turkey from various countries has increased in recent years. Animals imported from countries such as Brazil and Australia that are endemic for bovine babesiosis should be tested in order to determine their status before being admitted into Turkey [8,28]. Therefore, there is a need to develop pan-Babesia direct and serological assays to be implemented in the country. However, there is a possibility that animals test positive for Babesia because they are vaccinated and/or infected with a virulent strain, since vaccination is a common practice in such endemic countries. Regardless of the type of Babesia strain infecting these imported animals, they potentially pose an important risk for Turkey's cattle industry. In these cases, additional practices are needed, such as quarantine to evaluate clinical signs of acute disease and examination for tick infestation, among others. These animal health practices increase the costs for the cattle industry that should be partially or totally covered by government's funds, or otherwise consumers would have to pay for it. Differentiating infected from vaccinated animals (DIVA) vaccines and diagnostic tests that Microscopy analysis revealed that the incidence of B. bigemina is 0.6-18.8% in the Central Anatolian Region, 7.1-32.2% in the Black Sea Region, 1% in the South East Anatolia Region, and 11.6% in the Marmara Region. Similar to B. bovis, serological assays for B. bigemina have shown evidence of parasite exposure (0.9-80%) in all regions in Turkey. A study conducted across the country showed seroprevalence for B. bigemina to be 26.3%. The prevalence of B. bigemina in molecular studies has been reported at the rate of 0.6-18.2% (Table 2 and Figure 1).
There have been only a few studies performed so far in cattle designed to examine the prevalence of B. divergens in Turkey, and the parasite was detected microscopically in only one particular study [100]. Serological studies in cattle reported 75% positivity in the Black Sea region, 7.2% in the Mediterranean region, 18.5% in the Aegean region, and 9.4-48.9% in the Marmara region. B. divergens prevalence based on molecular techniques was reported as 3.4-7.4% and 1.1% in the Black Sea and Central Anatolian Regions, respectively (Table 2 and Figure 1). Abbreviations: ME, microscopic examination; SM, serologic methods; MT, molecular techniques.
No positive results have been reported so far regarding B. major by microscopy and serological methods in Turkey. In recent years, the molecular diagnostic of B. major based on RLB tests has been found a prevalence of 0.2% in the Ankara region [114] and 0.51% in the Eastern Black Sea Region [115] ( Table 2 and Figure 1). Similar to B. major, only molecular data are available for B. ocultans and it was reported a prevalence of 3% for this Babesia species using RLB technique in samples from the Black Sea region [100]. In the same study, the authors reported cases of clinical babesisosis caused from B. bovis, B. bigemina, and B. divergens species.
Additional studies have also showed the presence of new uncharacterized Babesia genotypes in cattle in Turkey. By using RLB technique, these studies have reported the occurrence of Babesia sp. CS58, Babesia spp., and Babesia sp. Sivas in cattle. Blast analysis indicated that their 18S sequences are 99-100% identical to the 18S gene of B. occultans [115][116][117] (Table 2). Considering these preliminary findings, further investigation is needed in order to precisely identify these currently uncharacterized Babesia organisms in the country and evaluate their implication on the development of bovine babesiosis.
Studies based on molecular methods have reported considerable sequence variations on some Babesia genes, such as RAP-1, MSA, and AMA-1. Using a combination of PCR and sequencing techniques, Duzlu et al. (2015) [118] showed that the sequences of the B. bovis MSA-2c gene in Turkish isolates are 7.5% different from other isolates in the world. This rate was found to be 0.5% in the B. bigemina RAP-1 gene. Another study focused on defining sequence polymorphisms of AMA-1 protein of in vivo and in vitro isolates of "Babesia bigemina Kayseri/Turkey" strain. The analysis revealed that nucleotide sequences of Kayseri/Turkey IV1 and Kayseri/Turkey IT2 isolates showed 99.7% identity to each other with a single nucleotide mutation at the position 103. Interestingly, this non-synonymous mutation results in the change of the amino acid at the position 35 (Phe 35 in Kayseri/Turkey IV1 and Leu 35 in Kayseri/Turkey IT2) [119]. Implications of this finding need to be investigated in the light of epidemiological and immunopathological view of the disease.
In summary, bovine babesiosis has been reported using different diagnostic methods in all of Turkey's seven geographical regions ( Figure 1). While B. bovis and B. bigemina are reported in all geographical regions, B. divergens has been found more in humid regions located near the seacoast. Despite these many epidemiological investigations, the number of case-based studies is very low. Therefore, prevalence and the actual economic impact of the disease on the livestock industry cannot be accurately assessed, due, in part, to the absence of a national program to systematically record cases of bovine babesiosis in Turkey. Consequently, more well-designed surveys based on highly sensitive assays are needed, especially in areas of the country that so far have received little attention. Although there is no study on annual economic losses associated with bovine babesiosis, the high incidence of the disease in geographical regions that are highly populated by cattle suggests that the disease is already causing serious costs to the livestock industry in Turkey. Clearly, all these findings together strongly suggest the need to organize coordinated efforts aimed at assessing the current status and the development of control measures of bovine babesiosis in Turkey.

