Bovine respiratory disease (BRD) incorporates all possible respiratory diseases in cattle and is characterised by abnormal clinical signs of the respiratory tract. Bovine respiratory disease refers to bacterial bronchopneumonia that may be complicated by previous, or concurrent, viral or Mycoplasma infection. The principal viruses involved in BRD include bovine herpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (PI-3) and bovine viral diarrhoea virus (BVDV). Despite advances in veterinary medicine, animal husbandry, and animal welfare, respiratory disease among dairy cattle continues to be a major problem in the dairy industry. In addition to enzootic calf pneumonia, outbreaks of respiratory disease in adult animals can have devastating economic outcomes for dairy owners.

Many studies have been performed to detect animal-level risk factors for respiratory disease in young calves, whereas the literature concerning BRD in adult dairy cattle is deficient. In adult dairy cattle, respiratory disease is less important than mastitis, lameness, or reproductive disorders as a cause of morbidity. According to the Annual Report of the Estonian Animal Recording Centre (EARC, 2009), BRD was the reason for culling dairy cows in 0.7% of cases. According to our experience, in most herds BRD occurs as a sporadic disease in adult dairy cattle. However, epidemic outbreaks occur with high morbidity accompanied with dramatic economic losses due to medication use and discarded milk, as well as cow fatalities.

The subclinical course of BHV-1 infection has been observed after the introduction of the virus to a naive herd, however high morbidity of BHV-1 outbreaks involving respiratory disease symptoms (lethargy, coughing, conjunctivitis and oculonasal discharge) was seen on a number of occasions. Outbreaks of severe respiratory disease due to bovine respiratory syncytial virus (BRSV) have been observed in dairy herds throughout Sweden, where adult cattle were most severely affected. Risk factors associated with acute bovine respiratory disease, especially with BRSV outbreaks, were larger herd size, as well as the type of the production with a higher risk in dairy herds compared to beef herds. Acute BRD has been found to occur mainly during cold months, with an epidemic peak in December. Despite the multifactorial nature of BRD, only limited research data is available on herd management-related risk factors for respiratory disease in adult dairy cattle.

Poor fertility is the leading cause of culling cows in Estonia (EACR, 2009). Problems associated with reduced fertility in dairy cattle are related to: diseases of the reproductive tract of the cow, bull fertility, breeding management, and the environment, as well as nutrition. Several infectious diseases are related to abortion in cattle, and BHV-1, BVDV and Neospora caninum are often diagnosed as causes of abortion in cattle world-wide. However, field studies estimating the effect of BHV-1 on herd level reproductive performance have given contrary results. Previous studies found no association between the proportion of calves with antibodies against BVDV or BHV-1 virus and reproductive performance in beef herds. A somewhat higher mean open days period was found in cows that were serologically positive for BHV-1 than in seronegative dairy cows, however no decrease in reproduction performance was found to occur during an outbreak of BHV-1 in a dairy herd. To our knowledge no epidemiological studies have been published to identify and quantify the association between herd BHV-1 seroprevalence and farm-level reproductive performance in dairy cattle.

The objective of this study was to ascertain the associations between herd BHV-1 seroprevalence and the occurrence of acute respiratory disease and reproductive performance in adult dairy cattle. The association between management-related factors and higher BRD occurrence was also estimated.

Three symptoms most commonly related to respiratory disease were asked to evaluate by the respondents. As relatively small number of herds had values equal or higher than 'up to 10%', the variables were dichotomised separating herds with high or low occurrence of that symptom. In the graphical display of MCA all three respiratory disease symptoms were closely linked meaning that those were present concurrently in most of the herds. This encouraged us to create one summary outcome variable describing the occurrence of respiratory disease. We find that asking more precise preliminary information from the respondents and controlling the explanation capability of the variables before combining those into one outcome variable has decreased the recall bias and gives easily interpretable results.

BHV-1, BVDV and BRSV are associated with a high occurrence of respiratory disease in Estonian adult dairy cattle, according to the results of the MCA. A high prevalence of BRSV (≥50%) was associated with a high occurrence of respiratory disease symptoms in cows and pregnant heifers in the MCA. When combining these three BRD symptoms into one outcome variable in logistic regression analysis, a low to moderate prevalence of BRSV (1-49%) among youngstock was significantly associated with a high occurrence of respiratory disease among cows and pregnant heifers. This discrepancy may arise from the fact that relatively small number of herds (n = 14) belong to the highest BRSV prevalence group. This may result in larger standard errors of the estimates in the logistic regression analysis affecting also the p-value of the predictor. MCA on the other hand is not as sensitive to sample size as conditional methods. Sampling antibodies from a small number of young animals that have lost maternal immunity indicates the recent spread of infection. However, some studies have shown that outbreaks of acute respiratory disease associated with BRSV in fully susceptible populations affect adult cattle, pregnant or newly calved cows, most severely. Thereafter the disease remains endemic, manifesting itself among younger animals that serve as sentinels. Given that the signs of respiratory disease reported in this study were those associated with the occurrence of respiratory disease in the previous two years, cows and pregnant heifers might have experienced disease caused by BRSV some time previously, following the active spread of the virus among youngstock detected in this study at the time of testing. In a severe outbreak of BRSV in Sweden it was found that concurrent infection with other viruses may affect the expression of disease. In addition to BRSV BHV-1 and BVDV were associated with higher occurrence of BRD in MCA. As associations between variables are not adjusted for the effects of other variables with this method we can't state that BHV-1 and BVDV are direct risk factors for BRD. However, apparent bivariate association between these variable gives a reason to suggest that BHV-1 and BVDV may participate in the expression of BRD rather as contributing agents.

Large herd size has been found to be a risk factor for the high occurrence of respiratory disease in many studies. Elvander has shown that BRSV spreads rapidly within the herd. In larger dairy herds there are more numerous between-animal contacts, increased inter- and intra-farm traffic by farm employees such as veterinarians and AI-technicians as well as potential higher animal densities allowing the more efficient spread of infectious agents. The number of animals susceptible to infections in large herds is also higher than in small herds contributing maintenance of infections within a herd over extended periods.

