Histopathological alterations associated with immunolocalization of Clostridium perfringens and Salmonella spp in neonatal bovine calves died of diarrhoea Raksha Suresh, Brar A.P.S., Sood N.K., Leishangthem Geeta Devi*, Jaiswal Vikas Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India *Address for Correspondence, Dr Geeta Devi Leishangthem, Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141012, Punjab, India, E-mail: drgeetapatho@gmail.com
Online Published on 28 July, 2022. Abstract Clostridium perfringens and Salmonella spp are the major bacterial enteropathogens responsible for causing diarrhoea in calves. The aim of the study was to detect Clostridium perfringens and Salmonella spp by immunohistochemical technique (IHC) and to study the associated histopathological changes in jejunum and ileum of calves that died of neonatal calf scours. Ninety samples of intestine from the diarrhoeic neonatal calves were collected and processed for routine histopathology and immunohistochemistry. Out of these 90 samples examined, 21 samples were positive for Clostridium perfringens, 22 were positive for Salmonella spp and 25 were found to be positive for both the organisms by immunohistochemistry. Histopathological alterations such as altered villi: crypt ratio, desquamation of epithelium, congestion, intactness of muscularis mucosae, crypt hyperplasia, crypt destruction, fibrosis and severity of inflammation were observed and scoring was done in relation to Salmonella spp and C. perfringens. The total histopathological score in samples of mixed infections was higher as compared to the total score of samples with single infection indicating co-infections of Salmonella spp and C. perfringens can lead to severe enteritis in calves. The study was highly useful in immunolocalization of C. perfringens and Salmonella spp and to ascrible the pathological lesions to the etiology. Top Keywords Clostridium perfringens, Histopathology, Immunohistochemistry, Neonatal calf diarrhoea, Salmonella spp. Top |
Introduction Calf diarrhoea is the major cause of mortality in young calves. It is believed that half of the deaths in unweaned calves happens due to diarrhoea1. The cause of diarrhoea in calves is often difficult to diagnose as it is almost always a reciprocal interaction between infectious and non-infectious causes. The non-infectious causes of diarrhoea include faulty genetic, managemental, nutritional and adverse climatic conditions2. These non-infectious factors increase the susceptibility of the calf for infectious diarrhoea. Thus, multiple aetiologies along with other non-infectious causes make the definitive diagnosis of diarrhoea difficult. Clinical signs observed in diarrhoea are also non-specific in nature. Hence, better knowledge of the frequency of the pathogens occurring in livestock farms would allow the practitioner to provide proper treatment to this very common disease in calves. Also, this would avoid failure in the treatment of neonatal diarrhoea, and also decrease antibiotic resistance due to rampant use of antibiotics. |
Amongst the major bacterial pathogens responsible for diarrhoea in neonatal calves, Salmonella spp and C. perfringens are two very important etiological agents of calf diarrhoea. C. perfringens is a gram-positive spore forming enteropathogen responsible for causing enterotoxemia in dairy calves3,4 which can be divided into 5 types from A to E, based upon the toxins produced5. Out of these types, C. perfringens type C is more common in calves and is responsible for causing enterotoxaemia in calves less than 3 months of age. The toxins of C. perfringens type C causes cell lysis through hydrolysis of membrane phospholipids and induces mucosal necrosis6. |
Salmonella is a gram-negative facultative anaerobe mostly causing enteric and septicaemic infections in dairy cattle and its severity of pathogenicity is considered only next to bovine viral diarrhoea7. Salmonella spp such as S. typhimurium and S. dublin are mainly responsible for causing diarrhoea in 2-12-week-old calves8. The enterotoxins of these organisms have entero-invasive property. They multiply and survive in phagocytic cells. |
Application of both histopathology and immunohistochemistry (IHC) together can be helpful in knowing the severity of the damage caused by each organism and also about its pathogenesis. IHC is a very useful tool to visualise the presence of the pathogen, its distribution in the tissues and the histological lesions produced by it. Although in dead animals, pathognomonic gross lesions may help in suspecting a particular etiological agent as a cause of diarrhoea but IHC can be a very important technique for its confirmation and visualisation in tissues. |
