VETERINARY MEDICINE: A textbook of the diseases of cattle, horses, sheep, pigs and goats

SWINE INFLUENZA

Synopsis

Etiology Influenza A virus subtypes H1N1, H1N2 and H3N2 of Orthomyxovirus

Epidemiology United States, England, Japan, Canada, Belgium. Young pigs. High morbidity, low mortality. During cold months. Antigenic diversity of virus. Aquatic birds natural reservoirs. Spread between pigs

Signs High incidence of anorexia, fever, thumps, muscle stiffness; recovery in several days

Clinical pathology PCR test to detect virus. Hemagglutination test and ELISA

Lesions Marked congestion of upper respiratory tract. Exudate in bronchi. Atelectasis. Suppurative bronchiolitis

Diagnostic confirmation Demonstrate virus in tissues

Differential diagnosis list

• Enzootic pneumonia

• Hog cholera

• Inclusion body rhinitis

• Atrophic rhinitis

Treatment Antimicrobials for secondary infection

Control No effective measures available. Vaccines are in use in certain parts of the world

INTRODUCTION

Swine influenza is an important cause of bronchointerstitial pneumonia throughout all pig keeping areas of the world. Real problems are associated with the changing viruses that cause disease.

ETIOLOGY

Classical swine influenza is associated with influenza A virus subtypes H1N1, H1N2 and H3N2 belonging to the Orthomyxovirus genus of the Orthomyxoviridae family. Other types have been isolated from pigs but as yet have not established as widespread endemic strains.

EPIDEMIOLOGY

The segmented nature of the viral genome is a critical structural feature that enables them to be reassorted. Since 1998, H, N and PB1 polymerase genes from human viruses, M, NS and NP genes from classical swine viruses and PA and PB2 polymerase genes from avian viruses have also been found.^1,²

Occurrence

There are three groups of viruses but only influenza A viruses are important in pigs. They occur in a large number of species including humans, primates, pigs, horses, sea mammals, and birds.³ When new variants occur in pig husbandry they are usually found in the pig population before they acquire the ability to spread rapidly and become associated with disease. They are named using the following convention: A/species/localization/isolate number/year of isolation, e.g. A/Wisconsin/125/98. If no species is indicated it is a human virus. They are described with reference to the hemagglutinin (HA or H) and the neuraminidase (NA or N) that project from the surface of the viral envelope. There are 15H and 9N forms that can be distinguished antigenically and genetically and all of these have occurred in waterfowl and shore birds. They provide a permanent source of infection as does the water on which they float.⁴ The H binds to sialic acid and mediates the virus infection of the host. It also contains most of the antigenic sites. The N protein catalyses cleavage of sialic acid.

Swine influenza first appeared in the United States immediately following the 1918 pandemic of human influenza, and it was generally believed that it was caused by adaptation of the human influenza virus to swine. Nucleotide sequencing of the genes coding for the internal virus proteins indicates that the human pandemic H1N1 strain and the classic swine strain H1N1 have a common avian ancestor. It is suggested that a virulent avian strain H1N1 entered the human population in 1918 causing the pandemic. The pandemic virus was then introduced into the swine population where it has persisted unchanged. In contrast, swine influenza was seen in the UK in 1941 but then disappeared until it was seen in Czechoslovakia in 1950 and Germany in 1959. Influenza was not seen again until observed in swine in Europe in 1979 possibly following importation of pigs from North America, associated with a virus antigenically related to contemporary avian H1N1 strains found in ducks.⁵ These avian like strains have been the most common since 1979.⁶

Swine influenza still occurs in the United States and viruses of the H1N1 lineage were the dominant cause of SIV from 1930 to the 1990s.² These were highly conserved (relatively unchanged) but new antigenic and genetic variants did occur.^2,⁷ Classical H1N1 viruses have also been isolated from pigs from South America, Europe, and Asia. Wild pigs also have H1N1.⁸ In the 1980s there were many genetic mixings between avian-like H1N1 and human-like H3N2 viruses.⁹ In 1992, many outbreaks of classical swine influenza occurred in England, associated with a group of H1N1 viruses that were distinguishable from classical swine viruses, the European swine viruses, and human H1N1 viruses, all of which are known to be circulating in pigs.¹⁰ Influenza A virus subtypes H1N1 and H3N2 are endemic in pigs in Great Britain.¹¹ Two distinct antigenic variants of H1N1 viruses have been associated with outbreaks of swine influenza, one of which was probably transmitted from birds to pigs in the early 1990s.⁶ The H1N2 subtypes isolated from pigs in Great Britain appear to have originated from a human H1N1 virus, which was circulating in the pig population in the 1980s, and from swine H3N2. It is suggested that the H1N1 viruses have disappeared from the human population, and the pig population provides a reservoir for the virus.¹¹ Serological surveys indicate that a swine H1N1 influenza virus has circulated in the swine population in North America for many years.¹⁰ Recent isolates from Quebec posses a hemagglutinin distinguishable from subtype H1N1.¹²

Epidemics of swine influenza have also occurred in Japan, Canada, Belgium, and France. In North America, human H3N2 have been found much less often than in the rest of the world but the very recent introduction of H3N2 from humans to pigs was probably the major factor in the emergence of the recent strains.¹

Mixtures of human and classical virus genes have been isolated from pigs in Asia and the USA.¹³ H3N2 viruses with human H and N genes and avian internal protein genes have been isolated from pigs in Asia¹⁴ and H3N2 has been found in Korea¹⁴ and are currently the dominant H3N2 viruses in pigs in Europe.¹⁵ Since 1998 double and triple re-assortants have been isolated from pigs in the USA. The N. Carolina virus had three human genes and five swine genes.¹⁶ They include human H and N genes, genes from swine H1N1 viruses and two others from avians.¹⁷^-¹⁹

Soon after the occurrence of the H3N2 viruses, new H1N2 viruses arose in the USA where the human H3 had been replaced by a porcine H1²⁰ and then spread.^21,²² They had been known elsewhere in the world for some time: Japan²³; France²⁴; Germany²⁵; Taiwan.²⁶ They were described in the UK where they were found to be the most severe cause of pathology associated with the SIV viruses.²⁷ These were all re-assortants between human H3N2 and classical H1N1.

Human H3N2 and avian H1N1 were isolated in the UK^3,⁶ and were then found to have spread to Europe.²⁸^-³² They are usually human H and N and the rest avian genes but one Italian virus has an avian H1.²⁸ They have shown considerable genetic drift in Europe.³³

Subtype H3N2 has been isolated in Canada from pigs with severe proliferative and necrotizing pneumonia (PNP),³⁴ although this PNP is probably associated with PRRS and PCV2. Serological surveys indicate the infection is widespread in the swine populations in some countries.

The first unusual virus to be found in pigs was an H9N2 introduced to pigs in SE Asia probably from land-based poultry.³⁵

Further problems occurred in the autumn of 1999 when an avian H4N6 was found in pigs with pneumonia on a commercial swine farm in Canada.³⁶ Since then the avian H5N1 has appeared in pigs in China and is being carried west by bird migrations into Russia. The potential of avian viruses to spread to pigs and persist in pigs is unknown. Even if the viruses do not replicate they can contribute viral genes to other pig viruses. This is the reason for continual surveillance of SIV viruses. These were wholly avian viruses that were of North American lineage. It was the first report of an interspecies transmission of an avian H4 virus to domestic pigs under natural conditions.

The disease usually affects young pigs, but all ages may be affected. Typically, sudden onset epidemics occur with a high morbidity rate but with a low case fatality rate of less than 5%. Loss of body condition is marked, which is usually the important cause of financial loss, although occasionally death losses may be extensive if the pigs are kept under inadequate conditions or if secondary bacterial infections occur. Abortions and deaths of newborn pigs have also been reported as causes of loss in this disease.

Risk factors

Animal risk factors

Young, growing pigs are most susceptible. The viral infection is commonly complicated by bacterial infection due to Haemophilus parasuis, A. pleuropneumoniae, and possibly other opportunists of the upper respiratory tract of the pig. When an epidemic occurs, most of the pigs in the herd are affected within a few days, which suggests that all animals are previously infected and that some risk factor, such as inclement weather, precipitates the epidemic.

The agent also contributes to the Porcine Respiratory Disease Complex. In a recent study in Korea 14 of 105 cases had SIV,³⁷ whereas in Iowa it has been reported in 19% of the case of PRDC.³⁸

Environmental risk factors

Epidemics occur mainly during the cold months of the year, commencing in the late autumn or early winter and terminating with a few outbreaks in early spring. Several days of inclement weather often precede an outbreak. Three risk factors for SIV were identified on a survey of Belgian finishing farms³⁹ were H1N1 were found in 71% and H3N2 were found in 22%. There was a close association between H1N1 and H3N2. H1N1 appeared to be associated with fully slatted floors, increasing numbers of pigs in the locality and dry feeding. H3N2 was associated with the purchase of pigs from more than two herds, increasing numbers of pigs locally, and natural ventilation.

Pathogen factors

Molecular microbiology has now revealed the antigenic diversity of the virus. Several different H and N antigens have been identified and grouped on the basis of serological tests, which refine the diagnosis and reveal more about the epidemiological relationships. The H3N2 strain similar to H3N2 strains found in the human population has been isolated from an outbreak in England.^2,³

Two antigenically distinct H1N1 influenza A viruses were isolated during an outbreak of respiratory disease in swine in Canada in 1990/91.^40,⁴¹ One is a variant of the swine H1N1 influenza virus that is widespread in the American Midwest, whereas the other is similar to the virus isolated from swine in 1930. This suggests that influenza viruses can be maintained for long periods in swine herds, especially in certain geographical areas. It is proposed that the antigenic diversity of these viruses may be due to the result of drifts in the population of circulating swine influenza viruses in an area.⁷ The antigenic diversity oligonucleotide analysis of strains isolated from outbreaks in Sweden indicated a similarity with the Danish strain. One of the Swedish strains was closely related to the US strain.

The H1N1 strain of the virus can be found in pig tissues at slaughter but it does not persist for more than 2–3 weeks in deep frozen or refrigerated storage.

Methods of transmission

The natural reservoir of influenza A virus is aquatic birds. Various subtypes have been established in other species, such as influenza A H1N1 viruses, which infect human and different animal species. The influenza viruses may be transmissible between humans and pigs. Swine are the sole animals known to be susceptible to influenza A viruses of human, swine, and avian origin. Swine may become infected with related type A human influenza strains during epidemics of human influenza, but show no clinical signs of infection. The human strains have been isolated from pigs in Hong Kong, and pigs may serve as a reservoir for pandemics in humans as well as a source of genetic information for recombination between human and porcine strains. In Japan, pigs may be seropositive to the H1N1 human viruses relative to human H1N1 influenza epidemics, and seropositive to human H3N2 viruses unassociated with human epidemics of disease.⁴² In Czechoslovakia, influenza A viruses are brought into pig herds by carrier people.

Pigs can be naturally infected with a range of avian influenza viruses. There have been at least three independent introductions of distinct wholly avian viruses into pigs.^43,⁴⁴ The virus in the late 1970s spread throughout Europe and the UK and became a major cause of SI.⁴⁵ These viruses also undergo drift.^45,⁴⁶

Elsewhere in the world antibodies against H4, H5, and H9 viruses have been isolated from Asian pigs⁴⁷ and avian H4N6, H3N3 and H1N1 viruses have been recovered from pigs in Canada.

Swine are susceptible to both human and avian viruses because they have receptors on their respiratory epithelial cells for both avian (receptor SA α 2, 3 Gal) and human (receptor SA α 2, 6 Gal).⁴⁸ Several re-assortants have been isolated from pigs in the USA and other parts of the world.^49,⁵⁰

Thus, swine have an important role in the ecology of influenza A viruses and are regarded as a ‘mixing vessel’ for the introduction of reassorted viruses into the human population.^40,^43,⁵¹

There is a report claiming that outbreaks of influenza in turkeys followed outbreaks of swine influenza in pigs from nearby swine herds.⁵² Swine and other influenza viruses have also been isolated from cattle, and experimental inoculation of calves has been successful. The swine influenza virus may cause natural infection in cattle and the virus can be transferred to uninoculated calves.

The primary route of infection is through pig to pig contact via the nasopharyngeal route. Peak shedding occurs 2–5 days post-infection (>10⁷ infectious particles/mL at a peak).^53,⁵⁴

The rapid spread of infection from pig to pig occurs by inhalation of infective droplets. The disease may appear almost simultaneously in several herds within an area following the first cold period in late autumn. The virus can persist in infected swine, which can act as convalescent carriers and be the reservoir of the virus between epidemics. However, the experimental inoculation of a swine influenza virus into specific pathogen-free (SPF) pigs resulted in a mild disease and the period of viral shedding was shorter than 4 weeks.

