The infection caused by Canine Herpesvirus (CHV), described for the first time by Carmichael et al. in 1965 and almost simultaneously by two other groups of researchers (Stewart et al., 1965; Spertzel et al., 1965), is responsible for abortions and deaths of puppies below three weeks of age. CHV belongs to the Herpesviridae family, subfamily of Alphaherpesvirinae. The species includes a single serotype, whose strain of reference is represented by CHV-1 F205V (Carmichael et al., 1965). The morphological characteristics of CHV are common to the family of belonging.
The viral particles are spherical, with an approximate diameter of 120-150 nm and have a trilaminar lipid envelope from which glycoprotein projections known as spikes or peplomers originate. Similarly to what is observed in the infection caused by alphaherpesvirus, a particularly important factor for the persistence and diffusion of CHV in the environment is represented by the phenomenon of latency in the nerve ganglia. Stressful conditions such as treatments with corticosteroids, transportation of the animal and immunosuppressive pathologies may lead to the reactivation and consequent shedding of the virus into the external environment.
EPIDEMIOLOGY
CHV is sensitive to the action of many disinfectants, such as quaternary ammonium salts, formaldehyde and phenolic derivatives. The virus is stable within pH values between 6.5 and 7.6 and is inactivated by acid pH. It is also sensitive to heat. In natural conditions, the main species receptive to the infection is the dog, exceptionally the coyote. Invitro, the majority of strains replicate on cell monolayers of canine origin, preferring primary cultures of the kidneys and lung macrophages, at a temperature of 34-35°C; higher temperatures slow down its development.
The cytopathic effect is characterised by cell rounding and the presence of Cowdry type A intranuclear inclusion bodies. The infection caused by CHV has a cosmopolitan distribution. Serological investigations conducted on different populations of dogs have found seropositivity rates included between 6 and 30%. However, in kennels where circulation of the virus can be encouraged by inadequate hygiene conditions and overcrowding, a high percentage of infection (95%) is usually found, even in the absence of clinical manifestations in newborn puppies (Rinaldo et al., 2000).
TRANSMISSION
The virus is transmitted via the oro-nasal, vertical and venereal route. In particular, newborn puppies may be infected through the digestive and inhalatory route, by ingesting or inhaling infected materials, transplacentally and in the vagina during the transfer into the birth canal (Rinaldo et al., 2000). In adult dogs the infection is primarily transmitted via the venereal, during mating, and oro-nasal route. The main sources of shedding of CHV into the external environment are represented by:
- nasal excretion: up to 15 days after infection;
- vaginal excretion: up to 20 days after infection in the male and up to 16 in the female;
- foetus and foetal membranes in the case of abortion;
- excretion by infected puppies (saliva, tears, urine and faeces).
PATHOGENESIS
The pathogenesis of the herpetic infection depends upon factors such as: method of infection, age, body temperature and immune status of the host. In puppies under the age of two weeks, the virus initially replicates in the nasal mucosa, pharynx and tonsils and then, spreading through the blood system, invades the liver, kidneys, lymphatic tissues, lungs and central nervous system. When litters over the age of two weeks are affected, the infection remains localised in the ocular and respiratory mucosae where the local temperature (35°-36°C) encourages the survival and replication of the virus. Generally, only one litter of the same female dog is affected as, after the first infection, the female dog develops immunity against CHV and subsequent litters are protected by the maternally derived antibodies taken with colostrum (Rinaldo et al., 2000). In the adult dog, the infection is limited to the genital tract, nasopharynx, tonsils, bronchial lymph nodes and occasionally to the lungs. The effects of transplacental infection vary based upon the stage of gestation in which the infection appears: if the pregnant female is infected during mid-late gestation, there have been reports of abortions, still-births or births of puppies with the absence of vital signs which after just a few days develop a systemic herpes infection. In this latter case, the majority of puppies die within the 2nd and 4th day of life, manifesting a severe state of hypothermia associated with nervous system disorders.
CLINICAL SIGNS
In dogs infected at more than two weeks of life the disease is generally asymptomatic, or the onset is in a subclinical form; occasionally conjunctivitis may be observed, combined with mild respiratory symptoms. In newborn puppies lacking maternal immunity the infection is on the other hand generally fatal. Puppies may be infected during the transfer into the birth canal in infected bitches, but more commonly through contact with oro-nasal secretions of the mother itself or of other dogs present in the breeding farm. In animals infected during the first two weeks of life the most frequently observed clinical signs are anorexia, depression, abdominal pain with greenish-yellow or bloody faeces, nausea, vomiting and constant whimpering; in addition, rhinitis with haemorrhagic or mucopurulent nasal discharge, dyspnoea, hypothermia, paddling and opisthotonus are often observed. Petechiae are also common at the level of the mucous membranes. The infection affects all puppies, which, generally, die within 24-48 hours.