Top Materials and Methods Collection of samples The present study was conducted on dead bovine calves suffering from diarrhoea which were less than 3 months of age. The samples were collected from August 2019 to December 2019. A total of 90 samples showing gross lesion in intestine were collected from dairy farms in and around Ludhiana district of Punjab and post-mortem hall of Department of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana. Histopathology Pieces of tissues from affected intestine were collected in 10% neutral buffered formalin. Fixation was done in 10% neutral buffered formalin. After fixation for 2 days, tissue samples were given overnight washings under tap water. Dehydration of samples was done through ascending grades of alcohol followed by clearing with acetone and benzene. Tissues were embedded in paraffin wax (Leica Microsystem, Paraplast tissue embedding medium, 56°C) for further processing and 4-5μ thin sections were cut. The paraffin sections were stained with routine hematoxylin and eosin technique. Special staining The intestinal samples were stained by Good pasture stain for differentiation of bacteria as described in the previous study9. Histopathological scoring Histopathological score was made on the 8 important parameters for affected intestinal samples. The criteria followed are given in Table 1. The parameter taken into consideration were villi: crypt ratio, congestion, desquamation of villous epithelial cells, crypt hyperplasia, crypt destruction, intactness of muscularis mucosae, severity of inflammation and fibrosis. Criteria followed for scoring was based on previous work done by other researchers10-12. Certain modifications were made in the scoring parameters based on the histopathological findings observed in the present study to make it more authentic. Immunohistochemistry protocol For immunohistochemical studies 4-5 μ thin paraffin embedded tissue sections were mounted on positively charged microscopic slides. After deparaffinization and rehydration, antigen retrieval was done in sodium citrate buffer (pH=6) at 95°C for three minutes, followed by 70°C for seven minutes in a microwave oven. To quench the endogenous peroxides, slides were kept in 3% H2O2 for 30 minutes. After washing with PBS, protein blocking was done by using 2.5% normal horse serum for 30 minutes in humidified chamber. The primary antibodies were diluted Table 2. Tissue sections were incubated with primary antibody at 37°C for 2hours in an incubator. After adequate washing by PBS, secondary antibody (ImmPRESS® HRP Universal Antibody (Anti-Mouse IgG/Anti-Rabbit IgG, Peroxidase Polymer Detection Kit, made in Horse) was put on tissue and incubated for 30 mins at room temperature. After washing, section was incubated with freshly prepared 3,3′-diaminobenzidine (DAB) (ImmPACT®DAB Peroxidase (HRP) Substrate) solution and counterstained with Gill’s Haematoxylin. Negative control of each tissue was run by incubating with PBS instead of primary antibody. Statiscal analysis The data was analysed using SPSS software and was expressed as Mean±SE. Top Results Gross findings Grossly, the intestines of most of the affected calves were distended and filled with gas along with watery yellowish fluid. The affected intestine showed congestion of varying intensity (Fig 1). Mesenteric lymph nodes were enlarged in most of the cases. The intestinal mucosa was strongly hyperaemic and covered with yellowish intestinal contents. Common gross changes in the intestine were catarrhal (Fig 2) and haemorrhagic enteritis. In some cases, mucosae were seen covered with fibrin (Fig 3) and in others ulcers were observed (Fig 4). Histopathology The histopathological changes observed in intestine were altered VC ratio (Fig 5), desquamation of crypt epithelium (Fig 6), engorgement of capillary plexus due to congestion (Fig 7), crypt destruction (Fig 8), destruction of muscularis mucosae (Fig 9), fibrosis (Fig 10), mononuclear cell infiltration in mucosa often extending till submucosal layer (superficial and deep enteritis), infiltration of mononuclear cells into crypt, congestion and haemorrhage. Good Pasture staining was also employed for demonstration of gram-positive and gram-negative bacteria in the histopathologic sections. The technique appeared