ANNUAL VARIATIONS OF THE SODIUM CHANNEL BLOCKING TOXIN IN VENEZUELAN CATTLE PRONE TO DEVELOP THE BOVINE PARAPLEGIC SYNDROME: ITS DEPENDENCE ON RAIN

Abstract

A characteristic of the bovine paraplegic syndrome (BPS) is ventral or sternal decubitus in animals that make unsuccessful efforts to stand when stimulated. Death occurs after a few days. In a previous work we showed the existence of a sodium channel blocking toxin (SCBT) produced by ruminal bacteria in cattle with or liable to develop BPS. The presence of SCBT in bovine plasma sampled monthly during the rainy and dry seasons in animals without BPS has now been observed. A positive correlation between rain precipitation and plasma toxin levels (Spearman's rank correlation coefficient=0.457, n=135, p<10<img SRC="http:/img/fbpe/jvat/v4n1/image522.gif"> ) was found. Precipitation was 194 (121-289) mm water/month (n= 162 months in 27 years, median and its 95% confidence interval) during the rainy season and 7 (0-35) mm water/month (n=109 months in 28 years) during the dry season [p < 10<img SRC="http:/img/fbpe/jvat/v4n1/image523.gif"> , Mann-Whitney (Wilcoxon) test]. Plasma toxin levels were determined by high performance liquid chromatography (HPLC). Toxin levels, expressed as peak units of area (PUA) at 340 nm, (1 PUA = 2.5x10<img SRC="http:/img/fbpe/jvat/v4n1/image523.gif"> units of absorbance/cm) were 5.5 (0.0-23.3) PUA/nl (n = 133) in the rainy season and 0.0 (0.0-1.1) PUA/nl in the dry season (n= 88, p=2x10<img SRC="http:/img/fbpe/jvat/v4n1/image523.gif"> ). The distribution of toxin concentration in both groups was also different (<img SRC="http:/img/fbpe/jvat/v4n1/image524.gif"> test, p=0.024, 7 degrees of freedom). It is then determined that toxin was not detectable in 84.1 % of the cows in the dry season (n = 88) and in only 38.3% of the cows in the rainy season (n = 133) (<img SRC="http:/img/fbpe/jvat/v4n1/image524.gif"> test, p<10<img SRC="http:/img/fbpe/jvat/v4n1/image523.gif"> ).

bovine paraplegic syndrome (BPS); cattle; sodium channel blocking toxin (SCBT); annual variation; rain dependence

Original paper

ANNUAL VARIATIONS OF THE SODIUM CHANNEL BLOCKING TOXIN IN VENEZUELAN CATTLE PRONE TO DEVELOP THE BOVINE PARAPLEGIC SYNDROME: ITS DEPENDENCE ON RAIN

C. SEVCIK

, G. D'SUZE

1 Laboratory of Cellular Neuropharmacology, Center of Biophysics and Biochemistry, Venezuelan Institute of Scientific Investigations (IVIC).

ABSTRACT. A characteristic of the bovine paraplegic syndrome (BPS) is ventral or sternal decubitus in animals that make unsuccessful efforts to stand when stimulated. Death occurs after a few days. In a previous work we showed the existence of a sodium channel blocking toxin (SCBT) produced by ruminal bacteria in cattle with or liable to develop BPS. The presence of SCBT in bovine plasma sampled monthly during the rainy and dry seasons in animals without BPS has now been observed. A positive correlation between rain precipitation and plasma toxin levels (Spearman's rank correlation coefficient=0.457, n=135, p<10 ) was found. Precipitation was 194 (121-289) mm water/month (n= 162 months in 27 years, median and its 95% confidence interval) during the rainy season and 7 (0-35) mm water/month (n=109 months in 28 years) during the dry season [p < 10 , Mann-Whitney (Wilcoxon) test]. Plasma toxin levels were determined by high performance liquid chromatography (HPLC). Toxin levels, expressed as peak units of area (PUA) at 340 nm, (1 PUA = 2.5x10 units of absorbance/cm) were 5.5 (0.0-23.3) PUA/nl (n = 133) in the rainy season and 0.0 (0.0-1.1) PUA/nl in the dry season (n= 88, p=2x10 ). The distribution of toxin concentration in both groups was also different ( test, p=0.024, 7 degrees of freedom). It is then determined that toxin was not detectable in 84.1 % of the cows in the dry season (n = 88) and in only 38.3% of the cows in the rainy season (n = 133) ( test, p<10 ).

KEY WORDS: bovine paraplegic syndrome (BPS), cattle, sodium channel blocking toxin (SCBT), annual variation, rain dependence.

