Global Veterinaria 19 (5): 627-632, 2017

ISSN 1992-6197
© IDOSI Publications, 2017
DOI: 10.5829/idosi.gv.2017.627.632

Isolation of Pasteurella maltocida and Mannheimia hemolytica

from Pneumonic Calves and Their Antibiotic Susceptibility in
Harar and Haramaya, Eastern Ethiopia

Musteria Muktar, Getnet Fikadu, Anteneh Wondimu and Yehualashet Bayu

College of Veterinary Medicine, Haramaya University, P.O. Box 138 Dire Dawa, Ethiopia

Abstract: Cross sectional study was conducted from November 2016-March 2017 on calve pneumonic
pasteuerellosis on dairy farms in Haramaya and Harar districts to determine the causative agents and their
antimicrobial susceptibility pattern. Laboratory examination of 120 nasal swab samples from pneumonic calves
was analysed to isolate Pasteurella. The results indicated that the overall prevalence of Pasteurella is 47(39.2%)
including 28(23.3%) in Harar and 19 (15.8%) in Haramaya. 74.5% of the isolates were M. haemolytica and 25.5%
were P. multocida. Pneumonic pasteurellosis was significantly associated with herd size and bedding.
According to this study antimicrobial susceptibility test results, chloramphenicol (89.4%) and tetracycline
(80.9%) were the most effective drugs, whereas ampicilin (53.2%) was the intermediate drug. Penicillin-G (10.6%)
and streptomycin (14.9%) were inefficient drugs; while vancomycin was almost inactive against both isolates.
In conclusion besides the use of best drug for the treatment of pasteuerellosis periodic bacterial isolation and
antibiotic susceptibility test should be done before treating with antibiotics except for critical ones.

Key words: Antibiotic Dairy Calf Pneumonia Pasteurellosis

INTRODUCTION Mycoplasma species. M. haemolytica and P. multocida

are the most common bacterial isolates from respiratory
Animal production is now facing new challenges disease cases than the others [5, 6, 7, 8].
since demographic growth, urbanization and economic Many factors have been associated with an increased
development are all contributing to the increasing in food risk of infectious disease during the first 90 days of life.
requirement of which animal products are very important. For example, lack of adequate passive immunity have
In Ethiopia cattle are important sources of dairy product increased death rate and cause calves more susceptible to
and the future of any dairy production depends on the infectious diseases [9]. Persistence of the agents that
successful raising of calves and heifers for replacement, caused calf disease in the environment was the major
Calves are the future herd and keeping them in a fit and reason for out broken of calf problems on the dairy
healthy condition not only makes an effect on a livestock production [10].
farms’ efficiency but also contributes to the economy and Vaccination and other preventative measures have
production outputs in a country [1]. been widely adopted for the control of calf disease
Despite the large livestock population of Ethiopia, worldwide, the benefit of appropriate antimicrobial
the economic benefits remain marginal due to prevailing therapy decreases treatment costs and, increases health
diseases, poor nutrition, poor animal production systems, and wellbeing but the emergence of microbial tolerance of
reproductive inefficiency, management constraints and different antimicrobial agents has become a well-known
general lack of veterinary care [2]. phenomenon, which represented a major concern [11].
The most frequent calf disease syndrome is Because of this antimicrobial susceptibility testing
pneumonia [3, 4]. Calves develop a bacterial pneumonia was very crucial in the selection of an appropriate
most often caused by Mannheimia Haemolytica, antimicrobial agent for the treatment of pneumonia.
Pasteurella Multocida, Haemophilus Somnus and Moreover in Ethiopia, calf disease and death were ranked

Corresponding Author: Anteneh Wondimu, College of Veterinary Medicine Haramaya University,

