The present work was conducted to study haematology and to identify the major bacterial and parasitic etiologic factors responsible for gastrointestinal (GIT) affections in bovine calves by evaluation of clinical samples from Palam Valley of Himachal Pradesh, India. Blood picture revealed a significant increase in total leukocyte count and neutrophilia was shown in differential leukocyte count from diarrhoeic cases. Out of 43 isolates from clinical cases, E. coli, Citrobacter and Salmonella were found 90.69%, 6.97% and 2.32% respectively. Disc diffusion test was used to check sensitivity of isolates from clinical samples. Cloxacillin was found most resistant drug while amoxy-sulbactum and gentamicin were most effective drugs against the E. coli isolates. In case of parasitic infestation, Out of 43 clinical cases, Eimeria spp. and Strongyloids spp. were present in 6.97% and 4.65% respectively. Also, overall incidence of enteritis was highest in winter season.
Calf plays an important role in dairy industry development and profitability. The future of a dairy herd solely depends upon the successful raising of young calves. Healthy calves are not only essential for sustenance of the dairy industry but essential for preserving and maintaining good quality germplasm also (Khatun et al., 2009). Unfortunately, it has been observed that large number of calves either get sick or die due to various diseases and managemental errors. Calf hood diseases have a major impact on the economic viability of cattle operations (Lorenz, 2011). Septicemic and enteric diseases are most common during early period, with respiratory disease being more common in later period of life. Enteric diseases are attributed to both infectious and non-infectious factors. Multiple enteric pathogens (e.g., viruses, bacteria, and protozoa) are involved in the development of this disease. GIT affections are the most common cause of calf mortality in worldwide. Out of all, diarrhoea has its major concern due to high morbidity and mortality rate during early period of life in calves. A variety of infectious agents are implicated in calf diarrhoea and co-infection of multiple pathogens is not uncommon in diarrheic calves (Susma et al., 2016). Bacteria, viruses and parasites by attacking the lining of intestine give rise to diarrhoea. This in turn decreases the absorption of essential nutrients from milk leading to weight loss and dehydration. In severe cases, calf may die.
The present study was conducted to determine the prevalence and factors responsible for GIT diseases in bovine calves in Palam Valley of Himachal Pradesh, India. The study was also focused on the haematology of affected bovine calves.
Materials and Methods
2ml of blood was collected from sick and apparently healthy calves in EDTA vials for performing haematological parameters i.e. Differential Leucocyte Count (DLC) and Total Leucocyte Count (TLC).
In present investigation a total of 88 samples were collected from, diarrhoeic (43) and apparently healthy (45) calves. Faecal samples were collected from the rectum of diarrhoeic and apparently healthy animals. Detail of places from where sample were collected has given below in Table 1.
Table1: Places of feacal sample collection
|S. No.||Source||Clinical Cases||Apparently Healthy||Total no. of Samples|
|1||H.P.K.V. Dairy Farm, Palampur, Kangra||21||9||30|
|2||Govt. Jersey Farm, Palampur, Kangra||3||2||5|
Faecal swabs were taken from the rectum of healthy and clinically sick animals and processed for bacterial identification and cultured invariably on the same day. The primary isolation was done on nutrient agar as well as on McConkey’s lactose bile salt agar (MLA) by streaking the swab over the agar plates. This was carried out under the laminar flow and incubated aerobically at 37°C for 16-24 hours to check for any microbial growth. The smears prepared from the purified colonies were subjected to Gram’s staining and the morphological features of isolates were studied under the microscope. The isolates were subjected to routine biochemical fermentation reactions for their confirmation. Motility was determined by hanging drop method.
Culture Sensitivity Test
Disc diffusion method was used to study the drug sensitivity pattern against the isolate obtained from the deceased animals (Cruikshank et al., 1975). Four to five colonies of the isolate were transferred to 5 ml of nutrient broth and incubated at 37°C for 24 hours. Subsequently, sterile cotton swab was dipped into the broth and evenly mopped onto the entire agar surface of Mueller Hilton Agar (MHA) plate. The antimicrobial discs were placed equidistant with 30 mm minimum center to center distance. These plates were incubated at 37°C for 24 hours and results were recorded according to the size of zone of growth inhibition. The following antimicrobials were used (Table 2) (Hi-Media Laboratories Pvt. Ltd. Mumbai India).