Immunity

Both cell-mediated immunity and humoral responses are important. They do not prevent infection but they can mediate the killing of infected cells. The immune response is rapid and completes elimination of the virus within 1 week. Antibodies decline by 8–10 weeks.⁵⁵ The IgA levels in nasal washes are the most important defence.⁵⁶ There is limited cross-protection between different viruses³¹ and protection after vaccination is more virus specific.⁵⁴

Maternal protection will last from 4–14 weeks with no pigs being completely protected from nasal virus shedding upon challenge but at least the lung is protected.^57,⁵⁸ Pigs with a high maternal antibody level did not develop an immune response.⁵⁸ Maternal antibody does not cross protect between subtypes.^57,⁵⁹

ZOONOTIC IMPLICATIONS

Swine influenzas pose a significant health risk to humans ever since the first human and porcine outbreaks in the USA in 1918.⁶⁰ By 1970 there was evidence that people who came into contact with pigs through their jobs became infected with the viruses and a virus was isolated from pigs and stockman.^61,⁶² There is very little evidence of maintenance of human H1N1 in the pig populations⁴⁴ but human H3N2 have been recovered regularly from pigs in Asia and Europe.^44,⁶³ The drift that has taken place in pigs of former human H3N2 has also been minimal compared to the rate of drift in the human population. The viruses from pigs found in humans have been reviewed.^3,^49,⁶²

PATHOGENESIS

Classical swine influenza was originally described as a disease of the upper respiratory tract, the trachea and bronchi being particularly affected, with secondary bacterial pneumonia due to Pasteurella multocida. However, recent descriptions of the lesions in naturally occurring cases and in the experimental disease indicate that the primary lesion is a viral interstitial pneumonia.⁶⁴ Viral replication takes place in the epithelial cells of the nasal mucosa, tonsils, trachea, lungs and tracheobronchial lymph nodes.⁶⁵ No other sites have been detected⁶⁶ and viremia is of low titer. Inoculation of the H1N1 strain of influenza virus isolated in England from pigs with clinical disease into 6-week-old pigs caused fever, coughing, sneezing, and anorexia.⁶⁴ A widespread interstitial pneumonia, with lesions in the bronchi and bronchioles, and hemorrhagic lymph nodes was characteristic. The H3N2 swine influenza virus isolated in Canada is associated with a proliferative and necrotizing pneumonia (PNP) of pigs, and there is evidence the strain may be related to A/Sw/Hong Kong/76H3N2 swine influenza virus.³⁴ There is recent evidence that this PNP is more a feature of PRRS and PCV2 than SIV. A new antigenic variant of H1N1 swine influenza A virus isolated in Quebec has been associated with proliferative and necrotizing pneumonia of pigs.^7,¹²

In the United Kingdom there has also been recorded an H1N7 which included both equine and human influenza genes. It was of low pathogenicity for pigs, found on only one farm, and did not establish in the pig world.⁶⁷ Re-assortant H3N1 viruses from human and classical swine H1N1 have also been seen in the UK and also in Taiwan.²⁶

The virus causes an acute infection with shedding beginning on day 1 and finishing by day 7.⁶⁶ Infected cells in the respiratory tract are reduced by 2–3 days post-inoculation. Most of the effects of the infection are caused by the production of proinflammatory cytokines (IFN-α, TNF-α IL-1, and IL-6.) These produce inflammation, fever, malaise, loss of appetite. The depth of infection in the lung probably determines how much of these cytokines are produced.^68,⁶⁹

Contrary to widespread belief there is no evidence that the virus causes reproductive failure in swine. The experimental inoculation of seronegative pregnant gilts did not reveal any evidence of transplacental transmission of the virus to the fetus.

CLINICAL FINDINGS

It is essentially a herd disease.^1,^13,^17,⁴⁴ The signs have not changed over the 80 years. After an incubation period of 1–7 days, (usually 1–3), the disease appears suddenly, with a high proportion of the herd showing fever (up to 41.5°C, 107°F), anorexia and severe prostration. The animal is disinclined to move or rise because of muscle stiffness and pain. Labored, jerky breathing (‘thumps’) is accompanied by sneezing and a deep, painful cough which often occurs in paroxysms. There is congestion of the conjunctivae with a watery ocular and nasal discharge. Sometimes there is open mouthed breathing and dyspnea especially if the pigs are forced to move. Morbidity is usually 100% but mortality is rarely above 1%. In general, the severity of the illness appears greater than, in fact, it is and after a course of 4–6 days signs disappear rapidly depending, in part, on the level of colostral antibody. However, there is much loss of weight, which is slowly regained. Clonic convulsions are common in the terminal stages in fatal cases. The condition may continue to affect the herd for several weeks as the disease spreads, especially so if the herd is outdoors and the population dispersed. The new H3N2 re-assortants in the USA have been associated with respiratory disease but also spontaneous abortion in sows and death of adult pigs.^13,¹⁷ The clinical signs are dependent on immune status, but are also influenced by age, infection pressure, concurrent infections, climatic conditions, housing and most of all on the secondary infections particularly bacteria. There is some question as to whether other viruses can predispose to SIV but experimentally infection with PRCV and H1N1 or H3N2 SIV has not shown this.⁷⁰ Pigs with both M. hyopneumoniae and SIV coughed more and had more pneumonia than either of the two agents on their own.⁷¹

CLINICAL PATHOLOGY

Within 24 hours of the onset of clinical signs there is a switch of cells in the bronchial lavage from macrophages to over 50% neutrophils.⁶⁸

Serological tests

After infection has ceased to circulate in the herd SIV AB could still be demonstrated after 28 months post-infection.⁷²

It is extremely important to make sure that the antigens that are used in the serological tests are contemporary to the viral strains that may be found in the country.^22,^33,^67,^73,⁷⁴

Diagnosis of acute SIV infections requires the use of paired serum samples. The hemagglutination inhibition test has been the recommended test for many years and still remains so. However, it is tedious and has only moderate sensitivity but high specificity. It has been adapted and modified.⁷⁵ One HI test for H1N1 will detect other H1N1 strains,⁷⁶ but this is not true for H3N2 when the Midwest strains are compared with the N. Carolina strains as they differ considerably.⁷⁷ Above 1:80 is usually considered positive and within 5–7 days the titers may reach 1:320-I:640 by 2–3 weeks post-infection. An ELISA-based test is now available to estimate the hemagglutination titer⁵ and can be used at the herd screening level.⁷⁸

Detection of virus

Virus is likely to be found in the nasopharyngeal area during the acute phase of the disease. Swabs should be taken on Dacron, placed in transport medium stored at 4°C for no more than 48 hours but if for longer freeze at −70°C. Viruses can also be isolated from trachea or lung tissues of pigs. They can be grown in hens’ eggs or increasingly in tissue culture. Samples need to be cool and moist. The virus is then detected by hemagglutinating activity in egg fluids about 5 days after inoculation. There are some strains that may not grow in hens’ eggs or require more than one cell line to isolate and identify the virus which may require 1–2 weeks.⁷⁹

Antigen detection

A PCR test can be used to detect virus in nasal swab specimens⁸⁰ and give results similar to virus isolation.^53,^66,^81,⁸² Recently, a gel based multiplex RT-PCR assay was developed to detect H1 and H3 subtypes of SIV.⁸³

The virus can be detected by direct immunofluorescence of lung tissue or lavage fluids.

Immunohistochemistry on fixed tissue is also useful. The positivity is mainly in the bronchial and bronchiolar epithelial cells and less intense in the interstitial cells and alveolar macrophages.⁸⁴

NECROPSY FINDINGS

Swelling and marked edema of cervical and mediastinal lymph nodes are evident. There is congestion of the mucosae of the pharynx, larynx, trachea, and bronchi. A tenacious, colorless, frothy exudate is present in the air passages. Copious exudate in the bronchi is accompanied by collapse of the ventral parts of the lungs. This atelectasis is extensive and often irregularly distributed, although the apical and cardiac lobes are most affected, and the right lung more so than the left. It may reach 50% by 4–5 days post-infection.^85,⁸⁶ The affected tissue is clearly demarcated, dark red to purple and often reminiscent of enzootic pneumonia. Surrounding the atelectatic areas the lung is often emphysematous and may show many petechial hemorrhages.

Histologically, in acute swine influenza the major feature is necrotizing bronchiolitis.⁸⁷ There is a suppurative bronchiolitis and widespread interstitial pneumonia characterized by the early appearance of neutrophils followed by the accumulation of macrophages and mononuclear cells in the alveolar walls.⁶⁴ After a few days there is a peribronchial and peribronchiolar infiltration of lymphocytes.⁸⁷ In the variant of H1N1 swine influenza in Canada, there is more diffuse damage to the respiratory epithelium, resulting in firm to meaty lungs that appear thymus-like on cut surface.¹²

Microscopically, there is marked proliferation of type II pneumocytes, in addition to the presence of macrophages and necrotic inflammatory cells in the alveoli. The influenza type A virus can be demonstrated by indirect immunofluorescence staining using monoclonal antibody directed to certain protein parts of the human type A influenza virus.¹² The influenza type A virus can be detected and differentiated from the virus of porcine reproductive and respiratory syndrome in formalin-fixed, paraffin-embedded lung tissue using immunogold staining.⁸⁸

Samples for confirmation of diagnosis

• These are best collected from animals with high fevers and clear nasal discharge. Most pigs may excrete virus for 5–7 days post-infection but the peak load may be around 24 hours post-infection

• Histology – formalin-fixed lung, trachea, turbinate (LM, IHC). After 72 hours there is little IFA or IHC positivity. Histopathology may help in the diagnosis for 2 weeks post-infection

• Virology – nasopharyngeal swab in viral transport media; lung and trachea (ISO, FAT, PCR) fresh chilled but not frozen. Keep cool. Do not use cotton.

DIFFERENTIAL DIAGNOSIS

The explosive appearance of an upper respiratory syndrome, including conjunctivitis, sneezing, and coughing, with a low mortality rate, serves to differentiate swine influenza from the other common respiratory diseases of swine.

Enzootic pneumonia of pigs is most commonly confused with swine influenza, but it is more insidious in its onset and chronic in its course.

Hog cholera is manifested by less respiratory involvement and a high mortality rate.

Inclusion body rhinitis in piglets may resemble swine influenza quite closely.

Atrophic rhinitis has a much longer course and is accompanied by characteristic distortion of the facial bones.

TREATMENT

No specific treatment is available. Treatment with penicillin, sulfadimidine, or preferably a broad-spectrum antibiotic, may be of value in controlling possible secondary invaders. The provision of comfortable, well-bedded quarters, free from dust, is of major importance. Clean drinking water should be available, but feed should be limited during the first few days of convalescence. Medication of the feed or water supplies with a broad-spectrum antibiotic for several days is a rational approach to minimizing secondary bacterial pneumonia.

CONTROL

There are only two options vaccination and biosecurity. Biosecurity is difficult as there is always the possibility of aerosol infections and wild fowl/poultry infections.

All in/all out systems may remove infection with each group of pigs and the subsequent disinfection may kill out the virus. Good housing and protection from inclement weather help to prevent the occurrence of severe outbreaks. Once the disease has appeared little can be done to prevent spread to other pigs. Recovered animals are immune to subsequent infection for up to 3 months.

Vaccines, both commercial inactivated and adjuvanted SIV for IM use are available in the USA and Europe. Active immunization occurs in the face of maternal derived antibody when titers are <10 for H1N1 and <40 for H3N2.⁶³ Some of the vaccines contain the original H1N1 viruses but others such as in the USA contain a monovalent H1N1 virus. Following the outbreaks of H3N2 in the USA in 1998 both monovalent and bivalent H1N1/H3N2 SIV vaccines became available. Autogenous vaccines are used in the USA.

In Europe although the viruses have changed the old vaccines are still used as they produce high antibody titers.^89,⁹⁰ There is a need to add H1N2 to the vaccines however as there is no cross protection between the European H1N2 and H1N1 and H3N2 viruses and because it was shown³³ that there is no current vaccine protection against H1N2. Evidence from the USA shows that there is cross protection with their strain of H3N2.⁹¹ Most animals with titers >160 are probably protected against viral replication in the lungs and disease.^70,⁹⁰ Sow vaccination is important in controlling infection in suckling pigs and often controls the infection in nursery pigs. Intranasal or IN/IM vaccination of pigs with formalin inactivated SIV induces very specific IgM, IgG and IgA antibodies in their nasal secretions and sera resulting in complete protection.⁹²

A recent trial of a new H1N1/H3N2 vaccine was successful,⁹³ with reduced viral shedding, and reduced clinical signs and pneumonia.

Experimental vaccines continue to be produced including a human adenovirus 5 recombinant expressing the hemagglutinin and nucleoprotein of H3N2 SIV has been used experimentally to provide protection against challenge with H3N2.⁹⁴ Complete protection was shown by lack of nasal shedding and by lack of lung lesions following subsequent challenge.

REVIEW LITERATURE

van Reeth K, Nauwynck H. Pro-inflammatory cytokines and viral respiratory diseases of pigs. Vet Res. 2000;31:187-213.

Brown IH. Molecular epidemiology and evolution of influenza viruses in pigs. In: Proceedings of the 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome, 29 June-2 July, 2003; pp. 245–249.

Olsen CW, Swayne D, Subbarao K. Human avian and swine viruses — past and current. In: Kawaoka Y, editor. Contemporary topics in influenza virology. Norwich: Horizon Scientific Press, 2004.

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63 Hoover TC, Fricker MD. Proc Am Assoc Swine Pract. 2004:271.

64 Brown IH, et al. Vet Rec. 1993;132:598.

65 Heinen PP, et al. Viral Immunol. 2000;13:237.

66 Choi Y-K, et al. Am J Vet Res. 2004;65:303.

67 Brown IH, et al. Arch Virol. 1997;142:1045.

68 van Reeth K, et al. J Inf Dis. 1998;177:1076.

69 van Reeth K, et al. Viral Immunol. 2002;15:583.

70 van Reeth K, et al. J Vet Med B. 2001;48:283.

71 Thacker EL, et al. J Clin Microbiol. 2001;39:2525.

72 Desrosiers R, et al. J Swine Hlth Prod. 2004;12:78.

73 Olsen CW. Vet Microbiol. 2000;74:49.

74 Richt JA, et al. J Clin Microbiol. 2003;41:3198.

75 Long BC, et al. J Vet Diag Invest. 2004;16:264.

76 Janke BH, et al. Proc Am Assoc Swine Pract. 1997:7.

77 Swenson SL, et al. Proc Am Assoc Vet Lab Diag. 1999:242.

78 Skibbe D, et al. J Vet Diag Invest. 2004;16:86.

79 Erickson G, et al. Proc Am Assoc Vet Lab Diag. 1999:45.

80 Schorr E, et al. Am J Vet Res. 1994;55:952.

81 Choi YK, et al. J Virol Methods. 2002;102:53.

82 Landolt GA, et al. Am J Vet Res. 2005;66:119.

83 Richt JA, et al. J Vet Diag Invest. 2004;16:367.

84 Jung T, et al. Vet Pathol. 2002;39:10.

85 Born B, et al. Proc Am Ass Swine Pract. 1998:63.

86 Richt JA. J Clin Microbiol. 2003;41:3198.

87 Done SH, et al. Eur J Vet Pathol. 1996;2:73.

88 Larochelle R, et al. Can Vet J. 1994;35:513.

89 Heinen PP, et al. Vet Immunol Immunopathol. 2001;82:39.

90 van Reeth K, et al. Vaccine. 2001;19:4479.

