Study on diagnosis and prognosis of bacterial respiratory disease in buffalo calves

Introduction

Bovine respiratory disease constitutes one of the key health issues and most costly disease occurring in calves in all parts of the world. Bovine respiratory disease is common in calves and causing gigantic economic losses due to reduced live weight gain and feed conversion efficiency, poor performance and cost of treatment. The success of any dairy enterprise depends upon the survival of the calf produced. Bacterial respiratory disease in calves is a multifactorial disease complex involving interaction between a wide range of pathogens, immune status and environmental stress. It is a syndrome of diverse etiologies that is caused by one or more of wide range of microorganisms including Pasteurella multocida, Manhemia haemolytica, Histophilus somnei and Mycoplasma bovis are most commonly reported (Godinho et al., 2007). Current data on the prevalence and relative importance of different bovine respiratory pathogens in preweaned dairy calves was scant and available data usually from large dairy farms. The diseased calves usually exhibit clinical signs of acute respiration distress, although, in some cases sudden death appear before the development of significant pulmonary lesions. Some of the pathogens are normal inhabitant of respiratory tract of buffalo calves. However, any activity that is precipitating stress leads to clinical disease conditions. The diagnosis of bovine respiratory disease in calves poses significant challenges to the clinicians. Bovine respiratory disease affected calves exhibit signs of fever, nasal discharges, depression, cough and separate themselves from the rest of the pen along with severe alteration in blood gases (Zeineldin et al., 2017). This generally occurs within the first 2 weeks of calves transported to the feedlot farms. Auscultation of the lung in BRD affected calves exhibit abnormal lung sounds including loud wheezing, crackling sound and moist rales due to the exudates produced by inflammatory cells and goblet cells as a result of pneumonia (Metwally et al., 2017). Haemotological picture of Bovine respiratory disease affected calves characterized by change in blood Hb, RBCs, PCV, Total WBCs and DLC count. Severe Biochemical alteration in bovine respiratory disease affected calves were also common and Might display reasonably predictable changes in response to inflammation and resulted in significant difference in protein profile. The inflammation Associated with bovine bacterial respiratory disease could lead to significant pulmonary damage and reduced lung function. Although various forms of calf respiratory diseases are known, their clinical manifestation is frequently less differentiated. Despite the great variety of combination of pathogens that have been implicated as the causes of this disease, the clinical entity of most manifestation is bronchopneumonia. Calves typically face two primary problems that contribute a high incidence of bacterial respiratory disease. First, stress associated with weaning and transportation has a negative effect on calf immune system. Second, this stress typically occurs when the animal is exposed to a variety of infectious agents as a result of marketing management producers. Nutrition can also interact with these two factors, most likely as a result of preweaned nutritional deficiencies or through decreased feed intake associated with stress.

Materials and methods

The study comprised apparently healthy buffalo calves and clinical cases. Ten apparently healthy buffalo calves were selected as control group for obtaining normal data for comparison of parameters under study. The study was conducted in 54 clinical cases brought to the large animal outpatient ward, Veterinary Clinical Complex (VCC), NTR College of Veterinary Science, Gannavaram, Veterinary Hospitals and farms nearby Vijayawada with clinical signs suggestive of bovine respiratory disease were screened by using specially designed data sheet and subjected to detailed clinical examination, haematology, serum biochemical profile, radiography, cultural studies of nasal discharges and blood to confirm the bovine bacterial respiratory disorders.

Clinical material

Clinical samples collected for the present study included nasal discharges, whole blood and serum.

Laboratory material

All the dehydrated media used in the present study were obtained from Hi-media Laboratories Pvt. Ltd., Mumbai, chemicals from S D Fine Chemicals Ltd, Mumbai. 2 points Calves were categorized "BRD positive" if their total score is ≥4.

