Pathological and molecular detection of diseases of cattle at slaughter

Background This study was carried out to identify important zoonotic diseases of beef cattle at slaughter in Bangladesh. Methods A total of 20 slaughtered cattle of both sexes, different ages and breeds were taken under investigation from Nilphamari, Rajshahi and Mymensingh district, Bangladesh during July to October, 2017. Detailed ante-mortem and post-slaughter lesions were inspected and the tissues from the lungs, liver, kidney and spleen were collected aseptically for histopathological examination. The tissue sections were processed and stained with hematoxylin and eosin (H&E) stain, acid fast stain and Gram stain. To detect the presence of specific microorganism in the tissues, the genomic DNA was extracted and polymerase chain reaction (PCR) was carried out.


Introduction
Cattle production is an essential phenomenon of our livestock economy in Bangladesh in terms of supplier of meat, milk, draught, hides, bone and other derivatives (Anon, 2008). Due to lack of awareness of meat-borne zoonoses the life of livestock producers, abattoir workers and the general public are at risk of infection (Maxwell, 2005;Ahmedullah et al., 2007;Mellau et al., 2010;Alawa et al., 2011). Records of antemortem and post-mortem inspections are useful epidemiological data for the evaluation of diseases at farm level and to verify the efficacy of prophylactic and therapeutic interventions (Ogunrinde, 1980;Antia, 1982). In this regards, slaughter houses can provide an excellent opportunity for detecting pathological lesions of both economic and public health importance (Vecerck et al., 2003;Raji et al., 2010). Frequent abattoir surveys have been conducted in different countries to detect the gross and microscopic lesions in lungs, liver, spleen and kidney of cattle  Alawa et al., 2011). However, the gross and microscopic lesions usually indicate the advanced stages of diseases. Therefore, along with the pathological findings, the use of specific laboratory tests and isolation and molecular detection of the infecting agents is necessary (Corner, 1994;Shitaye et al. 2006).
The daily encounters of diseases and the concern of the veterinary public health staff at abattoir convinced us to carry out a survey of diseases associated with slaughtered cattle at abattoir. It is necessary to know the extent to which the people are exposed to zoonotic diseases associated with abattoirs. But, little is known on the economic and public health aspects of carcass condemnation in Bangladesh. Few studies have been conducted in this regards and the frequently detected abattoir diseases or conditions were fascioliasis, taeniasis and hydatidosis (Affroze et al., 2013;Salmo et al., 2014;Islam et al., 2015). This study involved gross and histopathologic investigation of lungs, liver, kidney and spleen as well as polymerase chain reaction detection of important zoonotic diseases of cattle at slaughter.

Collection of sample
This investigation was carried out on randomly selected 20 slaughtered cattle of different ages, sexes and breeds from different slaughter houses of Nilphamari, Rajshahi and Mymensingh district. In every case, a detail ante-mortem and postmortem examination was performed to identify the gross pathological changes in lungs, liver, kidney and spleen. Tissue samples (lungs, liver, kidney and spleen) for histopathological examination were collected in 10% neutral buffered formalin and transported to the Department of Pathology, Bangladesh Agricultural University, Mymensingh. Tissues from lungs, liver, kidney and spleen were also collected in sterile tubes and stored at -20 °C for molecular detection of specific diseases. The formalin fixed tissues were processed, sectioned and stained with hematoxylin and eosin (H&E) stain, Gram stain and acid-fast stain (Luna, 1968).

Extraction of Genomic DNA
DNA was extracted by locally adopted conventional method from representative tissue samples of slaughtered cattle (Buckingham and Flaws, 2007). In brief, a total of 20 mg tissues sample was crushed in pestle and mortar in liquid nitrogen, crushed tissues was taken in microcentrifuge tube containing 600 μl of cell lysis buffer and vortexed. The solution was centrifuged at 5000g for 10 mins to collect the supernatant. Equal volume of phenol chloroform isoamyl (PCI) alcohol was added to the supernatant and vortexed. Then the solution was centrifuged at 10000g for 2 mins and the supernatant was collected. Thereafter 10μl of 5M NaCl was added on the 90 μl solution. Ice cool absolute alcohol of 250 μl was added, centrifuged at 13000g for 15 mins and the supernatant was collected. The supernatant was desalted twice by using 80% ethanol. The tube was allowed to air dry for 15 mins and 50 μl of nuclease free water was added to the tube and stored at 4°C. The concentration of genomic DNA achieved was between 50-100ng/μl in nuclease free and deionized distilled water. The purity and concentration of genomic DNA was measured in spectrophotometry (260°A/280°A) and agarose gel (1.5%) electrophoresis respectively (Spectronic R GeneticsTM New York, USA).

