Hematological and antimicrobial resistance patterns in buffalo calves with Hemorrhagic septicemia: A clinical study

Introduction

The pathogenic bacterium Pasteurella multocida (P. multocida) is a major concern in veterinary medicine because it can cause a variety of diseases in livestock, such as respiratory infections, pneumonia, and Hemorrhagic septicemia (Harper et al., 2006; Hill and Brown et al., 2011; Souza, 2009) [6,7,16] . This bacteria causes significant financial losses in the agricultural sector in India and is common in a variety of animal species, such as pigs, sheep, cattle, and buffalo (Govindaraj et al., 2017) [5] . Additionally, P. multocida's zoonotic potential poses a threat to public health, especially in areas where intimate human-animal interactions are common (Wilson and Ho, 2013) [19] . Antibiotic-resistant P. multocida strains are becoming more common, which is a serious problem since they make treating infections more difficult and raise the possibility that resistant strains could spread among animal populations (Oh et al., 2018) [10] . There have been reports of resistance to popular antibiotics including ampicillin, tetracycline, and streptomycin in different parts of India, which calls for a more thorough investigation of the underlying resistance mechanisms (Uddin et al., 2021) [17] .

The existence of integrative and conjugative elements (ICEs) and mobile genetic elements, which promote the horizontal transfer of resistance genes between bacteria, is frequently the cause of antibiotic resistance (Partridge et al., 2018) [13] . These genetic elements support the persistence and dissemination of antibiotic resistance in P. multocida, in conjunction with efflux pumps and other resistance mechanisms (Li et al., 2015) [9] . Knowing these processes is crucial for controlling existing infections as well as anticipating and stopping resistant strain outbreaks in the future. Whole-genome sequencing and molecular methods have emerged as crucial instruments for detecting P. multocida and describing its resistance patterns in order to solve this problem. These techniques enable the identification of integrative and conjugative elements (ICEs), mobile genetic elements, and resistance genes-all of which are crucial for the spread of resistance (Peng et al., 2019) [14] . These kinds of insights are essential for creating focused interventions to control and lessen the effects of P. multocida strains that are resistant to antibiotics. This study's main goal is to look at the genetic processes that underlie antibiotic resistance in P. multocida strains that have been isolated from different animal hosts in India. In order to better understand P. multocida resistance patterns and develop management and control measures, this study will analyze resistance genes, mobile genetic elements, and other genomic aspects.

Materials and Methods

The study was conducted on a total of 46 buffaloes, including 23 clinically affected animals showing symptoms of hemorrhagic septicemia (HS) that were presented at the Veterinary Clinical Complex, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya (Uttar Pradesh), India. Additionally, 23 apparently healthy buffaloes maintained at the Department of Livestock Farm Complex, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya (Uttar Pradesh), India, were included as controls. The study aimed to evaluate alterations in hematological parameters and to assess the antibiotic resistance patterns of the isolated organisms.

Blood Sample Collection

Blood Samples for Hematology 5 ml of blood was collected aseptically from the jugular vein using a sterile 18-gauge needle and transferred into an EDTA vacutainer for Complete Blood Count (CBC) analysis using an automated hematology analyzer.

Blood Samples for Bacterial Isolation

An additional 5 ml of blood was collected in a plain sterile vacutainer (without anticoagulant). Immediately after collection and before clotting, 1 ml of whole blood was aseptically transferred into 9 ml of Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 hours to promote bacterial growth.

Nasal Swab Collection

Using a sterile cotton swab, a nasal sample was gently taken from the same animal. The swab was inserted into the nasal cavity, rotated, and placed into transport media. Bacterial Isolation and Identification  After enrichment in BHI broth, a loopful of the culture was streaked on 5% sheep blood agar and incubated at 37°C for 24-48 hours under aerobic conditions.  Colonial Morphology: Typical colonies of Pasteurella multocida were identified based on their small, grayish, non-hemolytic, mucoid appearance.

Results and Discussion

Giemsa-stained blood smears revealed the presence of bipolar organisms consistent with Pasteurella multocida. Hematological analysis of 23 buffalo calves demonstrated significant deviations from standard reference ranges, indicative of active infection. The TEC (8.3 ± 0.2 million/cu mm) and Hb (12.1 ± 0.4 gm/dl) were within upper normal limits, suggesting uncompromised oxygen-carrying capacity. Elevated TLC (16,000 ± 500/cu mm) and marked neutrophilia (72.9 ± 3.5%) reflected acute inflammation, while lymphopenia (17.9 ± 2%) suggested immune suppression. Monocytes (0.1 ± 0.1%) were low, possibly due to tissue sequestration. Eosinophils (9.1 ± 0.8%) remained within normal limits but could suggest parasitic co-infections. RBC indices including MCV (46.7 ± 0.9 fl), MCH (14.6 ± 0.2 pg), and MCHC (31.3 ± 0.3 gm/dl) remained within expected ranges, indicating no macrocytic or microcytic changes. Platelet count (245,000 ± 12,000/cu mm) was also within normal limits, suggesting no coagulation abnormalities. Arif Zafar et al., (2009) [1] , also reported concentration of haemoglobin in HS suffering animals waslower than the reference (reference value: 12-15 g/ dl) values. Haematocrit was also decreasedthan the normal values (reference value: 30-35%) in diseased buffaloes. Cultural characteristics on nutrient agar and blood agar confirmed P. multocida, with Gram staining revealing gram-negative rods with bipolar morphology. Colonies exhibited the typical greyish-white appearance with "mousy" odor. After incubation for 24 h at 37 °C, surface colonies on sheep blood agar plates were smooth, spherical, shiny, grayish or yellowish, nonhemolytic, and 1-2 mm in diameter (Piorunek et al., 2023) [15] . The type of capsule and level of encapsulation determine colony size. In rich broth, good growth was obtained; on MacConkey agar, no growth was observed (Desem et al., 2023) [4] . The organism exhibits bipolar staining with Leishman, methylene blue, or Giemsa staining in fresh cultures, blood smears, exudates, and tissues of infected animals (Panna et al., 2015) [12] . Antimicrobial susceptibility testing of isolates from nasal swabs indicated high resistance to methicillin (95.65%), cefoxitin (82.60%), oxacillin (82.6%) and penicillin (78.3% [3,8] , confirming neutrophilia as a key inflammatory marker in HS. The lymphopenia and monocytopenia reflect acute-phase immune alterations. The antibiotic resistance trends echo global concerns regarding multidrug-resistant strains of Pasteurella multocida (Watts and Sweeney, 2017) [18] . Though azithromycin showed moderate resistance (39.1%) in this study, some reports indicate it's in vivo effectiveness. Continued surveillance and tailored antibiotic regimens are necessary. The Kirby-Bauer method remains a reliable tool for guiding treatment decisions and addressing antibiotic resistance in clinical settings (Bauer et al., 1966; O'Neill, 2016) [2,11] .