to be fairly useful in detecting the bacterial pathogens in the superficial and at times in deeper mucosa of the intestine (Fig 11). Histopathology scoring The parameters and criteria followed for Histopathological scoring is given in Table 1. Histopathological scores of samples which were only positive for Salmonella spp, only positive for C. perfringens and positive for both are given Table 3. The histopathological score includes the parameters such as VC ratio, desquamation of epithelium, congestion, intactness of muscularis mucosae, crypt hyperplasia, crypt destruction, severity of inflammation and fibrosis. Mean total histopathological score of samples which were positive for Salmonella spp only was found to be (15.31±0.7), 15.19±0.91 for C. perfringens only and 17.04±0.95 for mixed infection of both C. perfringens and Salmonella spp. In cases of mixed infection, the scores were significantly high in parameters like congestion and severity of inflammation. Even in other parameters as well there were non-significantly higher scores in mixed infection than by single infection alone. Detection of Clostridium perfringens and Salmonella spp by IHC Immunoreactivity to C. perfringens was observed in 46 samples mostly on superficial and deep mucosa (Fig 12). The bacteria were also demonstrated in between crypts and within intestinal crypts (Fig 13). In addition, the reactivity for C. perfringens was also found in the deep microvasculature of intestine in few cases (Fig 14). Immunoreactivity to Salmonella spp was observed in a total of 47 samples as yellow to brown coarse granules or bacilli in mucosal layer of intestine. The positive reaction for salmonella was also observed in the deeper intestinal mucosa (Fig 15), dilated microvasculature of the intestine and occasionally in the mononuclear cells (Fig 16). Top Discussion A total of 46 samples showed immunoreactivity to Clostridium perfringens mainly in the mucosal layer and sometimes inside the blood vesselspossibly explaining the cause of septicaemia. In the study conducted by Brar13, the prevalence of Clostridium perfringens was found to be 62.79% in the dead diarrhoeic calves by IHC in the same region. Diab and his co-worker14 reported a case of Clostridium perfringens type C enterotoxaemia in horses where they observed numerous gram-positive rods superficially on the mucosa by IHC. Silva and his co-worker15 reported necrotic enteritis along with bacterial colonies adhering to exposed lamina propria in C. perfringens associated neonatal diarrhoea of piglets which was confirmed by IHC. |
The alteration in architecture of tissue in samples positive for an etiological agent can be interpreted in many ways. Decreased VC ratio and increased desquamation of the villous epithelial cells can be attributed to the production of toxins in C. perfringens. The major toxin produced is type A toxin which is produced by all species of C. perfringens. But C. perfringens type A produces more amount of this toxin16. Type A toxin is a phospholipase enzyme which has preference to the constituent of cell membrane of enterocytes which is sphingomyelin phosphatidylcholine17. Along with this character, phospholipase C also induces matrix metalloproteases which disrupt epithelium of infected tissue and increase the subepithelial tissue destruction18. Tumour necrosis factor-alpha production is also induced by mononuclear cells which further aggravate the destruction and increases vascular permeability. Apart from the toxin, C. perfringens also release many enzymes like mucin degrading enzymes mainly sialase and mucinase, collagenases, hyaluronidase which flares up destruction and inflammation. |
A total of 47 samples showed immunoreactivity for Salmonella spp mainly in mucosal layer and in intracellularly in monocytes and enterocytes. In the study conducted by Brar13 the prevalence of Salmonella was found to be 62.79% by IHC in the same region in dead diarrhoeic calves. Similarly, Ries and his co-worker19 showed attachment of Salmonella to the domed villi which in cattle is composed almost exclusively of M cells by IHC and they also demonstrated within the macrophages. Watanabe and his co-worker20 observed strong immunoreactivity to Salmonella spp in the mucosa of the large intestine, enterocyte and macrophages in the sub-mucosa of intestine as well as lamina propria in 160 piglets. |
