10.4 Postmortem diagnosis

Detection of tuberculosis using pathological examination involves visual observation, palpation, and incision of organs and tissue to detect lesions. A presumptive diagnosis can be made on the basis of macroscopic granulomatous lesions (Figures 1 and 2). Differential diagnosis includes parasitic and mycotic granulomas and abscesses caused by other bacterial pathogens such as Actinomyces bovis, Actinobacillosis, and Trueperella pyogenes, as well as bovine lymphosarcoma [67, 68]. Further, very small lesions may be missed and may only be detected microscopically. Routine postmortem meat inspection has been found to detect approximately only 47% of presumptive lesions [69]. Direct smears of suspected lesions should be stained by the acid fast method and examined for acid-fast bacilli (Figure 5).

## 10.5 Culture and isolation of mycobacteria

Culture is considered the "gold standard" for detection of Mycobacteria [69]. Samples for culture are first homogenized and decontaminated with sodium hydroxide to inactivate any contaminant bacteria present in the sample, inoculated into solid or liquid media and incubated at 37°C. Solid media include egg-based Lowenstein-Jensen (L-J), Agar-based media such as Middlebrook 7H10, 7H11, and Stonebrink Leslie solid culture media. Solid media is prepared as slants in screwcapped bottles. In Lowenstein-Jensen media, malachite green dye (0.025 g/100 ml) is used as a selective agent. Isolation should target MTBC and MOTTs and it is recommended that each sample is inoculated into three tubes of LJ, one containing glycerol, another pyruvate, and the other PNB. Most mycobacteria are obligatory aerobic but M. bovis is microaerophilic. Screw caps should be loosened, to allow in oxygen, and the tubes incubated in a slanting position, to allow bacteria to seed onto Diseases Caused by Bacteria in Cattle: Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.82051

## Figure 5.

respectively [58, 64]. The CTT has higher specificity than the SITT since it can distinguish animals infected with nontuberculous mycobacteria, specifically the MAC complex, which include M. avium subsp. paratuberculosis, the causative agent of Johne's disease. Other MOTTs species with ability to cross-react with M. bovis have, however, been isolated from tuberculous lesion in cattle and related wild species. The advantage of the CITT over the SITT is therefore limited [5–7].

The gamma interferon assay (IFNγ) is an in vitro form of the CITT. It is based on detection of γ interferon produced by specific circulating lymphocytes upon stimulation of heparinized whole blood in vitro with PPD-B and PPD-A. Detection of IFNγ is carried by a sandwich ELISA, using two monoclonal antibodies to bovine gamma-interferon, after incubation of the blood for about 16–24 hours with PPD-B and PPD-A. The IFNγ test is reportedly more sensitive than the tuberculin test and can detect infected animals that are negative to the later. The sensitivity and specificity are estimated at 81.8 and 99.1% [65]. It has been observed that more infected cattle can be identified by using both the tuberculin and the IFNγ tests and it is recommended that both tests be conducted in parallel [66]. The advantage of the IFNγ is that infected animals are detected early and only one visit to the farm is required. It is particularly convenient for animals that are difficult to capture or handle, such as cattle reared in ranches or under nomadic pastoralism, or wildlife, as they need only to be captured once rather than twice. It however requires more technical expertise and facilities and is costly (approximately 10 USD, for consum-

Detection of tuberculosis using pathological examination involves visual observation, palpation, and incision of organs and tissue to detect lesions. A presumptive

(Figures 1 and 2). Differential diagnosis includes parasitic and mycotic granulomas and abscesses caused by other bacterial pathogens such as Actinomyces bovis, Actinobacillosis, and Trueperella pyogenes, as well as bovine lymphosarcoma [67, 68]. Further, very small lesions may be missed and may only be detected microscopically. Routine postmortem meat inspection has been found to detect approximately only 47% of presumptive lesions [69]. Direct smears of suspected lesions should be stained by the acid fast method and examined for acid-fast bacilli (Figure 5).

Culture is considered the "gold standard" for detection of Mycobacteria [69]. Samples for culture are first homogenized and decontaminated with sodium hydroxide to inactivate any contaminant bacteria present in the sample, inoculated into solid or liquid media and incubated at 37°C. Solid media include egg-based Lowenstein-Jensen (L-J), Agar-based media such as Middlebrook 7H10, 7H11, and Stonebrink Leslie solid culture media. Solid media is prepared as slants in screwcapped bottles. In Lowenstein-Jensen media, malachite green dye (0.025 g/100 ml) is used as a selective agent. Isolation should target MTBC and MOTTs and it is recommended that each sample is inoculated into three tubes of LJ, one containing glycerol, another pyruvate, and the other PNB. Most mycobacteria are obligatory aerobic but M. bovis is microaerophilic. Screw caps should be loosened, to allow in oxygen, and the tubes incubated in a slanting position, to allow bacteria to seed onto

diagnosis can be made on the basis of macroscopic granulomatous lesions

10.3 Gamma interferon assays

Bacterial Cattle Diseases

able materials per test).