Current Gaps on Bovine Babesiosis Research in Turkey and Suggestions for Intervention
Causative agents of transboundary animal diseases, such as bovine babesiosis, can be easily transported among countries with the increase in intercontinental animal trade [147][148][149]. The number of animals imported to Turkey from various countries has increased in recent years. Animals imported from countries such as Brazil and Australia that are endemic for bovine babesiosis should be tested in order to determine their status before being admitted into Turkey [8,28]. Therefore, there is a need to develop pan-Babesia direct and serological assays to be implemented in the country. However, there is a possibility that animals test positive for Babesia because they are vaccinated and/or infected with a virulent strain, since vaccination is a common practice in such endemic countries. Regardless of the type of Babesia strain infecting these imported animals, they potentially pose an important risk for Turkey's cattle industry. In these cases, additional practices are needed, such as quarantine to evaluate clinical signs of acute disease and examination for tick infestation, among others. These animal health practices increase the costs for the cattle industry that should be partially or totally covered by government's funds, or otherwise consumers would have to pay for it. Differentiating infected from vaccinated animals (DIVA) vaccines and diagnostic tests that can also differentiate imported vaccinated from naturally infected animals need to be urgently developed and implemented in the country. Importantly, tick control measurements need also to be performed in such imported animals. Lack of such control in the past resulted in the emergence of R. microplus, one of the most devastating vectors of Babesia species implicated in bovine babesiosis. Fortunately, R. microplus have not been found yet in Turkey, despite the importation of animals from places, such as Brazil and several African countries, where this tick species is endemic [150,151]. R. microplus has a major economic impact on milk and beef production in Brazil, and annual losses in this country were estimated at $3.24 billion US dollars [151]. R. microplus tends to replace the local boophilic fauna after entering a new region and affects the epidemiology of bovine babesiosis. Rapid spread of R. microplus was observed in South Africa, Zambia, and East Africa [5]. Therefore, introduction of R. microplus can potentially dramatically change the future status of bovine babesiosis in Turkey. Thus, prevention measures as well as novel research are needed to address gaps in our understanding on the biology and ecology of this tick species in exotic and non-endemic environments.
Many novel Babesia genotypes or species have been recently reported in diverse vertebrates hosts worldwide [6]. Some of these species have also been reported in cattle in Turkey, but no detailed studies on parasite biology and host-parasite-tick interactions have been performed [115][116][117]. While making novel genotype discoveries, the 18S gene region alone has been shown to be insufficient and additional sequencing analysis needs to be performed using other gene regions [5]. The impact, vectors, and pathogenicity of these newly reported genotypes or species of Babesia remain largely unknown in Turkey, and these factors need to be further characterized. A recent study in Sri Lanka reported the presence of a novel Babesia species, called Babesia sp. Mymensingh, which causes clinical babesiosis characterized by fever, hemoglobinuria, anemia, and jaundice in cattle [103,152]. Therefore, studies focused on detailed examination of biology, pathogenicity, host-parasite interactions will fill a large gap that, in turn, can help design strategies to control outbreaks of bovine babesiosis caused by newly, uncharacterized local parasites strains in Turkey.
Use of acaricides is the only method currently used for the prevention of bovine babesiosis in the country. Although there are studies on the efficacy of some acaricides (flumethrin, deltamethrin, cypermethrin) against ticks on cattle in Turkey, investigations on emerging acaricide-resistance in tick populations have not been performed [153,154]. Furthermore, acaricide applications in cattle are usually performed using poorly standardized and unhealthy procedures by breeders in Turkey, and this can potentially generate increased acaricide resistance by ticks due to dose errors as well as environmental and health problems. Livestock producers would benefit greatly from a national training program on the use of acaricides in cattle from the Turkish Ministry of Agriculture. In addition, acaricide applications can also be performed at certain intervals by the local official veterinarians in selected endemic areas. In this way, a more effective tick control program could provide a platform that, in turn, can help decrease the chances for development of acaricide resistance. Economic losses due to TBD can also be reduced by introducing defined policies and requirement for the application of acaricides to livestock, using the frame of existing support programs currently provided by the state, such as the Livestock Support Program.
To date, costs associated with bovine babesiosis in Turkey have not been yet estimated and this analysis is urgently needed. Proper estimation should include cattle mortality, abortions, losses in milk/meat production, loss of draft power, and expenses related to control measures, such as acaricide treatments, and the purchase of therapeutics, among other aspects. In addition, annual losses due to other costs related tick borne diseases (TBD) should also be calculated.
Functional, genetic, and immunological studies on Babesia genes and subunit vaccine candidate proteins will be needed in order to develop subunit vaccines, efficient and simpler point of care diagnostic tests, and other possible control methods. Such investigations would be much more relevant to Turkey if local parasites strains are included in comparison with other, more studied strains, such as the virulent T2Bo B. bovis strain [155]. Live vaccines are not currently available to prevent bovine babesiosis in the country; therefore, production of live vaccines based on parasites strains that are currently circulating in the country's provinces should increase control efficacy, prevent outbreaks, and decrease the use of acaricides. Thus, the need for such live vaccines to alleviate the load of bovine babesiosis in Turkey should be addressed and properly assessed.
As a road for future directions on Babesia research, well-known and novel, uncharacterized parasite species preserve their mystery like an unconquered land, even after many years of exploration. Numerous characterization studies have been performed up to now and will continue until effective eradication of this parasite is accomplished. Along with genome analysis, important advances have been made in the biology of Babesia parasites globally [59,[155][156][157]. Afterwards, transfection systems have been developed for gene modification and functional analysis to accelerate vaccine candidate discovery [156]. Together with novel gene editing technologies, these new approaches can potentially accelerate the development of efficient diagnostics and novel vaccine strategies, such as TBV based on Babesia sexual-stage antigens. It would be essential to develop a national research program in bovine babesiosis/Babesia to translate this new knowledge to the hands of research and field personnel involved in cattle production in Turkey. In vitro tick artificial feeding systems can also be developed and applied to better study tick-Babesia interactions [158]. With the development of this system, effectiveness of transstadial and transovarial transmission of many tick-borne pathogens can be determined. Importantly, tick artificial feeding systems can provide a framework for reducing and improving animal use to study ticks and tick-borne pathogens that are important for human and veterinary medicine [158]. Results of such research projects will greatly benefit the development and implementation of control strategies against bovine babesiosis in Turkey.
Subunit vaccines using recombinant antigens produced in Escherichia coli expression systems may or may not be efficient in eliciting protective immune responses against Babesia. Protection to Babesia is believed to be dependent on Th-1 helper cells and early production of IFNγ, which requires adequate antigen presentation by professional antigen-presenting cells via major histocompatibility complex (MHC) class II and effective T cell responses. Perhaps effective subunit vaccines will require the use of mammalian expression, or transfection-based homologous parasite-specific expression systems for antigen production. In addition, as far as vaccine efficacy is concerned, nanoparticles, such as Immune stimulating complexes (ISCOM), are thought to be effective adjuvants for Babesia subunit vaccines, which opens new possibilities for the testing of candidate antigens [59].
Novel non-sophisticated point of care diagnostic methods that can be performed by untrained personnel are needed for the rapid "on site" detection of the disease in field conditions and may accelerate the time of response in severe outbreaks of bovine babesiosis. On the other hand, state-of-the-art technologies, such as next-generation and nanopore sequencing approaches, can help our understanding of the composition of local strains, population dynamics of the parasites, and help identify virulence factors that can be used for developing novel vaccines. These techniques combined with gene manipulation techniques already available for the transfection of Babesia parasites [156,159] may also lead to better defined and non-tick transmissible live vaccines incorporating DIVA markers to differentiate vaccinated from naturally-infected animals. These DIVA vaccines are much needed in Turkey, especially considering the importation of a large number of live cattle from countries that are endemic for bovine babesiosis.
Cattle imported into Turkey are currently not subjected to strict sanitary controls, including checking for ticks and TBD, thus imposing an important risk for the development of cattle industries in the country. Such controls need to be incorporated in order to prevent the importation of exotic ticks and Babesia strains in the country. As stated before, the introduction of novel tick species that are competent for Babesia, such as R. microplus, would have devastating consequences, given the ability to these ectoparasites to develop resistance to acaricides, and the lack of alternatives for efficient anti-tick vaccines.
To eliminate the negative effects of acaricides, effective anti-tick vaccine formulations need to be developed based on protective antigens using new generation adjuvants, such as nanoparticles [160]. By combining protective epitopes from different tick proteins into a single chimeric antigen, new vaccines may be likely to target more than one ectoparasite species and host [161]. In addition, microspheres and other sustained-release technologies could also be integrated into acaricides to offer a potential solution for its downside effects. Importantly, new generation sequencing methods can also help identify parasites and other infectious organisms present in distinct tick populations and facilitate our understanding of the interplay among Babesia parasites and tick vectors in the ecosystem. Development of new methods for the detection of tick-infested cattle may also help in the control of TBD, including bovine babesiosis.
Collectively, these new technologies and research directions can potentially reveal bottlenecks on the Babesia lifecycle and tick ecology that will help implement strategies to control bovine babesiosis to benefit the cattle industry worldwide and particularly in Turkey.