91 Rapp-Gabrielsen VJ, et al. Proc 4th Int Symp Emerg Re-emerg Pig Dis 2003; p. 266.

92 Lim Y-K, et al. Jap J Vet Res. 2000;22:3427.

93 Graham M, Rossow K. Proc Allen D Leman Conf. 2004:69.

94 Wesley RD, et al. Vaccine. 2004;22:3427.

PORCINE CYTOMEGALIC VIRUS (INCLUSION BODY RHINITIS, GENERALIZED CYTOMEGALIC INCLUSION BODY DISEASE OF SWINE)

Inclusion body rhinitis, associated with a beta herpesvirus (family Herpesviridae), is an extremely common, but generally minor, disease in young pigs. It was first recognized in 1955. The virus is now called porcine herpesvirus 2. The disease has probable worldwide occurrence¹^-⁵ and has also been described in Canada⁶ and Japan⁷ and clinical and serological^8,⁹ observations suggest that it is present in most pig herds. SPF herds established by hysterectomy techniques are not necessarily exempt and congenital transmission of the virus has been demonstrated.¹⁰ When the virus first enters a susceptible herd then both trans-placental and horizontal virus transmission takes place. Antibody responses start quickly so there are often no clinical signs but widespread infection.

The virus is present in the upper respiratory tract of pigs and the major infection site is the conchal (turbinate) epithelium. High excretion occurs predominantly in the 2–4-week period after infection.¹⁰ Transmission is via the respiratory route through direct contact and aerosol infection, and possibly also via urine. The virus invades epithelial cells, especially those of the nasal mucous glands, to produce destruction of acinar cells and metaplasia of the overlying epithelium and the major clinical manifestation is that of upper respiratory disease. Following infection, the virus may become generalized. In older pigs, generalization is restricted to epithelial cells of other organ systems, especially those of the renal tubules, and is clinically inapparent. However, in very young pigs the virus also shows a predilection for reticuloendothelial cells, and generalization may result in further clinical abnormality.¹¹

Clinically, the disease affects piglets up to approximately 10 weeks of age but the age at manifestation in any herd can depend upon the method of husbandry. In the UK, it is assumed that approximately 50% of the herds are infected. Within the individual herd there may be up to 98% affected. The disease usually occurs when the virus is introduced into the susceptible herd or if for some reason there is a huge increase in the number of susceptible pigs. A wide age-spectrum of involvement may be seen initially when the disease is introduced into the herd for the first time. In most herds the disease affects pigs in the late suckler and early weaner stage. It is at its most severe in pigs under two weeks of age. Sneezing is the most prominent sign and frequently occurs in paroxysms and following play fighting. There is a minor serous nasal discharge which rarely may be blood-stained, and also sometimes muco-purulent with a brown or black exudation around the eyes. There may be coughing. The clinical course varies approximately from 2–4 weeks. All pigs within the group are affected but there is usually no mortality.

The virus also crosses the placenta so it is possible for intra-uterine infection to produce fetal death, and runting after birth as well as very early pneumonia, rhinitis and poor piglet weights at weaning.

Generalized cytomegalic inclusion body disease may occur in pigs exposed to intra-uterine infection and usually occurs as an outbreak involving several litters. The syndrome is characterized by sudden death and anemia. There is often a history of scouring within the group within the first week of life, and affected pigs show skin pallor and often superficially appear plump and well-developed due to edema, especially in the neck and forequarter regions. Death, resulting primarily from anemia, occurs during the 2nd–3rd week of life, and mortality within the group may approach 50%. Petechial hemorrhages have been a feature of the experimentally produced disease in gnotobiotic pigs¹¹ but do not necessarily occur in field outbreaks. A moderate anemia producing a check to growth, but without significant mortality, may also be seen in recently weaned pigs experiencing the disease. Many survivors may be stunted.

More serious effects from generalized infection are seen when piglets are exposed to heavy infection at a very young age. It also occurs when there are new imports and when intercurrent disease and poor nutrition reduce resistance. This commonly occurs in large herds with high density continual throughput farrowing and weaning houses. In addition to upper respiratory disease, infection at this age may result in enteric disease, sudden death, anemia and wasting, with a marked unevenness of growth within the litters.

There may be complete blockage of the nasal passages. It is believed that the olfactory epithelium may be damaged so that there is no sense of smell and that piglets may not then eat and that is the reason so that so many die.

Inclusion body rhinitis is not a primary cause of atrophic rhinitis. However, it is probably contributory in lowering local resistance to infection and in predisposing to more severe infection with Bordetella bronchiseptica and other respiratory pathogens.

The diagnosis of inclusion body rhinitis is commonly made following the demonstration of typical intranuclear inclusion bodies in histological sections from electively slaughtered piglets.¹² Large basophilic inclusion bodies are found in the mucous gland cells of the turbinate mucosa and may also be demonstrated in exfoliated cells obtained via nasal swabs from live pigs. Small intra-nuclear inclusion bodies are found in the reticulo-endothelial cells. These are best taken from several pigs at the height of clinical infection. Diagnosis by virus isolation is uncommon, as the virus has proved difficult to grow, but it will establish in porcine lung macrophage cultures.¹³ Antibody to infection may be detected by indirect immunofluorescent techniques.^8,⁹ An ELISA is also a sensitive reproducible and practical test.⁶ Recently, a PCR has been developed¹⁴ and this showed that 59% of pigs tested positive. However only 59% of PCR positive pigs had clinical signs and lesions consistent with inclusion body rhinitis.

There is no effective treatment and in most herds none is warranted. With severe rhinitis, antibiotics may temporarily reduce the severity of secondary bacterial infection. Control of severe disease rests with managemental procedures that avoid severe challenge to very young piglets. It is also possible to produce virus-free pigs from hysterotomy-derived pigs but it is necessary to monitor.

REFERENCES

1 Done JT. Vet Rec. 1955;67:525.

2 Cohrs P. Dtsch Tierärztl Wochenschr. 1959;66:605.

3 Corner AH, et al. J Comp Path. 1964;74:192.

4 Camon-Stephens ID. Aust Vet J. 1961;37:87.

5 Rundhuis PR, et al. Tijdschr Diergeneeskd. 1980;105:56.

6 Assaf R, et al. Can J Comp Med. 1982;46:183.

7 Tajima T, et al. Vet Med Sci J. 1993;55:421.

8 Plowright W, et al. J Hyg Camb. 1976;76:125.

9 Kanitz CL, Woodruff ME. Proc 4th Intern Cong Pig Vet Soc. 1976:9.

10 L’Ecuyer C, et al. Proc 2nd Intern Cong Pig Vet Soc 1972; p. 99.

11 Edington N, et al. J Comp Path. 1976;86:191.

12 Narita M, et al. Am J Vet Res. 1987;48:1398.

13 Watt RW, et al. Res Vet Sci. 1973;14:119.

14 Hamel AL, et al. J Clin Microbiol. 1999;37:3767.

ENZOOTIC PNEUMONIA OF CALVES

ETIOLOGY

The cause is multifactorial, associated with various species of viruses, bacteria, mycoplasma, and environmental and host risk factors contributing to the pathogenesis, severity and nature of the pneumonia. Pathogens like mycoplasma and viruses may act as primary pathogens, and certain bacterial species may cause secondary complications.

Mycoplasma spp.

The mycoplasma are considered as primary pathogens and probably the most important and most common etiological agents of calf pneumonia. Mycoplasma bovis is a major cause of calf pneumonia.¹ In addition, Ureaplasma diversum, Mycoplasma dispar,² and M. bovirhinis are isolated with a high frequency from the lungs of pneumonic calves.³ M. canis is being isolated with increased frequency. Acholeplasma laidlawii and M. arginini are also found but of dubious significance.¹ M. bovis produces clinical pneumonia experimentally in calves and has been isolated from severe epidemics of pneumonia in calves imported into one country from others.¹ The prevalence of nasal mycoplasmal flora in healthy cows and calves in the same herds is much lower.⁴

Parainfluenza-3 paramyxovirus

The evidence for viruses as primary etiological agents is based on virus isolation, serological evidence of active infection, lesions of viral pneumonia, and experimental infection. The parainfluenza-3 (PI-3) virus has been isolated most commonly from affected calves and inoculation of the virus into colostrum-deprived calves results in a pneumonia that resembles the naturally occurring disease.

Bovine respiratory syncytial virus

Bovine respiratory syncytial virus (BRSV) causes pneumonia in both dairy and beef cattle of all ages, but primarily in dairy calves under 6 months of age.^5,⁶ BRSV isolates belong to an antigenic grouping different from that of human respiratory syncytial virus (HRSV), and two distinct antigenic subgroups of BRSV exist.^5,⁶

Mixed viral and other pathogen infections

A survey of viral infections of the respiratory tract of calves over a 3-year period revealed that only BRSV, the PI-3 virus and bovine virus diarrhea virus (BVDV) were significantly associated with disease. Seroepidemiological and clinical surveys of calves raised as herd replacements in dairy herds commonly reveals evidence of PI-3 virus and BRSV infections associated with respiratory disease. The rhinoviruses, adenoviruses, reoviruses, and enteroviruses were also isolated but in much lower frequency, and were considered not to be important. Chlamydia spp. have been associated with respiratory disease in calves and usually as part of a mixed infection with viruses and bacteria. Bovine coronaviruses have been isolated from calves with respiratory disease but their significance is uncertain.⁷

Bacteria

Mannheimia haemolytica and Past. multocida may also be recovered from the lungs of calves with pneumonia and may act synergistically with the Mycoplasma spp. to cause a more severe and fatal pneumonia. Several different bacterial species including Streptococcus spp., Staphylococci spp., Arcanobacterium pyogenes, Histophilus somni and Fusobacterium spp. may also be recovered from pneumonic lungs.⁵

EPIDEMIOLOGY

Occurrence

Dairy calves

Enzootic pneumonia occurs most commonly in housed dairy calves from 2 weeks to 5 months of age being raised as herd replacements. Pneumonia can be responsible for up to 30% of all deaths of calves in dairy herds from birth to 16 weeks of age,⁸ second to enteritis which can account for 44% of all deaths. Some farms report cases of pneumonia, while others have none.

Pneumonia can be the single largest cause of death in veal calf farms.⁹ The calves are purchased at about 10 days of age, assembled into large groups of 25–50 per group and fed a milk substitute diet for about 16 weeks and then sent to slaughter. The peak incidence of disease occurs about 5 weeks after arrival in the calf house during which time PI-3 and BRSV are recovered most often.

BRSV appears to have assumed major importance as a causative agent of herd epidemics of pneumonia in housed dairy calves^5,⁶ weaned beef calves, and even adult cows. Serological surveys indicate that the prevalence of infection is high and varies from 60 to 80% of the cattle populations examined. Most mature cattle in some populations have seroconverted to the virus. However, the incidence of clinical respiratory disease associated with BRSV is much lower. Respiratory disease in weaned beef calves 6–8 months of age in North America has also been attributed to BRSV and is characterized by a sudden onset, commonly after the cold weather begins in the fall of the year.^5,⁶ Affected animals are commonly in good bodily condition and well-nourished. BRSV was the most commonly isolated viral agent in a series of 14 epidemics of pneumonia in housed dairy calves. The disease may be mild, moderate, or severe.

In dairy herds, clinical disease attributed to BRSV may appear initially in the youngest calves from 2 to 8 weeks of age in which the mortality will be highest, followed by disease in the mature lactating cows in which milk production will drop.

Beef calves

Enzootic pneumonia occurs in nursing beef calves and can account for significant reductions in weaning weight and a significant cause of economic loss due to disease in the neonatal period.¹⁰ In cow–calf herds in northwestern Quebec, one of the major causes of a low percentage of weaned calf crop was the occurrence of diarrhea and pneumonia in calves under 2 weeks of age.¹¹ Pneumonia can also occur after beef calves have been housed.¹²

Morbidity and case fatality

Morbidity rate and case fatality rates vary depending on the quality of housing and management provided, and the kind and concentration of viruses and bacteria that predominate in the environment at any one time. The morbidity rate may reach 100%, and the case fatality rate is usually less than 5%. In dairy calves under 3 months of age, the morbidity rate and case fatality risk for pneumonia were 25.6% and 2.25, respectively.¹³ The cumulative risk incidence of respiratory disease in dairy heifers from birth to 8 weeks of age was 8.4%.¹⁴ In acute respiratory syncytial viral pneumonia of calves there may be an unexpected acute onset of respiratory disease in which 80–90% of calves are affected, with a case fatality rate that may reach 30% or higher.

On veal calf farms, pneumonia can be the largest single cause of death, with mortality rates up to 3.7% and culling rates at 5.1%. Peak death and cull losses occur during the 7th and 8th week of production.⁹

In Ontario Holstein dairy herds, 15% of calves were treated for pneumonia before the age of weaning.¹⁵ Treatment rates for pneumonia increased slightly until about the 6th week of life and then declined until weaning. Calves that had pneumonia during the first 3 months of life had an increased risk of mortality before they reached calving age.¹⁶ In Holstein herds in New York, the crude incidence rate for respiratory disease within 90 days of birth was 7.4%.¹⁷ In those same herds, dullness of calves and unspecified diagnosis within 90 days of birth increased the hazard rate of death after 90 days of age 4.3-fold above that for heifers without dullness within 90 days of birth.¹⁸ These data indicate pneumonia in dairy calves in the first 3 months of age can have an adverse effect on long-term survival and subsequent growth rate.^19,²⁰

Methods of transmission

Aerosol infection and direct contact are the methods of transmission and both are accentuated in crowded, inadequately ventilated conditions. The principal mode of transmission of M. dispar among calves reared in dairy farms is the airborne route up to several meters in distance.²¹ Newborn calves raised in individual pens may become infected within 5–15 days after an experimentally infected calf is placed in the calf house.²¹

M. bovis has been isolated from calves exhibiting severe fibrinonecrotic bronchopneumonia that were imported directly from continental Europe into Northern Ireland.²

Risk factors

Because most of the pathogens described under etiology can be found in the respiratory tract of normal calves, it has been generally accepted that environmental risk factors, such as ambient temperature, relative humidity, air quality, and population density, are necessary to precipitate the disease. In addition, several animal risk factors make calves susceptible to the pathogens in their environment. There are also pathogen risk factors that determine the disease outcome.

