Evaluation of Internal and External Biosecurity Measures and Their Association with Mycoplasma gallisepticum Infection in Broiler Farms in Khartoum State, Sudan

The present study was conducted to investigate the internal and external biosecurity levels and their association with Mycoplasma gallisepticum infection in broiler farms in Khartoum State, Sudan. Sixty-six broiler farms were selected. A questionnaire of 77 questions on internal and external biosecurity measures was administered during each farm visit. A total of 360 blood samples were collected and used for serological examination to detect antibodies against M. gallisepticum . The overall score of biosecurity was 0.57, and the average score of internal biosecurity (0.75) was higher ( p <0.01) than the average score of external biosecurity (0.51). Poor internal biosecurity practices were observed in 23.4% of broiler farms, whereas most farms (93.6%) recorded poor external biosecurity. A significant ( p <0.01) positive correlation was encountered between the external biosecurity and the number of houses (0.62), as well as the number of workers ( r s = 0.54). Both internal and external biosecurity displayed significant positive correlations with the number of birds ( r s = 0.64, 0.35; p < 0.05). Fifty percent of the farms were positive for M. gallisepticum . The disease incidence correlated positively with the spread of manure on neighboring farms ( r s = 0.44, p <0.05). A negative correlation was observed between the presence of Mycoplasma and the distance between farms ( r s = -0.50, p <0.01) as well as farm enclosure ( r s = -0.44, p <0.05). It is concluded that broiler farms in Khartoum State exhibited low biosecurity measures and a high seroprevalence of M. gallisepticum infection. Further efforts are necessary to improve external biosecurity practices.


INTRODUCTION
Biosecurity is defined as the implementation of a set of measures that reduces the risks of the introduction and spread of disease agents, preventing infectious agents from entering or exiting the farm (Maduka et al., 2016).
Different studies have shown positive associations between biosecurity and some animal Biosecurity is subdivided into two major categories: external and internal.External biosecurity comprises all measures taken to prevent the introduction of off-farm pathogens.On the other hand, "internal biosecurity" includes all measures that address the spread of pathogens within the farm (Gelaude et al., 2014;Kim et al., 2015).
There are still great defects in the application of preventive measures on poultry farms, even with the recognized importance of biosecurity (Van Steenwinkel et al., 2011).This necessitates a better evaluation of biosecurity measures.For example, a substantial scoring system has been used in several studies conducted in Europe to quantify the level of farm biosecurity (  .This is mainly because of their design, either as descriptive studies or based on qualitative surveys, without using quantification tools. Mycoplasma gallisepticum is the causative agent of chronic respiratory disease in poultry.The disease causes immense economic losses as a result of the low performance of birds and increased medication costs.The infection with M. gallisepticum also shows immunosuppressive activities resulting in vaccination failure against virulent disease agents such as the Newcastle Disease virus.Serologic ELISA tests are commonly used for flock monitoring and the diagnosis of M. gallisepticum (Raviv and David Ley, 2013).It is noteworthy that in Sudan, vaccination against M. gallisepticum is commonly applied to breeders but not commercial flocks.However, the maternal-derived antibodies against M. gallisepticum in broilers could no longer exist by day 10 of age (Gharaibeh et al., 2013).Taken together, strict biosecurity measures are recommended as one of the practical solutions to prevent and reduce the adverse effects of M. gallisepticum (Awad et al., 2019).
The aim of this study was to quantify the internal and external biosecurity levels on broiler farms in Khartoum State, Sudan.A secondary objective was to conduct a Mycoplasma serological survey to assess the correlation between the existence of M. gallisepticum in broiler flocks and the biosecurity level of the farms.

MATERIALS AND METHODS
The study was carried out in Khartoum State, Sudan, from December 2015 to January 2019.A list of all broiler farms in the state was collated based on information gathered from different sources, including the Ministry of Agriculture, Animal Wealth and Irrigation (Khartoum State), as well as veterinarians and commercial companies working in poultry production.The list included 66 broiler farms distributed through 5 districts of Khartoum State, namely Khartoum North, Sharg En Nile, Omdurman, Jabel Awlya, and South Khartoum (Fig. 1).Only fortyseven farms (71.2%) were involved in the study (Table 1).

Study design
A questionnaire (in both Arabic and English) was designed according to the guidelines provided by Gelaude et al. (2014).General information about the farm (Table 2), along with a total of 77 questions on biosecurity measures, were included in the questionnaire.The questions were divided into two major categories, internal and external biosecurity, which in turn comprised several subcategories (Table 3).During each farm visit, the questionnaire was administered by a veterinarian using two combined methods: direct observation and an interview with either the farm owner or the supervisor/veterinarian of the farm.

