J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 1 Original Article Natural Babesia bovis Infection in Water Buffaloes (Bubalus bubalis) and Crossbred Cattle under Field Conditions in Egypt: a Preliminary Study Yasser Mahmmod 1, 2 1Department of Large Animal Sciences, Faculty of Health and Medical Sciences, Copenhagen University, Copenhagen, Denmark 2Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt (Received 13 Nov 2012; accepted 27 July 2013) Abstract Background: There is a little or no data available on the natural Babesia bovis (B. bovis) infection in water buffaloes (Bubalus bubalis) comparing to the available one for cattle. This study was conducted to investigate the natural B. bovis infection in water buffaloes in comparison to crossbred cattle under field conditions in Egypt. Methods: A total of 35 buffaloes and cattle were clinically and laboratory investigated from March to June 2008. Twenty-nine buffaloes and cattle out of 35 were naturally infected with B. bovis and showed signs of bovine babesiosis. Three cows and three buffaloes showed no clinical signs and were free from external, internal, and blood parasites served as control group. Results: Babesia bovis-infected cattle showed typical signs of bovine babesiosis while B. bovis-infected buffaloes showed a milder form (less severe) of the clinical signs. Advanced cases of cattle showed dark brown to dark red (coffee-color) urine, hemoglobinuria and nervous manifestations while these manifestations were not detected in the infected buffaloes. Hematological changes in both species however, these changes were less significant in buffaloes than those reported in cattle. Conclusion: This paper documents the first description of natural B. bovis infection in water buffaloes which were found to be more likely to be tolerant than cattle to the natural clinical infection with B. bovis and its subsequent haematological changes. Our finding may lead to a better understanding of the disease pattern of B. bovis infection under field conditions in buffaloes. Keywords: Babesia bovis, Natural infection, Water buffaloes, Clinical signs, Hematology Introduction Bovine babesiosis is economically the most important tick-borne disease of cattle worldwide including areas of Australia, Af- rica, South and Central America (Bock et al. 2004). In addition, the United States is con- tinuously under threat of reintroduction of the vector and the disease (Bock et al. 2004). Under natural conditions, Babesia bovis trans- mitted by the tick Rhipicephalus microplus, although transmission may occur by other tick species (Papadopoulos et al. 1996). The life cycle of B. bovis has two phases. In the vertebrate host they multiply by merogony in erythrocytes while in ticks by sporogony (Susan and Asa 1999). The disease is the most prevalent in tropi- cal and subtropical countries, affecting cattle industries causing a major economic impact worldwide (Böse et al. 1995, Nayel et al. 2012). Costs due to babesiosis are the results of high mortality, ill-thrift, abortions, loss of milk/meat production and draft power and from control measures such as acaricide treat- ments, purchase of vaccines and therapeutics (Bock et al. 2004). It was estimated that losses and control of babesiosis and anaplasmosis *Corresponding author: Dr Yasser Mahmmod, E-mail: yasser@sund.ku.dk, yasserpcr@gmail.com http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 2 in Kenya, Zimbabwe, Tanzania, South Afri- ca, China, India, Indonesia and Philippines cost 5.1, 5.4, 6.8, 21.6, 19.4, 57.2, 3.1 and 0.6 million US dollars annually, respectively (Bock et al. 2004). Kaufmann (1996) reported that the mortality rates in cattle infected by B. bovis without treatment could reach 70– 80%. The diagnosis of ruminant babesiosis is generally based upon the microscopic ex- amination of Giemsa-stained blood smears and clinical signs in acute cases. Previous stud- ies provide information on the relative sus- ceptibility of various breeds of cattle to Babesia infection (Bock et al. 1997). In Egypt, bovine babesiosis is caused mainly by B. bigemina and B. bovis and con- sidered as the most important and endemic parasitic disease affecting cattle (Nagati 1947, Adham et al. 2009). Bovine babesiosis has a significant impact