Animal risk factors

The onset of calf pneumonia occurs between 2 and 4 weeks of age when the concentration of serum IgG₁, IgG₂ and IgA in the nasal secretions are lowest.²² When the concentrations of serum IgG₂ begin to increase at about 2–4 months of age, the incidence rate of new cases of pneumonia begins to decline. The spectrum of colostral antibodies present in home-raised calves will depend on the spectrum of infection in the adult cows. Most calves that recover from clinical enzootic pneumonia are resistant to further attacks of the disease associated with the same infectious agents. Herd immunity to one or more viruses develops, and severe outbreaks of disease usually occur following the introduction of animals that may be carriers of infectious agents to which the resident animals are non-immune. In commercial veal calf units where market-purchased calves are being introduced on a regular basis, there is commonly a succession of minor epidemics of enzootic pneumonia. The incidence is highest in the recently introduced calves and the disease will occur in a small percentage of resident calves.

In a study of range beef calves from birth to 45 days of age, respiratory disease accounted for a total mortality of 1% and was associated with twins, which may result in a less viable calf at birth that may be neglected and abandoned.²³ The risk of respiratory disease was also higher for male calves. The recent advancement of calving dates of beef cattle herds in the cold areas of North America from April–June to January–March results in crowded conditions in calving yards, which creates the environmental conditions similar to those of housed dairy calves. This has increased the incidence risk for enzootic pneumonia in beef calves.

In herds infected with BRSV, newborn calves acquire colostral antibodies to BRSV, which declines to undetectable levels in an average of about 100 days with a range of 30–200 days.²⁴ It appears that colostral immunity does not protect calves from experimental or naturally occurring clinical disease, but active immunity from natural infection with or without evidence of clinical disease will protect the animals from clinical disease but not from reinfection upon later exposures to BRSV.

Environmental and management risk factors

Environmental risk factors, such as inadequate housing and ventilation are major contributors to the disease process.²⁵ These include calving area, calf housing, spatial separation between calves, mixing calves of different age groups, and seasonal effects. Dairy herds that do not house calves in groups prior to weaning, or that house calves in groups of seven or fewer calves per group, are less likely to be affected with high mortality rate due to respiratory disease.²⁶ The calving area and environment can affect calf health through stress and the degree of exposure to infectious agents. Inadequate ventilation, improper climate control, and poorly constructed facilities can induce stress in calves. Crowding results in close contact and promotes spread of infection, and also results in excess moisture which, in the presence of inadequate ventilation (movement of air) and supplemental heat, causes a high relative humidity and chilling of calves. Many calf barns are old, adapted barns which are occupied for several months without depopulation and disinfection. Monitoring 48 dairy herds over 1 year in the National Animal Health Monitoring System, revealed that mortality was lower in herds that used calf hutches compared to those that did not.²⁷ In commercial veal units, the longer the disinfection and vacancy break, up to 6–7 days, the lower the incidence of disease in new calf crops entering the unit. Ventilation is commonly inadequate because of poor design of the building.

Rapid changes in weather, particularly during the winter months, are often followed by outbreaks of acute pneumonia because of inadequate ventilation. A common practice during cold weather is to close the air inlets and turn off the ventilating fans in an attempt to maintain the inside temperature at a comfortable level. This results in increased relative humidity, condensation of moisture on walls and on the calves, leading to wet conditions, and the reduced ventilation results in an increase in the concentration of droplet infection.¹⁷ Attempts to correlate meteorological data with the daily morbidity rate have not yet provided evidence for the hypothesis that climatic factors have an influence on incidence. This may be due to the difficulties associated with accurately monitoring meteorological data, and the lack of a direct relationship between the environment outside a calf barn and the microclimate of the calf inside the barn. The disease appears to be most common during the winter months when calves are housed continuously and when ventilation is commonly inadequate.

Humid weather results in a marked increase in the percentage of bacterial colony-forming particles of less than 4–7 μm in size. This provides the beginnings of a sound physical framework for the explanation of this and other, as yet empirical, relationships between the microenvironment in calf barns and the etiology and epidemiology of calf pneumonia.

The management risk factors that can influence the incidence rate and mortality of calves with pneumonia include:

• Colostrum feeding practices

• General feeding practices

• Quality of perinatal care provided by the personnel

• Age at weaning

• Use of prophylactic antimicrobials

• Health management of the dams.²⁸

The feeding of a coccidiostat to preweaned calves may be associated with an increase in the risk of pneumonia because herds with a history of disease would be more likely to feed a coccidiostat.²⁹

Factors associated with mortality to 21 days of life in dairy heifers in the United States include:

• First colostrum-feeding method, timing and volume

• Time of separation from dam

• Calving ease

• Twin birth.³⁰

Up to 31% of mortality is associated with ineffective colostrum feeding. The longer the calf is left with the dam after birth, the greater the mortality, presumably due to greater exposure of the calf to pathogens harbored by the dam. Difficult calving also may interfere with the optimum ingestion of colostrum and absorption of immunoglobulins.

A path model of individual-calf risk factors for calfhood morbidity and mortality in New York Holstein herds indicated that management appeared to affect, directly and indirectly, the risk of respiratory disease within 90 days of birth.³¹ Being born in loose housing is strongly related to development of clinical signs of calf diarrhea within 14 days of birth, which in turn increases the risk of respiratory disease within 90 days of birth.

Calves reared as herd replacements may be born inside and raised indoors until they are about 6 months of age and then turned out to pasture for the summer. In the case of veal calf-rearing units, the calves are kept and fed indoors under intensive conditions from a few days of age until they reach 150 kg body weight (BW) at 12 weeks of age. In the barley-beef units, the calves are fed indoors on an intensive basis from weaning until they reach market weight at 10–12 months of age. In all of these situations, young, growing calves are raised together under confined conditions, which promotes the spread of bovine respiratory disease associated with several viruses, Mycoplasma spp., and Pasteurella spp. Based on serological surveys, most calves raised in close confinement will have become infected by several viruses, including the PI-3 virus, adenoviruses, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, and bovine viral diarrhea. If natural exposure to these viruses, Mycoplasma spp., and bacteria is so widespread and inevitable, it raises serious questions about the rationale for vaccination. In most cases the effects of the viruses and Mycoplasma spp. are minimal. The stress factors associated with inadequate ventilation, high relative humidity and chilling, and the secondary bacterial complications are responsible for the onset of clinical disease.

Pathogen risk factors

The infectious agents are ubiquitous in the respiratory secretions of the animals and in their environment, and more numerous in crowded poorly ventilated conditions. The spectrum of infectious agents that are present and acting in a calf population and the severity of clinical disease will vary between farms, between countries, and from season to season. It has been assumed that the older calves and mature animals in a herd are the source of infection for the young calves. this assumes major importance in control measures that are commonly designed to rear calves separate from older animals.

Mycoplasma dispar colonizes the respiratory tract of experimentally infected young calves for several months and can be isolated from nasal swabs and transtracheal samples throughout the period of colonization. M. dispar and Past. multocida have been cultured from transtracheal aspirates of dairy calves with pneumonia under 3 months of age.¹¹ In calves aged 1–5 months in calf-rearing farms that purchase calves from dairy farms, the prevalence and level of colonization of the respiratory tracts with Mycoplasma spp. can be more than 90% over a 2-year period.²¹ M. dispar, M. bovirhinis, and Acholeplasma laidlawii have all been isolated from such calves. A high degree of colonization with M. dispar among 1 to 2-month-old calves on these rearing farms indicates the ability of the pathogen to spread among the calves and colonize the respiratory tract. M. dispar is able to spread very rapidly among groups of calves, and airborne transmission is considered to be an important mode of transmission in addition to direct contact. The infection rate in the calves at the farms of origin is small.

The number and types of Mycoplasma spp. that colonize the nose and trachea of calves are influenced by the age of calves and not by the environmental temperature or relative humidity. Mycoplasma spp. start to colonize the upper respiratory tract of calves within days after birth, and the peak isolation rate from their nasal cavities occurs at about 2–6 weeks of age, and from the trachea at 6–8 weeks of age.³² Over 92% of calves collected from farms and reared in a controlled environment can harbor Mycoplasma spp. in their noses when they are 2 weeks of age. The rate of recovery falls gradually thereafter.

Parainfluenza-3 virus is commonly subclinical in a group of calves, and clinical disease may not occur until other pathogens are present or when adverse environmental conditions precipitate clinical disease. Following natural infection of young calves, the PI-3 virus may persist for several weeks. However, the presence of PI-3 infection may predispose to respiratory disease by interfering with normal pulmonary clearance mechanisms and allowing secondary invasion by bacteria or mycoplasmas.

Bovine respiratory syncytial virus.

Infection with BRSV may be subclinical, mildly clinical, or highly fatal. Raising calves in close proximity to older cattle may result in constant exposure to infectious agents to which the mature animals are immune. The disease may be endemic on particular farms in which almost every calf experiences clinical disease. Herd epidemics may occur following the introduction of a different virus, such as BRSV, or following a breakdown in the ventilation system. The disease occurs specifically in nursing beef calves from 1 to 4 months of age while on pasture.

Mixed flora.

While a mixed flora of viruses, mycoplasma and bacteria can be isolated from the respiratory tract of calves with pneumonia, and the unpassaged respiratory material can cause disease similar to the naturally occurring disease, the inoculation of pure cultures of M. dispar, M. bovis, and Ureaplasma spp., or pure cultures of BRSV or PI-3, into calves does not produce the severe clinical disease seen in the field. The failure of pure cultures of a pathogen to produce a severe pneumonia may be for one of three reasons:

• Combinations of organisms are required for disease

• Laboratory passage of the pathogens, necessary for purification causes their attenuation

• Material in the respiratory secretion other than the pathogens identified is required for disease, which may include agents that were not detected by routine culture techniques.

Economic importance

The economic losses associated with enzootic pneumonia may be considerable. One estimate reports that the disease accounts for 50% of all calf mortality and a reduction of 7.2% in liveweight gain. In commercial veal units, the presence of enzootic pneumonia may be associated with a prolonged time in the unit because of reduced daily liveweight gain.

The economic loss due to calfhood morbidity and mortality is well-recognized by the dairy industry. However, the long-term effects of morbidity from diseases such as enzootic pneumonia on health and performance may constitute an even greater economic loss to the herd. Calfhood diseases occurring in the first 3 months of life may have serious long-term consequences. Heifer calves that are treated for pneumonia during the first 3 months of life are 2.5 times more likely to die after 90 days of age than heifers that are not treated for pneumonia.¹⁶ Heifer calves without respiratory disease are twice as likely to calve, and calved for the first time 6 months earlier, when compared to calves with respiratory illness as calves.³³ Some studies have found no significant independent association with calfhood disease status with first lactation milk production.³⁴ However, the population selected did not include all heifers affected as calves; a heifer could have a suboptimal rate of growth or unthrifty appearance and would be removed from the herd before milk production was measured.

PATHOGENESIS

Mycoplasma

The endobronchial or intratracheal inoculation of gnotobiotic calves with Mycoplasma spp. does not usually result in significant clinical disease. However, 2 or 3 weeks following inoculation, there is microscopic evidence of pneumonia. The lesions produced by experimental inoculation of calves with M. bovis, M. dispar or Ureaplasma spp. are characterized by peribronchiolar and perivascular ‘cuffing’, catarrhal bronchiolitis, and atelectasis.¹ Intranasal inoculation of Ureaplasma diversum into SPF calves results in thick cuffs of round cells surrounding the bronchi, bronchioli and blood vessels, and a lobular catarrhal pneumonia.³⁵ However, clinical signs of pneumonia are not observed. Inoculation of M. canis results in only a slight pathological change which disappears 9 days after infection. M. dispar produces an alveolitis without cuffing lesions. It is thought that the Mycoplasma spp. are synergistic with each other, viruses, and bacteria in producing the lesions of subclinical and clinical enzootic pneumonia.

M. bovis pneumonia in calves is characterized by coagulative necrosis and accumulation of catarrhal exudate in the airways resulting in a sudden onset of marked dyspnea, fever, and poor response to therapy. The coagulative necrosis does not cross the interlobular septa, which is in contrast to Mann. haemolytica which crosses the septa.

Viruses

The respiratory viruses can cause a viral interstitial pneumonia affecting the cranial lobes of the lung which may be subclinical, mildly clinical or severe and highly fatal.

Parainfluenza-3

Subclinical viral pneumonia associated with the PI-3 virus uncomplicated by secondary bacterial invasion is usually of minor importance. In subclinical PI-3 infection in calves, seroconversion will occur and at necropsy there are microscopic lesions consisting of bronchiolitis, bronchial and bronchiolar epithelial hyperplasia, alveolar epithelialization, and giant cell syncytial formation. In the mild form there are slight clinical signs such as coughing and polypnea. In the severe form of viral pneumonia, such as in respiratory syncytial viral infection, there is severe dyspnea, with mouth-breathing and an expiratory grunt, but a marked absence of toxemia compared with a bacterial pneumonia. Death can occur without secondary bacterial bronchopneumonia. Atelectasis and consolidation of the anterior lobes of the lungs are characteristic and account for the loud bronchial tones audible on auscultation over the anterior ventral aspect of thorax.

The experimental intranasal inoculation of the PI-3 virus into colostrum-deprived calves results in a pneumonia that is grossly and histologically similar to the naturally occurring disease. Within 2–4 days following infection there is bronchiolitis and bronchitis and cellular exudate in the bronchiolar lumina. These lesions become more severe and are accompanied by alveolar cell thickening and hyperplasia. Beginning at about 14 days following infection, there is healing of the bronchiolar and alveolar lesions. The bronchiolar exudate becomes organized by fibroblasts, and mononuclear cells predominate in the alveolar exudate. Bronchiolitis obliterans is widespread but re-epithelialization of damaged bronchiolar mucosa and alveoli occur.

Experimentally, the PI-3 virus can affect alveolar macrophages, which may impair the lung clearance mechanisms and allow Mann. haemolytica to produce a secondary bacterial bronchopneumonia. However, aerosols of PI-3 followed by Mann. haemolytica 7 days later do not necessarily result in significant pulmonary disease.

After the primary viral pneumonia is established, bacterial invasion may occur and the resulting pneumonia will vary with the species of bacteria which are present. Secondary bacterial pneumonias usually respond to treatment, although relapses are common if the viral pneumonia is extensive. Viruses are capable of reducing the resistance of mucous membranes, allowing bacteria such as pasteurellae to invade tissues. They are also capable of destroying the cilia on the bronchial mucosa which act as an escalator and help to keep the lower respiratory tract free of potential pathogens. In animals where there is an uncomplicated viral pneumonia with very extensive lesions there may be minimal clinical signs and almost complete resolution.

