Isolation in Cell Culture

Chapter 1

Isolation in Cell Culture


Cell culture refers to the culture of nucleated (eukaryotic) cells under controlled conditions within the laboratory. Infectious agents that require living host cells for replication can only be isolated in cell culture. With the advent of molecular diagnostic assays based on nucleic acid detection, cell culture is being used less often for routine clinical diagnostic purposes, because of the long turnaround times (days to weeks), cost, and requirement for significant technical expertise to perform cell culture and interpret results (Table 1-1). Nevertheless, isolation of viral and intracellular bacterial and protozoal pathogens in cell culture remains an important technique for the discovery of new pathogens, identification of organisms involved in disease when the results of molecular testing or serology are unavailable or equivocal, the propagation of isolates for research purposes, the generation of organisms for vaccination purposes, and the establishment of the efficacy of novel antimicrobial drugs. Vaccines for dogs and cats that are propagated in cell culture include those for canine distemper, canine adenovirus infections, parvovirus infections, rabies, and feline viral and chlamydial respiratory tract disease. Veterinary clinicians should remain aware of situations where cell culture may be the best technique to identify the presence of an infectious agent and the optimum methods for collection and submission of specimens. Knowledge of cell culture methods can help veterinary clinicians to submit the optimum specimens and to understand laboratory turnaround times, potential complications, and how to interpret results.

Specimen Collection and Transport

Although cell culture can be used to propagate intracellular bacteria and protozoa, it is most often used by clinicians for the diagnosis of viral infections. Active communication between the clinician and the laboratory that performs viral isolation is recommended. Successful detection of viruses is highly dependent on (a) collecting the appropriate specimens, (b) the timing of specimen collection, and (c) rapid and proper specimen transport and processing. Thus the actions of the veterinary clinician play a critical role in ensuring positive test results when a virus is present.

The clinician should discuss with the laboratory what types of viruses are suspected in light of the animal’s clinical presentation. The patient signalment, history, clinical signs, immune status, travel history, and number of animals affected should be discussed to generate conclusions regarding the nature of the suspected infection (Box 1-1). Some viruses, such as feline coronavirus (FCoV), are difficult to isolate in cell culture or grow slowly, whereas others, such as feline calicivirus (FCV), replicate readily and rapidly in cell culture, and the sensitivity of cell culture is high. Viruses differ in respect to the cell type they prefer to replicate within. As a result, specimens should be sent to the laboratory with information on the specific viruses that are suspected.

The timing of specimen collection is particularly important for viral infections. Specimens should be collected as early as possible following the onset of clinical signs, optimally within the first week, because viral shedding may commence before the onset of signs and continue for only a few days. The duration of viral shedding depends on the type of virus and the anatomic site sampled. When multiple animals are affected, collection of specimens from more than one animal may increase the chance that an isolate will be obtained. If possible, antibody testing using acute and convalescent phase serology should be performed concurrently to help confirm the diagnosis (see Chapter 2).

Selection of the best specimen and collection site for culture is optimized based on knowledge of the pathogenesis of the infectious agent involved, because the optimum specimen collection site may not be the site where clinical signs are most severe. Attempts should be made during specimen collection to prevent contamination of the specimen with normal flora, although this is not always possible. Specimen size should also be maximized (for example, at least 5 mL of blood, body fluids, or lavage specimens, and ideally 8 to 10 mL of blood) to increase the chance of a positive isolation. In general, nasal or nasopharyngeal washes have been preferred over nasal swabs in human patients for isolation of respiratory viruses, but one study showed that nasal swab specimens were just as sensitive as nasopharyngeal washes for isolation of most respiratory viruses.1 Nasal or oropharyngeal swab specimens are collected by placing a long-shafted swab in the area to be sampled, rotating the swab against the mucosa, and allowing the secretions to be absorbed for approximately 5 to 10 seconds.

Swabs and small tissue specimens for virus isolation should be placed in buffered virus transport medium, which contains antibiotics and protein. This can be obtained from the laboratory or purchased from other commercial sources. It is important that the medium used has not reached its expiry date.

Liquid specimens such as blood, cerebrospinal fluid, and bronchoalveolar lavage fluid do not need to be placed in transport media. Blood samples should be collected using sterile technique, with antiseptic preparation of the site of venipuncture, and can be submitted in EDTA anticoagulant tubes.

All specimens should be refrigerated on collection and transported as quickly as possible (preferably within 24 hours) to the laboratory, because delayed transport can lead to loss of organism viability. If delays in excess of 2 to 3 days are anticipated, the specimen can be frozen. Freezing should be avoided whenever possible, as it may lead to dramatic loss of virus viability. If freezing is unavoidable, freezing at −70° C is preferable to freezing at −20° C, and shipping on dry ice is preferable, if possible. The laboratory’s submission guide should be checked for specimen handling recommendations.

