Phosphorus kinetics in calves experimentally submitted to a trickle infection with Cooperia punctata

Veterinary Parasitology 163 (2009) 47–51

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Phosphorus kinetics in calves experimentally submitted to a trickle infection with Cooperia punctata H. Louvandini a,*, R.R. Rodrigues b, S.M. Gennari c, C.M. McManus a, D.M.S.S. Vitti b a

Faculdade de Agronomia e Medicina Veterina´ria, Universidade de Brası´lia, Brası´lia, Distrito Federal, CP 04508, 70910-900, Brazil Centro de Energia Nuclear na Agricultura, Universidade de Sa˜o Paulo, Piracicaba-SP, CP 96, 13400-970, Brazil c Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, Av: Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universita´ria, Sa˜o Paulo, 05508-900, Brazil b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 December 2008 Received in revised form 23 March 2009 Accepted 8 April 2009

Ten male Holstein calves (74.3  3.2 kg LW) were used for a trial with trickle infection with Cooperia punctata to evaluate phosphorus (P) kinetics. Five calves were inoculated with 10,000 L3 stage larvae per week during 35 days, while the other group of five calves was kept as a control. On the 29th day each calf was intravenously injected with 29.6 MBq of a 32P solution. Blood samples were taken at 24 h periods for 7 days, after which all calves were slaughtered and worms burdens. Faeces, urine and tissue samples were taken for analysis using isotopic dilution and modeling techniques. The number of eggs per gram of faeces (EPG) was 1920  168 on 28th day and the total number of worms burdens was 11,131  1500. Infected calves showed lower feed intake and live weight gain, as well as lower P intake, absorption and retention than control calves. The P flows between body compartments were lower for blood to gastrointestinal tract (TGI), TGI to blood, blood to soft tissues, bone balance and soft tissue balance in infected calves when compared to the control. The trickle infection of C. punctata affected P metabolism due to the decrease in P retained and live weight due to fall in feed intake. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Model Nutrition Parasite Phosphate Radioactive-phosphorus

1. Introduction Intestinal parasites can impair livestock production by inducing loss of appetite, diarrhea, nutritional deficiency, reduced weight gain, and even death of young animals. Among the trichostrongylids that infect livestock, and preferentially parasitize calves, Cooperia punctata is the most prevalent intestinal nematode in Brazil, as observed by Bianchin et al. (1990), Honer and Vieira-Bressan (1992) and Lima (1998). More than 70% of parasites recovered from the intestine of tracer calves kept in naturally infected pastures belonged to genus Cooperia, and more than 92% of these were of the species C. punctata (Bianchin et al., 1990; Lima, 1998). This genus has a direct life-cycle in intestinal mucosa, where L3 (third stage larvae) develop into adults. The first description of lesions caused by C. punctata were reported

by Ransom (1920) and Hung (1926), who observed damage to intestinal mucosa of infected calves. The fixation site is the upper part of the small intestine (Rodrigues et al., 2004), which is also the main site of dietary P absorption (Pfeffer et al., 1970; Ben-Ghedalia et al., 1975; Schro¨der et al., 1995). Thus, damage caused in the intestinal epithelium by parasites could interfere with P absorption and metabolism. Wilson and Field (1983) and Bown et al. (1989) observed low P absorption in lambs infected with parasites of the Tricostrongylus genus, which occupies the same intestinal region. The aim of the present study was to evaluate P kinetics in calves submitted to a trickle infection by C. punctata, using the 32P isotopic dilution technique. 2. Materials and methods 2.1. Local, animals, infection and radioactivity procedures

* Corresponding author. Tel.: +55 61 33400537; fax: +55 61 32736593. E-mail address: [email protected] (H. Louvandini). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.04.021

The experiment protocol was approved by animal welfare committee and carried out in the Department of

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Table 1 Chemical composition of the calf diet.

2.2. Parasitological exam

Chemical composition (g kg1 DM)

Hay

Concentrate

Dry matter (DM) Crude protein Neutral detergent fiber Acid detergent fiber Extract etereo Ash Phosphorus