Bovine respiratory syncytial virus

Infection with BRSV in calves causes rhinitis, tracheitis, bronchitis, proliferative and exudative bronchiolitis, with accompanying alveolar collapse and multinucleated syncytial giant-cell formation in the epithelial lining and in the lumen of the bronchioles and alveoli.³⁶ The pulmonary function changes observed during the acute phase of the disease are consistent with a diffuse obstructive disease. This lesion could impair the lung clearance mechanism and predispose to bacterial bronchopneumonia. Experimental inoculation of gnotobiotic calves with BRSV produces macroscopic lesions of the lungs without clinical signs. The lesions consist of proliferative and exudative bronchiolitis, with accompanying alveolar collapse and mononuclear cellular infiltration of the peribronchiolar tissue and alveolar walls. Infected calves respond serologically by 11 days after inoculation. The experimentally induced lesions are commonly resolved by about 30 days after inoculation.

The effects of BRSV on bovine pulmonary alveolar microphage function have been studied and there is only limited impairment at the in vitro level.³⁷ In experimentally infected calves the virus can be detected in the epithelial cells of bronchioli and alveoli by immunofluorescence.³⁸

BRSV can replicate and induce cytopathological changes in airway epithelial cells which include bronchial ciliated and mucous cells and bronchiolar ciliated and non-ciliated epithelial (Clara) cells.³⁹ Syncytial epithelial cells may be observed in bronchi, bronchioles, alveoli, and in alveolar macrophages. However, syncytial cell formation is not unique to infection with BRSV since it may also occur in other viral infections of the lung. Whether or not BRSV can predispose to bacterial pneumonia is unknown, but the changes in lung tissue associated with the virus may affect lung clearance mechanism and predispose to secondary bacterial infection and inflammation.

The pathogenesis of acute fatal pneumonia due to the BRSV is not well-understood. The characteristic lesions are exudative or necrotizing bronchiolitis, atelectasis, interstitial edema, and emphysema. field observations have recorded that the acute fatal disease is commonly preceded by a mild respiratory disease several days previously, which suggests that hypersensitivity may be an important pathogenetic mechanism causing lung injury. The second stage may follow initial improvement or recovery from the first stage, and is associated with the onset of extreme respiratory distress. However, these observations have not yet been validated. The acute fatal disease attributed to BRSV has been reproduced experimentally in colostrum-fed, conventionally reared 1-month-old calves.⁴⁰

CLINICAL FINDINGS

Regardless of the identity of the causative pathogen, the clinical findings in almost all enzootic pneumonias of calves are similar. In the experimental viral pneumonia, a febrile reaction occurs on about the 5th day and is followed by the appearance of rhinitis, pneumonia, and mild diarrhea. The fever is only moderate (40–40.5°C, 104–105°F). A harsh, hacking cough, easily stimulated by pinching the trachea, is characteristic.

In naturally occurring cases, the clinical findings are similar, although the fever is usually higher. This may be due to bacterial invasion in the early stages. The nasal discharge is only moderate in amount and is mucopurulent. On ausculation of the thorax the major abnormalities can be detected over the ventral aspects of the apical and cardiac lobes. The breath sounds are loud and harsh and represent breath sounds transmitted through consolidated lung. The intensity of the heart sounds is increased because of shrinkage of lung tissue in the cardiac area. The usual course ranges from 4 to 7 days. Some peracute cases of uncomplicated viral pneumonia die within 1 day after the onset of signs. Infections with the PI-3 virus generally cause mild respiratory disease characterized by coughing, nasal discharge, slight fever, and recovery in a few days.

M. bovis pneumonia in young calves is characterized by the sudden onset of severe dyspnea, fever, and rapid deterioration in spite of therapy.

In BRSV pneumonia there may be a sudden onset of acute pneumonia in 80–90% of a group of calves. The clinical findings are characteristic of a severe viral pneumonia. Affected calves are usually mentally alert and there is only a mild fever. There is polypnea and dyspnea which in a few days become worse with mouth-breathing and an expiratory grunt. Loud breath sounds, indicating consolidation, are audible over the anterior lobes of the lung. Squeaky, wheezing sounds due to the bronchiolitis are also commonly audible over the periphery of the consolidated areas. Loud, crackling sounds due to interstitial emphysema may also be audible over the dorsal aspects of the lungs. Death may occur in 2–4 days in spite of intensive therapy.

When secondary bacterial bronchopneumonia occurs, the fever, dyspnea, and toxemia are usually more severe. When secondary infection with Pasteurella spp. occurs, the temperature rises to 41–41.5°C (106–107°F), the area of lung affected is much increased, and loud harsh breath sounds due to edema are followed by crackles and a pleuritic friction rub. These cases usually respond rapidly to adequate treatment. When Arcanobacterium pyogenes is the secondary invader, consolidation is marked, there is a profound toxemia and loud breath sounds. In cases where Fusobacterium necrophorum is present, the clinical findings are similar and pulmonary abscesses are likely to develop. Necrotic lesions are often present in the mouth and pharynx in these cases and the pulmonary infection probably originates from here. With both of these latter infections there may be some response to antibiotic treatment, but there is a predisposition to relapse soon after treatment is terminated. Coughing, dyspnea, anorexia and emaciation continue, and the animal eventually has to be destroyed.

CLINICAL PATHOLOGY

Isolation of pathogens

Nasopharyngeal swabs, transtracheal aspirates, and lung lavage samples^3,¹³ may be taken for isolation of viruses, mycoplasmas, and bacteria. Special laboratory media are required to isolate Mycoplasma spp.³⁵ Determination of drug sensitivity to the bacteria may be valuable, particularly when a number of calves are involved in an outbreak. The isolation of BRSV from natural infections is difficult due to the labile nature of the virus. The immunofluorescent antibody test for antigen detection is one of the most rapid, reliable, and sensitive tests for BRSV from tracheal aspirates, nasal swabs, and lung samples.

Serology

Serological tests have been more extensively used for confirmation of suspected BRSV infections. The standard serological test is a virus-neutralization test using microtiter plates.⁴¹ Others include a modified indirect fluorescent antibody test,⁶ indirect hemagglutination, and an ELISA test, the latter of which is considered to be sensitive and specific and has the advantage of giving test results within several hours whereas the virus-neutralization test requires 5–6 days for completion. The complement fixation test is less specific and less sensitive than the ELISA test.

NECROPSY FINDINGS

In uncomplicated viral pneumonia, irrespective of the specific cause, there are areas of atelectasis and emphysema in the apical and cardiac lobes, with little macroscopic involvement of the diaphragmatic lobes. In the later stages, a dark red consolidation featuring a hobnail appearance of the pleural surface affects most of the ventral portions of the apical and cardiac lobes. The lesions are always bilateral. Histologically, there is a bronchointerstitial pneumonia. Acute inflammation of the nasal mucosa, particularly on the turbinate and ethmoid bones, is usually accompanied by a marked, mucopurulent exudation. In PI-3 infection, intracytoplasmic inclusion bodies are widespread in the lungs; and after experimental infection, are present on the 5th day, but have disappeared by the 7th day after infection.

In respiratory syncytial viral pneumonia there is severe interstitial pneumonia and interstitial emphysema. Histopathologically, there is severe bronchiolitis, alveolitis with multinucleated syncytia (which often contain eosinophilic intracytoplasmic inclusion bodies), and alveolar epithelial cell hyperplasia.

When bacterial or mycoplasmal invasion has occurred, the lesions vary with the agent present. Extensive hepatization with mottled red and gray lobules, and considerable interlobular aggregations of serofibrinous fluid, often accompanied by a fibrinous pleuritis, is characteristic of Past. multocida infection. Extensive consolidation and suppuration occur with A. pyogenes and F. necrophorum infections. In the latter case there may be necrotic lesions in the mouth and upper respiratory tract.

Confirmation of this diagnosis at necropsy is somewhat awkward, as the population of pathogens responsible may change between the time of disease onset and the death of the calf. In severe outbreaks it may be necessary to euthanize animals early in the course of the disease, or to perform serological surveys for respiratory pathogens among surviving herdmates.

Samples for confirmation of diagnosis

• Histology – lung (several sections), trachea, turbinate (LM, IHC)

• Virology – lung (several sections), trachea (FAT, ISO)

• Mycoplasmology – lung (MCULT, FAT)

• Bacteriology – lung (CULT).

DIFFERENTIAL DIAGNOSIS

Clinically, the diagnosis of pneumonia is usually readily obvious, but the causative agents are usually not determined. Young calves raised indoors and affected with a cough, nasal discharge, and pneumonia are usually affected with enzootic pneumonia associated with the agents described under etiology. The common diseases of the respiratory tract of young calves which may resemble enzootic pneumonia include the following:

• Bacterial pneumonia due to Mann. haemolytica, Klebsiella pneumoniae or H. somni in young calves characterized by severe toxemia, fever, dyspnea, grunting, and a poor response to therapy

• M. bovis pneumonia is characterized by sudden onset of dyspnea, fever, depression, and poor response to therapy in a group of calves

• Calf diphtheria usually affects a single calf and is characterized by inspiratory dyspnea, stridor, toxemia, fever, and obvious lesions of the larynx

• Lungworm pneumonia occurs in young calves at pasture, and marked dyspnea, coughing, and a few deaths are characteristic. A fever is common in lungworm pneumonia and there are loud breath sounds over the ventral aspects of the lungs, and loud and moist crackles over the dorsal aspects

• Acute myocardial dystrophy in young calves, following turn out on pasture, is characterized by sudden onset of weakness, polypnea and dyspnea due to pulmonary edema and lesions of the diaphragm, tachycardia and arrhythmia, and skeletal muscular weakness

• Aspiration pneumonia occurs occasionally in calves that have been force-fed colostrum or milk. There is a sudden onset of marked dyspnea, anxiety and distress, and death may occur within a few minutes. However, some calves survive and there is marked dyspnea with abdominal breathing, and loud breath sounds and crackles over the dorsal and ventral aspects of both lungs. Some calves will recover completely in a few days

• BRSV interstitial pneumonia in weaned beef calves must be differentiated from pneumonic pasteurellosis. In BRSV pneumonia there is a sudden onset of marked dyspnea, fever, anxiety but not toxemia, mouth-breathing in advanced cases, loud breath sounds and wheezes over both lung fields especially over the ventral aspects, and subcutaneous emphysema. Several animals are usually involved. Affected animals fail to respond to treatment with antimicrobials and the case fatality rate is usually over 75%. There may be a history of mild respiratory disease in the affected group about 10 days previously. In pasteurellosis, depression, toxemia, fever, loud breath sounds over the ventral aspects of the lungs, and a favorable response to treatment are characteristic

• Chronic enzootic pneumonia is characterized by bronchiectasis and pulmonary abscessation, causing unthriftiness and a poor response to therapy.

TREATMENT

Antimicrobial therapy

Uncomplicated enzootic pneumonia associated with mycoplasma or viruses is unlikely to respond to treatment, but antimicrobial therapy daily for 3 days is indicated because of the high probability of secondary bacterial pneumonia. Any of the antimicrobials used commonly for the treatment of acute undifferentiated bovine respiratory disease are effective. The short-acting or long-acting oxytetracyclines, the trimethoprim-potentiated sulfonamides, and florfenicol are efficacious and are recommended. Penicillin, tilmicosin, and ceftiofur are also effective for the treatment of secondary bacterial pneumonia. Danofloxacillin has excellent in vitro activity against several field isolates of Mycoplasma spp. from cattle,⁴² and has potential for the treatment of respiratory infections associated with Mycoplasma spp.⁴² Early treatment is necessary to avoid the development of incurable secondary complications, such as pulmonary abscesses, pleuritis, bronchiectasis, and suppurative pneumonia. In commercial veal calf units, the case fatality rate can be kept to a low level by early and adequate treatment. In some cases it may be sufficient to treat animals once only, but a proportion of cases are likely to relapse after an initial response. Such cases require repeated daily therapy for 3–5 days. If the number of relapses in an area or on a farm is excessive, all cases should receive multiple treatments. M. bovis strains in Europe are becoming resistant to antibiotics traditionally used for mycoplasma infections in particular oxytetracyclines, florfenicol, tilmicosin, and spectinomycin^2,⁴³; only danafloxacin showed any antimycoplasma activity.⁴⁴ Spectinomycin had only partial therapeutic effect in calves experimentally infected with M. bovis.²⁴ The fluoroquinolones are still effective but their use in animals in controversial.²⁵

Valnemulin, a pleuromutilin antibiotic with high activity against mycoplasmas had some beneficial effect when added to the milk from four days of age for 3 weeks of one-month-old calves with respiratory disease from which M. bovis was isolated in about 80% of cases.⁴⁵ Treated animals had less severe clinical disease but individual treatment was still necessary.

Adjunctive therapy

Bronchodilators and NSAIDs as adjunctive therapy for enzootic pneumonia in calves are used but their efficacy is questionable.

Correction of adverse environmental conditions

The clinical management of an outbreak of enzootic pneumonia in calves must include correction of adverse environmental conditions, which may have precipitated the disease.

CONTROL

Environmental and managemental

Control of the disease in housed calves is dependent on effective animal and environmental management. Overcrowding, drafty or inadequately ventilated housing, exposure to inclement weather, and sudden changes in environmental temperatures are major risk factors. Recently purchased calves should be isolated for several weeks before being introduced to the group.

Ideal environmental conditions

Control is especially difficult and expensive in countries where the calves are housed for several months during the winter months in northern climates. The most comfortable ambient temperature for young calves ranges from 13 to 21°C (55–70°F) with a relative humidity of 70%. To achieve these environmental conditions requires a suitable insulation material in the walls and ceilings, ample bedding to absorb moisture from feces and urine, and adequate movement of air to remove aerosol particles that may be infectious. This requires an adequate air inlet and outlet system, adequate capacity fans, and supplemental heat during very cold periods. The installation of recirculating air filter units can lead to a substantial reduction in the concentration of airborne bacteria to which calves are exposed. field studies in veal calf units indicate that mean aerial bacteria concentration in filtered barns can be reduced by 45%, the number of calves requiring treatment reduced by 19%, the number of repeat courses of treatment and the total antibiotic usage reduced by 29% and 35%, respectively. At slaughter, the average area of lung consolidation in calves from filtered barns can be reduced by 35%. In general, air filtration can result in a reduction in both the incidence and severity of clinical and subclinical pneumonia in calves and in improved weight gain.