Table 2: Antimicrobials used in culture sensitivity test
Faecal Sample Processed for Parasitology
Faecal samples collected from clinically sick cases were subjected to faecal flotation method (FAO) for screening of parasitic oocysts and eggs under low magnification of microscope. Also, Modified Ziehl-Neelseen (MZN) staining of faecal samples was also done for identification of Cryptosporidium.
Results and Discussion
The present study was undertaken to explore the cause that are responsible for GIT affections in bovine calves and to identify the major bacterial and parasitic etiologic factors. Attempts were made for the isolation/identification of major bacterial and parasitic pathogens associated with diarrhoea and other GIT affection by using standard isolation/identification methods, wet smears and MZN stained smears. Blood samples from severely affected sick cases were subjected to haematologic analysis.
Haematological parameters are always helpful for not only giving prognosis but also to improve efficacy of a particular selected therapeutic regimen irrespective of causal agents (Kumar et al., 2012a, 2012b). In present study, there was significant increase in immature neutrophils (band cells) in diarrhoeic calves as compared to the healthy calves. Also, there was drastic increase in TLC values which might have occurred due to normal reaction of body defense mechanism against infection and these finding had close agreement with the findings of other study (Schalm, 1975). Previous studies also revealed leukocytosis mainly neutrophilia with no significant increase in other leukocytic series cells in haematological studies (Hoerstke et al., 1982; Shridhar et al., 1988). Other study also revealed increase in neutrophils in diarrhoeic calves (Malik et al., 2013). This marked neutrophilic response is characteristic of acute bacterial enteritis. Monocyte and eosinophils in diarrhoeic calves did not show any significant change.
Detection of Bacterial Isolates
A total of 43 samples of clinically sick and 45 samples from non-diarrhoeic cases from different farms and places were screened for the presence of enterobacteria. During analysis of samples, 39 E. coli (90.69%), three Citrobacter sp. (6.97%) and one Salmonella sp. (2.32%) were isolated from clinical cases and data was similar to the previous studies. In non-diarrhoeic cases 36 E. coli (80%) and 4 Cirobacter sp. (8.88%) were isolated. Five samples showed no growth in isolation. Among bacteria, E.coli is known to be the most common agents that cause diarrhoea in young calves and this might be the reason of high prevalence of E. coli in calves. The prevalence of Salmonella in the present study was less when compared to other study in India (5%) (Joon and Kaura, 1993). This might be due to different prevalence rates of Salmonella in diarrhoeic calves which can vary with geographical locations and managemental practices. Other study also revealed that E. coli and Salmonella were the most common identified pathogens in diarrhoeic calves of less than 2 months of age (Acha et al., 2004). Prevalence of isolates varied with geographical location of the farms, farm management practices, and herd size (Cho and Yoon, 2014). One Study demonstrated that faecal samples collected from 127 diarrhoeic calves up to 3 months of age and the prevalence of Salmonella and E. coli in diarrhoeic calves was 18.1% and 75.6%, respectively (Elshehedi et al., 2013) which was similar to present findings. One study also showed the prevalence of Citrobacter (8%) in diarrhoeic calves (Ambrosim et al., 2002).
The purified bacterial colonies were cultured on MLA plates. E.coli colonies were dark pink, Citrobacter sp. and Salmonella sp. produced pale colored colonies. The isolated bacterial colonies were stained with Gram’s stain and examined under microscope. The Gram negative rods were further processed for their biochemical characterization as given in Table 3.
Table 3: Biochemical features of bacterial isolates
+ = Positive, – = Negative
Bacterial colonies showing characteristics of E.coli were further streaked on EMB plates for confirmation (Fig. 1). All E.coli colonies produced metallic sheen. Pale non-lactose fermenting colonies on MLA were streaked on BGA plates (Fig. 2). The Salmonella sp. colonies looked like dewdrop on BGA and changed the color of medium to pinkish red (Fig. 3).
Fig1: Gram staining and growth of E. coli on different media- (A) E. coli on BGA (Changing color of media to yellowish) (B) E. coli on EMB (Metallic sheen) (C) Gram negative rods (rectangle) (330x).