Concluding Remarks
The world population has been increasing exponentially since the 1950s and nowadays, there is a need to efficiently produce high amounts of animal protein and fiber, and consequently, different human and animal health problems may arise. Protein sources are becoming more valuable every day and an effective and safe method of protection against Babesia parasites that affect millions of cattle every year has become more important than ever. Based on the limited data available, it is possible to infer that bovine babesiosis caused by distinct Babesia spp. as well as novel and newly identified Babesia genotypes/species is highly prevalent throughout Turkey, and safe and effective methods are urgently needed to combat this disease. For this purpose, firstly, multidisciplinary studies need to be performed nationally to address the economic and social impact of bovine babesiosis in Turkey. Secondly, the need for live vaccines should be carefully evaluated, and if needed, vaccines should be produced based on strains of the parasite that are circulating in the country. Use of acaricides, and combinations of these drugs, should be used consciously by technical personnel as part of developed control strategies and practices. In addition, further basic studies on parasite and tick biology need to be performed to develop rationally designed recombinant vaccines. We also should take advantage of newly emerging technologies in order to develop sophisticated tools required to control Babesia, a highly co-evolved parasite that is able to evade so efficiently the host immune system and optimize control measures that are currently available. The Turkish cattle industry and, in turn the entire country's economy, will greatly benefit from such control strategies against the devastating effects of bovine babesiosis.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Use of Amino Oligosaccharins and Alternaria Fine Protein in the Management of Crown Gall Disease in Roses

