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Isolation of Listeria monocytogenes and Listeria ivanovii and its Nitric Oxide Expression Level in Serum, Brain and Reproductive Organs of Sheep

Listeria spp. is a gram positive facultative intracellular and invasive bacterium. The genus Listeria contains 2 pathogenic species, L. monocytogenes and L. ivanovii (Seeliger et al., 1986; Lotfollahi et al., 2017). L. ivanovii infection is associated with fetal death, stillbirths and premature births in ruminants, although it is less common International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 8 (2020) Journal homepage: http://www.ijcmas.com


Introduction
Listeria spp. is a gram positive facultative intracellular and invasive bacterium. The genus Listeria contains 2 pathogenic than L. monocytogenes (Low and Donachie, 1997). They both invade host cells, replicate in the cytosol after phagosomal escape, and spread from cell to cell by polymerizing actin. However, each species appears to infect different hosts: L. monocytogenes infects humans and ruminants, whereas L. ivanovii is thought to infect ruminants only (Vázquez-Boland et al., 2001). It is an emerging foodborne zoonotic pathogen of high public health significance and is responsible for 60% of hospitalizations annual mortality of 20-40% (Low and Donachie, 1997). The ruminants are the major reservoir of L. monocytogenes in which infection is generally asymptomatic but could be manifested as encephalitis, abortion, septicemia and mastitis with considerable high economic loses (Low and Donachie, 1997).
It is known that NO can directly and indirectly modulate the immune response through diverse mechanisms within macrophages and other phagocytes. That is, it works as a microbiostatic or microbicidal agent or modulates biological activity of innate and acquired immunity cell lines (Heck et al., 2005). Basic function of NO in immune system is its contribution in protective immunity against intracellular pathogens including viruses, bacteria and protozoa (Nathan 1991, Fang 1997, De Groote and Fang 1995Singh et al., 2018a. Its mechanism depends upon production of NO and superoxide (O2) by activated macrophages, which causes formation of peroxynitrite anion (ONOO-), which causes effect on pathogens. (Hess et al., 2005;Sun et al., 2006). Thus the knowledge of NO is not only important in understanding immunology but it is also a foundation for the development of new approaches for the management and treatment of various diseases, including Listeria spp. Based on above information, the aimed of this study to isolation and monitor NO expression level in serum, ovary, uterus and brain of naturally affected slaughtered sheep with Listeria. spp. infection.

Materials and Methods
In the current study the (n=78) serum and organs (comprising of 26 ovaries, 26 brain and 26 uterus) samples from the local abattoir in and around Jammu were taken and evaluate for the presences of Listeria spp. in meat and the concentration of nitric oxide in the same samples were studied. Reproductive tracts along with the brain of same animals were collected and transported to the laboratory in Phosphate Buffered saline (PBS) in an ice box within 30 minutes. Hypothalamus was separated carefully from the brain, thoroughly rinsed in physiological saline and stored at -20°C till further use. Follicular fluid was aspirated by using a separate hypodermic tuberculin syringe from each ovary. It was collected by applying slight pressure to avoid additional traumatisation of follicles. Aspirated fluid was centrifuged at 3000 rpm for 15 minutes to remove cellular debris. The sample was kept at -20°C till further use. Samples of reproductive tissues (ovarian, oviductal and uterine tissues) were collected aseptically and kept at -20°C till further use.

Isolation of Listeria spp.
Isolation of Listeria species was based on two fold enrichment procedure as recommended by USDA 2009 and as described by Loncarevic, et al., (2006) with suitable modifications. Briefly, approximately 10 g of samples were directly inoculated into 90 ml of University of Vermont-1 (UVM-1, Himedia Labs, Mumbai, India) and incubated overnight at 30°C. The enriched UVM-1 inoculum (0.1 ml) was then transferred to 10 ml of fraser broth and incubated overnight at 30 °C. The inoculum from the enriched fraser broth was streaked on oxford agar and inoculated plates were incubated at 37 °C for 48 h. The typical small, round greyish blackish colonies of about 0.5 mm diameter surrounded by diffuse black zone of aesculin hydrolysis were presumed to be Listeria spp (Fig.1). The presumed colonies of Listeria (at least 3/plate) were further confirmed.