915.16 73.75 870.15 518.43 19.72 51.96 2.04

909.42 193.67 326.23 91.37 109.98 57.05 12.11

Preventive Veterinary Medicine and Animal Health of the Faculty of Veterinary Medicine of the University of Sa˜o Paulo, Pirassununga Campus, Brazil. Two days after birth, males Holstein calves were transferred to a collective stall and bottled fed using in natura milk, twice a day. At 2 months of age, 10 calves (74.3  3.2 kg LW) were weaned and placed in metabolic cages for 35 days. They received 1000 g of concentrate mixture, 1500 g of Cynodon dactilon hay and water ad libitum. The bromatological analysis of the calf diet is shown in Table 1. Five animals were inoculated weekly with 10,000 L3 C. punctata larvae; 5000 twice a week and the other five animals were not inoculated to form the control group. The calves were weighed weekly. Faeces samples were collected for EPG counts daily to determinate patency period. After that it were weekly, until the 32P injection due the faeces became radioactive. On the 29th day each animal was intravenously injected with 29.6 MBq of a 32P solution. Blood, faeces and urine samples were taken at intervals of 24 h for P determination, for the following seven days. The plasma was obtained by blood centrifugation and inorganic P was determined by colorimetric analyses (Fiske and Subbarow, 1925). Total protein and albumin serum were determinated by colorimetric methods using commercial kits (Labetst1, Brazil). Feed samples and 10% of total outputs (faeces and urine) were stored at 4 8C and P content was determined by a colorimetric method (Sarruge and Haag, 1974). The samples of feed and faeces were dried overnight (105 8C) and ashed (500 8C). Urine samples were acidified by using 100 mL of 12 N HCl, then dried (55 8C) and ashed (500 8C). Radioactivity of 32P was measured by using Cerenkov radiation on centillator. Plasma and urine samples (1 mL) were added to distilled water (19 mL) in counting vials. Ashed fecal samples (1 g) were dissolved in 18N H2SO4 and also placed in counting vials. Specific activities in plasma and faeces were determined according to Lofgreen and Kleiber (1953). All calves were slaughtered by 5 mL kg1 intravenous injection of mebezonic iodine, embutramide and tetracaine cloridrate, at the end of the trial. Liver, heart, kidney, muscle and bone samples were collected from three calves from each group (to reduce manipulation with radioactive tissues). These were cleaned, weighed, and autoclaved. The tissue samples were then ground and digested in 18 N H2SO4 and transferred to vials for radioactivity determination. For inorganic P determination, tissue samples were dissolved in concentrated HCl according to Sarruge and Haag (1974).

Number of the eggs per gram of faeces was in accordance with Leland (1995). All calves were used to determine worm burden. The small intestine was removed and opened to wash the content and mucosal scrapings. Duplicate samples of 10% of the total volume obtained were collected. The small intestine was then incubated in distilled water at 32.8 8C for 24 h to release the worms which adhered to the mucosa. All samples were preserved in 10% formalin until identification of larval stages and determine worm burden counts. 2.3. Mathematical model Fig. 1 illustrates the kinetic model involving 32P according to Vitti et al. (2000). The principles of mass conservation are the basis of the model. The differential equations which describe the behaviour of the P kinetics are assumed to be in partial equilibrium and the solutions for differential equations were obtained by equaling them to zero. These were then manipulating to obtain expressions of individual flows of interest. The flows utilized in Fig. 1 are described by the equations: F 12 ¼

s1 F˜10 ; s2  s1

(1)

F 21 ¼ F˜10 þ F 12  F˜01 ;

(2)

F 32 ¼

s3 Q 3 ; tðs2  s3 Þ

(3)

F 42 ¼

s4 Q 4 ; tðs2  s4 Þ

(4)

jF 23 þ F 24 j ¼ F˜02 þ F 12 þ F 32 þ F 42  F 21 ; ðs3þ4  s3 ÞjF 23 þ F 24 j ; s4  s3þ4 ¼ jF 23 þ F 24 j  F 24 ;

F 24 ¼ F 23

(5)

(6) (7)

1

where F (g d ) represents flow, s (dpm/g P) is the specific activity (SRA), t (day) is the time from the start of the experiment until the count and Q (g) is the total content of P in a certain compartment. The notation s3+4 represent the mean specific activity in compartments 3 + 4. The total endogenous P in the faeces was calculated as: F e01 ¼

F 12 F 01 s1 F 01 ¼ F 12 þ F 10 s2

(8)

where F10 is the consumption of P and F01 the P in the faeces. The true absorption of P was calculated as F abs ¼ F 10  ðF 01  F e01 Þ

(9)

2.4. Statistical analysis Experimental measurements were analyzed in a completely randomized design. The variables EPG, larval

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Fig. 1. Schematic mathematical model for phosphorus metabolism in calves, adapted from Vitti et al. (2000). Legend: P intake (F10), P in faeces (F01), P blood to TGI (F12), P in urine (F02), P TGI to blood (F21), P blood to bone (F32), P bone to blood (F23), P blood to soft tissues (F42) and P soft tissues to blood (F24).