Selection of primer for molecular detection of diseases
Oligonucleotide primer sequences for PCR were designed by selecting genes from NCBI data base to amplify genetic fragments of tuberculosis, toxoplasmosis, brucellosis and leptospirosis (Table 1).

Electrophoresis
The amplified PCR products were electrophoresed in 1.5% agarose gel, stained with ethidium bromide and examined under UV light using an image documentation system (Spectronic R GeneticsTM New York, USA).

Results and Discussion
In this study, 80 tissue samples from 20 slaughtered cattle including lungs, liver, spleen and kidney were examined. The causal agents targeted were Mycobacterium, Brucella, Leptospira and Toxoplasma species from the slaughter cattle of three cities. The H & E staining, acid fast staining, Gram's staining of tissue sections were carried out to aid in diagnosis. Further, the confirmation of the etiology of the diseases was attempted by using PCR technique.
In this study, the distribution of suspected lesions showing in the carcasses are shown in Table 2. The results indicated that the male cattle were affected more with various diseases than the female cattle; which could be due to the facts that they were not castrated, frequently move here and there, hunt for estrous females and spend little time to eat and drink. These may lead them hunger, catching more infections and disorders. Cattle aged between 3 to 6 years of age were the most vulnerable groups as they were infected more (   ., 2015). Hydatidosis commonly infect lung tissues and hence lungs stand second in terms of infectivity of organs. Lesions in kidneys stand third, cattle mostly reared on pasture land from where they may take up dirty or rotten food staff, hence the toxic ingredient of food may pass through kidney and affected thereafter. The spleen is the sites where most of the infectious agents were destroyed but predominant lesions suggestive for specific infectivity was absent. The spleen can be affected in clinically sick or sub-clinically infected cattle; noticeable hemorrhages were seen in the spleen of two cattle and these two cattle may have septicemia before death.
During necropsy, the study found nodular development with congestion and hemorrhages in lungs of one cattle (Fig. 1a). Bovine tuberculosis is usually characterized by the formation of granulomas (Singh et al., 2017) and the studied animal was suspected to be a case of bovine tuberculosis. In cattle, multiple proliferative necrotic granulomas in the lungs, liver and nervous system is characteristic sign of bovine toxoplasmosis (Bhopale, 2003;Dubey and Jones, 2008). However, such lesions were not found in any of the studied animals. Cystic structure embedded in lungs tissues was seen in three cases. On incision, clear watery fluid was coming out and suspected as a case of hydatidosis. Pipe stem liver was seen in the liver of three cattle and the liver was severely necrosed (Fig. 2c). Presence of pipe stem liver indicates fascioliosis. Chronic fascioliasis may provide risk for tuberculosis (Flynn et al., 2007) and the cattle showed tuberculous nodules also had pipe stem liver; a relation between fascioliasis and tuberculosis was interlinked. In this study petechial hemorrhage in the spleen was seen in two cases (Fig. 2a). There was hemorrhage in the upper respiratory tract and serosal surface indicating that the animal was suffering from septicemic disease immediately before slaughter. Petechial hemorrhages were common and may be extensive, and organs are often discolored due to icterus, such lesions may also be due to Leptospira infection (Levett, 2001) but was absent in this study.  However, at necropsy, most of the diseases could not be confirmed. Representatives sample were, therefore, studied using histopathology. The histopathological examination of tissue sections stained with H & E showed granular and nodular inflammation and abscess formation in lungs (Fig. 3a, b). The granular lesions may be due to tuberculosis. Filia et al. (2016) reported accumulation of live and dead macrophages and other inflammatory cells in bovine tuberculosis. There were plenty of macrophages in the lungs parenchyma. Furthermore, lung tissue sections stained with acid fast stain showed acid fast bacilli in a case (Fig. 3c), and the case was suspected as Mycobacterial infection. This result supported the findings of Rhyan et al. (1995) and stated that acid-fast bacilli were rare to numerous and were randomly scattered in the margin of caseous necrotic tissue. Inflammatory cells of tuberculosis infection were predominantly macrophages. In this study acid fast bacilli were seen in macrophages and inflamed tissues of lung. The routine H & E staining and acid fast staining of tissue sections were further aided by using Gram staining method. Tissue sections obtained from the cattle with abscesses formation showed plenty of Gram positive bacteria in the inflammatory zone and they were cocci in appearance (Fig. 4a, b, c). The species of incriminated bacteria was not confirmed, however, the bacterial species involved in abscesses formation may be either Streptococci or Staphylococci that will require further investigation.  Tissue sections of liver stained with H&E showed wide spread cirrhosis (Fig. 5a) and formation of granuloma (Fig. 5b). Nahar et al. (2011) reported cirrhotic liver with granulomatous reaction in tuberculosis infection in cattle. There was abscesses in liver, the liquefied necrotic mass disappeared at time of tissue processing and appeared empty at time of examination under microscope. The wall of the abscess stained intensely pink and consisted of neutrophils and fibrous connective tissues (Fig. 5c). Section of pipe stem liver stained with H & E showed the presence of flukes inside the intra-hepatic bile duct (Fig. 6a). There was hemosiderin pigment in the Kupffer cells of liver. Hyperplasia of bile duct and duct epithelium was also seen in the affected liver (Fig. 6b, c).  While, sections of spleen stained with H & E and Gram stains showed distended trabeculae with lymphocytic depletion indicating atrophy of the spleen in three cattle (Fig. 7a, b, c). Dey et al. (2013) reported in histopathological study that atrophy of spleen in cow and aborted fetus is related to Brucella infection. To detect bacteria in tissues, special staining is required to carry out. Gram staining of kidney section did not reveal any bacteria either in the tubular lumen or in the interstitium. The evidence of the presence of Brucella or Leptospira could not be confirmed by using Gram staining method.  In general nephritis (a, red arrow) and glomerulonephritis (b and c, arrows) was seen in all of the kidneys. Hemorrhages in kidney parenchyma (a, black arrow) was also seen.

