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Experimental infection of cattle with highly pathogenic avian influenza virus (HSN1).

Four calves were experimentally inoculated with highly pathogenic avian influenza avian influenza: see influenza.  virus A/cat/Germany/ R606/2006 (H5N1) isolated from a cat in 2006. All calves remained healthy, but several animals shed low amounts of virus, detected by inoculation inoculation, in medicine, introduction of a preparation into the tissues or fluids of the body for the purpose of preventing or curing certain diseases. The preparation is usually a weakened culture of the agent causing the disease, as in vaccination against  of nasal swab fluid into embryonated chicken eggs and onto MDCK MDCK Madin-Darby Canine Kidney Cells (virus tissue culture)  cells. All calves seroconverted.


Since 1997, an epidemic of highly pathogenic avian influenza virus (HPAIV) subtype (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T.  H5N1 has spread in Asia, causing fatal infections in poultry, wild birds, and mammals, including humans (1). Knowing the susceptibility to HPAIV (H5N1) of mammalian species living in close proximity to humans and poultry, such as members of the family Bovidae (e.g., cattle or water buffalo water buffalo: see buffalo.
water buffalo
 or Indian buffalo

Any of three subspecies of oxlike bovid (species Bubalus bubalis). Two have been domesticated in Asia since the earliest recorded history.
), would be helpful to those developing surveillance plans or determining risk areas. Serologic se·rol·o·gy  
n. pl. se·rol·o·gies
1. The science that deals with the properties and reactions of serums, especially blood serum.

 examinations have indicated that calves might be susceptible to influenza A influenza A
Influenza caused by infection with a strain of influenza virus type A.

influenza A Infectious disease An avian virus, especially of ducks–which in China live near the pig reservoir and 'vector';
 virus (2); however, so far only 1 strain (A/calf/Duschanbe/55/71 H3N2) has been identified as a cattle strain (3). A correlation between influenza A virus infection, reduced milk yield, and respiratory symptoms in dairy cows was assumed in the late 1990s (4,5) and has received recent attention (6,7). Nevertheless, to our knowledge, no data about the susceptibility of cattle to infections with HPAIV have been reported. Therefore, we experimentally infected bovine calves with HPAIV (H5N1) and collected data about clinical symptoms, viral excretion, and serologic reactions.

The Study

In 2007, 6 Holstein-Friesian calves, 3 month of age, were obtained from a breeder near Greifswald-Insel Riems, Germany. Their influenza A virus-free status was confirmed, and no avian influenza virus-specific genomes or antibodies were detected. All experiments were performed in the high-containment animal facility (Biosafety Level biosafety level Epidemiology A classification for the degree of caution required when working with specific groups of pathogens. See Maximum containment facility.  3+) at the Friedrich-Loeffler-Institut (trial approval no. LVLM-V/TSD/7221.3-1.1-003/07).

Four of the calves were intranasally inoculated with HPAIV (H5N1) strain A/cat/Germany/R606/2006, which had been isolated from a cat in 2006 (8,9). The virus was aerosolized Adj. 1. aerosolized - in the form of ultramicroscopic solid or liquid particles dispersed or suspended in air or gas

gaseous - existing as or having characteristics of a gas; "steam is water is the gaseous state"
 in 5 mL cell culture medium containing [10.sup.8.5] 50% egg infectious dose/mL (third egg passage). The other 2 calves (contacts) were not inoculated but were housed in the same containment room for the duration of the experiment. For 7 days all calves were monitored by physical examination, and pharyngeal pharyngeal /pha·ryn·ge·al/ (fah-rin´je-al) pertaining to the pharynx.

pha·ryn·geal or pha·ryn·gal
Of, relating to, located in, or coming from the pharynx.
 swabs were collected and examined for virus excretion.

