- How brucellaphages can fight a deadly cattle disease
By Arfat Yousaf Shaheen
Pakistan, being an agricultural country, has a large livestock population, which is a vital sub-sector of agriculture, with a pivotal position in the overall economic strategy of Pakistan. The sector contributes around 21 percent to GDP, out of which the share of livestock and dairy sector is 60.54 percent. The share of the livestock sector in exports is about 13. In the FAO ranking, Pakistan is ranked at the second position in buffalo population and fourth in global livestock population. The sector has potential to grow rapidly but currently it is facing challenges such as infectious diseases, management issues and farm biosecurity. Among various infectious diseases, brucellosis is one of the top ranked bacterial diseases prevalent in developing countries. Bovine brucellosis is an economically significant bacterial disease having zoonotic potential globally and is mainly caused by Brucella abortus.
It is a highly contagious chronic infectious disease in animals and is a major source of production loss to livestock owners, especially in developing countries, whose economics are largely dependent on livestock. This disease mainly affects cattle but it also affects other animals such as camels, water buffalo, bison, and elk. It causes abortions, retained placenta, infertility, orchitis and epididymitis, but its main effect is abortion in animals. The abortion rate in naive cattle is 30-80 percent. Brucellosis can be transmitted to people through direct contact with infected material from animals or indirectly by ingestion of contaminated dairy products. Once infected ruminants become chronic carriers though they are usually asymptomatic after their first abortion, and may shed the organism in the environment. Therefore, during subsequent pregnancies they continue to shed Brucella in milk as well as uterine secretions. Brucella also spreads through contaminated food and water. Various environmental factors, such as low temperatures, high humidity and no sunlight, favors survival of the bacteria in animal manures, dead fetal material, wool and hay, as well as equipment and clothes available in the vicinity of affected herds for several months. Brucella is tolerant to drying especially in the presence of organic material and can survive in soil.
Current use of antibiotics and vaccination strategies are not optimal and only culling of positive animals is recommended to control the disease. Culling of all Brucella-positive animals to eradicate the disease across the country is not practical due to limited resources and the high cost of dairy animals. WHO has recommended the use of smooth live vaccine (S19) and rough attenuated strain (RB51) for cattle, but both of these vaccines are not effective enough in controlling the disease. Further, being live vaccines, if injected in pregnant animals, they can cause abortions. Alternatively, bovine brucellosis could be controlled effectively using biological substances such as brucellaphages. The use of host-specific bacteriophages has been promoted as a cost-effective and adaptable approach to control zoonotic bacteria like Brucella. Bacteriophages are viruses which exclusively infect bacteria. These viruses attack specific bacteria and kill them by replicating inside bacterial cells and are also called bacterial viruses. Some studies demonstrated presence of bacteriophages in almost every known bacterial species and thus they can be found from vast habitats. Bacteriophages are found to be an effective agent to control and treat many bacterial diseases. These bacterial viruses are very specific to their host which only target the specific bacteria without impacting human or animal cells. The phages use the metabolic machinery and genes of host bacteria and establish a lytic cycle for their own replication and ultimately kill the bacterial cells. Recently, phage therapy has emerged as an effective alternative to antibiotics as the irrational use of antibiotics has led to resistance all over the world.
We offered a systematic approach for the isolation and propagation of brucellaphages. Consequently, these brucellaphages can be used for the effective bio-control of bovine brucellosis in fields to control the spread of brucellosis among dairy animals and also to reduce the economic losses due to this deadly disease of dairy sector
The first study has been conducted in Punjab, Pakistan. Brucella cells can be infected by lytic phages known as Brucellaphages. Bacteriophages have varied practical applications, so we did a basic study for physicochemical and molecular characterization of brucellaphages so as the indigenous isolates of brucellaphages may be available for future application in field. Brucellaphages were isolated from 50 slurry samples or semi-liquid mixtures of manure, collected from cattle farms. Procedures were developed and growth conditions were optimized for the isolation and propagation of lytic bacteriophages active against Brucella abortus. Seven samples were found positive in screening and two isolates were confirmed for brucellaphages. Physicochemical characterization of brucellaphages showed that they can withstand temperature upto 60oC but when exposed to 70oC their viability decreased and beyond 70oC they were completely deactivated in one-hour exposure. They have stability at pH 7 to 9, but acidic pH of 2 to 4 can decrease the phage viability in one-hour exposure. Evaluation of thermal stability and optimum pH conditions of brucellaphages are helpful to standardize the phage therapy as well as in food industry such as in pasteurized dairy products. Extreme resistance to temperature is advantageous for brucellaphages to apply in field conditions to keep phages working in harsh conditions.
Brucellaphages have considerable stability in fluorescent light and sunlight and it was observed that there is slight reduction in phage viability after one-hour exposure. However, ultraviolet light deactivated the phage within 15 minutes. Consequently, it is indicated, sunlight would have some reduction effect on survival of brucellaphage, therefore there is a need to keep a check on phage survivability with contact time if applied in the environmental conditions. Treatment with chloroform, Sodium Dodecyl Sulphate, EDTA, Lysozyme, and Proteinase K inactivated phages in 15 minutes, while normal saline, RNAse and Trypsin had no effect on brucellaphages. Growing interest in using bacteriophages for the control of diseases requires exploring the most suitable long-term storage temperature of bacteriophages. Storage stability of tested brucellaphages revealed that they have maximum stability at 4oC as they retain their viability up to 12 months. However, freezing temperatures found not to be favorable for storage of phages as they lost their viability to 100 percent after a 12-month period at -80oC and 50 percent at -20oC. In conclusion, we offered a systematic approach for the isolation and propagation of brucellaphages. Consequently, these brucellaphages can be used for the effective bio-control of bovine brucellosis in fields to control the spread of brucellosis among dairy animals and also to reduce the economic losses due to this deadly disease of dairy sector.
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