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Agricultural And Food Sciences
Environmental Science

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Effect of Soil Type: Qualitative and Quantitative Analysis of Phytochemicals in Some Browse Species Leaves Found in Savannah Biome of South Africa

In semi-arid regions, browse plant species are used as feed and for medicinal purposes for both animals and humans. The limitation of the utilization of these species to medicinal purposes or as feed for livestock is a lack of knowledge on the concentration level of phytochemicals and other bioactive compounds found in these plants. The study sought to assay the qualitative and quantitative bioactive constituents of some browse species found in the savannah biome of South Africa, viz. Adansonia digitate, Androstachys johnsonii, Balanites maughamii, Berchemia discolor, Berchemia zeyheri, Bridelia mollis hutch, Carissa edulis, Catha edulis, Colophospermum mopane, Combretum Imberbe, Combretum molle, Combretum collinum, Dalbergia melanoxylon, Dichrostachys cinerea, Diospros lycioides, Diospyros mespiliformis, Euclea divinorum, Flueggea virosa, Grewia flava, Grewia flavescens, Grewia monticola, Grewia occidentalis, Melia azedarach, Peltophorum africanum, Prosopis velutina, Pseudolachnostylis maprouneifolia, Pterocarpus rotundifolius, Schinus molle, Schotia brachypetala, Sclerocarya birrea, Searsia lancea, Searsia leptodictya, Searsia pyroides, Senegalia caffra, Senegalia galpinii, Senegalia mellifera, Senegalia nigrescens, Senegalia polyacantha, Strychnos madagascariensis, Terminalia sericea, Trichilia emetic, Vachellia erioloba, Vachellia hebeclada, Vachellia karroo, Vachellia nilotica, Vachellia nilotica subsp. Kraussiana, Vachellia rechmanniana, Vachellia robusta, Vachellia tortilis, Vachellia tortilis subsp. raddiana, Vangueria infausta, and Ziziphus mucronata. These browse species’ leaf samples were harvested from two provinces (Limpopo and North-West) of South Africa. The Limpopo province soil type was Glenrosa, Mispah, and Lithosols (GM-L), and the soil types in the North-West Province were Aeolian Kalahari Sand, Clovelly, and Hutton (AKS-CH). The harvested browse samples were air dried at room temperature for about seven days and ground for analysis. The methanol and distilled water extracts of the browse species leaves showed the presence of common phytoconstituents, including saponins, flavonoids, tannins, phenols, cardio glycosides, terpenoids, and phlobatannins, as major active compounds in browse species leaves. In the quantitative analysis, phytochemical compounds, such as soluble phenols, insoluble tannins, and condensed tannins, were quantified for common species found in both sites. Two-way ANOVA and multivariate analysis were used to test soil type and species effect on soluble phenols, insoluble tannins, and condensed tannins of woody species. Dichrostachys cinerea (0.1011% DM) in GM-L soil type and Z. mucronata (0.1009% DM) in AKS-CH soil type showed the highest (p < 0.05) concentration of soluble phenols. In AKS-CH soil type, D. cinerea (0.0453% DM) had the highest insoluble tannins concentration, while V. hebeclada had the lowest (0.0064% DM) insoluble tannins content. Vacchelia hebeclada had lower (p < 0.05) condensed tannins concentration levels than all other browse plants in both soil types. Under multivariate analysis tests, there was a significant effect (p < 0.001) of soil type, species, and soil type x species interaction on soluble phenols, insoluble tannins, and condensed tannins of woody species. In this study, most of the woody species found in GM-L soil type showed a lower amount of tannins than those harvested in AKS-CH soil type. There is a need to identify the amount of unquantified phytochemicals contained in these browse species and valorize the high-bioactive-compound browse species to enhance and maximize browsing of these browse species for animal production.

Introduction
Savannah woody species play an important role as the cheapest protein source for livestock in semi-arid regions [1]. Some of these plants are deciduous and semi-succulent. They can retain green leaves in certain seasons of the year, making them advantageous in responding to feed shortages during dry seasons, when the herbaceous species and other annual grasses wilt [2,3]. These plants' contributions to multi-purpose tasks (protein and medicinal sources) have been documented [4][5][6].
The chemical composition of browse species varies with plant species, growth, plant parts, soil type, temperature, altitude, light, and other anthropogenic factors, such as herbivory [7][8][9][10]. Some of these woody species contain plant secondary metabolites (phytochemicals) which are anti-nutritional elements, which may negatively affect an animal's health and nutrition [11,12]. A large number of bioactive compounds, including phenols, flavonoids, tannins, coumarin, lignin, terpenes, saponins, phlobatannins, and glycosides, are usually scattered in plant species [13]. Mazid et al. [14] pointed out that phenolic compounds are known to be abundant plant secondary metabolites in browse species which can contribute up to half of their organic matter.
Furthermore, Liu et al. [15] highlighted that phenolic compounds, structurally, are made up of aromatic benzene rings with more than one hydroxyl group, arranged from lower to higher polymerization levels. According to Mueller-Harvey [16], tannins, flavonoids, and phenolic acids are phenolic compounds which are abundantly constituted in savannah woody species, and they play a significant role in animal nutrition, health, and performance [17]. Flavonoids are a group of secondary metabolites found in plants with a lower molecular weight, and they are constituted with major classes, such as flavonols, flavones, and isoflavones.
Plant secondary metabolites (phytochemicals) offer important pharmacological and biological properties, including hypoglycemic, anti-inflammatory, antioxidant, anti-bacterial, and anti-carcinogenic properties, which can fight against oxidative cell damage [18][19][20]. These bioactive compounds are also essential sources for proper plant growth and defensive mechanisms against infection and plant cell damage. Salem et al. [21] indicated that the isolation and identification of browse plant phytochemicals have long been a difficult challenge for researchers. Different plant species can have variation in secondary metabolites produced through biosynthesis [22]. Though there are numerous research studies that have been conducted worldwide with special focus on phytochemical medicinal plants [23][24][25][26][27], little scientific validation has been carried out for browse species phytochemicals found in most semi-arid areas of South Africa. There is a need to perform screening of phytochemicals in order to establish the concentration levels of these browse species. This is a valuable step to serve as a guiding tool to identify and manipulate those browse species that might be toxic to ruminants due to high concentration of certain bioactive compounds. Therefore, the current study was designed to evaluate the existence of phytochemical components and assess the qualitative and quantitative aspects of phytochemicals present in browse species extracts. The study hypothesized that there was variation in the qualitative and quantitative aspects of phytochemicals present in browse plant species, given the therapeutic value of these widely accessible browse plant species. Hopefully, the study will contribute to the improvement of livestock production in savannah areas of South Africa.

