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Blood Protozoan Disease Theileriosis: Causes and Prevalence

The genesis of the problem has been the introduction of the 6 cows from Rajasthan, a hot and dry state of India in the Holstein cross bred herd in the Graphic Era University dairy at Dehradun capital of hill state of India. Suddenly after introduction of these Sindhi cows there was death of death of 8 cross breed cows in 2010. In spite all possible veterinary treatments. However we have examined the blood samples of these animals which were found positive for Theileria and the same was confirmed by Indian Veterinary Research Institute (IVRI) thus we have reported Theileria for the first time from the hilly region of Uttarakhand [39]. The native cows did not suffer of Theileria but crosses of Holstein do suffer and die.
Twenty year back in India the cow producing over 1 gallon of milk was considered to be high producing cows. Since 1971, the cross breeding with high producing cows of European and North American horizon has introduced. India’s cow population is 200 million out of this population 20 percent is cross bred, say 40 million. These cross bred produce over 15 litres of milk. Hence our milk production has increased but the introduction of cross bred animals has increased the vulnerability of herds to tick borne diseases (TBDs). The most common TBDs in India are tropical theileriosis, babesiosis and anaplasmosis. The most detrimental blood protozoan is Theileria. Our native cows have not been susceptible to Babesia and Theileria [12, 370, 371]. However the cross bred cows has been in danger. Theileria are obligate intracellular protozoan parasites that cause theileriosis. The babesia affects only adult animals and occurrence of Babesia is marked by red colour urine. So it has been diagnosed easily. The drug for treatment of Babesia is cheap and easily available in the market and animal can be easily saved. The Theileria causes very great damage to cross bred cows because it affects all ages of the cattle and it is transmitted in calves from mother. The tick positive with the Theileria has to be attached for 48-72 hours which has to be infected.
The Theileria protozoan affects the exotic animals but not the native animals. Exotic cattle (Bos taurus) are particularly susceptible with mortalities up to 40–80% in some areas, whereas indigenous cattle (B. indicus) generally suffer much lower mortalities (about 10%) confined mainly to calves [57]. This is the same as Negro has not affected by malaria where European has affected by malaria. Plasmodium falciparum, has inhibited in glucose six phosphate deanhydrose (G6PD)-deficient erythrocytes the parasite has very sensitive to oxidative damage and has killed at oxidative stress level that doesn’t affects the G6PD-deficient human (Negro) host. By this human population with G6PD-deficient genotype survives in malaria prevalent ares. The same is true about Theileria, exotic cow suffers due to Theileria but native do not.
The theileriosis has not been fatal in subclinical cases. It lowers production and reproduction this is because the tropical theileriosis resulting from the infection with the intracellular protozoan parasite Theileria annulata [379] has considered as aprogressive lymphoproliferative disease which imposes heavy losses due to decreased productivity and considerable mortality in cattle [380]. After the invasion of the parasites into lymphoid cells, they inrush the erythrocytes to complete part of their life cycle [364], whereby the occurrence of mild to severe anaemia would be inevitable [381]. Some recent studies have declared that the anaemia is probably a consequence of the oxidative damage in erythrocytes [382, 383] and indicated significant modulations in the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidise (GPX) [384] and catalase [385]. Furthermore, some studies suggested that erythrocytes destruction during oxidative stress has related to membrane lipid peroxidation [385]. This process might cause morphological changes in the cell surface and cellular osmotic fragility, whereby an increase in the erythrocytes susceptibility to phagocytosis would be predictable.
Dairy with Holstein cows has become a big enterprise but theileriosis have extensive prevalence and high mortality rates due to these cause economic losses, as in several countries [386]. We have reported incidence of theileriosis in the hot and humid month this is in agreement with other workers. The highest abundance of the ticks was reported in the month of July [239] whereas Hyalomma sp. of ticks is most abundant in June [369].
India is a vast country where tropical and Himalayan regions both present. Theileria has reported from many tropical regions of India but there has no report of theileriosis from the hilly states in India. However, we have reported theileriosis from sub Himalayan, sub tropical region. India theileriosis has been reported from Punjab, Haryana, Gujarat etc. geographical regions. In Bangalore north the occurrence of T. annulata among crossbred cattle has reported in 2009 [365]. In Northern Kerala 16 % positive cases of theileriosis has reported in crossbred cattle [222]. 37% cattle reported positive for the haemoprotozoan infection in Kaira and Anand District of Gujarat [387]. They have also reported that the higher incidence of Theileria has found in monsoon season. A case of tropical theileriosis has also reported from West Bengal in 2012 [223]. An outbreak of theileriosis in cattle has reported from Punjab with 4.86% mortality rate [388].