Methods

History and clinical examination

Collection of blood samples Blood and serum samples were collected aseptically from healthy animals and clinical cases for carrying out different laboratory and haemato-biochemical examination by standard tests. Blood samples were collected aseptically using sterile EDTA vacuutainers from jugular vein and transported on ice to the laboratory. Blood smears were prepared and fixed with methanol. Five ml of blood was collected into a sterilized test tube for bacterial isolation Separation of serum Approximately 5 ml of blood was collected in safe lab clot accelerator for biochemical analysis. Collected serum samples were kept in a slant position with an angle of 20-30 degrees and keeping it undisturbed for 2-3 hrs. Then they were subjected to centrifugation @ 2000 rpm for 15 minutes and serum was separated and stored in Eppendorf tubes at -20 °C until further assay.

Collection of nasal discharges

Deep nasal discharges were collected from buffalo calves with clinical signs suggestive of bacterial respiratory disease.

Preparation of glassware For 24 hours, all the glassware used in this analysis was dipped in 1 per cent hydrochloric acid, then washed under running tap water, soaked again for 24 hours in teepol solution, cleaned with brush and washed under running water. The glassware was then rinsed in glass distilled water and dried. Then carefully packed and sterilized at 160ᵒC for one and half hour. The sterilized glassware were used for cultural and biochemical analysis.

Preparation of media

All media obtained as dehydrated powders were rehydrated by adding distilled water to dissolve as directed by the manufacturer. The media were then sterilized at 15 lb of pressure for 15 minutes by autoclaving. The blood agar was prepared in the laboratory as per the procedure described by Quinn et al. (1999).

Isolation of bacteria

Nasal swabs collected from calves affected with Bovine respiratory diseases were inoculated into TSB broth and incubated at 37 0 C for 24 hours aerobically. Then a loopful of broth culture was streaked on BHI agar plates. Based on morphology and Gram's staining properties, cultures were inoculated into specific or selective media like MacConkey agar, Mannitol salt agar, Nutrient agar, Blood agar, Eosine-Methylene-Blue agar. For isolation of Streptococcus spp the nasal swabs were inoculated into the Streptococcus selective broth and incubated at 37 0 C in an anaerobic jar for 24 hours. After incubation in SSB, the broth was examined for the presence of Streptococcus spp by gram staining and further inoculated onto Streptococcus selection agar.

Identification of bacteria

Primary identification was done by Gram's staining, colony morphology and type of haemolysis on blood agar of different bacteria. Pure cultures were identified upto genus level as per the Bergey's Manual of Determinative Bacteriology (Holt et al., 1994)

Gram's staining

On a clean microscopic slide bacterial smears were prepared by mixing 24 hour old cultures with sterile saline. The smear was then air dried, heat fixed over flame and then stained with Gram's stain and examined under oil immersion. The bacteria were identified as Gram positive or Gram negative, cocci, bacilli or coccobacilli.

Motility test by hanging drop technique

A drop of 6-8 hour old broth cultures was placed on a cover slip and the cover slip was then the cover slip was placed upside down on a microscopic slide containing a circular depression. Then the motility of bacteria was examined under high power. This technique is known as hanging drop technique.

Catalase activity

This was tested by placing a looful of bacterial colony on microscopic slide and then mixed with a drop of three percent hydrogen peroxide solution. Production of gas bubbles after few seconds indicated the bacteria as catalase positive whereas absence of gas bubbles was taken as negative.

Cultural characters on blood agar

Blood agar plates containing 5-10 per cent of defibrinated sheep blood were used for this test. The bacteria were grown on these plates and the type of haemolysis was recorded after 24 hours of incubation at 37ᵒC. The appearance of zones of clear circumscribed areas around the colonies was taken as indication of occurrence of haemolysis.

Indole test

The medium was inoculated with the culture and incubate for 48 hours at 37ᵒC. After incubation 0.5 ml of Kovacs reagent was added and shaked gently. A red colour indicated a positive reaction

Methyl Red (Mr) Test

The medium was inoculated from a young agar slope culture and incubated at 37ᵒC for 48 hr. Five drops of the methyl red reagent was added, mixed and read the results immediately. Positive results are bright red and negative results are yellow.