MCA has spotlighted several other management practices as possible risk factors for BRD in adult dairy cattle. Although these associations are not conclusive due to the limitations of MCA, they are worthwhile to mention here as factors likely contributing to the disease and requiring attention. First, loose housing of cows was associated with a higher level of BRD in cows and pregnant heifers. We may suggest that more direct contacts between the animals, and the frequent regrouping of animals in loose housing barns, create greater possibilities for the direct transmission of the infectious agents over the whole farm.

Second, housing youngstock in a separate building from six months of age until service was associated with a higher occurrence of BRD. In order to maintain an immunizing infection, the susceptible pool must be replenished via recruitment. Depending on the pattern of infectious disease epidemiology within the herd, commingling animals with different immunity status to specific infections may predispose the active circulation of the virus.

Newly purchased animals can be the source of BRSV infection, which was confirmed in a Swedish study in which outbreaks of BRSV occurred most often after the introduction of purchased animals.

High occurrence of respiratory disease was present in 19% of herds included in this study. Due to sporadic nature of the disease the sample size evaluating risk factors for high occurrence of BRD in adult dairy cattle should be larger. Therefore the results of this study give first insight about the risk factors associated with the disease and some factors might have been missed as significant influencing factors.

Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production, as it is one of the most important causes of lower respiratory tract infections in calves. In dairy cattle, BRSV infection usually occurs in young calves aged between 2 weeks and 9 months. Adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [1, 4, 5].

BRSV, bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV) and bovine parainfluenza type-3 (PI-3) are considered primary agents involved in the bovine respiratory complex. Additionally, secondary infection by Pasteurella multocida, Histophilus somni and mycoplasmas contribute to the aggravation of the disease. Clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. However, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against BRSV infection.

Considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of BRSV. In Europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant BVDV infection [7–11]. Similar studies have also been performed in some Latin American countries and they showed that most of the animals probably have already been exposed to the virus with consequent high BRSV prevalence in cattle herds. In these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with BRSV infection [12–16].

In Brazil, BRSV was first diagnosed in calves in the state of Rio Grande do Sul and some studies have shown that BRSV infection is widespread in Southern and Southeastern Brazil, with high serological prevalence rates [18–20]. Nevertheless, research has not been conducted in order to verify possible risk factors involved in BRSV epidemiology. Due to this, the current study aimed to determine antibody prevalence against BRSV and investigate some risk factors associated with BRSV seroprevalence in herds of an important milk producing region in São Paulo State, Brazil.

Pneumonia in pre-weaned calves is a multi-factorial disease involving a well known group of viruses (bovine herpesvirus 1, BoHV1; bovine respiratory syncytial virus, BRSV; parainfluenza 3 virus, PI3) and bacteria (Mycoplasma bovis, Pasteurella multocida, Mannheimia haemolytica, Histophilus somni), as well as calf-related and environmental risk factors. Bovine viral diarrhoea virus (BVDV) appears to play an important role, both in terms of immunosuppression and synergistic effects with other pathogens and also as a primary pneumopathogen. Accumulating evidence has been found in recent years that bovine coronavirus plays a role in bovine respiratory disease.

Many aspects of respiratory disease in cattle have recently been reviewed, including issues specific to beef and dairy calves.

A number of factors are known to modify the risk of pneumonia in calf populations. Prior to weaning, single-suckled beef calves outdoors are at the lowest risk of pneumonia. Outbreaks can occur due to sudden inclement weather. However, if suckler calves are born and reared indoors, the incidence of pneumonia can be considerable. In the period following weaning, pneumonia has a high prevalence in weaned beef calves. A large national survey of the US beef industry found that 14.4% of cattle placed in US feedlots acquire the disease. The population wide-characteristics of weaned cattle of high respiratory disease risk include animals being of light weight, cattle from multiple origins, previous history of disease and cattle experiencing long journeys before arriving at a feedlot.

According to recent US data, respiratory disease is responsible for nearly a quarter of pre-weaned calf deaths and nearly half of weaned calf deaths in dairy replacement heifers. Dairy calf rearing management and facilities vary widely between farms. Therefore, each of the previously mentioned risk factors at calf and environment level has to be considered when faced with an outbreak of calf pneumonia. Additional factors identified to increase pneumonia risk in housed calves include shared airspace with older animals, overcrowding, and power-washing of calf facilities while calves are still present.

BPI-3V sometimes cause severe disease as a single agent and can predispose the animal to bacterial infections of the lung. Our results revealed high BPI-3V seroprevalence (47.1%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. These results agree with those by Carbonero et al., who found high seroprevalence values in cattle of Yucatan, Mexico. However, the results obtained in this study differ with those published by Betancur et al., who reported lower seroprevalence values (13.5%) in cattle from Monteria, Colombia. The high seroprevalence of BPI-3V found in this research is in agreement with the ubiquitous nature of the virus and with its worldwide distribution. In this research, the seroprevalence was higher in the age group of >24 months of age (Table-4). This age group was a significant risk factor for BPI-3V transmission (OR=3.5). Possibly, due to the presence of some stress factor in these animals that favors reinfections with or without respiratory signs. In adults, especially BPI-3V, it is subclinical unless it is part of concomitant infections with other viruses and bacteria such as Pasteurella multocida, Mannheimia haemolytica, Mycoplasma spp., and immunosuppressive factors. With regard to the clinical signs, conjunctivitis had a statistical association with the BPI-3V seroprevalence values, and regarding sex, female was a significant risk factor for BPI-3V infection (OR=3.6). This result differs with those published by Betancurt et al., who found no statistical association between BPI-3V infection and sex.

Pneumonia is a significant cause of morbidity and mortality in calves, both during the pre-weaning period and shortly following weaning. A range of events are linked with increased disease risk, including weaning management, painful procedures, housing systems and ventilation and effective preventive measures have been demonstrated. The management of pneumonia in calves is reliant on a sound understanding of aetiology and of relevant risk factors and of effective approaches to diagnosis and treatment.

This is the first epidemiological study to assess risk factors for BRSV seroprevalence carried out in Brazil. Even though BRSV prevalence of 79.5% in the animals sampled was similar to that estimated, the prevalence in adult animals was higher than that expected, reaching 87% of samples. In calves, the seroprevalence was lower than that found in adult animals (62.8%) and could be even lower once VNT does not allow the distinction between antibodies from colostrum and natural infection. Thus, this study demonstrated that the prevalence of BRSV antibodies was higher in adult animals, as previously reported in other countries [13, 16].