and worm burden counts were transformed by log(x + 10). A comparison of means between each treatment was carried out using the general linear model (GLM) procedure of SAS (2000). Treatment means were assessed using the least significant difference method when overall treatment effects were P  0.05. 3. Results No marked changes were noted in faeces consistency, which were soft at the beginning of trial, but became normal after a few days. The animals, however, were seen to be depressed. The EPG counts for days 0, 7, 14, 21 and 28 were 0, 0, 1150  35, 2470  393 and 1920  168, respectively. The EPG increased until 21 days, when it started to decline. After the 32P injection the faeces became radioactive, thereby no longer used for EPG. The mean total parasite number at slaughter was 11,131  1500 (9702  1470 adult worms with 1229  76 immature forms). The variation in live weight of calves during the experiment is shown in Fig. 2. A decrease in weight in

Fig. 2. Live weight variation in experimental control and calves infected with C. punctata, trickle infection (10,000 L3 per week for 28 days).

Table 2 Intake, daily gain, total protein, albumin and P metabolism in control and calves infected with C. punctata, trickle infection (10,000 L3 per week for 5 weeks). Variables

Dry matter intake (g d1) P intake (g d1) Dairy gain (g d1) Plasma P (mg 100 mL1) Total protein (g 100 mL1) Albumin (g 100 mL1) Endogenous P in faeces (g d1) Dietary P absorbed (g d1) P faeces (g d1) P retained (g d1) Biological availability (%) a

Treatments Control (n = 5)

Infected (n = 5)

2157a 18.13a 282a 7.39 7.10 3.10 1.50 15.40a 4.41 13.72a 84.94

1670b 12.56b 68b 7.74 7.00 3.10 1.34 10.86b 3.15 9.41b 86.46

and b: means with different letters in the same row are significantly different (P < 0.05). * Mean standard error.

S.E.*

Probability

6.17 8.51 0.01 0.30 0.23 0.05 0.41 0.54 0.65 0.66 4.15

0.01 0.01 0.0001 0.40 0.81 0.44 0.79 0.01 0.19 0.01 0.42

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Table 3 P Input and output (g d1) from body compartments of control and calves infected with C. punctata, trickle infection (10,000 L3 per week for 5 weeks). Variables

Flow

P P P P P P P P P P P

intake faeces urine blood to TGI TGI to blood blood to bone bone to blood bone balance blood to soft tissues soft tissues to blood soft tissues balance

F10 F01 F02 F12 F21 F32 F23

a

and b: means with different letters in the same row are different (P  0.05). * Mean standard error.

F42 F24

Treatments Control (n = 3)

Infected (n = 3)

18.14a 3.52 0.00 15.17a 29.86a 20.71 8.43 12.28a 3.95a 1.61 2.34a

12.57b 2.41 0.00 6.61b 16.77b 19.67 9.49 10.18b 2.04b 2.06 0.02b

infected calves submitted to a tickle infection can be observed fall from 72.0 to 70.2 kg LW), while control calves maintained their weight gain (77.6–84.8 kg LW) (P < 0.05). The means of daily dry matter (DM) and P intake, and P metabolism parameters are shown in Table 2. Higher values of DM intake, P intake, P absorption and P retention were observed in the control compared to the infected calves (P < 0.01). According to data shown in Table 3, some fluxes between body compartments were changed due to trickle infection. Fluxes (F10, F12, F21 and F42) and tissue balances (bone and soft) were higher in control when compared to infected calves (P < 0.01). 4. Discussion In this experiment calves showed average EPG and total worm burden counts average 1847 and 11,131, respectively, denoting a severe acute infection according to Ueno and Gonc¸alves (1998). The difference observed in live weight between control and infected calves was a response to the DM intake. Infected animals showed no weight gain due to low DM intake. According to Kertz (2002), based on NRC (2001), P requirements for calves of about 100 kg of body weight is 0.31% of DM intake, i.e. 8.3 g d1. In the present experiment, P intake was 0.84 and 0.75% of DM intake for control and infected calves respectively, much higher than recommended for calves with weight varying from about 65 to 85 kg LW, indicating the level of P in the diet was not responsible for weight loss of infected calves. Weight gains observed here were 282 g d1 for control calves and 61 g d1 for infected calves. Some authors have reported weight loss or impaired gain due to maintenance or decrease of DM intake as result of trickle infection. Coop and Field (1983) observed significant differences (P < 0.05) in DM intake and weight gain in lambs experimentally infected with 2500 L3 of Trichostrongylus vitrinus for 12 weeks. Armour et al. (1987) reported a decrease in voluntary intake for experimentally infected calves with 60,000 L3 of C. oncophora during a 6-week-period. Possible explanations for the decrease in DM intake by infected animals involve increase in gastrin and cholecystokinin secretion, hormones related to control of feed intake (Soulsby, 1982). This may be due to morphological