P.O. Box: 138 Dire Dawa, Ethiopia. Tel: +251910172163.
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next to mastitis as the second biggest problem for dairy swabs by using sterile test tubes with peptone water up
production [12]. But there was a gap on identifying the to 5ml which were transported to Haramaya University,
major bacteria, involved in calf disease particularly on Veterinary Microbiology Laboratory. Collected samples
pneumonia and antimicrobial susceptibility testing for the were clearly labeled including the information on the name
appropriate treatment of the case. Therefore, the of farms, date of sampling and tag number of the calves
objectives of this paper are: To identify and rank and samples were processed as soon after collection.
associated risk factors according to their contribution to Major risk factors including lactation, sex, age, exotic
pneumonic calves’ morbidity and to indentify and genetic influence, colostrum feeding time and method of
determine antibiotic susceptibility testing of bacterial colostrum feeding, quantity of milk feeding, feeding and
isolates. type of feeds, watering, health care and management
practices were also recorded during sample collection.
MATERIALS AND METHODS
Detection of Pasteurella Species: Sediments mixed
Study Animals: The sampling units for the study were with 5% peptone water was inoculated onto
calves of up to six month of age. All dairy calves, most MacConkey agar plate, then, further sub culturing to 5%
bred (Local Zebu, Holstein-Friesian and cross) born in ovine blood agar plates (Oxoid) for examining of
farm during the study period would be enrolled. The hemolysis after incubation at 37°C for 24 hours. A single
sample size was determined based on the availability of colony was sub-cultured and tested by Gram staining
the suspected case with pneumonia having the sign of and biochemical tests including catalase and oxidase
cough and nasal discharge. A total 120 calves were test [13]. Isolate, characteristic of Gram negative,
suspected and the samples were collected from nasal coccobacilli shape, catalase (3% H2O2) positive,
cavity for further laboratory examinations. cytochrome oxidase positive and having the typical
colony morphology of Pasteurellaceae, was taken into
Study Design and Sampling Technique: Cross sectional consideration for identification procedure. Differentiation
type of study was conducted from November 2016 to between P. Multocida and M. Hemolytica were
April 2017. Total of 18 dairy farms, composed of 10 determined according to hemolysis on blood agar
smallholder and 8 large dairy farms, were included. (Blood agar base No: 2, Oxoid, containing 5% ovine
The farm was purposively selected based on the blood), growth on Mac Conkey agar (Oxoid), catalase
availability of calf, the willingness and support of the (3% H2O2) and oxidase (After incubation 37°C for 24
owners to participate on this research activity. All hours). Motility was examined in SIM medium (Oxoid);
suspected pneumonic calves in selected farms were urease activity was tested in Urea Medium (Urea agar
incorporated in the study. The health status of each calf base, Oxoid); sugar fermentation, indole production was
was evaluated by clinical examination. Calves free from also examined by adding Kovac’s reagent to a 48-hours of
nasal discharges and coughing were classified as healthy cultured samples [13, 14].
whereas sick calves showed abnormal body condition or
erected hair coat, nasal discharge, coughing and Antimicrobial Susceptibility of Pasteurella: Antibiotic
weakness. Laboratory examination of nasal swabs from susceptibility test was done by the disc diffusion
sick calves for the diagnosis of disease causative agents technique [15, 16]. The pure culture colony suspension of
was done and each bacterial isolate was tested for the isolate was made using sterile physiological saline and
antimicrobial susceptibility. In addition questionnaire adjusted to 0.5 McFarland standards then spread to
survey was also conducted to assess the farm Muller Hinton agar using sterile cotton swap and allowed
management practices (Including, herd size, animal to stand for 3-5 minutes to observe any excess moisture
housing, cleaning and disinfection, feeding of the calves from the medium before the antimicrobial discs were
and colostrum feeding). applied. And the a ring of each disc (Oxoid, England)
containing single concentration of each antimicrobial
Sample Collection Procedure: Nasal specimens were agent was then placed onto the inoculated surface using
collected from all suspected pneumonic calves aged from sterile forceps, gently pressed with the point of the
newborn to six month present on the farm at the time of forceps to ensure complete contact with the agar surface
the visit. Samples were collected directly from nasal cavity and left for 30 minutes for diffusion of the antibiotics in
of suspected none treated pneumonic calves taking nasal the disc. The plates were inverted upside down and