All 6 calves remained healthy; no specific clinical signs were observed. All nasal swabs were tested with real-time reverse transcription-PCR specific for subtype H5N1 (10), and the genomic load was semiquantified by using threshold cycle values. Infectious virus was detected by inoculation of swab fluid into embryonated chicken eggs with 1 subsequent passage and onto MDCK cells (collection of cell lines in veterinary medicine veterinary medicine, diagnosis and treatment of diseases of animals. An early interest in animal diseases is found in ancient Greek writings on medicine. Veterinary medicine began to achieve the stature of a science with the organization of the first school in the , Friedrich-Loeffler-Institut, Isle of Riems, RIE n. 1. See Rye.
Rie grass
a - (Bot.) A kind of wild barley (Hordeum pratense
b - Ray grass.
- Dr. Prior.

Nasal swabs from all inoculated calves collected at 1 day postinoculation (dpi) were positive for viral RNA RNA: see nucleic acid.
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
, and 3 of 4 calves shed infectious virus, detected by virus isolation in embryonated chicken eggs (Table 1). Isolation of infectious virus in cell culture was successful in 2 of 4 samples. Furthermore, of 4 inoculated calves, 2 were positive for HPAIV (H5N1) genome copies at 2 dpi, and 1 shed infectious virus. From 3 dpi through 7 dpi, samples from nasal swabs of all animals were negative for viral genome, and all nasal swabs of the 2 contact calves remained negative for HPAIV (H5N1) RNA during the experiment (Table 1).

Assuming that susceptible animals should mount an antibody response, we looked for antibodies against the highly conserved and immunogenic im·mu·no·gen·ic
Producing an immune response.


producing immunity; evoking an immune response.
 nucleoprotein nucleoprotein

Macromolecular complex consisting of a protein linked to a nucleic acid, either DNA or RNA. The proteins that combine with DNA are generally of characteristic types called histones and protamines.
 (NP) of type A influenza viruses (11). Heat-inactivated (30 min at 56 [degrees] C) serum samples collected at 0, 7, 14, 21, 28, and 91 dpi were tested for NP-specific antibodies with a licensed commercial ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.

 (avian influenza A--blocking ELISA, Pourquier, Montpellier, France) according to the manufacturer's instructions. Serum samples from 50 HPAIV (H5N1)-negative, nonrelated cattle were used as controls and to confirm the specificity of the ELISA (data not shown).

To quantify the serologic response, we performed virus neutralization neutralization, chemical reaction, according to the Arrhenius theory of acids and bases, in which a water solution of acid is mixed with a water solution of base to form a salt and water; this reaction is complete only if the resulting solution has neither acidic nor  (VN) and hemagglutination hemagglutination /he·mag·glu·ti·na·tion/ (he?mah-gloo-ti-na´shun) agglutination of erythrocytes.

 inhibition (HI) tests with homotypic virus. The VN test was modified according to a previously described procedure (12). In brief, bovine serum samples were heat inactivated inactivated

rendered inactive; the activity is destroyed.

inactivated viruses
treated so that they are no longer able to produce evidence of growth or damaging effect on tissue.
 for 30 min at 56[degrees]C, and 3-fold serial dilutions were performed in a 50-[micro]L volume of cell culture medium in 96-well plates. The diluted serum samples were mixed with an equal volume of media containing homotypic influenza virus at a concentration of 102 50% tissue culture infectious dose/well. After 1 h incubation at 37[degrees]C in a 5% C[O.sub.2] humidified atmosphere, 100 [micro]L of Vero cells (African green monkey kidney, collection of cell lines in veterinary medicine, Friedrich-Loeffler-Institut, Isle of Riems, RIE228) at 1.5 x [10.sup.5]/mL was added to each well. The plates were incubated for 3 days at 37[degrees]C and 5% C[O.sub.2]. Viral replication was assessed by visually scoring the cytopathic effect without staining. Each assay was validated by comparison with positive and negative control serum from chickens and cattle as well as back titration of the used virus dilutions. Previous treatment with periodate Per`i´o`date

n. 1. (Chem.) A salt of periodic acid.
 (13) had no influence on the neutralizing titers. Serum samples were also tested for H5-specific antibodies by HI test with 4 hemagglutinating units of homotypic virus as antigen according to standardized methods (14). All serum samples were treated with periodate and heat inactivated to eliminate serum inhibitors. The HI tests were performed with a starting dilution of 1:8 by using a 1% suspension of chicken erythrocytes Erythrocytes
Red blood cells.

