PREVALENCE OF ENTEROPATHOGENS ASSOCIATED WITH NEONATAL CALF SCOUR IN CATTLE AND BUFFALO CALVESUSING (FASTEST® STRIPS) RAPID FIELD TEST

GHADA A. ABOU EL-ELLA^**; AMR M. MOHAMED^** and AHMED A. AAMER^*

^* Clinical Laboratory Diagnosis, Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Egypt. ^** Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Saudi Arabia.

INTRODUCTION

Neonatal calf diarrhea, also known as calf scours, is a common disease affecting the newborn calf and characterized by diarrhea (scouring), progressive dehydration and death (Radostits et al., 2007). Each year thousands of neonatal calves are lost as a result of calf diarrhea. Calf scours result in losses not only by increasing calf fatality, but also by a decrease in the calf’s ability to gain weight. (Frank and Kaneene, 1993).

Infectious agents are the most significant causes of calf scour. The pathogens most commonly incriminated in neonatal calf scour include bacteria as Escherichia Coli, rotavirus (RV), bovine corona virus (BCV) or enteric protozoa as Cryptosporidium parvum. E. coli causes calf diarrhea in the first ten days of life and is characterized as a pasty to a fluid diarrhea. Generally E. coli diarrhea affects a small percent of herd unless sanitation is extremely poor. RV and BCV result in scours from 3 to 21 days. And characterized by explosive episodes in a herd and involve a high percentage of the calves. Cryptosporidiosis usually occurs between one and three weeks of age and characterized by bloody and pasty diarrhea (de la Fuente et al., 1998; Reynolds    et al., 1986; Waltner-Toews D., 1986).

Several risk factors contribute to the problem of neonatal calf scour including health and nutrition of cows, herd management before and after calving as well as feeding of neonatal calves. However, the most common locally encountered risk factor ishaving cows and heifers together in the same area they will be calving, which is a common practice among small scale owners as it was the case in the current study. This results in accumulation of manure with the subsequent increase in the number of pathogens in the calf’s environment. It was shown that calves born in this environment are 3 times more likely to get scours than calves born in an area that did not have cows or heifers living together in the calving area. (Frank and Kaneene, 1993; Langoni et al., 2004).

Identification of the possible causative agent in outbreaks of diarrhea is important because it allows targeted preventative measures, such as vaccination, and identification of possible risk factors or sources of infection. Conventional diagnostic methods to detect enteropathogens in calves require time, experience and specialized laboratory equipment. The practicing veterinarian, however, requires rapid, reproducible, sensitive and simple diagnostic tests for quick decisions on therapeutic and prophylactic strategies. Therefore, rapid immune-chromatographic assays have been developed to detect different enteropathogens in calf feces within a few minutes in the field. Advantages of rapid assays are that a large number of samples can be processed quickly with minimum effort and intervention strategies can be implemented immediately and the need for technological expertise or specialized laboratory equipment is minimal (Klein et al., 2009).

Recently, a commercial antigen-capture immune-chromatographic assay (FASTest^® D4T cattle test kits) has been developed and marketed for rapid detection of the 4 major bovine enteric pathogens (BCV-1, RV, E. coli K99⁺, and C. parvum) in feces from diarrheic calves. The principle of the kit, that is designed as a rapid test for field diagnosis, is based on a lateral flow immune-chromatography assay that captures target antigen (s) within a fecal sample (Al-Yousif et al., 2002). The assay was evaluated for detecting infections with BCV, RV-A and C. parvum from fecal samples of 180 calves using reverse transcriptase polymerase chain reaction (BCV and RV) and sedimentation-flotation technique (C. parvum) as gold standards. High specificity (96.4% and 95.3%) was recorded for the detection of BCV and RV, respectively. However, a relatively low sensitivity (60.0% and 71.9%, respectively) was recorded. On the other hand, sensitivity and specificity for detection of C. parvum were high (100% and 94.6%, respectively) (Klein et al., 2009).

Identification of the possible causative agent is crucial for the control of neonatal calf diarrhea. The different infectious agents of neonatal calf scour without specific symptoms make the etiologic differential diagnosis such a difficult process for veterinarians. In this context, scarce information is available about the prevalence and identification of different causative agents associated with diarrhea in neonatal calves in Egypt and particularly in Assiut. Therefore, the aim of the current study was to implement the recently described and recommended rapid field assays to investigate the prevalence of BCV, RV, C.parvum and E.coli F5 among diarrheic neonatal calves under field conditions at local districts of Assiut, Egypt.

MATERIALS and METHODS

Sample collection:

Fecal samples were collected from a total of 124 diarrheic calves from 2 adjacent districts (Alfateh and Sahel Seleem) of Assiut, Egypt during the period from June to August 2012. This included 76 cattle calves and 48 buffalo calves. Inclusion criteria included diarrheic calves aged from 1-35 days old that were selected from small size herds (10-30 cows) owned by local villagers. All selected calves had signs of systemic disease as pasty–watery feces, poor appetite, reduced suckle reflex and dehydration.  Approximately 15-20 mL of fecal material was collected in sterile containers directly from the rectum of diarrheic calves by direct digital stimulation using a disposable latex glove. Samples were then kept in ice box till reach the diagnostic laboratory of the faculty of Veterinary Medicine, Assiut University. Samples were then centrifuged and the resulted supernatant were collected in sterile containers and kept at -20 till time of use.

