Mycobacterial infections in carcasses of ruminants slaughtered at the two slaughterhouses of Kassala, Sudan

Tuberculosis (TB) is a chronic infectious disease of both humans and animals. It is characterized by progressive development of granulomatous lesions in affected tissues and organs (Shitaye et al., 2006; Theon et al., 2006). TB is caused by slow growing non-pigmented bacilli that belong to the Mycobacterium tuberculosis complex (MTBC). Mycobacterium bovis, M. caprae, and M. tuberculosis are responsible for causing TB in cattle, goats, and humans respectively and occasionally in other animal species (Malone et al., 2003; Smith et al., 2006; Radostits et al., 2007; Marianelli et al., 2010; Mamo et al., 2011). Moreover, different species of non-tuberculous mycobacteria are able to cause infections in different species of animals (Radostits et al., 2007; Asil et al., 2013; Aljameel et al., 2014). ■ S A N TÉ A N IM A LE E T ÉP ID ÉM IO LO G IE

Worldwide prevalence of TB in cattle and other ruminants varies greatly by region and country.The disease has been effectively controlled in many countries but it is still prevalent in many others (Schiller et al., 2010).In Africa, around 85% of cattle and 82% of human populations live in areas where bovine TB is poorly controlled.In these areas, zoonotic TB caused by M. bovis is common (Ayele et al., 2004;Smith et al., 2006;Kulchavenya, 2014).Human TB is endemic in Eastern Sudan, however, knowledge on the prevalence of TB in ruminants is scarce and its epidemiology is not very well understood.This is attributed to the lack of systemic surveys and monitoring programs (Asil et al., 2013).Inadequate animal health infrastructures, traditional extensive grazing system for rearing animals, and a lesser interest in TB are other significant reasons (Asil et al., 2013;Ehsan and Nganwa, 2014).
Several diagnostic techniques can be used to investigate TB prevalence in livestock and to understand better its epidemiology and dynamics (Ramos et al., 2015).Tuberculin skin test and postmortem inspection remain the most important and economically affordable techniques, especially in resource-limited countries (Schiller et al., 2010;Ramos et al., 2015).Recently, few studies have investigated the prevalence of TB-suggestive lesions in cattle carcasses in the central and western parts of Sudan (Osman, 2007;Manal et al., 2010;Asil et al., 2013;Ehsan and Nganwa, 2014;Aljameel et al., 2014).The aim of the present study was to provide a better understanding of the epidemiology of TB and other mycobacterial infections in carcasses of ruminants slaughtered in Kassala in Eastern Sudan, as well as to identify the Mycobacterium species causing the infections.

Study area
Kassala State is located in the eastern part of Sudan at latitude 14°-17° N, and longitude 34°-37° E (Figure 1 and Buchroithner, 2006).During the rainfall season, Kassala State is rich in fodder, grazing areas, by-products of sorghum and sesame.Moreover, the State has abundant amounts of water.Gaash, Siteet and Atbrah seasonal rivers, and Halfa Al-Gadeeydah canal are full of water during the rainfall season.Port Sudan, the main port for exportation of livestock and livestock products, is located 575 kilometers to the northeast of Kassala.Mixed crop-livestock, nomadic and semi-nomadic production systems predominate in the State.Animals are bred in Kassala for domestic consumption and for exportation to international markets (Omar et al., 2009).The State has an estimated livestock population of about 4.5 million head (MARF, 2008).