Preliminary Qualitative Phytochemical Analysis
Results of phytochemical screenings of browse species found in GM-L soil type are shown in Table 1. Phytochemical compounds such as saponins, flavonoids, tannin, phenols, glycosides, terpenoids, and phlobatannins were screened and observed in methanol and distilled water extracts. Phlobatannins were found to be absent in distilled water extracts for all browse species harvested in both soil types. Terpenoids were present in both methanol and distilled water extracts, except for S. galpinii, which was found to be negative in the distilled water extract. Negative results for tannins and phenols were found in most browse leaves from distilled water extracts. Vachellia hebeclada showed negative results for phlobatannins, glycosides, phenols, and tannins in both methanol and distilled water extracts. Saponins and flavonoids were present in all browse species from distilled water extracts, except for C. edulis, T. sericea, and V. tortilis subsp. taddiana.  In Table 2, for AKS-CH soil type, terpenoids were present in both methanol and distilled water extracts, except only for V. nilotica subsp. Kraussiana, which were absent in the water extract. Saponins were not present only in S. molle when using both methanol and distilled water extracts. Senegalia galpinii and V. hebeclada were found negative in tannins, phenols, glycoside, and phlobatannins in both methanol and distilled water extracts. Flavonoid, saponins, tannins, and phenols were present in most browse species from methanol extracts.  Table 3 showed the findings of the effect of species and soil type on the soluble phenols, insoluble tannins, and condensed tannins of browse plants leaves. There were significant differences (p < 0.05) observed on the effect of species, soil type, and the interaction between species and soil type on soluble phenol, insoluble tannin, and soluble condensed tannin concentrations of browse leaves found in communal rangelands. In GM-L soil type, V. nilotica subsp. Kraussiana and T. sericea had a higher concentration of insoluble tannins (0.0386% DM), while in AKS-CH soils, D. cinerea had a higher concentration of insoluble tannins (0.0453% DM) when compared to all other species in the same harvesting site. Melia azedarach, D. cinerea, S. mole, S. leptodictya, V. hebeclada, and V. karroo in GM-L soil type had higher insoluble tannin concentration when compared to all other species in the same harvesting site. In both sites, V. hebeclada browse leaves had the lowest (p < 0.05) insoluble tannin (0.0041% DM, GM-L and 0.0064% DM, AKS-CH soil type) and condensed tannin (0.0137 AU 550 /200 mg, GM-L and 0.0138 AU 550 /200 mg, AKS-CH soil type) concentrations. In both sites, V. hebeclada browse leaves had the lowest (p < 0.05) concentration levels of soluble phenols (Limpopo-0.0160% DM) and (North-West-0.0334% DM). Within GM-L and AKS-CH soil types, V. tortilis has shown no significant difference in soluble phenolic content. Again, in the concentration of CTs, Z. mucronate, V. hebeclada, P. africanum, and T. sericea did not show any significant differences. Table 3. Soil type and species effect on soluble phenols (SPhs-% dry matter), insoluble tannins (ITs-% dry matter), and condensed tannins (CTs-AU 550 /200 mg) of woody species found in AKS-CH and GM-L soil types. The results on multivariate tests on the effect of species and soil type on the soluble phenols, insoluble tannins, and condensed tannins of browse plants leaves are shown on Table 4. All of the multivariate tests displayed significant differences (p < 0.001) on the effect of species, soil type, and the interaction between species and soil type on soluble phenol, insoluble tannin, and condensed tannin concentration of browse leaves found in communal rangelands.

Phytochemical Screening of Browse Species
In almost all cases, the methanol extract showed the presence of a rich variety of secondary metabolites when compared to the distilled water extract. This may be due to the fact that methanol extract tends to dissolve many organic compounds more quickly than in water extract. Again, Paulsamy and Jeeshna [28] highlighted that methanolic solvent tends to draw a higher variety of plant compounds than other solvents. Ghasemzadeh et al. [29] explained that the variation in browse species' nature and polarity level plays a major role in extracting plant secondary metabolites. Similar results were also reported by Shen and Shao [30] and Ingle and Padole [27].
Plant secondary metabolites, which are chemical compounds responsible for biological activities, are commonly found in browse plants [31]. From phytochemical screening in the current study, we observed that the methanol and distilled water extracts from browse species gave positive results in the major phytoconstituent compounds. In this study, Catha edulis leaves from GM-L and S. molle leaves from AKS-CH soil type reported negative in saponin concentration using both methanol and distilled water extracts. The frothing tests were used in the present study to confirm the presence of saponins in the methanol and distilled water extracts on browse species leaves. Okwu [32] indicated that saponins have the distinctive properties of precipitating and coagulating red blood cells and also help in regulating the immune response. These results are in line with those reported by Chaudhary et al. [33] that saponins induce the degradation of cell wall enzymes and the leakage of proteins from the cell.
In this study, flavonoids were found to be moderately present when tested with distilled water extract as compared to those tested with methanol extracts, which were more abundant, and these results are in agreement with those reported by Ajiboye et al. [34] and Senguttuvan et al. [24]. Flavonoids are polyphenolic compounds that are water-soluble and are extensively dispersed in browse plants species, with over 5000 flavonoids discovered [35]. Flavonoids have anti-allergic, anti-inflammatory, tumor inhibitory, antiviral, and anti-cancer properties in addition to antioxidant activities [36,37]. These effects have been associated with health-promoting properties and a wide range of chemical and biological functions, including vasoprotection and free radical scavenging, which can prevent oxidative cell damage [36]. Rauf et al. [38] also reported that flavonoid-rich browse plants are diuretics, whereas others are anti-spasmodic and antibacterial.
Browse plants usually generate phenolics, which are aromatic benzene ring molecules containing one or more hydroxyl groups, mostly to protect plants against stress [39]. Tannin accumulation levels on browse plant species play a critical role in animal health, because most plants frequently produce these secondary metabolites either in high or low volume. Hoste et al. [40] indicated that tannins as polyphenolics in natural plants may have detrimental effects on animals consuming high amounts (>6%) of tannins in their dietary feed. In this study, tannins and phenols were found negative in V. hebeclada from both solvent (methanol and distilled water) extracts. Vachellia hebeclada can be used as a beneficial browse plant for animal consumption due to the fact that it has low levels of tannins and phenols, which are known to be anti-nutritional to animal diet when above 6 g/kg of the total intake diet [41]. From the present study, negative results for tannins and phenols were found in most browse leaves from the distilled water extract. Nevertheless, tannins and phenols are not only considered to have detrimental effects, but they also have some beneficial effects that have been documented. Okwa and Josiah [42] reported that tannins have a critical role in promoting healing of wounds and inflamed mucous membranes.
In this study, cardiac glycosides were present, except in V. hebeclada (GM-L and AKS-CH soil types) and M. azederach (GM-L soil type) in both methanol and distilled water extracts. These results may be influenced by browse plant genetic makeup, because polarity did not show any influence in the results reported by Senguttuvan et al. [24] when using methanol and water solvents to test cardiac glycosides. Konstantinou et al. [43] indicated that cardiac glycosides have a direct effect on the heart; they normally aid in restoring a failing heart by maintaining the contraction rate and heart strength. Browse plants rich in glycosides are known to strengthen the immune system, making them useful as dietary supplements. In general, glycosides are present in every woody plant species and play a significant role in therapeutic efficacy. From the present study, terpenoids were found to be present in both methanol and distilled water extracts, except for S. galpinii (GM-L soil type) and V. nilotica subsp. krassianna (AKS-CH soil type), which were found to be negative in the distilled water extract. Several authors, such as Wagner and Elmadfa [44] along with Rabi and Bishayee [45], have found that terpenoids have great potential as beneficial remedies for treating and preventing various ailments in both livestock and humans. Again, Wagner and Elmadfa [44] also highlighted that terpenoids have several functions, such as anti-parasitic, antifungal, antimicrobial, and anti-inflammatory properties. Furthermore, terpenoids tend to have insecticidal characteristics, and they can be used as protective chemicals in the storage of agricultural products.
Phlobatannins, in the current study, were found to be absent when using distilled water extract for all woody species leaves in both soil types. These findings may be influenced by the polarity level of water, which was not suitable for drawing phlobatannins in the selected browse leaves. Setchell [46] indicated that phlobatannins in browse plant species have astringent properties, such as anti-inflammation and antiviral as well as antioxidant activities. Moreover, the antimicrobial, antifungal, and anti-parasitic activities of woody species as well as the presence or dispersion of phytochemical compounds appeared to be partly dependent on the host plant species.