Pathogen detection and identification:

All samples were tested with FASTest^® D4T cattle test kits (MegaCor Diagnostik GmbH, LochauerstraBe 2•A - 6912 – Horbranz, AUSTRIA). The test kit included 4 rapid immune-chromatographic test strips for the detection of cattle BVC, RV, C. parvum and E.coli-K99 (F5). The tests were carried out according to the manufacturer’s guidelines. Briefly, one sample tube (P) and one test tube (R) were labeled for each fecal sample. Fecal samples were mixed homogenous and using the spoon on the cap of the sample tube, 2 spoons from each fluid-watery fecal sample were added to the diluents buffer of the corresponding test tube. Test tubes were then closed tightly and rotated in a slight and circular way for homogenous mix of the fecal sample with the diluents buffer. Then the feces-buffer diluents were added vertically to the opened corresponding test tube. Then after adding the sample mix to the test tube, the cap of the test tube was turned until hearing a clicking noise for two times. The test tube was kept on flat surface to allow the absorption of the feces-buffer diluents by the test membranes. Test result was read after 5 minutes and before 10 minutes. Correct test procedure is proved by the occurrence of a second pink/purple CONTROL line. The appearance of a clearly signed weak to strong intensive pink/purple TEST line in the TEST zone and a clearly signed weak to strong intensive pink/purple CONTROL line in the CONTROL zoneindicated positive result. On the other hand, theabsence of a pink/purple TEST line in the TEST zone with the presence ofclearly signed intensive pink/purple CONTROL line in the CONTROL zone indicated negative results.

Analysis of data:

Investigated calves from both cattle and buffalo species were divided into four age groups for analysis: 0–4 days old, 5–14 days old, 15–21 days old and >21 days old. The age categories were selected based on the pathophysiology of some of the pathogens and the age groups that have been previously described (de la Fuente et al., 1998; de la Fuente et al., 1999) The age distributions of calves as well as the number and prevalence of causative enteropathogens in different age groups were compared in both cattle and buffalo calves using chi square (SPSS, 16.0).

RESULTS

Age distribution of investigated diarrheic cattle and buffalo calves:

A total of 124 diarrheic calves (76 cattle calves and 48 buffalo calves) were collected from small sized herds of 2 adjacent districts. The mean age of the calves sampled was 13.9 days, with a SD of ± 9.06. The median age was 11 days with an interquartile range of 13 days, a minimum of 1 day and maximum of 35 days. Diarrheic calves from both cattle and buffalo species were divided into four age groups; 0–4 days old, 5–14 days old, 15–21 days old and <21 days old. The majority of calves of both cattle and buffalo herds were in the 5–14-day-old age group followed by the 15-21-day-old age group (Table 1).

Prevalence of enteropathogen infection among diarrheic claves:

Enteropathogens were isolated from 88.7% of investigated diarrheic calves (93.4% of cattle calves and 81.3% of buffalo calves) as shown in figure 1. Out of 124 diarrheic claves, 14 (11.3%) were free of enteropathogens, 78 (62.9%) were infected with single pathogen, 21% with double mixed infection and 4.8% with triple mixed infection (table 2). Significant increases (P < 0.001) in the frequency of single infection as compared to mixed infections were recorded among both cattle and buffalo calves. Significant increase (P < 0.05) was also recorded in the frequency of single infection among buffalo calves as compared to cattle calves. In addition,a significant increase (P < 0.05) in multiple infection rates among cattle calves as compared to buffalo calves was also evident. Regarding type of mixed infection, current results revealed that C. parvum infection was more frequently encountered (P < 0.05) among single infection group of both cattle and buffalo calves. On the other hand, C. parvum and rotavirus mixed infection was the most frequently identified concurrent infection (P < 0.05) among the double mixed infection group in cattle calves     (Table 3).

Prevalence of different enteropathogens in relation to age of investigated diarrheic calves:

Cryptosporidium parvum was the most common pathogen identified from all investigated calves (51.6%) followed by rotavirus (29.8%), E. coli (21.8%) and coronavirus (16.9%) (Figure 2). With regard to age groups, the obtained results revealed that C. parvum was the most frequently identified pathogen from all four age groups except for the first age group (0-4 days old), where E. coli was more frequently identified with an overall rate of 50% of investigated cases (44.45 from cattle calves and 57.1% of buffalo calves). A significant increase (P < 0.05) was recorded for C. parvum infection in cattle calves as compared to buffalo calves. On the other hand, a significant increase (P < 0.05) was recorded for the rate of E. coli infection in the first age group of both cattle and buffalo calves as compared to the other three age groups (Table 4).

Table 1: Age distribution of investigated diarrheiccattle and buffalo calves.

Table 2: Number of diarrhea-associated enteropathogens from investigated Bovine and buffalo calves.

^a Significant increase (P ˂ 0.001) in the frequency of single infection as compared to mixed infections; ^b significant increase (P ˂ 0.05) in the frequency of single infection among buffalo calves as compared to Bovine calves; ^c significant increase      (P ˂ 0.05) in multiple infection rates among Bovine calves as compared to buffalo calves.

Table 3: Types of diarrhea-associated enteropathogens in relation to single and mixed infections among investigated Bovine and buffalo calves.

^a Significant increase (P ˂ 0.05) in the frequency of C. parvum infection among single infection group of both Bovine and buffalo calves as compared to other types of infection.^b significant increase (P ˂ 0.001) in the frequency of C. parvum and rotavirus infection as compared to other types of double mixed infections in Bovine calves. Rotavirus (RV), Bovine coronavirus (BCV).

Table 4: Frequency of different enteropathogens among diarrheic calves in relation to age groups.

^aA significant increase (P ˂ 0.05) for the rate C. parvum infection between Bovine calves and buffalo calves in all age groups.^bA significant increase (P ˂ 0.05)for the rate of E. coli infection in the first age group of both Bovine and calves as compared to the other three age groups.

DISCUSSION

Diarrhea is a leading cause of economic losses to the beef industry and major cause of calf mortality and morbidity during the first few weeks of life in most countries (Radostits et al., 2007). Defining the map of calve diarrhea-causing enteropathogens allows for application of effective and targeted preventative measures. The objective of the current study was to determine the prevalence of enteric pathogens associated with neonatal calf diarrhea among both cattle and buffalo calves. Immuno-chromatographic rapid tests (FASTest® Strips) were used for the detection of BCV, RV, C. parvum and E.coli-K99 (F5) from diarrheic cattle and buffalo calves.

