Introduction

Caseous lymphadenitis (CLA) is a chronic contagious disease caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) affecting sheep and goat and able to infect human, leading to severe economic losses like reduction in the milk production, weight loss, and low-quality meat and wool ( Dorella et al. 2006; Dorneles et al. 2014; Droppa-Almeida et al. 2016). Ten cases have been registered in Australia of human lymphadenitis due to C. pseudotuberculosis. Most of the patients were exposed to sheep on an occupational basis. The primary basis of management is surgical excision of the affected lymph glands, and antibiotic therapy is supplementary therapy (Peel et al. 1997). Although the disease is chronic, many cases do not show any clinical signs and appear subclinical in sheep and goats around the world (Dorella et al. 2006; Selim et al. 2016). The disease has a visceral form characterized by a severe decline in body weight and respiratory manifestations with chronic ruminal tympany (Al-Gaabary and El-Sheikh 2002; Oreiby 2015). Rarely, CLA may cause mastitis, orchitis, cellulitis, or in some death times (Sunil 2006; Baird and Fontaine 2007).

Diagnosis of CLA in sheep is mainly depending on the distinctive clinical signs of the disease and the isolation and identification of C. Pseudotuberculosis from the abscesses of the affected animals (Baird and Fontaine 2007). Subclinical infected animals or carriers are considered important sources of infection to healthy animals and cannot be diagnosed by clinical examination (Kaba et al. 2001). These cases without clinical signs require other diagnostic techniques for diagnosis of the infection such as enzyme-linked immunosorbent assays (ELISAs) (Binns et al. 2002). ELISA is the most significant serological test used to detect immune responses against C. pseudotuberculosis (Chirino-Zárraga et al. 2009; Hoelzle et al. 2013).

Caseous lymphadenitis is endemic in Egypt and this disease is found in Egyptian sheep herds; few epidemiological studies have been carried out in this country (Mubarak et al. 1999; Al-Gaabary and El-Sheikh 2002; Al-Gaabary et al. 2009; Oreiby et al. 2014). To the best of the authors’ knowledge, the present study considers the first one to investigate the seroprevalence of CLA in Egypt, whereas many previous studies (Al-Gaabary et al. 2009;Al-Gaabary et al. 2010) determined the clinical prevalence of CLA in sheep in Egypt, while there is paucity in the risk factor data associated with this infection. Identification of risk factors associated with CLA either at farm or animal level helps in constructing a potential control system for such high economic important infection. Therefore, in this study we investigated the seroprevalence of CLA caused by C. pseudotuberculosis among sheep reared in Dakahlia Governorate, Egypt, and study the potential risk factors associated with the disease. The area of the Middle East and developing countries has similar breeding and husbandry practices, and the disease prevalence in these countries is close to each other. Our results could be upscaled to other endemic countries with similar way of animal breeding in the Middle East and Mediterranean basin countries.

Materials and methods

Ethical approval

All experimental protocols in this study were approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Mansoura University, Egypt (Approval No. 04-33).

Area of study

This study was conducted at four districts (Mansoura, Dekernes, Belkas, and Sherbin) in Dakahlia Governorate, which is situated in the central area of Egyptian Delta. This area contains a lot of cultivated lands; therefore, the farmers let their animals to graze at day light; then, animals are collected in a fixed yard at night.

Sample collection

A cross-sectional study was conducted to determine the prevalence of CLA among sheep flocks of the Dakahlia governorate, Egypt. According to the census of the Dakahlia governorate, the number of sheep was 119,850. The total number of required animal samples was determined by using a random sampling design on Win Episcope 2 program using the following formula: n = t2 × p (1 − p)/m2: n = required sample size, t = confidence level at 95% (standard value of 1.96), and m and p are the accepted error and the expected prevalence which were set at 5.5% and 15% according to previous study (Al-Gaabary et al. 2009) respectively. The total number of samples was estimated at 162 and we inflated it to 346 sheep. These animals were divided among the 4 main districts of the governorate, Mansoura (total number of sheep: 9900), Dekernes (total number of sheep: 8300), Belkas (total number of sheep: 9720), and Sherbin (total number of sheep: 12,000). According to the total number of sheep in each of the selected districts, the classification was conducted; therefore, the total number of samples per each district was 104 samples from Sherbin, 72 from Dekernes, 84 from Belkas, and 86 from Mansoura. In each of the selected districts, the total number of samples was divided from five to seven places and the college campaigns have occurred in these places. Animals in each campaign were randomly selected by using a systematic sample wherein each flock at every other number of sheep one was chosen (Thrusfield 2007). Level of risk factors in animals included age and gender. Also, farm-level risk factors included the type of housing (open or closed), rearing with a goat, movement of the sheep through the year, treatment of the infected sheep, and handling of the infected sheep (keep or discard) through questionnaires.