Phytochemical Quantification of Browse Species
Secondary plant compounds, which included tannins and phenolics, normally serve important roles in tropical animal productivity, because animals in semi-arid regions rely on woody species as a source of daily feed, and this browse forage tends to accumulate highconcentration levels of phytochemical compounds [41]. In this study, the findings of phytochemical compounds (soluble phenols, insoluble tannins, and condensed tannins) were significantly different among woody species harvested in both sites. Ravhuhali et al. [47] highlighted that woody species found in various locations had a wide range of chemical constituents. Browse species, soil type, and their interaction had an impact on the concentration levels of soluble phenolic (SPhs), insoluble tannins (ITs), and condensed tannins (CTs). The concentration of SPhs on woody species leaves from both soil types was between 0.0160 and 0.1011% DM, and those woody species in AKS-CH soil type had higher values than those from GM-L soil type. This might be due to different altitudes and soil profiles, because plants growing in the poor soil profiles are known to accumulate high levels of phenolics [48], and, in this study, AKS-CH soil type is assumed to have a poor soil profile when compared to GM-L soil type. Reed [49] highlighted that soluble phenols have a moderate impact on ruminant nutrition when compared to tannins. The leaves of V. hebeclada had the least soluble phenols in both sites. These results might have been influenced by plant genetic make-up. According to Ibrahim et al. [50], plant genetic make-up has a large impact on concentration levels of phenolics, way more than other abiotic elements (altitude, temperature, light, and soil type) that aid in the secretion of bioactive compounds in woody plants. Schinus molle leaves from AKS-CH soil type had the highest soluble phenolics (0.1000% DM), while in GM-L soil type they had the least soluble phenolics (0.0377% DM). These results could be due to herbivorous activities, as browse plants trees tend to produce and secrete their defensive chemicals throughout the plants' parts as a form of defense mechanism against herbivorous activities [51]. This might warrant the investigation to assess the most preferred plant by livestock ruminants in the study areas. Mnisi and Mlambo [3] highlighted that dietary intake and digestibility of plant-based feed for livestock can be determined by the number of phenolic compounds constituted in the feed diet.
The concentration of insoluble tannins (ITs) in this study ranged from 0.0037% DM (S. leptodictya) to 0.0386% DM (T. sericea and V. nilotica subsp. krassianna) in GM-L soil type, and in AKS-CH soil type the levels ranged from 0.0064% DM (V. hebeclada) to 0.0453% DM (D. cinerea). Within GM-L soil type, S. leptodictya, V. hebeclada, S. molle, and M. azedarach had the lowest insoluble tannins content. With these results, it can be suggested that these browse species can be regarded as a suitable feed source for ruminants due to their lower tannin concentration [52][53][54].
In this study, condensed tannin (CT) concentration was influenced by species and their environments. Vachellia hebeclada, M. azederaach, S. molle, and S. leptodictya concentration levels of condensed tannins in this study are similar to the findings reported by Mlambo et al. [55] (3-6%) on the same woody species and this range is regarded as the best concentration level for herbivores. The highest concentration of CTs reported by Mokoboki et al. [11] was in V. karroo leaves, which agrees with the findings of this study. The majority of the selected woody species, regardless of their harvesting sites, had a lower concentration of CTs in this study, which is likely considered to be nutritionally safe. Makkar and Singh [56] indicate that the presence of condensed tannins in large quantities (>4-9%) in fiber fractions may influence their digestion kinetic characteristics, causing the link between kinetic measures and fiber composition to be muddled. This is in line with the findings reported by Foo et al. [57], Dube et al. [58], and Mlambo et al. [41], who highlighted that the concentration of CTs accumulated in the animal diet tends to interfere with intake and digestion, while very low concentration has a beneficial effect in ruminants. The variance in condensed tannins content across plant species might be related to genetic make-up, altitude, and herbivory, which results in different response mechanisms to spatial variation [47,59,60]. However, to improve the utilization of tanniniferous browse species, tannin-inactivating treatments are required. Various studies have already indicated the use of PEG (polyethylene glycol) and WA (wood ash) to deactivate the protein tannin bonds on various plant parts as a remedy to high-tannin browse plants [61][62][63].
The results showed that with the use of multivariate analysis tests, a significant effect of soil type, species, and interaction between soil types and browse species on soluble phenols, insoluble tannins, and condensed tannins of woody species. In this study, CTs, ITs, and SPhs concentration was influenced by species and their environments. Indeed, species from different locations tend to have variation in concentration level, and related results were also reported by Schleppi et al. [64] and Suárez et al. [65]. The positive view on the interaction between biotic and abiotic components with living organisms was also highlighted by Hervé et al. [66].

Limpopo
The harvesting sites were in the Thulamela and Makhado local municipalities, with a probably high number of livestock, such as goats, sheep, donkeys, and cattle that completely rely on these communal rangelands. The browse tree species were harvested in Lamvi  [67]. The study site temperatures range from 13 • C to 34 • C and with rainfall range between 200 mm to 500 mm annually, respectively.

