Canine parvovirus infection is an infectious and contagious disease commonly known as “canine haemorrhagic gastroenteritis”. It generally affects puppies between 30 days and 3 months of age and the clinical course may be severe and at times fatal. Recently, disease episodes have also been reported in 7-8 month-old subjects.
The disease was reported for the first time almost simultaneously in Europe and North America between 1978 and 1979; a parvovirus, not correlated with other canine parvoviruses (type 1), known as Canine Minute Virus and responsible for respiratory and/or gastroenteric diseases in puppies up to one week of age, was identified as the responsible agent. The new virus was labelled Canine parvovirus type 2 (CPV-2).
VIRUS PROPERTIES
CPV-2 belongs to the Parvoviridae family. It is a small virus (26 nm) with a single stranded DNA. The capsid has icosahedral symmetry and is formed by 32 capsomeres; in view of the absence of an envelope, it can survive for long periods (months) in the external environment, even under highly unfavourable conditions. In fact, it can survive for 1 hour at 56°C and is resistant to pH variations and to lipid solvents. CPV-2 is sensitive to soda, sodium hypochlorite, beta-propiolactone, formalin and UV rays. It possesses an haemagglutinating activity for erythrocytes of swine, monkeys, cats and horses at a temperature of +4°C. This characteristic is exploited for diagnostic purposes. The genome of the virus is completely absent in genes that encode for replication enzymes, consequently the virus uses cellular enzymes, which are very active in cells in the S-phase of mitosis. For this reason, the virus shows a preference for replicating in cells undergoing active replication such as, for example, in cells of the gastroenteric mucosa.
In laboratory tests, CPV-2 propagates well on primary and secondary cultures of canine and feline kidney cells, on continuous cell lines such as CrFK (feline kidney cells) and A-72 (canine fibroma cells). The cytopathic effect of the virus is not very evident, however the supernatant of cell cultures shows haemagglutinating activity, and inclusion bodies can be identified in the cell nuclei. The interesting evolution of the virus has been described in detail.
PATHOGENESIS
Thanks to the use of more sophisticated laboratory techniques (PCR, real-time PCR) new and important information have been acquired on the pathogenesis of CPV-2 infection; to some degree, such information has substantially changed our understanding of this virus.
Penetration of the virus is via the oro-nasal route and, after an initial and brief replication in the oropharynx (approx. 1 day), it invades the bloodstream (where it remains for around 30 days) and the small intestine. The virus then colonises, via the bloodstream, the other lymphoid organs (spleen, lymph nodes, thymus, Peyer’s patches), where it causes tissue depletion and necrosis. It is during this phase (4-5 days after infection) that the virus colonises the cells of the intestinal crypts, triggering, as a result, the appearance of diarrhoea which, in many cases, is haemorrhagic. The virus is shed with the faeces before clinical signs appear, reaching elevated values (109 particles/gram of faeces) between 5 and 8 days from the infection.
The clinical course is generally rapid (4-5 days) and is characterized by pronounced leucopaenia/lymphopenia (necrosis of the lymphoid tissues). The outcome may be death or a very slow recovery. In subjects that survive, the virus is shed - even if at low titres – for up to 40 days after the infection. Subclinical or asymptomatic infections, characterized by mild leucopaenia and viral excretion in the faeces, are frequent.
The main factors which may condition the evolution of the infection and of the disease are basically the age of the subject (puppies up to 2 months of age are more sensitive) and the immune status (antibody level) at the time of exposure to the virus.
Regardless of the final outcome, as early as 7 days after exposure high levels of specific antibodies may be detected, with a maximum peak (1:2500-1:5000) 14 days after exposure.
The most significant recently acquired data are related to the long duration of the viraemia (around 30 days) and the viral excretion through the faeces (for around 40 days).