The cross bred animals are most sensitive to heat [389]. They further stated that the climate temperature control the activity of both Theileria as well as the vector of this protozoan disease. In indigenous cattle no extra care is needed to control tick infestation. Mortality is usually low or insignificant due to tick-borne diseases in indigenous cattle because they are resistant to ticks responsible for transmission of paraites [390, 391, 392, 393]. The 0% mortality and low incidence of theileriosis in Sahiwal suggests that this breed exhibits a high level of resistance to ticks and ultimately to tick transmitted diseases.
We have found thermal humidity index (THI) for the 2 consecutive years. Surprisingly in this doing so we have found out that the milk production in cows which are low producers (15 lt). The cross bred animals are most sensitive to heat [389]. In this connection we have measured the thyroid hormones also which was found very high during winter months and low in summer months.
In our study we included. 40 HF cross bred cows for the period of 10 months i.e. from June to March. The 40 lactating HF cows have assigned to 2 groups according to milk production i.e. high milk producers and low milk producers. As THI increases milk yield decreases. This decrease in the milk production can range from 35-40%. In the high yielding cows once the milk production decline it has not been rise and there has been loss of 8-10 litre milk everyday and this has irreversible loss. In the low yielding cows producing less than 8 or 10 litre of milk, they has not much affected by increase in thermal humidity index or stress level.
We carried out our study in cross bred cattle in Dehradun district, Uttarakhand during summer, rainy, winter and spring season. Since then we have been diagnosing Theileria all around the Dehradun, the capital of Uttarakhand. Microscopically we have examined a total of 301 cattle by using Giemsa’s stained blood smear method. Giemsa’s-stained blood films contained Theileria piroplasms, including cocci, rod, stick, comma, fusiform, racquet-shaped, signet-ring, and pear-shaped forms with diameter of 0.5-1.5 micrometer. The ring form has found to be the most common in present study. Microscopic examination of blood smears have revealed 27.2% (82) overall prevalence of theileriosis. Season wise 9.0% cows have found positive for Theileria as per blood smear in spring season, 19.6% have found positive in summer season, 45.4% have found positive in rainy season and 8.8% have found positive in winter season. Highest numbers of positive cases have obtained in rainy season which corresponds to months between July and October when the THI has also found high i.e. above 80. Similar observations have observed in previous reports [365, 366]. High incidences of tropical theileriosis in cross bred cattle were found during summer and monsoon season [367]. The result coincides with the study conducted in Zimbabwe and Rhodesia [30]. They found that peak activity of tick occurred in humid season and the seasonal activity has greatly influenced by temperature, humidity and day length. Similar observations have made by Flach who studied the epidemiology of tropical theileriosis in an endemic area of Morocco [100]. He reported adult Hyalomma detritum detritum peak numbers at the end of June. The activity period of Hyalomma detritum detritum was between September and early December and the highest numbers of vector ticks were found in late October. The data collected indicated that H. detritum detritum delays either egg laying in summer or larval host searching in autumn.
There is a new biotechnology innovation which has been recently come. This innovation is outcome of recognition of endonuclease by Hamilton smith in 1971 and recombinant technology initiated by Herbert Boyer and Stanley Cohen in 1973 and polymerase chain reaction by Kary Mullis in 1983. The enzyme linked immunoassay for antibody in immunodiagnosis was tool of diagnosis in sick animals. The antibodies are not 100% correct. Because the antibody cross react. Sample size should be large. The DNA does not cross react. Small sample size required. In nut shell the results are quick and reproducible. However it is bit expensive.