Previous evaluation studies of the use of rapid tests for field diagnosis of calve diarrhea-causing agents revealed high sensitivity and specificity for detection of C. parvum and E. coli-K99 (Cho et al., 2012; Klein et al., 2009). However, regarding the detection of BCV and RV, rapid tests showed high specificity but a relatively low sensitivity (Klein et al., 2009; Luginbuhl et al., 2005). Despite of that, the use of rapid tests was recommended with standard methods in calves with acute diarrhea where the specificity might be of higher importance than the sensitivity, which could be increased by testing on a herd basis (Luginbuhl et al., 2005). In general, previous evaluation studies suggested that rapid field test could be considered as a helpful tool for fast and effective diagnosis of common enteropathogens associated with neonatal calve diarrhea (Cho et al., 2012; Klein et al., 2009; Luginbuhl et al., 2005).

In spite of the complex etiology of calf diarrhea, microbial infections represent the main cause especially in neonatal calves (Bazeley, 2003; Boyed et al., 1974). This notion was confirmed in the current study, where 88.7% of investigated cases of calve diarrhea was proved due to microbial agents. The majority of those microbial infections were recorded as single-pathogen infections (78 out of 110). This could be attributed to the age factor as in current study most enteropathogen infections were recorded among early age groups where previous study recorded the presence of a significant age-associated decrease in the detection rate of mixed infections (de la Fuente et al., 1999). Although there was a significant increase in the overall rate of single infection as compared to mixed infection as revealed in the current study, this rate was significantly higher among buffalo calves as compared to cattle calves, or in other word the rate of mixed infection was significantly higher among cattle calves as compared to that in buffalo calves. This finding could be attributed to the lower susceptibility of buffalo calves to attract mixed infection as compared to cattle calves due to different susceptibilities (Malik et al., 2012). In addition, it could be also related to the diversity of the natural immunity of the two different species as manifested in other diseases (Yang et al., 2012).

The overall results from all diarrheic calves revealed that C. parvum was the most frequently isolated pathogen (51.6%), followed by rotavirus, E. coli-K99 (F5) and coronavirus with an overall rate of 29.8%, 21.8% and 16.9%, respectively. Although similar findings were reported in central Spain (de la Fuente et al., 1999), the currently reported prevalence of C. parvum was higher than has been reported earlier in Europe (Bordas et al., 1985; Reynolds et al., 1986; Snodgrass et al., 1986). Moreover, the currently recorded higher prevalence of C. parvum as compared to rotavirus contrasts findings of recent study in Australia, which reported higher prevalence of RV than that of C. parvum (Izzo et al., 2011). This discrepancy may in part reflect the lower sensitivity of the rapid assay used in the current study as compared to the more sensitive qRTPCR (Gutierrez-Aguirre et al., 2008) used in the Australian study for detection of rotavirus. However, one disadvantage of the qRTPCR higher sensitivity is the ability to detect very low viral numbers that may not be of clinical significance. The different geographic areas and the different conditions of the studies may represent another possible explanation of this discrepancy especially with regard to C. parvum for which the sensitivity and specificity were proved high using the rapid assay (Cho et al., 2012; Klein et al., 2009; Trotz-Williams et al., 2005). In general, the high rate of C. parvum infection recorded in the current study could have attributed to several factors. The resistance of the oocysts and the great proliferative capacity of the parasite (Trotz-Williams et al., 2007), in addition to the poor hygiene and sanitary conditions found on many ruminant exploitations contribute to the important presence of the parasite on such farms, particularly those that raise sheep and goats, which serve as asymptomatic carriers of the disease (O'Donoghue, 1995; Robertson et al., 1992).

In relation to age of investigated diarrheic calves, current study showed that, with exception of the first age group (0-4 days old) in buffalo calves where E. coli was more frequently encountered, C.parvum was the most frequently detected enteropathogen in all age groups of both cattle and buffalo calves. This finding could be attributed to the limited ability of E.coli to affect large percent of herds unless sanitation is extremely poor and its tendency to cause its characteristic pasty to fluid diarrhea during the first ten days of life and not in later ages on the contrary of to C. parvum, which has the ability to produce progressive infection at all ages (de la Fuente et al., 1998; Reynolds et al., 1986; Waltner-Toews D., 1986). Although C.parvum showed the higher infection frequency approximately in all age groups, the rate of infection was highest at the 2nd age group (4-15 day old) and reduced as the age of investigated calves get older. This observation was in agreement with many studies that reported the highest prevalence of C. parvum in animals of less than a month of age (Huetink et al., 2001; Santin et al., 2004; Sturdee et al., 2003).

In conclusion, C. parvum was reported as the most frequently encountered causative agent among diarrheic cattle and buffalo calves whilst coronavirus infection seemed to be of minor importance in the investigated population. The higher rate of C. parvum infection was recorded among the second age group (5-14 day old) of cattle calves and the forth age group (> 21 day old) of buffalo calves, whilst  the highest rate of E.coli infection was recorded among the first age group (0-4 days) of both cattle and buffalo calves. Cattle calves were more likely to have mixed multiple infections than buffalo calves.

REFERENCES

Al-Yousif, Y.; Anderson, J.; Chard-Bergstrom, C. and Kapil, S. (2002): Development, evaluation, and application of lateral-flow immunoassay (immunochromatography) for detection of rotavirus in bovine fecal samples. Clin Diagn Lab Immunol9, 723-725.

Bazeley, K. (2003): Farm Animal Practice: Investigation of diarrhea in thr neonatal calf. In Practice25, 152-159.

Bordas, C.; Billardon, G.; Dellamaggiore, P. and Gerster, F. (1985): Changes in newborn calf gastroenteritis from 1976-1984 in the Nievre department. Bull Mensual Societ Vet Prat France69, 89-111.