Polymerase Chain Reaction (PCR)
To detect specific etiology of Mycobacterium, Brucella, Leptospira and Toxoplasma in tissues, DNA was extracted from lungs, liver, kidney and spleen. Specific primers were used in PCR to identify these organisms. Out of 20 cattle investigated, one cattle showed positive amplification (346 bp) for Mycobacterium spp (Fig. 9). The primer was designed targeting the H37Rv strain of M. tuberculosis and therefore, the organism detected reasonably was M. tuberculosis. This study provides evidence that PCR is a sensitive screening assay for the detection of M. tuberculosis with DNA obtained from lungs and can generally be used to diagnose bovine tuberculosis under field condition (Sreedevi andKrishnappa, 2003, Taylor et al., 2007). However, existence of M. bovis or other species of Mycobacterium was not validated. Sequencing of the PCR amplicons and sequence analysis may enable us to confirm the species specificity of the organism targeted. Four sets of primers were designed to detect Mycobacterium, Brucella, Leptospira and Toxoplasma. However, none of the tested samples showed specific amplification in PCR for brucellosis (alkB/621), leptospirosis (HP/323) and toxoplasmosis (B1/512). It needs to test large number of samples in PCR settings and other molecular technique like ELISA to know the presence of Mycobacterium, Brucella, Leptospira and Toxoplasma organisms in various tissues of slaughtered cattle to unveil their existence of those diseases in slaughtered cattle.

Conclusions
Fascioliasis, hydatidosis and tuberculosis like zoonotically important diseases are prevalent in slaughtered cattle of Bangladesh. PCR was found as a sensitive and specific screening test to diagnose tuberculosis. Screening of large number of samples is necessary to detect the presence of important zoonotic diseases and their level of infectivity in slaughtered cattle in order to design future preventive or control strategies.