Table 1-2 provides a guide to the recommended specimen types for isolation of viruses or obligate intracellular bacteria from companion animals. Specimens should be labeled with the patient data, the site(s) from which the specimen(s) was collected, specific organisms suspected, and the time and date of specimen collection. Contained specimens should be placed inside leak-proof triple packaging and transported on wet ice or cold packs to the laboratory, especially if transport is expected to take longer than 1 hour. Absorbent materials should be placed within the secondary container in order to absorb any spills. If specimens are to be shipped, the specimen must be labeled and handled according to governmental and International Air Transport Association (IATA) regulations for shipping materials known to contain infectious substances, which are categorized as Category A or Category B. Category A infectious substances are those capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy animals and humans.2 Most specimens submitted by veterinarians fall under Category B, which are those that do not fall under the criteria for inclusion in Category A. Updated documents providing guidance on regulations for the transport of infectious substances are provided online by the World Health Organization (WHO).2 Import permits may be required for interstate and international transportation.


Specimen Collection Guide for Diagnosis of Viral and Intracellular Bacterial Infections of Companion Animals

System Affected Possible Agents Specimen Type
Respiratory tract Dogs: coronaviruses, canine adenovirus, influenza viruses, parainfluenza virus, CDV, canine herpesvirus
Cats: FHV-1, FCV, influenza viruses, FCoV
Oropharyngeal swabs
Nasal flushes, transtracheal wash or bronchoalveolar lavage specimens: ideally 5 to 10 mL of fluid
Lung tissue obtained at biopsy or necropsy, including an area adjacent to affected tissue
Eye Dogs: canine herpesvirus, canine adenovirus
Cats: FHV-1, FCV, Chlamydia felis
Conjunctival swab, scraping or biopsy
Central nervous system Dogs: CDV, West Nile virus, arboviruses Cerebrospinal fluid: ideally at least 0.5 to 1 mL
Blood: 8 to 10 mL
Brain at necropsy
Gastrointestinal tract Dogs: CDV, CPV, rotaviruses, canine coronavirus
Cats: FCoV, FCV, FeLV, rotaviruses, toroviruses
Feces: ideally an olive-sized portion of formed feces or 10 mL of liquid stool
Intestinal biopsies obtained using endoscopy or surgery, or intestinal tissue obtained at necropsy
Genital Dogs: canine herpesvirus
Cats: Chlamydia felis
Vesicle scrapings, vaginal swabs
Congenital and perinatal Dogs: canine herpesvirus
Cats: FHV-1, FeLV
Blood, tissues obtained at necropsy
Blood Dogs: Anaplasma phagocytophilum, Rickettsia rickettsii, Ehrlichia canis
Cats: FeLV, FIV, FCoV
Blood: ideally 8 to 10 mL

CDV, Canine distemper virus; CPV, canine parvovirus; FCoV, feline coronavirus; FCV, feline calicivirus; FeLV, feline leukemia virus; FHV-1, feline herpesvirus-1; FIV, feline immunodeficiency virus.

Diagnostic Methods

Maintenance of Cell Cultures in the Laboratory

In general, cells are grown as a monolayer on a plastic plate. The cells in the monolayer can be derived directly from an animal (primary cell culture), which tend to have a limited life span, or they may be immortalized (continuous cell lines). Primary cell cultures are needed for the isolation of some viruses, because the cells more closely resemble those present in vivo, and the replication of these viruses occurs more efficiently in primary cell lines than in continuous cell lines. Further subculture of primary cell lines often reduces their sensitivity to viral infection. Primary cell cultures are generated by placing tissues in cell culture media, often after treatment of the tissue with an enzyme such as trypsin or collagenase. Primary white blood cell cultures (such as peripheral blood mononuclear cell cultures) are generated by separation of the white cells from the other cellular elements using density gradient centrifugation, and adding them to a culture medium. Ficoll, a highly branched polysaccharide, is an example of a medium used commonly for density gradient centrifugation. Primary cell cultures have been used widely for the isolation of intracellular pathogens of dogs and cats.36

Low-passage cell lines remain viable and sensitive to viral infections for 20 to 50 passages. Continuous cell lines are the type of cell line used most commonly for diagnostic, research, and commercial purposes. These are derived from cancer cells (such as the widely used HeLa cell line, derived from human cervical cancer cells of a patient named Henrietta Lacks),7 or they result from experimental induction of cellular mutations (for example, using a carcinogen). Continuous cell lines representing a wide variety of cell types are available from commercial suppliers (Table 1-3). Laboratories that perform virus isolation for disease diagnosis may need to simultaneously inoculate multiple cell lines, because different viruses prefer to replicate in differing cell types. Mixed cell cultures are also now available commercially to simultaneously facilitate isolation of multiple different viral pathogens.

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Jul 10, 2016 | Posted by in INTERNAL MEDICINE | Comments Off on Isolation in Cell Culture

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