Rose (Rosa hybrida Vill.) production is limited by a variety of factors such as poor mineral nutrition, high salinity, pests and diseases. Crown gall disease caused by Agrobacterium tumefaciens causes a significant damage to roses in Kenya. The study was carried out in Winchester farm (Nairobi) and Bahati farm (Nakuru) to determine the effects of a mixture of amino oligosaccharins and Alternaria fine proteins on crown gall disease in roses. The experiment was conducted on Rosa hybrida var. Mariyo in a Randomized Complete Block Design with four replications. The treatments comprised of different rates 0.5, 1, 1.5g/L of water of the product at 3% concentration applied as foliar spray and a commonly used product copper sulphate pentahydrate (Mastercop) produced by ADAMA applied at 2ml/L as the standard and water as a negative control. Crown gall tissues were collected from four different roses per treatment from the two sites, counted and used for biochemical tests in Kabete laboratory. Bacteria were isolated by culturing and the representative colony types growing on nutrient agar media selected and sub-cultured by successive streaking on nutrient agar media. The biochemical test for the different isolates was done to identify the bacterial isolates. Application of the mixture of amino oligosaccharins and Alternaria activated protein at the rate of 1.5g/L had significant effects on galls formation and reduced the numbers significantly at both farms. The gram reaction indicated that the selected isolates were gram negative and were positive for motility, catalase, oxidase, lactose, mannitol, and salt tolerance tests. There was a significant reduction in the number of galls and size following the application of amino oligosaccharins and Alternaria fine proteins as well as improvement in plant growth. The ability to manage the disease can be attributed to enhanced defense enzyme activity enhanced by amino oligosaccharins and Alternaria fine proteins. Current Agriculture Research Journal www.agriculturejournal.org ISSN: 2347-4688, Vol. 8, No.(2) 2020, pg. 118-127 CONTACT Emmanuel Wesonga Atsango atsangoe@gmail.com Department of Plant Science and Crop Protection, University of Nairobi, Nairobi, Kenya. © 2020 The Author(s). Published by Enviro Research Publishers. This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY). Doi: http://dx.doi.org/10.12944/CARJ.8.2.07 Article History Received: 07 April 2020 Accepted: 30 July 2020