Besides 82 positive samples, we found that 16 other samples were also detected as positive for the disease in the polymerase chain reaction test. These cattle were categorized as “carrier cattle”. So Theileria genus specific PCR could detect 98 samples (32.5 %) as positive. Thus it has confirmed that PCR test is more sensitive in detecting low grade of infections in carrier animals and hence is more suitable for epidemiological surveys as compared with microscopic blood and lymph node smear examination. Similar observations were made that carriers are important contributors to the infection within Hyalomma ticks [36]. Detection of carrier animals is very important to control the spread of infection. However, due to low number of infected erythrocytes in carriers it is difficult to identify them by Giemsa staining of blood smears. The efficacy of PCR was also reported which concluded PCR as a versatile method for the identification of multiple tick-borne infections in cattle [139]. Similar observations were reported by Aktas in their survey to check the prevalence of Theileria by PCR conducted in eastern Turkey [162]. They reported that out of 252 blood samples examined, 41 (16%) have positive for piroplasms by microscopy, whereas 114 (45%) have positive for the presence of at least one species of Theileria by PCR. The percentages of positive animals for Theileria annulata and benign Theileria species (Theileria sergenti/buffeli/orientalis) have 39% (99/252) and 7% (18/252), respectively. The sensitivity and specificity of PCR test has also confirmed [152, 155, 171, 177].
The specificity of the primer set N516/N517 is confirmed by PCR test. Expected band was generated on agarose gel of size 721-bp and it was confirmed by running 100 bp ladder DNA alongwith sample. Theileria-specific small subunit rRNA primers 989 and 990 have also used and expected 1098 bp fragment of DNA was amplified. These findings were in accordance to Allsopp who differentiate six species of Theileria by PCR test [26]. The expected fragment of size 721- bp has been shown on agarose gel by electrophoresis which corresponds to 19 parasites per ml. These findings were in accordance to Roy who also detected Theileria annulata carrier cattle by polymerase chain reaction (PCR).
Some cattle after treatment in early stage of the infection become immune carriers. No schizonts were found in the lymph node of the carrier but few Theileria piroplasms still resides in erythrocytes. Therefore, such carrier animals have been not detected by microscopic examination as well as serological tests. All the infected animals during field and experimental study showed clinical manifestations ranging from mild to severe reactions. The clinical signs included high fever, swelling of submandibular and sub scapular lymph nodes, weakness, increased respiration and pulse. Anorexia, anaemia and loss of condition also occurred. The first symptom of fever was manifested by the animal in theileriosis [368]. The clinical signs recorded in the present study were also reported [6]. Similar findings were reported Theileria annulata infection in cross bred animals at Faisalabad with clinical signs of enlarged lymph nodes and spleen, oedema of lungs and hemorrhages in the abomasums [12]. Swelling of lymph nodes was the first sign in all calves along with lacrimal and nasal secretions [113]. This study also reported that initially animals were constipated and had mucus covered feaces, this has followed by diarrhoea which resulted in emaciation and weakness. In the last stage of the disease calves showed laboured respiration, recombancy, depression and all infected animals died.
Today because of the availability of the sexed semen and environmentally controlled housing the dairying has become big enterprise with big profit. However the problem of Theileria remains to be solved. The Theileria can be diagnosed microscopically in clinical cases. However, the subclinical cases, which has not been apparently sick and cannot be diagnosed microscopically. They need to be diagnosed by the Polymerase Chain Reaction.
Vaccines for this disease are available but that has to be kept in liquid nitrogen and -197 degree Centrigrade and whole lot of 300 doses has to be purchased which has been available at around rupees one lakh. .
It has reported recently even in the sub clinical cases if vaccination is done then Theileria flares up plus those animals which has vaccinated always found positive microscopically (personal communication with Dr. Rajat Garg of Indian Research Institute).
In this connection we have diagnosed Theileria microscopically by which only 50% of the cases were diagnosed. The present study has been focused on blood protozoan disease theileriosis and its prevalence as well as finding out the “carriers”. This study was carried out for ten months (March to December) and the cases positive for theileriosis showed that there was a high prevalence of these haemoprotozoan, especially in the rainy season, in Dehradun district. This is in agreement with previous research [394, 395]. PCR has allowed the development of sensitive and specific diagnostic assays for Theileria [35]. It is also reflected from our study that the entire positively stained samples were confirmed positive by PCR but an additional 16 samples were also detected as positive (“carriers”). As Dehradun is a hilly region, so it was thought that the occurrence of these haemoprotozoan is very less because of the temperate climate. But our study suggests that this problem is spreading in Dehradun area and also in areas located in middle Himalaya region (especially the valleys in central Himalaya) where farmers have introduced exotic cattle breed as an attempt to increase their dairy yields. So it is suggested that essential screening should be done before introduction of the cross bred cows.