Serological test for the detection of mycoplasma
A total of 360 blood samples were collected from 24 farms (15 blood samples from one broiler house on each farm) using a simple random sampling technique.Samples were collected from flocks aged 3 weeks or more.About 1 ml of the blood was collected from the wing veins of each bird in a sterile syringe.The serum was then separated and stored until it was used for serological examination.An indirect enzymelinked immunosorbent assay (ELISA) test was performed to detect the antibodies against M. gallisepticum using ELISA test kits (Synbiotics Corporation,USA,.The test was applied, and the sample/positive (S/P) ratio was calculated according to the manufacturer's instructions.The samples with S/P ratios <0.50 were deemed negative, whereas those with S/P ratios >0.50 were deemed positive.A farm was considered to be infected with mycoplasma if one or more of the fifteen samples tested positive.

Statistical analysis
Statistical analysis was applied utilizing the Statistical Package for the Social Sciences software (SPSS/PC version 25 for Windows).The collected data were coded and analyzed using descriptive and comparative statistical analysis.The questions were assessed and each subcategory of biosecurity was given a score between 0.00 (absence of biosecurity measure) and 1.00 (full presence of biosecurity measure).The average subtotal scores of internal and external biosecurity [mean (lower and upper 95% confidence interval)] were then calculated, and the overall average of biosecurity [mean (lower and upper 95% confidence interval)] score was determined as well.The scores of all subcategories, along with the subtotal scores of internal and external biosecurity of the broiler farms, were classified as poor (< 0.50), good (0.50-0.59), very good (0.70-0.79), and excellent (> 0.90); the frequency of the broiler farms was calculated accordingly.A student's t-test was used to compare the average scores of internal and external biosecurity.Spearman's Rho Coefficient Correlation Test (rs) was applied to examine the correlation between internal and external biosecurity, as well as the correlation among all subcategories.The test was also used to determine the correlation between the different subcategories of biosecurity and the incidence of mycoplasma on broiler farms.A P-value <0.05 was considered statistically significant.

RESULTS
Table 3 shows the scores of internal and external biosecurity on broiler farms.The average score of biosecurity was 0.57, while 76.19% (10/13) of the subcategories exhibited scores less than 0.70.The average score of internal biosecurity (0.75) was higher (p<0.01)than the average score of external biosecurity (0.51).Poor internal biosecurity practices were observed in 23.4% of broiler farms, whereas almost half of the farms (48.9%) revealed good internal biosecurity with an average score of 0.75.Unlike internal biosecurity, the majority of the farms (93.6%) recorded low scores and poor external biosecurity.
As shown in Fig. 2, all broiler farms involved in this study displayed poor practices concerning the supply of materials, infrastructure, and biological vectors.More than 80% of the farms also revealed poor biosecurity measures related to the export of live animals, the removal of manure and dead birds, and the entrance of visitors and personnel.The subcategory purchase of one-day-old chicks in external biosecurity recorded the highest score (0.7), whereas the supply of materials had the lowest score (0.06).The disease management subcategory in internal biosecurity displayed the highest score (0.84) as compared to all subcategories in both internal and external biosecurity (Table 4).The external and internal biosecurity showed moderate correlation (R = 0.44) (Fig. 3).As shown in Table 5, significant positive correlations were also detected between external and internal biosecurity and some of the subcategories of biosecurity.Correlations among the subcategories were also observed.External biosecurity had significant positive correlations with several biosecurity items.The strongest positive correlation (rs = 0.81) was observed between external biosecurity and the entrance of visitors (p<0.001).A moderate positive correlation (rs= 0.50) was recorded between the external biosecurity and both the removal of manure and dead animals, as well as materials and measures between compartments (p<0.01).Internal biosecurity revealed a moderate correlation (rs= 0.49) with cleaning and disinfection (p<0.01).Exports of live animals correlated positively (rs= 0.42) with materials and measures between compartments.The later has also shown a moderately significant correlation (rs= 0.51) with the removal of manure and dead birds and with the entrance of visitors and personnel (rs= 0.41).Table 6 displays the correlation of basic information with internal and external biosecurity.There is a significant (p<0.01)positive correlation between the external biosecurity and the number of houses (0.62), number of workers (rs= 0.54), and number of birds (0.64).The internal biosecurity showed a significant (p<0.05)positive correlation with the number of birds (rs= 0.64, 0.35).Serological tests for the detection of Mycoplasma demonstrated that half of the farms (50 %) were positive for Mycoplasma (Table 7).The incidence of Mycoplasma was positively correlated with the subcategories separated dead birds' storage (rs = 0.46, p<0.05), and spread of manure on neighboring farms (rs = 0.44, p<0.05).A negative correlation was observed between the presence of Mycoplasma and the distance of the nearest farm (rs = -0.50,p<0.01) as well as farm enclosure (rs = -0.44,p<0.05).