Mentioned in: Bartonellosis

erythrocytes (ē·rithˑ·rō·sīts), red blood cells.
 in a 0.85% saline solution saline solution
A solution of any salt, usually an isotonic sodium chloride solution. Also called salt solution.

Saline solution
A solution of sterile water and salt used in a variety of medical procedures.

The commercial NP-specific ELISA detected influenza A virus-specific antibodies at 14 dpi in 2 of the 4 inoculated animals (Table 2). All 4 animals had positive scores for neutralizing antibodies against the homologous virus at 14 dpi; specific titers ranged from 16 to 51. Furthermore, the HI test detected titers of 8 at 28 dpi in the 4 inoculated calves. At 21 dpi, VN testing indicated that 1 of the contact calves was positive for subtype H5Nl-specific antibodies, NP-ELISA results for this calf were questionable, and HI testing did not detect any hemagglutinating antibodies. The other contact calf remained serologically negative throughout the experiment. Finally, 3 months after inoculation, VN test and ELISA clearly indicated seroconversion seroconversion /se·ro·con·ver·sion/ (-con-ver´zhun) the change of a seronegative test from negative to positive, indicating the development of antibodies in response to immunization or infection.  by all inoculated calves and 1 of the contact calves. In contrast, HI results were negative for all but 2 inoculated animals (Table 2).


Our findings show that HPAIV (H5N1) has the potential to infect bovine calves, at least after high-titer intranasal in·tra·na·sal
Within the nose.
 inoculation, and that conventional HI tests may underestimate such infections. Furthermore, asymptomatic shedding of HPAIV (H5N1) by infected calves and subsequent seroconversion seem to be possible, and even low levels of HPAIV (H5N1) might be sufficient to induce a detectable antibody response in contact calves. However, the possibility that the infectivity detected in the contact calf at 1 dpi was the result of residual inoculum inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula   material used in inoculation.

n. pl.
 cannot be ruled out. Although the question whether calf-to-calf transmission of HPAIV (H5N1) occurs could not be definitely answered by our study, bird-to-calf transmission resulting in seroconversion is probable.

The incidence of clinical infections of cattle with HPAIV (H5N1) in disease-endemic regions should be low. However, our data indicate that serum from bovine species would be a valuable source of additional information about transmission events, especially in regions like Asia and Egypt, where HPAIV (H5N1) is endemic and probability of contact between poultry and cattle is high. The NP-ELISA is currently the assay of choice for the evaluation of bovine serum, and the VN test should be used for confirmation.

DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.3201/eid1407.071468


We thank M. Grawe for excellent technical assistance, and T. Arnold and G. Bauer for caring for the cattle during the challenge study.

This study was funded by the Federal Ministry of Food, Agriculture and Consumer Protection, Germany (FSI FSI Foreign Service Institute
FSI Fluid Structure Interaction
FSI Fuel Stratified Injection
FSI Federazione Scacchistica Italiana (Italian Chess Federation)
FSI Free Standing Insert
FSI Flight Simulator
 project no. 1-4.1).

Dr Kalthoff is a veterinarian veterinarian /vet·er·i·nar·i·an/ (vet?er-i-nar´e-an) a person trained and authorized to practice veterinary medicine and surgery; a doctor of veterinary medicine.

 at the Institute of Diagnostic Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression , FLI FLI - Flash Lights Impressively.  Insel Riems. Her research interests are the pathogenesis of HPAIV in different bird and mammalian species and vaccine development.