74

10.4 Postmortem diagnosis

10.5 Culture and isolation of mycobacteria

Direct smear of a tuberculous lymph node lesion from a cow, showing presence of acid-fast bacilli (arrowed). ZN 1000. ©2018. JKN Kuria.

the media. Thereafter, the caps are tightened and the tubes incubated vertically for six 12 weeks. Liquid media include BACTEC 460, Mycobacterial Growth Indicator Tube (MGIT), which have enriched Middlebrook 7Ha with antibiotics and growth promoters are added. BACTEC 460 MGIT media is fully automated and can monitor the growth of mycobacteria by the use of oxygen quenching or fluorescent sensor. Mycobacteria may not be recovered in the cultures for a number of reasons:


Cultures suspected to be mycobacteria are then stained by acid fast method for confirmation. All laboratory procedures must be conducted in a class II biosafety cabinet in a laboratory environment that has been found safe and secure following risk assessment.

## 10.6 Molecular diagnosis

Molecular tools for differentiating Mycobacterium species have been developed [70]. Polymerase chain reaction (PCR) technique involves detection of the genetic material that is unique and specific to a species. Convenient commercial kits are available. Genotype Mycobacterium (Hain, Nehren, and Germany) are line probe assays available in three different formats: Genotype MTBC differentiate species in MTBC; GenoType Mycobacterium common mycobacteria (CM) detects most frequently encountered Mycobacteria species and Genotype Mycobacterium additional species (AS) detects less frequently encountered Mycobacteria species. This kit uses reverse hybridization technology on a solid membrane matrix consisting of nitrocellulose strips. The DNA probes are immobilized on parallel lines on the strips. Biotinylated DNA apricon fragments of the 16S-23SrRNA spacer region are incubated with the labeled strips and hybridization detected colorimetrically by addition of an enzyme, Streptavidinalkaline phosphatase, and a chromogenic substrate. A precipitate is formed on

the membrane, where hybridization takes place. Another line probe assay is INNOLiPa Mycobacteria (Innogenetics, Ghent, Belgium). Line probe assays are convenient in that they can detect many species of mycobacteria simultaneously. The strips can also be conveniently dried and preserved.

and pasteurization of milk is an effective method of preventing infected animal products from entering the food chain. Meat inspection can allow trace-back to the herd of origin, which can then be tested and eliminated. Individual testing of cattle and removal of infected and in-contact animals, coupled with animal movement controls reduces prevalence [28]. Testing and slaughter may, however, not be tenable in poor countries because of insufficient financial resources, pastoral production method that is characterized by uncontrolled movement of animals, weak veterinary institutions and political instability [28]. Further, in developing countries especially in Africa, cattle are raised together with sheep and goats, which act

Department of Veterinary Pathology, Microbiology and Parasitology, Faculty of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Veterinary Medicine, University of Nairobi, Nairobi, Kenya

\*Address all correspondence to: jknkuria@uonbi.ac.ke

provided the original work is properly cited.

as reservoirs and are not targets for test and slaughter.

Diseases Caused by Bacteria in Cattle: Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.82051

Author details

Joseph K.N. Kuria

77

AccuProbe (GEN-Probe, San Diego, California, USA), is an in-solution hybridization assay. DNA probes consisting of species-specific, single-stranded DNA oligonucleotides are prepared complementary to ribosomal RNA released from bacterial cultures and labeled with acridinum ester (chemiluminescent). Hybridization is measured by chemiluminescence using a luminometer and expressed as relative light units (RLU). The test can be performed on culture growing from broth or solid media and will detect all members of MTBC but without differentiating the species. However, since no nucleic acid amplification occurs in the assay, identification requires sufficient growth.

Real-time commercial PCR kits are also available for direct detection in clinical specimens and pathological specimens but can also be used for identification of cultures. The current available kits detect MTBC but not individual species.

Restriction fragment length polymorphism (RFLP) or spoligotyping distinguishes between phenotypically different strains of M. bovis [71]. It is designed to detect the unique spacers within the direct repeat (DR) locus of the M. bovis genome [72] and is a useful epidemiological tool, in that it indicates strains circulating in a population, and therefore the transmission patterns.

DNA tests are more rapid and reliable than the conventional identification methods, but are still limited to the postmortem diagnosis of the infection, in that, tissue samples or isolates are still required. Extraction and detection DNA in nasal swab samples, milk, lymph node aspirates may however be achieved [73].