Study design, sample size and sampling
A cross-sectional study was conducted from June to November 2014 in Kassala State.This State was selected as it is one of the animal production sites in Sudan.The main town of the State has two slaughterhouses: East and West Gaash.On average, around 7500 head of animals are slaughtered per month in these slaughterhouses, among which about 2500 cattle, 4000 sheep, 950 goats, and 50 camels.The two slaughterhouses were conveniently included in this survey.
The sample size (n) was calculated based on the standard formula of Thrusfield (2007).The parameters of the formula were as follows: 95% confidence level, ± 5% desired level of precision, an assumed expected prevalence of 50% as the prevalence of TB in ruminants was unknown in Kassala.We used: where n was the required sample size, (1.96) 2 the constant, P exp the expected prevalence rate of 50%, and d the desired precision level of ± 5%.
The required sample size n was determined to be 384 animals from each of the two slaughterhouses.This number was inflated threefold to account for the effect of randomness and representativeness (Thrusfield, 2007).Thus, total n was 1152 animals from each slaughterhouse.
Convenient stratification according to species and systematic random sampling were used to select animals (Thrusfield, 2007).Selected animals were identified using permanent markers, kept in a separate place and released for slaughter one after another.The sampling interval was obtained by dividing the total number of slaughtered animals on that day by the estimated daily sample size (Thrusfield, 2007).Four animals were selected each study day from each of the four species.Each of the two slaughterhouses was visited 72 times (three visits per week).Therefore, the total number of animals slaughtered on a particular day from a particular species was divided by four and every n th animal was selected after random selection of the first animal.Accordingly, n comprised a total of 710 cattle, 729 sheep, 700 goats, and 165 camels.Fewer than four camels were slaughtered per day in each slaughterhouse.To account for this, the required n per day was achieved by selecting more animals from other species.

Postmortem inspection
Postmortem inspection was performed as described by Corner et al. (1990), andCorner (1994).This included visual inspection, palpation and in situ slicing of lymph nodes, organs of the thoracic and abdominal cavities, and the head.Additionally, lymph nodes of the fore and hind limbs and the inguinal region were also inspected.

Laboratory procedures
Samples that were collected from the suspected granulomatous lesions were frozen at -20°C until shipping to the National Reference Laboratory (NRL) for mycobacteria, Research Center Borstel (FZB), Germany.At NRL, samples were minced, then homogenized and  (2011).Smears were prepared from the decontaminated and resuspended specimens according to Kinyoun's method in conjunction with an automated staining system (ZN Aerospray TB Slide Stainer/ Cytocentrifuge, Wescor, Logan, USA).Moreover, cultures were made by inoculation into a mycobacteria-growth indication tube (MGIT) liquid medium (Bactec MGIT 960 system) (Becton-Dickinson, Heidelberg, Germany) and onto solid Löwenstein-Jensen and Stonebrink media (own production).Cultures were incubated at 37°C and 31°C for six (MGIT) or eight (solid media) weeks, respectively.
For molecular characterization of the isolate of the positive cultures, DNA was extracted with Tris-EDTA (TE) boiling/sonication extraction technique or the cetyltrimethylammonium bromide (CTAB) method, respectively.Commercially available line probe assay (LPA) (GenoType Mycobacterium CM, HAIN Lifescience GmbH, Nehren, Germany) and 16S rDNA and ITS gene sequencing were conducted from TE buffer prepared samples.In addition, whole genome sequencing (WGS) was carried out with Illumina MiSeq sequencer, Nextera XT library preparation kits, and MiSeq reagent kits according to the manufacturer's instructions (Illumina, San Diego, CA, USA).Classification of the reads was done by Kraken and reads were mapped to the M. tuberculosis H37Rv genome and M. intracellulare MOTT-64 with the exact alignment program Saruman.

