Seroprevalence of hemorrhagic septicemia in buffalo and cattle in flood , irrigated and sandy areas of Punjab , Pakistan

Hemorrhagic septicemia (HS) is an acute, fatal, and septicemic disease of cattle and buffalo caused by Pasteurella multocida. It is particularly important in the tropical regions of the world, especially in Asia and Africa. The study was conducted to record the HS disease prevalence rate in different areas (flood, irrigated and sandy), species of animals, and different age groups. Moreover, the antibody titer against HS of vaccinated and nonvaccinated animals (cattle and buffalo) in different areas against HS disease. Morbidity, mortality, and case fatality rate (CFR) due to HS disease in the different animal population (cattle and buffalo), age groups (young and adult), various areas (flood, irrigated and sandy) and vaccinated and unvaccinated animal groups. It can be concluded from the present investigation that HS is endemically occurring in cattle and buffalo. The mean of morbidity, mortality and CFR in young buffalo (12.56%, 22.44%, and 40.44%) and adult buffalo (0.94%, 2.82% and 25.68%) higher than young cattle (2.42%, 6.46%, 28.74%) and adult cattle (0.54%, 1.97% and 17.63%), which shows that buffalos are more susceptible of disease as compared to the cattle. Moreover, the young population of buffalo and cattle highly affected (morbidity, mortality, and CFR) of HS as compared with the adult stock. It was also noted that the morbidity, mortality, CFR was greater in flood area (7.78%, 13.95%, and 39.05%), and lesser in the sandy area (0.84%, 3.76%, and 11.84%). The comparative value of GMT against HS, deducted with IHA test both for cattle and buffalo, and it was noted that GMT in vaccine status and category of animals showed that it was higher in vaccinated animal than nonvaccinated animal population. Moreover, the GMT was higher in the sandy area as compared with flood and irrigated area. Furthermore, the GMT for vaccinated buffalo was lesser as compared to the vaccinated cattle making them more susceptible to the disease.


Introduction
Pakistan is an agricultural country, livestock and dairy farming plays a major role in the economy of Pakistan [1, 2].Livestock is the sub-sector of agriculture and its share in agriculture's GDP is round about 58.3% and 11.4% of the total GDP of Pakistan during the year 2016-17 compared to 58.3% and 11.6% during the corresponding last year respectively.Livestock revenue increased from Rs. 1288 billion  to Rs. 1333 billion .The above picture depicted that 3.4% increase during the last year.Livestock share in the economy of Pakistan may be seen because there are round about 8 million families being involved in the livestock sector, generating 35% of their income from the livestock production In Pakistan farmers mainly rely on their animal's income for their survival and it is reflected annually in GDP of Pakistan.Farmers are frequently facing many problems which affect the income of the farmers due to certain hurdles such as the onset of infectious disease, duration of illness of animals, nutrition costs and vaccination costs [7].Previously due to five economical endemic diseases like HS, FMD, Rinder Pest, Anthrax and Black Leg 58.7% deaths reported [8].It was observed during participatory disease surveillance in Karachi that HS has higher economic losses than FMD due to higher mortality rate [9].The morbidity losses due to HS considered 23% of the total losses and mortality losses contributed 77% of the total losses.The share of calves was round about 74.8% of the total losses due to HS disease [10].The prevalent serotyping of Pasteurella multocida causing Hemorrhagic Septicemia (HS) is listed as B disease i.e B2 (Asian Type) E2 (African Type).Other serotypes reported are A (1), A (1, 3), A (4), B (1), B (2, 5), B (3, 4), E (2, 5), F (3), F (3, 4) [11].HS is mostly the disease of river valleys and case reported mostly from the Delta of South East Asia among the buffalo [12,13].
District Muzaffar Garh (M.Garh) have cattle (1.436), buffalo (0.647), sheep (0.441), goat (0.994) Million respectively [14].District M. Garh is situated B/w River Chanab, and River Indus in the form of a strip and these rivers pass along the eastern and western borders of the district respectively.District M. Garh has extreme hot and cold climate, and due to river access, some parts are flooded every year [14,15].Therefore, disease outbreak risk of HS disease in such areas is remaining every year in flood areas, so it is imperative to follow the better vaccination strategies of animals especially living in flood areas to combat the outbreak of HS disease.In some areas of Punjab and North West Frontier Post, study on seroprevalence of Hemorrhagic Septicemia (HS) was being done in cattle and buffalo [12,16].In another study, Antibody titer in cattle with the alum-precipitated vaccine was monitored [15,17].Whereas, the routine vaccination without monitoring their efficacy (antibody titer) is the whole problem of L&DD Department Punjab.Therefore, an epidemiological investigation, in the present study was designed.The primary objective of this investigation was, therefore, to evaluate the prevalence of HS disease and antibody titer against HS in cattle and buffalo in Dist.M. Garh.Moreover, affects of the various risk factor were studied on the frequency of HS disease outbreak.