Confirmation of the isolates
These isolates were further subjected to different biochemical tests according to standard procedure described by Cruickshank et al., (1975) and Cowan (1977). Typical colonies were verified by Gram's staining, catalase reaction, tumbling motility at 20-25 °C, methyl red-Voges Proskauer (MR-VP) reactions, CAMP test with Staphylococcus aureus and Rodococcus equi, nitrate reduction, fermentation of sugars (rhamnose, xylose, mannitol and α-methyl-D-mannoside) and hemolysis on 5% sheep blood agar (SBA). The isolates were further tested for their pathogenicity.

Pathogenicity test
Phosphatidylinositol-specific phospholipase C (PI-PLC) assay. All the biochemically characterized Listeria isolates were assayed for PI-PLC activity as per the method of Notermans et al., (1991) and Ottoviani et al., (1997) with certain modifications.
The Listeria isolates were grown overnight onto L. mono Confirmatory Agar Base supplemented with L. mono Selective Supplement I & II and L. mono Enrichment Supplement II at 35-37°C. Phospholipase C enzyme produced by L. monocytogenes and L. ivanovii hydrolyses the purified substrate (FD227) added to the medium and results in the formation of an opaque halo around the colonies (Fig.2 &3).
It also contains alpha-Methyl D-mannoside, its fermentation is shown by L. monocytogenes.
Thus, Differentiation between L. monocytogenes and L. ivanovii is achieved on the basis of alpha -Methyl Dmannoside utilization and PIPLC activity.

Determination of total nitrate
The total nitrate and nitrite estimation in hypothalamus, follicular fluid, ovarian tissue, fallopian tube and uterus was carried out as per the modified method described by Sastry et al., (2002). The method performed is as follows: Reproductive tissues (ovarian, oviductal and uterine tissues) and hypothalamus were weighed and homogenized in ice-cold 1.0M PBS with a pH of 7.4. The homogenate was then subjected to centrifugation at 4000 g for 10 minutes at 4 0 C. The supernatant was then collected in fresh tubes. Alongside test samples, standards were also processed. 100µL of supernatant (test sample) and standard solutions were taken in test tubes and to them 400 µL of 50 mM carbonate buffer was added to each test tube.

Activation of copper-cadmium alloy
Copper-cadmium alloy filings were washed twice with 100 ml of deionised distilled water in a 150 ml Erlenmeyer flask. After discarding the supernatant, the filings were washed with 100 ml 0.5N HCl. This washing removed the hydroxide of cadmium Cd(OH) 2 , which resulted in a change in texture and color. The filings appeared gray and slightly buoyant. These activated filings are quickly washed twice with 100 ml of 0.1N HCl and stored in 50 ml of 0.1M HCl at 2-8°C till further use. After activation approximately 150 mg of dried copper-cadmium alloy was added in the test and standard sample tubes followed by incubation of test tube for 1hr at 37 0 C with thorough shaking. This incubation causes full conversion of nitrate to nitrite. After incubation, 100µL of 0.35 M Sodium hydroxide was added to halt the reaction; followed by adding 400 µL of 120 mM Zinc sulphate solution on a vortex mixture. Zinc sulphate leads to deproteinization (white precipitation was seen). The test tubes were allowed to stand for 10 minutes. The tubes were centrifuged at 4000g for 10minutes at room temperature. Precipitate settled down and clear transparent supernatant stood up. Then these samples were subjected to macro assay.

Macro-assay
Aliquots (500µL) of clear supernatant were transferred to another test tube and 250µL of 1.0% Sulfanilamide (prepared in 3N HCl) and 250µL of 0.1% N-naphthylethylenediamine (prepared in water) were added with shaking. After 10minutes the absorbance was measured at 545 nm against a blank containing the same concentration of ingredients without any test sample.