on meat and milk pro- duction and consequently, on livestock man- agement (Adham et al. 2009). The rapidly changing patterns of demand for cattle and its products point to cattle production being an important and increasing component of the Egyptian agriculture economy which re- quired improving cattle health. Egyptians farmers cross between the Holstein-Friesian breed and a native local breed known as Baladi cattle breed (Bos taurus) to improve production and disease resistance. Water buffaloes (Bubalus bubalis) repre- sent an important source of various human needs, such as meat, horns, hides, milk and milk products, leather, land plowing, and transportation of people and crops (Somparn et al. 2004). Due to the fact that water buf- faloes are raised together with cattle, among which bovine babesiosis is highly prevalent (Iseki et al. 2010), they might be potential carriers for Babesia parasites. Babesia bovis infection was experimentally investigated in splenectomised buffaloes (Mahmoud and Abou-Zeina 2008). Yao et al. (1997) reported the clinical findings on buffaloes after ex- perimental infection with cryopreserved B. bovis parasites. However, efforts to furnish information about natural infection with B. bovis in water buffaloes as well as crossbred cattle are necessary for better understanding of disease pattern under uncontrolled field conditions and subsequently, implementation the suitable policy for treatment and control. The objective of the present study was to investigate the natural B. bovis infection in water buffaloes in comparison to crossbred cattle under field conditions in Egypt. Materials and Methods Animals and sampling Blood samples were collected from the jugular vein into EDTA-containing tubes from 35 animals (23 cattle and 12 buffaloes) of both sexes and aged 2–5 years, and were orig- inating from different villages: El-Aslogy, Shobk Basta and Tel Basta around Zagazig city, Sharkia province. These animals were divided into 2 groups, the field-exposed (diseased) group comprised of 20 cattle and 9 water buffaloes which was examined at the Veterinary Teaching Hospital, Faculty of Vet- erinary Medicine, Zagazig University, during the period from March to June 2008 and resulted to have persistent fever, anemia and anorexia. The control group (3 cows and 3 buf- faloes) was carefully examined clinically and parasitologically and found healthy and free from external, internal, and hemoparasites. The common available foods for animals under the present study were mainly con- sisted of Barseem (Trifolium alexandrinum), rice or wheat straw and concentrate mixture (1–2 kg/head/day). Crossbred cattle were resulted from crossbreeding between the imported Holstein-Friesian breed cattle and Egyptian Baladi cattle breed (Bos taurus). Samples collection, handling and examina- tion of cattle and buffaloes under the current study were done after approval of animals’ owners. http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 3 Clinical examination Animals were subjected to clinical and he- matological examinations at Veterinary Teach- ing Hospital, Faculty of Veterinary Medi- cine, Zagazig University. The filed-exposed group showed various degrees of bovine babesiosis such as high fever (>40 °C), ano- rexia, hemoglobinuria (bloody urine), ane- mia, and jaundice. They were also infested with ticks to various degrees. The control group was examined thoroughly for presence of any abnormal clinical changes and exter- nal parasites, and was thoroughly examined by different laboratory techniques such as direct smear, flotation, sedimentation and Barmen’s techniques and blood film to con- firm the absence of any internal parasites and/or hemoparasites (Rosenberger 1990). Microscopic examination Thin blood films were prepared immedi- ately after taking the blood samples directly from the ear vein in the field to allow these smears to dry by air then fixed by using methanol for about 3–5min, allow them to dry by air after fixation step then stained with Giemsa stain diluted at 8% with dis- tilled water for about 30–45min. They were dried by air and examined on Olympus mi- croscope using oil immersion lens at x1000 magnification (Kelly 1984). Blood film was examined for B. bovis at 1/4–1/2 inch from the end of the