ACUTE CPV-2 MYOCARDITIS
Acute myocarditis was a frequent finding following the initial circulation of CPV-2 (in the late ‘70s). Today, acute CPV-2 myocarditis is rarely observed. This disease is correlated with the replication of CPV-2 which, as already mentioned, has a marked tropism for cells with intense mitotic activity. Until 14 weeks of age the intestinal cells of puppies undergo intense replication and are thus fully receptive to the action of the virus. The cells of the myocardial syncytium also undergo an intense mitotic activity up to the 3rdweek life and are thus an excellent target for CPV-2, which can cause acute myocarditis.
Today, CPV-2 myocarditis is a very rare finding in the dog, unlike what used to occur at the time of the initial spread of the virus, when entire litters were affected by the pathogen. The explanation lies in the fact that, at the beginning of the epidemic, the majority of dogs had no specific antibodies and hence bitches could not transfer colostral immunity to their puppies. These puppies, in the early days of life, could easily contract the infection, developing a fatal myocarditis before the appearance of gastrointestinal symptoms.
For several years now, both in view of the spread of the infection and of the large-scale use of vaccines, almost all dogs are instead immune to CPV-2. In puppies, therefore, the cardiac syncytium can no longer be attacked by CPV-2, as it stabilizes before the disappearance of the protection provided by colostral antibodies.
CLINICAL SIGNS
The course of parvovirus infection varies from subject to subject, even within the same litter. Two clinical forms are essentially recognized: a gastroenteric form (of variable severity) and a cardiac form; a third, subclinical, form is also recognized.
Gastroenteric form
In the gastroenteric form after a short period of incubation vomiting and diarrhoea appear, with the presence of blood. Subjects are anorexic and exhibit sensory depression. Dehydration sets in quickly, sometimes accompanied by a slight hyperthermia. Leucopaenia is constant (<3000 cells/mm3), with pronounced lymphopenia. The course is variable; a slow healing or a worsening of the general conditions with death of the affected subjects may be observed.
Cardiac form
In the cardiac form the clinical evolution is rapid and in subjects 2-4 weeks of age the outcome is fatal with signs of acute myocarditis.
Subclinical form
This is perhaps the relatively most frequent form. It is characterized by a mild sensory depression and/or anorexia, mild diarrhoea and modest leucopaenia/lymphopenia. The development of the subclinical form, which is sometimes asymptomatic, is closely correlated to the animal’s degree of immunity at the time of viral exposure. The case is obviously one of partial immunity, which does not prevent replication and excretion of the virus. The epidemiological importance of this clinical form varies depending on the lifestyle of the animal. In a puppy, living in a home environment, the epidemiological impact of the infection is limited; on the contrary, the same situation projected into breeding kennel implies a totally different scenario, as the infected puppy sheds the virus with the faeces and can transmit the infection to other subjects.
DIFFERENTIAL DIAGNOSIS
It should be considered that the clinical course of parvovirus infection is not always the classic one and that subclinical forms may often be present. In addition, it is necessary to differentiate parvovirus from other important infectious diseases of the dog, like distemper, coronavirus infection, infectious hepatitis and coccidia infestation.
| Laboratory tests are essential for diagnostic certainty |
DIAGNOSTIC APPROACH
The typical clinical and, sometimes, pathognomonic signs of CPV-2 infection make it possible to formulate a diagnosis which, nevertheless, must always be confirmed by specific laboratory tests. In living animals, besides the white blood cell count, a faecal sample must be submitted to laboratory tests. At necropsy, apart from the faeces, portions of intestine or lymphoid organs may be collected. To this purpose, it is worth recalling that it is always advisable to collect faecal samples from the ampulla of the rectum since, especially in subjects that have had a long clinical course, the virus might no longer be present in the intestinal segments presenting lesions.
Clinical diagnosis
The clinical suspicion of parvovirus infections can be formulated in the presence of:
- haemorrhagic diarrhoea,
- vomiting and rapid dehydration,
- leucopaenia (< 4000 WBC/mm3).
The degree of leucopaenia may also be important for advancing a prognostic hypothesis. Values around 3000-4000 WBC/mm3, associated with pronounced haemorrhagic diarrhoea, allow to anticipate a favourable outcome of the disease. Vice versa, a leucopaenia with values < 2000 WBC/mm3 is usually of unfavourable prognostic significance.