Results and Discussion
Listeria spp. are ubiquitous organisms and an increasing number of recent reports show prevalence of L. monocytogenes. Listeria spp. have been isolated from various different environments, including soil, surface water, vegetation, sewage, animal feeds, farm environments, and food-processing environments (Lotfollahi et al., 2017). Of the six bacteria species within the genus, L. ivanovii is one of the two pathogenic species (the other being L. monocytogenes). It behaves like L. monocytogenes, but is found almost exclusively in ruminants (mainly sheep).
In the present study, out of (n=78) serum and organs (comprising of 26 uterus, 26 ovaries and 26 brain) samples, L. monocytogenes was detected in 5 samples i.e. 3 in uterus, 1 in ovary and 1 in brain, with the over-all prevalence of 6.41%. On other hand, L. ivanovii was isolated only in 3 uterus sample with the prevalence of 3.85%.  The prevalence of other Listeria spp. in the sample was 3.85%. All the isolates were gram positive, cocco-bacillus, positive for catalase, Methyl Red and Voges-Proskauer reaction. They showed negative reaction for indole, oxidase, H 2 S, urea hydrolysis, nitrate reduction and gelatin liquefaction. But showed characteristic tumbling motility when kept in the broth at 25°C for 18-24 hrs. In our study 8 samples showed typical β-haemolysis when streaked on HBA (5%), for 24-48 hr at 37ºC. But no hemolysis pattern was observed in 3 isolates. All the 8 positive haemolytic isolates were subjected to CAMP test and Phosphatidylinositol-specific phospholipase C (PIPLC) assay. 5 isolates showed enhancement on Staphylococcus aureus streak and 3 on R. equi streak on HBA plate. Similar results were obtained on PIPLC assay, thus 5 isolated showing yellow colonies and halo zone in PIPLC assay were confirmed as L. monocytogenes and the 3 isolates showing purple colonies with halo were confirmed as L. ivanovii.
The prevalence recorded in present investigation is lower as compared to the reported 30% prevalence of Listeria spp. from 69 brain samples of goats collected at the time of post-mortem (Shivasharanappa et al., 2014). L. monocytogenes can also be isolated from the surface and underground waters, improperly fermented silage, sewage sludge, slaughter wastes, animal and human faeces, foodstuffs, and food industry plants (Ivanek et al., 2006). In the current study it was found that there was high concentration or expression of NO in Listeria infected tissues in comparison to the tissues where Listeria was not isolated (Table.1).
Our findings are similar to the reports given by Brosnan et al., (1994) and Moncada et al., (1991), which suggests the involvement of NO (central nervous system) in pathological sequelae. It was found that (sample 7), there is increase in nitric oxide concentration in the brains of Listeria infected sheep. Therefore, nitric oxide produced by neutrophils and macrophages may play an important role in eliminating L. monocytogenes and in the histopathology of brain tissue. Even though Listeria spread to other organs but powerful anti-listerial mechanisms of NO led to acomplete elimination of L. monocytogenes from the brain and other organs. There was isolation of L. monocytogenes from sample 18 (uterus), but the concentration of NO was found very high in the serum in comparison to the tissue. It may suggest chronic infection in the uterus, but the high NO level of serum does not result in spread of infection to other organs. However some earlier workers suggest that the role of NO˙ as anti-listerial activity is controversial (Ohya et al., 1998;Samsom et al., (1996). But Remer et al., (2001) had reported that there is neutralize of L. monocytogenes by the production of NO from macrophages. The role of NO generation in ruminant listeriosis remains to be studied. Another question raises that whether NO generation is essential for the elimination of L. monocytogenes in the brain. However some studies show that killing of L. monocytogenes in-vitro was dependant on NO (Fehr et al., 1997). Although L. monocytogenes are by far the leading cause of human and animal's listeriosis, and its role of high NO in infected sheep. But, our report shows that L. ivanovii infected can also cause enhance expression of NO levels. As we know, Listeriosis causes sudden death in animals, thus the functional role of nitric oxide (either iNOS or nNOS) in Listeria spp. affected animals needs to be further studied.