Adult animals are associated with high seroprevalence of BRSV as consequence of a repeated exposure to the virus infection throughout their life and possibility of reinfections. Similarly, the highest antibody titers were associated with non-vaccinated adult cattle, probably due to the exposure to successive viral reinfections, which results in a booster effect on antibody titers. Other factor related to high antibody titers is recent BRSV infections, which can be confirmed only by paired serology, antibody screening in calves after the period of colostral antibody detection or viral detection by direct methods. As respiratory disease was not reported in half of the herds studied, it is indicative that BRSV infection can be subclinical. This is consistent with previous reports. Herds can remain free of clinical BRSV infection for many years even in areas of high prevalence of the virus.

The presence of other pathogens is also associated with the prevalence of BRSV [8, 11, 14, 16]. This information explains the association of BRSV serological prevalence with the prevalences of BoHV-1 and BVDV-1. The infection by these viral agents is also reported in Brazilian herds, with high prevalences [27, 28]. BVDV infection can cause impairment of the animal’s immune function and thereby decrease resistance to other infections. The synergistic effects of BVDV with other respiratory pathogens have been observed [29, 30]. Thus, health status of the herds may also be affected indirectly by BVDV control measures.

Dairy cattle herds in São Paulo State usually have poor biosecurity measures, such as the lack of quarantine of newly purchased animals, lack of diagnosis of respiratory diseases (particularly for BRSV) and vaccination is rarely performed against these viruses. Therefore, we hypothesized that risk factors for the seroprevalence of BoHV-1, BVDV-1 and BRSV in the studied population likely to overlap.

Despite the logistic regression not confirming “type of calves feeding” variable as a risk factor for high prevalence of BRSV, the Fisher’s exact test detected “natural suckling” as a protective factor. “Natural suckling” would be important as it may be able to reduce the risk of calves becoming infected by BRSV. Weaning can be stressful and results in impaired immune function, which may further exacerbate a BRSV exposure. Suckling reduces the occurrence of diarrhea, prevents the abnormal behavior of cross-suckling of other calves and improves animal health [31, 32]. Prior to the current study there have been no report about “natural suckling” and its relationship with BRSV seroprevalence or its role as a protective factor, therefore, based on the results presented, it has the potential to decrease seroprevalence to BRSV.

Similarities were observed among the results found at the present study and those previously obtained by others conducted in Brazil [18–20]. In Latin America countries, equivalents prevalences of BRSV have also been reported [12, 14–16], as well as difficulties in detecting the risk factors involved in the dissemination of the agent, even using different forms of sampling and analyzing a considerable number of variables. Thus, the dynamics of infection may differ even in a particular country or geographic area.

The high serological prevalence of BRSV found in this study shows the importance to know more about this infection since it is not considered important in the country, mainly due to the lack of diagnosis. The awareness of the risk factors involved in the BRSV dissemination can allow understanding its mechanisms, even though, as in other studies, these factors were not very clear. Thereby, further studies as a complement to the current one should be performed until concrete information has been found.

Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen in cattle, detrimentally affecting the economy and animal welfare. The virus is distributed worldwide and is a major pathogen of the bovine respiratory disease complex [1, 2]. Viral respiratory infections are also of concern with regards to antibiotic resistance, as they predispose cattle to secondary bacterial infections that are commonly treated with antibiotics. Bovine respiratory disease is traditionally handled with management measures, vaccination and metaphylactic antibiotic treatment. Another possible strategy is to prevent inter-herd transmission of the main pathogens by increasing biosecurity measures at herd level. Because live animal transport is considered one of the main modes of BRSV transmission between herds [5, 6], proper mitigation must ensure that live animal transport be performed without compromising biosecurity. This requires knowledge on transmission risk associated with animal contact at different stages of infection. Knowledge of BRSV shedding related to clinical features would also be useful in order to assess the transmission risk of an infected herd without the use of viral diagnostic assays. For both of these areas, several knowledge gaps exists. Although way of infection may affect both viral shedding and clinical signs compared to naturally exposed animals, challenge studies are superior in the sense that aetiology and time of exposure is known and clinical features and virus excretion can be followed closely. Challenge studies, many of them aiming to evaluate the efficacy of vaccines [7–11] seldom last longer than one to two weeks. Grissett et al. and Gershwin concluded that shedding of BRSV begins on day three or four post-infection (p.i.) and usually lasts until day nine or ten. Grissett et al. summarized that the median time to appearance, peak and resolution of clinical signs was 3, 6 and 12 days, respectively, based on information from 22 inoculation studies [7–11, 14–22]. As studies outlasting the acute phase of infection are lacking, it is not known how long an animal can transmit infectious viruses to other animals. Appearance of clinical signs is usually the only information available in the field, and finding a clinical parameter that indicates shedding of infectious BRSV would be valuable. The existence of chronic or persistent infections in individuals is likewise still unclear [23–26].

During the acute phase of a BRSV infection, immunological protection develops, but it is assumed to be short-lived. This might enable early reinfection and new shedding of the infective virus, which complicates the risk assessment. A few BRSV studies have been performed to shed light on this. In a study by Kimman et al. they reported a strong local IgA response in the respiratory tract, but no virus shedding, when calves were re-exposed 3–4 months after primary BRSV infection. Stott et al. indicated, referring to their own unpublished results, that reinfection in calves and heifers may occur as early as three weeks post-infection. However, early reinfection with BRSV is not well-documented, and more precise knowledge of the occurrence is needed.

The existing literature on BRSV shedding and transmission is based on various laboratory methods, such as detection of viral RNA and culturing of the virus. Although resource-demanding, virus transmission studies are preferably performed using live animals in sentinel trials.

The aim of the present study was, therefore, to study basic features of BRSV infection in calves infected by exposure to BRSV-shedding calves. This was performed by:Investigating the shedding of viral RNA and infective virions:related to clinical outcome during the experimental period, lasting for two monthsin calves rechallenged by inoculation seven weeks p.i.Investigating whether the calves and their environment are not infectious to naïve in-contact calves four to nine weeks post-infection despite rechallenge with BRSV and mild stress induction.