Serological analysis

Blood samples were collected under the complete aseptic condition from sheep jugular vein into 5-ml plain vacutainer tubes (B.D. Bioscience, Germany). Serum was collected from clotted blood by centrifugation at 3000 rpm and stored at -20°C until examination. ELISA procedures were applied according to Menzies et al. (2004). Preparation of exotoxin antigen was performed according to Tahoun et al. (2015) from local isolates of C. pseudotuberculosis isolated from CLA abscess of native sheep. Antigen was standardized and quantified according to Bradford (1976). Two negative control and two positive control serum samples were kindly provided by the Department of Animal Medicine, Faculty of Veterinary Medicine, Kafr Elsheikh University, Egypt.

Statistical analysis

Statistical analysis was performed by using SPSS software program windows version 22.0. Descriptive statistics and the distribution of risk factor, among cases of caseous lymphadenitis, were obtained. A chi-square test was used to perform comparative prevalence. Logistic regression analysis on animal levels was performed to check the relationship between the incidences of infection and the potential risk factors. Firstly, univariate logistic regression statistics were performed. The infection status of sheep was the dependent variable and classified into the infected or non-infected, but the recommended risk factors were the independent variables. Consequently, the independent factors with P value (P < 0.1) were incorporated in the multivariate logistic regression analysis. In each variable, the parameters incorporated in the results were P value, confidence interval (CI: 95%), and odds ratio (OR). Results were considered to be significant at P < 0.05.

Results

Under this study, ELISA was used to determine the prevalence of C. pseudotuberculosis in sheep. The cut-off point for ELISA plates was 0.298 and determined by the ROC curve at sensitivity 72.2% and specificity 73.7% (Fig. 1). The number of positive animals, from each district by an indirect ELISA test, was presented in Table 1. Out of 346 samples, 256 were serologically positive with a prevalence of 73.98%. The highest prevalence was found among flocks of Sherbin (83.65%) followed by Mansoura (74.41%), then Dekernes (69.44%), and Belkas (65.47%).

Fig. 1
figure 1

ROC curve analysis showing sensitivity and specificity

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Table 1 Seroprevalence of CLA-infected sheep according to localities
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When analyzing epidemiological data in univariable analysis between independent variables and CLA status of the examined animals by using ELISA, we found that occurrence of CLA infection increased with ages. Furthermore, the highest incidence was observed in the age group between 2 and 3 years (79.66%) and the lowest incidence in animals less than 1 year (57.4%, P = 0.02, OR = 0.44, 95% CI: 0.21–0.89) (Table 2). Interestingly, a statistically significant association between type of housing (open, closed) and the occurrence of CLA was found, and the higher incidence of CLA infection was observed in open housing (82.57%, P = 0.007, OR = 2.16, 95% CI: 1.27–3.69) (Table 2).

Table 2 Univariate logistic regression for the association between potential risk factors and individual sheep CLA ELISA seropositive
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Concerning movement and occurrence of CLA, the higher prevalence was found in sheep that moved through the year (79.21%, P = 0.023, OR = 1.76, 95% CI: 1.07–2.8) than the non-movable flocks (Table 2). Additionally, the higher incidence of CLA infection was observed in in herds that keep diseased animals (76.66%, P = 0.004, OR = 2.53, 95% CI: 1.33–4.80) (Table 2). On the contrary, gender, treatment, and breeding with goats were not associated with a high prevalence of CLA in sheep (P > 0.05) (Table 2).

Results of multivariate analysis are presented in Table 3; treatment variable with P value > 0.05 was removed from the final model; furthermore, age variable was found a confounder for handling of diseases animals and movement variable was found a confounder for housing variable and so both age and movement variables were removed from the final multivariate model. Final results showed that animals found in open housing systems and those animals found in herds that keep diseased animals have a significant higher chance to get CLA infection than those found in closed houses and those found in herds which discard diseased animals (P = 0.033 and 0.047, respectively, and OR = 1.84, 95% CI: 1.05–3.88 and 1.98, 95% CI: 1.01–3.23, respectively) (Table 3).

Table 3 Multivariate logistic regression for the association between potential risk factors and seropositive CLA sheep by ELISA
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Discussion

CLA is a worldwide chronic infectious disease of sheep and goat, which is characterized by pyogranulomas formation in superficial lymph nodes and internal organs may be affected (Paule et al. 2004; Baird and Fontaine 2007). Importantly, the disease is endemic in Egypt (Oreiby et al. 2014; Oreiby 2015). The spread of C. pseudotuberculosis, among the flock, is very high; subsequently, CLA remains a cause of concern for sheep and goat breeders around the world (Williamson 2001; Paton et al. 2003).