A decreased VC ratio in Salmonella positive cases can be attributed to its ability to destroy enterocytes. Epithelial shedding observed in the present study can also be due to factors such as pro-inflammatory cytokine (TNF), bacterial lipopolysaccharide (LPS)and toxins21. In the present study, the organism penetrated the lamina propria of the intestine and was engulfed by the mononuclear cells. Similarly, it has been reported that the organism penetrates the lamina propria in the distal small intestine and colon where they are engulfed by the inflammatory cells which help them to disseminate throughout the body22. Lymphoid tissue is the predilection site for S almonella and it disseminates mainly through M-cells, and are found in the high numbers in the Peyer’s patches and mesenteric lymph nodes7. The inflammatory reaction starts in mucosa and the organism disseminates via lymphatics and may eventually lead to bacteraemia. In an experimental study conducted by Santos and his co-worker8, infection of bovine Peyer’s patches by S. typhimurium resulted in an acute severe infiltration of neutrophils. This can be due to increased expression of anti and proinflammatory cytokine by enterocytes. |
Crypt hyperplasia observed in the present study may be due to increased expression of Wnt/β-catenin pathway as observed in an experimental study where 3-day old chicks were inoculated with S. pullorum which resulted in crypt hyperplasia in chicken intestine 23 |
In the present scenario, fibrosis in the intestinal tissue might be due to injury to mesenchymal cells caused by toxins and infectious agents which activates mesenchymal cells to fibrogenic phenotype which can be followed by normal healing or fibrosis24. Further, endotoxins have been known to cause extensive congestion in the splanchnic bed25. |
The histopathological score in mixed infections of C. perfringens and Salmonella showed higher total score when compared to samples of single infection. Significantly higher scores were observed in parameters like congestion and severity of inflammation. Presence of both C. perfringens and Salmonella might have flared up the inflammatory process and might have also increased the histopathological alterations in the clinical samples. |
Top Conclusion The study was highly useful to detect immunolocalization of C. perfringens and Salmonella spp and to ascrible the pathological lesions to specific etiology. It indicates that both the agents together cause more severe damage in the jejunum and ileum leading to the death of calf. This may probably be the first study that uses scoring of the histopathological changes in different segment of the intestine caused by C. perfringens and Salmonella spp in bovine calf diarrhoea. |
Top Figures | Fig. 1.: Gross specimen of intestine showing severe congestion
|  | |
| | Fig. 2.: Gross specimen of intestine showing catarrhal enteritis
|  | |
| | Fig. 3.: Gross specimen of intestine showing fibrinous type of enteritis
|  | |
| | Fig. 4.: Gross specimen of intestine showing ulcers and congestion on mucosal surface
|  | |
| | Fig. 5.: Histopathology of the intestine showing VC ratio <1:1, Score 3 (H&E Bar=100 μm)
|  | |
| | Fig. 6.: Histopathology of the intestine showing desquamation of crypt epithelium, Score 3 (H&E Bar=100 μm)
|  | |
| | Fig. 7.: Histopathology of the intestine showing engorgement of capillary plexus due to congestion, Score 3 (H&E=100 μm)
|  | |
| | Fig. 8.: Histopathology of the intestine showing complete destruction of crypts, Score 3 (H&E; Bar=50 μm)
|  | |
| | Fig. 9.: Histopathology of the intestine showing complete destruction of muscularis mucosae (H&E; Bar=100 μm)
|  | |
| | Fig. 10.: Histopathology of the intestine showing complete fibrosis, Score 3 (H&E; Bar=100 μm)
|  | |
| | Fig. 11.: Section of intestine showing blue coloured gram-positive and pink coloured gram-negative bacteria (Good Pasture stain; Bar=20 μm)
|  | |
| | Fig. 12.: Section of intestine showing immunoreactivity for Clostridium perfringens in the mucosal layer (IHC, Bar=20 μm)
|  | |
| | Fig. 13.: Section of intestine showing immunoreactivity to Clostridium perfringens in between and within crypts (IHC; Bar=20 μm)
|  | |
| | Fig. 14.: Section of intestine showing immunoreactivity to Clostridium perfringens inside the blood vessel (IHC; Bar=20 μm)
|  | |
| | Fig. 15.: Section of intestine showing immunoreactivity to Salmonella spp in mucosa (IHC; Bar=50 μm)
|  | |
| | Fig. 16.: Section of intestine showing immunoreactivity to Salmonella spp in enterocytes and mononuclear cells (IHC; Bar=20 μm)
|  | |
|
Tables | Table 1.: Scoring criteria for different parameters.