North-West Sites
The North-West province harvesting sites were Mahikeng and Ratlou local municipalities, with high numbers of goats, sheep, and cattle grazing in these communal areas. In Mafikeng, the browse tree leaves were harvested from Six Hundred (25 • 42 43 S, 25 • 37 32 E and 1300 m above sea level) and Tsetse (25 • 44 07 S, 25 • 39 40 E and 1296 m above sea level), with both constituted with clay-loamy soil structure, and, meanwhile, Loporung (25 • 45 37 S, 24 • 59 54 E and 1162 m above sea level), with red-brown sandy soil structure, is situated in Ratlou municipality in North-West Province, South Africa. These communal areas have Aeolian Kalahari Sand, Clovelly, and Hutton (AKS-CH) soil type, based in Mafikeng Bushveld, Eastern Kalahari Bushveld, and Thornveld vegetation types [67]. These areas receive mean annual rainfall varying from a minimum of 400 mm to 500 mm. The study sites have winter seasons with sunny dry days and cold nights with an annual average temperature ranging between 2 • C and 36 • C, with a rainfall ranging from 250 mm to 450 mm annually, respectively.

Cold Extraction of Secondary Metabolites from Browse Plant Species
Approximately 10 g of finely ground browse leaves powder was weighed into a separate 250 mL conical flasks and then about 100 mL of each solvent (methanol, distilled water) was added to the flask and sealed with laboratory film (parafilm, Bemis, Brampton, ON, Canada). The flasks were kept at normal room temperature for about 7 days, and the flasks were daily shaking for a period of 10-15 min. After 7 days, the extracts were then filtered by using Macherey-Nagel No.1 filter paper under vacuum and then air dried at room temperature in a Petri dish. The weight of each Petri dish was noted prior to drying of the extracts. The weight of the extracts was then calculated from the difference following the procedure demonstrated by Harborne [68].

Preliminary Qualitative Phytochemical Screening of Crude Methanolic and Water Extract
Qualitative phytochemical analyses were carried out using the cold extracts from browse plants following the standard procedure [69] to identify the constituents as demonstrated by Harborne [68], Trease and Evans [70], and Sofowora [71]. The tests for the presence or absence of various plant secondary metabolites (PSM) are summarized below.

Test for Saponins
About 0.5 g of the crude plant extracts was dissolved with distilled water (10 mL) in a test tube. The suspension was shaken in a test tube for about 10-15 min. A 2 cm layer of foam was taken as preliminary evidence for saponins [72]. The layer of foam was used to determine the concentration level of saponins, whereby the higher level of foam indicated a higher level of saponins.

Alkaline Reagent Test (Flavonoids)
Alkaline reagent test: Exactly 0.5 g of plant extract was firstly dissolved with solvent (methanol and distilled water) up to 10 mL in a test tube, and, secondly, an aqueous solution of the extracts was then treated with 10% ammonium hydroxide solution. The yellow fluorescence indicates a positive test for flavonoids [73,74]. The highest level of yellow fluorescence in the test tube symbolized the reflection of high flavonoids.

Test for Tannins
About 0.5 mg of crude plant extracts was added to 10 mL of their freshly prepared solvents (methanol, distilled water) in a test tube and shaken to dissolve. A few (2-4) drops of 0.1% ferric chloride was added and produced brownish green or a blue-black coloration, which confirms the presence of tannins [69,74]. The darkest blue-black color was regarded as a reflection of high concentration of tannins.

Test for Phenols
Ferric chloride test: Aqueous plant extracts were treated with 3-4 drops of 0.1% ferric chloride solution. The formation of a bluish green or black color was confirmed as a positive presence of phenols [75]. The darkest black color was regarded as a reflection of high concentration of phenols.

Test for Cardiac Glycosides
Keller-Killani test: About 0.5 g of crude plant extract was dissolved with 5 mL of distilled water in the test tube and shaken. Each 5 mL extract in the test tube was treated with 2 mL of glacial acetic acid containing a few drops of 0.1% ferric chloride solution. This was underplayed with 1 mL of concentrated sulfuric acid (H 2 SO 4 ) along the side of the test tube. The formation of a brown ring at the interface indicates deoxysugar characteristics of cardenolides, which gives a positive presence of cardenolides. The appearance of a violet-green ring below the brown ring in the acetic acid layer indicates a positive result for cardiac glycosides [76].

Test for Terpenoids
Salkowski test: The crude plant extract (5 mL) was separately shaken with 2 mL of chloroform and followed by careful addition of concentrated sulfuric acid (2 mL) along the test tube to form a layer. A reddish brown coloration of the interface formed, confirming a positive presence of terpenoids [69,76,77].

Test for Phlobatannins
About 2 mL of extract of each plant sample was boiled with 1% aqueous hydrochloric acid. Appearance of a red precipitate was taken as evidence for the presence of phlobatannins [69].

Quantitative Phytochemical Analysis
The quantitative assay was carried out using the prepared plant extract as described by Makkar [78]. The extraction preparation is described below, and procedures for determination for phenols, tannins, and condensed tannins are summarized below.

Extract Preparation
Finely ground browse plant material (200 mg) was weighed into a small (25 mL) glass beaker; 10 mL of aqueous acetone (70%) was added, and then the glass beaker was suspended in an ultrasonic water bath (Branson 5800) for about 20 min at room temperature [78]. The content in the glass beaker was then transferred into small centrifuge tubes and subjected to centrifugation for about 10 min at speed of 3000 rpm, with temperature set at 4 • C. After centrifugation, the supernatant was transferred into large test tubes, and the solid particles were left in the small centrifuge tubes. The collected supernatant was kept on ice for further analyses.

Determination of Total Phenolic Content
A 0.01 mL aliquot of tannin-containing extract was pipetted into a test tube. Distilled water (0.49 mL) and 0.25 mL of the Folin-Ciocalteu reagent were added to the test tube and then followed by 1.25 mL of sodium carbonate solution [79]. The test tubes were then vortexed (Lasec Vortex Mixer) and then allowed to stand for 40 min at room temperature. The absorbance was recorded at 725 mm wavelength using a spectrophotometer (T60 UV-Visible Spectrophotometer, PG Instruments). A standard curve was developed using tannic acid (0.5 mg/mL), and concentration of tannic acid equivalent in browse leaf samples was predicted from this curve. Concentration of tannic acid in dry matter was expressed as [Tannic acid in grams/Dry matter%] × 100.

Determination of Total Tannins
Polyvinyl-pyrrolidone (PVP) was used to bind tannins for determining total tannins (TTs), as demonstrated by Makkar [78]. A 100 mg sample of polyvinyl pyrrolidone was weighed into a glass laboratory test tube; 1.0 mL of distilled water and 1.0 mL of the tannin-containing extract were added, and this was stored at a temperature of 4 • C for about 10-15 min. Afterwards, it was centrifuged at 3000 rpm for 10 min. Subsequently, the supernatant was collected with a simple phenolic other than tannins, and the absorbance or UV reading was recorded as mentioned above and expressed as tannin acid equivalent (TAE). Total tannin content was calculated as a difference between simple phenols and total phenol (TP) content in the extract.