To the best of our knowledge, the present study is the first one to investigate the farm and animal risk factors associated with CLA in sheep herds in the Dakahlia governorate in Egypt. The serological results in this present study indicate a high frequency of seropositive animals (73.98%), and the previous study made in Egypt revealed the prevalence of CLA among slaughtered animals was 26.92% and 25.05% based on a gross and bacteriological examination respectively (Al-Gaabary et al. 2010). Additionally another study reported a prevalence (43.8%) in Egypt based on bacteriological examination (Algammal 2016). The serological results in this study indicate a high frequency of seropositive animals, and this is not surprising as the Dakahlia governorate is an endemic region in Egypt and agrees with previous studies reporting similar results. Guimarães et al. (2011) reported high prevalence (75.8%) in an endemic region in Brazil, and Aslan et al. (2016) identified prevalence (63.6%) in Turkey. On the other hand, Moura et al. (2020) reported lower prevalence (34.07%) in Ceará state Brazil and this may be due to the lack of managemental procedures, hygienic measures, and cleaning. We thought that due to the low sensitivity and specificity percentages of ELISA, the true seroprevalence could be higher and this gives an indication of the dangerous situation of caseous lymphadenitis in Egypt and findings may be upscaled to other endemic countries in the Middle East and Mediterranean basin countries with similar animal breeding methods. The highest prevalence was recorded among flocks of Sherbin and Mansoura while the lowest prevalence was recorded in Belkas. Low prevalence in Belkas other than locality and seroprevalence differences in several localities may be due to minor biosecurity measures and breeding habits, and most of the Belkas flocks were fixed not movable so the chance to contact with other flocks is very low.

Risk factor identification analyses showed that treatment, keeping of affected animals in the herd, and other preventive precautions are not offered by many farmers, which is a dangerous situation for more spreading of the disease among the flocks. According to age, we found that the highest prevalence was in the age group 3 (2–3 years) and this is in agreement with (Al-Rawashdeh and Al-Qudah 2000; Al-Gaabary et al. 2009; Zavoshti et al. 2012; Oreiby et al. 2014) and this might be attributed to shearing of wool which is the major route of transmission of disease not occurring in young animals less than 1 year. Additionally, we found lower prevalence in animals less than 1 year and this agrees with the results obtained by Windsor (2011) who stated that the prevalence of CLA is very low in sheep under 1 year of age, although the sex of animals is not statistically significant for the higher prevalence in females (74.75%) than males (68.88%) and this is in accordance with Musa (1998), Al-Gaabary et al. (2009), and Chikhaoui and Khoudja (2013). They reported a higher CLA occurrence in females in comparison with a lower frequency in males.

Regarding the breeding system, it is found that the prevalence was higher in mobile flocks that are moving all the year than fixed ones and this agrees with Gerges (2015) who recorded the same results and disagreed with the results reported by Oreiby et al. (2014) who said that the fixed flocks are at high chance to get the infection. This difference may be due to the mobile flocks having a greater chance to come in contact with other flocks, which supply the free flocks with a source of infection (Baird and Malone 2010). In Dakahlia Governorate, the grazing area is restricted, so is considered the source of infection due to its ability to persist for long period in the environment and the type of feeding on hard and thorny plants increases the opportunity of wound and abrasion, which give a higher chance to disease transmission among sheep (Moura-Costa et al. 2008).

Concerning housing of animals, we found a higher prevalence in open housing than closed ones and this is due to the structural materials of the open housing. Some materials like straw and pus contain the C. pseudotuberculosis bacteria which can survive on hay, straw, and wood for up to 2 months. Also, it can survive in the soil for up to 8 months (Augustine and Renshaw 1986). In open housing, we noticed that the owners did not pay more attention to the hygienic measures and cleaning. Keeping infected animals in the flock rather than discarding them and not following treatment regimens for wounded cases can result in the increased infection rate of the herd as these animals consider a source of infection to all herd (Baird and Fontaine 2007).

The limitations of this study include that the investigated area is limited to one governorate in Egypt. Therefore, other future studies are required to investigate the prevalence of C. Pseudotuberculosis in other governorates in north and south of Egypt highlighting the risk factors associated with such important infectious animal disease. Furthermore, the diagnosis of the infection in the present study was restricted on using serological test with no identification of the infection by using molecular techniques and sequencing. Subsequently, further studies should be done to identify the infection using molecular techniques.

Conclusion

The present study determined the prevalence of C. Pseudotuberculosis in sheep in Egypt based on the serological analysis. Farmers should establish accurate preventive measures through avoiding potential risk factors associated with CLA that include keeping diseased animals for breeding and prevent introduction of newly purchased animals from flock of unknown status of infection especially old animals.