Introduction
Globally, the Kenyan flower industry is the third major flower exporter by value and volume just behind the Netherlands and Colombia on a global level 1 while in Africa, it is the largest producer and the leading supplier of Fair trade flowers to Europe accounting for 50-60% of total volumes. 2 The flower production in the country is mainly large scale and export oriented. 3 Cut flower is one of the two commodities where large-scale production dominates in the country and roses make up over 70% of all buds, trees, roots, flowers and foliage that is exported from Kenya. 4 Roses alone account for about 35% of the total value of horticultural exports and therefore form the most important export product. 5 In commercial production of roses, yield is restricted by an assortment of variables such as light, humidity, mineral nutrition, temperature, salinity and biotic factors. 6 The common biotic factors in cut-flower roses include pests and diseases. Crown gall caused by A. tumefaciens causes a significant damage to roses in Kenya. The bacterium is found in the soil and it is responsible for the tumorous growth found in infected plants. 7 The pathogen is widespread, naturally occurring soil bacterium that causes crown gall in numerous plant species in nurseries, commercial production and uncultivated areas and has the capacity to introduce new genetic material into the plant cell. Significant annual losses of between 5-6% are faced by the flower growers worldwide due to the disease in the form of lack of vigor, reduction in foliage, low quality rose flower, low productivity and increased susceptibility of infected plants to pathogens and environmental stress. 8 In other instances, losses of between 10-30% in nursery stalk have been reported in fruit trees. Both yield loss and stunting of growth may occur when seedlings or young cuttings are infected in the early stages of plant growth. 8 Many strategies have been used in the management of crown gall disease including the use of chemicals, pre-plant application of soil sterilants, soil solarization, use of herbicides and soil amendments. 9 Despite losses incurred in flower exports due to effects of the disease and high residual effect of pesticides, research especially focusing on management of A. tumefaciens is limited. The use of oligosaccharins and alternaria fine protein has the potential to be one of the safest means to manage the plant disease. Oligosaccharins induce responses that may help the plant to resist disease and have positive effects on growth and development while the alternaria fine protein accelerates plant growth vigor, increases proline content and cellulase strength. Therefore, this study sought to investigate effects of amino oligosaccharins and alternaria fine protein on crown gall disease of roses.

Materials and Methods
A-tailing is the mixture formulation using the active ingredient (A.I) Fine alternaria activated protein and amino oligosaccharins. The content of each active ingredient is three (3%) and the product is available in formulation specification as wettable powder (WP) and currently registered in China.

Experimental Materials, Design and Treatment Application
The experimental material used was Rosa hybrida var. Mariyo both at Winchester and Bahati farms. The variety was chosen because it's high yielding and has a continuous market as compared to other varieties whose market is seasonal. 5 Its susceptibility to Agrobacteria made it appropriate for this research therefore. An existing infected crop was used for the research and was laid out in a randomized complete block design. Two-year-old rose of variety Mariyo planted in pumice (an inert growing media that is a by-product of volcanic ash) was pruned and sprayed at two weeks' intervals with oligosaccharins and fine Alternaria protein at various concentrations. The treatment concentrations comprised of a mixture of oligosaccharins and fine Alternaria protein at 3% concentration applied as foliar spray at 0.5, 1, 1.5g per litre of water. Commonly used product in the market, Mastercop, was applied at 2ml/L and was used as the standard and a negative control. Reapplication of the treatments was done at fortnight intervals. Six plants from each plot were randomly selected from each treatment and used for data evaluation.

Collection of Crown Gall Samples
Ten galls of diameter 3-10cm were collected and put in sterile bags from two different rose farms (Winchester farm and Bahati roses) located in Nairobi and Nakuru, respectively. The samples were labeled and transferred under a cool box to the laboratory in Kabete for isolation.