film by visually scanning from one side of the film to other (cross–sectional method) to give constant and representative examination. Each blood film and at least twenty microscopic fields of each slide were examined twice before being considered negative. Hemogram Parameters Approximately 5ml of blood was taken from the jugular vein of all animals with a syringe containing EDTA. The blood sam- ples were subjected to hematological param- eters analysis (Schalm et al. 1975, Coles 1986), that is, red blood cell (RBC) and white blood cell (WBC) counts were made with improved Neubauer hacmocytometers, Hemoglobin con- centration (Hb) by Sahli's haemoglobinometer and packed cell volume (PCV%) by mic- rohematocrit tubes. Differential WBC counts were performed on thin blood smears by the Battlement technique. Statistical analysis The obtained data were statistical ana- lyzed by mean of computer based statistical program, SPSS (Borenstein et al. 1997). Data were analyzed using Student’s t-test to com- pare the mean data between groups. The results obtained were expressed as mean ±SD. Differences were considered statisti- cally significant based on P< 0.05. Results Clinical Findings Cattle infected with B. bovis showed typical clinical signs of babesiosis, Table 1. Briefly, highly rise in body temperature (40– 41.5 °C), conjunctival and vaginal mucous membranes were anemic and the clinical severity was ranged from paleness in mild cases to severe yellow discoloration (icterus) in more progressive cases, dark brown to dark red (coffee-color) urine, hemoglobinuria was common sign in cattle with severe clinical manifestation and accelerated heart and respiratory rates. Some cases showed nervous manifestations in advanced stages such as incoordination and head pressing. Various degrees of tick infestations were pre- sent around groins, horns, Inter-mandibular space, and ears. Water buffaloes infected with B. bovis showed a milder form (less severe) of clin- ical signs of infection in comparison to the clinical signs appeared on B. bovis-infected cattle. These clinical signs were in the form of highly rise in body temperature (40–41.5 http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 4 °C), and conjunctival and vaginal mucous membranes were mainly anemic and pale in color and loss of body condition. Icterus, hemoglobinuria and nervous manifestations were not detected/ observed in the affected buffaloes. Hematological Findings Giemsa-stained blood smears from B. bovis infected animals showed intra-erythrocytic piroplasms of B. bovis that were in the form of pyriform or pear-shaped, Fig. 1. Blood smears from B. bovis progressive cases of cattle showed severe hemolytic anemia with abnormalities in cell size (Anisocytosis) and cell shape (Poikilocytosis) of erythrocytes, Fig. 2. Giemsa-stained blood smears from B. bovis uninfected cattle and buffaloes showed no parasites or erythrocytic changes. The control group resulted to be healthy on clin- ical and laboratory examination and free from external, internal and hemoparasites. The mean values of RBCs, hemoglobin amount, PCV %, WBCs, and differential leucocytic count are listed in Table 2. Brief- ly, the important findings can be summa- rized as follows; there is a clear significant difference in the haematological parameters between B. bovis-infected buffaloes and B. bovis-infected cattle in comparison to con- trol group at P-value (≤ 0.01) and (≤ 0.001), respectively. Haematological changes for B. bovis-infected buffaloes were less significant than their changes for B. bovis-infected cattle. Table 1. Clinical findings of Babaesia bovis (B. bovis)-infected cattle and buffaloes in comparison to control group under natural field conditions Parameters Field-exposed animals (n= 29) Control group (n= 6) B. bovis infected cattle (n= 20) B. bovis infected buffloes (n= 9) Temperature (°C) 40.8 (40.3–41.4) 40.6 (40.2–41.1) 38.5 (38.1–38.8) Appetite anorexia anorexia Normal Haemoglobinuria Present in advanced acute cases No haemoglobinuria Straw yellow Mucus membranes Varied from paleness in mild cases to severe yellow discoloration in progressive ones paleness and anemic of mucous membranes Bright red, moist and no lesions Icterus Marked and characteristic No No Nervous signs