Determination of Condensed Tannins
The same acetone extract used in the soluble phenolics assay was used to assay for soluble/extractable-condensed tannins (SCT) using the butanol-HCl reagent (95:5 v/v) [80]. Aqueous acetone extract (0.5 mL) was pipetted into a glass screw cap test tube and 5 mL butanol-HCl reagent added. The test tube was closed and then placed on a heating block at 100 • C for 1 h. Absorbance was measured after the test tubes had cooled to room temperature. The measurements were reported as absorbance units (au) per 200 mg sample at 550 nm wavelength using the spectrophotometer as prescribed by Makkar [78] (Supplementary Materials).

Statistical Analysis
Data on quantitative phytochemical composition (insoluble phenols, soluble tannin, and soluble condensed tannin) were analyzed using two-way analysis of variance (ANOVA) that used general linear model (GLM) procedures of SAS 9.3 [81] for a factorial treatment arrangement in a completely randomized design (CRD). Factors such as browse species and soil type were considered in this study. The following general linear model was used: where Y ijk is the dependent variable, µ is the overall mean, B i is the effect of browse species, Lj is the effect of two different locations (soil type), (B × L) ij is the interaction effect between browse species and two different locations, and ε ijk is the random error term associated with observation ijk and assumed to be normally and independently distributed. Where significant variation was detected, multiple comparisons of treatment means were carried out using the probability of difference (pdiff) option of the GLM procedure of SAS [81]. In addition, multivariate analysis was used to test soil type and species effect on soluble phenols, insoluble tannins, and condensed tannins of woody species found in AKS-CH and GM-L soil types.

Conclusions
The methanol and distilled water extracts of browse species examined in this study contain a variety of phytochemicals that could be responsible for their medicinal, antimicrobial, and antioxidant properties. The most important chemicals are saponins, alkaloids, flavonoids, glycosides, phlobatannins, and terpenoids, which have a metabolic role in biological systems and perform a protective role in livestock. Furthermore, most of the species analyzed had low concentrations of chemical constituents that are essential for maintaining good health. Though there were some discrepancies, the findings suggest that different sites (soil type) and species affected the phytochemical constituent content. To enhance and maximize utilization of these browse plant species, there is a need for various studies to identify all other bioactive compounds constituted in these plant species that may have detrimental effects on animals, e.g., alkaloids, glucosinolates, and terpens. All browse species exposed to animals can be subjected to screening to determine the concentration level of phytochemicals. Multivariate analysis tests revealed a significant effect of both factors and their interaction on soluble phenols, insoluble tannins, and condensed tannins of woody species. To those browse species (S. caffra, V. karroo, and D. cinerea) having high concentration levels of tannins, there is a need to use wood ash or PEG as a remedy to deactivate high tannin protein bonds that reduce the availability of nutrients to the ruminal microbes. The outcomes of this study will provide important information and knowledge on how to improve livestock health and production in semi-arid regions.


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Distribution of Theileria orientalis in Virginia Market Cattle, 2018–2020

Theileria orientalis, genotype Ikeda, was recently detected in North America. Determining the emerging distribution of this pathogen is critical for understanding spread and developing management strategies. Whole blood samples were collected from cattle at Virginia livestock markets from September 2018 through December 2020. Animals were tested for T. orientalis using a universal and then genotype specific real-time PCR based on the MPSP gene. Prevalence for each genotype was analyzed for temporal trends and mapped by county. Spatial patterns were compared between genotypes and assessed for associations with habitat features, cattle movements through cattle markets and county proximity. Overall, 212 of 1980 samples tested positive for T. orientalis with an overall prevalence of 8.7% (172/1980) for genotype Ikeda, 1.8% (36/1980) for genotype Chitose, 0.2% (3/1980) for genotype Buffeli. The Ikeda genotype increased over time in northern and southwestern Virginia markets. The Ikeda and Chitose genotypes occurred in different regions, with little overlap, but for each genotype, spatial distribution was associated with a combination of cattle movements and environmental factors. Genotype specific qPCR testing and surveillance of cattle from across a wide area of Virginia are providing information on temporal, spatial, and other patterns for this emerging disease.

Introduction
Theileria orientalis is a parasitic, unicellular alveolate originally confined to East Asia and Oceania [1,2], but increasingly diagnosed as a cause of bovine infectious anemia in the Mid-Atlantic region of North America [3]. The disease is an economic concern in endemic regions, causing anemia, abortion, and failure to thrive in cattle [2,4] with reduction in the growth rate of beef cattle [5] and substantial reduction in milk volume and quality in dairy cattle [6]. There is currently no approved treatment for T. orientalis infection in the USA. Virulence varies widely between the 11 distinct genotypes [7,8] causing asymptomatic to severe disease; the Ikeda genotype is the most clinically concerning [4]. The distinct Chitose and Buffeli genotypes [9], are often found alongside Ikeda variants, though they pose lesser risk to the cattle.
Although the Buffeli genotype has been detected in numerous eastern states since the turn of the century [10][11][12], the more pathogenic Ikeda and Chitose genotypes had not been identified in North America prior to 2017 [3,4] although all three genotypes co-occur in Australasia [13]. The origin of the US introduction remains a mystery, though some have suggested it coincided with the arrival of the Asian Longhorned Tick, Haemaphysalis Pathogens 2022, 11, 1353 2 of 13 longicornis [14][15][16], which serves as the primary vector for T. orientalis in the Eastern Hemisphere [1,17,18]. Since the first confirmed identification in North America in the early 2010s, H. longicornis has been found across the East Coast from Connecticut to North Carolina, with widespread distribution in western Virginia and neighboring states [19]. Given the agricultural impact of theileriosis, it is critical to understand the factors which underlie the emerging distribution and spread of this pathogen in North America. Using a recently developed qPCR assay and samples from market cattle collected across the Commonwealth of Virginia, we characterized the prevalence, demographic, and distributional features of T. orientalis in cattle in Virginia.

Sampling and Blood Collection
Whole blood samples were collected by Virginia Department of Agriculture and Consumer Services (VDACS) staff beginning in August 2018 with five sites (three Northern, one Central, one Southwest) and scaled up, in January 2019, with seven additional sites (one Northern, three Central, three Southwest). These included 11 of the 12 regularly scheduled, weekly Virginia Livestock Auction sites as well as one additional weekly auction. To gain wide geographic coverage with continuity over time, approximately five adult animals from different lots were sampled during each visit. Fewer samples were collected if there were less than five lots of cattle presented and when staff travel or auction sales were interrupted, mainly due to pandemic COVID restrictions. Whole blood was collected in purple-top BD Vacutainer ® blood collection tubes containing EDTA anticoagulant (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Sex, breed, and radio frequency identification (RFID) tag were recorded. Breeds were also coded as groups (beef, dairy or unknown). Origin of each animal was approximated based on address of the owner, abstracted from market sales records, and geocoded using the Google Maps geocoder API (Available online: https://developers.google.com/maps/documentation/geocoding/overview, accessed on 5 August 2021).