Bovine respiratory disease is amongst the most significant causes of health problems and reduced welfare and profitability in the cattle industry. Decreases in production and profitability are associated with factors such as reduced growth rate, treatment costs and increased mortality. In dairy herds, one may also observe reduced milk production, milk quality and reproductive performance [1–5].

Bovine respiratory syncytial virus (BRSV) is an important etiological agent in bovine respiratory disease. The infection can be subclinical, or result in mild to severe clinical signs. Disease might be caused by BRSV directly or in combination with secondary bacterial infections which occurs frequently [7, 8]. The morbidity in a BRSV outbreak is reported to be between 60 and 80 %, and the mortality between 0 and 20 %.

Many experimental studies have been conducted to describe the clinical and pathological consequences of BRSV infection [7, 9–13]. However, experimental infection usually results in less severe disease compared to natural outbreaks, fewer animals are usually included, and the animals are monitored for a relatively short period after virus exposure. Studies on natural BRSV outbreaks [14–19] often lack relevant information on the situation prior to the outbreak, and suitable control groups are usually not available. Furthermore, such studies often provide little information on the production after the outbreak.

Large scale epidemiological studies from data records investigating production losses and economic impact of bovine respiratory disease are usually based on clinical diagnosis, without specific information about the etiological agent [1, 2, 4]. For BRSV specifically, reduced milk yield [5, 20] and reduced semen quality are amongst the reported effects. Studies on negative consequences of this infection, relevant for rearing of young stock, are scarce and represent an important knowledge gap in the estimation of the total consequences of BRSV.

The Norwegian cattle population is currently free from several globally important respiratory pathogens such as bovine herpes virus type 1 and bovine viral diarrhoea virus, and Mycoplasma bovis has never been detected. This makes the Norwegian cattle population a suitable population for studying the impact of BRSV. The prevalence on herd level is estimated to be between 34 and 41 %. BRSV has been found to be the main cause of outbreaks of respiratory disease, either acting as a single agent or in combination with other pathogens. Other viral pathogens known to cause respiratory disease in Norwegian cattle, are bovine corona virus (BCoV) and bovine parainfluenza virus type 3 (BPIV3).

To calculate the cost-efficiency of preventive strategies for BRSV, accurate estimates of the potential losses are essential. The aim of the present study was to provide robust estimates of such losses by analysing the association between exposure to BRSV, weight gain and feed conversion rate, quantify any reduction in these parameters, and estimate the duration of decreased production. Furthermore, it was an objective to illustrate a method for estimating the effect of disease outbreaks in beef herds by employing intra-herd comparisons based on health and production records.

Our results revealed high BRSV seroprevalence (98.6%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. The herd seroprevalence of BRSV (100%) found in this research is consistent with published data of Solis-Calderon et al., Saa et al., and Carbonero et al., who reported a herd prevalence of 90.8% (Mexico), 91.3% (Ecuador), and 95.8% (Argentina), respectively. However, these results differ from those reported by Obando et al. Contreras and Parra, who found lower seroprevalence values in other studies. The individual seroprevalence of BRSV (98.6%) agrees with the findings of Saa et al. who reported 80.4% of seroprevalence in herds of Ecuador. This result also agrees with those of Betancur et al. and Betancur et al. who found 13% and 31% of seroprevalence in dairy cows and calves, respectively, in herds of Montería state, Colombia.

Nevertheless, these results differ from those published by Carbonero et al., who reported 46.6% of seroprevalence in Argentina. The results obtained demonstrate that BRSV is widespread among animals and dual-purpose cattle herds in Cesar department. Probably, after the initial infection occurs in some animals, the virus is rapidly spread throughout the animals, probably by aerosols, particularly in herds without prior exposure to the virus, increasing seropositivity. The several herds in Colombia are not being vaccinated against BRSV and result from this research demonstrates that this virus circulates among the animals and herds from the three municipalities. It would be important to include BRSV in vaccination programs with the aim of controlling infection in this region.

Regarding the age, BRSV infection was observed in both age groups in this research. Although the analysis was not done in younger animals, as reported in the literature, the clinical disease is more frequent in calves. This seroreactivity in adult animals suggests possible reinfections during the course of their life. The result obtained in this research agrees with those reported by Betancur et al., who found no statistical association between infection and age group. Nevertheless, the results obtained differ from those published by Bidokhti et al., who found statistical differences with respect to the age of the infected animals. They demonstrate that after infection with BRSV, the animals will remain seropositive for several years. The older cows were seropositive while the younger cows were seronegative, i.e., there had been no virus circulating for several years. In this study, municipality, sex, and herd size were not a significant risk factor (Table-4). Regarding the clinical signs, animals with respiratory problems (34.9%) and conjunctivitis (38.5%) were found (Table-3). However, there was no statistical association (p>0.05) between seroprevalence values and respiratory signs in tested animals. These results are due to BRSV, which is observed in any age group, but infections that result in severe clinical disease are typically observed in calves. Nevertheless, there was no sampling in calves in this research.

Ureaplasmas, also belong to the same family as the Mycoplasmas, and are pathogenic bacteria which were initially associated with urogenital tract infections but have also been isolated from pneumonic bovine lungs [10, 68, 75]. The species Ureaplasma diversum has been associated with clinical respiratory disease [75, 76]. As Ureaplasma was found to be present in pneumonic lung tissue samples from calves which died from BRD, this genus may be an important contributor to BRD which is often overlooked.

Respiratory syncytial virus (RSV) infection is a major cause of death in the first year of life, with especially high mortality rates in African and Asian countries,,. Upper respiratory tract infections (URTI) with RSV generally cause relatively mild disease that does not require treatment. These infections can progress, however, into lower respiratory tract infections (LRTI) and cause severe disease, especially in pre-mature infants. RSV can also cause recurrent infection of the upper respiratory tract.

An important complication of upper respiratory infections is middle ear inflammation and-related deafness,, and RSV infections are associated with the development of asthma at a later age,,.

Other common causes for respiratory tract infections are Haemophilus influenzae type b (Hib), parainfluenza virus, rhinovirus and influenza virus

[9],,.

Protective immunity against respiratory pathogens like influenza and RSV is mediated by IgG and IgA,,,,. RSV-specific serum IgG levels in neonates are inversely associated with increased prevalence of RSV infections,, and breastfeeding reduces the incidence and severity of RSV infection,. In addition, the levels of anti-influenza IgA in breast milk correlates with decreased frequency of respiratory illness with fever. These findings indicate that pathogen-specific antibodies are crucial for protection against respiratory infections, and that orally ingested immunoglobulins (like breastmilk-derived IgA) may contribute to immunity to airway infections.