Prediction of the complete sequence of 16S gene by WGS
To predict the complete sequence of 16S gene, de novo genome assembly was carried out.Reads were trimmed with Trimmomatic version 0.35 for Q30 in a sliding window of 20 bp and nextera adapter contamination discarding all reads shorter than 100 bp (Bolger et al., 2014).Trimmed paired and unpaired reads were assembled with SPAdes version 3.6.2using the built-in read error correction based on k-mer frequencies and the 'careful' option (Bankevich et al., 2012;Caverly et al., 2016).The 16S gene sequence was predicted by the RNAmmer Prediction Server from the assembled contigs which were filtered for containing ribosomal sequences (Lagesen et al., 2007).
■ RESULTS In total 40,089 animals were slaughtered in the two surveyed slaughterhouses during the study period.More than three quarters (81.0%) were slaughtered at East Gaash slaughterhouse and 19.0% at West Gaash.The male to female ratio was 1:1 for cattle, goats and camel, whereas it was 1:2 for sheep.Only animals older than one year in the case of cattle, sheep and goats, and older than five years in the case of camels had been slaughtered.The animals were mostly of indigenous breeds, e.g.Gaash cattle, and Garrage, Dubassy and Gaash sheep, Baladi, Garrage and Nubi goats, and Bushari and Anaafy camels.A few were exogenous from Eritrea and Ethiopia, e.g.Karour and Barka cattle, and Arrit and Horro sheep.
The overall prevalence of granulomatous lesions in all inspected carcasses was 0.1% (2/2304).The two carcasses that had suggestive TB lesions were carcasses of sheep.No TB-suggestive lesions were detected in carcasses of cattle (0/710), goats (0/700), and camels (0/165).The detected granulomatous lesions were typical caseous necrosis, with a whitish or yellowish color.They were of various sizes congregating together, or scattered and enclosed in light congested grey fibrous tissue.They were found in the liver and lungs, and in the peritoneal cavity covering the whole abdomen and viscera (Figure 2).
Cultures revealed growth of mycobacteria from samples collected from one carcass.Samples collected from the other carcass were extensively contaminated with bacteria and yeast, and no mycobacterial growth could be found.Furthermore, MTBC isolates including M. bovis, M. caprae, and M. tuberculosis could not be grown from any of the samples.
LPA revealed a banding pattern (bands 1, 2, 3, and 10) that was not specific for any of the mycobacterium species identified by the assay (Figure 3).Moreover, 16S rDNA and ITS sequences of the isolated mycobacteria did not align with any known validly described Mycobacterium species in NCBI Blast database.
The fully sequenced genome had 4.8 mbp.Metagenomics classification by Kraken showed that 71.6% of the reads were unclassified and the closest relative was Mycobacterium sinense JDM601.Moreover, Kraken showed that only 0.2% and 0.1% of the sequenced genome was identical to the genome of M. tuberculosis H37Rv and M. intracellulare MOTT-64, respectively.
The complete sequence of 16S allocated this isolate to slow growing mycobacteria with the closest similarity to members of the M. terrae group.The alignment of the sequence of 16S in the NCBI Blast database is shown in Figure 4.
TB and other mycobacterial infections in animals are economically significant.The consequential production losses from these infections include lower milk production, loss of body weight, and condemnation of infected organs and carcasses.Animal reproduction and breeding as well as livestock trade at national and international levels are directly and adversely affected by mycobacterial infections (Radostits et al., 2007;Ehsan and Nganwa, 2014;Ramos et al., 2015).TB and other mycobacterial infections are quite prevalent in dairy growing mycobacterium species have also been detected (Sulieman and Hamid, 2002;Oloya et al., 2007;Osman, 2007;Manal et al., 2010;Asil et al., 2013;Aljameel et al., 2014;Nalapa et al., 2017).Environmental risk factors, malnutrition, presence of other diseases, and heavy infestation with endoparasites are important factors that increase the susceptibility of animals to TB (Radostits et al., 2007;Ehsan and Nganwa, 2014).Environmental risk factors have not been investigated in this study; therefore, any solid conclusion regarding their effect in the low observed prevalence of granulomatous lesions could not be made.On the other hand, emaciated animals that are likely to have TB-suggestive lesions in their organs had been excluded during antemortem examination and were not slaughtered for human consumption, in accordance with public health and animal welfare regulations.Nonetheless, some of these excluded emaciated animals might be illegally slaughtered and prepared for human consumption.This practice is common in Sudan and is locally called 'Keery slaughtering'.TB-suggestive lesions might be more prevalent in carcasses of ruminants that are illegally slaughtered for human consumption.