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incubated at 37°C for 18 to 24 hours. The result is Laboratory examination of 120 nasal swab samples
evaluated for clear zones produced by antimicrobial from pneumonic calves indicated 47 (39.2%) of overall
inhibition of bacterial growth were measured in mm using Pasteurella, with 28 (23.3%) in Harar and 19 (15.8%) in
a measuring caliper and interpreted as susceptible, Haramaya. From these total isolates of Pasteurella 74.5%
intermediate and resistant according to Clinical and of the isolates was M. haemolytica and 25.5% was P.
Laboratory Standards Institute break points [16]. Each multocida.
isolate was tested against commonly used antimicrobials Antimicrobial susceptibility testing was performed for
for treatment of pneumonia. These antimicrobials were all bacteria isolated from pneumonic calves. As shown on
chloramphenicol, penicillin-G, streptomycin, vancomycin, table 3, high resistance by Pasteurella isolates was seen
ampicillin and tetracycline against vancomycin (93.6%), penicillin (87.2%) and
streptomycin (72.3%). On the other hand, 89.4, 80.9 and
Data Management and Analysis: Data obtained from the 53.2% of isolates were susceptible to chloramphenicol,
research was exported to SPSS 20.0 for appropriate tetracycline and ampicillin, respectively.
statistical analysis. The occurrence of Pasteurella from the
total pneumonic calves was determined by using DISCUSSION
descriptive statistics. Chi square ( 2) was used to measure
the association between the different risk factors and In all study dairy farms, there were no professionals
occurrence of Pasteurella causing calf pneumonia. Effects animal health technicians either as fully employed or part
were reported as statistically significant if p-value is less time employed to deal with health aspects of the farms.
than 5 % (P<0.05). Rather the farms call private veterinary practitioners
whenever their animals face health problems. From farm
RESULTS managers or owners that mentioned calf health problems
as a problem in dairy production and the majority of them
Most of the farms kept exotic Holstein Friesian (HF) complained calf pneumonia as major cause of calf death in
(76.6%), cross breed (HF and local) (23.4%) and local the younger age. In this study, Pasteurella species were
breed (0%) calves. In most of dairy farms, male calves isolated in 39.2% of the calves, which was taken from
were vended soon after birth and females were kept for dairy farms. M. haemolytica was accounted for 74.5% and
replacement stock (Table 1). Regarding different it was highly prevalent than P. multocida. Pneumonic
management system, some of the farm practiced navel pasteuerellosis was significantly associated with herd size
treatment during birth of calves; bedding was provided (0.046) and bedding (p-0.009) and not significantly
for calves in all large dairy farms and in none of the associated with the sex, age, breed, CFT and DFT of
smallholder dairy farms. Most of the farms kept their suspected calves (Table 2).
calves’ in-group pens separating from the cows, while In this particular study the recruitment of animals was
others kept calves together with cows due to lack of based on their clinical signs and symptoms to pneumonia
enough space. Almost 98% of the dairy farmers had and the identification of Pasteurella species among these
knowledge of the advantage of colostrum feeding but animals could be reliable as causative agents. Comparing
only 23.4% were feed colostrum with in less than 6 hours the two Pasteurella spp, M. haemolytica constituted
of birth and just at 6 hours 40.4%, while 36.2%, were feed 74.5% of the total indicated that, M. haemolytica was the
after 6 hours of birth. All study farms fed whole milk for major causative agent involved in calve pneumonic
calves two times daily by bucket feeding with the pasteurellosis. This is consistent with previous reports
exception of few smallholder farms that allowed calves to [17, 18]. M. haemolytica, which is a normal flora of the
suckle their dams. No special starter feed was used in upper respiratory tract, but in animals with suppressed
any of the farms; rather the same feed given to cows was host defence mechanism, most commonly in young
used for calves. These include straw, hay and growing cattle under certain conditions associated with
concentrates. Weaning age varied from farm to farm; most debilitation, nutrition and climatic factors may lead to
(74.5%) of farms weaned calves at the age of 4 month bronchopneumonia [19, 20, 21]. Although the percentage
whereas few smallholder farms (25.5%) weaned at above isolation was relatively low (12.5%), the possible role of P.
4 months of age. In general, the weaning age was lower multocida in calve pneumonia should not be under
for male calves, mostly under three months (Table 2). estimated.

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Table 1: Prevalence of calves’pneumonia according to breed, age and sex categories

Category P. Haemolytica P. Multocida Negative Chi-Square (P-value)
Breed Exotic 25(69.4%) 11(30.6%) 58(79.5%) 4.062(0.398)
Cross 10(90.9%) 1(9.1%) 13(17.8%)
Local 0 0 2(2.7%)
Total 35 12 73(100%)
Age <3month 21(72.4%) 8(27.6%) 34(46.6%) 2.783(0.249)
>3month 14(77.8%) 4(22.2%) 39(53.4%)
Total 35 12 73(100%)
Sex Female 27(71.1%) 11(28.9%) 63(86.3%) 2.052(0.358)
Male 8(88.9%) 1(11.1%) 10(13.7%)
Total 35 12 73(100%)