Intermittent Fasting Benefits and Risks

So far, I have shared with you my own story, and details of the success that I achieved personally through a program of intermittent fasting. I hope that you have found my story interesting, perhaps even inspiring, and that you feel positive towards introducing a regime of intermittent fasting in your own life.
However, you may be wondering whether one person’s achievement can translate into success in other people – all with different bodies, lifestyles, and attitudes. And, you would be right to wonder. It is important before you embark on such a regime that you have confidence in the system, and that you believe it will work for you. To address this concern, I can offer two thoughts. Firstly, not only have I benefited through this regime myself, I have also helped hundreds of direct clients, and many thousands of readers of my blog, all achieve their best condition ever. This is a program that works for all types of body. Secondly, and just as importantly, the program of intermittent fasting that I describe in this book is supported by compelling, well-conducted, and published scientific evidence.
Intermittent fasting is not a fad or a craze dreamt up without basis in fact, but a regime that is founded on hard science. The purpose of the following chapter is to present and summarize the scientific literature that is available regarding the benefits of intermittent fasting, and to allow you to draw your own conclusions. I feel strongly that it is important for you to be satisfied with the science and convinced by the data before you begin your own regime.
The studies discussed in the sections below have been identified by a comprehensive search of the literature. They are not studies that have been selected because they fit a particular thesis. The full reports of many of these studies are freely available online to any reader with an interest in exploring the subject further. If the full paper is not available free of charge, a summary (or “abstract”) of the paper almost certainly will be. The most convenient way to access the abstract (or in some cases, full paper) is through the PubMed interface to the Medline database, available at
Health benefits of intermittent fasting A significant proportion of people who embark on a program of intermittent fasting undoubtedly do so in order to achieve a reduction in body weight. And, this is a perfectly reasonable goal in its own right. However, you will find as you progress with your regime that a number of other health benefits become apparent. For example, you may find that you have more energy, that you suffer fewer coughs and colds, and that you feel generally in a better state of health. There are a number of further advantages to this program which may not be immediately noticeable but which will improve your underlying health in general. For example, data show that intermittent fasting has positive effects in terms of cardiovascular health, neurological function, protection against disease, insulin sensitivity, and hormonal responses. The scientific evidence to support the benefits of intermittent fasting in these different areas is discussed over the following sections.
Body weight A number of clinical research studies have evaluated the effects of different regimes of intermittent fasting on body weight.
In one recent study (2009) investigating a regime similar to the one that I follow (1), subjects were asked to observe a 12-14 hour fast every day. In addition, participants were required to perform prolonged, moderate-intensity aerobic exercise during this fasting period, on at least three to five occasions each week. No restrictions were placed on total daily calorie consumption. During the 12-week study, the average fat loss in the 27 participants was an impressive 7.4 kg, equivalent to one-quarter of the fat mass at baseline. Importantly, the rate of fat loss was at least as great in the second six weeks as in the first, suggesting that fat loss might have persisted for some time if the study had run over a longer period.
A slightly earlier study showed a similar benefit in terms of weight loss through a related regime of alternate-day fasting (2). In this study, 16 non-obese men and women who fasted every other day for 22 days lost on average a total of 2.5% (±0.5%) of their initial body weight and 4% (±1%) of their initial fat mass. These findings are even more impressive when you consider that this group of participants was not particularly overweight to begin with. Furthermore, improvements in fat oxidation were reported over the duration of the study. However, although this study demonstrates the benefits of intermittent fasting, the particular regime followed, alternate-day fasting, does not appear optimal. In particular, an increased hunger was reported on the first day, which did not decline throughout the remainder of the study. This observation suggests that a regime of alternate-day fasting may lead to problems of compliance.
A third study was performed to investigate whether an intermittent, very low calorie diet improves weight loss more than caloric restriction alone in diabetic patients or overweight individuals (3). This study found that subjects who followed a very low calorie diet for five consecutive days every five weeks lost more weight than those participants who received a standard reduced-calorie (1,500-1,800 kcal/day) diet continuously for the same duration. The intermittent group also revealed improvements in markers of diabetes, notably glycosylated hemoglobin (HbA1c) levels, compared with the constant calorie intake group.
