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Diagnostic notes
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Non refereed |
Diagnosis of Haemophilus parasuis in affected herds
and use of epidemiological data to control disease
Simone Oliveira, DVM, MS; Carlos Pijoan, DVM, MS, PhD
Department of Clinical and Population Sciences, University
of Minnesota, St Paul MN 55108. E-mail: oliv0107@tc.umn.edu.
Oliveira S, Pijoan C. Diagnosis of Haemophilus parasuis
in affected herds and use of epidemiological data to control
disease. J Swine Health Prod. 2002;10(5):221-225.
Haemophilus parasuis is a commensal
organism in the upper respiratory tract of pigs.1 This
organism is particularly interesting due to its ability to invade
the host and cause severe lesions characterized by fibrinous polyserositis,
arthritis, and meningitis. Little is known about the pathogenesis,
virulence factors, and immunogenicity of H parasuis, which
makes control of systemic infections a difficult task.
Diagnosis of Haemophilus parasuis as a primaryagent
causing disease in a herd requires evaluation of the herd's health
status and association of H parasuis with characteristic
clinical signs and lesions. Control of disease, on the other hand,
depends on characterization of the strains involved in systemic
disease and an understanding of the epidemiology of the agent
within and between herds. In this article, we will discuss how
to improve H parasuis diagnosis in affected herds, how
systemic strains may be characterized, and how strain-characterization
data may be used in disease control.
Clinical characterization
and lesions
Haemophilus parasuis usually affects young pigs after
4 to 6 weeks in the nursery. Affected animals may show a variety
of clinical signs and lesions, depending on the immune status
of the herd, virulence of the strain, and stage of infection.
Systemic infection by H parasuis in naive animals is characterized
clinically by high fever (41.7°C), lethargy, swollen joints,
coughing, abdominal breathing, and central nervoussystem signs,
such as trembling and paddling. Lesions are characterized by fibrinous
exudate on the pericardium, pleura, peritoneum, meninges, and
joints.2 These clinical signs and lesions generally
occur when highly virulent H parasuis strains are introduced
into a susceptible herd, or when concurrent infections, for example,
porcine reproductive and respiratory syndrome virus (PRRSV), are
affecting the herd.
In endemically infected, stable herds,
H parasuis usually affects older animals. In these herds,
occurrence of disease is more sporadic and less severe than in
naive populations, and lesions may be restricted to the lung.
Pneumonia is characterized by antero-ventral consolidation, with
purulent exudate in the bronchi and bronchioli. Occasionally,
arthritis may occur in adult populations, especially in sow herds.
Genotyping and serotyping
H parasuis isolates
Haemophilus parasuis field isolates may be characterized
by serotyping or genotyping. Serotyping is based on the reaction
between serovar-specific polyclonal antiserum and heat-stable
antigens extracted from the bacterial cultures, either in an agar
gel precipitation test (AGPT) or an enzyme-linked immunosorbent
assay (ELISA).3,4 Genotyping of H parasuis isolates
has been performed by repetitive-element-based PCR (Rep-PCR).5,6,7
The Rep-PCR is a molecular-based technique that uses repetitive
sequences within the bacterial genome (enterobacterial repetitive
intergenic consensus (ERIC) sequences) to produce strain-specific
fingerprints. The ERIC-PCR technique is schematized in Figure
1. Primers targeting the ERIC sequence align to the bacterial
DNA and new double-stranded DNA is produced (Figure 1A). The repetitive
sequences are randomly distributed in the bacterial genome, and
the distance between two repetitive sequences may vary between
different strains. This results in the production of several amplicons
with different sizes (Figure 1B). The PCR products are then separated
by size through electrophoresis in an agarose gel (Figure 1C).
The resulting DNA pattern is called a fingerprint, and the process
is called genotyping. Similar strains show comparable DNA bands,
while different strains have varied distribution of the DNA bands
(Figure 1D).8
Fifteen serovars of H parasuis have been identified
using the AGPT.9 A recent epidemiological study characterized
98 H parasuis field isolates recovered from 15 North American
herds between 1999 and 2001.10 Isolates were serotyped
using the AGPT and genotyped using the ERIC-PCR technique. Serovar
4 (38.8%) and non-typable serovars (27.6%) were the most prevalent
among the evaluated isolates. Other serovars that were identified
included serovars 3 (8.2%), 1 (7.1%), 12 (7.1%), 2 (4.1%), 14
(3.1%), 5 (2%), 7 (2%) and 13 (1%). The ERIC-PCR technique detected
a high genetic diversity within serovars. Twelve different strains
were identified among isolates from serovar 4 (Figure 2). Non-typable
isolates were divided into 18 genotypes. Genetic diversity was
also observed within isolates from serovars 1, 3, and 7 (two strains
each), serovars 12 and 14 (three strains each), and serovar 2
(four strains).