Molecular Detection and Genotyping of T. orientalis
Animals were classified as infected with T. orientalis using duplex, real-time PCR, which simultaneously tested for Anaplasma marginale, performed as previously published [20]. Briefly, DNA was extracted using the Qiagen DNeasy ® Blood and Tissue kit (Qiagen LLC, Germantown, MD, USA). Primers and probes targeted T. orientalis MPSP, A. marginale msp1b, and a VetMAX™ Xeno™ internal positive control (ThermoFisher Scientific, Waltham, MA, USA), and probes were labeled with NED, FAM and VIC, respectively. Samples were run on an Applied Biosystems™ 7500 Fast Real-Time PCR System (ThermoFisher Scientific) for 45 cycles with an annealing and extension temperature of 60 • C. Samples that were positive for the universal T. orientalis qPCR (quantification cycles of less than 45) were then genotyped using a previously published protocol and the same equipment and parameters as above [20,21]. Samples that were positive only after more than 45 amplification cycles were considered equivocal, potentially representing a related gene or an extremely low parasitemia. Samples that were positive using the universal qPCR but negative for all three genotypes were tested with conventional PCR and Sanger sequencing to attempt to characterize their 18s ribosomal RNA subunit and MPSP.

Statistical Analyses
Prevalence estimates, with 95% confidence intervals (CIs), were calculated for each T. orientalis genotype for cattle demographic groups. Prevalence Ratios (PR), with 95% exact, unconditional CIs [22], were determined for male versus female, beef versus dairy, breed relative to Angus (the most common breed in our sample) and grouped known breeds (Angus, Hereford, etc.) versus grouped crossbreds. Analyses were completed with R version 4.1.0 [23] using the Exact2x2 package version 1.6.6 [24]. Prevalence for each genotype and the total number of samples collected, by month, were plotted in time for each of the three Virginia cattle market regions [25], because there were too few samples to assess each market individually. Prevalence over time within each market region was assessed for linear trend and then for inflections in slope using adaptive logistic regression splines [26,27] with the Earth 5.3.1 package in R [28].

Spatial Analyses
The number of cattle tested varied between counties; some counties had zero or very few animals tested. Therefore, counties were classified and mapped using ArcMap 10.7 (Esri, Inc., Redlands, CA, USA) for each genotype as having at least one positive sample; having only negative samples; or as unsampled. Spatial codistribution between Ikeda and Chitose genotypes was tested with Tjostheim's Coefficient [29] after removing counties where neither genotype was found so as to limit artificial similarity due to shared absences [30]. This was done using the SpatialPack package [31] in R.
A network defining connectivity between counties, based on cattle movement through markets, was estimated by first calculating the proportion of geocoded cattle that originated from each county for each market. Since buyer information was not available, bidirectional proportionality of county origin and destination was assumed. Flow was estimated as the product of proportions for each pair of counties divided by the total of these proportions to remove circular movement back to county of origin. Total annual cattle sales per market [32] were then used to scale for market cattle volume differences. Weighted cattle numbers were summed for each county pair across all markets and divided by total market cattle to give the relative weight between each pair of counties. This network was graphed as a force-directed layout to optimize visualization of community structure [33] using Gephi 0.9.2 [34]. The optimized number of communities (clusters) within the network was determined using the Girvan-Newman algorithm [35]. More localized cattle movement was defined by a dichotomous matrix in which counties were considered connected if they shared a border and disconnected if they did not.
Similarity in relevant habitat between pairs of counties was assessed through ecological niche models separately created for the Ikeda and Chitose genotypes using generalized additive models [36] in the ENMTools package in R [37]. Individual geocoded herds with at least one positive animal were used as Theileria genotype specific presence points for selecting predictive geospatial variables, while herds with exclusively negative tests were considered to be absence points. Potential predictors included elevation at 30-arc second resolution and 19 remotely sensed bioclimatic variables [38], as well as three new habitat variables (forest, field, and edge densities). The 2019 National Land Cover Dataset [39] was reclassified into forest (NLCD codes 41, 42 and 43) and fields (NLCD codes 71, 81 and 82). Edge-habitat was present where any forest and field blocks intersected [40]. Forest or field area or total linear edge habitat was summed for a 42.45 arc-second diameter circle from the centroid of each bioclimatic cell to cover the entirety of the 30 × 30 arc-second raster cells. Each bioclimatic and habitat variable was resampled and registered to match the resolution of the elevation data using ESRI ArcMap 10.7. Overall similarity was compared between the two genotype niche models using Warren's I [41], again using the ENMTools package in R. Composite ecological suitability scores for each county and differences between these scores for county-to-county pairs were determined for each genotype.
Join count statistics for dichotomous spatial data [42] were used to assess clustering between counties classified as positive for T. orientalis, by genotype, using the SpDep Package for R [43]. The join count test compares the observed number of joins between similar areas to the expected based on total number of areas and possible joins and is more appropriate than continuous autoregressive measures, such as Moran's I, for binary data [44]. Significance of county connectivity was tested using a series of spatial weight matrices that varied the contribution of cattle movements through markets, local cattle movements, and similarity in genotype ecological niche. The percentage distribution of each was varied from 0 to 100%, always requiring the three to total to 100%, to determine where the combined influence correlated most strongly with the county-level distribution for the Theileria genotype [45]. Results were plotted as smoothed spline ternary surface using ESRI ArcMap 10.7.