One objection that is sometimes raised concerning intermittent fasting is whether the body is physically capable of withstanding the effects of fasting, or whether such a regime could actually cause damage to the systems of the body. For obvious reasons, very few studies investigating the effects of prolonged fasting have been conducted. However, one study in a group of eight hunger strikers who refused food for 43 days revealed a decrease in body fat of approximately 60% and a reduction in body mass index (BMI) of 18% (4). By the end of the starvation period, BMI (21.5 ±2.6 kg/m2) and body composition measurements were still within acceptable limits, suggesting that the body had been surprisingly efficient in conserving muscle mass. The researchers concluded that a regime of complete food withdrawal had a far greater impact on body fat than on muscle mass, and that patients were not morphologically malnourished after 43 days of fasting. From this extreme example we can be confident that fasting for a relatively short duration every day offers no threat to physiological safety.
From the results of the studies discussed above, it becomes apparent that intermittent fasting offers significant benefits in terms of weight reduction and fat loss and that a regime involving of daily periods of fasting appears superior to alternate-day fasting in terms of reduced sensations of hunger and consequent compliance.
Health markers As well as a clear, and expected, benefit in terms of body weight, intermittent fasting also offers advantages in relation to some of the underlying physiological processes that keep us healthy without our being aware of it. Although these benefits may not be so readily apparent as weight loss, they can be measured by following certain health “markers” that provide an insight into the underlying processes. The effects of intermittent fasting on lipid profiles and inflammatory parameters, two examples of health markers, will be discussed in detail below.
Lipid profile “Hyperlipidemia” is a condition in which excessive quantities of fatty substances known as “lipids” are present in the blood. Lipids, including cholesterol, triglycerides, cholesterol esters, and phospholipids, are transported in the blood as part of large molecules called “lipoproteins”. The blood concentration of the different lipoproteins is collectively described as the “lipid profile”.
The presence of hyperlipidemia places you at greater risk of “atherosclerosis”, a condition in which fatty deposits accumulate on the inside of your blood vessels. There is overwhelming evidence to demonstrate that atherosclerosis increases your risk of heart disease, stroke, high blood pressure (“hypertension”) and other problems.
Hyperlipidemia can be controlled to a large degree through diet and exercise. The greatest effects result from reducing intake of saturated and trans fats; increasing intake of polyunsaturated and monounsaturated fats; fortifying foods with plant stanols or sterols; and adopting a Mediterranean, low-carbohydrate, or low-fat diet (5). However, there is also growing evidence to suggest that your lipid profile can be affected not only by what you eat, but also when you eat.
Evidence that a program of intermittent fasting can improve lipid profiles has been found in animal studies (6). For example, in one study of 144 mice, triglyceride levels were found to decrease significantly in mice that were fed hunger-resistant food on alternate days compared with those mice who were allowed to eat ad libitum (7). This is an important observation as it demonstrates the effect of an eating schedule on lipid levels. However, while I am sure that such a finding is of major interest to the academic community, I am equally confident that you would be more convinced by a demonstration of a similar benefit in humans.
Interestingly, a number of studies conducted in humans also show a benefit of intermittent fasting in terms of lipid profiles. Several of these studies have been conducted among Muslims observing the period of Ramadan (8). As we saw during Chapter 1, Ramadan is a time of religious observance during which Muslims abstain from eating and drinking from sunrise until sunset. The fasting period during Ramadan is approximately 13-16 hours, depending on the time at which participants stop eating. Although drinking is prohibited during the fasting periods of Ramadan (an approach which is certainly not advocated by proponents of intermittent fasting), Ramadan fasting may be considered a good model for the approach discussed in this book. In particular, the length and frequency of the fasting periods during Ramadan is very close to the regime I follow myself.
A comprehensive review of all studies cataloged in Medline [1] , three international congresses on health and Ramadan, and several cases from local journals (8), concludes overall that Ramadan-style fasting results in an improvement in lipid profiles that could be beneficial for the cardiovascular system. In particular, the review finds that levels of “good” cholesterol, known as “high-density lipoprotein” (or “HDL”) increase while levels of “bad” cholesterol, known as “low-density lipoprotein” (or “LDL”) decrease as a result of fasting during Ramadan.