Study area
This project was designed to examine in the area of District M. Garh.Muzaffargarh is situated in Southern Punjab Province, almost exact center of Pakistan.The area is spread over 8435 km 2 , it forms a strip between river Chenab in the east and river Indus in west district Multan and Khanewal are present on the eastern side of the district M. Garh while on western side D. G. Khan district, on north side district Layyah and south side district Bahawalpur is present.District M. Garh has four tehsils as Kot Addu, Ali Pur, Jatoi and M. Garh and in the study different UC's of all these tehsils were randomly selected.During the monsoon of 2010, almost 50% population of people and animals were mostly affected by the flood.The study was carried out in cattle and buffalo (young and adult) found in different areas (flood area, irrigated area, and sandy area) to determine the prevalence of HS and its morbidity, mortality, and CFR.Whereas, different risk factors like housing, feeding, and watering of the animals in these areas were also studied.The serum samples were also analyzed to check the antibody titer of the vaccinated and nonvaccinated animals of these different areas.

Sampling strategy
Two groups of cattle and buffalo vaccinated and nonvaccinated animals against HS disease were selected.The blood samples from cattle and buffalo were collected from the flood, sandy and somewhat marshy areas of District M. Garh 5 ml blood was collected from each animal, the sample was kept for 12 hr. in a slanting position at room temperature for the collection of required serum samples and was stored serum at -20 °C.The antibody titer of the vaccinated and nonvaccinated animals was checked by Indirect Hemagglutination Test (IHA).

Population under study
Sero-prevalence of HS was done in the area of district M. Garh to understand the disease prevalence and economic losses due to a disease outbreak.Randomly ten villages were selected from 3 different areas as flood area, irrigated area and the sandy area from four Tehsils of District M. Garh (Kot Adu, Alipur, Jatoi and M. Garh.The total population of animals from these areas selected 4533 having 2614 cattle and 1919 buffalo.Total 2359 vaccinated and 2174 not inoculated animals were studied (Table1 & 2).From the study area, ten farmers were randomly selected for data collection from each village and each Union Council level, ten villages were selected randomly.10villages further subdivided into vaccinated and nonvaccinated groups (animals of First 5 villages were vaccinated, and other five were not vaccinated).Each farmer of different villages from all areas under study was interviewed, and by using proportional piling and seasonal calendar, data was collected on the prescribed questionnaire.Further 200 serum samples were taken from each area, 50 samples were collected from vaccinated, and 50 nonvaccinated animals from cattle and buffalo of that respective area and their antibody titer was checked against HS disease.

Research design
The study area is categorized into three zones that flood area, irrigated area, and sandy area.From each area 10 villages were selected, from each village of each area, 10 farmers were randomly selected of which five were vaccinated and five nonvaccinated.Mortality rates, Morbidity rate, and CFR were observed in different animal population (cattle and buffalo), age groups (young and adult), areas (flood, irrigated and sandy).After blood sample collection from cattle and buffalo, further study was done in M. Garh Disease Diagnostic Laboratory, Livestock, and Dairy Development Department.Antibody titer by IHA test of serum samples was performed.

Serological analysis
Antibodies against Pasteurella multocida were measured by indirect hemagglutination test (IHA) using human blood group 'O' [16,18].Briefly, two-fold dilutions of the test sera starting from 1:5 to 1:640 were made in normal saline using microtiter plates (96 wells) and added the 25µl amount to all the wells of plate except those of column 11 and 12 which served as control.First four wells (A-D) of column 11 were added with known negative serum and last four wells (E-H) with the known positive serum.Sensitized RBC's (1%) was added in equal amounts (25µl) to all the wells of the plate so that column 12 served as a control for the RBC's.The plates were incubated at room temperature for two hours, and the observations were recorded.
After that, the plates were kept re-incubated under refrigeration for overnight, shaken vigorously, allowed to resettle and were read again.The IHA test was performed as formerly reported [18-20].Results were interpreted as positive (no bead formation) or negative (bead formation with sharp, clear margins).The IHA antibody titers against HS were converted into geometric mean titers (GMT) for each group [19][20][21].

Statistical analysis
The statistical analysis was performed by SPSS 16.0 (SPSS Inc., Chicago, IL, USA).The results of all experiments were analyzed analysis of variance (ANOVA), followed by Latin square design (LSD) allpairwise comparisons test to compare the significance (P) between the means of morbidity, mortality and CFR of different groups (young and adult; buffalo and cattle; vaccinated and nonvaccinated; flood, irrigated and sandy areas).P ˂ 0.05 was considered to indicate a significant difference between the values compared.