These results demonstrate the limitations of serotyping for
characterizing and differentiating H parasuis strains.
However, serotyping may still be useful for selection of commercial
vaccines. Haemophilus parasuis isolates of the same serovar
may induce satisfactory homologous protection, while heterologous
protection is restricted or absent.11,12 Commercial
vaccines generally produce inconsistent results between herds,
possibly because prevalent serovars differ among herds. Another
hypothesis is that different strains of the same serovar do not
induce cross-protection. Although H parasuis is highly
prevalent among US herds, different herds are infected by different
strains (Figure 2), and it is unknown whether or not a commercial
vaccine containing a unique strain can protect herds affected
by heterologous strains of the same serovar. Further studies are
necessary in order to test this hypothesis.
Genotyping provides an accurate characterization of any H
parasuis isolate, including non-typable isolates, and differentiates
between strains of the same serovar. This information is very
useful for selection of strains to be included in autogenous vaccines.
Moreover, genotyping may be used to compare H parasuis
strains isolated from recipient and supplier herds and to monitor
the introduction of new strains into the herd.8
Diagnosis of H parasuis: Selection of animals and tissues
for sampling
Selection of appropriate animals and tissues for sampling is
essential for the correct diagnosis of H parasuis. Sample
animals that are showing characteristic signs of H parasuis
infection. Better results are obtained if samples are collected
from animals with both respiratory signs and arthritis. Select
animals in the early stages of infection (newer cases) that have
not been treated with antibiotics for at least 1 week. Euthanize
clinically affected animals and using a swab, sample the fibrinous
exudate covering the surface of the pericardium, pleura, and peritoneum,
or joint and cerebrospinal fluids. Sampling dead animals significantly
reduces the chances of isolating H parasuis. Keep samples
refrigerated at 4°C until shipment and submit them to a diagnostic
laboratory as soon as possible, using ice packs and a Styrofoam
container.
Haemophilus parasuis can be isolated from the nasal
cavity, tonsils, and trachea of healthy animals. Occasionally,
H parasuis may be isolated either from healthy lungs or
lungs with severe pneumonia lesions. Major differences exist among
strains of H parasuis isolated from the upper respiratory
tract, from lung samples, and from systemic sites. Strains that
cause systemic infection have a low prevalence in the upper and
lower respiratory tracts. Isolates from nasal cavities and tonsillar
areas are particularly diverse serotypically and genotypically,
and are usually of low virulence. Lung isolates are less diverse
than nasal isolates, but may show a higher genetic variability
than systemic strains. Strains isolated from pneumonic sites may
or may not include the strains that are actually causing disease
in the herd.7 Considering these factors, only samples
from non-respiratory tract sites should be submitted for H
parasuis isolation and diagnosis.
Interpreting results and deciding on disease control
Once H parasuis is isolated from affected animals, evaluate
herd health status to decide whether H parasuis is a primary
or a secondary problem. Herds experiencing an active PRRSV infection
may have a significant increase in nursery mortality due to secondary
agents such as Streptococcus suis and H parasuis.
Until PRRSV infection is controlled, management of secondary agents
through vaccination is generally unsuccessful. In some herds,
stabilizing PRRSV infection is the key to H parasuis control.
When H parasuis is isolated from systemic sites in diseased
nursery pigs in a PRRS-stable herd, efforts should be concentrated
on disease prevention through vaccination or treatment. Control
of H parasuis depends on understanding the epidemiology
of the agent within and between herds.8 The genetic
variability of systemic isolates is generally limited within herds,
with only one or two strains involved in systemic disease.7
Characterization of strains isolated from related herds (supplier
and recipient) is very useful for identifying potential sources
of pathogenic strains. In some herds, use of either commercial
or autogenous vaccines controls disease. Commercial vaccines contain
several strains of different serovars, including non-typable strains.
Autogenous vaccines usually include one or two prevalent strains
recovered from the affected herd. In herds experiencing mortality
due to H parasuis, try to characterize as many isolates
as possible by serotyping and genotyping, and continue to do so
as new cases occur after the vaccination program has been implemented.