Determination of size of galls
Size of Agrobacteria galls formed on rose plants were measured and recorded fortnightly. This was used for disease severity determination.

Isolation of Bacteria from the Gall
Agrobacteria gall samples from treated and untreated plots having young tumors were transferred to the laboratory for isolation. Small fragments of tumor tissue were chopped and crushed with sterile scalpel blade in a few drops of sterile distilled water. The suspension was then left to stand for approximately 15 min and 100μL was spread with a loop onto the nutrient agar (NA), which contains 0.5% peptone, 0.3% beef extract, and 1.5% agar, and incubated at 28°C. Representative colony types growing on nutrient agar media were selected from each gall sample by use of a sterilized wire loop and sub cultured by successive streaking on new nutrient agar media.

Disease Assessment
Rose plants were randomly inspected for the assessment of crown gall disease using disease incidence and severity levels. Disease incidence was determined using the formula by. 10 Disease incidence = (Number of infected plants)/(total number of plants)×100 While severity was assessed based on the size of the infection where mild; 1-3 cm, moderate;4-6 cm and severe; above 6 cm.

Characterization of Agrobacterium tumefaciens Biochemical Test
Biochemical test for the distinctive isolates was carried out following the Bergey's manual of Determinative Bacteriology. 10 Motility, gram stain, catalase, oxidase production, utilization of lactose, mannitol and salt tolerance (2%) tests were conducted.

Gram Staining
Gram staining was done by making bacterial smears from two days old cultures on sterile microscope slides. The smears were air-dried and then after, heat fixed by passing the slides over a flame and then Gram stained. The slides were observed under a compound microscope at magnification of × 1000.

Utilization of Lactose
Phenol red mannitol broth with 1% lactose was used. The broth contains peptone, phenol red(indicator) a Durham tube and carbohydrate(lactose). Each test tube was aseptically inoculated using an inoculating loop and incubated at 37 o C for 24 hours. A positive result was indicated by gas production (bubbles) on inverted Durham tube.

Motility Test
A semi solid agar medium was prepared in a test tube and inoculation done by use of a wire loop to make a single stab at the centre of the tube. Incubation at 37 o C (conditions favoring motility) were carried out and examined at intervals (6, 24 and 48 hours).

Growth on MacConkey agar
Isolates were introduced into agar plates containing MacConkey agar by use of a sterile wire loop. The agar is selective and differential medium that is designed to isolate and differentiate gram-negative bacteria based on their ability to ferment lactose. Pink colonies on the MacConkey agar confirmed the bacteria to be gram negative.

Salt Tolerance
The nutrient broth was prepared by use of sodium chloride (65 g) (1, 2, 3, 4, and 5%) and dextrose (1.0 g) and allowed to settle at room temperature. Two to three colonies of the isolates were picked and inoculated into the broth and incubated. Turbidity was observed after 12 and 24 hours.

Oxidase production
A filter paper was soaked with the substrate (tetramethyl-p-phenylenediamine dihydrochloride) and moistened with sterile distilled water. The bacterial colony was then picked using a wooden loop and smeared on the filter paper. Appearance of deep-purple colour was observed after 15 seconds.

Catalase Test
Slide method test was used for catalase test. Small amount of bacterial colony was transferred to the surface of a clean, dry glass slide by use of a sterile wire loop and a drop of 3% hydrogen peroxide added onto the slide and mixed. The rapid elaboration of oxygen bubbles after five seconds confirmed the bacterium catalyzes the breakdown of oxygen from hydrogen peroxide.

Pathogenicity Test
Isolates suspected to be A. tumefaciens were inoculated onto an indicator carrot disc bioassay and potato disc bioassay. 10 The same was inoculated onto healthy rose plants and observed.

Carrot Disc Assay
Carrots were obtained from a local market in Rongai. The collected carrots were then sterilized with 1-2% sodium hypochlorite and rinsed in three changes of sterile distilled water. Aseptically, the discs were prepared by chopping into smaller discs using a sterile surgical blade. The discs were placed in sterile petri dishes lined with sterile filter paper. Soriful procedures were followed and one hundred microliters of the inoculum was overlaid on each disc and sealed with parafilm and incubated in growth chamber at 28 0 C for two weeks with continuous observation for tumor formation from meristematic tissues around the vascular system.