Comparison of means of different parameters under investigation
The comparison of the means presented in (Table 5).It was noted that among the age group the morbidity, mortality and case fatality in young buffalo was 12.56%, 22.44%, 40.44% which was significantly higher as compared to the young cattle except case fatality (28.74%) which has no significant difference with young buffalo.However, the adult group the morbidity (0.94% in buffalo and 0.54% in cattle) has no significant difference, whereas the mortality and case fatality was significantly higher in adult buffalo (2.82% and 25.68%) as compared with cattle (1.97% and 17.63) (Table 5).Moreover, the nonvaccinated animal population has significantly higher morbidity, mortality and case fatality (6.89%, 12.13%, and 39.99% respectively) than vaccinated animal population (1.33, 4.71 and 16.25 respectively) (Table 5).Furthermore, the morbidity (7.78%, 3.71%, and 0.84%) in different areas (food, irrigated and sandy) have no significant difference among them.However, the mortality, case fatality (13.95%, 39.05%) was significantly higher in flood area, and lesser in the sandy area (3.76% and 11.84%) (Table 5).

IHA Titer against HS in various areas
The flood area nonvaccinated buffalo in flood area the GMT was recorded 6.97 while in vaccinated buffalo recorded 45.16 (Table 6), whereas, GMT in nonvaccinated cattle recorded 8.29 while in vaccinated cattle observed 49 (Table 6).In the irrigated area, the GMT in nonvaccinated buffalo was noted 8.88, while in vaccinated buffalo it was recorded 45.16 (Table 6).The GMT in nonvaccinated cattle was 8.29 whereas, in vaccinated cattle was 49 (Table 6).However, in the sandy area, the GMT among nonvaccinated buffalo was 13.54, while in vaccinated buffalo it was noted 47.33 (Table 6).Whereas the GMT in nonvaccinated cattle recorded 10.24 while in vaccinated cattle it was recorded at 47.95 (Table 6).Discussion Hemorrhagic septicemia is a globally significant livestock disease.However, due to poor livestock farm practices, it is more severe in Asia and Africa.The climate change for the animal is important, as the particular breed rearing under adverse environmental conditions or environment, influence the exposure to the disease and cause substantial economic losses to the livestock breeders [22].This is the first report on seroprevalence of HS disease in changing climate condition (flood, irrigated and sandy) in M. Garh.Moreover, the study was carried out to check the prevalence of HS disease in district M. Garh in different geographical areas (flood, irrigated and sandy) and different age groups (young and adult).The population of animals in district M. Garh was 4533 in which young animals were 1347 while adult animals were 3186.The seroprevalence of HS in various age group was already investigated in different districts of Pakistan [12, 16], whereas in different geographical region or condition were also investigated [13].
In our study area vaccinated animals in flood areas were 657, in irrigated areas were 940 and in the sandy area were 762 while nonvaccinated animals in flood area were 813, in the irrigated area were 526 and in the sandy area were 835.In another study, animals were selected in towns, small villages, and big villages.The animals were compared regarding the HS disease outbreaks in these different areas [12,13].area (Table 4).The same was observed previously that in young animal population the morbidity, mortality and case fatality was higher than adult animal population [16] this may be due to the better immune response or having already exposure of the microorganism from the environment or by vaccination.
It was noted that among the age group the morbidity, mortality and case fatality in young buffalo was significantly higher than young cattle except case fatality which has no significant difference with young buffalo.However, the adult group the mortality and case fatality was significantly higher in adult buffalo as compared with cattle [11].Moreover, the nonvaccinated animal population has considerably higher morbidity, mortality and case fatality than vaccinated animal population (Table 5) [9].Furthermore, the mortality, case fatality was significantly higher in flood area, and lesser in the sandy area (Table 5).
The comparative value of GMT against HS deducted with IHA test both for cattle and buffalo presented in (Table 6).GMT in vaccine status and category of animals showed that it was higher in vaccinated animal than nonvaccinated animal population.Moreover, the GMT was greater in the sandy area as compared with flood and irrigated area.Furthermore, the GMT for vaccinated buffalo was lesser as compared to the vaccinated cattle making them more susceptible to the disease.The results are in line with previously noted finding, the higher GMT values for cattle as compared to those of buffalo [16,18].

Conclusion
The highly endemic nature of HS disease indicates its important in cattle and buffalo in Asia and Africa.The mortality and case fatality were high in HS disease unlike other livestock diseases, indicates the importance of disease in maintaining the overall animal assets and lively hood of livestock farmers.The control option mitigates the disease especially in more susceptible species (buffalo and cattle) and area (flood, irrigated and sandy) and if vaccination is planned and implemented based on the epidemiological results, the disease burden can be minimized.It can be concluded from the present investigation that HS is endemically occurring in cattle and buffalo.Buffalo are more susceptible of disease as compared to the cattle, moreover, the young population of buffalo and cattle highly affected (morbidity, mortality, and CFR) of HS as compared with the adult stock.It was also noted that the morbidity, mortality, CFR was greater in flood area, and lesser in a sandy area.
In Pakistan, there are vast numbers of cattle and buffalo population (41.2 million & 35.6 million respectively) [5].Another report indicated that annual beef production in Pakistan was 1.6 million tons while milk production was 43.6 million tons [6].