Boyed, J.W.; Baker, J.R. and Leyland, A. (1974): Neonatal diarrhea in calves. Vet Rec95,      310-313.

Cho, Y.I.; Sun, D.; Cooper, V.; Dewell, G.; Schwartz, K. and Yoon, K.J. (2012): Evaluation of a commercial rapid test kit for detecting bovine enteric pathogens in feces. J. Vet. Diagn Invest24, 559-562.

De la Fuente, R.; Garcı́a, A.; Ruiz-Santa-Quiteria, J.A.; Luzón, M.; Cid, D.; Garcı́a, S.; Orden, J.A. and Gómez-Bautista, M. (1998): Proportional morbidity rates of enteropathogens among diarrheic dairy calves in central Spain. . Prev. Vet. Med. 36,         145–152.

De la Fuente, R.; Luzon, M.; Ruiz-Santa-Quiteria, J.A.; Garcia, A.; Cid, D.; Orden, J.A.; Garcia, S.; Sanz, R. and Gomez-Bautista, M. (1999): Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain. Vet Parasitol80, 179-185.

Frank, N.A. and Kaneene, J.B. (1993): Management risk factors associated with calf diarrhea in Michigan dairy herds. J. Dairy Sci.76,     1313–1323.

Gutierrez-Aguirre, I.; Steyer, A.; Boben, J.; Gruden, K.; Poljsak-Prijatelj, M. and Ravnikar, M. (2008): Sensitive detection of multiple rotavirus genotypes with a single reverse transcription-real-time quantitative PCR assay. J. Clin. Microbiol46, 2547-2554.

Huetink, R.E.C.; Van der Giessen, J.W.B.; Noordhuizen, J.P.T.M. and Ploeger, H.W. (2001): Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm. Vet. Parasitol.102, 53-67.

Izzo, M.M.; Kirkland, P.D.; Mohler, V.L.; Perkins, N.R.; Gunn, A.A. and House, J.K. (2011): Prevalence of major enteric pathogens in Australian dairy calves with diarrhoea. Aust Vet. J. 89, 167-173.

Klein, D.; Kern, A.; Lapan, G.; Benetka, V.; Möstl, K.; Hassl, A. and Baumgartner, W. (2009): Evaluation of rapid assays for the detection of bovine coronavirus, rotavirus A and Cryptosporidium parvum in faecal samples of calves. Vet J. 182(3): 484182, 484.

Langoni, H.; Linhares, A.C.; De Avila, F.A.; Da Silva, A.V. and Elias, A.O. (2004): Contribution to the study of diarrhea etiology in neonate dairy calves in Sao Paulo state, Brazil. . Braz J Vet Res Anim Sci41, 313-319.

Luginbuhl, A.; Reitt, K.; Metzler, A.; Kollbrunner, M.; Corboz, L. and Deplazes, P. (2005): Field study about prevalence and diagnostics of diarrhea causing agents in the new-born calf in a Swiss veterinary practice area. Schweiz Arch Tierheilkd147, 245-252.

Malik, S.; Verma, A.K.; Kumar, A.; Gupta, M.K. and Sharma, S.D. (2012): Incidence of Calf Diarrhea in Cattle and Buffalo Calves in Uttar Pradesh, India. Asian Journal of Animal and Veterinary Advances7, 1049-1054.

O'Donoghue, P.J. (1995): Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol25, 139-195.

Radostits, O.M.; Gay, C.C.; Hinchcliff, K.W. and Constable, P.D. (2007): Veterinary medicine : a textbook of the diseases of cattle, sheep, pigs, goats, and horses, 10^th Edition. Elsevier Saunders, New York, xxii, 2156 p. pp.

Reynolds, D.J.; Morgan, J.H.; Chanter, N.; Jones, P.W.; Bridger, J.C.; Debney, T.G. and Bunch, K.J. (1986): Microbiology of calf diarrhoea in southern Britain. Vet Rec119, 34-39.

Robertson, L.J.; Campbell, A.T. and Smith, H.V. (1992): Survival of Cryptosporidium parvum oocysts under various environmental pressures. App Environm Microbiol58, 3494-3500.

Santin, M.; Trout, J.M.; Xiao, L.; Zhoub, L.; Greiner, E. and Fayer, R. (2004): Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Vet. Parasitol.122, 103-117.

Snodgrass, D.R.; Terzolo, H.R.; Campbell, D.; Sherwood, I.; Menzies, J.D. and Synge, B.A. (1986): Aetiology of diarrhoea in young calves. Vet Record119, 31-34.

Sturdee, A.P.; Bodley-Tickell, A.T.; Archer, A. and Chalmers, R.M. (2003): Long-term study of Cryptosporidium prevalence on lowland farm in the United Kingdom. Vet. Parasitol.116, 97-113.

Trotz-Williams, L.A.; Jarvie, B.D.; Martin, S.W.; Leslie, K.E. and Peregrine, A.S. (2005): Prevalence of Cryptosporidium parvum infection in southwestern Ontario and its association with diarrhea in neonatal dairy calves. Can Vet. J.46, 349-351.

Trotz-Williams, L.A.; Wayne Martin, S.; Leslie, K.E.; Duffield, T.; Nydam, D.V. and Peregrine, A.S. (2007): Calf-level risk factors for neonatal diarrhea and shedding of Cryptosporidium parvum in Ontario dairy calves. Prev. Vet. Med. 82, 12-28.

Waltner-Toews, D.; Martin, S.W. and Meek A.H. (1986): An epidemiological study of selected calf pathogens on Holstein dairy farms in south-western Ontario. Can J. Vet. Res. 50, 307–313.

Yang, J.; Fu, Z.; Feng, X.;Shi, Y.; Yuan, C.; Liu, J.; Hong, Y.; Li, H.; Lu, K. and Lin, J. (2012): Comparison of worm development and host immune responses in natural hosts of Schistosoma japonicum, yellow cattle and water buffalo. BMC Vet. Res.8, 25.