INTRODUCTION

Bovine paraplegic syndrome (BPS) is a disease known to occur in cattle in the South American plains from Venezuela to Paraguay(8). BPS(1) occurs in healthy heifers and cows, mostly in lactating or pregnant cows. The zebus (Bos indicus) and their crossbreeds are the most affected animals. It is not clear, however, if the high occurrence among the zebus is due to this species being the most common in the area BPS occurs. Most of the cases happen in the beginning of the rainy season (May-June) and at end of the rainy season (October-November) in Venezuela. At the onset of the disease, the animals present uncoordinated gait which is most evident in the hind legs. Ventral or sternal decubitus is always present, and thus, the animals cannot stand without help even after being stimulated to do so. Most of the clinical histories report normal deglutitory reflex and absence of both mydriasis and of tongue protrusion. There is no visual impairment and the animals are usually alert. The head and neck are usually held in a normal position, although in a few cases, the head lies on the ground or on the scapula, and no macroscopic alterations are found in autopsies.

Previous work showed a toxin that can block sodium channels (SCBT) in cows downed by BPS (8). This toxin is produced by anaerobic bacteria living in the ruminal fluid (2). Our work suggested that the toxin can be found both in cows with BPS and in apparently healthy animals (8). Toxin concentration, however, was higher in tissues of animals stricken with BPS.

We have now observed that the plasma concentration of the sodium channel blocking toxin in apparently healthy zebu cattle or their crossbreeds living in Venezuelan areas where BPS is not frequent and now depends on rainfall.

MATERIALS AND METHODS

ANIMALS AND SAMPLE COLLECTION: We observed the plasma concentration of SCBT by sampling plasma once a month. Samples were collected on the Turagua ranch (Apure State, Venezuela, 68° 19' 27" W, 7° 48' 15" N). The data presented here were sampled from August 1993 to September 1994. Turagua ranch has a herd of more than 10,000 head of cattle; 205 animals were studied during the rainy season, 88 during the dry season and 38 animals were sampled during the intermediate months. Five-milliliter blood samples were drawn from the jugularis externa vein (6) of cows using evacuated blood collection tubes with EGTA (Vacutainer ). The blood was kept on ice for a couple of hours until being centrifuged ( 800 g, 25 min) to separate plasma. Plasma was kept in a dry ice chest in the field or at -20°C in the laboratory.

PROCESSING OF THE PLASMA SAMPLES: All the chemical products used for the extractions were of analytical grade; HPLC grade reagents were used in the chromatographies. Thawed samples of plasma were mixed 1/1 v/v with methanol and centrifuged at 1,000 g for 25 min to separate proteins. The supernatant was then treated with trinitrobenzesulfonic acid (TNBS), a reagent for primary amines(5,7,8). TNBS-treated samples were centrifuged at 10,000 g for 30 min to reduce contamination of the high performance liquid chromatography (HPLC) columns.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: Analysis of TNBS-treated samples was on a reverse phase phenylborasil column 10µ particles, 4x390 mm (µBONDAPAK Phenyl) with a constant regime of 40% methanol in water, at a flow rate of 1 ml/min at 20°C as described by Sevcik et al.(8). Area of eluted peaks was determined automatically by the analyzer in µV [1 µV = 1 peak unit of area (PUA) = 2.5x10 units of absorbance/cm at 340 nm].

STATISTICAL PROCEDURES: Data were processed using nonparametric statistical methods(4). Data are presented as medians and their 95% confidence interval calculated by the procedure of Hodges and Lehmann. Statistical significance of differences was determined by the Mann-Whitney (Wilcoxon) test. Comparisons among multiple samples were made by the Kruskall-Wallis test. Ratios among data in two samples and their 95% confidence interval were calculated using the method of Moses. Linear regressions were carried out according to the Theil procedure and correlation expressed by the Spearman's rank correlation coefficient. Proportions were compared using the distribution by the Yates(9) correction as suggested by Fleiss(3). Differences among these statistical treatments were considered significant when p < 0.05 (two tails).

RESULTS

RAINFALL ON THE TURAGUA RANCH: Figure 1 presents data on rainfall on the Turagua ranch from January 1969 to January 1996. The data show that the rainfall on the Turagua ranch varies randomly to some extent, but follows a single, roughly bell shaped yearly cycle. In this study, we divided the year into three classes: a dry season with a rainfall average of 7 (0-35) mm water/month (n= 109 months), a rainy season with a rainfall average of 194 (121-289) mm water/month (n= 162 months) and an intermediate period that flanks the rainy season with a rainfall of 83 (40-131) mm water/month (n= 54 months). Statistically, the rainfall differences in these three classes were highly significant when compared with by the Kruskall-Wallis test (p<10 ).

DEPENDENCE OF SCBT PLASMA CONCENTRATION ON RAINFALL: The data in Figure 2a and Figure 2b show that the plasma levels of SCBT were as follows: dry season 0 (0-1.1) PUA/nl (n= 88); intermediate period 0.8 (0-9.4) PUA/nl (n= 55); and rainy season 5.5 (0-26.3) PUA/nl (n= 133). The median plasma levels of SCBT during the dry and intermediate seasons were distinct from the median levels during the rainy season when compared by the Kruskall-Wallis test (p < 2x10 ).

To evaluate the effect of rainfall on SCBT plasma levels in apparently healthy cattle, we studied the distribution of the proportion of animals with different toxin concentrations. The distributions observed during the dry and rainy seasons are depicted in Figure 3. The two distributions were distinct (p= 0.024, = 16.108, 7 degrees of freedom). Likewise, toxin was not detected in 74 of the 88 animals (84.1%) in the dry season, but only in 45 of the 133 animals (33.8%) in the rainy season; the two proportions are distinct (p< 10 ).