The degree of leucopaenia varies depending on the ability of the virus to replicate in the lymphoid organs. In its turn, the virus manages to replicate more or less intensely based on the degree of immunity of the puppy at the time of infection. In a subject without specific immunity, CPV-2 can therefore replicate massively and without obstacles, causing pronounced leucopaenia and severe clinical symptoms and signs; in a subject with partial immunity, on the other hand, the virus generally causes a subclinical infection with moderate leucopaenia ( 3000-4000 WBC/mm3).
Anatomopathological lesions
The anatomopathological lesions present in parvovirus infection vary depending on the course of the disease. The lesions are especially obvious in the gastrointestinal tract, where a severe form of haemorrhagic enteritis may be observed (Figs. 1 and 2). Haemorrhages may at times be present also in mesenteric lymph nodes. At microscopic examination, nuclear inclusions may be seen in the intestinal crypt cells. Severe tissue depletion and necrosis is detected in the lymphoid organs. In the cardiac form of the infection the carcass is not visibly altered. In cases in which the course of the myocarditis has not been fulminant, pulmonary oedema, hydropericardium, ascites and flaccid myocardium with infarctions may be detected.
Fig. 1 Fig. 2
Virologic diagnosis
Several laboratory tests may be used to diagnose CPV-2 infection, among which:
Direct electron microscope examination of faecal extractsfor direct identification of the virus
The faecal sample is diluted in distilled water and then subjected to ultracentrifugation and to negative colouring with phosphotungstic acid. To facilitate the diagnosis, an antiserum specific for CPF-2 can be added to the suspension being examined in order to obtain the formation of more easily identifiable immune complexes (immune electron microscopy).
Faecal haemagglutination (HA) test.
HA is the first-choice diagnostic test for parvovirus infection. The test presents several advantages: simplicity, rapid execution, easy interpretation and contained costs. The faecal sample is homogenised and centrifuged to remove debris. The supernatant is diluted on plates to which a 1% suspension of red blood cells (usually of swine) is added; the reading is performed after incubation for 2 hours at 4°C. A negative result to the HA test is not sufficient to exclude the presence of the virus. Samples drawn in a late phase of the infection may give false negative results, both because a small amount of virus is present and because the faeces may already contain antibodies which, having complexed the virus, prevent bonding with the red blood cells. In the presence of strong clinical suspicion, other tests must therefore be used to confirm the negative results.
ELISA (Enzyme-linked immunosorbent assay)
This is a plate enzyme immunoassay that uses monoclonal antibodies capable of “capturing” any virus present in the sample. A positive result is indicated by a colourimetric reaction following the addition of other enzyme-marked antibodies (peroxidise or alkaline phosphatase).
Immunochromatographic test
Assays that allow a rapid diagnosis of CPV-2 are currently available on the market. This test allows the detection of the antigen in canine faecesand it is used in veterinary clinics because it is both practical and quick. The sensitivity of the test is limited and, in the event of a negative result, the same considerations already expressed for the HA test are valid.
Isolation on cells
Suspensions of faecal extracts or of intestinal mucosal scrapings are treated with antibiotics and inoculated on cell lines A-72 and CrFK immediately after trypsinisation. Viral growth is detected after 2-7 days of incubation, at 37°C, with an immunofluorescence test or via the detection of nuclear inclusions. The sensitivity of the test is quite high however, being difficult to perform, it is restricted to specialised laboratories.
PCR
PCR is based on the in vitro amplification of a specific nucleotide sequence of viral DNA and is used on faecal samples of animals with suspected infection. The test is extremely sensitive and it is capable of detecting positivity even in samples with a low viral titre or which resulted negative to other diagnostic tests.
Real-time PCR
Real-time PCR allows the detection of both the presence and the quantity of viral DNA. For this reason it is often used in studies on the pathogenesis of CPV-2 infection.