Incoordination, head pressing No No Body condition Thin/emaciation and anemic Weak to moderate Good Lymph nodes Normal Normal Normal Respiration Exaggerated/ accelerated Exaggerated/ accelerated Normal Giemsa-stained blood film Intraerythrocytic piroplasms of B. bovis in the form of pyriform or pear-shaped Advanced cases showed severe haemolytic anemia with anisocytosis and poikilocytosis Intraerythrocytic piroplasms of B. bovis in the form of pyriform or pear-shaped No parasites, normal RBCs http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 5 Table 2. Hemogram findings of B. bovis-infected cattle and buffaloes in comparison to control group under natural field conditions (mean ±S.E) Parameters Field-exposed animals (n= 29) Control (n= 6)B. bovis infected buffaloes (n= 9) B. bovis infected cattle (n= 20) RBCs 1 (106 /ml) 3.4±0.1 a ** 2.6±0.2 b *** 6.9±0.9 c Hb 2 (g/dl) 7.3±0.2 a ** 5.5±0.3 b *** 12.7±1.2 c PCV 3 (%) 24.6±1.1 a ** 18.1±1.5 b *** 36±1.3 c WBCs 4 (103 mul-1) 7.3±0.2 a* 7.2±0.5 a * 9.3±0.5 b Lymphocytes (103 mul- 1) 4.1±0.1 a* 3.8±0.5 a * 4.9±0.8 b Monocytes (103 mul-1) 0.46±0.07 a 0.44±0.9 a 0.46±0.01 a Neutrophils (103 mul-1) 3.1±0.4 a 2.9±0.5 a 3.2±0.6 a Eosinophils (103 mul-1) 0.2±0.01 a 0.2±0.02 a 0.2±0.04 a Basophils (103 mul-1) 0.02±0.001 a 0.02±0.002 a 0.02±0.003 a *Values with different superscripts are significantly different from each other (*P≤ 0.05, **P≤ 0.01, ***P≤ 0.001). 1 RBCs= Red Blood Cells, 2 Hb= hemoglobin, 3 PCV= packed cell vol- ume, 4 WBCs= White Blood Cells Fig. 1. Giemsa-stained blood smear showing intra- erythrocytic Pyriform (Pear-shape) of Babesia bovis in pairs Fig. 2. Giemsa-stained blood smear showing severe hemolytic anemia with abnormalities in cell size (Anisocytosis) and cell shape (Poikilocytosis) of erythrocytes from advanced cases of cattle naturally infected with Babesia bovis Discussion Babesia bovis is one of the most im- portant blood parasites affecting cattle and buffaloes and in its acute forms, it lowers the productive performance of the affected ani- mals (Talkhan et al. 2010, Ziapour et al. 2011). Most of the previous studies de- scribed the clinical findings of B. bovis in- fection in cattle of different breeds. To the best of our knowledge, this is the first study which investigating thoroughly the clinical and hematological pictures of natural B. bovis infection in water buffaloes under un- controlled field conditions. The reported clinical findings of B. bovis infection in cat- tle come in agreement with what was previ- ously described by Brown and Torres (2008), Georgi et al. (1990) and Kaufmann (1996). The demonstrated high fever could be at- tributed as response to the effect of un- specific toxic substances produced during the metabolism of Babesia on thermoregu- latory (Radostits et al. 2000). It was notable that water buffaloes identi- fied as B. bovis-infected showed a milder fgdfdd http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 6 form (less severe) of clinical signs of B. bovis infection in comparison to the clinical signs appeared on B. bovis-infected cattle. This variation was represented in appearance of icterus, hemoglobinuria and nervous man- ifestations in clinically infected cattle while they were not reported in infected buffaloes. This finding may propose that buffaloes may have more tolerance to clinical infection with B. bovis than cattle. Tolerance means that the host is infected by the pathogen, but suffers little adverse effect (FAO 2007). It could be argued that buffaloes may have acquired natural immunity/tolerance to some extent against B. bovis infection. Genetic variations within the host between cattle and buffaloes may explain the variation in their susceptibility. This finding is inconsistent with the experimental findings by Yao et al. (1997) who found that B. bovis produces acute, often fatal, infections in buffaloes. The proportion of buffaloes identified as B. bovis-infected was (31.1%) while the pro- portion of cattle identified as B. bovis-in- fected was (68.9%) within the same period of the study. This finding could suggest that water buffalos have more tendencies to be carriers (apparently healthy) than showing clinical manifestations. This finding support- ed by Ferreri et al. (2008) who noticed that