S.E.*

Probability

0.04 1.76 0.00 5.34 7.00 11.75 11.74 0.47 1.41 2.43 0.53

0.01 0.28 – 0.03 0.02 0.87 0.86 0.03 0.05 0.73 0.03

and biochemical changes in epithelial cells as a result of parasite activities in intestine mucosa (Holmes and Coop, 1994). Dynes et al. (1998) observed an increase in feed intake two hours after intracerebral infusion of loxiglumid, a cholecystokinin antagonist, in lambs experimentally infected with Trichostrongylus colubriformis. Although the action of hormones has a real influence on inappetence genesis, they are only part of the cause. Another important factor is abdominal pain, as discomfort can lead to a decrease in feed intake. Some situations regarding parasitism in ruminants, such as grinding teeth and depression, were observed in some of the infected animals here, and are signs of pain. These factors, however, are difficult to evaluate due to their highly subjective nature (Holmes, 1985). P absorption and P retention (g d1) were higher in control calves than infected (P < 0.05), but absorption efficiency was similar between control (0.85) and infected (0.86) calves. These values are considered high (Braithwaite, 1981) and expected in young animals (Vitti et al., 2000). This suggests that, in the case of imposed chronic infection the ability to absorb the mineral was maintained, possibly due to the lower quantity of P in the diet (F01) and endogenous P (F21), lower degree of lesions in the intestinal mucose membrane or a more efficient compensatory system. In another experiment evaluating P kinetics, but with a single infection of 45,000 L3 of C. puctata in calves (66 kg LW) and consumption of 12.43 g d1 P, the absorption efficiency was lower (0.76) (Louvandini person communication). Therefore, the time and quantity of parasitic infection in this case may act on the mucose in a more severe manner, thereby affecting mineral absorption. The decrease in DM and P intake in infected calves changed the flows of P through the body compartments. Flows F10 (intake), F12 (blood to TGI), F21 (TGI to blood) and F42 (blood to soft tissues) were significantly higher (P < 0.05) in control calves. The lower F12 flow in infected animals, 56.63% lower than in healthy calves, should decrease losses of endogenous P, which are associated with the ingestion of feeds and in particular the production of saliva in ruminants. The F21 flow was lower in infected calves, and allowed a positive balance in the bones, but not in the soft tissues, which was reflected

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by weight loss in infected calves. This indicated that the P present in soft tissues may also be used to control mineral homeostasis. Thus, the importance of hidden parasitic infections is underlined. Damages caused by intestinal parasites are not easily detected without the means used in the present experiment, and may impair animal metabolism. The fall in feed intake caused by trickle infection with C. punctata led to impaired P metabolism in experimentally infected calves and worsened the problem. The secondary action of the parasite causing a reduction in appetite led to a greater difference in retention between infected and control animals, which was 4.31 g d1 compared to calves with a single infection where animals showed no loss in appetite and the difference in retention between healthy and infected animals was only 1.71 g d1 (Louvandini personal communication). This fact, as well as P deficiency in soils and pasture (Tokarnia et al., 1988) and the high prevalence of the C. punctata in bovines (Bianchin et al., 1990; Honer and Vieira-Bressan, 1992; Lima, 1998), may limit P intake and absorption by infected animals under field conditions. In conclusion, P metabolism in calves submitted to a trickle infection with C. punctata negatively affected the P kinetics, as a consequence of lower DM and P intake, as well as P retained and especially led to loss of weight. Acknowledgement We would like to thank FAPESP (project number00/00640-0) for the financial support. H. Louvandini, S.M. Gennari, C.M. McManus and D.M.S.S. Vitti are in receipt of a fellowship from CNPq - Brazil. References Armour, J., Bairden, K., Holmes, P.H., Parkins, J.J., Ploeger, H., Salman, S.K., Mcwilliam, P.N., 1987. Pathophysiological and parasitological studies on Cooperia oncophora infections in calves. Research in Veterinary Science 42, 373–381. Ben-Ghedalia, D., Tagari, H., Zamwel, S., Bondi, A., 1975. Solubility and net exchange of calcium, magnesium and phosphorus in digesta flowing along the gut of the sheep. British Journal of Nutrition 33, 87–94. Bianchin, I., Honer, M.R., Nascimento, Y.A., 1990. The epidemiology of helminths in Nellore beef cattle in the cerrados of Brazil. In: Epidemiology of bovine nematode parasites in the Americas. 16th World Buiatrics Congress, Salvador, Bahia, Brazil. Bown, M.D., Poppi, D.P., Sykes, A.R., 1989. The effects of a concurrent infection of Trichostrongylus colubriformis and Ostertagia circumcincta on calcium, phosphorus and magnesium transactions along the digestive tract of lambs. Journal of Comparative Pathology 101, 12–20.

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