Table 2: Prevalence of calves’ pneumonia based on management of risk factors

P. haemolytica P. multocida Negative Chi-Square (P-value)
Herd Size Large 20(90.9%) 2(9.1%) 31(43.5%) 6.156(0.046)
Small 15(60%) 10(40%) 42(57.5%)
Total 35 12 73(100%)
Navel Untreated 18(64.3%) 10(35.7%) 41(56.2%) 3.858(0.145)
Treated 17(89.5%) 2(10.5%) 32(43.8%)
Total 35 12 73(100%)
CFT <6 hours 9(81.8%) 2(18.2%) 20(27.4%) 0.964(0.915)
At 6 hours 14(73.7%) 5(26.3%) 31(42.5%)
>6 hours 12(70.6%) 5(29.4%) 22(30.1%)
Total 35 12 73(100%)
DCF 24 hours 16(88.9%) 2(11.1%) 35(47.9%) 5.427(0.246)
24hr-4days 13(72.2%) 5(27.8%) 24(32.9%)
>4days 6(54.5%) 5(45.5%) 14(19.2%)
Total 35 12 73(100%)
Weaning <4 month 27(77.1%) 8(22.9%) 51(68.9%) 0.782(0.677)
>4 month 8(66.7%) 4(33.3%) 22(30.1%)
Total 35 12 73(100%)
Housing Separate 21(80.8%) 5(19.2%) 39(54.4%) 1.251(0.535)
Non separate 14(66.7%) 7(33.3%) 34(46.6%)
Total 35 12 73(100%)
Bedding <once/week 2(33.3%) 4(66.7%) 8(11%) 13.569(0.009)
>Once/week 28(90.3%) 3(9.7%) 53(72.6%)
once/week 4(44.4%) 5(66.6%) 12(16.4%)
Total 35 12 73(100%)
TCF1st = Time of first colostrum feeding, DCF = Duration of colostrum feeding

In our study, according to the antimicrobial [19]. However, this result contradicts the findings of
susceptibility test results, chloramphenicol (89.4%) and Aschalew [18] who reported tetracycline as ineffective
tetracycline (80.9%) were the most effective drugs; where drug of choice.
as ampicilin (53.2%) was the intermediate drug while One of the interesting findings of the present study
penicillin-G (10.6%) and streptomycin (14.9%) were demonstrates the highest resistance of Pasteurella
inefficient drugs and vancomycin was totally inactive isolates against vancomycin (93.6%). In contrast
against both isolates (Table 3). Increase in resistance Esra et al. [24] reported that vancomycin 95 and 90% as
against antibiotics in both P. multocida and M. the most effective antibiotics against M. haemolytica
haemolytica isolates have been reported by the work of isolates. This might be due to difference in the strain of
many scholars [6, 22, 23]. The result is in line with the the isolate or due to the existence of host factors that may
literature which state as chloramphenicol is highly affect the action of drug in bovines. Besides in this study
effective and well-tolerated broad spectrum antibiotic to P. multocida showed resistance to penicillin-G, in
many genera of Gram-positive and Gram-negative bacteria contrary to literature [25] which indicates that most strains

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Table 3: Antibiotic susceptibility of Pasteurella

P. Haemolytica P. multocida Total
Penicillin G Resistant 31(66%) 10(21.3%) 41(87.2%)
Intermediate 0(0%) 1(2.1%) 1(2.1%)
Susceptible 4(8.5%) 1(2.1%) 5(10.6%)
Total 35(74.5%) 12(25.5%) 47(100%)
Streptomycin Resistant 24(51.1%) 10(21.3%) 34(72.3%)
Intermediate 4(8.5%) 2(4.3%) 6(12.8%)
Susceptible 7(14.9%) 0(0%) 7(14.9%)
Total 35(74.5%) 12(25.5%) 47(100%)
Chloramphenicol Resistant 2(4.3%) 0(0%) 2(4.3%)
Intermediate 2(4.3%) 1(2.1%) 3(6.4%)
Susceptible 31(66%) 11(23.4%) 42(89.4%)
Total 35(74.5%) 12(25.5%) 47(100%)
Vancomycin Resistant 32(68.1%) 12(25.5%) 44(93.6%)
Intermediate 3(6.4%) 0(0%) 3(6.4%)
Susceptible 0(0%) 0(0%) 0(0%)
Total 35(74.5%) 12(25.5%) 47(100%)
Tetracycline Resistant 1(2.1%) 3(6.4%) 4(8.5%)
Intermediate 4(8.5%) 1(2.1%) 5(10.6%)
Susceptible 30(63.8%) 8(17%) 38(80.9%)
Total 35(74.5%) 12(25.5%) 47(100%)
Ampicillin Resistant 9(19.1%) 3(6.4%) 12(25.5%)
Intermediate 8(17%) 2(4.3%) 10(21.3%)
Susceptible 18(38.3%) 7(15%) 25(53.2%)
Total 35(74.5%) 12(25.5%) 47(100%)

of P. multocida are susceptible to penicillin-G. This ACKNOWLEDGEMENTS

difference strengthens the recommendation. Kaan [26]
stated that “Antibiotic susceptibility profiles of P. The researchers acknowledge Haramaya University
multocida and M. haemolytica help veterinarians to for laboratory facilities support during the study.
choose appropriate antibiotic against bovine respiratory
disease; however, antibiotic susceptibility studies should Competing Interests: The authors declare that they have
be renewed periodically”. no competing interests.

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