Dogs that survive showsome persisting neurological signs, such as: ataxia, blindness, sensory depression and cerebellar vestibular dysfunction incompatible with life. The genital infection, which appears in adult dogs during mating, leads to the occurrence of lymphofollicular lesions. In particular, in males, balanoposthitis may be observed, while in females, vesicular lesions and hyperaemia of the vaginal mucosa may develop, occasionally associated with petechial haemorrhages. Vesicular lesions become evident during proestrus and tend to regress in anoestrus.
In the adult dog, in which the infection appears through the respiratory system, the clinical signs most frequently reported are represented by dry cough, nasal discharge and tonsillitis; in some cases, due to the intervention of secondary pathogens, bronchitis or bronchopneumonia may be observed. Studies conducted in this regard have shown that CHV forms part of the group of pathogens responsible for kennel cough syndrome, along with canine adenovirus type 2, canine respiratory coronavirus, parainfluenza virus and bacteria such as mycoplasma and Bordetella spp.
DIFFERENTIAL DIAGNOSIS
Neonatal mortality has become an emergency particularly with the appearance of endemic herpetic infections (CHV); however, the causes that might lead to the death of many small puppies or newborns are varied. Non-infectious causes of neonatal death during the first three weeks of life of the puppy are often linked to problems that arose while the foetus was in utero or are related to whelping: uterine inertia or difficulty passing through the canal, congenital anomalies, colds, malnutrition, hypocalcaemia or hypoglycaemia. Infectious causes are known to be both viral and bacterial, but also parasitic: parvovirus (CPV1) and CHV are the aetiological factors that most often lead to the death of a puppy between the age of 0 and 12 weeks. However, bacterial infections caused by Escherichia coli, Staphylococcus spp. and Streptococcus spp. are also a cause of stillbirths that should not be underestimated.
ASPETTI DIAGNOSTICI
Asymptomatic dogs or females suffering from uterine infection may maintain a latent infection with shedding of the virus for about a week through nasal or genital secretions and thereafter with shedding at unpredictable intervals, for months or even years. 
The latent virus may be shed again during times of stress (moving, shows, pregnancy) or, experimentally, with the use of immunosuppressive drugs (corticosteroids). From the anatomical pathology perspective, in puppies, the predominant lesions are disseminated focal necrosis and haemorrhages. The organs which are most affected are the kidneys (“pathognomonic” lesions) (Fig. 1), in which subcapsular haemorrhages may be present with red areas surrounded by a white halo of necrotic tissue, and which often extend to the cortex and medulla. Multifocal areas of necrosis and haemorrhage may be observed also in other organs such as the lungs, liver, spleen (Fig. 2), brain and intestine. Meningoencephalitis is also common. Occasionally, within the necrotic area, it is possible to observe microscopically the presence of intranuclear inclusions, but this is rare. The direct laboratory diagnosis is based upon isolating the virus from the organs of dead puppies or from vesicular lesions on the genitalia of a
dult dogs; in addition, molecular biology methods such as the polymerase chain reaction (PCR) may be used which, compared to traditional techniques, guarantee greater sensitivity and specificity. A latent virus may be evidenced using PCR, as it persists in the trigeminal ganglia as well as in the lumbar and sacral glands, tonsils and parotid salivary gland. However, as viral shedding is intermittent, a negative outcome should not be taken as sure evidence that the animal is actually negative. In some works it is suggested, in any case, to conduct sampling for PCR during proestrus or immediately before birth in order to ensure that the animal does not shed the virus during whelping. An indirect diagnosis, aimed at identifying the anti-CHV antibodies, may be carried out via an indirect seroneutralisation or immunofluorescence assay to be conducted on duplicate or triplicate samples at intervals of 15 and 21 days to assess that the seroconversion has occurred. It should be noted that a drop in the antibody titre has been observed during dioestrus, while the higher titres are observed during the stages at the end of anoestrus or proestrus.
PROPHYLAXIS
Direct prophylaxis against CHV infection is based upon conducting regular examinations aimed at identifying carriers selected for reproduction. These controls should be based on a careful physical examination, which allows to verify the presence of vesicular lesions to the genital organs, and on serological sampling necessary for indirect seroneutralisation or immunofluorescence assays. Alongside direct prophylaxis interventions a subunit vaccine is also currently available for annual administrations to dogs used for breeding. In Europe, including Italy, an inactive subunit vaccine is on the market; it is to be used in female dogs close to conception and 1-2 weeks before birth, in order to prevent puppy mortality, clinical symptoms and lesions caused by herpesvirus contracted from the mother in utero or in the initial days of life. However, the low incidence of clinical epidemics and the limited duration of protection induced by the vaccine do not allow to recommend its routine use, other than in specific cases, such as, for example, in breeding farms with proven presence of the virus, even though also in these circumstance the role of canine herpesvirus in cases of neonatal death and reproductive disorders has not been fully confirmed (breeding farms with a seroprevalence of 100% do not present problems of stillbirths). However, when a non-immune female is introduced into these breeding farms, severe episodes of mortality may occur in her puppies.