PREVALENCE OF ENTEROPATHOGENS ASSOCIATED WITH NEONATAL CALF SCOUR IN CATTLE AND BUFFALO CALVESUSING (FASTEST® STRIPS) RAPID FIELD TEST

GHADA A. ABOU EL-ELLA^**; AMR M. MOHAMED^** and AHMED A. AAMER^*

^* Clinical Laboratory Diagnosis, Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Egypt. ^** Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Saudi Arabia.

INTRODUCTION

Neonatal calf diarrhea, also known as calf scours, is a common disease affecting the newborn calf and characterized by diarrhea (scouring), progressive dehydration and death (Radostits et al., 2007). Each year thousands of neonatal calves are lost as a result of calf diarrhea. Calf scours result in losses not only by increasing calf fatality, but also by a decrease in the calf’s ability to gain weight. (Frank and Kaneene, 1993).

Infectious agents are the most significant causes of calf scour. The pathogens most commonly incriminated in neonatal calf scour include bacteria as Escherichia Coli, rotavirus (RV), bovine corona virus (BCV) or enteric protozoa as Cryptosporidium parvum. E. coli causes calf diarrhea in the first ten days of life and is characterized as a pasty to a fluid diarrhea. Generally E. coli diarrhea affects a small percent of herd unless sanitation is extremely poor. RV and BCV result in scours from 3 to 21 days. And characterized by explosive episodes in a herd and involve a high percentage of the calves. Cryptosporidiosis usually occurs between one and three weeks of age and characterized by bloody and pasty diarrhea (de la Fuente et al., 1998; Reynolds    et al., 1986; Waltner-Toews D., 1986).

Several risk factors contribute to the problem of neonatal calf scour including health and nutrition of cows, herd management before and after calving as well as feeding of neonatal calves. However, the most common locally encountered risk factor ishaving cows and heifers together in the same area they will be calving, which is a common practice among small scale owners as it was the case in the current study. This results in accumulation of manure with the subsequent increase in the number of pathogens in the calf’s environment. It was shown that calves born in this environment are 3 times more likely to get scours than calves born in an area that did not have cows or heifers living together in the calving area. (Frank and Kaneene, 1993; Langoni et al., 2004).

Identification of the possible causative agent in outbreaks of diarrhea is important because it allows targeted preventative measures, such as vaccination, and identification of possible risk factors or sources of infection. Conventional diagnostic methods to detect enteropathogens in calves require time, experience and specialized laboratory equipment. The practicing veterinarian, however, requires rapid, reproducible, sensitive and simple diagnostic tests for quick decisions on therapeutic and prophylactic strategies. Therefore, rapid immune-chromatographic assays have been developed to detect different enteropathogens in calf feces within a few minutes in the field. Advantages of rapid assays are that a large number of samples can be processed quickly with minimum effort and intervention strategies can be implemented immediately and the need for technological expertise or specialized laboratory equipment is minimal (Klein et al., 2009).

Recently, a commercial antigen-capture immune-chromatographic assay (FASTest^® D4T cattle test kits) has been developed and marketed for rapid detection of the 4 major bovine enteric pathogens (BCV-1, RV, E. coli K99⁺, and C. parvum) in feces from diarrheic calves. The principle of the kit, that is designed as a rapid test for field diagnosis, is based on a lateral flow immune-chromatography assay that captures target antigen (s) within a fecal sample (Al-Yousif et al., 2002). The assay was evaluated for detecting infections with BCV, RV-A and C. parvum from fecal samples of 180 calves using reverse transcriptase polymerase chain reaction (BCV and RV) and sedimentation-flotation technique (C. parvum) as gold standards. High specificity (96.4% and 95.3%) was recorded for the detection of BCV and RV, respectively. However, a relatively low sensitivity (60.0% and 71.9%, respectively) was recorded. On the other hand, sensitivity and specificity for detection of C. parvum were high (100% and 94.6%, respectively) (Klein et al., 2009).

Identification of the possible causative agent is crucial for the control of neonatal calf diarrhea. The different infectious agents of neonatal calf scour without specific symptoms make the etiologic differential diagnosis such a difficult process for veterinarians. In this context, scarce information is available about the prevalence and identification of different causative agents associated with diarrhea in neonatal calves in Egypt and particularly in Assiut. Therefore, the aim of the current study was to implement the recently described and recommended rapid field assays to investigate the prevalence of BCV, RV, C.parvum and E.coli F5 among diarrheic neonatal calves under field conditions at local districts of Assiut, Egypt.

MATERIALS and METHODS

Sample collection:

Fecal samples were collected from a total of 124 diarrheic calves from 2 adjacent districts (Alfateh and Sahel Seleem) of Assiut, Egypt during the period from June to August 2012. This included 76 cattle calves and 48 buffalo calves. Inclusion criteria included diarrheic calves aged from 1-35 days old that were selected from small size herds (10-30 cows) owned by local villagers. All selected calves had signs of systemic disease as pasty–watery feces, poor appetite, reduced suckle reflex and dehydration.  Approximately 15-20 mL of fecal material was collected in sterile containers directly from the rectum of diarrheic calves by direct digital stimulation using a disposable latex glove. Samples were then kept in ice box till reach the diagnostic laboratory of the faculty of Veterinary Medicine, Assiut University. Samples were then centrifuged and the resulted supernatant were collected in sterile containers and kept at -20 till time of use.