Human and bovine milk contain high levels of immunoglobulins, which are important for protecting the infant from infections with bacteria and viruses,. Cross-species activity between human and bovine immune-related milk proteins has been reported before,, and cow’s milk contains bovine IgG (bIgG) that binds to gastrointestinal pathogens that also infect humans, such as Shigella flexneri, Escherichia coli, Clostridium difficile, Streptococcus mutants, Cryptosporidium parvum, Helicobacter pylori, and rotavirus.

At present there is, however, no information on binding of bovine IgG to human respiratory viruses.

Most infant nutrition is bovine milk-based, but lacks intact bIgG as a result of heat treatment during processing. To investigate if bIgG would be a useful ingredient in these formulas, the aim of the present study was to investigate the specificity and functional relevance of bIgG against RSV and other human respiratory pathogens, the ability of bIgG to bind to human Fcγ receptors, and the induction of effector functions in human myeloid cells.

All experimental procedures were performed with approval and under the guidelines of the US Meat Animal Research Center (USMARC) Institutional Animal Care and Use Committee (IACUC approval numbers 5438–31,000–082-04 (24) and 3040–32,000–031-07 (5)).

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality for all production classes of cattle and calves in the U.S., causing losses to the cattle industry in excess of $1 billion dollars annually [1, 2]. Multiple etiologies, including both viral and bacterial, contribute to BRD. Those generally accepted to be important contributors to BRD include the viral pathogens bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus types 1 and 2 (BVDV), bovine respiratory syncytial virus (BRSV) and parainfluenza-3 virus (PI3); and the bacteria Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis [2, 4]. BRD is frequently initiated by a viral infection that disrupts local defenses and/or causes immune suppression, allowing opportunistic bacterial pathogens that are in healthy animals as normal nasophayngeal commensals to proliferate and infect the lungs [2, 4]. Superimposed environmental or management related stress (such as adverse weather, shipping, and commingling) can further suppress the host immune system, increase pathogen exposure, and may be important co-requisites in many BRD outbreaks. Although vaccines and antibiotic treatments are readily available to prevent and treat infection caused by common BRD pathogens, the incidence of disease remains high.

In recent years, bovine coronavirus (BCV) has been implicated as an important contributor to BRD. Although initially described as being associated with calf diarrhea, BCV has been found to infect the upper and lower respiratory tract and has been isolated from pneumonic lungs alone or in combination with other respiratory pathogens [7–12]. In addition, results of multiple studies indicate that groups of cattle with high titers of serum antibodies to BCV at the time of feedlot entry are less likely to shed BCV and develop BRD than those with low anti-BCV serum antibody titers [7, 13–15]. Taken together, it appears that BCV contributes to feedlot BRD, and high titers of serum anti-BCV antibodies associate with reduced risk of BCV infection and disease. However, it remains unknown whether the serum antibodies themselves are immune correlates of protection, or whether they simply reflect prior exposure to the virus.

The relationship between BCV and BRD in pre-weaned beef calves has not been comprehensively evaluated. Though BCV is frequently detected in nasal swabs from nursing calves with BRD, subclinical BCV infections are also common in young dairy calves, even in the presence of relatively high anti-BCV antibody titers [16, 17]. These results raise questions about the association between anti-BCV antibody titers and BCV shedding with the risk of developing BRD in nursing dairy calves. Similarly, in a 2014 study, our group sampled four research herds (n = 890) at predefined times from birth through their fifth week in the feedlot. This study revealed that the herds in which BCV was detected in nasal sections during the pre-weaning period also had the highest incidence of pre-weaning BRD; however, nasal swabs were not collected at the time of treatment to diagnose the pathogens associated with those pre-weaning BRD cases. This study also reported that serum anti-BCV antibody abundance did not correlate with BCV shedding prior to weaning. Thus, while mounting evidence suggests that anti-BCV antibodies protect weaned feedlot cattle from BRD associated with BCV infection, the relationship between humoral immunity to BCV, virus shedding, and the risk for developing BRD in nursing calves remains unclear. This represents a major obstacle in the development of effective control strategies to reduce the impact of BCV-related respiratory disease in cattle, which is significant given that there are currently no licensed BCV vaccines in the United States to aid in the prevention of BRD.

To address this knowledge gap, the present study serially sampled 817 calves from three herds of beef cattle from birth through weaning to determine whether shedding of BCV is associated with BRD and whether levels of anti-BCV serum antibodies associate with BCV shedding or BRD incidence in pre-weaned beef calves. Sequence analysis of the virus strain(s) circulating in each herd and the prevalence of common opportunistic bacterial pathogens (M. haemolytica, P. multocida, H. somni and Mycoplasma bovis) in the upper respiratory tract of sick and apparently healthy cattle were also evaluated to account for potentially confounding factors that could influence BRD development in these populations.

Mycoplasma was one of the most abundant OTUs present in the post-mortem lung and lymph node samples. It was found to be more abundant among the tissue samples from dairy calves which died from BRD relative to the tissue samples from the clinically healthy, lung lesion-free, H-F calves. This is consistent with previous studies which reported Mycoplasma to be one of the dominant genera in nasopharyngeal swab samples from cattle at feedlot [26, 33], despite being infrequent in cattle at feedlot entry. Additionally, this result is concordant with previous observations which report that Mycoplasma species bovis, dispar and bovirhinis, were identified more often in pneumonic lungs and respiratory tracts compared with clinically healthy lungs and respiratory tracts [25, 62, 68, 69].

Although Mycoplasma bovis is a recognised BRD pathogen and is commonly screened for in veterinary diagnostic laboratories [71, 72], the other major Mycoplasma species are not generally screened. However, they may be responsible for BRD as Mycoplasma dispar is a recognised pathogenic Mycoplasma species, capable of colonising the lower respiratory tract and caused pneumonia when inoculated into gnotobiotic calves. Moreover, it has been cultured from the respiratory tracts of calves presenting with BRD [63, 74] and has been isolated from lavage fluids of calves with recurrent respiratory disease. Furthermore, although Mycoplasma arginine and Mycoplasma bovirhinis did not cause pneumonia following inoculation into gnotobiotic calves, these species have also been isolated from lavage fluids of calves with recurrent respiratory disease and pneumonic lungs [68, 69] and have been suggested to act as co-pathogens which may intensify respiratory disease symptoms [69, 74].

