(a) Describe the etiology, clinical signs, postmortem lesions and control of hemorrhagic septicemia in buffaloes. (20 marks) (b)…

(a) Haemorrhagic Septicaemia (HS) in Buffaloes

The primary etiological agent of HS in Asian buffaloes (Bubalus bubalis) is Pasteurella multocida, a Gram-negative coccobacillus, specifically serotype B:2 (Asian serotype; Heddleston type 2, Carter type B), while serotype E:2 is prevalent in Africa. Buffaloes are more susceptible than cattle, and young adults of 6 months to 2 years are most affected. The organism survives in the nasopharynx of healthy carrier animals and spreads by ingestion or inhalation of their secretions. Environmental stressors such as the onset of the monsoon, high humidity, transport, and malnutrition lower host immunity, triggering outbreaks.

The clinical course is peracute to acute. Clinical signs begin with sudden high fever (104°F to 106°F), profound depression, and anorexia. This is rapidly followed by warm, painful, gelatinous subcutaneous inflammatory edema in the submandibular region, throat, and brisket, which extends to the parotid area. Animals display severe respiratory distress characterized by stertorous breathing, open-mouth respiration with extended head and neck, profuse frothy salivation, conjunctival congestion, and recumbency, with death typically occurring within 6 to 36 hours due to endotoxemia and asphyxiation; the swelling, hot and painful at first, later becomes cold and painless, and recovery is rare once signs are advanced. Diagnosis rests on history and signs, bipolar-staining coccobacilli in blood smears (Leishman or methylene blue) from a fresh case, culture and PCR. Anthrax (whose carcass must not be opened), black quarter, and snake bite are the main differentials.

Postmortem lesions reveal extensive petechial, ecchymotic, and suffusion hemorrhages across serous membranes, particularly on the pericardium, pleura, and endocardium. Severe straw-colored gelatinous infiltration is evident in the subcutaneous connective tissue of the throat, neck, and intermandibular space. The lungs exhibit acute fibrinous pneumonia, congestion, marked pulmonary edema, and thickened interlobular septa with serofibrinous pleural and pericardial exudation.

Control requires mass pre-monsoon vaccination with killed Pasteurella multocida B:2 vaccines: the alum-precipitated vaccine protects for about 6 months, and the oil-adjuvant vaccine (developed at ICAR-IVRI) for about 12 months, so calves are vaccinated from about 6 months of age and boosted every year before the rains. Vaccination is supported under the state component of the Livestock Health and Disease Control programme. In early stages of an outbreak, prompt parenteral antimicrobial therapy using oxytetracycline (10 mg/kg), enrofloxacin (5 mg/kg), or ceftiofur sodium, combined with non-steroidal anti-inflammatory drugs, is effective. Sanitary measures include deep burial or incineration of carcasses with quicklime, disinfection of sheds with 2% sodium hypochlorite, and quarantine of in-contact herds.

(b) Methods and Procedures of In Vitro Staining in Histology

In vitro staining is the staining of tissues or cells after removal from the body (fixed sections, smears), as distinct from vital or in vivo staining of living cells (for example trypan blue or supravital new methylene blue). It may be routine (H&E), special (for particular components), histochemical (detecting chemical groups, such as PAS or Perls' Prussian blue for iron), enzyme histochemical, or immunohistochemical (antibody-based detection of antigens).

Tissue preparation involves tissue fixation (10% neutral buffered formalin), dehydration through ascending grades of alcohol (70% to 100%), clearing in xylene, paraffin infiltration, embedding, and sectioning (4–5 µm). Sections are mounted on slides, deparaffinized in xylene, and rehydrated through descending alcohols to water before staining.

Routine Hematoxylin and Eosin (H&E) staining relies on the basic dye hematoxylin binding to acidic nuclear chromatin (staining nuclei blue-purple) and the acidic counterstain eosin staining basic cytoplasmic and extracellular proteins pink. In the regressive method, sections are overstained in Harris's hematoxylin (about 5–10 minutes), washed, differentiated in 1% acid alcohol to remove excess dye, 'blued' in running tap water or Scott's solution, counterstained in 1% eosin (about 1–2 minutes), then dehydrated, cleared and mounted; progressive hematoxylins such as Mayer's are simply stained to the desired intensity without differentiation. Frozen sections cut on a cryostat allow rapid staining, and fat stains such as Oil Red O or Sudan III need them because processing dissolves lipids.

Special staining techniques differentiate specific cellular and extracellular components: Periodic Acid-Schiff (PAS) oxidizes glycol groups to stain glycogen, mucins, and fungal walls magenta; Masson’s Trichrome stains collagenous connective tissue blue/green, muscle red, and nuclei black; Giemsa stain identifies hemoparasites (Babesia, Theileria) and mast cell granules; Gram staining differentiates Gram-positive (violet) and Gram-negative (red) bacteria in tissue sections; and Ziehl-Neelsen (ZN) stain identifies acid-fast bacilli (Mycobacterium spp.) in red against a methylene blue background. Other useful stains are Perls' Prussian blue for haemosiderin, Congo red for amyloid, von Kossa for calcium and toluidine blue for mast cells. Stained sections are dehydrated, cleared in xylene, and permanently mounted under a coverslip using DPX mountant, then examined under the light microscope.

(c) Pharmacology and Clinical Use of Atropine in Animals

Atropine, a tertiary amine alkaloid, functions as a competitive, reversible antagonist at postganglionic muscarinic acetylcholine receptors (M₁, M₂, M₃). Obtained from Atropa belladonna and Datura species, it is well absorbed from all routes, crosses the blood–brain barrier and placenta, and is partly metabolised in the liver and excreted in urine. It blocks parasympathetic transmission, causing tachycardia by inhibiting vagal tone at the sinoatrial node, reducing salivary, lacrimal, gastric, and respiratory secretions, relaxing bronchial smooth muscles, decreasing gastrointestinal motility, and causing passive mydriasis with cycloplegia by blocking the pupillary sphincter.

Clinically, atropine is widely used in Indian veterinary practice as a pre-anesthetic medication (0.02–0.04 mg/kg IM/SC) to inhibit excessive salivation and prevent reflex vagal bradycardia during endotracheal intubation. It is the specific antidote for organophosphate (OP) and carbamate pesticide toxicities, at about 0.2–0.5 mg/kg, a quarter given intravenously and the rest intramuscularly or subcutaneously, repeated until complete atropinization (cessation of hypersecretion, dry mouth, mydriasis, bronchodilation). It relieves muscarinic signs only, so in OP poisoning it is combined with pralidoxime (about 20 mg/kg), which reactivates acetylcholinesterase before ageing, whereas oximes are not needed in carbamate poisoning. It is also indicated for managing symptomatic sinus bradycardia and incomplete atrioventricular heart blocks, and topically for ophthalmic examination. In ruminants it is used cautiously as a pre-anaesthetic because it makes saliva more viscous and causes rumen atony and bloat, and in horses because it can precipitate colic. Atropine is contraindicated in tachycardia, glaucoma, and gastrointestinal stasis/equine colic; furthermore, its efficacy is reduced in many rabbits due to endogenous atropinesterase. Overdose or Datura poisoning causes dry mucous membranes, dilated pupils, tachycardia, hyperthermia, excitement and ileus, treated with physostigmine and supportive care.