(1.) Webster RG, Peiris M, Chen H, Guan guan: see curassow.  Y. H5N1 outbreaks and enzootic en·zo·ot·ic
Prevalent among or restricted to animals of a specific geographic area. Used of a disease.

An enzootic disease.


peculiar to or present constantly in a location. See also endemic.
 influenza. Emerg Infect Dis. 2006;12:3-8.

(2.) Lopez JW, Woods GT. Influenza virus in ruminants: a review. Res Commun Chem Pathol Pharmacol. 1984;45:445-62.

(3.) Campbell CH, Easterday BC, Webster RG. Strains of Hong Kong influenza Hong Kong influenza
Influenza caused by a serotype of influenza virus type A; it was first identified in Hong Kong during the 1968 epidemic. Also called Hong Kong flu.
 virus in calves. J Infect Dis. 1977;135:678-80.

(4.) Brown IH, Crawshaw TR, Harris PA, Alexander DJ. Detection of antibodies to influenza A virus in cattle in association with respiratory disease and reduced milk yield. Vet Rec. 1998;143:637-8.

(5.) Gunning RF, Brown IH, Crawshaw TR. Evidence of influenza A virus infection in dairy cows with sporadic milk drop syndrome. Vet Rec. 1999;145:556-7.

(6.) Graham DA, Calvert V, McLaren E. Retrospective analysis of serum and nasal mucus from cattle in Northern Ireland for evidence of infection with influenza A virus. Vet Rec. 2002;150:201-4.

(7.) Crawshaw TR, Brown IH, Essen SC, Young SC. Significant rising antibody titres to influenza A are associated with an acute reduction in milk yield in cattle. Vet J. 2007 Sep 7 [Epub ahead of print]. DOI: 10.1016/j.tvjl.2007.07.022

(8.) Weber S, Harder T, Starick E, Beer M, Werner O, Hoffmann B, et al. Molecular analysis of highly pathogenic avian influenza virus of subtype H5N1 isolated from wild birds and mammals in northern Germany. J Gen Virol. 2007;88:554-8. DOI: 10.1099/vir.0.82300-0

(9.) Giese M, Harder TC, Teitke JP, Klopfleisch R, Breithaupt A, Mettenleiter TC, et al. Experimental infection and natural contact exposure of dogs with avian influenza virus (H5N1). Emerg Infect Dis [serial on the Internet]. 2008 Feb [cited 2008 Apr 15]. Available from http://

(10.) Hoffmann B, Harder T, Starick E, Depner K, Werner O, Beer M. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J Clin Microbiol. 2007;45:600-3. DOI: 10.1128/JCM.01681-06

(11.) Paltrinieri S, Spagnolo V, Giordano A, Moreno Martin A, Luppi A. Influenza virus type A influenza virus type A
A myxovirus of the genus Influenzavirus,antigenically varying from influenza virus types B and C, that causes acute respiratory illness in humans and infections in birds and certain other animals.
 serosurvey in cats [letter]. Emerg Infect Dis [serial on the Internet]. 2007 Apr [cited 2008 Apr 15]. Available from

(12.) Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, Lim W, et al. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol. 1999;37:937-43.

(13.) Anders EM, Hartley CA, Jackson DC. Bovine and mouse serum beta inhibitors of influenza A viruses are mannose-binding lectins Lectins

A class of proteins of nonimmune origin that bind carbohydrates reversibly and noncovalently without inducing any change in the carbohydrate. Lectins bind a variety of cells having cell-surface glycoproteins (carbohydrate bound proteins) or glycolipids
. Proc Natl Acad Sci USA. 1990;87:4485-9. DOI: 10.1073/ pnas.87.12.4485

(14.) World Organization for Animal Health. Avian influenza. In: Manual of diagnostic tests and vaccines for terrestrial animals, 5th ed. Updated Aug 7, 2005 [cited 2008 15 Apr]. Available from http://www.