Pathogen detection and identification:

All samples were tested with FASTest^® D4T cattle test kits (MegaCor Diagnostik GmbH, LochauerstraBe 2•A - 6912 – Horbranz, AUSTRIA). The test kit included 4 rapid immune-chromatographic test strips for the detection of cattle BVC, RV, C. parvum and E.coli-K99 (F5). The tests were carried out according to the manufacturer’s guidelines. Briefly, one sample tube (P) and one test tube (R) were labeled for each fecal sample. Fecal samples were mixed homogenous and using the spoon on the cap of the sample tube, 2 spoons from each fluid-watery fecal sample were added to the diluents buffer of the corresponding test tube. Test tubes were then closed tightly and rotated in a slight and circular way for homogenous mix of the fecal sample with the diluents buffer. Then the feces-buffer diluents were added vertically to the opened corresponding test tube. Then after adding the sample mix to the test tube, the cap of the test tube was turned until hearing a clicking noise for two times. The test tube was kept on flat surface to allow the absorption of the feces-buffer diluents by the test membranes. Test result was read after 5 minutes and before 10 minutes. Correct test procedure is proved by the occurrence of a second pink/purple CONTROL line. The appearance of a clearly signed weak to strong intensive pink/purple TEST line in the TEST zone and a clearly signed weak to strong intensive pink/purple CONTROL line in the CONTROL zoneindicated positive result. On the other hand, theabsence of a pink/purple TEST line in the TEST zone with the presence ofclearly signed intensive pink/purple CONTROL line in the CONTROL zone indicated negative results.

Analysis of data:

Investigated calves from both cattle and buffalo species were divided into four age groups for analysis: 0–4 days old, 5–14 days old, 15–21 days old and >21 days old. The age categories were selected based on the pathophysiology of some of the pathogens and the age groups that have been previously described (de la Fuente et al., 1998; de la Fuente et al., 1999) The age distributions of calves as well as the number and prevalence of causative enteropathogens in different age groups were compared in both cattle and buffalo calves using chi square (SPSS, 16.0).

RESULTS

Age distribution of investigated diarrheic cattle and buffalo calves:

A total of 124 diarrheic calves (76 cattle calves and 48 buffalo calves) were collected from small sized herds of 2 adjacent districts. The mean age of the calves sampled was 13.9 days, with a SD of ± 9.06. The median age was 11 days with an interquartile range of 13 days, a minimum of 1 day and maximum of 35 days. Diarrheic calves from both cattle and buffalo species were divided into four age groups; 0–4 days old, 5–14 days old, 15–21 days old and <21 days old. The majority of calves of both cattle and buffalo herds were in the 5–14-day-old age group followed by the 15-21-day-old age group (Table 1).

Prevalence of enteropathogen infection among diarrheic claves:

Enteropathogens were isolated from 88.7% of investigated diarrheic calves (93.4% of cattle calves and 81.3% of buffalo calves) as shown in figure 1. Out of 124 diarrheic claves, 14 (11.3%) were free of enteropathogens, 78 (62.9%) were infected with single pathogen, 21% with double mixed infection and 4.8% with triple mixed infection (table 2). Significant increases (P < 0.001) in the frequency of single infection as compared to mixed infections were recorded among both cattle and buffalo calves. Significant increase (P < 0.05) was also recorded in the frequency of single infection among buffalo calves as compared to cattle calves. In addition,a significant increase (P < 0.05) in multiple infection rates among cattle calves as compared to buffalo calves was also evident. Regarding type of mixed infection, current results revealed that C. parvum infection was more frequently encountered (P < 0.05) among single infection group of both cattle and buffalo calves. On the other hand, C. parvum and rotavirus mixed infection was the most frequently identified concurrent infection (P < 0.05) among the double mixed infection group in cattle calves     (Table 3).

Prevalence of different enteropathogens in relation to age of investigated diarrheic calves:

Cryptosporidium parvum was the most common pathogen identified from all investigated calves (51.6%) followed by rotavirus (29.8%), E. coli (21.8%) and coronavirus (16.9%) (Figure 2). With regard to age groups, the obtained results revealed that C. parvum was the most frequently identified pathogen from all four age groups except for the first age group (0-4 days old), where E. coli was more frequently identified with an overall rate of 50% of investigated cases (44.45 from cattle calves and 57.1% of buffalo calves). A significant increase (P < 0.05) was recorded for C. parvum infection in cattle calves as compared to buffalo calves. On the other hand, a significant increase (P < 0.05) was recorded for the rate of E. coli infection in the first age group of both cattle and buffalo calves as compared to the other three age groups (Table 4).

Table 1: Age distribution of investigated diarrheiccattle and buffalo calves.

Table 2: Number of diarrhea-associated enteropathogens from investigated Bovine and buffalo calves.

^a Significant increase (P ˂ 0.001) in the frequency of single infection as compared to mixed infections; ^b significant increase (P ˂ 0.05) in the frequency of single infection among buffalo calves as compared to Bovine calves; ^c significant increase      (P ˂ 0.05) in multiple infection rates among Bovine calves as compared to buffalo calves.

Table 3: Types of diarrhea-associated enteropathogens in relation to single and mixed infections among investigated Bovine and buffalo calves.

^a Significant increase (P ˂ 0.05) in the frequency of C. parvum infection among single infection group of both Bovine and buffalo calves as compared to other types of infection.^b significant increase (P ˂ 0.001) in the frequency of C. parvum and rotavirus infection as compared to other types of double mixed infections in Bovine calves. Rotavirus (RV), Bovine coronavirus (BCV).

Table 4: Frequency of different enteropathogens among diarrheic calves in relation to age groups.

^aA significant increase (P ˂ 0.05) for the rate C. parvum infection between Bovine calves and buffalo calves in all age groups.^bA significant increase (P ˂ 0.05)for the rate of E. coli infection in the first age group of both Bovine and calves as compared to the other three age groups.

DISCUSSION

Diarrhea is a leading cause of economic losses to the beef industry and major cause of calf mortality and morbidity during the first few weeks of life in most countries (Radostits et al., 2007). Defining the map of calve diarrhea-causing enteropathogens allows for application of effective and targeted preventative measures. The objective of the current study was to determine the prevalence of enteric pathogens associated with neonatal calf diarrhea among both cattle and buffalo calves. Immuno-chromatographic rapid tests (FASTest® Strips) were used for the detection of BCV, RV, C. parvum and E.coli-K99 (F5) from diarrheic cattle and buffalo calves.