Results
A total of 1993 blood samples were collected from cattle at Virginia markets from August 2018 through December 2020. Thirteen animals were excluded from further analyses because qPCR results were equivocal; positive amplification for T. orientalis occurred, but only above the established qPCR cut-off. Of the remaining 1980 samples, 765 were from southwest markets, 744 from northern markets, and 471 from central markets. These were collected on 399 different sampling dates (19 samples were missing collection dates), with an average of 37 sampling dates and 165 samples per market. Samples were collected from southwest markets on 161 different visits, from northern markets on 173 visits, and from central markets on 109 visits. Cattle sampled in Virginia markets came from 82 of the 133 counties/county-equivalents in the Commonwealth and 80 counties in adjacent states ( Figure 1). Using qPCR, T. orientalis was detected in 212 (10.7%) samples, from 11 of the 12 sale barns surveyed. The Ikeda genotype was the most common, representing 172 (81.1%) of the genotyped samples, present in 8.7% (95% CI = 7.5%, 10.0%) of the 1980 classified samples. The Chitose genotype was found in another 36 cattle, representing 17.0% of the genotyped samples and 1.8% (95% CI = 1.3%, 2.5%) of the classified samples, while the Buffeli genotype was detected only three times, representing 1.4% of the genotyped samples and 0.2% (95% CI = 0.02%, 0.5%) of the classified samples. The genotype of one T. orientalis positive sample was not identifiable with the 3 genotype probes nor with conventional PCR for the 18s ribosomal RNA subunit or MPSP. No animals in the study group were found to be infected with multiple genotypes. Among samples with unequivocal test results, 1889 (95.4%) were able to be geocoded, representing 1318 unique herd locations. Ikeda was detected in 132 of these herds (10.0%; 95% CI = 8.5%, 11.8%) and Chitose in 27 herds (1.9%; 95% CI = 1.4%, 3.0%). One unique herd location (<0.1%) had both Chitose and Ikeda positive animals present at the same site. The three Buffeli samples were all collected at the same market on the same date, but the herd locations remained undetermined.   Ikeda positive herds were found in 52 (30.8%), Chitose positive herds in 20 (11.8%), with six (3.6%) counties having a mixture of herds positive for Ikeda or Chitose. Counties with cattle that tested positive for Ikeda were primarily found in the Appalachian region, running from northeast to southwest along the Interstate 81 corridor on the western side of Virginia. The ecological niche for the Ikeda genotype, developed from locations of individual positive and negative herds, was predominantly in areas with lower forest density that were located in regions with cooler/drier summers ( Figure 2, Table S1, Figure S1). Counties with cattle that tested positive for Chitose were primarily concentrated in southcentral and southeast Virginia. The ecological niche for the Chitose genotype, based on locations of individual positive and negative herds, covered lower elevation areas of low forest-herbaceous edgedensity, mainly contiguous agricultural fields ( Figure 3, Table S1, Figure S2). The countylevel distribution of the Ikeda and Chitose genotypes showed significantly less overlap than expected by chance (p < 0.001) and the ecological niche patterns were dissimilar (p < 0.05).        cattle markets and also were more likely to be adjacent to other positive counties. This relationship became stronger when the counties had similar proportions of forested area and the cool/dry summers characterized by the ecological niche profile (Figure 4a). The strongest association was with a contribution of 34.5% for market movements, 22% for adjacency, and 43.5% for niche similarity. Counties with at least one Chitose positive herd had similar proportions of the lower elevation, agricultural field ecological niche, with connectivity through cattle markets and local flows also statistically significant (Figure 4b).
The strongest relationship was with a contribution of 62.5% for niche similarity, 22.5% for market movements, and 15% for local movements.
Counties were extensively linked through shared cattle markets ( Figure S3), wi each county connected to a median of 40 other counties with a range of 7 to 113 links p county. Weights between pairs of counties ranged from 0 to 832, with a median of 0, sin only 28% of county-pairs were connected through cattle markets. Counties with at lea one Ikeda positive herd had significantly greater connectivity with other positive counti through cattle markets and also were more likely to be adjacent to other positive countie This relationship became stronger when the counties had similar proportions of forest area and the cool/dry summers characterized by the ecological niche profile (Figure 4 The strongest association was with a contribution of 34.5% for market movements, 22 for adjacency, and 43.5% for niche similarity. Counties with at least one Chitose positi herd had similar proportions of the lower elevation, agricultural field ecological nich with connectivity through cattle markets and local flows also statistically significant (Fi ure 4b). The strongest relationship was with a contribution of 62.5% for niche similarit 22.5% for market movements, and 15% for local movements.  . The color at each point represents the join count statistic, measuring the spatial autocorrelation for the combination of factors. The brightest peaks represent the optimal contribution that best explained the observed spatial association for each genotype. The number of animals that tested positive for T. orientalis genotypes was small, limiting our power to assess demographic differences. Although beef cattle made up the majority of animals testing positive for Ikeda, prevalence was higher among dairy cows, approaching significance (p = 0.06; Table 1). Among individual breeds, prevalence was significantly higher in cross-bred cattle than among Angus (p < 0.01), with increased prevalence in Holstein cattle approaching significance (p = 0.09). With only 36 positive Chitose results, we lacked power to detect demographic differences, however the direction of the prevalence relationships tended to be opposite of those seen with Ikeda. In fall 2018, at the start of the study, the Ikeda genotype was detected in northern markets and the Chitose genotype in northern and central markets. Ikeda and Chitose were detected in fall 2019 in the southwestern markets after sampling began there in January 2019. Temporal prevalence patterns differed between Ikeda and Chitose and between market regions of Virginia. In the northern markets, the prevalence of the Ikeda genotype increased each month (Figure 5a) by about 14% of the previous month (OR = 1.14; 95% CI = 1.10, 1.19). In the southwestern markets (Figure 5b), Ikeda prevalence also increased about 14% each month from August 2018 through April 2020 (OR = 1.14; 95% CI = 1.02, 1.26), followed by a sharper increase of about 38% compared to the previous month from April 2020 through December 2020 (OR = 1.38; 95% CI = 1.32, 1.83). For the Chitose genotype, there were only sporadic positives in the northern and southwestern regions with no consistent trends. For the central group of markets, there were no significant trends for either genotype (Figure 5c).

Discussion
We investigated the occurrence and distribution of T. orientalis, an emerging cause of bovine infectious anemia, in samples from cattle marketed across Virginia. We found the Ikeda, Chitose, and Buffeli genotypes in Virginia and neighboring states with Ikeda in 8.7% of our samples and Chitose in just under 2%. No cattle were co-infected with more than one genotype in our study. The prevalence of the Ikeda genotype increased over time in northern and southwestern Virginia cattle markets. The Ikeda and Chitose genotypes occurred in different regions, with little overlap, and with disparate environmental predictors. Characteristics of infected cattle also varied for the two genotypes. Despite these differences, connectivity based on cattle movements, through markets and locally, and on environmental factors contributed to the distribution of T. orientalis, for both genotypes.
T. orientalis Ikeda was first recognized in the USA as a clinical outbreak in a beef herd in west-central Virginia in fall 2017 [3]. Our detection of both the Ikeda and Chitose genotypes from multiple cattle across Virginia in fall 2018 and the temporal patterns in market groups show that both genotypes are established, and that Ikeda is spreading. This increasing presence of a new pathogen may not have been recognized in other herds, as asymptomatic infection is common [4] and anaplasmosis, which has similar clinical signs, is also present in Virginia [46]. In New Zealand, T. orientalis Ikeda was first detected in spring 2012 and spread rapidly across much of the northern island within two years [47]. In Australia, the Ikeda genotype was initially detected in 2006 and spread rapidly across most states [48].

