The present epidemiological study was an attempt to evaluate and compare the seroprevalence of MAP with ELISA method in the cattle, sheep and goats following the detection of clinical cases. Moreover, the relationship between the infection with determinants such as species, age, gender and geographical location was investigated. In MAP infection, there is a relationship between active infection and shedding with high serum antibody titre [33–35]. Therefore, in this study, the results of ELISA indicated antibodies in cattle, sheep and goats that might have active infection and possibly shed MAP in their faeces and milk; however, animals with subclinical infection may be missed. Collins et al. (2005) showed that there is a direct relationship between the magnitude of ELISA results and the odds of a cow shedding MAP [33]. The results indicated that following the detection of clinical cases, the apparent and true seroprevalence of MAP respectively is 4.34% and 7.59% in cattle, 6.87% and 13.34% in sheep, and 7.07% and 13.68% in goats. Thus, in an endemic area, the rate of infection is similar in cattle, sheep and goats and so the species is not a risk factor for it. Also, Cramer’s V coefficient showed that there is a very slight to slight relationship in seropositivity rates between these species in 4 cities, including Susangerd, Ahvaz, Dezful and Hendijan.
In addition, there are reports that indicate the prevalence of infection in herd and livestock in different contries; however, because different methods have been used for detection of MAP antibodies and the difference existing in host and environmental dereminants, it is difficult to compare them with each other [7, 36]. For example, the frequency of MAP infection in slaughtered cattle in Ahvaz abbatoir was 3% and 2% by ELISA and Ziehl-Neelsen staining methods, respectively [22, 29, 30] but in sheep and goats in the same slaughterhouse by Ziehl-Neelsen staining, was 1.4% and 0.96%, respectively [31]. In other areas of Iran, the MAP infection rate in cattle was 3.6–25% by ELISA, PCR, culture and Ziehl-Neelsen staining methods which sera, milk, and feces were used [21, 23–28]. The prevalence of MAP infection in cattle from other countries has been reported to be 2.31 to 70.4% [37–46]. The MAP infection rate in goats from some area of Iran was 37% and 17.3% by PCR and culture methods, respectively [32] but it was 0.3–45.1% in other countris [47–56]. The prevalence of infection in sheep has been reported as 2.4–21.1% [48–50, 53, 57, 58]. A quick glance at the results of these studies shows that they are different. This may be due to the difference in the sample size, sampling method, methods of examination, herd size and managment, environmental and host determinants. In order to diagnose the infection to MAP, different methods such as fecal culture, Ziehl-Neelsen staining (different parts of the intestine, lymph node of ileocecal valve and other parts of intestine and fece), PCR, ELISA, CFT and AGID are used. Both the sensitivity and specificity of each of the above methods are different. That is why, the type of diagnostic test used in the study of could be referred to as one of the basic reasons of the difference existing in the frequency of infection to MAP. Moreover, scince the cattle in a herd infected by MAP are divided into four groups as those: 1- exposing clinical symptoms and excreting the bacteria; 2- being subclinically infected and excreting the bacteria; 3- being infected by MAP but not excreting the bacteria to the extent that it could be traced; 4- not being infected [7], therefore, in addition to the difference in sensitivity and specificity, the above categorization can also affect the general outcome of using a specific method. For example, the sensitivity of fecal culture is different depending on the stage of disease. Different methods of serology seem to be different from the standpoint of sensitivity and specificity as follows: CFT 90% and 70%, AGID 96% and 94% and ELISA 45% and 99%, respectively (Constable et al., 2017). In general, studies show that the specificity of the ELISA kit is very high, but the sensitivity is low [59, 60]. In diagnosing the infection in young and newly infected animals, because of the lack of enough antibody production, the ELISA has less sensitivity; thus, the diagnosis power of ELISA increases with advancement of the disease and the increase of antibody production [61]. Juste et al. (2005) examined the power of both ELISA and PCR in detecting MAP in cattle and sheep. They showed that out of 136 samples that had a positive result by ELISA or PCR, only 10 samples were positive by both methods, whereas 70 samples were positive only by ELISA and 56 samples by PCR. Therefore, agreement between the two tests was low. In fact, each method would detect different stages of MAP infection because their respective targets (bacteria and antibodies) might not have parallel dynamics. The young animals were more easily diagnosed by PCR than by ELISA, possibly because of the rapid recirculation of MAP-loaded phagocytic cells from the intestinal lymphoid tissue into other lymphoid tissues after the infection, reinfection, or reactivation [62]. This should be expected to be more frequent among young animals newly exposed to MAP than in adults known to be more resistant to infection. In contrast, because of the antibody response is slow to develop and highly dependent on the total number of mycobacteria, the most advanced cases should have detectable antibody responses [63–64]. In general, microscopic examination of feces and different parts of intestine mucus, using Ziehl-Neelsen staining method is not reliable enough to trace the MAP [7].
MAP is mostly transmitted to susceptible animals via ingestion of contaminated milk, water, and other feed products or uptake from the environment. With newborn calves being the most susceptible age group for MAP infection, contaminated colostrum and milk are considered a primary source of infection. MAP is introduced into milk and colostrum either via contaminated teats or direct shedding of the organism into the colostrum/ milk. In the same line, infected cattle and other species excrete MAP directly into the milk during at least the late disseminated stage of the infection. Therefore, there is resistance to disease increases with age and older animals appear susceptible to infection but relatively resistant to progression to disease. [65]. Experimental and field studies showed that infection becomes more difficult when calves are 4 months or older, and susceptibility to infection from 1 year of age on appears to be similar to that of adult animals [7]. The age of all the three examined species was more than 6 months old and had the same susceptibility to infection. For this reason, as results showed there was no significant relationship between age and infection in cattle, sheep and goats and most infections occurred in the early stages of life. Besides, in this study, the average age of infected cattle, sheep and goats was similarly about 4 years. In fact, the age of infection was the same in these species. Stau et al. (2012) and Morales-Pablos et al. (2020) also proved that there was no relationship between age and infection in sheep and goats [53, 58]. However, Cetinkaya et al. (1996), Woodbine et al. (2009), Weber et al. (2010), Fecteau et al. (2010), Attili et al. (2011) and Karimi et al. (2012) showed that age was significantly related to infection [6, 26, 66–69]. This reported relationship may be due to bias types including, selection, information or measurement, confounders such as husbandry and management and selected statistical method.
In this study, the relative frequency of positive cases in females and males was the same. Also, Kimberling (1988), Anderson et al. (1992), Karimi et al. (2012), Stau et al. (2012), Constable et al. (2017) and Morales-Pablos et al. (2020) showed that there is no statistically significant relationship between infection and gender [7, 26, 53, 58, 70, 71]. Generally, the MAP, not attending to a specific gender, infects both males and females and infection is not related to sex determinants such as hormonal, occupational, behavioral and genetic determinants.
The effect of geographical region on MAP infection rate might be due to the difference in animal management such as herd size, health, feeding and stress. In this study, the relationship between geographical location and infection in cattle, sheep and goats was not statistically significant. However, there was a significant relationship between geographical location and infection in cattle by univariate analysis, which might have appeared due to bias and confounding factors such as age. Therefore, this reported spurious association was eliminated by controlling the confounding factors in multivariate regression. In line with the results of the present study, Cetinkaya et al. (1996), Lombard et al. (2006), and Morales-Pablos et al. (2020) indicated that there is no significant relationship between geographical location and infection [58, 66, 72]. However, Singh et al. (2014) proved that there is a significant relationship between the above mentioned variable [73].