In spite of ideal hygiene and management it may not be possible to prevent the development of new cases if the infection already exists in a herd, or if cattle from other herds are moved into the herd. At present, it is feasible only to be vigilant and treat new cases urgently and vigorously, because a strict hygiene program may not be feasible in the average commercial herd. If management is inadequate and the general resistance of the animals is low, losses due to calf pneumonia with significant bacterial or mycoplasmal invasion can be sufficient to make calf-rearing unprofitable.

Calf barns or hutches

Where economics permit, the ideal situation is to construct a calf barn completely removed from the main adult cow barn to minimize the spread of infection from adults that may be symptomless carriers. After the colostral feeding period, calves are removed from the calving barn and placed in individual pens in the calf barn. The raising of young calves outdoors in calf ‘hutches’ or ‘igloos’ is highly satisfactory and economical, even in countries where the outside temperatures go well below freezing. With adequate bedding, protection from the prevailing winds and adequate nutrition, calves will grow satisfactorily. Dairy herds that have had difficulty controlling enzootic pneumonia of calves have found this system to be an excellent alternative to the construction of a stand-alone, well-ventilated calf house. Nutritional deficiencies, usually of energy and protein, are common in young calves and often accentuate the severity of the pneumonia. Young calves should receive a balanced calf starter grain ration supplemented with essential vitamins and minerals, and good quality hay beginning by at least 3 weeks of age.

Vaccines and immunization

There is insufficient information available from field trials to make recommendations for the use of vaccines for the control of enzootic pneumonia in calves. It is difficult to evaluate the results of vaccination trials because investigators use so many combinations of vaccines, different vaccination schedules, and there are many different management variables and differences in methods of evaluation. In addition, most vaccination trials are not randomized controlled trials.

Any successful vaccine would have to be multivalent and would have to be effective when given before 2 months of age or earlier to coincide with the decline in immunity and the occurrence of enzootic pneumonia in calves. A study of vaccination of calves in a commercial calf-rearing unit that compared the use of no Vaccine intranasal infectious bovine rhinotracheitis (IBR), intranasal IBR–PI-3, and intranasal IBR–PI-3 plus BRSV on three occasions at 7, 10, and 16 weeks did not have a significant effect on growth rates during a 10-month period to slaughter.⁴⁶ There is good field evidence that the colostral immunological status of the calf has a significant effect on the susceptibility of the calf to pneumonia.⁴⁷ There is a clear association between low levels of IgG₁, IgG₂ and IgA of calves at 2–3 weeks of age, and subsequent susceptibility to pneumonia at 2–3 months of age. Calves with signs of pneumonia had low levels of IgG₁ compared with non-pneumonic calves which had relatively higher levels. In addition, calves with high levels of serum immunoglobulin do not respond normally to vaccine and any vaccine for enzootic pneumonia would have to be administered during this relatively refractory period. However, for veal calves, which are purchased at a few days of age and with low levels of immunoglobulin, this may not be a problem.

The intranasal inoculation of calves with virulent or a modified strain of PI-3 virus stimulates the development of both serum antibody and nasal secretion antibody. The nasal secretion antibody is dose-dependent. Challenge exposure of these calves provides protection against clinical disease. These factors should be considered in the development and administration of PI-3 viral vaccines if the objective is to establish an optimal concentration of antibody in the nasal secretion. The parenteral administration of two sequential doses, 2 weeks apart, of an inactivated PI-3 virus vaccine with adjuvant will induce high levels of serum antibody and prevent virus excretion in nasopharyngeal secretions after challenge. Successful immunization of calves against PI-3 infection may be useful for protection against pneumonic pasteurellosis if PI-3 precedes the bacterial infection. This is presented in greater detail in the section on pneumonic pasteurellosis.

A quadrivalent vaccine containing the inactivated antigens of BRSV, PI-3, M. dispar, and M. bovis, or a vaccine containing only BRSV, given to two vaccinated groups respectively, and compared to controls, provided protection against naturally occurring pneumonia and non-fatal respiratory disease in a large beef-rearing unit over a period of 2 years in the United Kingdom.⁴⁸ Calves were collected from farms in the first few weeks of life and reared in the unit in groups of 100 until slaughter at about 18 months. The proportion of calves receiving treatment for respiratory disease was 38% in the control group, 25% in those vaccinated with the quadrivalent vaccine and 27% in those vaccinated with the BRSV vaccine. Mortality in the control group was 9%, 2% in the quadrivalent vaccine group, and 3% in the BRSV-vaccinated group.

A single dose of an experimental vaccine for M. bovis pneumonia, inactivated with saponin, given subcutaneously to 3 to 4-week-old calves followed by experimental challenge 3 weeks later with a virulent strain of M. bovis provided protection against clinical pneumonia.²² Unvaccinated calves developed clinical signs of disease due to lung lesions. The vaccine also reduced the spread of M. bovis to internal organs. Calves tested 6 months after immunization had high levels of humoral immunity. The successful use of saponin in vaccines has been demonstrated for other mycoplasma infections such as contagious caprine pleuropneumonia (CCPP) and contagious agalactia. The saponin may preserve the major antigens seen in untreated whole cells.⁴⁹

The evaluations of BRSV vaccines are currently in progress and the preliminary results are inconclusive. A combined vaccine containing Mann. haemolytica, H. somni, and a modified live-virus for the control of enzootic pneumonia in young beef calves vaccinated at 3 and 5 weeks of age reduced the number of calves requiring treatment.⁵⁰

REVIEW LITERATURE

Baker JC, Ellis JA, Clark EG. Bovine respiratory syncytial virus. Vet Clin North Am Food Anim Pract. 1997;13:425-454.

Larsen LE. Bovine respiratory syncytial virus (BRSV): A review. Acta Vet Scand. 2000;41:1-24.

Nicholas RAJ, Ayling RD. Mycoplasma bovis: disease, diagnosis and control. Res Vet Sci. 2003;74:105-112.

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VIRAL PNEUMONIA IN OLDER CALVES, YEARLINGS AND ADULT CATTLE (BOVINE RESPIRATORY SYNCYTIAL VIRAL PNEUMONIA)

Synopsis

Etiology Bovine respiratory syncytial virus (BRSV). Subtypes A, B

Epidemiology Prevalence of infection high; disease most common in cattle under 6 months of age but adult cattle also affected; recurrent infections and disease in herds common; persistent infection in few seropositive cows. Immunity following natural infection or vaccination short-lived. Antibodies following natural exposure are different than those following experimental infection or vaccination. Maternal antibody does not prevent infection but high levels decrease severity of clinical disease

Signs Mild, moderate, or severe dyspnea, fever, agalactia, coughing, wheezes of lungs, most animals recover, small percentage develop severe fatal viral interstitial or bacterial pneumonia.

Outbreaks occur in cattle under 6 months of age and also in adult cattle

Clinical pathology Difficult to isolate or detect virus in tissues. Immunohistochemical tests of nasopharyngeal swabs and lung tissue. Serology

Differential diagnosis Pasteurella pneumonia. Other viral interstitial pneumonias. Infectious bovine rhinotracheitis. Lungworm pneumonia

Treatment Nothing specific

Control Minimize stressors. Control by natural exposure and treat secondary bacterial pneumonia. Modified live-virus and inactivated virus vaccines available but efficacy uncertain because lack of field trials

ETIOLOGY

The bovine respiratory syncytial virus (BRSV) is a cause of a viral pneumonia primarily in cattle under 6 months of age, and also yearlings and adult cattle. The BRSV is a pneumovirus in the family Paramyxoviridae.¹ Isolates of the virus from calves with severe respiratory disease in a region of Britain are more closely related by genetic analysis sequences to US isolates than to earlier British and mainland European isolates.² Importation of live cattle from North America to Britain may explain the relatedness of the isolates. The literature of the history and taxonomy of the virus has been reviewed.¹

EPIDEMIOLOGY

Occurrence

Prevalence of infection

The virus is ubiquitous in the cattle population and new infections occur most commonly in autumn and winter annually and may result in severe respiratory disease. In longitudinal studies in dairy herds, 90% of primary infections occur in calves and heifers; very few occur in cattle over 2 years of age, and all cattle in the herds are seropositive when they are over 3 years of age.^1,³ Recurrent infections occurring annually at the same time, and in cows of all ages, without new introductions into the herd, are characteristic of BRSV infections in a herd.³ There is evidence that the virus circulates during spring or summer at very low levels or not at all. Persistent BRSV infection in some of the cows in a herd might be a means for the virus to survive during the summer, but a steady state of reinfection of seropositive cows throughout the year at a low level might also maintain a reservoir of infection. Monthly data on the prevalence of BRSV antibodies in dairy herds suggest that persistent infection in seropositive cows is more likely than population persistence.³

While the prevalence of infection in the cattle population is high, the incidence rate of clinical disease is much lower.³ It can be assumed that most mature cattle have been exposed to the virus. Surveys in the United States, England, Denmark, Sweden, and France found seropositive rates in herds ranging from 50 to 80%. Cattle entering feedlots may seroconvert to the virus, which may be associated with an increased risk to subsequent treatment for respiratory disease.³ A high percentage of young beef bulls aged about 6 months and entering performance test stations may seroconvert to BRSV and adenovirus, both of which may be associated with clinical respiratory disease.

Occurrence of clinical disease

In general, calves under 6 months of age are most commonly affected and some BRSV infections are undoubtedly associated with enzootic pneumonia of housed dairy calves. In dairy herds, recent introductions of young cattle purchased from public saleyards may introduce the infection to home-farm cattle that have had no previous exposure to the viruses, or in which their immunity to a previous infection with the virus has declined. Thus, adult dairy cows may be affected with a highly fatal pneumonia due to the virus.⁴ A high prevalence of infection exists in Swedish cattle and annual outbreaks of disease have occurred in adult cattle, pregnant or recently calved cows being most severely affected.⁵ Outbreaks have occurred in beef cattle on pasture.⁶ The disease occurs in nursing beef calves 1–8 months of age on pasture with their dams without any history of previous stress. A common occurrence is in weaned beef calves 6–8 months of age within 2–3 weeks following weaning and commingling in confinement.⁶ Yearling cattle in feedlots are also susceptible.

In North America, herd epidemics of clinical disease usually occur during the fall and winter months.³ However, nursing beef calves may be affected with clinical disease during the summer months. Some outbreaks have occurred in nursing beef calves between 1 and 2 months of age while they are still in nursery pastures or in the calving areas.

A spontaneous outbreak of respiratory disease in goats due to the BRSV has been described,⁷ and sheep can be infected with the virus.⁸

An epidemic of acute respiratory disease associated with the BRSV occurred during the winter and spring of 1995 in Norway.⁹ Data from 431 cattle herds were collected. The risk of acute respiratory disease occurring in cattle was related to herd size and type of production and an expressed interaction between these two variables. The risk of a herd outbreak in a mixed herd of 20 animals was estimated to be 1.7 times greater than in a dairy herd, and 3.3 times greater than in a beef herd of comparable size. On increasing herd size to 50 animals, the risk increased 1.3 fold for a mixed herd, 3.3 fold for a dairy herd, and 2.1 fold for a beef herd, compared to a risk for a corresponding type of herd of 20 animals. The disease spread initially from one location to another during the first 6 to 9 weeks, where the rate of transmission between neighboring farms seemed to be higher than for the other districts included in the study. It was hypothesized that one common source of infection was involved in the outbreak and the case herds were clustered in time as well as spatially.¹⁰ The average daily milk loss was estimated to be 0.70 kg per cow for 7 days after a herd outbreak compared with the period one week before.¹¹

Morbidity and case fatality

The morbidity rate in herd epidemics of clinical disease can vary from 30 to 50% or higher. The case fatality rate is usually low, 3–5%, but may be higher.³

Methods of transmission

The mode of transmission has not been defined but aerosol infection and direct contact are probable. Infection spreads rapidly among susceptible cattle.

Risk factors

Animal risk factors

Naturally occurring BRSV infection affects both dairy and beef cattle and those under 6–10 months of age are most susceptible to clinical disease. Nursing beef calves with colostral BRSV antibody are not protected from infection but the incidence and severity of clinical disease is inversely related to the level of maternal antibodies in calves younger than 3 months.² The highest percentage of reinfections occurs most commonly in cows during their first lactation.¹² Older animals may have a more effective immunity because of previous exposure.

Seroepidemiological surveys in feedlot cattle found that seroconversion to the virus may occur in up to 70% of animals within 1 month after arrival.¹³ Animals with low titers to the virus on arrival are at increased risk of subsequent treatment for respiratory disease, which suggests that the virus may be a factor in bovine respiratory disease. In some situations in feedlot cattle, high BRSV serum antibody levels on arrival were related to a lower risk of respiratory disease.⁶

Environmental risk factors

The highest incidence of clinical disease occurs in autumn and winter months. Outbreaks have been associated with changes in weather, especially declining ambient temperatures and atmospheric pressure.

Pathogen risk factors

BRSV has a narrow host range, affecting primarily cattle. Important antigenic differences between BRSV isolates have been described. Subgroups A and AB are associated with severe respiratory disease and circulate in the Dutch cattle population.¹ In natural outbreaks of infection in closed dairy cattle herds and veal calf units in Denmark, using DNA sequence data, identical viruses were isolated within a herd during outbreaks, but viruses from recurrent infections varied by up to 11% even in closed herds.¹⁴ It is possible that a quasispecies variant swarm of BRSV persisted in some of the calves in each herd and that a new and different highly fit virus type (master and consensus sequence) became dominant and spread from a single animal in connection with each outbreak.

Antigenic subtypes may have relevance both in explaining differences in virulence between subtypes and in the development of new vaccines for the control of clinical disease.¹⁵ The production and characterization of monoclonal antibodies to a vaccine strain of BRSV has been described.¹⁶ The respiratory syncytial virus of goats and sheep, caprine respiratory syncytial virus and ovine respiratory syncytial virus are antigenically related, but not identical, to BRSV.