Discussion
We investigated the occurrence and distribution of T. orientalis, an emerging cause of bovine infectious anemia, in samples from cattle marketed across Virginia. We found the Ikeda, Chitose, and Buffeli genotypes in Virginia and neighboring states with Ikeda in 8.7% of our samples and Chitose in just under 2%. No cattle were co-infected with more than one genotype in our study. The prevalence of the Ikeda genotype increased over time in northern and southwestern Virginia cattle markets. The Ikeda and Chitose genotypes occurred in different regions, with little overlap, and with disparate environmental predictors. Characteristics of infected cattle also varied for the two genotypes. Despite these differences, connectivity based on cattle movements, through markets and locally, and on environmental factors contributed to the distribution of T. orientalis, for both genotypes.
T. orientalis Ikeda was first recognized in the USA as a clinical outbreak in a beef herd in west-central Virginia in fall 2017 [3]. Our detection of both the Ikeda and Chitose genotypes from multiple cattle across Virginia in fall 2018 and the temporal patterns in market groups show that both genotypes are established, and that Ikeda is spreading. This increasing presence of a new pathogen may not have been recognized in other herds, as asymptomatic infection is common [4] and anaplasmosis, which has similar clinical signs, is also present in Virginia [46]. In New Zealand, T. orientalis Ikeda was first detected in spring 2012 and spread rapidly across much of the northern island within two years [47]. In Australia, the Ikeda genotype was initially detected in 2006 and spread rapidly across most states [48].
The Ikeda genotype is found in highest cattle density areas in Virginia, composed mainly of cow-calf operations but also including those counties with the highest numbers of dairy cows [49]. Scotch-Irish and German settlers in this region historically purchased and fattened cattle, providing beef for northern markets through cattle drives to Baltimore and Philadelphia [50]. The steep, lush pastures and limited workforce led to similar management practices across this region of Appalachia [51]. This continuing practice of buying and assembling cattle to feed and resell later can amplify disease spread. The strong association of Ikeda with cattle movements, through cattle markets or county adjacency, matches dissemination in New Zealand, where both long distance and local cattle translocations were important [47]. One advantage of testing cattle moving through the market system, as in our study, is that these represent animals with potential to enter a new herd. In this region, bulls are generally also moved into cow herds for the breeding season, providing another possible route of spread through cattle movement.
Although counties with Ikeda positive cattle were significantly linked through cattle movements, these associations were strongest for counties sharing similarity in the forest dense, cooler/drier summer niche. The importance of both movements and environmental features has been shown for T. orientalis in New Zealand and Australia [2,47] as well as for other vector-borne diseases of cattle [44,52]. The direct effect of climate should be limited on the parasite itself, which can spend the entirety of its lifecycle within a host and, potentially, within a vector. While the ecological niche could reflect locations favorable for cattle rearing, we contrasted locations of Ikeda positive and negative herds to control for overall effects of cattle distribution in environmental factor selection. These niches could reflect locational suitability for other important hosts and/or vectors within the pasture-based management in this region. H. longicornis has been collected from Virginia animals with theileriosis [20] and is a demonstrated vector of the Ikeda genotype, both internationally and using Virginian strains of both tick and parasite [4]. Before the start of our survey in 2018, the tick had already been passively reported from 20 counties along Virginia's northernmost to southwestern border and is currently found in 38 counties [19], mainly in the same western region as the Ikeda positive animals described in this study. In New Zealand, the most rapid dissemination of the Ikeda genotype was in areas where tick vectors were already established. Recently, Cumbie et al. [53], in a longitudinal study of H. longicornis at sites in central Appalachian Virginia, found forested habitats, temperature, and precipitation important predictors of tick abundance, aligning with our associations for the presence of Ikeda infections in cattle. Since pasture grazing still predominates in this region, even for certain production intervals for dairy cattle, opportunities for exposure to vectors and other cattle are abundant.
Though Chitose commonly co-occurs with Ikeda in other countries [54], we found no co-infected animals in our study. The Chitose genotype was first identified in Virginia through our study, so its historical distribution in the US is unknown. In New Zealand, Chitose was recognized as causing mild disease in 1982 with Ikeda likely introduced decades later, but in the same regions [47]. In Australia, one of the two genetic clusters of Chitose (B) was reported more often alone, while in Queensland, a second cluster (A) was more commonly found in cases of co-infection after the introduction of Ikeda [55]. The patterns in the US may also represent separate introductions of Chitose and Ikeda at disparate locations and times.
Chitose genotype clustered among counties that were significantly connected with each other through cattle movements but separate from the Ikeda counties. Cattle in southcentral Virginia were introduced by English settlers from the coast [50] and remain at a lower density, separated by mountain ridges and limited transportation routes from the Ikeda-rich Appalachian region [49]. This may have slowed dissemination of this genotype and may also account for the trend toward different breed associations than those found for Ikeda.
Overall, the Chitose genotype was less prevalent than Ikeda within Virginia market cattle. H. longicornis has been shown to have lower vector competence for Chitose than for Ikeda in mixed infections [55], which could lead to lower prevalence. Alternatively, since the Chitose distribution and the ecological niche factors, specifically lower elevation and more contiguous agricultural fields, did not overlap with those reported for H. longicornis, this may indicate a different vector or different means of transmission for Chitose. Other Theileria species are spread by Amblyomma, Dermacentor, and Rhipicephalus species, including those found in Virginia: A. americanum, D. variabilis, and R. sanguineous [56]. Although only studied with Ikeda, transmission directly using blood was slower and less effective than tick transmission [4], which could also account for lower prevalence in areas with fewer tick vectors.
Although this study provides important, initial information about the distribution of T. orientalis genotypes in this region, the limited number of cattle sampled and the selection of one animal from only up to five lots at a sale makes it likely that the breadth of occurrence was underestimated. However, most studies of emerging animal diseases are based on smaller samples of clinical cases or within a few herds, so this large, geographically broad, surveillance study capturing more than 1300 herds shows the importance and wide distribution of this recently recognized infection. Virginia dairy cattle were also underrepresented at market sales but those sampled tended toward having a higher prevalence, so this production system should have further study. If infection was associated with loss of condition, abortion, or calf loss, prevalence could be higher in culled market cows than those remaining in herds. Additionally, detection of Theileria in healthy US market cattle could represent animals with recent infections, with mild disease, or recovered carriers, making the temporal pattern for onset of individual, herd, or regional spread unknown. Study of clinical cases and outbreaks of theileriosis and surveys of veterinarians and producers about knowledge, occurrence, and management practices across affected and unaffected regions of Virginia are needed to better understand and manage this disease.
This study provides an initial description of occurrence patterns of T. orientalis genotypes Ikeda and Chitose in Virginia and neighboring states. Surveillance for an introduced livestock disease is challenging because the prevalence and determinants of distribution may differ from those in other parts of the world. These genotypes, especially Ikeda, have significant negative economic and welfare effects on cattle internationally and this information will help with development of management strategies for the US. Availability of a duplex test for A. marginale and multiplex test for T. orientalis genotypes [20] provides an important tool for detecting, differentiating, and managing both diseases Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens11111353/s1, Table S1: Response curves for the environmental factors relating the presence and absence of the Ikeda genotype in the generalized additive model; Figure S1: Response curves for the final variables in the generalized additive model relating the presence and absence of the Ikeda genotype to environmental factors; Figure S2: Response curves for the environmental factors relating the presence and absence of the Chitose genotype in the generalized additive model; Figure S3

Institutional Review Board Statement:
There were no human subjects in this research. The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Tech (protocols 18-143 and 21-248, approved 12/2018).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are not publicly available due to the sensitive nature of the disease diagnoses. Even though the dataset has limited identifiers, we believe that there remains the possibility of deductive disclosure of herds due to market and county locators and some breed characteristics. Portions of the data can be shared through a data use agreement.