Previous evaluation studies of the use of rapid tests for field diagnosis of calve diarrhea-causing agents revealed high sensitivity and specificity for detection of C. parvum and E. coli-K99 (Cho et al., 2012; Klein et al., 2009). However, regarding the detection of BCV and RV, rapid tests showed high specificity but a relatively low sensitivity (Klein et al., 2009; Luginbuhl et al., 2005). Despite of that, the use of rapid tests was recommended with standard methods in calves with acute diarrhea where the specificity might be of higher importance than the sensitivity, which could be increased by testing on a herd basis (Luginbuhl et al., 2005). In general, previous evaluation studies suggested that rapid field test could be considered as a helpful tool for fast and effective diagnosis of common enteropathogens associated with neonatal calve diarrhea (Cho et al., 2012; Klein et al., 2009; Luginbuhl et al., 2005).

In spite of the complex etiology of calf diarrhea, microbial infections represent the main cause especially in neonatal calves (Bazeley, 2003; Boyed et al., 1974). This notion was confirmed in the current study, where 88.7% of investigated cases of calve diarrhea was proved due to microbial agents. The majority of those microbial infections were recorded as single-pathogen infections (78 out of 110). This could be attributed to the age factor as in current study most enteropathogen infections were recorded among early age groups where previous study recorded the presence of a significant age-associated decrease in the detection rate of mixed infections (de la Fuente et al., 1999). Although there was a significant increase in the overall rate of single infection as compared to mixed infection as revealed in the current study, this rate was significantly higher among buffalo calves as compared to cattle calves, or in other word the rate of mixed infection was significantly higher among cattle calves as compared to that in buffalo calves. This finding could be attributed to the lower susceptibility of buffalo calves to attract mixed infection as compared to cattle calves due to different susceptibilities (Malik et al., 2012). In addition, it could be also related to the diversity of the natural immunity of the two different species as manifested in other diseases (Yang et al., 2012).

The overall results from all diarrheic calves revealed that C. parvum was the most frequently isolated pathogen (51.6%), followed by rotavirus, E. coli-K99 (F5) and coronavirus with an overall rate of 29.8%, 21.8% and 16.9%, respectively. Although similar findings were reported in central Spain (de la Fuente et al., 1999), the currently reported prevalence of C. parvum was higher than has been reported earlier in Europe (Bordas et al., 1985; Reynolds et al., 1986; Snodgrass et al., 1986). Moreover, the currently recorded higher prevalence of C. parvum as compared to rotavirus contrasts findings of recent study in Australia, which reported higher prevalence of RV than that of C. parvum (Izzo et al., 2011). This discrepancy may in part reflect the lower sensitivity of the rapid assay used in the current study as compared to the more sensitive qRTPCR (Gutierrez-Aguirre et al., 2008) used in the Australian study for detection of rotavirus. However, one disadvantage of the qRTPCR higher sensitivity is the ability to detect very low viral numbers that may not be of clinical significance. The different geographic areas and the different conditions of the studies may represent another possible explanation of this discrepancy especially with regard to C. parvum for which the sensitivity and specificity were proved high using the rapid assay (Cho et al., 2012; Klein et al., 2009; Trotz-Williams et al., 2005). In general, the high rate of C. parvum infection recorded in the current study could have attributed to several factors. The resistance of the oocysts and the great proliferative capacity of the parasite (Trotz-Williams et al., 2007), in addition to the poor hygiene and sanitary conditions found on many ruminant exploitations contribute to the important presence of the parasite on such farms, particularly those that raise sheep and goats, which serve as asymptomatic carriers of the disease (O'Donoghue, 1995; Robertson et al., 1992).

In relation to age of investigated diarrheic calves, current study showed that, with exception of the first age group (0-4 days old) in buffalo calves where E. coli was more frequently encountered, C.parvum was the most frequently detected enteropathogen in all age groups of both cattle and buffalo calves. This finding could be attributed to the limited ability of E.coli to affect large percent of herds unless sanitation is extremely poor and its tendency to cause its characteristic pasty to fluid diarrhea during the first ten days of life and not in later ages on the contrary of to C. parvum, which has the ability to produce progressive infection at all ages (de la Fuente et al., 1998; Reynolds et al., 1986; Waltner-Toews D., 1986). Although C.parvum showed the higher infection frequency approximately in all age groups, the rate of infection was highest at the 2nd age group (4-15 day old) and reduced as the age of investigated calves get older. This observation was in agreement with many studies that reported the highest prevalence of C. parvum in animals of less than a month of age (Huetink et al., 2001; Santin et al., 2004; Sturdee et al., 2003).

In conclusion, C. parvum was reported as the most frequently encountered causative agent among diarrheic cattle and buffalo calves whilst coronavirus infection seemed to be of minor importance in the investigated population. The higher rate of C. parvum infection was recorded among the second age group (5-14 day old) of cattle calves and the forth age group (> 21 day old) of buffalo calves, whilst  the highest rate of E.coli infection was recorded among the first age group (0-4 days) of both cattle and buffalo calves. Cattle calves were more likely to have mixed multiple infections than buffalo calves.

REFERENCES

Al-Yousif, Y.; Anderson, J.; Chard-Bergstrom, C. and Kapil, S. (2002): Development, evaluation, and application of lateral-flow immunoassay (immunochromatography) for detection of rotavirus in bovine fecal samples. Clin Diagn Lab Immunol9, 723-725.

Bazeley, K. (2003): Farm Animal Practice: Investigation of diarrhea in thr neonatal calf. In Practice25, 152-159.

Bordas, C.; Billardon, G.; Dellamaggiore, P. and Gerster, F. (1985): Changes in newborn calf gastroenteritis from 1976-1984 in the Nievre department. Bull Mensual Societ Vet Prat France69, 89-111.