The BRSV may act synergistically with a concurrent experimental challenge of the virus and 3-methylindole to produce more severe pulmonary disease similar to BRSV pneumonia seen in feedlot cattle, than either agent alone.¹⁵ But vaccination of cattle with BRSV vaccines does not protect the potential synergism between the 3-MI and BRSV infection.¹⁷

Whether or not the virus persists in individual animals in spite of the presence of maternal or naturally-acquired antibodies has been a major question. Serological findings indicate persistence of the virus but the virus could not be detected in lung lavage fluid or nasal swabs.¹⁸ Experimentally, the virus persisted in tracheobronchial and mediastinal lymph nodes for up to 71 days after infection.¹⁹ In vitro, the virus was still able to replicate in bovine B-lymphocyte cell lines 6 months after infection. This may explain the absence of the virus between epidemics, recurrent infections in the same individuals and inapparent reinfection of adults.

Immune mechanisms

After a natural BRSV infection, the protection is short-lived and multiple reinfections are common.^1,³ In endemic areas, the absence of BRSV-associated disease in adult cattle is possibly due to repeated infections. This places a constraint on vaccine development, because one or two vaccinations would have to induce immunity that only repeated natural infections can provide. BRSV infections can occur in the presence of high to moderate levels of maternal antibodies.³ Maternal antibodies, which are directed against the F, G and N proteins of BRSV, are commonly present in calves, but do not protect against infection.^1,³ However, the incidence and severity are inversely related to the level of specific maternal antibody,³ and natural infection does not prevent reinfection but appears to offer good protection against clinical disease after infection.

The BRSV colostral antibody of dairy calves varies dependent on season of the year the calves are born.²⁰ Dairy calves born during the winter months in the Netherlands have lower BRSV colostral antibody titers than those born during the summer months. This may be due to the seasonal periodicity of BRSV circulation or by other factors influencing antibody development or colostrum intake is uncertain. Calves born in the summer have higher antibody titers at 14 to 19 weeks of age most likely attributable to BRSV exposure. Calves born during the season of infection may be primed with BRSV field virus during the period of maternally derived immunity and may be better protected against disease by cellular immunity during the next season of infection.

IgM and IgA are the predominant antibody isotypes in the respiratory tract after BRSV infection, with IgA especially prominent after reinfection.³ Both serum antibody responses and local antibody responses are suppressed by maternal antibodies.³ After natural BRSV infection of cattle, antibodies are predominantly directed against the epitope A, whereas after experimental infection, or vaccination with an inactivated vaccine, the antibody responds against epitope B and non-neutralizing epitope C are markedly increased compared with the same epitopes in naturally infected cattle.¹

The subgroups of the virus are based on antigenic differences of the G protein, and BRSV infection protects against reinfection by homologous strains of the virus. It is also known that a complete BRSV can partially protect against a BRSV infection with a strain that contains an antigenic dissimilar G protein.¹ Therefore, incorporation of representative viruses of different BRSV subgroups in vaccines for cattle does not seem necessary to achieve cross-protection. A gE-negative bovine herpesvirus-1 (BHV-1) strain can successfully be used as a vector for development of combination vaccines against bovine respiratory disease, and the G protein can induce significant protection against BRSV infection in calves.²¹

Vaccination of calves with a formalin-inactivated BRSV vaccine followed by challenge exposure to virulent virus increased the severity of clinical disease and lesions compared to calves non-vaccinated and challenged.²¹ Vaccination did not induce neutralizing antibodies, but IgG antibodies were detected with ELISA. Immunization with formalin-inactivated BRSV vaccine mainly primes a Th2-like inflammatory response characterized by a significant eosinophilic influx in the bronchia alveolar lung field and lung tissues and high levels of immunoglobulin E serum antibodies.²²

PATHOGENESIS

BRSV causes rhinitis, tracheitis, bronchitis, bronchiolitis, and mild interstitial pneumonia. In naturally occurring cases, the principal lesions are bronchitis and bronchiolitis in the cranioventral portions of the lungs combined with widespread emphysema and edema throughout the lungs.^1,³ BRSV infection causes airway obstruction and hyperactivity that may persist for up to 30 days following viral exposure.³ In naturally occurring cases, the cranioventral lung fields are particularly poorly ventilated and there is arterial hypoxemia associated with mismatching of ventilation and perfusion.³ Radiographic and radionuclide lung perfusion imaging reveals the presence of bullous emphysema and areas of marked atelectasis.

The pathogenesis of acute fatal pneumonia due to BRSV is not clear. The characteristic lesions are exudative or necrotizing bronchiolitis, atelectasis, interstitial edema, and emphysema. The acute fatal disease is commonly preceded by a mild respiratory disease several days previously, which suggests that hypersensitivity may be a pathogenetic mechanism causing lung injury. The second stage may follow initial improvement or recovery from the first stage and is associated with the onset of extreme respiratory distress. The virus-specific IgE antibody may play a role in the pathogenesis of the severe disease as part of a hypersensitivity reaction.²³ The IgM and IgA antibodies are not involved in a hypersensitivity reaction. In experimentally induced infection in calves, there is considerable injury to bronchiolar epithelium including hypertrophy, hyperplasia, and formation of syncytia.²⁴ In the alveoli, BRSV infection results in necrosis of type I pneumocytes; the response of type II pneumocytes includes hypertrophy, hyperplasia, and syncytial formation. It is suggested that an immune-mediated mechanism may be responsible for the widespread lesions over the entire lung.

The severe highly fatal form of the disease, also known as the ‘malignant’ form, or the paroxystic respiratory dis-tress syndrome (PRDS) is associated with extensive pulmonary mast cell degranulation.²⁵ In a series of naturally-occurring paroxystic respiratory disease in calves, paired serum samples were taken three weeks apart, and lungs examined at necropsy. The serum concentration of tryptase was used as a marker of mast cell degranulation. Tryptase is a preformed serine protease stored in mast cell granules and causes significant changes in the respiratory tract smooth muscle tone and vascular permeability. The substances released by the mast cells are at least partially responsible for the pulmonary edema, in particular by means of venoconstriction and the increase in the vascular permeability induced by the histamine. (In neonatal calves and young adult cattle, histamine affects respiratory function by contraction of the trachea and pulmonary veins which increases total pulmonary resistance and induce venous hypertension.²⁶ This would likely result in increased pulmonary capillary pressure and the development of alveolar edema. The main physiopathologic target of pulmonary mast cells in cattle is the pulmonary vein.)

The edema and bronchoconstriction caused by the mast cell leucotrienes impede bronchiolar flow, which causes ventilatory asynchronism. The mechanical constraints caused by the asynchronism are aggravated because the bovine lungs consist of a number of compartments, which prevents any collateral ventilation and any dissipation of interlobular pressure gradients. The breaking point is reached when the level of the mechanical constraints exceeds the level of tissue resistance causing interstitial emphysema.

Calves which die from the BRSV-associated PRDS have a uniform pattern of gross lesions. The trachea and bronchi are filled with a white-to-pink froth and the lungs are heavy and voluminous and fail to collapse. The most characteristic lesions were the dramatic lung distension by edema, alveolar hyperinflation and severe interstitial and subpleural emphysema, often with large dissecting bullae on the dorsal edge of the diaphragmatic lobes.

Microscopically, the most characteristic lesions are bronchitis, bronchiolitis, and alveolar edema, mononuclear cell infiltration, hyaline membrane deposition and scattered hyperplasia of type-2 pneumocytes. There is a clear gradient in the severity of inflammatory changes in the airway along a cranio-caudal axis, lesions being more frequent and severe in the cranial parts except for hyperinflation and emphysema. Extensive mast cell degranulation occurs in the diaphragmatic lobes where neither the virus nor the epithelial syncytia, nor the bronchiolitis, typically observed in cranioventral zones are found.

Experimental reproduction of BRSV pneumonia

Experimental reproduction of the disease has been difficult; in most cases, infection results in only mild clinical disease with limited lesions.

Severe respiratory tract disease and lesions can be reproduced experimentally in conventionally reared calves and the virus can be recovered from tissues.²⁴ Severe disease similar to the naturally-occurring disease can be induced with a single aerosol of a low-passage clinical isolate of the virus.²⁴ Moderate to severe BRSV-induced pneumonia can be reproduced in colostrum-fed calves, and nasal shedding of the virus and demonstration of the antigen in the lungs at necropsy provides evidence that the virus causes the disease.²⁷

In neonatal calves with experimental acute infection with BRSV, there is increased pulmonary resistance and decreased compliance, which explains the severe dyspnea observed in some calves. There is no evidence that transplacental infection occurs.³ Experimental infection of young lambs with BRSV can result in severe pathological changes with only mild clinical disease.²⁸

In experimentally infected calves, the virus can be detected in the bronchiolar epithelial cells and in alveolar cells, including bronchial ciliated and mucous cells, and bronchiolar ciliated and non-ciliated epithelial (Clara) cells. Syncytia are often observed in the bronchiolar walls and in the alveoli, and such syncytia were always replicating the virus.²⁹ However, syncytial cell formation is not unique to infection with BRSV since it may also occur in other viral infections of the lung.

Ultrastructural studies of experimental BRSV pneumonia reveal that ciliated and non-ciliated bronchiolar epithelial cells and alveolar type II pneumocytes are targets of the virus.^1,³ BRSV infection of ciliated cells in the airway can result in the loss of cilia and ciliated cells, which may interfere with lung clearance mechanisms and predispose to bacterial pneumonia. The experimental inoculation of lambs with both BRSV and Mannheimia haemolytica results in a more severe acute respiratory disease than that produced by either agent alone, and the severity of the disease may be a result of synergistic action of the two agents.

Experimental BRSV infection in calves, induces an acute phase protein response.²⁸ Strong and reproducible acute phase proteins haptoglobulin and serum amyloid A will peak at 7–8 days after inoculation of the virus. The proinflammatory cytokine, tumor necrosis factor (TNF-α), can be detected in the broncho alveolar lung lavage fluids and high levels appear on the days when severe lung lesions and clinical signs are obvious.³⁰ It may be involved in mechanisms leading to increased permeability of endothelium.

CLINICAL FINDINGS

The clinical findings vary considerably from herd to herd and from year to year. In dairy cattle, disease occurs most commonly in young calves under 6–10 months of age, although outbreaks of severe disease in mature dairy cattle also occur. Clinical signs of infection in older cattle, particularly those with previous exposure to the virus, are less severe. In large dairy herds, episodes of infection are usually mild and often unnoticed, despite cattle having a fever, slight inappetence, and a corresponding decrease in milk production which lasts 3–5 days.³¹ Primary infections in lactating dairy cattle may cause a considerable decrease in daily milk production. However, reinfections are not associated with an important loss of milk production.

A sudden outbreak of acute respiratory disease in a group of animals is a characteristic of a primary BRSV infection. The disease is more severe in animals with no previous exposure to the virus. A dry, non-productive cough, severe dyspnea and polypnea, and bilateral nasal discharge are characteristic. A fever of 40–42°C (104–108°F) is common and milk production in lacatating cows declines markedly. Feed consumption in the affected group declines for a few days. The fever usually persists for 3–5 days in spite of therapy with antimicrobials. Toxemia is not a feature unless there is secondary bacterial pneumonia. On auscultation of the lungs there are loud breath sounds over the ventral aspects indicating consolidation, and wheezes indicating bronchiolitis. These are the findings of a viral interstitial pneumonia. Most animals recover within 5–7 days. About 1–2% of affected animals will develop a fatal viral pneumonia characterized by severe dyspnea with abdominal breathing and an expiratory grunt, mouth-breathing with foamy salivation, marked anxiety, persistent fever, and death within 2–5 days after onset.^4,⁵ Feed and water consumption are decreased because of severe dyspnea, which results in a gaunt abdomen and dehydration. Affected animals are reluctant to move or lie down. The loud breath sounds audible over the ventral two-thirds of both lung fields, indicating that extensive consolidation is becoming pronounced. Subcutaneous emphysema over the withers may also occur. Occasionally, some animals that are not being observed closely will die with peracute pneumonia within a few days and represent the index case of an outbreak.

In outbreaks of BRSV infection in young dairy cattle under 12–16 months of age, the first clinical abnormalities usually noticed by the owner are coughing and a mild nasal discharge in 50–75% of the animals. Inappetence with a fever of 40°C (104°F) or higher lasts for about 3 days followed by recovery in most cases. Coughing, nasal discharge, and conjunctivitis may persist for several days or a few weeks in 10–30% of the animals with no long-lasting complications. Abdominal breathing, and loud and abnormal lung sounds may occur in about 50% of the animals but these commonly resolve within 10 days.

In an outbreak of BRSV in recently weaned beef calves 6–8 months of age, nasal and lacrimal discharge, polypnea and dyspnea, fever of 40–42°C (104–108°F) decreased feed intake, coughing, and lethargy are common. In a small percentage of affected animals, within a few days the dyspnea becomes marked with mouth-breathing and the production of frothy saliva created by the labored respirations. Subcutaneous emphysema over the withers due to severe, interstitial emphysema also occurs. Loud breath sounds, wheezing, and crackling sounds are audible over the ventral aspects of the lungs. Death may occur within a few days after the onset of the dyspnea. Secondary bacterial bronchopneumonia may occur but is uncommon.

CLINICAL PATHOLOGY

It is difficult to obtain a definitive etiological diagnosis of BRSV infection because the virus is highly labile in tissue samples and virus detection in specimens is poor because of inadequate laboratory techniques. The virus replicates slowly, classical virus isolation is laborious and several blind passages are often necessary before any cytopathic effect can be seen. Nasopharyngeal swabs for virus isolation and paired serum samples are necessary to make a definitive etiological diagnosis. Successful laboratory diagnosis of BRSV is generally based on one of four criteria:

• Virus isolation

• Detection of BRSV antigen in suspected tissues

• Indications of BRSV seroconversion

• Histopathology.

The high prevalence of antibody titers to the virus, and the need for skilled personnel to process and interpret the diagnostic tests, have hindered development of a routine diagnostic test. Successful isolation of the virus from typical clinical cases of disease is often unsuccessful and can take 11–21 days because of the late appearance of any noticeable cytopathic effect. Because of these difficulties, isolation of the virus is not commonly recommended as a routine diagnostic approach.

Virus isolation or detection

The ideal sample for isolation of the virus is a transtracheal aspirate in the very early stages of the disease. The sample also provides cells for immunofluorescent antibody (IFA) staining.^1,³ Nasopharyngeal swabs are also useful, but sampling technique must insure good contact with the most caudal part of the pharyngeal cavity and the samples must be placed in viral transport medium and shipped on cold packs and not frozen.