Bovine respiratory disease complex (BRDC), a multi-factorial disease, is an economically important health problem of cattle worldwide. The disease is commonly referred to as “Shipping fever” and causes an increase in morbidity mortality rates. The multiple factors that cause BRDC include stress, infectious agents, immunity, and housing conditions. The infectious agents associated with BRDC include viruses, bacteria, and mycoplasmas. While most acute infections with uncomplicated infectious agents are sub-clinical, they can cause respiratory disease characterized by a cough, fever, and nasal discharge. Mixed infections with two or more infectious agents are thought to contribute to BRDC. The primary viral infectious pathogens that cause BRDC are bovine parainfluenza virus 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BHV-1), bovine coronavirus (BCV), and so forth.

Bovine parainfluenza virus type 3 (BPIV3) was one of the most important viruses associated with BRDC in cattle. It was first isolated in 1959 and first identified in cases of BRDC. BPIV3 is an enveloped, non-segmented negative-strand RNA virus within the genus Respirovirus. BPIV3 induces respiratory tract damage and immunosuppression. More severe secondary bacterial and mycoplasma infections are caused in susceptible animals in instances of high stress, such as transportation and feedlot situations.

Up to now, based on phylogenetic analysis, BPIV3 has been divided into three genotypes: Genotype A, genotype B, and genotype C. Multiple BPIV3 genotype A strains have been isolated in USA, China, Argentina, and Japan. Genotype B was initially identified in Australia. Isolation of BPIV3 genotype C, first identified in China, has also been conducted in South Korea, Japan, Argentina, and USA. A high seropositivity rate for BPIV3 in dairy cattle indicated that a high level of BPIV3 infections occurs. Many efforts have been made focusing on the prevention and control of BRDC in order to reduce production losses in the livestock industry.

Here, we describe the cell culture isolation and genomic sequencing of a BPIV3 genotype A strain isolated from cattle in China. Although BPIV3 is endemic in cattle, little is known about the pathogenesis of this virus and information regarding antigenic variation owing to the genetic variability is rare. The phylogenetic comparison of our isolated strain with strains previously characterized in China indicated the presence of divergent strains of genotype A circulating in the country. The diversity of BPIV3 in China seems to mirror the diversity of this virus, which is observed in the USA. In addition, the full characterization of our BPIV3 genotype A strain will lend support to molecular diagnoses and to future studies aimed at developing an efficient vaccine against multiple viral lineages.

Bovine corona virus (BCV) and bovine respiratory syncytial virus (BRSV) are two worldwide distributed viruses. BCV causes diarrhoea in calves, winter dysentery in adults and various degrees of respiratory symptoms. BRSV is regarded as one of the most important causes of respiratory tract disease, especially in young calves. An infection can cause respiratory distress, fever, anorexia and subcutaneous emphysema and can lead to secondary bacterial pneumonia and death. Outbreaks of BCV and BRSV occur mainly in autumn and winter. These infections are common in dairy herds; in a nationwide survey in England and Wales the prevalence of antibodies to these viruses in bulk tank milk (BTM) was 100%. Swedish studies have shown a prevalence of 70-100% for BCV and 41-89% for BRSV, with the higher prevalence in southern parts. In a more recent study in a high animal-density area in south-west Sweden, the prevalence in BTM was 100% for both BCV and BRSV.

Previous studies have shown that BRSV and BCV infections are effectively spread within the herd. It has also been shown that acquired antibodies remain detectable for years, even without reinfection, whereas maternal antibodies are only detectable for a few months. Spot samples from a few young animals can thus be used to reflect recent infections of BRSV and BCV in a herd, whereas bulk tank milk samples mirror the long-term history. Spot sampling has previously been described for bovine virus diarrea virus (BVDV).

Despite the importance of these viruses and the fact that they are widely spread, little is known about transmission routes and management risk factors. Introduction of new animals and indirect spread via people and equipment are believed to be important and airborne transmission has been shown to occur for BRSV, at least under experimental conditions. Studies have been carried out to determine the relationship between herd health, reproduction efficiency and milk production and seropositivity to other viruses, for example bovine viral diarrhoea virus and bovine leukemia virus. Similar studies for BRSV and BCV have, as far as we know, not been conducted and it is therefore difficult to quantify their effect on the farm efficiency and economy. The purpose of this study was to explore if there were any associations between antibody status to BCV and BRSV and disease incidence, reproduction and some herd characteristics in dairy herds. A secondary aim was to investigate if there were any difference in proportion antibody positive herds between two neighbouring areas.

Bovine respiratory disease (BRD) affects the lower respiratory tract of cattle, causing high mortality and carcasses of lower quality. The syndrome has a multifactorial etiology, including infectious agents, host and environmental factors, with particular emphasis on transport stress. The latter is indeed responsible for physiological changes that favor pathogen proliferation and invasion of tissues by opportunistic pathogens. Viruses and stress-related behavior interfere with the mucociliary clearance of the respiratory tract and dysregulate the tracheal antimicrobial peptides of the innate defenses, allowing opportunistic bacteria to cause pulmonary infections. Infectious agents of BRD include both viral and bacterial agents such as bovine herpesvirus type 1 (BoHV-1), bovine adenovirus (BAdV), bovine viral diarrhea virus (BVDV), bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus (BPiV), Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Mycoplasma bovis.

It has been shown that, to minimize the incidence of transport-related respiratory disease, antibiotics and vaccines are widely used both before and after transport. However, the data on the effectiveness of these preventative methods are conflicting. The hypothesis of this work was that there would be a change in the nasal microbiota, with an increase in the prevalence of bacteria and virus involved in BRD, in beef steers subjected to long distance transportation and not treated before the journey.

Despite the high number of trucks transporting livestock from North Europe to Italy, to the authors’ knowledge, there are no data available in Italy regarding the potential associations between long-distance transport and the onset of BRD after feedlot arrival. Consequently, the aim of this pilot study was to document the prevalence of the multiple pathogens involved in BRD after a long-distance travel from France to southern Italy through investigation of the nasal microbiota.