Conclusion

In summary, the hematological findings in this group of buffalo calves strongly support the presence of an acute bacterial infection, aligning with the clinical presentation of Haemorrhagic Septicemia. Marked neutrophilia, lymphopenia, and elevated total leukocyte counts were consistent with the systemic inflammatory response observed in septicemic conditions caused by Pasteurella multocida. Early detection through hematological profiling, as demonstrated in this study, is crucial for timely intervention and reducing HS-associated mortality. Furthermore, antimicrobial susceptibility testing revealed substantial resistance to commonly used antibiotics such as methicillin, penicillin, and oxacillin. However, promising efficacy was observed with ceftriaxone-sulbactam, ciprofloxacin, amikacin, gentamicin and azithromycin indicating their potential utility in therapeutic management.

References

1. Arif Z, Muhammad MG, Iqbal Z, Riaz M. Effects of hypertonic saline solution on clinical parameters, serum electrolytes and plasma volume in the treatment of Haemorrhagic septicaemia in buffaloes. Pak Vet J. 2009;30(2):95-99.
2. Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966;45(4):493-496.
3. Borah P, Nath R, Das A. Hematological changes in buffalo calves suffering from hemorrhagic septicaemia. J Vet Anim Sci. 2014;2(1):23-28.
4. Desem MI, Handharyani ES, Setiyono A, Safika S, Subekti DT, Ekawasti F. Morphology, biochemical, and molecular characterization of Pasteurella multocida International Journal of Veterinary Sciences and Animal Husbandry https://www.veterinarypaper.com causing hemorrhagic septicaemia in Indonesia. Vet Med Int. 2023;2023:7778707.
5. Govindaraj G, Krishnamoorthy P, Nethrayini KR, Shalini R, Rahman H. Epidemiological features and financial loss due to clinically diagnosed Haemorrhagic septicaemia in bovines in Karnataka, India. Prev Vet Med. 2017;144:123-133.
6. Harper M, Boyce JD, Adler B. Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiol Lett. 2006;265(1):1-10.
7. Hill WA, Brown JP. Zoonoses of rabbits and rodents. Vet Clin North Am Exot Anim Pract. 2011;14(3):519-531.
8. Kumar V, Kumar A, Singh V. Antibiotic resistance in Pasteurella multocida isolated from buffalo calves. J Vet Pharmacol Ther. 2020;43(2):151-155.
9. Li XZ, Plésiat P, Nikaido H. The challenge of effluxmediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev. 2015;28(2):337-418.
10. Oh YH, Moon DC, Lee YJ, Hyun BH, Lim SK. Antimicrobial resistance of Pasteurella multocida strains isolated from pigs between 2010 and 2016. Vet Rec Open. 2018;5(1):e000262.
11. O'Neill J. Tackling drug-resistant infections globally: Final report and recommendations. London: Review on Antimicrobial Resistance; 2016.
12. Panna SN, Nazir KHMNH, Rahman MB, Ahamed S, Saroare G, Chakma S, et al. Isolation and molecular detection of Pasteurella multocida type A from naturally infected chickens and histopathological evaluation of artificially infected chickens in Bangladesh. J Adv Vet Anim Res. 2015;2(3):338-345.
13. Partridge SR, Kwong SM, Firth N, Jensen SO. Mobile genetic elements associated with antimicrobial resistance. Clin Microbiol Rev. 2018;31(4):e00088-17.
14. Peng Z, Wang X, Zhou R, Chen H, Wilson BA, Wu B. Pasteurella multocida: Genotypes and genomics. Microbiol Mol Biol Rev. 2019;83(4):e00014-19.
15. Piorunek M, Brajer-Luftmann B, Walkowiak J. Pasteurella multocida infection in humans. Pathogens. 2023;12(10):1210.
16. Souza MJ. Bacterial and parasitic zoonoses of exotic pets. Vet Clin North Am Exot Anim Pract. 2009;12(3):401-415.
17. Uddin TM, Chakraborty AJ, Khusro A, Zidan BR, Mitra S, Emran TB, et al. Antibiotic resistance in microbes: History, mechanisms, therapeutic strategies and future prospects. J Infect Public Health. 2021;14(12):17501766.
18. Watts JL, Sweeney MT. Antimicrobial resistance in bacteria from animals. J Vet Pharmacol Ther. 2017;40(3):281-294.
19. Wilson BA, Ho M. Pasteurella multocida: From zoonosis to cellular microbiology. Clin Microbiol Rev. 2013;26(3):631-655.
20. World Health Organization. Global antimicrobial resistance surveillance system (GLASS) report. Geneva: World Health Organization; 2017.