Sensitivity of inspection for detection of TB-suggestive lesions in animal carcasses is between 28% and 64% (Biffa et al., 2010;Schiller et al., 2010;Okoro et al., 2014;Ramos et al., 2015;Worku and Abreham, 2016).However, this technique is cost-effective for passive surveillance and TB monitoring in animals.Besides, it is affordable in resource-limited countries (Schiller et al., 2010).The very low prevalence of TB-suggestive lesions in animal carcasses reported in this study could be attributed to the limited sensitivity of postmortem inspection.Furthermore, this study was conducted over a period of six months.Such period is short, especially in case of seasonal variations in the prevalence of animal TB.The sensitivity of the postmortem inspection technique is improved when animals are tested using the tuberculin skin test before slaughtering.Granulomatous lesions are likely to be detected in internal organs of animals that are positive by the tuberculin skin test.Moreover, extended slaughterhouse-based surveys should explore whether there are seasonal variations in the prevalence of granulomatous lesions suggestive of TB or not.
In this study, neither M. bovis nor M. caprae nor M. tuberculosis have been isolated from the detected granulomatous lesions.It suggests that zoonotic transmission of these mycobacteria from animals to humans via the consumption of contaminated meat is unlikely, although it cannot be completely ruled out, especially when the meat is prepared through unsupervised Keery slaughtering.A very slight potential risk of zoonotic transmission of unknown species of mycobacteria to humans via consumption of contaminated meat exists.It is not likely that local and international trade in livestock from Eastern Sudan is going to be restricted or hindered because of animal TB.Nevertheless, occurrence of this disease in animals that are reared on farms and in animals that are grazing in pastures should be monitored.
There is no control program directed against TB in animals in Kassala.Conversely, none of the carcasses inspected at the two slaughterhouses of Kassala had been infected with any of the MTBC bacteria.Radostits et al. (2007) indicate that the overall incidence and prevalence of TB are low in indigenous animals such as zebu type cattle and this might be due to natural resistance.Furthermore, these authors specify that low overall incidences of TB have also been observed in countries where cattle are at pasture all year round.These might probably explain the findings of this study.In Sudan, most of the slaughtered ruminants are indigenous breeds, and animals are at pasture for the whole year in search of fodder and water.
One of the strengths of this study was the investigation of a large number of different animal species at the two slaughterhouses of Kassala.Therefore, the findings might be generalized to the same animal species slaughtered and prepared for human consumption in other slaughterhouses in Eastern Sudan.Nonetheless, the study had limitations.
No dairy farms have been investigated, hence, TB prevalence could and meat animals in countries where control and management measures are not in place (Cousins, 2001;Humblet et al., 2009).
This study revealed a very low prevalence of granulomatous lesions (0.1%) in carcasses of cattle, sheep, goats, and camels slaughtered at the two slaughterhouses of Kassala in Eastern Sudan.Overall in Sudan, the prevalence of granulomatous lesions suggestive of TB in slaughtered ruminant carcasses has been reported to range from 0.01% to 8.0% in different parts of the country.Lesions have been found in organs or lymph nodes, or generalized in the whole carcass (Manal et al., 2010;Asil et al., 2013;Aljameel et al., 2014;Ehsan and Nganwa, 2014).With the exception of one study that reported a small number of dairy cattle (11/569) that were positive by the tuberculin skin test in Eastern Sudan (Ayman et al., 2014), prevalence of TB in dairy and fattening farms has not been investigated in Sudan.Moreover, in neighboring and other African countries, prevalence of granulomatous lesions in slaughtered food animal carcasses has been up to 16% (Mohamed et al., 2009;Biffa et al., 2010;Manal et al., 2010;Kassa et al., 2012;Youssef and Ahmed, 2014;Okoro et al., 2014;Worku and Abreham, 2016;Nalapa et al., 2017).
Different Mycobacterium species have been isolated from granulomatous lesions collected from carcasses in Sudan and elsewhere.The main identified bacteria are M. bovis, M. farcinogenes, M. tuberculosis, M. avium, and Nocardia species.Unidentified rapidly and slowly not be compared between animals raised for meat and animals raised for milk.Additionally, the study was conducted in a short period.Perhaps TB prevalence was underestimated because of seasonal variations.Furthermore, only postmortem inspection was used as a mean to investigate TB, but granulomatous lesions are small during early stages of the disease and might not be visible with naked eyes.