The fluorescent antibody test for virus detection is one of the most rapid, reliable, and sensitive tests for the diagnosis of BRSV infection.¹ For tracheal aspirates, an aliquot of the sample is centrifuged onto a microscopic slide to obtain a cell preparation for the IFA test.

The PCR assay is rapid and sensitive and can be recommended as the method of choice in the analysis of clinical specimens.³ The presence of the virus can be determined by using PCR on nasal swabs taken in the acute phase of a suspected outbreak. The virus can be detected and quantified in cell cultures using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays.³² A sensitive RT-PCR assay for detection of the virus in lung tissues from calves with natural or experimental infection has been developed.³³

The virus can be detected in tissues with monoclonal or polyclonal antibodies and avidin–biotin complex immunohistochemistry.³ This is typically done on formalin-fixed, paraffin-embedded tissues.

Serology

The standard serological test for specific BRSV antibodies is the virus-neutralization (VN) test, usually done with microtiter plates.^1,³ Paired acute and convalescent samples from both affected and normal animals in the herd are desirable. The indirect ELISA is a rapid and reliable test for detecting antibodies to BRSV in milk, bulk tank milk, and serum.³⁴ A microneutralization ELISA has been developed which correlates well with other assays and is useful in assessing antibody responses to the virus both in naturally occurring disease and in vaccination studies.³⁵

A leukopenia and neutropenia are common and are aids to diagnosis.

NECROPSY FINDINGS

Affected lungs are voluminous and heavy, and fail to collapse when the thoracic cavity is opened. The cranioventral portions of the lung are consolidated and usually dark red or plum-colored. The interlobular septa are edematous, and mucoid exudate can often be expressed from small bronchi. Severe interstitial emphysema and edema are prominent over the dorsal and caudal lobes. Subpleural emphysema is often obvious in the cranial and caudal lobes. The caudodorsal lung regions may be ‘meaty’ in consistency. The caudal lobes are often markedly distended because of interstitial emphysema, and large bullae are common. The interlobular septa of the caudal lobes are usually distended because of emphysema and edema. Subcutaneous emphysema over the withers, thorax, and neck are common. Secondary bacterial bronchopneumonia with pleuritis may occur.

Histologically, there is bronchiolitis and bronchitis. Large multinucleated syncytia are present, projecting from the bronchiolar walls or lying free in the lumen. Hyperplasia or necrosis of the bronchiolar epithelium are common. Exudates consisting of neutrophils, macrophages, desquamated epithelial cells, and syncytia are present in the bronchiolar lumina. Small airways are often occluded with exudate. Alveolar changes include cellular infiltration and thickening of alveolar septae with multinucleate giant-cell syncytia in the alveoli. Epithelial syncytia containing eosinophilic intracytoplasmic inclusion bodies are often present on alveolar walls. The presence of epithelial syncytia is a useful feature but the numbers and prominence of these structures can vary considerably. Other viruses can also induce these syncytia. In the caudodorsal lung regions, there is severe emphysema, often with rupture of alveolar walls, alveolar edema, sometimes with hyaline membrane formation and swelling of alveolar epithelial cells.⁸

In experimental BRSV pneumonia, the findings include bronchitis, bronchiolitis, proliferative and necrotizing bronchiolitis, interstitial pneumonia with areas of atelectasis and alveolar edema, epithelial syncytium formation on bronchiolar and alveolar walls, and pneumocyte hyperplasia. The virus antigen can be demonstrated by immunoperoxidase or immunofluorescent staining of bronchiolar and alveolar epithelium.

Isolation of the BRSV from natural field cases has always been difficult because of the long duration required for the appearance of characteristic cytopathic effects. Fluorescent microscopy can be used for detection of the antigen in the cranioventral lung areas but PCR is a more sensitive technique. It is advisable to collect and sample several areas of lung as viral antigen/nucleic acid will be most abundant in areas of acute infection. The virus can also be demonstrated in formalin-fixed paraffin-embedded bovine lung tissue using immunohistochemical techniques.³²

Samples for confirmation of diagnosis

• Histology – fixed lung (several sites) (LM, IHC)

• Virology – chilled lung (several sites) (FAT, PCR); nasal swab (ELISA, PCR).

DIFFERENTIAL DIAGNOSIS

The differential diagnosis includes those infectious diseases of the respiratory tract of young cattle that commonly affect groups of animals in a short period of time.

It is not usually possible to make a definitive etiological diagnosis based on the clinical findings. However, the combination of the epidemiological and clinical findings are usually suggestive of an acute viral respiratory disease. It is not usually possible to be more specific than making a clinical diagnosis of acute undifferentiated respiratory disease.

• Acute respiratory disease due to BRSV infection in weaned beef calves is characterized by marked dyspnea, anorexia, mouth-breathing, fever, subcutaneous emphysema, loud breath sounds, and death in a small percentage of animals in a few days or less. In some cases there may be a history of respiratory disease in the affected group several days previously³⁶

• Infectious bovine rhinotracheitis (IBR) is characterized by outbreak of coughing, profuse nasal discharge, fever, inappetence, the presence of typical nasal lesions; pneumonia is not common. Recovery occurs in several days

• Pneumonic pasteurellosis is characterized by anorexia, toxemia, fever, abnormal lung sounds, coughing, nasal discharge, and response to treatment with antimicrobials. fibrinous pneumonia at necropsy is typical

• Lungworm pneumonia occurs most commonly in groups of young cattle on summer pasture and is characterized by coughing, nasal discharge, tachypnea, abdominal breathing, fever and inappetence, and increased breath sounds with crackles. A necropsy diagnosis is usually necessary

• BRSV infection in mature dairy cattle may be mild and is characterized by a slight drop in milk production, fever for a few days, inappetence, and recovery in a few days. Adult cattle lacking immunity may develop severe fatal pneumonia which must be distinguished from pneumonic pasteurellosis, infectious bovine rhinotracheitis, and other causes of interstitial pneumonia.

TREATMENT

Antimicrobial therapy

Broad-spectrum antimicrobials given daily for 3–5 days for secondary bacterial pneumonia are commonly administered but may not be necessary. Recovery usually occurs gradually over a period of 3–5 days. Severely affected animals will become worse in spite of therapy.

Non-steroidal anti-inflammatory agents

These are used for their anti-inflammatory effect but are unnecessary and there is no evidence that they are efficacious.

CONTROL

Reliable control measures are unavailable. The ubiquitous nature of the virus, the persistency of infection in herds, the movement of cattle between herds, the expansion of herds and the replacement practices used in herds, and recurrent infections make control difficult.

Management

A rationale approach to control would be management of the herd to minimize stressors such as inadequate ventilation. Herd replacements brought into the herd should be quarantined from the rest of the herd for 2–3 weeks before mixing with the remainder of the herd.

Vaccines and immunization

Several modified live virus and inactivated virus vaccines are available for the control of respiratory disease due to BRSV infection, but there are few randomized clinical trials evaluating the efficacy of the vaccine under naturally occurring conditions against BRSV infection or clinical disease. Because calves under 6 months of age are most frequently infected with BRSV despite the presence of colostral antibodies, there is a need for a vaccine which is effective in calves. The presence of passively derived antibodies to BRSV interferes with immunization by vaccination of young calves with commercially available inactivated vaccines. Vaccines must therefore be effective at an early age and be able to overcome the immunosuppressive effects of colostral antibodies.

Protection has been reported in experimental challenge models in cattle for several vaccines. field trials, with live or inactivated BRSV vaccines revealed different levels of protection, while others found that vaccination enhanced disease in calves.³⁷ The intramuscular administration of the vaccine in calves which had colostral antibodies, was least efficacious, and intranasal inoculation of live virus in colostrum deprived calves proved most effective.³⁷

Cattle vaccinated with MLV BRSV vaccines generally develop high concentrations of virus neutralizing antibodies (VN) and fusion inhibiting antibodies, compared with low to moderate concentrations of total BRSV-specific IgG.³⁸ In contrast, cattle receiving inactivated virus vaccines develop lowered concentrations of VN antibodies and high concentrations of virus-specific (non-neutralizing) IgG.

Formalin-inactivated BRSV vaccines have not been successful when tested by experimental challenge of vaccinated calves.^21,³⁹ This is similar to the enhanced disease which may occur in children vaccinated with a formalin-inactivated alum adjuvanted vaccine.³⁹ However, one adjuvanted inactivated BRSV vaccine did provide protection in vaccinated calves challenged by experimental infection.³⁸

A BRSV vaccine inactivated with beta-propiolactone and adjuvanted provided calves with colostral antibodies to the virus with protection against challenge with the virus.⁴⁰ A schedule of three vaccinations in calves with high levels of antibodies or two vaccinations in calves with moderate levels provided protection for nearly 6 months. However, in one study in beef herds, calves vaccinated with an inactivated BRSV vaccine developed clinical, serological, virological and pathological evidence of BRSV pneumonia 2 months after vaccination.³⁷

A modified-live BRSV vaccine provided protection in calves against a challenge model that mimics severe naturally occurring disease.⁴¹ Vaccinated calves shed reduced numbers of virus, had less pulmonary disease, and had vaccine-induced cell-mediated immune responses. The cell-mediated immunity was a more consistent measure of protection than prechallenge serum antibody.

A single intranasal dose of MLV BRSV vaccine protected calves from experimental challenge and the vaccine induced cell-mediated immunity characterized by the production of BRSV-specific interferon-gamma (IFN-γ) by mononuclear cells isolated from various tissue specimens.⁴²

Subunit vaccines which can overcome maternal antibodies have been examined. The use of immunostimulating complexes (ISCOMs) against BRSV has been evaluated and compared to a commercial inactivated vaccine in calves with BRSV-specific maternal antibodies.⁴³ Following experimental challenge, vaccinated calves remained healthy, while control calves developed severe clinical disease. Significantly higher BRSV-specific nasal IgG, serum IgG1, and IgG2 titers were detected before and after challenge in calves vaccinated with ISCOMs compared to the results in calves vaccinated with the commercial vaccine. BRSV was isolated from the nasopharynx of control calves none from the calves vaccinated with ISCOMs. The vaccine overcame the suppressive effect of colostral antibodies and induced a strong clinical and virological protection against a BRSV challenge.

Vaccination of dairy heifers with a vaccine containing chemically altered temperature-sensitive infectious bovine rhinotracheitis (IBR), PI-3 viruses, two strains of killed bovine virus diarrhea viruses, and a modified live virus BRSV, compared to a similar vaccine but without the BRSV, twice at a 2-week interval with the second vaccine administered at 2–3 weeks before calving, may increase milk production and first insemination conception rates.³¹ Milk production was increased in first-parity cows during the first 21 weeks of lactation. Vaccination did not have any effect on milk production after the first 21 weeks of lactation in cows of any parity.

REVIEW LITERATURE

Baker JC, Ellis JA, Clark EG. Bovine respiratory syncytial virus. Vet Clin North Am Food Anim Pract. 1997;13:425-454.

Larsen LE. Bovine respiratory syncytial virus (BRSV): A review. Acta Vet Scand. 2000;41:1-24.

REFERENCES

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3 Baker JC, et al. Vet Clin North Am Food Animal Pract. 1997;13:425.

4 Ellis JA, et al. Can Vet J. 1996;37:103.

5 Elvander M. Vet Rec. 1996;138:101.

6 Martin SW, et al. Can Vet J. 1999;40:560.

7 Redondo E, et al. J Vet Med B. 1994;41:27.

8 Bryson DG. Vet Med. 1993;88:894.

9 Norstrom M, et al. Prev Vet Med. 2000;44:87.

10 Norstrom M, et al. Prev Vet Med. 2000;47:107.

11 Norstrom M, et al. Prev Vet Med. 2001;51:259.

12 van der Poel WHM, et al. Vet Quart. 1995;17:77.

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14 Larsen LE, et al. J Clin Microbiol. 2000;38:4222.

15 Bingham HR, et al. Am J Vet Res. 1999;60:563.

16 Underwood WJ, et al. Vet Microbiol. 1995;43:261.

17 Bingham HR, et al. Am J Vet Res. 2000;61:1309.

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43 Hagglund S, et al. Vaccine. 2004;23:646.

EQUINE HENDRA VIRUS INFECTION (FORMERLY EQUINE MORBILLIVIRUS)

ETIOLOGY

EPIDEMIOLOGY

Transmission

Zoonotic potential

CLINICAL SIGNS

CLINICAL PATHOLOGY

NECROPSY

TREATMENT AND CONTROL

CAPRINE HERPESVIRUS-1 INFECTION

ETIOLOGY

EPIDEMIOLOGY

Occurrence

Transmission and experimental reproduction

Economic importance

PATHOGENESIS

CLINICAL SIGNS

Adults

Newborn kids

CLINICAL PATHOLOGY

NECROPSY FINDINGS

Adults

Newborn kids

Samples for confirmation of diagnosis

TREATMENT AND CONTROL

SWINE INFLUENZA

INTRODUCTION

ETIOLOGY

EPIDEMIOLOGY

Occurrence

Risk factors

Animal risk factors

Environmental risk factors

Pathogen factors

Methods of transmission

Immunity

ZOONOTIC IMPLICATIONS

PATHOGENESIS

CLINICAL FINDINGS

CLINICAL PATHOLOGY

Serological tests

Detection of virus

Antigen detection

NECROPSY FINDINGS

Samples for confirmation of diagnosis

TREATMENT

CONTROL

PORCINE CYTOMEGALIC VIRUS (INCLUSION BODY RHINITIS, GENERALIZED CYTOMEGALIC INCLUSION BODY DISEASE OF SWINE)

ENZOOTIC PNEUMONIA OF CALVES

ETIOLOGY

Mycoplasma spp.

Parainfluenza-3 paramyxovirus

Bovine respiratory syncytial virus

Mixed viral and other pathogen infections

Bacteria

EPIDEMIOLOGY

Occurrence

Dairy calves

Beef calves

Morbidity and case fatality

Methods of transmission

Risk factors

Animal risk factors

Environmental and management risk factors

Pathogen risk factors

Economic importance

PATHOGENESIS

Mycoplasma

Viruses

Parainfluenza-3

Bovine respiratory syncytial virus

CLINICAL FINDINGS

CLINICAL PATHOLOGY

Isolation of pathogens

Serology

NECROPSY FINDINGS

Samples for confirmation of diagnosis

TREATMENT

Antimicrobial therapy

Adjunctive therapy