Boyed, J.W.; Baker, J.R. and Leyland, A. (1974): Neonatal diarrhea in calves. Vet Rec95,      310-313.

Cho, Y.I.; Sun, D.; Cooper, V.; Dewell, G.; Schwartz, K. and Yoon, K.J. (2012): Evaluation of a commercial rapid test kit for detecting bovine enteric pathogens in feces. J. Vet. Diagn Invest24, 559-562.

De la Fuente, R.; Garcı́a, A.; Ruiz-Santa-Quiteria, J.A.; Luzón, M.; Cid, D.; Garcı́a, S.; Orden, J.A. and Gómez-Bautista, M. (1998): Proportional morbidity rates of enteropathogens among diarrheic dairy calves in central Spain. . Prev. Vet. Med. 36,         145–152.

De la Fuente, R.; Luzon, M.; Ruiz-Santa-Quiteria, J.A.; Garcia, A.; Cid, D.; Orden, J.A.; Garcia, S.; Sanz, R. and Gomez-Bautista, M. (1999): Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain. Vet Parasitol80, 179-185.

Frank, N.A. and Kaneene, J.B. (1993): Management risk factors associated with calf diarrhea in Michigan dairy herds. J. Dairy Sci.76,     1313–1323.

Gutierrez-Aguirre, I.; Steyer, A.; Boben, J.; Gruden, K.; Poljsak-Prijatelj, M. and Ravnikar, M. (2008): Sensitive detection of multiple rotavirus genotypes with a single reverse transcription-real-time quantitative PCR assay. J. Clin. Microbiol46, 2547-2554.

Huetink, R.E.C.; Van der Giessen, J.W.B.; Noordhuizen, J.P.T.M. and Ploeger, H.W. (2001): Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm. Vet. Parasitol.102, 53-67.

Izzo, M.M.; Kirkland, P.D.; Mohler, V.L.; Perkins, N.R.; Gunn, A.A. and House, J.K. (2011): Prevalence of major enteric pathogens in Australian dairy calves with diarrhoea. Aust Vet. J. 89, 167-173.

Klein, D.; Kern, A.; Lapan, G.; Benetka, V.; Möstl, K.; Hassl, A. and Baumgartner, W. (2009): Evaluation of rapid assays for the detection of bovine coronavirus, rotavirus A and Cryptosporidium parvum in faecal samples of calves. Vet J. 182(3): 484182, 484.

Langoni, H.; Linhares, A.C.; De Avila, F.A.; Da Silva, A.V. and Elias, A.O. (2004): Contribution to the study of diarrhea etiology in neonate dairy calves in Sao Paulo state, Brazil. . Braz J Vet Res Anim Sci41, 313-319.

Luginbuhl, A.; Reitt, K.; Metzler, A.; Kollbrunner, M.; Corboz, L. and Deplazes, P. (2005): Field study about prevalence and diagnostics of diarrhea causing agents in the new-born calf in a Swiss veterinary practice area. Schweiz Arch Tierheilkd147, 245-252.

Malik, S.; Verma, A.K.; Kumar, A.; Gupta, M.K. and Sharma, S.D. (2012): Incidence of Calf Diarrhea in Cattle and Buffalo Calves in Uttar Pradesh, India. Asian Journal of Animal and Veterinary Advances7, 1049-1054.

O'Donoghue, P.J. (1995): Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol25, 139-195.

Radostits, O.M.; Gay, C.C.; Hinchcliff, K.W. and Constable, P.D. (2007): Veterinary medicine : a textbook of the diseases of cattle, sheep, pigs, goats, and horses, 10^th Edition. Elsevier Saunders, New York, xxii, 2156 p. pp.

Reynolds, D.J.; Morgan, J.H.; Chanter, N.; Jones, P.W.; Bridger, J.C.; Debney, T.G. and Bunch, K.J. (1986): Microbiology of calf diarrhoea in southern Britain. Vet Rec119, 34-39.

Robertson, L.J.; Campbell, A.T. and Smith, H.V. (1992): Survival of Cryptosporidium parvum oocysts under various environmental pressures. App Environm Microbiol58, 3494-3500.

Santin, M.; Trout, J.M.; Xiao, L.; Zhoub, L.; Greiner, E. and Fayer, R. (2004): Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Vet. Parasitol.122, 103-117.

Snodgrass, D.R.; Terzolo, H.R.; Campbell, D.; Sherwood, I.; Menzies, J.D. and Synge, B.A. (1986): Aetiology of diarrhoea in young calves. Vet Record119, 31-34.

Sturdee, A.P.; Bodley-Tickell, A.T.; Archer, A. and Chalmers, R.M. (2003): Long-term study of Cryptosporidium prevalence on lowland farm in the United Kingdom. Vet. Parasitol.116, 97-113.

Trotz-Williams, L.A.; Jarvie, B.D.; Martin, S.W.; Leslie, K.E. and Peregrine, A.S. (2005): Prevalence of Cryptosporidium parvum infection in southwestern Ontario and its association with diarrhea in neonatal dairy calves. Can Vet. J.46, 349-351.

Trotz-Williams, L.A.; Wayne Martin, S.; Leslie, K.E.; Duffield, T.; Nydam, D.V. and Peregrine, A.S. (2007): Calf-level risk factors for neonatal diarrhea and shedding of Cryptosporidium parvum in Ontario dairy calves. Prev. Vet. Med. 82, 12-28.

Waltner-Toews, D.; Martin, S.W. and Meek A.H. (1986): An epidemiological study of selected calf pathogens on Holstein dairy farms in south-western Ontario. Can J. Vet. Res. 50, 307–313.

Yang, J.; Fu, Z.; Feng, X.;Shi, Y.; Yuan, C.; Liu, J.; Hong, Y.; Li, H.; Lu, K. and Lin, J. (2012): Comparison of worm development and host immune responses in natural hosts of Schistosoma japonicum, yellow cattle and water buffalo. BMC Vet. Res.8, 25.