Fig 2: Biochemical and IMViC results of E.coli– (A) Catalase test: Positive test shows bubble formation. (B) Oxidase test: Positive test shows violet color reaction on oxidase disc (C) Methyl red test (Positive with red ring formation) (D) Citrate utilization test (Positive with change in color to blue) (E) Indole test (Positive with red ring formation)
Fig 3: Growth of Salmonella sp. on different media: (A) Salmonella on MLA (non-lactose fermenting colonies) (B) Salmonella on BGA (Changing color of media to pinkish red)
Depending upon the severity of condition and also availability of trend personnel, antimicrobials are commonly used in therapy of diseased calves either orally or parentally and sensitivity/resistance of those antimicrobials are the matter of concern. In present study, a total of 30 E. coli isolates from clinical cases were subjected to Culture sensitivity test (CST) against 8 antibiotics. Cloxacillin was found most resistant drug while amoxy-sulbactum and gentamicin were most sensitive drugs (Fig. 4). High resistance was perhaps due to gross misuse and inappropriate usage of the antibacterial agents. So, appropriate antibiotic should be used to reduce antimicrobial resistance. The previous study revealed the presence of cloxacillin and oxytetracycline resistance in E. coli isolates recovered from diarrhoeic calves (Malik et al., 2013). In present investigation, amoxy-sulbactum and gentamicin were the most effective drugs against the isolates of E. coli from faecal samples of clinically sick animals. Previous study also revealed the same findings showing the sensitivity of amoxicillin and gentamicin against E. coli isolates (Malik et al., 2013). Another study also investigated the amoxicillin sensitivity against the E. coli isolates (Constable, 2004).
Fig.4: Histogram showing sensitivity pattern of E. coli isolates from faecal swab of clinical cases
Detection of Parasites
The study further investigated the presence of parasites which were responsible for the calf diarrhoea. A total of 88 faecal samples (43 clinical and 45 apparently healthy) were screened for parasitic infestation. Out of 43 clinical cases, three (6.97%) samples were positive for Eimeria spp. infestation, two (4.65%) samples were positive for Strongyloids spp. infestation (Fig. 5). Out of 45 apparently healthy samples, three (6.66%) cases found positive for Strongyloids spp. infestation and one (2.22%) case for Eimeria spp. infestation. In previous studies, different Eimeria species viz. E. zurnii and E. bovis have seen in neonates and young calves having diarrhoea and data was in harmony with present findings (Yasar et al., 2006). Eimeria spp. and Strongyloids spp. infestations were widely distributed in bovine calves and were characterized by gastroenteritis (Sharma and Busang, 2013).These reports were coincide with the present study which also showed the presence of Eimeria spp. in faecal samples of clinically sick animals. Cryptosporidium was considered an important protozoan species responsible for inducing neonatal diarrhoea in animals (Yong and Kyong, 2014) but in present investigation, none of MZN stained smears found positive for Cryptosporidium.
Fig 5: Microphotography of parasitic eggs– (A) Strongyloids spp. egg (enlarged view) (B) Eimeria spp. oocyst (circle) (66x).
Non infectious factors like insufficient intake of colostrum, stress, poor sanitation and erratic weather conditions such as low temperatures, rain, heavy snow, wind and high levels of moisture act as stress factors to young calves and increase the susceptibility of calves to diarrhoea (Carroll and Forsberg, 2007). Present investigation showed highest incidences (57.5%) of calf diarrhoea in winters. Neonatal calves are not able to effectively regulate their body temperature when exposed to extreme weather conditions. Season has significant effect on calf mortality as well as absorption of immunoglobulin (Ig) in neonatal calves (Fink, 1980). Titer of IgG colostrum from dairy cows in winter is lower than IgG concentration in other seasons (Gulliksen et al., 2008) which may predispose calves to diseases. All these previous findings coincide with present investigation.
In conclusion, the present work was undertaken with an aim to study the haematology and to identify bacterial and parasitic etiologic factors responsible for GIT affections in calves in Palam Valley of Himachal Pradesh, India. In accordance with that, the blood picture revealed the significant increase in immature neutrophils (band cells) and TLC values in diarrhoeic calves as compared to the healthy calves which suggested a responsive defense mechanism of animals against infection. E. coli was the most prevalent pathogen associated with enteritis with overall incidence of 90%. Pathogens were found resistant to cloxacillin antibiotic while amoxy-sulbactum and Gentamicin were most sensitive drugs. The incidence of parasitic infestation in clinical and apparently-healthy animals was 11% and 9% respectively. Eimeria spp. and Strongyloids spp. were found common with highest incidences of calf diarrhoea in winters.
I would like to thank faculty of Department of Pathology and Microbiology and Dean, CSKHPKV Palampur for providing necessary facilities and resources for this work.