DISCUSSION

In our previous work(8), we attempted to use blood samples to detect the SCBT plasma levels in non-BPS cows. However, the results obtained showed that the toxin concentration in plasma of non-BPS animals fluctuated considerably and was relatively high [12.5 (6.6-41.2) PUA/nl, n=21]. The blood samples were obtained during the rainy season in regions with high occurrence of BPS(8). We have now detected the plasma levels of SCBT in apparently healthy animals from a region with low occurrence of BPS, in which BPS has not been a problem during the last 4 years.

This paper shows that SCBT plasma level obtained from apparently healthy animals is at any time very low. If we pool all the results on plasma level of SCBT of the 276 cows studied under any rain condition, the SCBT plasma level is 1.2 (0-22.3) PUA/nl that is lower (P= 10 ) than the SCBT plasma level of non-BPS cows mentioned above. The present results show that when the toxin in BPS cows is studied in a large sample, the toxin is a rare finding in apparently healthy animals. The toxin-free, healthy animals comprised 33.8% in the rainy season and 84.1% in the dry season. In addition, this paper shows a significant tendency to find higher plasma concentrations of SCBT in apparently healthy animals during the rainy season, precisely when the disease is observed.

One important point is the possibility to predict the risk of BPS occurrence in cattle herd using an assay to measure plasma levels of SCBT. We are not sure to have a final answer, but considering that our sampling of the Turagua ranch during the rainy season had the highest values and the animals were not downed; this may be a guide for estimating the animal tolerance considering the SCBT plasma level concentration. Thus, a herd of cattle may be apparently healthy showing a median SCBT plasma concentration below 12 PUA/nl. This conclusion is supported by our prior finding that the blood concentration of SCBT in BPS cows in Venezuela was 33.4 (19.4-43.3) PUA/nl (n= 8)(8). As discussed in our previous paper(8), the value detected in blood samples of Venezuelan BPS was probably underestimated. Venezuelan and Paraguayan values did not differ (p= 0.95), however, these values were different comparing with those of non-BPS cattle during the rainy season on the Turagua ranch (p < 10 ).

FIGURE 1.
Monthly rainfall on the Turagua ranch during 27 years. The ordinate is the monthly rainfall in mm/water.

FIGURE 2. Rainfall and sodium channel blocking toxin plasma levels of apparently healthy cattle during the rainy, dry and intermediate seasons on the Turagua ranch.

FIGURE 3. Distribution of plasma toxicity of apparently healthy animals on the Turagua ranch.

ACKNOWLEDGEMENTS

Supported in part by grants from the International Foundation for Science (IFS), Sweden, (No. B/2039-1, GD'S), Petróleos de Venezuela (PDVSA, Grant No. 4.2, CS) and the Consejo Nacional de Investigaciones Científicas (CONICIT), Venezuela (Grant No. S1-2086, CS). The field work was supported by Agropecuaria Flora (branch of The Union International Ltd., UK) on their Turagua ranch.

REFERENCES

01 CHICCO RC., LINARES T. Avances en el estudio de la caracterización del síndrome parapléjico del bovino. PROGRAMA DE COOPERACIÓN AGRÍCOLA CONVENIO MAC/PDVSA SÍNDROME PARAPLÉJICO DEL BOVINO Y MEJORAMIENTO INTEGRAL DE LA GANADERÍA. Caracas, 1992. (Boletín 3)

02 DOMÍNGUEZ-BELLO MG., LOVERA M., SEVCIK C., BRITO J. Characterization of ruminal bacteria producing a toxin associated with a bovine paraplegic syndrome. Toxicon, 1993, 31, 1595-600.

03 FLEISS JL. Statistical methods for rates and proportions. New York: Wiley, 1973.

04 HOLLANDER M., WOLFE DA. Nonparametric statistical procedures. New York: Wiley, 1973.

05 OKUYAMA T., SATAKE K. On the preparation and properties of 2,4,6-trinitrophenil-amino acids and peptides. J. Biochem.,1960, 47, 454-66.

06 POPESKO P. Atlas de anatomía topográfica de los animales domésticos. Salvat: Barcelona, 1990, 1: 25.

07 SATAKE K., OKUYAMA T., OHASHI M., SHINODA T. The spectrophotometric determination of amine, amino acids and peptides with 2,4,6-trinitrobenzene-1- sulfonic acid. J. Biochem., 1960, 47: 654-60.

08 SEVCIK C., BRITO JC., D'SUZE G., DOMÍNGUEZ-BELLO MG., LOVERA M., MIJARES AJ., BONOLI S. Toxinology of the bovine paraplegic syndrome. Toxicon, 1993, 31, 1581-94.

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Received 07 February 1997

Accepted 21 July 1997

Publication Dates

• Publication in this collection
  26 Nov 1998
• Date of issue
  1998

History

• Accepted
  21 July 1997
• Received
  07 Feb 1997