| | Parameters | Score 0 (Normal) | Score 1 (Mild) | Score 2 (Moderate) | Score 3 (Severe) | | VC Ratio | 6:1 | 5:1 to 4:1 | 3:1 to 2:1 | <1:1 | | Desquamation of epithelium | No desquamated cells | Few desquamated cells of tip of villous epithelium | Marked desquamation of villous epithelial cells | Severe desquamation of crypt epithelium | | Congestion | No evidence of congestion in any vessel | Evidence of congestion in few blood vessels | Evidence of congestion in moderate number of blood vessels | Evidence of congestion in moderate number of blood vessels | | Crypt hyperplasia | <3 crypts show hyperplasia | 3 to 5 crypts show hyperplasia | 5 to 8 crypts show hyperplasia | >8 Crypt show hyperplasia | | Crypt destruction | 0 crypts are destructed | <5 crypts are destructed | 5 to 8 crypts are destructed | >8 Crypts are destructed | | Intactness of muscularis mucosae | Muscularis mucosae is intact throughout | Muscularis mucosa is not intact a some places | Muscularis mucosa layer is completely lost | | Severity of inflammation | No infiltration of inflammatory cells | Inflammatory cells only in the mucosal layer | Inflammatory cells in mucosa and submucosal | Transmural-when the cells are present in all the layers | | Fibrosis | No evidence of fibrosis | Evidence of fibrosis <3fields/hpf | Evidence of fibrosis seen >3fields/hpf | Complete fibrosis |
| | | Table 2.: Antibodies used for Immunohistochemistry.
| | S.No. | Antibody | Clone | Manufacturer | Dilution | Antigen retrieval | | 1 | Rabbit Salmonella Polyclonal Antibody | Polyclonal | Mybiosource | 1:500 | HIER | | 2 | Rabbit anti – Clostridium perfringens | Polyclonal | Biorad | 1:500 | HIER |
| | | Table 3.: Comparison of histopathological score with etiological agents.
| | Parameters | Score of histopathology (Mean±SE) | | Salmonella | Clostridium | Clostridium + Salmonella | | Villi-crypt ratio | 2.45±0.13 | 2.23±0.16 | 2.48±0.15 | | Desquamation of epithelium | 2.36±0.17 | 2.14±0.18 | 2.4±0.16 | | Congestion | 1.31±0.14 | 1.47±0.17 | 2.16±0.16* | | Intactness of muscularis mucosae | 0.90±0.15 | 0.85±0.18 | 1.12±0.14 | | Crypt hyperplasia | 2.63±0.12 | 2.52±0.15 | 2.6±0.14 | | Crypt destruction | 2.27±0.15 | 2.33±0.16 | 2.32±0.16 | | Severity of inflammation | 1.81±0.16 | 2.23±0.16 | 2.4±0.12* | | Fibrosis | 1.54±0.17 | 1.38±0.19 | 1.56±0.21 | | Total score | 15.31±0.7 | 15.19±0.91 | 17.04±0.95 |
|
| Significant at p≤0.05 (Clostridium + Salmonella) Vs Salmonella alone and Clostridium | |
|