Potato Disc Assay
Potatoes were obtained from local market in Rongai. The collected potatoes were sterilized with 1-2% sodium hypochlorite and rinsed in three changes of sterile distilled water. Aseptically, the discs were prepared by chopping into smaller discs using a sterile surgical blade. The discs were placed in petri dishes containing water agar. According to10 recommendations, each disc was overlaid with 100μL of the inoculum and sealed with parafilm and incubated in growth chamber at 28 0 C for two weeks with continuous observation. The discs were then stained with 5% Lugols Iodine (Iodine 5%, and potassium iodide 10%) for thirty (30) minutes and examined under microscope for tumor formation, where there was tumor formation, the starch was utilized.

Data Analysis
Quantitative data was analyzed using statistical analysis software Genstat Discovery Edition 15 (VSN International Ltd. 2015) while qualitative data was assessed visually ant through the microscope. Treatment means for quantitative data were compared using revised LSD test at the 0.05 level of probability using Fisher's protected LSD.

Effect of Amino Oligosaccharins and Alternaria Fine Protein on Incidence and Severity of Crown Gall
Disease severity was categorized into mild, moderate and severe (Table 1). Rose plants treated with various rates of amino oligosaccharins and alternaria fine protein had mild severity rates compared with the control plots in both sites. The incidence of the disease was assessed fortnightly for ten weeks ( Table 2). In Winchester, there was no significance differences in plots applied with amino oligosaccharin and alternaria fine protein at the rate of 1.5g/L and plots applied with copper based fungicide. These plots showed a significant reduction in number of galls after the tenth week. In Bahati however, plots treated with amino oligosaccharin at 1.5g/L showed reduced number of galls on the tenth week after application compared with other treatments. The least incidence was observed in control plots in both farms.
The incidence and severity of the disease was assessed in each plot and control plots had the greatest incidence. Based on the progress of the disease after ten weeks of assessment, the least incidence was reported on plots treated with amino oligosaccharin and alternaria fine protein at 1.5g/L and were not significant different from the plots treated with copper based fungicides. The severity index shows infection being severe in control plots after ten weeks of assessment.

Effect of Amino Oligosaccharins and Alternaria Fine Protein on the Galling and Size of Galls
There were significant differences in number of galls in each treatment and in the two sites (P≤0.05) ( Table 3). Application of the different rates of amino oligosaccharins had greater effect on the number of galls in Bahati than in Winchester. There was an increase in the number of galls in all the treatments in the second and fourth week after treatment application. However, decline in the number of galls was noticed sixth week after treatment application and it was least in plots treated with amino oligosaccharins at the rate of 1.5g/L. Application of amino oligosaccharins at 1.5g/L had greater effect on galling formation reducing the number significantly from 2.49 in the fourth week to 1.40 in the eight week and to 1.08 in the tenth week in Winchester while in Bahati, the number reduced significantly from 1.54 to 1.03 ten weeks after treatment application. The results were not significant different from the plots treated with copper at 2 mL/L in both farms. However, control plots had the number of galls increasing every week in both farms. Means followed by the same letters along the columns are not significant different at 5% level of probability Means followed by the same letters along the columns are not significant different at 5% level of probability Means followed by the same letters along the columns are not significant different at 5% level of probability Means followed by the same letters along the columns are not significant different at 5% level of probability There were significant differences in the size of galls before and after treatment applications (Table 4). There was significant reduction in sizes of galls after treatment application with the highest percentage reduction observed in plots treated with amino oligosaccharins at the rate of 1.5g/L and amino oligosaccharins at the rate of 1g/L in both sites. Application of copper fungicide as the standard also resulted in reduced gall sizes in both sites. There was significant increase in gall sizes in control plots.

Biochemical Tests
The biochemical tests for the different isolates of A. tumefaciens are presented in Table 5. The gram reaction indicates that the selected isolates were gram negative. The isolates were gram positive for motility, catalase, oxidase, lactose, mannitol, and salt tolerance tests. In terms of shape, the colonies were circular and slightly raised and were cream white in colour with smooth margins ( Table 6)

Pa t h o g e n i c i t y Te s t fo r A g ro b a c t e r i u m Tumefaciens Isolates
A. tumefaciens isolates from both farms produced tumors when inoculated on carrot and potato discs. Young galls (tumors) were observed developing at the central part of the carrot and potato discs two weeks after inoculation. However, no symptoms were observed in control discs.

