Faecal prevalence and histopathological evaluation of coccidiosis in bovine calves

Bovine coccidiois, caused by Eimeria spp. is widely prevalent around the globe and responsible for huge economic losses by causing morbidity and mortality among young calves. The present study was designed to evaluate the prevalence as well as to evaluate histopathological alterations associated with it. The faecal samples were collected from 700 bovine calves upto two month of age from August 2019 to July 2021 and screened for Eimeria oocycts. The intestinal tissue samples of 37 calves were also collected which died during the study period after showing symptoms of diarrhea and examined for histological lesions. The faecal prevalence of Eimeria observed in our study was 2.29% (16/700) while in tissue samples only two out of 37 were found positive for Eimeria infection. Tissue sections revealed various stages of Eimeria gametogony, variable congestion, haemorrhage, and necrosis along with cryptic dilatation and mononuclear cell infiltration. Coccidia was not found to be associated with season, age and sex of calf. Bovine coccidiosis was found to be endemic with low prevalence but severe onset characterized by moderate to severe congestion and inflammatory reaction mainly in the ileum and caecum.


Introduction
Eimeria is an obligate, intracellular, monoxenous, protozoan parasites of the phylum Apicomplexa and family Eimeriidae. It is one of the most important parasitoses reported in bovines around the globe and responsible for enormous economic losses in dairy industry (Li et al. 2021). E. bovis E. zurenii and E. bareillyi are three most common and most pathogenic species (Mundt et al. 2005;Manya et al. 2008;Ramirez et al. 2009;Das et al. 2015;Nain et al. 2017;Pyziel et al. 2020). It mainly exerts its pathological effect by causing damage to intestinal mucosa of large and small intestine during its sexual reproduction. The infection occurs through faeco-oral rout by ingestion of sporulated oocysts which are very resistant and remain viable in the environment for months in adverse climatic conditions (Cruvinel et al. 2018). Several risk factors such as seasonality, humidity, age, sex, change in feed and management system etc. were reported by previous researchers (Girma 2017; Das et al. 2015). There is paucity of reports especially in regards to histopathological evaluation of naturally infected Eimeria calves in India, so the current study was planned not only to estimate the prevalence but also to investigate the histopathological lesions in naturally infected bovine calves.

Material and methods
A total 700 faecal samples were collected from bovine calves from August 2019 to July 2021. The samples were collected from organized and unorganized farms in and around Ludhiana district of Punjab which is located between 30° -33', 31° − 1' north latitudes and 75° − 25' and 76° − 27' east longitudes, elevation ranges from about 216 m in the west to about 268 m in the east. It lies in humid subtropical climate with three distinct seasons; summer, winter and monsoon. Faecal smears were screened for oocysts after staining with Leishman and modified Ziehl Neelsen stain as per standard protocol. During the study period thirty seven calves were found dead after showing symptoms of diarrhea. Post mortem examination were carried out and gross lesions were recorded. The intestinal tissue samples were collected and fixed in 10% neutral buffered formalin. Paraffin embedded tissue Sects. (4 micron) were stained with hematoxylin and eosin stain by standard procedure (Luna 1968).The H&E stained slides were examined under microscope (Olympus BX53) and microphotography was carried out by using DP27 Olympus camera and cellsens software.

Results
The clinical signs like diarrhea, dysentery, dehydration, lethargy, inanition, emaciation, rough hair coat, anemia of varying severity were observed in most of the cases. In the current study, only 2.29% (16) faecal samples were found positive for coccidian oocyst out of 700 samples. Two species were identified on the basis of morphometric examination (Rehman et al. 2011) consisting of E. bovis (Fig. 1) and E. subspherica (Fig. 2). Out of 16 Eimeria positive cases 14 (87.5%) were of E. bovis while two (87.5%) were infected with E. subspherica. Mean oocyst size of E. bovis measured in our study was 20.24 µm X 28.29 µm while E. subspherica was 10.78 µm X12.22 µm. We also detected three cases of mixed infection of with Cryptosporidium (Fig. 3). These all three cases were found to be co infected with E. bovis only. Prevalence and results of Chi square test in relation to season, age and sex have been shown in the Tables 1 and 2 respectively. We observed highest prevalence of Eimeria in winter (2.80%) followed by summer (2.40%), monsoon (2.38%) and post monsoon (1.60%) season. Sex wise prevalence was higher in male calves (2.72%) than female calves (1.80%). Age wise prevalence was highest in age group IV (12.31%) followed by age group V(12.00%), age group III (5.91%), age group II (0.32%) and age group I (0.00%). However, Chi square test has shown non-significant association with all these three variables (Table 2).
Gross lesions of intestinal tissue samples observed in current study were congestion and haemorrhage in different parts of small and large intestine with highest severity in ileum and caecum. The mucosa was oedematous thickened, and covered with necrotic debris material. Ballooning of intestine with gas and diarrheic faeces was evident. Microscopic examination revealed moderate to high congestion of mucosa and submucosa, goblet cell hyperplasia (Fig. 4), infiltration of inflammatory mononuclear cells in lamina propria and Lymphoid cell depletion in peyers patches (Fig. 5). There was variable necrosis of mucosa and accumulation of debris material in the lumen (Fig. 6). Destruction of villi led to stunting and alteration in crypt-villi ratio (Fig. 5). In addition, mucosal haemorrhages and disruption of muscularis mucosae were also seen at places. Different stages of parasitic gametogony were distinctly visible (Fig. 7) in ileum and caecum. The parasite not only confined to mucosa but also invaded the submucosa and nodules of parasitic aggregation (coccidial nest) were seen in some of the Payer's patches (Fig. 5). Both male and female gamonts were detected in the same mucosal crypts of affected area. Sexually differentiated macrogamonts (female gamonts) were identified on the basis of centrally placed nucleus surrounded by large eosinophilic amylopectin granules (wall form bodies) while microgamonts (male gamonts) withnumerous nuclei (Fig. 7). Histology of mesenteric lymph node reveled lymhadenitis with infiltration of neutrophils and macropahges at places in one case while varying depletion of lymphoid cells and congestion in both the cases.