water buffaloes seem to be unapparent car- riers of the parasite. The marked anemia and hemoglobinuria in cattle could be attributed to the severe haemolytic process associated the presence of Babesia piroplams inside the erythrocytes and destruction of large numbers of these erythrocytes by the parasite resulting in hemoglobinaemia and consequently hemog- lobinuria (Georgi et al. 1990, Fujinaga 1981), the physical effect of parasite multiplication (Wright 1981), the increase of phagocytosis of erythrocytes by activated macrophages (Shoda et al. 2000, Court et al. 2001), the production of an anti-erythrocyte antibody (Goe′s et al. 2007) and the increase in the erythrocytic membrane permeability (Alkhalil et al. 2007). Hematological findings showed a signifi- cant decrease in the RBCs, WBCs counts, Hb concentrations and PCV% in the B. bovis-infected animals in comparison to the control group, these observations were similar to what were reported by Col and Uslu (2007) and Durrani et al. (2006). It seems that the immune response to the babesial antigen causes a significant lymphocytosis. This comes in agreement with what was described previ- ously by Schalm (2000). Hematological chang- es resulted from B. bovis infection in buffa- loes are less significant than the hematologi- cal changes of B. bovis infection in cattle, table 2. This finding reflected clinically on B. bovis-infected buffaloes which showed a milder form of clinical picture of B. bovis infection than B. bovis-infected cattle. The hemolytic anemia due to the break- down of erythrocytes membranes leading to release of hemoglobin and manifested by the presence of free hemoglobin resulting in the discoloration of the plasma (Sowemimo-Coker 2002). Extensive lipid peroxidation in bio- logical membranes causes disturbances of its structural integrity, loss of fluidity, decrease in membrane potential, and increased perme- ability to ions (Gutteridge 1995). These chang- es lead to rupture of the membrane and re- lease of cell contents (Halliwell and Chirico 1993). Babesia parasite (Alkhalil et al. 2007), and B. bovis (Aikawa et al. 1985) dramati- cally alters the permeability of its host eryth- rocytes to various organic solutes. B. bovis infection is associated with impairment of blood parameters and subsequently, hemato- logical examination may be a useful tool for confirmation the clinical diagnosis of bovine babesiosis. Conclusion Water buffaloes showed a milder form of B. bovis infection than cattle suggesting that http://jad.tums.ac.ir Published Online: December 18, 2013 J Arthropod-Borne Dis, June 2014, 8(1): 1–9 Y Mahmmod: Natural Babesia bovis… 7 buffaloes may be more tolerant to the clini- cal infection with B. bovis than cattle. He- matological changes as a result of B. bovis infection in buffaloes are less significant than hematological changes of B. bovis in- fection in cattle. B. bovis infection might be associated with severe clinical and hemato- logical changes especially in cattle, which might be of bad prognosis. With respect to the study population, fu- ture studies should consider a larger sample size for cattle and buffaloes for the robust- ness of the findings. Recent molecular tech- niques such as PCR showed many ad- vantages with regard to the sensitivity and specificity for detection and surveillance of hemoparasites (Nayel et al. 2012, Hüe et al. 2013, Ybañez et al. 2013). Hence, it would be advisable for future studies to use such techniques for investigating the B. bovis in- fection in buffaloes. Acknowledgements This study was accomplished and funded by Faculty of Veterinary Medicine, Zagazig University, Egypt. Author is grateful to Prof Farouk Elbalkemy and Dr Ahmed Abdelaal, Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, for their support. Special thanks for Prof Ahmed Badwy, Department of Parasitology, Faculty of Veterinary Medicine, Zagazig University for his help in the lab examination. The author of this paper has not a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. References Adham FK, Abd-El-Samie EM, Gabre RM, El-Hussein H (2009) Detection of tick blood parasites in Egypt using PCR as- say I-Babesia bovis and Babesia bige- mina. Parasitol Res. 105(3): 721–730. 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