The present study shows that intra-herd comparison of health and production data can be a valuable approach to estimate the costs associated with reduced health status. More specifically, the study shows that BRSV affects the growth performance of bulls for several months after infection. The effect is most pronounced in animals that are seriously affected during infection, but a significant loss of performance can also be detected in animals showing mild or no signs of disease, and despite apparent recovery. This amounts to a significant part of the costs associated with BRSV, however they may easily be underestimated. Such losses are of great concern to the farmer and must be taken into account in any cost-benefit analysis of preventive measures towards BRSV infection, whether on herd or population level.

Nasopharyngeal aspirates were collected from patients and control group according to Svensson et al.,. Sterile normal saline solution was instilled in one nostril while occluding the other nostril, using a sterile blunt-tipped disposable syringe. Then the patient was instructed to forcibly exhale through the lavaged side into a sterile specimen cup. The sequence was then repeated in the other side of the nose. NPA specimens were examined microbiologically immediately and part of the specimens was stored in aliquots at -70°C for PCR. Two ml blood samples from both patients and control were collected into vacutainer, centrifuged and serum was separated and stored at - 20°C for HBoV-IgM antibodies by ELISA.

All IG and EG animals had mild pathological lesions in the respiratory tract, with bronchointerstitial pneumonia, ranging from mild to moderate. Five out of eight calves had a mucopurulent bronchitis and/or bronchiolitis, six had a mild bronchiolitis obliterans, seven had mild to pronounced atelectasis and one calf had pleuritis. All the calves had mild to pronounced bronchus-associated lymphoid tissue hyperplasia and mild to moderate reactive hyperplasia of the lymphatic tissue of nasopharynx and the regional lymph nodes of the lungs. Bacterial culture from lung tissue showed no growth, except from one calf, where sparse occurrence of Trueperella pyogenes was identified.

DNA was extracted from 200 μl of the NPA samples using the High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany) according to the instructions of the manufacturer.

Amplification of hBoV DNA was performed using 50 μl elution volume with the NP-1 primers BoV188F and BoV542R described by Allander et al., (table 8) using the HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany). PCR reactions were carried out in a 50 μl volume consisting of 5 μl extracted DNA, 1× Qiagen HotStar buffer, dNTPs at a final concentration of 200 μM each, 200 pmol of each primer, and 1.5 U of Taq Polymerase. The cycling conditions were 50 cycles (94°C 30 s, 53°C 40 s and 1 min at 72°C) after a preheating step of 10 min at 95°C.

After amplification, PCR products were visualized by staining with ethidium bromide on agarose gels. A PCR reaction was considered as positive when a band of the expected size (354 base pairs) was visible.

A plasmid containing the PCR product cloned in the vector PCR 2.1-TOPO (Invitrogen) was used as positive control.

Along with equine rhinitis virus (ERV) and foot and mouth disease virus (FMDV), bovine rhinitis A and B viruses (BRAV and BRBV, respectively) are species in the genus Aphthovirus, family Picornaviridae. Two serotypes of BRAV have been identified, BRAV1 and BRAV2, while BRBV consists of a single serotype. The BRAV1 strain SD-1 was isolated in Germany in 1962 from nasal secretions from a calf with rhinitis. Additional BRAV1 strains were subsequently isolated from both healthy and diseased bovines in England, Japan, Italy and the U.S. and shown to cross react in serum neutralization assays [3–6]. The sole BRBV isolate EC-11 was isolated in England in 1964 by Reed from the lung of a specific pathogen free calf with respiratory disease. Likewise, BRAV2 consists of a single specimen, strain H-1, isolated from an outbreak of respiratory disease in cattle in 1984. Despite numerous studies on bovine rhinitis viruses (BRV) in the 1960’s through mid-1980’s, little work has been published on their epidemiology and ecology the past several decades.

Bovine respiratory disease complex (BRDC) is the most economically significant disease of the cattle industry, leading to losses due to mortality, morbidity, treatment costs and feed inefficiency in excess of $750 million dollars per year in the U.S. alone. BRDC has a multifactorial etiology involving a variety of bacteria and viruses in addition to host and environmental factors. Numerous commercial vaccines including both killed and attenuated live bacteria are available. Viruses commonly included in commercial vaccine include bovine viral diarrhea virus (BVDV), bovine herpes virus 1 (BHV1), parainfluenza virus 3 (PI3) and bovine respiratory syncytial virus (BRSV). Despite their widespread use, BRDC incidence has increased over the past 20 years. BRDC pathogenesis often involves a primary viral infection which damages respiratory mucosa and alters host immune responses leading to secondary bacterial pneumonia caused by commensal bacteria already present in the respiratory tract.

Both BRAV and BRBV are established but rarely studied etiologic agents of BRDC. Experimental inoculation of calves with BRAV1 via intranasal (IN) or intratracheal (IT) routes, either singly or in combination, resulted in variable clinical signs of respiratory disease and histologic lesions consistent with pneumonia. BRAV1 was also recovered from nasal swabs of IN inoculated animals and all animals inoculated or exposed by contact seroconverted to BRAV1 by day seven post inoculation. A similar experiment using a different BRAV1 strain (RS 3x) and colostrum deprived calves failed to reproduce clinical disease but was successful in isolating BRAV1 from nasal swabs post inoculation and found histological lesions of focal rhinitis and a neutralizing antibody response in all inoculated calves. BRBV pathogenesis was investigated using intranasal inoculation of gnotobiotic calves. Clinical signs including fever, nasal discharge and increased respiration rate were observed. Foci of epithelial necrosis were observed histologically in the turbinates and trachea and interstitial pneumonia was evident in the lungs. Virus was isolated from multiple tissues and was neutralized by convalescent antiserum. In addition to controlled studies, numerous investigations of acute respiratory disease in cattle resulted in the isolation of bovine rhinitis viruses where paired acute and convalescent sera suggested a causative role for bovine rhinitis virus.

Metagenomic sequencing on nasal swabs obtained from BRDC diagnostic submissions were performed to survey viruses present. Contigs with high identity to BRAV2 and BRBV were identified in one swab. To further our understanding of the epidemiology and ecology of bovine rhinitis viruses in BRDC, a more comprehensive survey was performed.