Discussion
In the current study, we observed only 2.27% (16) Eimeria prevalence which was in line with the finding of Brar et al. (2017) and Hussain et al. (2017) who reported only 4 and 2.9% prevalence respectively. In contrast to our finding, most of the researchers around the globe reported much higher prevalence ranging from 6.0 to 91.7% (Manya et al. 2008;Das et al. 2015;Girma 2017;Nain et al. 2017;Morgoglione et al. 2020;Li et al. 2021). The reason for getting very low prevalence in our study may be the age of the calf from which samples were collected and prepatient period of parasite (Mundt et al. 2005;Li et al. 2021). In our study we collected 87.14% samples from less than three week of age group animals. We did not find any positive case in first week of age and only one case in second week. Another reason may be easy availability of effective drugs against this parasite in this area and increased awareness about hygienic practices. Li et al. (2021) did meta-analysis of online available data of coccidial prevalence in China upto February  Das et al (2015) reported highest prevalence of E. bovis in pre monsoon (62.8%), followed by monsoon (58.6%), winter (55.4%) and summer season (50.8%). Higher prevalence observed in male calves in current study is in agreement with most of the previous researchers (Nain et al. 2017;Heidari et al. 2014). Though we found higher prevalence in older group of animals and male sex but statistically (Chi square test) it is non-significant. Direct life cycle of this parasite (without any intermediate host), short life span and autoinfection might be the cause of the non-significant association of the Cryptosporidium with above the risk variables. Similar to our study Mundt et al. (2005) also reported watery to haemorrhagic diarrhea but they observed lesions starting from terminal jejunum upto mid colon in experimentally infected calves. We observed highest severity of gross lesions in ileum and caecum. In contrast, Pyziel et al. (2020) recorded gross lesions mainly in large intestine. Hussain et al. (2017) noted specific transverse zebra striping on the mucosal surface in all the case along with haemorrhage and necrosis. Variable pathology of different species of Eimeria has been reported by various scientists (Das et al. 2015;Cruvinel et al. 2018). White raised nodules have been reported in small ruminants (Satish et al. 2019) but non apparent in bovine coccidiosis.
Our histological findings are in line with Hussain et al. (2017) who also documented mononuclear cellular infiltration in the lamina propria, desquamation of epithelial lining, and severe hemorrhages with mucosal scars and coccidial cells nested in the lamina propria in all infected animals. Mundt et al. (2005) reportedmicroscopic lesions starting from initial mucosal hyperemia to acute typhlitis and necrotising colitis. They also observed infiltration of granulocytic series and proliferation of goblet cell. However, Sultana et al. (2015) reported highest severity both in caecum and colon. They observed mild hyperemia along with granulocytic mucosal infiltration at 15 day post infection (dpi) and acute sever lesion (degeneration, shortened and necrosis of villi, haemorrhages in mucosa and sub-mucosa, widening of crypts of lieburkuhn and oedema of lamina propria) at 25 dpi. Stockdale (1977) also reported similar findings along with fibrin deposits and diphtheritic membrane on mucosa at 18dpi followed by resolution and mucosal regeneration commencing from 22 to 32nd dpi but they observed lesions mainly in large intestine. Kawahara (2016) also observed similar lesion produced by gametogony both in small and large intestine. The lesions observed in our study, such as severe congestion and destruction of villus epithelium were mainly attributed to gametogony which can be seen in Fig. 5 by presence of different developmental stages of Eimeria deep into submucosa. As a result of gametogony there was massive destruction and stunting of villi. In an effort of regeneration, there was proliferation of crypt epithelium. Oedema of mucosa appeared due to damage of villus epithelium as well as leaky blood vessels of lamina propria. The absorptive mature villus epithelium was replaced by immature crypt epithelium with secretory activity which causes diarrhea and when there is destruction of villi, it leads to haemorrhage and dysentery. Previous researches have not reported the invasion of Eimeria to submucosal payer patches of the current study. Most of the scientists reported mucosal involvement only (Mundt et al. 2005;Sultana 2015;Hussain et al. (2017).

Conclusion
Bovine coccidiosis is an endemic with low prevalence but with severe onset of disease. Histology revealed its predilection of distal small intestine and proximal large intestine. The gross pathology revealed lesions varying from mild congestion to haemorrhagic enteritis, necrosis, ballooning and enlargement of mesenteric lymph node. Microscopically, lesions include congestion, mucosal oedema, mononuclear cell infiltration, destruction and stunting of villus epithelium. Parasitic gametogony responsible for villus destruction, cryptic dilatation, accumulation of necrotic debris and replacement of mature villus epithelium with immature secretory cryptic epithelium leading to malabsorption, diarrhea, dehydration and death. Coccidial prevalence was not found to be associated with age, sex and season.