■ CONCLUSION
TB was not detected in carcasses of slaughtered ruminants in Kassala.This indicates that TB incidence in animals is very low in Kassala, and zoonotic transmission of MTBC bacteria is unlikely.One of the carcasses that was found with typical granulomatous lesions was infected with an unknown Mycobacterium species.This isolate could be pathogenic to humans.Slaughterhouse-based surveys combined with pre-slaughtering testing using the tuberculin skin test would help to explore TB epidemiology in animals in Sudan.La tuberculosis (TB) es una enfermedad bacteriana crónica de humanos y animales.Se caracteriza por un desarrollo progresivo de lesionas granulomatosas específicas en los órganos afectados.La TB humana es endémica en el est de Sudan.Sin embargo, los conocimientos de la epidemiología de TB en rumiantes son raros.En un estudio de seis meses, de junio a noviembre 2014, 2304 carcasas de ganado, ovejas, cabras y camellos sacrificados en los mataderos de Gaash Este y Oeste en Kassala, fueron inspeccionadas para investigar la prevalencia de TB.Solamente 0,1% (n = 2) de las carcasas presentaron lesiones sospechosas de TB.Estas lesiones solo se encontraron en carcasas de ovejas, en hígado, pulmones y cavidad peritoneal.Las muestras colectadas en estas lesiones fueron investigadas para la presencia de micobacteria.Se encontraron en una de las dos carcasas.Este aislamiento se sometió a un ensayo de sonda lineal (GenoType Mycobacterium CM), y a secuencias genéticas ITS y 16S rDNA, así como secuencia genómica completa (WGS).Sin embargo, ninguno de estos métodos identificó esta cepa como una especie válida de Mycobacterium.No se encontró ni Mycobacterium bovis ni M. caprae ni M. tuberculosis en las lesiones granulomatosas colectadas.En conclusión, la prevalencia general de lesiones sugestivas de TB en rumiantes en Kassala fue muy baja.Estudios extensivos, combinando el uso del test de tuberculina dérmica y una investigación centrada en los mataderos, debe ser adoptarse para la supervisión de TB en los rumiantes de Sudan.
Palabras clave: rumiante, Mycobacterium, tuberculosis, matadero, Sudan ).It is delimited by a border with Eritrea (235 kilometers) in the east, and by national borders with the Red Sea State in the north, Khartoum State and the River Nile State in the west, and Al-Gadarif State in the southwest.It covers an area of 42,282 square kilometers.It falls within the Sahelian climate zone of Africa.Soils are dark, heavy, deep cracking Vertisol.Rainfall is concentrated in a single relatively short autumn season from June to September and reaches about 680 millimeters per year.Temperature ranges from a mean minimum of 17°C in January to a mean maximum of 47°C in April and May (Sulieman

Figure 1 :
Figure 1: Location of the study area in Eastern Sudan.

Figure 2 :Figure 3 :
Figure 2: Granulomatous lesions in the liver and lungs (left), and peritoneal cavity (right) of two carcasses of slaughtered sheep in Eastern Sudan.

Figure 4 :
Figure 4: Alignment of the 16S rRNA gene sequence of the isolate (Query) and Mycobacterium sinense strain JDM601 (Sbjct) sampled from the carcass of a slaughtered sheep in Eastern Sudan.