Voges -Proskauer (VP) test

The test was done to detect the acetone production of the organisms by growing in MR-VP medium incubated at 37ᵒC for 48 hrs add 1 ml of 40 per cent potassium hydroxide and 3 ml of 5 per cent solution of alpha Naphthol in absolute Ethanol. A positive reaction is indicated by the development of pink colour in 2-5 minutes, becoming Crimson in 30 minutes. The tube can be shaken at intervals to ensure maximal aeration. A uniform dark brownish colour was considered as negative.

Citrate utilization test

The bacterial use of citrate was tested on simon's citrate agar slants. A light suspension of the test organisms in sterile saline was inoculated in linear streaks in citrate agar slants. The slants were then incubated at 37 0 C for 48 hours. Growth associated with a colour change to Prussian blue from green colour was regarded as positive where as a green colour media was considered as negative.

Nasal discharges culture

For isolation of bacteria, the nasal discharges were streaked on blood agar, nutrient agar, streptococcus selection broth and MacConkey agar. As per standard methods, culture plates were incubated. Presumptive and definitive identification of pathogens were done by staining characteristics, colony morphology and standard biochemical tests (Barrow and Feltham 1993 and Quinn et al., 1999) [17] .

Haematology

Five ml of venous blood was collected in two vacationer tubes containing K3 EDTA as anticoagulant for haematological investigations. Haematological parameters including packed cell volume (PCV), haemoglobin (Hb), red blood cell (RBC), white blood cell (WBC) and differential counts (DC) were done as per standard methods (Jain, 1986)

Molecular characterization of bacterial isolates

The TSB medium was inoculated with the isolates and incubated at 37℃ for 16 to 18 hr and overnight cultures adjusted to 0.5 McFarland standards were used for DNA isolation. Deoxyribonucleic acid (DNA) from bacterial cell mass was extracted by the high salt method of DNA extraction (Aravindakshan et al., 1997) [8] with suitable modifications (Anand Kumar, 2009) [6] Quality and purity of extracted DNA was checked by agarose gel electrophoresis. The concentration of the DNA obtained was measured on ethidium bromide stained agarose gel by comparing with different concentrations of DNA marker and the DNA extracted was used as template in PCR (Anand Kumar, 2009) [6] .

PCR and Detection of PCR Products

The PCR tests were carried out in Proflex PCR system, Applied Biosystems. All the reactions were carried out in a volume of 25 μl in 0.2 ml PCR tubes. The composition of Master Mix (2X) is shown in Table 6. The PCR amplicons were analysed by electrophoresis on 1.7 perrcent agarose gel stained with 0.5 μg of ethidium bromide/1 ml in Tris-Borate EDTA (TBE) buffer. Electrophoresis was carried out at 70V for 50 min in submarine gel electrophoresis unit (BIORAD, UK) and the PCR products were visualized in BIORAD molecular imager XR+. The sizes of PCR products were verified by comparison with quantitative DNA ladder (SRL, Mumbai). Negative control with sterile distilled water was maintained in each PCR. Staphylococcus species and Streptococcus species were detected using genus specific oligonucleotide primers and E. coli was detected using species specific oligonucleotide primers in PCR. Oligonucleotide primers and Thermal cycling conditions required for detecting the genes responsible for molecular characterization of Staphylococcus spp, Streptococcus spp and E. coli

Serum biochemistry

Serum biochemical parameters like serum total protein (g/dl), AST(U/L), ALP (IU/L), albumin (g/dl), total bilirubin (mg/dl), BUN (mg/dl) and creatinine (mg/dl) were estimated by using a Thermo Scientific Instrument (Multiskan GO Analyser) using standard kits manufactured by M/S Erba-Mannheim Pvt. Ltd., Mumbai.

Cardiac biomarkers

Qualitative measurement of cardiac troponinI and estimation Creatin kinase MB were done as per the standard methods.