Table 1 shows the number of positive animals in each truck at T0 and T1 for each pathogen tested. Neither BVDV nor BoHV-1 were detected, either at T0 or T1.

There was an association between day and H. somni, and a higher prevalence was found in DNS collected from animals travelling in the third shipment (Day 3) (90%, 38/42) in comparison with the first (60%, 24/40) and second (60%, 18/30) days (OR = 6.3, 95% CI = 1.8–21.2, p = 0.006). There were no other changes in the prevalence of the other pathogens among days.

The results of the univariate logistic regression analyses with time (before and after journey: T0 and T1, respectively) as a fixed factor are presented in Table 2. Higher odds of BCoV, BRSV. M. haemolytica M. bovis, and P. multocida were found in DNS collected four days after arrival (T1) compared with DNS collected before departure (T0). There was no difference in the prevalence of BAdV, BPiV, H. somni in the DNS collected at T0 and T1 (p > 0.05).

The DNS collected from the two steers reared at the arriving farm in southern Italy during the period from February to April, resulted positive only for BCoV.

Bovine Respiratory Disease (BRD) is a multifactorial disease characteristic of a viral-bacterial synergistic infection with predisposition from environmental stressors. The disease constitutes a major source of economic loss through mortality, clinical disease and the associated treatments and long lasting reduced growth performance of infected young stock [2, 3]. The annual cost of BRD is estimated at $1billion in the USA, with preventative measures contributing a further $3billion [4, 5]. Vaccines are commonly used for controlling BRD viral pathogens, but despite seasonal vaccination, animals can become infected with each new outbreak, maintaining the infection within the population. The viral pathogens associated with BRD [Bovine Parainfluenza Virus type-3 (BPI3V), Bovine Respiratory Syncytial Virus, Bovine Viral Diarrhoea Virus and Bovine Herpes Virus-1] impair immune responses in infected animals and damage the respiratory tract allowing the establishment of secondary infections, that may develop further into bacterial pneumonia. However, vaccinated animals can successfully clear viral infections faster than non-vaccinated animals through immune memory response, reducing the associated viral tissue damage or impairment of immune functions preventing the establishment of secondary bacterial and mycoplasma infections. During disease outbreaks, identification of unvaccinated animals at the early stages of infection could provide a window for effective treatment and facilitate the removal of animals that pose a greater risk of becoming infected and transmitting the infection to more susceptible juvenile stock. Furthermore, halting viral disease progression to more severe and costly secondary bacterial infections through the identification of vaccine failure animals during infection outbreaks would reduce the level of antibiotic use in the agricultural industry.

The only definitive method for successfully identifying vaccinated animals in the presence of an active viral infection is to determine the rate of viral shedding by virus isolation, cytokine/interleukin profiling or virus neutralization assay. These types of analysis require repeated sampling, a period for seroconversion and are expensive compared to serology based ELISA, and are therefore not routinely employed during endemic viral infection outbreaks. Differentiating infected from vaccinated animals (DIVA) marker vaccines (e.g. a modified wild type virus with a gene deletion resulting in the absence of a particular diagnostic antigen) can be employed to differentiate vaccine antibody responses from that of wild type virus. Companion serology based tests rely on seroconversion, and upon exposure to wild type virus the antibody response to DIVA vaccines will be masked by that of the wild type virus. Vaccine DIVA functionality is often limited to large viruses with increased potential for gene deletion and removal of redundant expressed antigens. Therefore, for viruses with small genomes such as paramyxoviruses (e.g. BPI3V and Bovine Respiratory Syncytial Virus of the BRD complex) where gene deletion of neutralizing antigens may reduce vaccine efficacy, alternative approaches are required to provide DIVA functionality. One approach is to design molecular DIVA vaccines that contain a marker nucleotide sequence differing from the wild type virus that can be employed in combination with PCR-based molecular diagnostics to differentiate between vaccine and wild virus strains [10, 11]. Successful differentiation of vaccinated from non-vaccinated animals using this technique requires concurrent vaccination and infection [12, 13], with a narrow diagnostic window post-infection for detection of DIVA vaccine and viral genetic material. Furthermore, detection of vaccine genetic material only demonstrates exposure to the vaccine and not the successful generation of immune protection, limiting functionality in assessment of herd level immunity. Consequently, there is a clear need for alternative diagnostic methods that can assess efficacy of vaccines and vaccination status of animals exposed to BRD viral pathogens at the early stages of infection prior to seroconversion and which do not require repeated sampling. Additionally, the lower initial exposure rates to viral infections in field settings combined with variation in strain nucleotide sequences and short periods of virus secretion highlights the requirement for a DIVA approach with a long diagnostic window which is not strain specific.

A potential approach that can meet these needs is based on the application of metabolomics to identify metabolites or ‘small molecules’ in biological samples that are signatures that correlate or provide some evidence of immune protection. These metabolites are often the end stage products of biological processes and therefore provide an accurate representation of an organism’s homeostatic status at time of sampling [14, 15]. Metabolomic analysis of bio-fluids has provided new insights to the understanding of the patho-physiological processes involved in disease establishment, development and diagnosis [16–19]. Whilst metabolomics has had limited application in the field of veterinary research, several studies have demonstrated the potential of this technique in the prediction of BRD disease outcome, differentiation of stress from viral infection responses, and characteristic of immune responses following vaccination. This study focuses specifically on BPI3V due to its endemnicity within cattle populations and absence of clinical symptoms which still predispose animals to more severe bacterial infections. Due to its small genome and absence of non-redundant proteins suitable for removal in DIVA vaccines, BPI3V is an excellent model for assessing the potential of metabolomics to establish vaccination status in infected animals. The aims of the current study were therefore to assess the performance of Reverse Phase (RP) and Hydrophobic Interaction Liquid Chromatography (HILIC) separation methods for Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) metabolomic profiling of bovine plasma and identify plasma metabolomic markers capable of differentiating between vaccinated and non-vaccinated calves following intranasal challenge with BPI3V. This work for the first time reports the metabolomic responses following challenge with BPI3V and demonstrates how the application of metabolomic profiling may help overcome current limitations in DIVA diagnostics by identifying markers capable of differentiating between vaccinated and non-vaccinated animals, and importantly allow the development of better tools to assess the performance of vaccines.