Relationship Among Flower Growth Parameters and Crown Galls
From correlation analysis, significant positive correlation was displayed between total number of galls and the size of gall (0.6416, P≤ 0.05). However, there was a significant negative correlation between the number of galls and shoot length (-0.695, P≤ 0.05).

Discussion
Application of amino oligosaccharins had greater effect on the number of galls; however, the effect depended on the rate of application. There was increase in the number of galls in all the treatments in the second and fourth week after treatment application. However, reduction in the number of galls was noted in the sixth week after treatment application and it was highest in plots treated with amino oligosaccharins at the rate of 1.5g/L. Amino oligosaccharins and alternaria fine proteins can constitute an important control agent for crown gall in roses. Similar results were reported by. 11 In their findings, 11 found chitosan and its derivatives to inhibit the growth of A. tumefaciens and Erwinia spp and displayed highest antibacterial activity against A. tumefaciens, Erwinia spp with MIC 500 mg/L and 480 mg/L, respectively. Similarly, when oligosaccharin derivatives chitosan were used in controlling Xanthomonas vesicatoria, infections were greatly reduced by above 60% 12 , reported significant reduction of the disease incidence and delayed symptoms development and the size of the lesions were smaller in comparison with other treatments. The reduction of number of galls was due to oligosaccharides derivatives unique elicitation of physiological and biochemical changes in roses resulting to induced resistance. 11 There is an enhanced activity of enzymes that are linked to defense against plant diseases in response to application of oligosaccharins. Enzymes such as Chi, β-1,3-Glucanase(GLU), peroxidase (PO), phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) are activated and are able to eliminate infectious agents such as Agrobacterium tumefaciens. 12 Increase in Phenylalanine ammonialyase (PAL) enzyme activity is important as it manages pathogenic infection. 1 Working on tomatoes reported enhanced activities in response to Ralstonia solanacearum inoculation in tomato pre-treated with chitosan.
According to 14,12 oligosaccharins act by inducing several activities of defense enzymes in roses that are involved in pathogen defense. Alternaria fine protein accelerates plant growth vitality, increase proline content, strengthen cellulase and promote the growth of plant cells, strengthen root activity, thereby managing plant diseases. Chitosan, a biopolymer amino polysaccharides has been reported to have strong antibacterial activities against plant pathogenic bacteria such as A.
tumefaciens, Erwinia spp, 11 Streptomyces scabies, 15 Xanthomonas spp, 16 Pseudomonas syringae, 17 However, the activity of chitosan oligosaccharins depends on the concentration 11,18 and molecular weight 19 and the type of bacteria. 20 The mechanism of antimicrobial activity of chitosan and its derivatives remains a mystery as it has not been understood well and it is therefore considered to be complex. However, according to, 21 at the initial stages chitosan ties with negatively charged components on the bacterial surface by means of electrostatic interactions which changes the permeability of bacterial wall and permits chitosan to get to the internal cell targets subsequently closing down cell division causing death. 22 In this study, bacteria were isolated using nutrient agar media which has been previously used for isolation of A. tumefaciens from rose samples with crown gall. A series of biochemical tests such as motility, gram staining, oxidase production and catalase, utilization of lactose and mannitol, salt tolerance (2%) potato and carrot disc assay revealed that the isolated bacterium was gram negative and had the capacity to cause tumor in plant disc sample. The biochemical approaches have been utilized in the past investigations for the recognizable proof of A. tumefaciens from crown gall samples from diverse plant species. 14 Tumor forming ability of the isolates confirmed that they were virulent, however, according to, 25 virulence of A. tumefaciens was because of the nature of the host, internal physiology of the strains and environmental conditions. Crown gall disease can be severe in rose plantations if young trees are infected. A. tumefaciens was found on root surfaces and was effectively isolated from galls. The bacterium was isolated and confirmed using different morphological and biochemical tests.

Conclusion
The study demonstrates the ability of amino oligosaccharins and alternaria fine proteins to be used as an alternative to management of plant diseases. A significant reduction in the number of galls and size following application of amino oligosaccharins and alternaria fine proteins was observed as well as improvement in plant growth. The ability of the treatments to manage the disease can be attributed to enhanced defense enzyme activity. Therefore, this study provides good support for incorporation of amino oligosaccharins and alternaria fine proteins in the management and yield enhancement of roses.
I wish to acknowledge also the contribution of CCIPT Director Mr. Fan Liqiang who provided the samples that were used in this research.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.