Comparison of bacterial cultivation, PCR, in situ hybridization and immunohistochemistry as tools for diagnosis of Haemophilus somnus pneumonia in cattle
Introduction
Haemophilus somnus is a Gram-negative bacterium capable of inducing a variety of bovine diseases including pneumonia and thrombotic meningoencephalitis (Humphrey and Stephens, 1983). In Denmark, H. somnus is one of the most common bacteria isolated from severe cases of calf pneumonia (Tegtmeier et al., 1999a).
Diagnosis of field cases of bacterial calf pneumonias is normally based on bacterial cultivation. However, bacterial cultivation is suspected to have a low sensitivity, as attempts to isolate pathogenic bacteria from cases of pneumonia often fail, even though their presence might be expected based on clinical and histopathological observations (Tegtmeier et al., 1999a).
In the diagnosis of calf pneumonia, especially isolation of H. somnus can pose a problem. H. somnus is particularly susceptible to antibiotics (Martin and Meek, 1981), and it is recommended that specimens are protected from drying and cultured as soon as possible (Quinn et al., 1994). No or very few live bacteria may otherwise be present, at the time of cultivation, as the bacteria have succumbed during transportation to the laboratory under sub-optimal conditions. Most isolates of H. somnus also demand CO2 for growth, and grow as tiny pin-point colonies on blood agar plates with variable haemolysis. These colonies can consequently be overgrown due to the post-mortem contaminating bacteria, e.g. Proteus species. Furthermore, isolation of bacteria is often hampered if intensive antibiotic treatment has been given to the animals prior to death. In a recent study, antibacterial substances were detected in 32% of examined bovine lungs (Tegtmeier et al., 1999a), and the difficulty of isolating H. somnus in animals treated with antibiotics has previously been shown (Martin and Meek, 1981). For these reasons, supplementary tools should be considered in addition to cultivation, in order to enhance the detection rate of H. somnus. Today, a variety of methods are available for detection of micro-organisms, including immunohistochemistry (IHC) (Larson, 1989), in situ hybridisation (ISH) (Brown, 1998) and polymerase chain reaction (PCR) (Tang and Persing, 1999). A major advantage in common for ISH and IHC is that a topologic assessment of the examined tissue is possible. This is not the case when detection is made by PCR. The PCR-technique has proven to be a very sensitive diagnostic tool (Angen et al., 1998), but it may be difficult to draw conclusions about the pathological significance of a positive PCR test. Previous studies have evaluated the potentials of such newer techniques in the diagnosis of different micro-organisms in various animals and tissues. These methods have often been found more sensitive as compared to bacterial cultivation (Thoresen et al., 1994; Boye et al., 2000) or virological examination (Burgesser et al., 1999; Larsen et al., 1999). The aim of the present study was to compare the sensitivity of bacterial cultivation, PCR, ISH and IHC in the diagnosis of H. somnus when applied to pneumonic bovine tissue.
Section snippets
Lung material
Pneumonic lung tissue from 65 calves originating from 56 herds was included in the study. All samples originated from field cases with pneumonia of unknown aetiology submitted to the Danish Veterinary Laboratory (DVL) between October 1997 and May 1998.
Necropsy and sample collection
All cases were examined pathologically and material for bacterial cultivation (BC), histopathology, IHC, ISH and PCR was collected from the same area in each lung (Fig. 1). In order to obtain comparable results, careful effort was made to ensure
Results
The results of the different detection methods and the relationship between the positive cases determined by each technique and the histopathological diagnosis are given in Table 1. In total, H. somnus was detected by one or more techniques in 33 of the 65 cases (51%). All 65 lung sections were examined histologically and divided into fibrinous/necrotizing bronchopneumonia (n=32) and suppurative bronchopneumonia (n=33), according to the guidelines previously described (Tegtmeier et al., 1999a).
Discussion
In the present study, the PCR-detection proved to be the most sensitive method, both when performed directly on swabs from either the cut surface (swab-PCR) or from a bronchus (bronchus-PCR), and when performed on the growth from agar plates (plate-PCR). As the plate-PCR and BC were performed on virtually identical material, the higher number of positive plate-PCR cases (32%) could be interpreted as a higher sensitivity of PCR as compared to traditional inspection of plates (15%), probably
Acknowledgements
The excellent technical assistance of Ulla L. Andreasen, Annie Ravn and Jannie Jensen is gratefully acknowledged. This study was financed by the Research Secretariat of the Danish Ministry of Food, Agriculture and Fisheries (Grant No. SUN94-SVS-6).