Minor groove binder (MGB) probes
Researchers in Bari, Italy, have recently developed two real-time PCR tests for the molecular characterisation of CPV-2 variants, based on MGB (minor groove binder) probe technology. MGB probes hybridise with the DNA of the sample in a very specific way and are even capable of recognising the mutation of a single nucleotide (single nucleotide polymorphism, SNP) in the region of attachment. The MGB tests used to characterise CPV-2 exploit the SNPs existing in variants 2a and 2b (4062A→G) and in variants 2b and 2c (4064T→A), allowing the typing of a large number of samples within a few hours. The test is only performed in specialised laboratories and is extremely useful as it allows the rapid characterisation of variants without having to resort to nucleic acid sequencing. Similar tests have also been developed to allow the distinction between vaccine and field strains. An additional use of these tests is to exclude the false positive results that may be seen with common diagnostic tests in the first 7-10 days following vaccination, which result from the faecal excretion of the vaccine virus, which, being alive, can replicate in the intestinal tract.
Serological diagnosis
Serological tests are of limited diagnostic value as a large portion of the canine population has vaccine- or field virus-induced antibodies. In addition, when the clinical symptoms appear, the animal already has high antibody titres, making a correct interpretation of the result impossible. Serological tests, instead, are very useful for assessing the immune status of the animal and the degree of protection against the infection; these tests are essential for a rational and effective vaccine protocol in puppies, as they allow to identify the best time for vaccination. The most often used serological test is haemagglutination inhibition (HI). Having the availability of monoclonal antibodies this test can be used to characterise CPV-2 variants (2a, 2b, 2c).
DISINFECTING THE ENVIRONMENT AGAINST PARVOVIRUS
In the presence of a parvovirus infection infected dogs should be isolated immediately, especially when other subjects are present. In view of CPV-2’s high degree of resistance in the external environment and the massive spread of the virus through the faeces, rooms in which infected dogs have stayed should not house new puppies for a long time. After the removal of all faecal material and washing of the floors with suitable products, a careful and complete disinfection of the rooms with sodium hypochlorite-based products is essential.
PROPHYLAXIS
- Sanitary prophylaxis: prophylaxis is of a certain importance in kennels and breeding establishments, albeit it is often not very effective because of the resistance properties of parvovirus in the environment.Some prophylaxis measures are necessary, bearing in mind that:
- a healed dog continues to shed the virus with the faeces for a very long time, consequently measures should be taken to prevent it from being a source of contagion for other dogs;
- the virus survives in the environment for as long as 4-5 months.
- Vaccine prophylaxis
THERAPEUTIC APPROACH
Treatment essentially consists in the correction of the hydrolytic deficit and of electrolytic alterations, a preventive treatment with antibiotics and the use of antiemetics.Substantial improvements have been obtained with the early use of feline interferon.
Symptomatic treatment
Correction of fluid and electrolyte deficits
- Rehydration, i.v. (750 ml glucose + 250 ml lactated Ringer's solution® + 26 mmol/l of Kcl) at a dose of 50 ml/Kg/24 hours, during the first 8 hours
- Monitoring of hydration, weight and kalaemia (every 6 hours at first and then every 12 hours)
- Rehydration, i.v. for the maintenance phase (0.9% NaCl, 5% glucose + 26 mmol/l Kcl)
- Antibiotic therapy (marbofloxacin + cefalexin i.v./12 hour)
- Antiemetic treatment (metoclopramide initially and then acepromazine until the disappearance of vomiting)
- After the first 24 hours, oral administration of a gastrointestinal protector (neomycin, kaolin, pectin).
Antiviral treatment
Antiviral treatment with the use of feline interferon-omega has proven effective. The earlier the treatment is begun following the first clinical signs (as primary treatment), the greater the efficacy. The drug must ALWAYS be administered together with a supportive therapy. This interferon not only significantly reduces the risk of death, but it also allows a quicker healing of the animal. Recommended dosage: 2.5 MU/kg i.v. every 24 hours for 3 consecutive days.
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