To further investigate the benefits of Ramadan-style fasting on lipid profiles, I will briefly consider three studies conducted in healthy observers of Ramadan. As mentioned in Chapter 1, these studies are observational in nature rather than strictly controlled, as the researchers do not intervene in the dietary practices and not all of the studies involve a comparator group. However, there is still considerable useful information to be gained from studying Ramadan in this way.
The first of these studies involved 40 healthy male and female observers of normal weight who fasted during Ramadan and another 28 healthy volunteers who did not fast (but who were age- and BMI-matched) (9). This study found that the ratio of total cholesterol (TC) to HDL cholesterol was decreased during and after Ramadan in both men and women who fasted, while no such changes were observed in the non-fasting group. In the second of the Ramadan studies (10), conducted in 50 healthy subjects, the ratio of LDL cholesterol (“bad” cholesterol) to HDL cholesterol (“good” cholesterol) ratio was significantly decreased during the month of Ramadan (p<0.05). Finally, in a third study investigating the effects of Ramadan-style fasting in healthy observers (11), HDL levels were significantly elevated during Ramadan (p<0.001) and 20 days after Ramadan (p<0.05). The findings of these three studies suggest that a 13-16-hour period of fasting every day for a prolonged period causes beneficial changes in lipid profiles which predispose to an improved cardiovascular risk profile.
Another interesting study of Ramadan-type fasting was conducted in 64 religious observers who had previously been diagnosed with diabetes or hyperlipidemia (12). This is an important area in relation to intermittent fasting because individuals with nutritional disorders of this type are normally required to comply strictly with advice on the timing and composition of food. Indeed, it could be anticipated that prolonged daylight fasting during the month of Ramadan may even produce undesirable biochemical consequences in these individuals. In the hyperlipidemic subjects without diabetes, intermittent fasting had no detrimental effect on triglyceride, TC and LDL levels, contrary to some expectations. In diabetic patients, TC levels increased somewhat following Ramadan, but not by a significant amount. It would be interesting to investigate the effects of intermittent fasting in diabetic patients further, but certainly in patients with hyperlipidemia, the findings of this study suggest that intermittent fasting may improve lipid profile and therefore favorably influence risk of coronary heart disease.
One final study to be considered in relation to lipid profiles was conducted in patients with asthma (13). The regime followed in this study was an 8-week program of alternate day calorie restriction, in which overweight subjects could eat ad libitum on alternate days while consuming less than 20% of their normal calorie intake on the intervening days. Although this scheme differs from the regime that I follow personally, it nevertheless provides us with a great deal of useful information. This study will be discussed again at greater length later in this chapter, particularly in relation to the observed benefits in terms of asthma symptoms. However, among the many interesting findings reported, the researchers found that those subjects who adhered to the diet not only lost an average of 8% of their initial body weight, but also presented with decreased levels of serum cholesterol and triglycerides at the end of the study.
The various studies discussed above would seem to indicate strongly that a regime of intermittent fasting achieves a beneficial effect in terms of lipid profiles, and resulting reduction in risk of consequences of atherosclerosis, including heart disease, stroke, hypertension and other conditions. This seems to be a very powerful argument for the introduction of a regime of intermittent fasting, in addition to the more obvious benefits of weight loss.
Inflammatory markers Most people associate inflammation with a condition that affects the joints and connective tissues of the body. Perhaps rheumatoid arthritis comes to mind, or other diseases of that type. However, recent studies have linked inflammation of the arteries to heart attacks and strokes. Indeed, in perhaps up to 50% of cases of heart attack and stroke, patients do not present with the classic risk factors such as hypertension or raised lipids. Research is beginning to suggest that in these cases the underlying cause may be inflammation of the arteries (14). Inflammation of the arteries can be measured using inflammatory markers, including soluble tumor necrosis factor (TNF-α), C-reactive protein, and interleukin-6.
Once again, a number of studies have investigated the potential benefit of intermittent fasting on inflammatory markers. In a study described in the previous section, the ratio of total cholesterol to HDL cholesterol was found to decrease in 40 healthy male and female observers of Ramadan (9). However, this study also measured the effects of Ramadan fasting on serum interleukin-6, and C-reactive protein levels and found that both were significantly lower (p < 0.001) during Ramadan in the fasting subjects of both genders when compared to basal values (one week before Ramadan).