References (16)
- et al.
Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures
Vet. Microbiol.
(1998) - et al.
Pathological, immunocytochemical and microbiological findings in calf pneumonias associated with Haemophilus somnus infection
J. Comp. Path.
(1990) - et al.
Microscopic lesions associated with the isolation of Haemophilus somnus from pneumonic bovine lungs
Vet. Pathol.
(1985) - et al.
Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence and peroxidase–antiperoxidase assays in formalin-fixed paraffin-embedded tissue sections from pigs
J. Vet. Diagn. Invest.
(2000) - et al.
Comparison of PCR, virus isolation, and indirect fluorescent antibody staining in the detection of naturally occurring feline herpesvirus infections
J. Vet. Diagn. Invest.
(1999) In situ hybridization with riboprobes: an overview for veterinary pathologists
Vet. Pathol.
(1998)- et al.
Haemophilus somnus: a review
Vet. Bull.
(1983) - et al.
Diagnosis of enzootic pneumonia in Danish cattle: application of the reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle
J. Vet. Diagn. Invest.
(1999)
Cited by (36)
Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex
2020, Veterinary JournalCitation Excerpt :The field diagnosis of BRD is based on clinical signs which can be supported by microbiology of clinical specimens such as tracheal aspirate fluid, nasal swabs and lung tissue samples. However, false negative results are commonly encountered as the causal bacteria may be fastidious or slow growing, and possibly overgrown by other organisms (Bell et al., 2014; Kugadas et al., 2014; Shanthalingam et al., 2014; Tegtmeier et al., 2000). While serological assays such as ELISA are frequently used to indicate prior infection with M. bovis, false negative results in early infection occur as seroconversion normally takes two weeks or more (Howard et al., 1986; Petersen et al., 2018).
Immunological and molecular techniques used for determination of serotypes in Pasteurellaceae
2020, Methods in MicrobiologyCitation Excerpt :The genetic background for the serotypes in H. somni has not been investigated. Immunohistochemistry and in situ hybridization have been used for the identification of H. somni in clinical samples (O’Toole & Sondgeroth, 2016; Tegtmeier, Angen, & Ahrens, 2000). A species-specific PCR test has been developed for H. somni targeting the 16S rRNA gene (Angen, Ahrens, & Tegtmeier, 1998).
Evaluation of the diagnostic value of fluorescent in situ hybridization in a rat model of bacterial pneumonia
2013, Diagnostic Microbiology and Infectious DiseaseCitation Excerpt :It may be directly performed on sectioned lung and is not susceptible to contaminants, providing that only tissue-embedded colonies are evaluated. In animal models or in veterinary context of pneumonia, FISH has been successfully used to identify intraparenchymal bacteria such as Streptococcus suis, Pasteurella multocida, and Haemophilus somnus, in pig, chicken, and cattle (Boye et al., 2000; Mbuthia et al., 2001; Tegtmeier et al., 2000). For clinical lung biopsy specimens, it has been suggested that FISH could be an important diagnostic tool (Ruiz et al., 2000).
Pathogenesis and pathology of bovine pneumonia
2010, Veterinary Clinics of North America - Food Animal PracticeCitation Excerpt :Technique sensitivity and specificity differ among these tests, and results may vary depending on the test applied. For example, H somni was cultured from only 10 of 65 cases of pneumonia, yet the bacterium was demonstrated by immunohistochemistry, in situ hybridization, and PCR in 17, 19, and 29 of the cases, respectively.40 In feedlots in particular, there is strong correlation between the time of onset of pneumonia, acuteness of the lesion, and the etiologic agents that can be isolated from that lesion.
Isolation and characterization of Histophilus somni (Haemophilus somnus) in semen samples of rams with epididymitis
2005, Small Ruminant Research
- 1
Present address: DAKO A/S, Produktionsvej 42, DK-2600 Glostrup, Denmark.