Address for correspondence: Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald-Insel Riems, Germany; email: Bund

The German government's federal bond. The bund is issued to the public as a way for the German government to finance its spending.

The bund is like the Treasury bonds in the U.S. They are government-backed instruments of the highest quality.

Donata Kalthoff, * Bernd Hoffmann, * Timm Harder, * Markus Durban, * and Martin Beer *

* Friedrich-Loeffier-Institut, Greifswald-lnsel Riems, Germany
Table 1. Detection of highly pathogenic avian influenza virus
(H5N1) in nasal swabs from calves, Germany, 2007 *

                  Day postinoculation

                 0                  1

Calf            PCR        PCR     Egg    Cell

  Al            >40        20-30   -      -
  A2            >40        30-40   +      +
  A3            >40        20-30   +      +
  A4            >40        30-40   +      -
  K1            >40        >40     ND     ND
  K2            >40        >40     ND     ND

                  Day postinoculation

                            2             3-7

Calf            PCR        Egg     Cell   PCR

  Al           30-40       -       -      >40
  A2           30-40       +       -      >40
  A3           >40         ND      ND     >40
  A4           >40         ND      ND     >40
  K1           >40         ND      ND     >40
  K2           >40         ND      ND     >40

* Inoculated calves received highly pathogenic influenza virus
(H5N1) strain A/cat/Germany/R606/2006 (8,9); contact calves were
not inoculated but were housed with the inoculated calves. Egg,
embryonated chicken eggs; cell, MDCK cell culture; +, virus detected;
-, virus not detected; ND, not done.

([dagger]) H5-specific real-time reverse transcription-PCR; results
are shown as range of detected threshold cycle values.

Table 2. Serologic testing results for highly pathogenic avian
influenza virus (H5N1) in calves, Germany, 2007 *

                              Day postinoculation


                             VN                             7
                 NP       ([double        HI
Calf         ([dagger])   dagger])    ([section])   NP     VN    HI

  Al             98          2.3          <3        90    3.3    <3
  A2            114          0.4          <3        117    3     <3
  A3            118          <1           <3        90    2.7    <3
  A4            102          0.7          <3        122    2     <3
  K1             96          1.3          <3        88     <1    <3
  K2            120          0.7          <3        98    2.3    <3

                              Day postinoculation

                             14                            21

Calf             NP          VN           HI        NP     VN    HI

  Al             42           4           <3        26     4     <3
  A2             69           5           <3        27     4     <3
  A3             28          5.7          <3        29     5     <3
  A4             25          5.3          <3        24     5     <3
  K1             73           2           <3        44     4     <3
  K2             89          <1           <3        68     <1    <3

                              Day postinoculation

                             28                            91

Calf             NP          VN           HI        NP     VN    HI

  Al             24          6.2           3        20     7     <3
  A2             22          6.2           3        18    6.7     3
  A3             21           7            3        15     7      3
  A4             24          5.8           3        19    6.3    <3
  K1             40          4.5          <3        45     5     <3
  K2             76          0.7          <3        50     <1    <3

* Inoculated calves received highly pathogenic influenza virus (H5N1)
strain A/cat/Germany/R606/2006 (8,9); contact calves were not
inoculated but were housed with the inoculated calves. Positive results
are in boldface.

([dagger]) NP, avian influenza A- blocking ELISA against nucleoprotein
(Pourquier, Montpellier, France) inhibition % (<35, positive; >45,
negative; 35-45, questionable).

([double dagger]) VN, virus neutralization test (ND1001092); values
[greater than or equal to] 4 are considered positive.

([section]) HI, hemagglutination inhibition (1092); values >3 are
considered positive.
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Title Annotation:DISPATCHES
Author:Kalthoff, Donata; Hoffmann, Bernd; Harder, Timm; Durban, Markus; Beer, Martin
Publication:Emerging Infectious Diseases
Article Type:Disease/Disorder overview
Geographic Code:4EUGE
Date:Jul 1, 2008
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