Radiography

Thoracic radiography in affected calves was performed as described by Masseau et al. (2008).

Electrocardiography

Electrocardiography in affected calves' war performed by standard method.

Echocardiography

Echocariography in affected calves was performed by standard method.

Statistical analysis

Data collected were subjected to statistically analysis as per described by Snedecor and Cochran (1994)

Results and discussion

The occurrence of respiratory disease in buffalo calves was observed to be 21. 25 [26] ,

Griffin (2010), Richeson et al. (2013) and Allen et al. (1991) [2] .

Fever is an indication of microbial infection. It is one of the mechanism that immune system utilizes to get rid the body from invading pathogens and as a normal response to immune stimulus. The change in attitude, appetite, rectal temperature and mucus membrane congestion of the buffalo calves might be attributed to toxemia which was associated with the release of bacterial endotoxins (Attia et al., 2016) [10] . Marked dyspnea with open mouth breathing was observed in 11.11 per cent of cases might be due to advanced pulmonary parenchymal destruction. The observed nasal discharges and ocular discharges was also attributed to inflammation changes in nasal and ocular mucous membrane as reported by Ramadan et al. (2019). The prominent physical examination findings were dyspnea, tachycardia, tachypnea, crackles and wheezes. These findings were similar to the observation made by previous researchers (Aslan et al., 2002; Radostits et al., 2007 and Griffin, 2010) [9] . Bovine respiratory disease affected calves with shallow rapid respiration due to hypoxia and dyspnea in some cases might be attributed to severe inflammation in bronchioles and alveoli that interfere with gas exchange and respiration (Ramadan , 2004). Anemia could be attributed to reduction of energy and protein intake or sequestration of iron in bone marrow macrophages and hepatocytes during infection, thus become unavailable for utilization in hemoglobin synthesis leading to inhibition of erythropoiesis (Aytekin et al., 2011) [15] . In Bovine respiratory disease affected calves when compared to control calves, there was a significant increase in both WBCs count and neutrophils whereas significant decrease in lymphocytes. These results gained support from the findings of Bednarek et al. (2003) [20] , [12] The significant increase in WBCs and neutrophils occurred in pneumonia might be attributed to inflammatory lesions and presence of bacterial infection (Sayed et al., 2002 and Smith., 2014). On the other hand, the significant decrease of lymphocytes might be attributed to stimulation of adrenal gland with the tissue invaded by bacterial toxins or might be resulted from immune suppression as a result of various stress conditions (El-Naser and Khamis (2009) and Aytekin et al., (2011). [14] No significant difference (P>0.05) was observed among eosinophil concentration in infected calves when compared with control group. Kumar et al., 2018). Others suggested that certain bacterial toxins increase capillary permeability and permit escape of plasma proteins in tissues, so osmotic pressure of proteins is increased in tissue fluids and at the same time decreased in blood (Omran et al., 2005). Albumin is also considered a negative acute phase proteins and its value frequently and markedly declines during inflammation. (Ceron et al., 2005 [25] ; Eckersall and Bell, 2010 and Georgieva et al., 2011) [24] . The significant elevation in AST, ALP levels in this investigation could be attributed to dysfunction of liver due to hepatic degenerative and necrotic changes caused by bacterial infection and toxins (Raghib et al., 2004 and Aytekin et al., 2011) [15] . The prominent thoracic radiographic findings were consolidation, pulmonary infiltration, pulmonary edema of the lung Masseau et al. (2008) reported similar clinical findings in bacterial respiratory disease in buffalo calves. In the present study the radiographic appearance of pulmonary parenchyma was interstitial pattern, bronchial pattern and alveolar pattern had been identified on thoracic radiography. Thoracic radiography was a sensitive in identifying interstitial pattern in buffalo calves having interstitial pneumonia, embolic pneumonia and chronic pneumonia.

Acknowledgement

AcknowledgementsWe are thankful to Sri Venkateswara Veterinary University, Tirupati for providing adequate facilities and SERB, New Delhi for providing financial support.

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