DISCUSSION
The present investigation covered about seventy percent of broiler farms located in five districts of Khartoum State.This is due to the challenges experienced in accessing all broiler farms.Firstly, some farms were not working because of either a shortage in day-old chick supplies or financial difficulties.Secondly, some farm supervisors or owners refused to participate in the study, possibly to maintain the confidentiality of their production information and flock health status.The obstacles in this study were similar to those faced in an earlier study dealing with poultry farms in Khartoum State (Sirdar et al., 2012).
The present study showed a significant difference between internal and external biosecurity scores.There was a low overall score for biosecurity, even though a high score was recorded for internal biosecurity.Similar results on broiler farms were reported by Gelaude et al., (2014) in Belgium.Poor external biosecurity practices are therefore responsible for the reduction of the overall scores.One possible explanation for the difference between the external and internal biosecurity scores in this study is that farm supervisors give less attention to external biosecurity measures, most probably due to the increase in cost as compared to internal biosecurity.More efforts are needed to improve such external biosecurity practices on broiler farms.The high score of the subcategory purchase of one-day-old chicks in this study may imply that the breeding companies applied strict biosecurity measures.In addition, the study revealed that most broiler farms used to buy their chicks from one source.This practice is supposed to reduce the risk of the introduction of disease-causing agents as stated by Hege et al., (2002).
It has become increasingly clear that human traffic and the unauthorized entrance of visitors are the key factors responsible for the spread of pathogens onto poultry farms (Thomas et al., 2005;Vieira et al., 2009).The present study showed poor measures related to the entrance of visitors; the suboptimum measure also had a strong positive correlation with external biosecurity practices.This may indicate that biosecurity hygiene measures for visitors were not properly applied at the entrances of these farms.The current investigation revealed a positive correlation between certain inadequate practices, such as the storage of dead birds, the spread of manure on nearby farms, and the occurrence of mycoplasma infection.This agrees with Dhuha (2024), who emphasized the role of inadequate biosecurity measures in increasing infection rates of M. gallisepticum.In addition, the present findings may suggest that manure is a potential vector for disease transmission.The negative correlation between M. gallisepticum infection and distance between farms and farm enclosures in the present study suggests that these factors may have a protective effect in reducing the spread of infection.

CONCLUSION
The present study revealed low biosecurity measures on broiler farms located in Khartoum State.More efforts are needed to improve farm biosecurity, with more concentration given to external biosecurity practices such as hygiene locks at main gates and the proper removal of manure and dead animals.The present study revealed that the broiler farms had a significant seroprevalence of M. gallisepticum, which was positively correlated with the spread of manure on nearby farms, indicating a potential route for disease transmission.The negative correlation between infection and farm enclosure and the distance between broiler farms suggests that these factors may play a protective role in reducing the spread of infection.

Fig. 1 :
Fig.1: The map of Khartoum State, Sudan displays the five districts of the study: Khartoum North; East Nile; Omdurman; Jabel Awlya; and South Khartoum.

Fig. 3 :
Fig. 3: Correlation between internal and external biosecurity of broiler farms in Khartoum State, Sudan using Spearman's Rho Coefficient correlation test.
A similar account has been reported by Souillard et al., (2024) in Europe.Thus, effective hygiene locks are required at the main gates of broiler farms.It is well known that the routine pickup and transport of manure and dead birds from poultry farms can spread pathogenic microorganisms and present a substantial threat (Blake et al., 1992; Ngogang et al., 2021).Poultry manure is an affordable organic fertilizer and the main fertilizer used in several African countries (Ngogang et al., 2021).The present study showed poor practices in terms of the removal of manure and dead animals.Similar findings have been reported in Nigeria (Adedayo, 2012; Moreki and Keaikitse, 2013).The poor results may indicate inefficient methods of manure removal and dead animals.It has been suggested that the dead birds' storage area must be in such a way that the rendering company can collect the mortalities without entering the farm (McQuiston et al., 2005).Mycoplasma spp. is an example of a densitydependent pathogen that can spread via the wind (Bradburry and Morrow, 2008).In the present study, serological tests for the detection of M. gallisepticum demonstrated that half of the farms (50%) were positive.Similarly, recent studies revealed a high seroprevalence of M. gallisepticum among broilers in different African countries (Mera and Mudasir, 2019; Shiferaw et al., 2022; Dhuha, 2024).Moreover, the present findings showed a negative correlation between the presence of M. gallisepticum infection and the distance between farms.It is well documented that the high density of poultry farms in a certain area is an important factor in the transmission of pathogens that are density-dependent (Truscott et al., 2007).Thus, Kammon et al., (2017) highlighted the necessity of implementing an appropriate buffer zone between poultry farms.

Table 1 :
Distribution of selected broiler farms in different districts of Khartoum State, Sudan (2016).

Table 2 :
General information of broiler farms (n=47) in Khartoum State, Sudan *

Table 3 :
Ranks of internal and external biosecurity scores [Mean (lower and upper 95% confidence interval)] of broiler farms in Khartoum State, Sudan (n=47).

Table 5 :
Correlation between internal/external biosecurity and some items of biosecurity using Spearman's Rho Coefficient correlation test.

Table 7 :
Seroprevalence of Mycoplasma gallisepticum and correlation (using Spearman's Rho Coefficient correlation test) between the infection and some biosecurity measures in broiler farms in Khartoum State, Sudan.