Serotyping is useful for selection of commercial vaccines, while
genotyping can be used to select prevalent strains to be included
in autogenous vaccines. After prevalent pathogenic strains have
been characterized, decide whether to use a commer- cial or an
autogenous vaccine. Either provides satisfactory results if the
vaccine strains are similar to those causing disease in the affected
herd, but heterologous protection is very limited. Non-typable
isolates may be responsible for severe outbreaks, and it is difficult
to match them with vaccine strains. It is not known how the high
genetic diversity within serovars and within non-typable isolates
relates to cross-protection. If commercial vaccines are not effective,
use of autogenous vaccines should be considered. Strains to be
included in autogenous vaccines should be the prevalent genotypes
affecting the herd.
Haemophilus parasuis isolates genotyped by Rep-PCR can
be compared using dendrograms (Figure 3), which may be constantly
updated with new isolates as new cases appear in the herd. Clusters
of prevalent strains are easily identified. Autogenous vaccines
should include a representative strain from each major cluster.
Always select strains isolated from systemic sites, such as pericardium,
pleura, peritoneum, joints, or meninges.
Timing of vaccination is very important. Implementation of
an appropriate vaccination protocol will depend on the epidemiology
of the disease in the herd. Although disease caused by H parasuis
may be observed as early as 2 weeks after placement in the nursery,
most affected herds will have a peak of infection after 4 to 6
weeks in the nursery, when maternal immunity has declined and
previously exposed animals infect naive animals. In herds experiencing
disease late in the nursery, vaccinate at weaning and again 2
weeks later. In herds experiencing significant death losses soon
after weaning, vaccinate prior to weaning. Although sows may also
be vaccinated, results are better with pig vaccination. When sows
are vaccinated, maternal immunity in pigs may persist until 6
to 7 weeks of age, and interferes with development of active immunity.
Never vaccinate sows and piglets at the same time.
Summary
Control of H parasuis depends on correct diagnosis of
the agent, accurate characterization of the strains causing systemic
disease in the herd, and selection of an appropriate vaccine and
vaccination protocol. Understanding the epidemiology of H parasuis
in a particular swine population is still the key to control in
affected herds.
References - refereed
1. Møller K, Kilian M. V factor-dependent members of
the family Pasteurellaceae in the porcine upper respiratory
tract. J Clin Microbiol. 1990;28:2711-2716.
2. Vahle JL, Haynes JS, Andrews JJ. Experimental reproduction
of Haemophilus parasuis infection in swine: clinical, bacteriologic,
and morphologic findings. J Vet Diagn Invest. 1997;7:476-480.
3. Morozumi T, Nicolet J. Some antigenic properties of Haemophilus
parasuis and a proposal for serological classification. J
Clin Microbiol. 1986;23:1022-1023.
5. Versalovic J, Schneider M, de Bruijn FJ, Lupski JR. Genomic
fingerprinting of bacteria using repetitive sequence based PCR
(rep-PCR). Meth Cell Mol Biol. 1994;5:25-40.
6. Rafiee M, Bara M, Stephens CP, Blackall PJ. Application
of ERIC-PCR for the comparison of isolates of Haemophilus parasuis.
Aust Vet J. 2000;78:846-849.
7. Ruiz A, Oliveira S, Torremorell M, Pijoan C. Outer membrane
proteins and DNA profiles in strains of Haemophilus parasuis
recovered from systemic and respiratory sites. J Clin Microbiol.
2001;39:1757-1762.
9. Kielstein P, Rapp-Gabrielson VJ. Designation of 15 serovars
of Haemophilus parasuis based on immunodiffusion using
heat-stable antigen extracts. J Clin Microbiol. 1992;30:862-865.
12. Rapp-Gabrielson VJ, Kocus GJ, Clark JT, Muir SK. Haemophilus
parasuis: immunity in swine following vaccination. Vet
Med. 1997;92:83-90.
References - non refereed
4. Lin BC, Cobb S. A "fuzzy" ELISA for serotyping
Haemophilus parasuis. Proc IPVS. Bankok, Thailand.
1994;156.
8. Oliveira S, Pijoan C. Haemophilus parasuis: Improvement
of diagnosis by a molecular-based technique. Proc AASV.
Nashville, Tennessee. 2001;479.
10. Oliveira S, Blackall P, Pijoan C. Characterization of Haemophilus
parasuis isolates by serotyping and genotyping. Proc IPVS.
Ames, Iowa. 2002;224.
11. Oliveira S, Pijoan C. Use of an uncapsulated Haemophilus
parasuis vaccine in pigs. Proc AASV. Kansas
City, Missouri. 2002;319.
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